5

OVERVIEW OF THE IMMUNE SYSTEM

All multicellular organisms have developed various mechanisms for defending
themselves against foreign invaders; collectively, they constitute the immune sys-
tem.

The immune system in host defense has been described as follows: A crim-
inal invades a town, threatening its safety; but before he can do any damage,
he is surrounded by police and then hauled off and confined in the local jail.


Immunity is derived from the Latin word immunis, which in
ancient Rome, referred to the exemption from various civic
duties and legal prosecution granted to Roman senators
during their terms in office.




Multicellular Organisms




Invertebrates
[Protozoa, Arthropods, etc]

Innate Immunity

Vertebrates

Innate Immunity
Adaptive Immunity


Innate Immunity (Natural, native, or nonspecific immunity)

Innate immunity is present in all individuals or animals at all times, hence, it is the
initial response to microbial invasion. Innate immunity does not improve on re-
peated exposure to a given pathogen [ie, no memory]; and does not discrimi-
nate between pathogens.

Constitutes the first and second lines of host defense.

Adaptive (Acquired or specific immunity)

The adaptive immune response [mediated by T and B lymphocytes] is highly
specific for a particular pathogen. In general, it takes several days for it to be-
come fully functional when a pathogen invades the body.

Unlike the innate immune response, the adaptive response improves with
each successive encounter with the same pathogen [ie, immunologic me-
mory]. Constitutes the third line of host defense.

6
Infection Reinfection
Innate Immunity
Disease
Adaptive Immunity
Recovery
Specific Immuno-
logic Memory
No Disease
Interaction of Innate and Adaptive Immune Responses

When a microbe eludes innate immune responses, adaptive immune response is
then enlisted. However, acquired immunity does not operate independently of in-
nate immunity; rather, it supplements and augments the nonspecific defense
mechanisms, producing a more effective total response.














Components of Innate Immunity

The first line of defense against microorganisms is the intact skin and mucous
membranes lining the gastrointestinal, respiratory, and genitourinary tracts. If a
microbe breaches this line and enters the body, then the other components of the
innate arm, eg, neutrophils, macrophages, are available to destroy the invader.

First line of defense Second line of defense
Intact skin
Mucous membranes and their
secretions, eg, mucus
Normal flora
Phagocytosis
Inflammation and fever
Antimicrobial substances

Age. The very young and the very old are much more susceptible to infectious
agents because their immune responses are suboptimal.

Poor nutrition. As individuals age, they sometimes lose some sense of smell
and taste. As a result, appetites may decrease and vitamin deficiencies in-
crease. Dietary components such as protein and vitamin A, D, C, and B com-
plex are necessary for healthy immune responses.

Skin. Mechanical barrier. The continuous sheets of tightly packed epithelial
cells and keratin layer making up the epidermis, act as a formidable barrier to the
entrance of microorganisms. Epithelia also produce peptides that have a natural
antibiotic function, eg, defensins [see Inflammation and Phagocytosis, page 50].
In humans, the epidermis is completely renewed every 15-30 days [so-called
epithelial turnover].
7
Sebum (produced by sebaceous [oil] gland). Forms a protective film over the
surface of the skin. Contains lactic and fatty acids that inhibit the growth of
many microorganisms [pH 3-5].

Perspiration. Flushes microorganisms from the surface of the skin. Sweat
also contains lysozyme.

Oral cavity. Saliva washes microorganisms from teeth and gums. It also con-
tains antibacterial agents, eg, lysozyme.

Gastrointestinal tract

Low pH of the stomach.
Normal flora.
Peristaltic movement, eg, vomiting and diarrhea.
Proteolytic enzymes, bile acids, and pancreatic secretions.

Respiratory tract

Mucociliary escalator.
Coughing and sneezing [speeds up the escalator].
Alveolar macrophages.

Eyes. Flushing action of tears. Tears also contain lysozyme.

Genitourinary tract

Urine. Flushing action of urine; acidity of urine. Urine contains lysozyme.
Vaginal lactic acid.

Normal flora. These are the microorganisms [mostly bacteria, fungi, protozoa]
that colonize a host without causing disease. However, under certain circum-
stances, some flora can cause disease. They contribute to host defense by pre-
venting potential pathogens from colonizing the host.

Competition for attachment sites and nutrients [competitive exclusion].

Produce substances that are harmful to pathogens, eg, bacteriocins [antimi-
crobial peptides produced by bacteria that kill or inhibit other bacteria]. In the
large intestines, E. coli produce colicins that inhibit the growth of Salmonella
spp. and Shigella spp.

Altering conditions that affect the survival of pathogens, eg, pH and O
2
availa-
bility. Lactobacillus acidophilus in the vagina alters its pH to prevent over-
population by Candida albicans.

8
Iron-binding proteins. These are proteins that sequester iron, thereby reducing
iron available to a pathogen, eg, transferrin, lactoferrin, and haptoglobin.

Iron plays a critical role in bacterial respiration as a component of the cyto-
chromes and the iron-sulfide proteins involved in electron transport.

A number of pathogenic bacteria produce iron-chelating compounds such as
siderophores which can remove iron from iron-binding proteins.

Oxygen tension. Inhibits the growth of obligate anaerobic bacteria, especially in
the lungs.

Complement. Plasma and cell surface proteins associated with lysis of Gram-
negative bacteria, chemotaxis of phagocytes, and opsonization [the coating of an
antigen or particle that facilitates its uptake into a phagocytic cell].

Interferons. Low molecular weight glycoproteins produced by certain cells in
response to viral infections. IFNs have antiviral and immune regulatory activity.

Temperature. Body temperature inhibits replication of some pathogens; fever
enhances phagocytosis. Also antibody production and T cell proliferation are
more efficient at higher body temperatures than at normal levels.

Fever can be induced by bacterial endotoxin (lipopolysaccharide [LPS]) and
interleukin-1.

Inflammation. This is a process which begins following sublethal injury to tissue
and ends with complete healing. Cause may be microbiological, physical, or
chemical. Inflammation results in the bringing of blood components and cells of
the immune system to the site of tissue damage.

Components of Specific Immunity

Antibody-mediated [humoral] immunity. Antibodies are soluble proteins pro-
duced as a result of interaction between a B lymphocyte and an antigen. The
antibody has the ability to combine with the antigen that stimulated its production.

Antibodies are found in plasma, lymph and tissue fluids of the body. They are
most effective in eliminating extracellular antigens and bacterial toxins.

Cell-mediated immunity. An adaptive immune response in which antigen spe-
cific T cells play the main role. Macrophages and natural killer cells, although
nonspecific cells, are included with T cells in cell-mediated immune responses.

CMI responses are most important against intracellular parasites, in allograft
rejection, and in delayed hypersensitivity reactions.
9
Phases of Specific Immune Responses

The adaptive immune response is divided into three phases: the recognition of
antigen by antigen-specific lymphocytes, the activation of the lymphocytes, and
the effector phase which results in destruction of the antigen.

Activation of lymphocytes results in clonal expansion. Clonal expansion is the
proliferation of antigen-specific T and B lymphocytes in response to antigenic
stimulation and precedes their differentiation into effector cells and memory
cells. It is a critical step in adaptive immunity, allowing rare antigen-specific T
and B cells to increase in number so that they can effectively combat the pa-
thogen that elicited the response.

Following elimination of the antigen, the immune response subsides and ho-
meostasis is restored.
Cells Involved in the Immune Response

The cellular elements of blood, including the white blood cells of the immune
system, platelets and red blood cells, are derived from the same progenitor cells,
ie, hematopoietic stem cells, in the bone marrow. There are two main lines of
differentiation of pluripotent stem cells:
10
The myeloid lineage produces monocytes, neutrophils, basophils, eosino-
phils, and other cells.

The lymphoid lineage produces lymphocytes.

Comparison of Innate and Adaptive Immunity
Characterisitic Innate Immunity Adaptive Immunity
Onset

Specificity

Memory

Response to vaccination

Cells
Rapid [minutes to hours]

Antigen nonspecific

No

No

Neutrophils, macrophages,
NK cells, dendritic cells, etc
Slow [days to weeks]

Antigen specific

Yes

Yes

T and B lymphocytes

Plasma Proteins

Plasma protein represents a complex mixture of a number of proteins of different
structural and functional properties. The major plasma proteins are albumin,
11
fibrinogen, and globulin. Virtually all the albumin and fibrinogen, and 50% to 80%
of the globulins are produced in the liver. The remaining globulin proteins [gam-
ma globulins] are produced in the lymphoid organs and tissues.

Globulins. Globulins are principally responsible for the bodys innate and
adaptive immune responses against invading pathogens. They comprise of
complement proteins and antibodies.















Immunopathology

Although host immune response is critical to the maintenance of health, on
occasion, the immune system itself is a cause of disease or other undesirable
effects. Host immune responses can result in the following outcomes:

Autoimmunity: The response is directed against self-antigens.
Immunodeficiency: Ineffective immune response.
Hypersensitivity: Overactive immune response resulting in allergies.
Transplantation reaction. Rejection of allograft.

Cluster of Differentiation (CD) Molecules

These are cell surface molecules expressed on a variety of cell types in the im-
mune system. The cell surface molecule is designated CD followed by a number
[eg, CD1, CD2, CD3, etc].

CD molecules are identified using monoclonal antibody. They may be used
as markers to differentiate different cell populations, such as CD4
+
T cell [T
helper cell] or CD8
+
T cell [cytolytic T lymphocyte].
Diffusion of plasma, interstitial
fluid, and dissolved constituents
through the walls of the capillary
and through the interstitial spac-
es.
12
ANTIGENS (antibody generator) AND HAPTENS

Antigens. Substances that can induce humoral and/or cell-mediated immune
response[s] when introduced into an individual or animal. The antigen must be
capable of reacting specifically with the antibodies or sensitized T cells produced
against it.

Microbial antigens. Bacteria, fungi, viruses, protozoa, and helminth parasites.

Nonmicrobial antigens. Foreign proteins, food antigens, plant antigens [eg,
pollen], cell surface proteins [eg, red blood cell antigens], etc.

Requirements for Antigenicity

The degree of antigenicity of a molecule is influenced by several factors. Among
them are the following:

Foreignness (Self vs Nonself). The macromolecule must come from a foreign
source, either an entirely different species or an animal of the same species that
is antigenically different from the animal being immunized. The more distant or
foreign the antigen source, the more vigorous the immune response.

Autologous antigens. Antigens found within the same individual.

Syngeneic [isogeneic] antigens. Antigens found in genetically identical indivi-
duals, eg, identical twins or inbred strains of mice.

Allogeneic antigens (alloantigens). Antigens found in genetically dissimilar
members of the same species, eg, blood-group antigens.

Xenogeneic antigens. Antigens found in different species.

Chemical complexity. The more complex a molecule, the more varied the epi-
tope [antigenic determinant] composition, hence the more likely different [indivi-
dual] immune responses will be induced.

Proteins. Proteins are the most effective antigens. They are often composed
of 18 or more amino acids. This diversity imparts epitopes of differing specifi-
cities to the protein.

Polysaccharides. Most polysaccharides are nonantigenic because they do
not possess sufficient chemical diversity; additionally, they are rapidly degra-
ded before the immune system has had time to respond to them.

More complex polysaccharides are antigenic, eg, capsular polysaccha-
rides and lipopolysaccharides.
13
Antigenicity of polysaccharides is enhanced if they are coupled to proteins
as glycoproteins.

Lipids. Weak antigens due to their structural simplicity and rapid metabolism.
However, immune responses to lipids may be enhanced when they are conju-
gated to proteins [lipoproteins] or polysaccharides [glycolipids].

Molecular size. The most potent antigens are proteins with high molecular
weights, ie, above 100,000. In general, molecules with molecular weight below
10,000 are weakly antigenic, and very small ones, eg, an amino acid, are non-
antigenic.

The number and variety of epitopes increase proportionately with the size of
the protein. With carbohydrate antigens, the number of epitopes may in-
crease with size but the diversity usually does not.

Moderate to large size molecules are readily internalized and processed by
antigen-presenting cells (see Major Histocompatibility Complex [MHC] and
Antigen Presentation, page 74).

Stability. Lymphocyte antigen receptors recognize an antigen by its shape.
Thus, highly flexible molecules that have no fixed shape are poor antigens.

Degradability. T cells respond only to processed antigens. Antigen-presenting
cells must first degrade the antigen before they can express antigenic peptides
noncovalently bound to MHC molecules, on their cell surface. Macromolecules
that cannot be degraded and presented with MHC molecules are poor antigens.
[see MHC and Antigen Presentation, page 74].

Genetic makeup of the host. The genetic constitution of the host determines
whether a given molecule will stimulate an immune response. Different strains of
the same species of animal may respond differently to the same antigen.

Method of administration. Dosage and route of administration also affect anti-
genicity.

Dosage. Low doses of an antigen may not stimulate an immune response ei-
ther because the amount given fails to activate enough lymphocytes or ren-
ders the lymphocytes unresponsive. On the other hand, very high doses of
antigen may lead to immune paralysis [see Autoimmunity, page 227].

Route. The route of antigen administration determines which organs and cell
populations will initiate the immune response. Antigens administered subcu-
taneously [beneath the skin], usually elicit the strongest responses. The anti-
gens are taken up by Langerhans cells present in the skin, carried to local
lymph nodes, where they are processed and presented to T cells.
14
Antigenic Determinants (Epitopes)

Epitopes are the sites on or within the antigen that stimulate the immune res-
ponse and against which that response is directed. Thus, epitopes determine the
specificity of the antigen molecule. Internal epitopes are expressed only after the
antigen has been partially degraded in vivo by antigen-presenting cells.

Antigens are polyvalent [many epitopes of different specificities] or multi-
valent [many epitopes of the same specificity]; thus, simultaneous immune
responses may be mounted against the various epitopes on these antigens.

Antigen-Antibody Binding


Accessibility of the epitope. Since hydrolytic products of the antigen gener-
ated by antigen presenting cells contain various epitopes, immune responses
are generated against both internal and surface epitopes.

Only those epitopes on the outside of the parent molecule are strongly im-
munogenic; epitopes within the molecule are weakly immunogenic.

Cross-Reactivity (Heterophile Antigens)

Sometimes antibody elicited by one antigen can cross-react with an unrelated
antigen. Such cross-reactivity is possible because the two unrelated antigens
share one or more identical or very similar epitope[s]. In the case of similar epi-
topes, the affinity [binding strength] of the antibody for the cross-reacting epitope
is usually less than that for the original epitope.


Antiserum to protein A
reacts with A and B.

Antiserum to protein B
reacts with A, B, and C.

Antiserum to protein C
reacts with B and C.
15
Cross-reactivity provides the basis for the following: Some autoimmune dis-
eases [molecular mimicry, see Autoimmunity, page 229]; heterologous vac-
cines [measles vaccine protects against canine distemper [see Vaccines and
Vaccination, page 168]; and false-positive diagnosis.

Antigen-Binding Molecules in Adaptive Immunity

B cell receptor [BCR; see page 92]. This is the cell surface receptor of B cells
that recognizes a specific antigen. It consists of a membrane immunoglobulin
molecule in association with the signal transduction molecules Ig and Ig.

T cell receptor [TCR; see page 78]. This is the cell surface receptor of T cells
that binds to antigenic peptide presented in association with major histocom-
patibility complex molecule. It is made up of and protein chains that asso-
ciate with the signal transduction molecules CD3 plus [zeta].

Major histocompatibility complex [MHC] molecules [see page 66]. These are
proteins encoded by MHC genes. They are classified as class I, class II, and
class III MHC molecules. Class I and class II MHC molecules are cell surface
molecules that present antigenic peptides to T cells.







Haptens (Greek haptein, to grasp or fasten)

A hapten is a nonantigenic molecule, usually of low-molecular-weight, that by it-
self, cannot induce an immune response, but can react with the products of that
response. Haptens include some antibiotics, analgesics, poison ivy, etc.

To induce an immune response, a hapten must always be coupled to a carrier
substance, preferably protein antigen [called carrier protein]. The hapten acts
as a new epitope of the carrier protein and an immune response is generated
to both the hapten and native epitopes of the protein. Carrier proteins include
serum albumin, globulins, and synthetic polypeptides.

Material injected Antibody formed

Hapten None

Carrier protein Carrier antibody

Hapten-carrier Carrier antibody
protein conjugate Hapten antibody
16
Antibody specific for a given hapten can recognize and bind the hapten, whe-
ther the hapten is free or bound to a carrier substance.

Significance of Haptens. Karl Landsteiner is credited with most of the work
with haptens. His findings illustrate the diversity of the immune response and
specificity of antigen-antibody and antigen-T cell reactions, ie, Paul Ehrlichs
lock-and-key phenomenon.

Reactivity of Antisera [Antibodies] with Various Haptens



Autocoupling Haptens

These are a unique class of haptens which possess the ability to form sponta-
neous covalent bonds with self proteins to create neoantigens [new antigens] in
vivo. These conjugates represent novel antigens to the animal or individual, and
the animal or individual responds with an immune response.

Unlike the usual response to antigen-hapten complexes, the response to au-
tocoupling haptens can have serious consequences in the body.

Poison ivy [see Type IV Hypersensitivity, page 222]. The resin of poison
ivy [urushiol], binds to any protein it comes in contact with, including skin
proteins. The modified skin proteins are regarded as foreign and attacked
by lymphocytes, and an allergic contact dermatitis results.

17
CYTOKINES

Cytokines are soluble proteins secreted by the cells of innate and adaptive immu-
nity that stimulate [and occasionally suppress] the growth, maturation, and func-
tioning of the cells of the immune system. Cytokines usually act locally, however,
on occasion, they may exhibit endocrine action. Cytokines act by binding to spe-
cific cytokine receptors on the cells that they affect.


Terminology

Lymphokines. Cytokines primarily produced by lymphocytes.

Monokines. Cytokines primarily produced by monocytes and macrophages.

Interleukins (Abbreviated IL). Cytokines produced by leukocytes that act on
other leukocytes.

Properties of Cytokines

Cytokines are produced during the activation and effector phases of innate and
adaptive immune responses. They are active in very low concentrations.

Cytokine secretion is a brief, self-limited event [they are not stored as pre-
formed molecules]. Cytokines often influence the synthesis of other cytokines.
18
Cytokine receptors. Cytokines exert their biological effects by binding to
specific high affinity cytokine receptors [a cell may express up to 1000 re-
ceptors], triggering a series of biochemical events that influence the activities
of immune cells and organs.

- The expression of many cytokine receptors is regulated by specific sig-
nals. The signal may be another cytokine or antigen binding to the cell.

- The cytokine receptors can have circulating forms that block the cytokine
before it reaches its cellular target [regulates the activity of the cytokine].

Sources. Individual cytokines may be produced by multiple cell types.



Pleiotropism. A single
cytokine may have mul-
tiple activities.



Redundancy. A single
activity can be caused
by multiple cytokines.




Synergism. A cytokine
may work best in asso-
ciation with another cy-
tokine.



Antagonism. A cyto-
kine may antagonize
the effects of another
cytokine.



PROINFLAMMATORY CYTOKINES

These cytokines contribute to the initiation of a wide spectrum of activities during
the inflammatory response, such as fever, acute phase response, etc, [see
Inflammation, page 39]. They include IL-1, IL-6, and tumor necrosis factor-o.

Proinflammatory cytokines act in concert with chemokines and hematopoietic
cytokines to ensure the development of physiologic responses to a wide
variety of stimuli, such as microbial infections and tissue injury.
19
Cytokine-Induced Fever



Interleukin-1 (IL-1)

Sources. Activated macrophages; endothelial cells, Langerhans cells, B cells,
epithelial cells (eg, keratinocytes), dendritic cells, etc..

Types. There are two principal forms of IL-1: IL-1o and IL-1|.

IL-1o binds to the macrophage membrane, enabling macrophages to activate
lymphocytes that come in contact with them. IL-1| is secreted by cells, thus,
most of the IL-1 found in the circulation is IL-1|. Both forms bind to the same
IL-1 receptors and mediate the same biologic activities.

IL-1 receptor antagonist (IL-1ra). This is a third form of IL-1 that binds to IL-1
receptors but is biologically inactive so that it functions as a competitive inhi-
bitor of IL-1. It is an endogenous regulator that serves to prevent excessive
IL-1 driven inflammatory response.

Principal activities

Lymphocyte activation. Major co-stimulator of T
H
2 cells (T
H
1 cells lack IL-1
receptors and do not respond to IL-1).
20
Acts on endothelial cells to increase expression of adhesion molecules (E se-
lectin, ICAM-1, -2, VCAM-1) that mediate leukocyte extravasation and secrete
chemokines (CXCL8) that activate leukocytes.

Stimulates the production of neutrophils and platelets by the bone marrow.

Acute-phase reaction. Stimulates hepatocytes to synthesize increased levels
of acute-phase proteins in response to inflammation.

Nervous system. Acts on the brain to cause fever [endogenous pyrogen], le-
thargy, malaise and lack of appetite.

- Fever. IL-1 acts on the thermoregulatory center in the anterior hypothala-
mus, resulting in prostaglandin-induced (PGE
2
) fever.

Initiates metabolic wasting [mobilization of amino acids from muscle].

Interleukin-6 (IL-6)

Sources. Activated macrophages; endothelial cells, T
H
2 cells, etc.

Principal activities

T cells. Promotes IL-2 and IL-2R production and T cell differentiation.

B cells. Differentiation of B cells into plasma cells and antibody production.

Stimulates synthesis of acute-phase proteins; endogenous pyrogen.

Bone marrow. Stimulation of hematopoiesis; in concert with colony-stimu-
lating factors, it promotes the production of neutrophils and platelets.

Tumor Necrosis Factor-o (TNF-o)

Sources. LPS-activated macrophages; activated T cells, NK cells, mast cells,
etc. It occurs in cell membrane-bound forms and secreted forms.

Principal activities

Vascular endothelium. Expression of adhesion molecules and synthesis of
chemokines [eg, CXCL8].

Activates neutrophils and macrophages, enhancing their microbicidal activi-
ties.

Stimulates synthesis of acute-phase proteins; potent endogenous pyrogen.
21
Pathologic abnormalities [prolonged production or high concentrations].

- Intravascular thrombosis. TNF stimulates endothelial cell expression of
tissue factor, a potent activator of coagulation. Thrombosis of tumor blood
vessels accounts for its tumor killing activity.

- Cachexia. Characterized by wasting of muscles and fat cells [due to TNF-
induced appetite suppression]. TNF is referred to as cachectin.

- Principal mediator of septic shock in Gram-negative septicemia. Very
large amounts of TNF result in inhibition of muscle tone and cardiac con-
tractility, resulting in decreased blood pressure and shock [see page 40].

CHEMOKINES (Chemotactic Cytokines)

Chemokines comprise a large family of small proteins [8 to 12 kD in size] that
are involved in the migration of leukocytes [especially phagocytes and lympho-
cytes] from the blood to tissues and activation of leukocytes. They play a cen-
tral role in inflammatory reactions.

Chemokines are produced by leukocytes and various tissue cells such as
fibroblasts, endothelial cells, and epithelial cells.

Secretion of chemokines is induced by pathogens and by proinflammatory
cytokines, principally TNF-o and IL-1. However, some chemokines are pro-
duced constitutively in lymphatic organs, where they aid in the traffic of lym-
phocytes through the organs in the absence of inflammation.

Chemokine receptors. They consist of seven-transmembrane polypeptide
chains [the chains traverse the plasma membrane seven times] and belong to
the large family of G-protein-coupled receptors. They are classified according
to the type of chemokine[s] they bind, eg, CXCRs, CCRs, etc.

Chemokines are classified into subfamilies based on the position of two of the
four highly conserved NH
2
-terminal cysteine residues they possess.











Classification of chemokines
based on the position of two
of the four conserved N-termi-
nal cysteine residues.
22
CXC chemokines (o-chemokines). They have their first two cysteines sepa-
rated by one amino acid. They mostly attract and activate neutrophils. The
most important is CXCL8 [IL-8].

- CXCL8 is chemotactic for neutrophils and effector T cells. It activates
neutrophils, increasing their expression of integrins and enhances degra-
nulation, the respiratory burst, etc.

CC chemokines (|-chemokines). They have two adjacent cysteine residues.
They mostly recruit and activate monocytes, lymphocytes, basophils, NK
cells, and eosinophils. They include:

- CCL3/CCL4 (Macrophage inflammatory proteins [MIP-1o; MIP-1||), CCL2
(monocyte chemoattractant protein-1, MCP-1), CCL11 (eotaxin), CCL5
(RANTES, regulated upon activation, normal T cell expressed and
secreted), etc.

HEMATOPOIETIC CYTOKINES

Various cytokines produced in the immune response stimulate the growth and
differentiation of bone marrow precursor cells. Some hematopoietic cytokines:

Interleukin-3 (IL-3). Sources: Activated T cells, macrophages, mast cells,
etc.

- Promotes the growth and differentiation of stem cells into all known ma-
ture cell types (multilineage colony-stimulating factor; multi-CSF).

Interleukin-7 (IL-7). Sources: Bone marrow and thymic stromal cells.

- Growth of T- and B-cell progenitors [called lymphopoietic cytokine].

Stem cell factor (SCF). SCF is constitutively produced by bone marrow stro-
mal cells. Active as membrane-bound form or secreted form.

- Required for the earliest stages of leukocyte development in the bone
marrow. It may also play a role in sustaining the viability and proliferation
of immature T cells in the thymus and of mast cells in mucosal tissues.

GM-CSF; M-CSF; and G-CSF. Sources: Activated T cells, macrophages,
endothelial cells, mast cells, and bone marrow stromal cells.





Granulocyte-monocyte colony-stimulating factor; Monocyte-CSF;
Granulocyte-CSF. CSFs: so-called because these cytokines are
assayed, in vitro, by their ability to stimulate the formation of cell
colonies in bone marrow cultures.
23
- CSF cytokines promote the expansion and differentiation of bone marrow
progenitor cells.

SOME CYTOKINES OF ADAPTIVE IMMUNITY

Interleukin-2 (IL-2)

Sources. The main source of IL-2 is naive CD4
+
T cells and T
H
1 cells. CD8
+
T
cells produce and secrete low levels of IL-2.

Principal activities. Stimulates growth and differentiation of T cells [CD4
+
and
CD8
+
T cells], B cells and NK cells.

NK cells. Proliferation and enhanced cytolytic activity (so-called lymphokine
activated killer [LAK] cells; see Tumor Immunotherapy, page 158).

Interleukin-4

Sources. Activated T
H
2 cells; mast cells and basophils.

Principal activities

Stimulates the development and clonal expansion of T
H
2 cells from naive
CD4
+
T cells.

B cells. Stimulates B cell differentiation and antibody synthesis.

- It is the principal cytokine that stimulates B cell immunoglobulin class
switching to the IgE isotype.

T
H
1. Down-regulates IFN- synthesis by T
H
1 cells, thereby inhibiting macro-
phage activation and suppressing cell-mediated immune responses.

Stimulates growth of mast cells.

Interleukin-5 (IL-5)

Sources. Activated T
H
2 cells.

Principal activities

Eosinophils. The principal cytokine that stimulates growth and differentiation
of eosinophils; activation of mature eosinophils.

B cells. Growth and differentiation. Promotes 5-10-fold increase in IgA pro-
duction in B cells that have already isotype switched to IgA.
24
Transforming Growth Factor-| (TGF|)

TGF-| was discovered as a product of tumor cells that promotes survival of tu-
mor cells in vitro.

Sources. Activated T cells, activated macrophages and other cell types.

Principal activities. Functions to inhibit immune and inflammatory responses.

Inhibits the proliferation and effector functions of T cells; inhibits macrophage
activation.

B cells. Inhibits proliferation of B cells; however, it induces switch to IgA
production [see Isotype Switching, page 98].

Anti-inflammatory cytokine. It acts on neutrophils, endothelial cells, and other
cell types to counteract the effects of proinflammatory cytokines.

Promotes wound healing. Proliferation of fibroblasts and enhanced synthe-
sis of collagen and other proteins; stimulation of angiogenic factors.

Interleukin-12 (IL-12)

Sources. Activated macrophages and dendritic cells.

Principal activities

Stimulates the induction of T
H
1 subset from naive CD4
+
T cells.

T
H
1 cells, CD8
+
T cells, and NK cells: stimulates production of IFN-. En-
hances the proliferation and cytolytic activities of CD8
+
T cells and NK cells.

Activation of macrophages [via IFN-] and killing of intracellular microbes.

B cells. Inhibits IgE isotype switch by suppressing IL-4 synthesis.

Interferon- (Type II Interferon)

Sources. T
H
1 cells, CD8
+
T cells (cytotoxic T cells), and NK cells.

Principal activities. It is the principal activator of macrophages.

Induces class I and II MHC expression.

Promotes B cell IgG isotype switch.

25
Promotes proliferation and cytolytic activities of NK cells and CD8
+
T cells.

It acts on T
H
2 cells to inhibit the production of IL-4, thus blocking IgE isotype
switch by B cells [antagonized by IL-4 and IL-10].

INTERFERONS (IFNs)

In 1957, Isaacs and Lindenmann reported on the antiviral activity of IFN. IFNs
are a family of glycoproteins synthesized by somatic cells in response to virus
stimulation, immune stimulation, mitogens, LPS, and synthetic dsRNA.

Type I Interferons

Type I IFNs inhibit virus replication and increase expression of MHC class I mole-
cules. They also inhibit the proliferation of many cell types, including tumor cells.
IFN-o. Secreted by macrophages and other leukocytes. Activates NK cells.

IFN-|. Secreted by fibroblasts and epithelial cells. Activates NK cells.

REGULATORY CYTOKINE

Interleukin-10 (IL-10)

Sources. Activated macrophages and activated T
H
2 cells

Principal activities. It is an immunoregulatory cytokine that immunosuppresses
CMI responses (called cytokine synthesis inhibitory factor).

T
H
1 cells. Inhibits the synthesis of IFN-.

Macrophages. Inhibits macrophage activationacts on activated macro-
phages to inhibit the secretion of cytokines (IL-1, IL-12, TNF-o, etc).

- IL-10 also inhibits the expression of MHC II molecules and co-stimulators
on macrophages, thus, inhibiting T cell activation.

CYTOKINE ANTAGONISTS

These are proteins that inhibit the biological activity of cytokines. Some are of
microbial origin. They function in one of two ways: (1) they can bind directly to
a cytokine receptor without activating it [eg, IL-ra] or (2) they can bind directly to
a cytokine, inhibiting its activity.

Viroceptors. These are secreted viral proteins that inhibit cytokine signaling
by mimicking host cytokine receptors. Several poxviruses encode homologs
of IFN-R, soluble TNF-binding protein, and a soluble IL-1 binding protein.
26
Virokines. Secreted viral proteins that mimic host cytokines. Epstein-Barr
virus encodes a homolog of IL-10.

Soluble cytokine receptors. These are generated by enzymatic cleavage of
the extracellular domains of cytokine receptors. They have been identified in
the bloodstream and extracellular fluid. They include IL-2 receptor [sIL-2R],
sIFN-R, etc. The soluble receptors can bind with their respective cytokines,
preventing the cytokine from interacting with the membrane-bound receptor.

- sIL-2R. It is used as a clinical marker of chronic T cell activation and has
been observed in various pathologies, including autoimmune disorders,
allograft rejection, and AIDS.

TOXIC EFFECTS OF CYTOKINES

High concentrations of cytokines or chronic exposure to a cytokine, can result in
various toxicities.

High levels of IL-2 results in fever, chills, and vascular leak syndrome.

Toxic shock syndrome. Enterotoxins produced by some strains of S. aureus
can act as superantigens and trigger polyclonal T cell response. Cytokines
secreted by these cells [eg, TNF-o], can cause fever, hypotension, and da-
mage to the liver and kidney [see Bacterial Superantigens, page 145].

CYTOKINE THERAPY

Cytokine-based therapy includes the use of monoclonal Abs produced against
specific cytokines, recombinant cytokines, and soluble cytokine receptors.

IL-ra. It is under investigation as a possible treatment for chronic inflamma-
tory diseases.

Erythropoietin [EPO]. Used in cats and dogs with nonregenrative anemia due
to endogenous EPO deficiency in chronic renal failure.

Colony stimulating cytokines, such as GM-CSF [Sargramostim] and G-CSF
[Filgrastim] given after chemotherapy or radiation therapy improve bone
marrow recovery [stimulate production of myeloid progenitor cells].

Interferon-o |IFN-o]. Induces an antiviral state in host cells; has antitumor ac-
tivity; and promotes processing and presentation of cytosolic proteins.

- Horses: Treatment of inflammatory airway disease; cats: treatment of fe-
line leukemia; together with corticosteroids, used in the treatment of effu-
sive feline infectious peritonitis.
27
THE COMPLEMENT SYSTEM

The complement system consists of approximately 20 heat-labile serum and cell-
surface proteins, many of which are enzyme precursors that must be cleaved to
form active enzymes. Complement proteins are normally found in plasma [they
make up about 10% of the total plasma - and globulins]; they are also among
the plasma proteins that leak out of the capillaries into the tissue spaces. Com-
plement proteins are synthesized mainly by the liver and macrophages.

In 1895, shortly after the discovery of antibodies, Jules Bordet demons-
trated that Vibrio cholerae were lysed when they were exposed to fresh
specific antiserum. However, if the antiserum was heated to 56
o
C for 30
minutes or aged for a few weeks, it could no longer cause lysis but would
instead agglutinate the bacteria since antibodies are heat-stable. When
fresh guinea pig serum was added to the heated or aged antiserum, the
ability to lyse Vibrio cholerae was regained. He concluded that the lytic
effect required two factors: (1) specific antibody, and (2) a heat-labile
component present in normal serum. Later, Paul Ehrlich called the labile
component, complement, since it was thought to assist or complement the
lytic function of antibodies.

There are two main pathways for complement activation: (1) the classical
pathway, which is initiated by antigen-antibody [IgM or IgG] complexes, and
(2) the alternative pathway, which is initiated by microbial surfaces.

Both pathways lead to the production of C3b, the central molecule of the
complement cascade. C3b can combine with other complement compo-
nents to generate C5 convertase, the enzyme that leads to the terminal
pathway that produces the membrane attack complex (MAC).

Classical pathway
(Ag-Ab complex) MAC
C3 C3b C5b C9

Alternative pathway
(Microbial surface)

When the complement system is activated, multiple products are formed that
perform a variety of biological functions, including cell membrane lysis.

Complement Proteins

Complement proteins are either labeled numerically with the prefix C [C1-C9] or
designated by letters of the alphabet [B, D, etc]. In humans, the serum concen-
tration of complement proteins vary between 20 g/ml for C2 and 1200 g/ml for
C3 (the most abundant complement protein).

The enzyme cascades are generated by the sequential proteolytic cleavage
of enzyme precursors [proenzymes or zymogens] to generate enzymes with
28
proteolytic activity. In the process, an inhibitory fragment [small fragment
( a )] is removed, exposing the active site on the major fragment ( b ).

Regulatory proteins present in serum and on host cell membranes inhibit or
minimize complement-mediated damage. Since microorganisms lack these
regulatory proteins, complement activation is allowed to occur on microbial
surfaces.

Proteins of the classical pathway: C1 (C1qr
2
s
2
); C2; C3; and C4.

Proteins of the alternative pathway: C3; Factor B; Factor D; Properdin.

The Classical Complement Pathway

The pathway is initiated by binding of C1 to the Fc regions of IgM or IgG mole-
cules that have bound to antigen [see Immunoglobulins, page 107]. C1 is com-
posed of 3 subunits: C1q and two molecules each of C1r and C1s (C1qr
2
s
2
) held
together by noncovalent bonds and stabilized by calcium ions [Ca
2+
].

C1q. Looks like six globes held on
slender shafts that fuse into a common
base. The globes serve as the recog-
nition unit. When IgM or IgG binds to
its antigen, its active site on the Fc re-
gion is exposed to C1q. C1q cannot
bind to free or soluble IgM or IgG.

Each IgM or IgG Fc region possesses
only one C1q-binding site, and each
C1q molecule must bind to two Fc re-
gions to be activated. The pentameric
IgM structure readily provides two
closely spaced complement-activating
sites. In contrast, several molecules of IgG must bind to the target cell so that
two IgG molecules are close enough to each other [ 30 to 40 nm apart] to
have the same effect. Thus, IgM is a much more efficient complement-bind-
ing antibody than IgG.

C1r (serine protease). Binding of two or more of the globular heads of C1q to
IgG or IgM induces a conformational change in C1r, converting C1r to an ac-
tive protease enzyme that is able to cleave and activate C1s.

C1s (serine protease). Activated C1s cleaves C4 into a larger C4b fragment
and a smaller C4a fragment. The C4b fragment has an internal thioester
bond that binds to the target cell membrane in the vicinity of C1; unbound
C4b decays in the medium. C4a is released into the fluid phase.
29
C2. C2 proenzyme binds to the exposed binding site on the cell-bound C4b
to form C4bC2 complex; C2 is cleaved by a nearby C1s molecule into a
soluble C2a fragment and C2b. C2b remains bound to C4b to form an en-
zyme complex, C4b2b, also called classical C3 convertase. C1s cannot act
on C2 unless it is bound to C4b [this is called substrate modulation].

C3. In order for the complement cascade to proceed, C3 must be activated
by proteolytic cleavage. C4b2b binds to and proteolytically cleaves C3 into
C3b and a small C3a fragment. A single C3 convertase enzyme can gener-
ate more than 200 molecules of C3b, resulting in tremendous amplification at
this step of the complement cascade.

C3b may bind to the C3 convertase to form the C5 convertase [C4b2b3b];
C3b may also bind directly to cell membrane in the vicinity of C4b2b.
Some C3b diffuse away and coat immune complexes [antigen-antibody
complexes] and particulate antigens, eg, bacteria, functioning as opsonin
and promoting phagocytosis.

C5 convertase [C4b2b3b]. The C3b component of the complex binds C5
and alters its conformation, so that the C4b2b component can cleave C5 into
C5a, which is released into the fluid phase, and C5b, which remains attached
to the C5 convertase, initiating the terminal pathway of complement activa-
tion.

The Alternative Complement Pathway

The alternative pathway was originally described by Pillemer and his associates
in 1954. This is a system for activating complement beginning at C3, which does
not involve an Ag-Ab reaction but by the binding of complement protein C3b to
the surface of a pathogen. Microbial structures that can activate the alternative
pathway include lipopolysaccharide (LPS; gram-negative bacteria), teichoic acid
[gram-positive bacteria], zymosan [fungal cell walls], viral envelopes, etc.

The alternative pathway is more important than the classic pathway the first
time an individual or animal is infected by a microorganism, because the IgG
or IgM required to trigger the classic pathway is not present.

C3. In normal plasma, C3 is slowly and continuously hydrolyzed to C3a and
C3b due to H
2
O-induced cleavage of an unstable thioester bond in C3 [re-
ferred to as C3 tickover]. The thioester bond can also be cleaved by plas-
min, phagocyte proteases, trypsin, etc.

The C3b fragment can bind to proteins and carbohydrates on host cell
surfaces or to foreign antigens, eg, bacteria, virus particles, etc.

C3b binding to host cell surface is inactivated by factor H and factor I.
30
Factor H has affinity for sialic acid [derivative of neuraminic acid, a nine
carbon sugar] constituents of host cell membrane glycoproteins and this
increases its binding to any C3b deposited on host cells. The cell bound
C3bH is then cleaved by factor I to its inactive derivatives, eg, inactive
C3b [iC3b], etc. As a result, activation of the alternative complement path-
way is stopped.

Conversely, if C3b binds to microbial surface it is not cleaved because micro-
bial membranes either lack sialic acid or have only low levels of sialic acid. In
that case, it can bind a protein called factor B to form C3bB complex.

Some bacteria may express high levels of sialic acid on their cell surfaces
or, alternatively, scavenge sialic acid from the host and enzymatically
transfer the sugar to their cell surfaces. This can inhibit the alternative
pathway by enhancing the binding of factor H to C3b on the bacterial
membrane.

Factor D [serine protease]. Factor B bound to C3b is cleaved by factor D to
yield a soluble fragment, Ba, and a larger fragment, Bb, that remains at-
tached to C3b. The C3bBb is the alternative C3 convertase, and it cleaves
more C3 molecules, initiating an amplification sequence for C3b.

C3bBb is extremely unstable [half-life is 5 minutes] unless it binds to pro-
perdin [factor P]. The half-life of C3bBbP is about 30 minutes.

C5 convertase. C3b generated by the C3 convertase can bind to the con-
vertase. This results in the formation of a C3bBb3b complex, which functions
as the alternative C5 convertase to cleave C5 and initiate the terminal path-
way.

The Membrane Attack Complex (MAC; C5b6789)















31
C5 convertases [C4b2b3b or C3bBb3b] cleave C5 into a soluble C5a and
C5b. While still attached to the C5 convertase, C5b first binds to C6 and then
C7.

The C5b67 complex undergoes a conformational change, causing its re-
lease from the C5 convertase and exposing hydrophobic regions that
enable the complex to bind to membrane phospholipids. Membrane-
bound C5b67 binds to C8.

The C5b678 complex is responsible for slow membrane leakage. A fully ac-
tive MAC is accomplished by the polymerization of up to 18 molecules of C9
to form a tubular pore in the cell membrane, allowing the entry of water and
ions.

Osmotic swelling [due to influx of water] occurs, resulting in cell lysis. Extra-
cellular calcium also enters the cell, and in nucleated cells, high calcium con-
centrations can induce apoptosis.

Complement Receptors

Complement receptors are cell-surface proteins on various cells which recognize
and bind to various complement fragments. This facilitates the effector functions
of complement.

Cell Distribution of Complement-binding Receptors
Receptor Ligand[s] Cell Distribution Functions
CR1(CD35)




CR2 (CD21)



CR3
(CD11b/CD18)


CR4
(CD11c/CD18)

C3a receptor



C5a receptor
C3b, iC3b, C4b




C3d, iC3b



iC3b



iC3b


C3a, C4a



C5a
Monocytes, B cells,
macrophages, FDCs,
neutrophils, RBCs


B cells, FDCs



Monocytes, NK cells,
macrophages, FDCs,
neutrophils

Macrophages, NK cells,
neutrophils, monocytes

Mast cells, basophils,
endothelial cells, pha-
gocytes

Mast cells, basophils,
endothelial cells, pha-
gocytes
Phagocytosis; clearance
of immune complexes;
promotes C3b and C4b
decay

Part of B cell coreceptor;
trapping of antigen in ger-
minal centers by FDCs

Phagocytosis; leukocyte
adhesion to endothelium;
antigen trapping by FDC

Stimulates phagocytosis


Degranulation; activation
of endothelial cells and
phagocytes

Degranulation; activation
of endothelial cells and
phagocytes
NK cells: Natural killer cells; FDC: Follicular dendritic cell
32
The Biologic Functions of Complement

Most of the effector functions of complement are mediated by the binding of pro-
teolytic fragments of complement proteins to various cell surface receptors; only
cell lysis is mediated by the MAC.

Cell membrane lysis

Insertion of the MAC into the cell membrane leads to killing or lysis of many
cell types, including erythrocytes [see Transfusion Reactions, page 203],
Gram-negative bacteria, tumor cells, and enveloped viruses.

Gram-positive bacteria are not susceptible to the MAC because their cell
membranes are protected by the thick peptidoglycan layer [see Host res-
ponse to Bacterial Infections, page 144].

Chemotactic fragments

C5a, C3a, C5b67. Chemotaxis of monocytes/macrophages and polymorphs.

Opsonization and phagocytosis

C3b, C4b, and iC3b bound to microbial surfaces can simultaneously bind to
complement receptors on neutrophils and macrophages, stimulating engulf-
ment and intracellular killing [see Inflammation and Phagocytosis, page 48].

Stimulation of inflammatory responses

C5a>C3a>C4a (Anaphylatoxins). Bind to mast cells and blood basophils and
induce degranulation, with the release of histamine and other vasoactive me-
diators. The mast cell reactions result in influxes of phagocytic cells and
plasma proteins to the site of microbial invasion.

Anaphylatoxins can also bind directly to smooth muscle cells of the bron-
chioles and cause bronchospasm [see Type I Hypersensitivity, page 188].

C5a and C3a. Bind to C5a or C3a receptors on endothelial cells, inducing the
expression of P-selectin [promotes neutrophil binding] and vascular permea-
bility, events that promote leukocyte extravasation into tissue.

Activation of phagocytes [neutrophils and macrophages]

Rearrangement of integrin adhesion molecules [increase adherence to
endothelial cells].

Increased expression of CR1 and CR3 [enhanced phagocytosis].
33
Stimulation of the respiratory burst and production of reactive oxygen
intermediates [ROI].

Release of inflammatory cytokines, proteases, etc.

Bb. Activates macrophages, causing them to adhere to and spread on sur-
faces, thus inhibiting macrophage migration from the site of antigen depo-
sition [see Immobilization of Macrophages, page 53].

Clearance of immune complexes. Clearance of immune complexes [Ag-Ab
complexes; see Immune Complex Hypersensitivity, page 214].

Enhancement of antibody production

C3d, a breakdown product of C3b, covalently attaches to antigen. B cells can
bind the antigen via their antigen receptors and simultaneously bind the at-
tached C3d via CR2 (CD21). The combined signals result in B cell activation
and antibody production [see B Cell Coreceptor Complex, page 96].

Antigen with bound C3b, C4b, or iC3b are also bound by follicular dendritic
cells in the germinal centers of lymphoid organs. FDCs display the bound
antigens to daughter B cells; this process is critical for the selection of high-
affinity B cells that will become plasma cells and memory B cells.









Regulation of the Complement System

Uncontrolled activation of complement can rapidly deplete complement proteins;
lead to formation of the MAC on normal host cells [bystander lysis]; and result in
excessive generation of inflammatory mediators.

Regulation of complement is mediated by circulating and cell-surface pro-
teins, as a result, a delicate balance of activation and inhibition of the com-
plement cascade is achieved which prevents damage to host cells and tis-
sues but promotes the effective destruction of foreign organisms.

Spontaneous decay of activated complement components, including time-
and temperature-dependent dissociation of some of the active complexes,
such as C4b2b, C3bBb, and C5b67 complexes.
The Central Role of C3b

Binds to factor B, C5, and cell surface proteins and polysaccharides
Nonspecific opsonin
Clearance of immune complexes from the circulation
Selection of B cells in germinal centers
Source of iC3b, C3d, etc
34
Soluble C1 inhibitor (C1 INH). Covalently binds to active C1r
2
and C1s
2
and
dissociates them from C1q, thus stopping activation by the classic pathway.






Membrane bound decay-accelerating factor (DAF). Displaces Bb from C3bBb
or C2b from C4b2b, the alternative and classic pathway C3 convertases.












Soluble anaphylatoxin inactivator. Proteolytically removes terminal arginine
residues and inactivates the anaphylatoxins [C3a, C4a, and C5a].

Fluid phase [soluble] Factor I. Cleaves cell-associated C3b and C4b. C3b is
cleaved to yield iC3b, C3c, C3d, C3f, and C3dg. Factor I is active only in the
presence of regulatory proteins or cofactors.

Membrane bound membrane-cofactor protein (MCP, CD46) and comple-
ment-receptor 1 (CR1). They block the formation of C3 convertase by
binding C4b or C3b. Cofactor for factor I-mediated cleavage of C3b or
C4b.

35
Soluble Factor H. Cofactor for cleavage of C3b by factor I.

Soluble C4b-binding protein [C4BP]. Binds to C4b on cell surface and
competitively inhibits the binding of C2; prevents formation of the C3
convertase. Cofactor for factor I-mediated cleavage of C4b into C4c and
C4d.

S protein [Vitronectin]. Binds to C5b67 complex, preventing the complex from
inserting into cell membranes.

In this way, S protein can diminish the potentially indiscriminate lysis of
host cells [so-called bystander lysis] by insertion of soluble C5b67 com-
plexes released from other activating surfaces.













CD59 (Protectin). Formation of the MAC is inhibited by a widely distributed
cell membrane protein on normal host cells called CD59. CD59 works by
binding to C5b678 on the cell surface and preventing C9 polymerization.

Most nucleated cells can endocytose C5b-8 or the entire MAC. Therefore,
if C5b-8 is removed early enough, the cell is able to repair any membrane
damage and restore its osmotic stability.

S protein in the plasma
inhibits bystander lysis]
36
INFLAMMATION AND PHAGOCYTOSIS

Inflammation is defined as the reaction of vascularized living tissues to local in-
jury. The vascular and cellular events that highlight acute inflammation serve to
deliver mediators of host defense [eg, leukocytes and plasma proteins] to sites
of microbial invasion and/or tissue injury.

Inflammation is always an evoked response set in motion by some kind of a
stimulus, eg, tissue injury. It occurs only in living tissue.

Inflammation is fairly stereotypical regardless of the inducing stimulus.

Inflammation is an overlapping series of events that form a continuum.

acute vascular
(minutes)
acute cellular
(hours)
chronic cellular
(days/weeks/months)
repair

The inflammatory response may have three outcomes: (1) elimination of the
causative agent; (2) walling off of the inflammation from the rest of the body
(delays the spread of bacteria or toxic products), with subsequent healing of
the lesion, and (3) persistence of the causative agent, leading to chronic in-
flammation or spread throughout the body.

Although inflammation is characterized by the controlled passage of cells,
plasma and plasma components into the injured tissue, sometimes it can be
more harmful than the initiating stimulus.

Cardinal Signs of Acute Inflammation

Rubor (redness). Caused by increased blood flow, dilation of arterioles and
vascular perfusion of the area.

Tumor (swelling, edema). Caused by diapedesis of blood cells and plasma
from the postcapillary venules [vascular permeability] into the damaged tis-
sue.

Calor (heat). Local increase in tissue temperature [due to vasodilation].

Dolor (pain). Caused by stimulation of neuronal pathways by bradykinin,
histamine, serotonin, and prostaglandin.

Functio laesa (loss of function).
37
Systemic Effects of Inflammation

The local inflammatory response is associated with systemic changes collectively
called the acute phase response or the systemic inflammatory response syn-
drome, [SIRS]. These systemic responses to infection are due to the combined
action of proinflammatory cytokines such as tumor necrosis factor, interleukin-1,
and interleukin-6 released by activated macrophages, mast cells, etc.

Other cytokines that also participate in the acute phase response include colony
stimulating factors [CSFs] that stimulate the bone marrow to produce more white
blood cells. The acute phase response includes the following:

Fever

Leukocytosis [hematopoiesis]

Increased synthesis of acute phase
proteins.

Decrease in the plasma concentration
of iron. High concentration of free iron
enhances bacterial replication.

Decrease in appetite. May deprive pa-
thogens of nutrients, especially mine-
rals, needed for their proliferation.

Increased secretion of many hor-
mones, notably ACTH and cortisol.

Acute Phase Proteins

Acute phase proteins are plasma proteins whose concentrations change by at
least 25% during inflammation. Most of them increase in concentration and are
called positive acute phase proteins. Some proteins decrease in plasma concen-
tration and are called negative acute phase proteins, eg, albumin.

Acute phase proteins are synthesized mostly by the liver. The proteins play
various roles in the innate immune response to infectious agents. They in-
clude:

Complement proteins.

C-reactive protein. So-called because of its ability to bind to the C-protein
of pneumococci. Binds to bacteria and promotes their uptake by phago-
cytic cells, a process referred to as opsonization.
38
Mannose-binding protein. Binds to mannose residues on the surface of a
bacterial cell, resulting in its opsonization.

Can activate complement in a manner homologous with the classical
pathway of complement activation.

Iron-binding proteins, eg, haptoglobin.

Lipopolysaccharide-binding protein [LBP]. Enhances the ability of macro-
phages and other cells, eg, endothelial cells, to detect and respond to
Gram-negative bacteria.

EVENTS IN ACUTE INFLAMMATION

Microvasculature

The microvasculature consists of the afferent arterioles, the capillary networks,
and the efferent venules. Following tissue injury, initiation of the inflammatory
response occurs at the level of the capillary and postcapillary venules.

Arterioles. The initial response of arterioles is transient vasoconstriction, me-
diated by neurogenic and chemical stimuli. Within a few minutes, however,
vasodilatation occurs, with an increase in blood flow to the inflamed area.

Capillaries. The reaction of endothelial cells in the capillaries to vasoactive
mediators leads to EC retraction, gap formation, and increased permeability.

Postcapillary venules. The venules are the primary anatomic site for infla-
mmation-related leakage. The junctions between the endothelial cells are
more permeable than those in the capillaries; additionally, the endothelial
cells are more sensitive to vasoactive mediators.


39
TISSUE INJ URY
Infectious agent
(bacterium, fungus, virus, parasite)
Trauma
Neoplasm, etc
PRODUCTION OF INFLAMMATORY
MEDIATORS
CHEMOTACTIC FACTORS
C5a
Leukotriene B
4
[LTB
4
]
Formylated peptides
Chemokines, eg, IL-8
PAF
VASOACTIVE MEDIATORS
Histamine
Serotonin
Bradykinin [kallikrein-kinin system]
Nitric oxide
Leukotriene C
4
, D
4
, E
4

Prostaglandins [PGD
2
, PGE
2
and PGI
2
]
Platelet-activating factor [PAF]
Anaphylatoxins [C5a/C4a/C3a]
Lipopolysaccharide [LPS]
Cytokines, eg, IL-1, TNF-
Vasodilatation
Increased vascular permeability
Endothelial expression of adhesion
molecules.
Chemical Mediators of Inflammation

Chemical mediators are chemical messengers that enhance blood flow, increase
vascular permeability, or induce the emigration of leukocytes from the blood-
stream to the site of tissue injury. Some mediators come from exogenous
sources, eg, bacterial lipopolysaccharide [LPS]. Endogenous mediators originate
from both plasma and host cells.

Cellular sources: Platelets, connective tissue mast cells, basophils, neutro-
phils, endothelial cells, fibroblasts, monocyte/tissue macrophages, injured tis-
sue, etc. Individual mediators may have multiple sources.



































Recruitment and stimulation
of inflammatory cells
40
Many mediators carry out their biologic activity by binding to specific recep-
tors on target cells. Most functions can be elicited by multiple mediators and
most mediators serve multiple functions.

Many mediators exert their effects locally and do not circulate systemically in
high concentration [difficulty in regulating and limiting the inflammatory res-
ponse] except in unusual circumstances.

Many mediators have short tissue half-lives. They quickly decay [eg, ara-
chidonic acid derivatives] or are inactivated by enzymes [eg, kininase inac-
tivates bradykinin], or inhibited [eg, regulatory proteins of the complement
system that break up and degrade activated complement components]. This
system of checks and balances limits the extent of the inflammatory response
and hence tissue injury.

Lipopolysaccharide (LPS; see page 142). LPS released from the cell wall
of Gram-negative bacteria is recognized and bound by lipopolysaccharide-
binding protein [LBP] present in plasma. LBP enhances the efficiency [100-
to 1000-fold] of transfer and presentation of LPS to the CD14 receptor on
macrophage cell membrane, resulting in macrophage activation.

The activated macrophage synthesizes and secretes vasoactive media-
tors of inflammation, including IL-1, IL-6, CXCL-8, TNF-, PAF, etc.

Nitric oxide [NO]. This is an important mediator of inflammation. It causes
vasodilation by relaxing vascular smooth muscle, reduces platelet aggrega-
tion and adhesion, and is a potent microbicidal agent. It is produced by acti-
vated endothelial cells and activated macrophages.

Leukocyte Recruitment (Extravasation)

The process of leukocyte recruitment to the site of an inflammatory response
begins with the local dilation of postcapillary venules, followed by [1] leukocyte
rolling, [2] activation, [3] margination/pavementing, and [4] transendothelial mi-
gration. Subsequent movement through the extracellular matrix to the site of
inflammation or tissue injury occurs under the influence of chemotactic factors.

The Marginal and Circulating Pools of Neutrophils in the Peripheral Vasculature

41
Leukocyte Adhesion to Vascular Endothelium followed by Emigration into the Tissues












Mechanisms of Leukocyte Adherence

The selective adhesion of leukocytes to vascular endothelium is achieved with a
myriad of complementary cell adhesion molecules possessed by endothelial cells
[ECs] and leukocytes. The 4 major families of adhesion molecules are:

Selectins: L-selectin; P-selectin; and E-selectin
Addressins: GlyCAM-1; CD34; PSGL-1; ESL-1; MAdCAM-1
Integrins: VLA-4; CD11aCD18; CD11bCD18; etc
Immunoglobulin superfamily: ICAM-1, ICAM-2, ICAM-3, VCAM-1, PECAM,
CD2, CD58, etc.

Leukocyte and Endothelial Cell Adhesion Molecules





















42
The Selectin Family

Selectins are a family of adhesion molecules expressed on platelets, leukocytes,
and endothelial cells that promote the initial localization and rolling of leukocytes
along endothelium at sites of tissue injury. Each selectin molecule is a single
chain transmembrane glycoprotein with an extracellular lectin domain.

P (Platelet)-selectin (CD62P). P-selectin is preformed and is
stored within Weibel-Palade bodies of ECs and the -gra-
nules of platelets. When EC is activated by histamine, etc,
the granule fuses with the cell membrane and P-selectin is
expressed on the cell surface.

P-selectin binds to an addressin on neutrophil, monocyte,
and T lymphocyte cell surfaces called P-selectin glyco-
protein ligand-1[PSGL-1].

E (Endothelial)-selectin (CD62E, ELAM-1). E-selectin is synthesized and ex-
pressed by endothelial cells within 1 to 2 hours after proinflammatory cytokine
[eg, IL-1 and TNF-] activation.

E-selectin binds to E-selectin ligand-1 [ESL-1] on the surfaces of granu-
locytes, monocytes, and T lymphocytes.

L (Leukocyte)-selectin (CD62L). It is expressed on lymphocytes, granulo-
cytes, monocytes and macrophages. It binds to several receptors on ECs in-
cluding CD34, glycan-bearing cell adhesion molecule-1 [GlyCAM-1], and mu-
cosal addressin cell adhesion molecule-1 [MAdCAM-1].

Addressins (Mucin-Like Family)

Vascular addressins are mucin-like molecules that possess carbohydrate regions
that bind the lectin domain of selectins. Addressins are expressed on the sur-
faces of leukocytes and endothelial cells.

Selectin-addressin binding is not a strong bond (one
author describes the bond as having the staying power
of a Post-it Note). If the inflammatory stimulus is weak
and integrin-mediated attachment of the cell does not
occur, the cell breaks away after a few seconds or mi-
nutes and returns to the circulation.





43
The Integrin Family

Integrins are transmembrane adhesive proteins expressed on leukocytes. They
are composed of and subunits, arranged as heterodimers. There are several
subfamilies of integrins, and members of each subfamily express a conserved
chain [eg,
1
or CD29;
2
or CD18;
3
or CD61, etc] associated with different
chains.

The major functions of integrins are to mediate adhesion of
leukocytes to endothelial cells, extracellular matrix proteins
[cell migration], and adhesion of T cells to antigen-presenting
cells [APCs, see page 84] or target cells [see page 124].
Some integrins bind to proteins that participate in the inflam-
matory response [eg, complement proteins].

Integrins are always present on the surface of leukocytes but
are quickly up-regulated in numbers and adhesiveness following activation of
leukocytes by chemoattractants [CXCL8, etc] secreted by activated ECs
and/or tissue cells [some tissue-derived mediators may diffuse from the tissue
into the blood vessels, bind onto the surface of ECs, and directly contact the
leukocytes]. The adhesiveness occurs via a conformational change in the
integrin.

The up-regulated, sticky integrins on activated leukocytes then bind to
their counterreceptors [eg, ICAM-1 or ICAM-2] on ECs, resulting in the
stable adhesion of the leukocytes to the endothelium, ie, pavementing.

The significance of integrin proteins in leukocyte extravasation is demon-
strated by leukocyte adhesion deficiency disease, which is characterized by
recurrent bacterial infections [see Immunodeficiency Diseases, page 234].


Subunits Name WBCs Counterreceptor Functions

2

L

x


VLA-1 (CD49aCD29)

VLA-4 (CD49dCD29)


CD11aCD18 (LFA-1)


CD11bCD18 (MAC-1,
CR3)



CD11cCD18
T , M

L, M, E (not N)


All WBCs


N, B, E, M,
NK cells



N, M, NK cells,
Dendritic cells
Collagen, laminin

VCAM-1, fibronectin


ICAM-1, -2, -3


ICAM-1, fibrinogen,
iC3b



iC3b, fibrinogen
Leukocyte-matrix adhesion

Leukocyte -EC adhesion
Leukocyte -matrix adhesion

Leukocyte-EC adhesion
T cell-APC adhesion

Leukocyte-EC adhesion
Leukocyte-matrix adhesion
Phagocytosis of iC3b -
bound particles.

Same as CD11bCD18
B, Basophils; E, Eosinophils; L, Lymphocytes; M, Monocyte/Macrophage; N, Neutrophils; T, T cells;
EC, Endothelial cell; APC, Antigen-presenting cell; LFA, Lymphocyte function-associated antigen; ICAM,
Intercellular adhesion molecule; VCAM, Vascular cell adhesion molecule; VLA, Very late activation antigen
44
The Immunoglobulin Superfamily

This is a family of proteins that share partial amino acid sequence homology and
tertiary structural features [at least 15%] that were originally identified in immuno-
globulin [Ig] heavy and light chains [see Igs, page 107]. Some members of the
family are expressed on the cell surface of cytokine-stimulated endothelial cells
and leukocytes and help localize leukocytes to areas of tissue damage.

ICAM-1 (CD54). Expressed by proinflammatory cytokine activated ECs. It is
also expressed by a variety of cells including B lymphocytes and mono-
cyte/macrophages.

ICAM-2 (CD102). Constitutively expressed at low levels on some ECs. Also
expressed by monocyte/macrophages and some T cells.

VCAM-1 (CD106). Expressed by activated ECs. Also expressed by macro-
phages and other cells.

Platelet-endothelial cell adhesion molecule [PECAM-1, CD31]. Present on
neutrophils, platelets, monocytes, B cells; and found within the junctional
complex of activated ECs. PECAM-1 molecule on one cell binds to PECAM-1
molecule on another cell, ie, displays homotypic binding.

DIAPEDESIS (Transendothelial Migration)

Following margination, diapedesis is enhanced by the binding of PECAM-1 on
neutrophils to its twin, PECAM-1, expressed on ECs close to the inter-endothelial
cell junction. This acts as an exit signal for the neutrophils. They squeeze
through the gaps and crawl into the perivascular tissue.

Once in the tissue, leukocytes migrate by interacting their integrin proteins
with the proteins of the extracellular matrix [collagen, laminin, fibronectin, etc],
preferentially toward gradients of chemotaxins formed within the tissue.

LEUKOCYTE ACCUMULATION

Leukocyte aggregation at the site of inflammation follows a fairly predictable pat-
tern. In most bacterial infections, neutrophils predominate during the first 6 to 24
hours of the inflammatory reaction. In most species, other than pigs and cattle,
neutrophils are the most numerous WBCs in circulation. Neutrophils also res-
pond more rapidly to chemotactic agents.

In the later stage [24 to 48 hours] of the inflammatory reaction, monocytes
and lymphocytes predominate. However, there are exceptions to this pattern
of leukocyte accumulation. In immediate hypersensitivity reactions and para-
sitic infestations, eosinophils predominate.
45












CHEMOTAXIS

Chemotaxis is the energy-dependent, unidirectional migration of cells toward in-
creasing concentrations of a soluble chemotactic agent [random, excited move-
ment of cells is called chemokinesis]. Chemotaxis is a receptor-mediated event.

Chemotactic agents are low-molecular-weight soluble compounds that are
generated in high concentrations at sites of tissue injury, with a decreasing
gradient away from the injured tissue. In some cases, chemotactic agents
bind to extracellular matrix protein, thereby creating a fixed chemotactic gra-
dient [haptotaxis].

Chemotaxis is effective up to 100 m away from an inflamed tissue; because
almost no tissue area is more than 50 m away from a capillary, the chemo-
tactic signal readily brings hordes of leukocytes into the inflamed area.

Leukocyte chemoattractants can be divided into two basic types:

Classical leukocyte attractants. They act broadly on several cell types, in-
cluding monocytes, macrophages and neutrophils. They include:

Bacterial chemotaxins, especially N-formylated peptides
Dead cells [necrotaxis]. Mitochondrial N-formylated peptides
Plasma-derived: C5a/C3a/C5b67 and fibrinopeptides
Platelet-activating factor
Leukotriene B
4
[LTB
4
]

Chemokines [see Chemokines, page 21]. They are the most important
group of chemotactic agents

CXC chemokines. Potent attractants and activators of neutrophils; the
most significant is CXCL8 [IL-8].

CC chemokines. They attract various leukocytes but not neutrophils.
Leukocyte accumulation. In many
inflammatory reactions, neutrophils
arrive first, followed by mononu-
clear cells.
46
PHAGOCYTOSIS (Gk, eating by cells )

This is the process by which certain cells of the innate immune system, including
macrophages and neutrophils [the professional phagocytes], engulf large parti-
cles [>0.5 m in diameter], such as bacteria, fungi, protozoa, etc.

The Russian zoologist Elie Metchnikoff is credited with discovering pha-
gocytosis in 1882. While studying the behavior of mobile cells in the trans-
parent starfish larva, he observed that the introduction of a sharp thorn into
the body of the larva caused mobile cells to surround the foreign body and
apparently to attack it.

In later experiments, he observed that the mobile cells were able to ingest
and destroy foreign matter, including bacteria. From his own observations
and those of Julius Cohnheim, who had earlier seen white blood cells
migrating from tissue capillaries to form pus at sites of injury, Metchnikoff
concluded that inflammation served as an important defense reaction of the
body and played a major part in bringing about recovery from bacterial
infections. In 1908, Metchnikoff was awarded the Nobel prize for his
discovery.


Steps in the Phagocytic Process


POLYMORPHONUCLEAR NEUTROPHILS (PMNs)

Neutrophils make up 40-70% of the total blood leukocyte population in most
animal species [the most abundant white cells in peripheral blood] except in pigs
and cattle [lymphocytes are the predominant peripheral blood cells].
47
Majority of PMNs are stored in the bone marrow and are released when pro-
inflammatory mediators reach the bone marrow via the bloodstream, and
there, act on the marrow capillaries and on stored PMNs to mobilize them into
the circulating blood. Once released from the bone marrow, neutrophils do
not have the ability to divide and give rise to new cells [ie, terminally-differ-
entiated cells]. An adult human produces over 1 x 10
11
neutrophils per day.

Normally, neutrophils circulate in the blood for about 4 to 8 hours and then
migrate into tissues where they may survive for up to 4 to 5 days. Once a
neutrophil enters tissue, it does not return to the bloodstream. If a circulating
PMN is not recruited into a site of inflammation [they are the first circulating
phagocytic cells to gather at sites of acute inflammation] it undergoes pro-
grammed cell death and is usually phagocytosed by resident macrophages in
the liver or spleen.

In circulation, a neutrophil is spherical, with a diameter of approximately 10-12
m, but can reduce its width to 1 m during diapedesis.

The neutrophils of chickens, rabbits, and guinea pigs contain large, reddish
staining cytoplasmic granules, hence they are referred to as heterophils.




















Adherence and Opsonization (Immune adherence)

Once a neutrophil encounters the microbe, it must bind to it. Attachment be-
tween the neutrophil and the microbe is mediated by the interaction between cell
surface receptors and ligands on the microbe.

Neutrophils carry many cell sur-
face receptors, including those
that mediate attachment to endo-
thelial cells and opsonization.
48
Neutrophils and macrophages recognize conserved microbial struc-
tures that are relatively invariant within a pathogen class. These struc-
tures are termed pathogen-associated molecular patterns [PAMPs],
eg, LPS, lipoteichoic acid, peptidoglycan, mannose-rich oligosaccha-
rides, etc. The receptors on phagocytes that recognize them are called
pattern recognition receptors [PRRs].

For example, the mannose re-
ceptor on a macrophage binds
terminal mannose and fucose
residues of glycoproteins and
glycolipids, which are typically
found on microbial cell walls.

Opsonins are naturally occurring [nonspecific] or acquired [specific] substances
that coat microbes and render them more susceptible to phagocytosis. Being
positively charged proteins, opsonins also help to neutralize the negative charges
on foreign particles, enabling them to bind to negatively charged phagocytes.

Specific opsonin [IgG]. Fab portions bind to epitopes on the microbe; Fc
portion binds to Fc receptor on phagocyte [see Immunoglobulins, page 107].

Nonspecific opsonins. Complement proteins C3b, iC3b, C4b and a number
of plasma proteins including fibronectin, mannose-binding lectin [MBL], and
C-reactive protein.

Nonspecific opsonization is less effi-
cient than IgG coating but can en-
hance phagocytosis early in the
course of infection before specific anti-
bodies are produced.








Engulfment

Following attachment, the adherent particle stimulates pseudopodia formation of
the cell membrane. The pseudopodia meet each other on the opposite side and
fuse. This creates an enclosed chamber that contains the phagocytized particle.
Then the chamber breaks away from the cell membrane to form a free-floating
phagocytic vesicle [also called a phagosome] inside the cytoplasm.
49
Killing and Degradation of Phagocytosed Microbes

Following engulfment, lysosomal granules migrate through the cytoplasm and
fuse with the phagosome to form a phagolysosome, where microbicidal mole-
cules and the proteolytic enzymes stored in lysosomes come into contact with
and destroy the phagocytized microbe. Microbial killing occurs within 10 to 30
minutes.

Some of the Chemicals present in the 1
o
and 2
o
Lysosomal Granules of Neutrophils

Respiratory Burst (Oxygen-Dependent Killing)

Phagocytosis stimulates a series of O
2
-dependent biochemical events occurring
at the plasma membrane and within the phagolysosome, which promotes des-
truction of the ingested particle. It is characterized by:

Two- to three-fold increase in O
2
consumption by the cell, (b) increased glu-
cose oxidation via the hexose monophosphate shunt, and (c) generation of
microbicidal reactive oxygen intermediates [ROIs; also called reactive oxygen
species, ROS]: superoxide anion [
.
O
2
-
], hydrogen peroxide [H
2
O
2
], hypochlo-
rous acid [HOCl], hydroxyl radical [OH
.
], and singlet oxygen [
.
O
2
].

The H
2
O
2
-myeloperoxidase-halide system is the most potent microbicidal
system in neutrophils.


NADPH oxidase Superoxide dismutase Myeloperoxidase
O
2

. .
O
2
-
H
2
O
2
HOCl
Plasma Superoxide Cl
-

membrane anion Chloride ion
enzyme


.
O
2
OH
.

Singlet oxygen Hydroxyl radical
50
Oxygen-Independent Killing

Killing of microbes can also occur by oxygen-independent mechanisms, through
the action of chemicals in phagocyte granules. The acidic [pH 4-5] environment
prevailing in the phagolysosome, a result of phagocytosis, enhances the activi-
ties of the chemicals.

Lysozyme. A mucopeptidase found in serum, sweat, tears, saliva, mucus,
nasal secretions, phagocytes, and tissue fluids.

Breaks down bacterial cell wall peptidoglycan by hydrolyzing the glycosi-
dic bond between the monosaccharides of the peptidoglycan backbone,
thereby rendering the bacterium susceptible to osmotic lysis. Effective a-
gainst Gram-positive bacteria [see Bacterial Cell Wall, page 144].

Defensins [cationic peptides]. A group of small peptides found in phago-
cytes and certain intestinal cells. Defensins form ion-permeable channels in
microbial cell membrane, resulting in escape of essential metabolites.

Defensins can kill or inactivate a wide variety of bacteria, fungi, and some
enveloped viruses.

Bactericidal permeability increasing protein (BPI; 1
o
granules). Binds spe-
cifically to the outer membrane of Gram-negative bacteria [see page 144].

BPI induces phospholipase activation, phospholipid degradation, and in-
creased permeability in the outer membrane of the bacterium. Although
the inner [cytoplasmic] membrane remains intact, bacteria exposed to BPI
for only a few seconds irreversibly lose their ability to replicate.

Bacterial Degradation

Many of the lysosomal enzymes do not have direct bactericidal activity but are
rather digestive, ie, function after killing of the microbe.

Neutral and acid hydrolases. Degrade and digest killed bacteria within the
phagolysosome.

Fate of Neutrophils

Neutrophils lack mitochondria, thus they possess a limited supply of energy re-
serves which cannot be replenished.

Following phagocytosis, neutrophils rapidly undergo apoptotic cell death and
are ingested by macrophages.

51
MONOCYTE-MACROPHAGE SYSTEM (MMS)

The MMS [syn. mononuclear phagocyte system] consists of monocytes, mobile
macrophages, and resident or fixed tissue macrophages. Upon release from the
bone marrow, monocytes circulate in the blood for 10 to 20 hours before migrat-
ing into tissues, where they undergo maturation to become tissue macrophages.




Resident or Fixed Tissue Macrophages and Macrophage-Like Cells

A large portion of monocytes, after becoming macrophages, become attached to
the tissues and remain attached for months to years; they form the basis of the
tissue macrophage system. Resident macrophages and macrophage-like cells
are strategically placed at all the sites where microorganisms may gain entry into
the host, thereby providing a continuing defense in the tissues against infection
[they serve as the first line of defense when microorganisms breach the physical
barriers].

Cell Location
Histiocytes
Mesangial cells
Kupffer cells
Osteoclasts
Microglial cells
Synovial type A cells
Fixed tissue macrophages
Pleural/peritoneal macrophages
Alveolar macrophages
Pulmonary intravascular macrophages
[ruminants, cats and pigs]
Connective tissue
Kidneys
Sinusoids of the liver
Bone
Central nervous system
J oints
Spleen, bone marrow, lymph nodes
Serous cavities
Lungs
Capillaries of the lungs

Mobile or Free Macrophages

Free macrophages derived from blood monocytes constitute the majority of ma-
crophages at inflammatory sites. A small proportion may come from fixed macro-
phages which can undergo cell division when appropriately stimulated.
52
Characteristics of Macrophages

Macrophages have been referred to as the most dynamic and gifted of the leuko-
cytes; they play a central role in innate and adaptive immunity and are important
effector cells for the elimination of microorganisms. Macrophages can live for
months to years unless they are destroyed by performing phagocytic functions.

Macrophages do not possess 1
o
and 2
o
granules; however, the lysosomal
contents of macrophages are similar in many ways to those of neutrophils.
Unlike neutrophils, macrophage lysosomes also contain large amounts of
lipases, which digest the thick lipid membranes possessed by some bacteria.

Similar to neutrophils, macrophages possess several cell membrane recep-
tors, including cytokine re-
ceptors, complement recep-
tors, antibody receptors, and
integrins.

Mannose-binding recep-
tors on cell membrane
bind to mannose or fu-
cose residues in the cap-
sule or LPS of invading
bacteria, allowing macro-
phages to bind directly to
nonopsonized bacteria.





Macrophages respond to the same chemoattractants as neutrophils, but in
addition, they respond to CC chemokines, cationic peptides released by dead
and dying neutrophils, and lymphokines released by activated T cells, eg, mi-
gration inhibition factor (MIF) and macrophage chemotactic factor (MCF).
Unlike neutrophils, macrophages are sluggishly motile.

Macrophages are more powerful phagocytes than neutrophils, capable of
sustained, repeated phagocytic activity.

Macrophages have the ability to engulf much larger particles, such as in-
tact red blood cells, neutrophils, necrotic debris, etc. Compared to neutro-
phils, a macrophage is capable of phagocytizing as many as 100 bacteria.

Intracellular killing is similar to neutrophils; in addition, activated macro-
phages can synthesize nitric oxide, a very powerful microbicidal agent.
53

L-arginine +O
2
+NADPH NO +L-citrulline +NADP
Inducible nitric oxide
synthetase

If the inflammation is short-lived and the microbe is eliminated, macrophages
eventually disappear, either by dying or making their way into the circulatory
system via the lymphatic system. In chronic inflammation, macrophage accu-
mulation will persist; this is mediated by multiple mechanisms:

Recruitment of monocytes from the circulation.

Macrophage proliferation. Unlike neutrophils, macrophages are not ter-
minally differentiated cells and can proliferate in situ at sites of chronic
inflammation and form still more macrophages.

Immobilization of macrophages. Some cytokines, eg, migration inhibition
factor, complement fragment Bb, etc., can immobilize macrophages at the
site of inflammation.





















When stimulated, macrophages may increase in size and form clusters of epi-
thelioid cells [resemble epithelial cells] or several may fuse to form multinu-
cleated giant cells.

Both cell types are found only in pathological conditions, such as chronic
inflammatory reactions.
iNOS
54











Activated macrophages [see Cell-Mediated Immunity, page 130] are major
secretory cells, able to synthesize over 100 products, including several immu-
noinflammatory mediators, such as complement proteins, cytokines, protein-
ases, etc and lipid mediators of inflammation [leukotrienes, prostaglandins,
PAF, etc].

Macrophages play a crucial role in tissue reorganization and wound healing.

Macrophage proteinases, eg, elastases, collagenases, degrade connec-
tive tissue; the cellular debris is then phagocytized. They also synthesize
and secrete many growth factors that promote the proliferation of fibro-
blasts and the growth of new blood vessels [angiogenesis factor].

The Many Functions of Macrophages






















55
Tissue Injury

When neutrophils and macrophages engage in phagocytosis, they can injure nor-
mal host tissues by release of ROI and lysosomal enzymes.

Antiproteinases [synthesized by the liver, leukocytes, and connective tissue
cells] present in plasma and tissues, eg,
2
-macroglobulin and
1
-antitryp-
sin, inhibit leukocyte proteinases, thereby preventing and/or minimizing tissue
damage.

Escape of reactive oxygen metabolites. Cytosolic and plasma molecular
scavengers of oxyradicals limit damage to cells and tissues.

Catalase and glutathione peroxidase, both present in cells, convert es-
caped H
2
O
2
to H
2
O and O
2
:

2H
2
O
2
+Catalase =O
2
+2H
2
O

H
2
O
2
+Reduced glutathione +Glutathione peroxidase =Oxidized glutathione +2H
2
O

Cytotoxicity. Membranolytic substances such as bacterial leukocidin, urate
crystals, can damage the membrane of the phagolysosome, spilling out po-
tent hydrolytic enzymes, which can then kill the leukocyte, and, in turn, are
released into the tissues.

Regurgitation during feeding. When large numbers of bacteria are present,
enhanced feeding may result in the fusion of the lysosome with the develop-
ing phagosome, prior to the complete closure of the phagosome.

In such an event, the lytic enzymes present in the lysosome have direct
access to the extracellular environment.

Frustrated phagocytosis. Sometimes a phagocyte encounters phagocytic
stimuli that are too large to be internalized, eg, helminth larvae, bacteria im-
mobilized against a fibrin meshwork, antigen-antibody immune complexes
fixed against a basement membrane or endothelium or joint surface, etc.

Under these circumstances, release of lysosomal enzymes by the phago-
cyte into the surrounding environment occur, leading to tissue injury, eg,
vasculitis, etc. [see Immune Complex Hypersensitivity, page 212].
56
THE LYMPHOID SYSTEM

The cells involved in the adaptive immune response [lymphocytes, accessory
cells, eg, antigen-presenting cells] are localized and concentrated in anatomi-
cally defined organs and tissues that constitute the lymphoid system. They are
also the sites where foreign antigens are transported and concentrated.

Sources of Lymphocytes

Lymphoid stem cells are produced by the yolk sac mesoderm and fetal liver in
the very young fetus. In older fetuses and neonates, the bone marrow is the
main source of lymphoid stem cells.

Classification of Lymphoid Organs and Tissues

The classification is based on (1) the level to which they participate in the ma-
turation of lymphocytes and (2) provision of a suitable environment for the inter-
action between foreign antigen and antigen-sensitive T and B lymphocytes.
Central (Primary or Generative) Lymphoid Organs

Central lymphoid organs and tissues [thymus, bone marrow, Peyers patches,
and bursa of Fabricius] regulate production and maturation of lymphocytes, ie,
generate lymphocytes that are individually different to meet the threat posed to
an animal or individual by the large number of microbial pathogens, the concept
being one cell, one specificity.

Central lymphoid organs develop early in embryonic life and involute after pu-
berty, except bone marrow.

Mature lymphocytes acquire specific antigen receptors and other phenotypic
characteristics and learn to discriminate between self-peptides, which are to-
lerated and foreign antigens, which are not, ie, self vs nonself.
57
Maturation and differentiation of lymphocytes occur independent of foreign
antigenic stimulation.

Upon removal early in life, there is loss of lymphocytes, resulting in loss of
immune responsiveness.

Thymus

The thymus is the site of T cell development and maturation. It is found in the
anterior mediastenal space. In horses, pigs, sheep, cattle, and chickens, it also
extends up the neck as far as the thyroid gland. It is organized into lobules se-
parated by connective tissue septae. The thymus has a rich blood supply and
efferent lymphatic vessels that drain into mediastinal lymph nodes.

The role of the thymus in host immune responses was determined following
neonatal thymectomy of day-old mice.

Dramatic decrease in circulating T lymphocytes [lymphopenia] and total
loss of the cell-mediated immune response.

Cross Section of a portion of the Thymus


















Cortex [outer compartment]. Contains about 85% of the total thymocytes
[immature lymphocytes]. During different stages of their maturation in the
cortex, thymocytes acquire antigen receptors [TCRs] and other functional and
phenotypic characteristics of mature T cells [see Thymus-Derived Cells, page
77]. As lymphocytes mature, they migrate toward the medulla.

Medulla [inner compartment]. Sparsely populated with mostly mature lym-
phocytes.
58
A network of epithelial cells, macrophages and dendritic cells are found
throughout the thymus, especially at the corticomedullary junction.

These cells provide the stimuli, such as cytokines [esp. IL-7], thymic hor-
mones [thymosin, thymic humoral factor, thymopoietin, thymulin, etc], and
major histocompatibility complex [MHC] molecules, that are required for
the proliferation and maturation of thymocytes.

T cell selection [see Negative and Positive selection, page 82]. Developing
thymocytes [pre-T cells] acquire TCRs by gene rearrangement: TCRs are
selected for both self-MHC restriction [see MHC restriction, page 68], and
self-tolerance, ie, ability to distinguish between self and nonself by self-
MHC-self-peptide complexes expressed on thymic stromal cells.

Using this selection process, the thymus induces the death, by apoptosis,
of thymocytes whose TCRs either cannot recognize antigen-MHC com-
plexes or whose TCRs react strongly with self antigen-MHC complexes.
As a result, more than 95% of developing thymocytes die by apoptosis.

In humans, thymic involution begins at puberty and continues throughout
adulthood. Thymic involution begins within the cortex which may disappear
completely, whereas medullary remnants persist. Cortical atrophy is related
to corticosteroid sensitivity [lysis] of the immature cortical thymocytes.














Bursa of Fabricius (BF)

The BF was described in the 16
th
century by Hieronymus Fabricius. It is a lym-
phoepithelial organ found in birds but not in mammals; it is a sac-like structure
dorsal to the cloaca.

The bursa reaches maximum size 1-2 weeks after hatching, followed by gra-
dual involution; by the time the bird reaches sexual maturity (6 months), only
atrophied vestiges remain of the BF and thymus.
Thymic involution with age.
Decrease in size and cellu-
larity of the thymus.
59
The function of the BF was determined following bursectomy of day-old
chicks.

Only a slight drop in the numbers of circulating lymphocytes; however,
there is a dramatic decline in antibody immune response.

B cell differentiation and maturation. Majority of B cells [90-95%] die
through apoptosis [a reflection of negative selection of self-reactive B cells,
etc]. Mature B cells begin to migrate from the bursa to the peripheral lym-
phoid organs a few days prior to hatching.

Bone Marrow

The bone marrow is the site of generation of all circulating blood cells [hemato-
poiesis] and a fat depot [as an individual ages, more than 50% of the marrow
becomes filled with fat]. In mammals, it is both a 1
o
and 2
o
lymphoid organ.

The bone marrow is divided into two compartments: hematopoietic and vas-
cular compartments. The hematopoietic compartment contains precursors of
all the blood cells [self-renewing stem cells] as well as stromal cells, macro-
phages and lymphocytes. Hematopoietic cytokines produced by these cells
stimulate the proliferation and maturation of precursor cells.

In mammals, the bone marrow is the site of early events in B cell develop-
ment [see B cells, page 89]. Stromal cells: (1) secrete various cytokines
that are required for B cell development, and (2) selection of immature B cells
[immature B cells that acquire self-reactive antigen receptors are allowed to
die].

The bone marrow contains numerous long-lived plasma cells that produce
antibodies for many months or years. These plasma cells are produced in
peripheral lymphoid organs as a result of antigenic stimulation of B cells and
then migrate to the bone marrow.

Peyers Patches (PP)

PP are aggregations of lymphatic nodules that are found in the walls of all three
segments of the small intestine. Two types of PP have been described:

Numerous discrete lymphatic follicles that function as peripheral lymphatic
organs. They contain B cells, T cells, antigen-presenting cells, etc. They per-
sist throughout the life of the individual or animal.

60
Ruminants, pigs, and dogs. In addition to the discrete lymphatic follicles in
the jejunum, there is a single large ileal PP that functions as a primary lym-
phatic organ. It is the site of early B cell development [populated by IgM
+
im-
mature B cells]. It undergoes involution by 1 years of age.





Peripheral (Secondary) Lymphoid Organs

Peripheral lymphoid organs arise late in fetal life and persist through adulthood.
Removal of any of them does not significantly reduce an individual or animals
immune capacity.

They are the sites where mature lymphocyte responses to foreign antigens
are initiated and develop.





























The 3 major portals of antigen entry
into the body: skin, respiratory tract,
and gastrointestinal tract. Blood-
borne antigens are filtered by the
spleen; tissue antigens are carried by
lymphatic fluid to regional lymph
nodes.
61
Lymph Nodes

Lymph nodes are the only lymphatic organ with both afferent and efferent lymph
vessels. They are the sites where immune responses to antigens entering the
body via the skin and mucosa, or from parenchymal organs and connective
tissues, are initiated. The antigens are carried to the nodes by lymphatic fluid.
All lymph nodes eventually drain into the thoracic duct system and back to the
peripheral blood.

Cortex. B cells are organized into 1
o
and 2
o
follicles within the cortex.

Primary follicles. Follicles without germinal centers; they contain mostly rest-
ing mature B cells, follicular dendritic cells (FDCs), and macrophages.

Secondary follicles. Primary follicles with germinal centers (GCs). GCs de-
velop in response to antigenic stimulation of B cells; thus, B cell proliferation,
selection of B cells producing high affinity antibodies, and generation of me-
mory B cells, occur in the germinal center (see B cell activation, page 96).

Paracortex. Contains mostly T cells, some macrophages and dendritic cells.
Lymphocytes enter the node from the circulation through the high endothelial
venules (HEVs) in the paracortex.

Medulla. Contains mostly macrophages, antibody-secreting plasma cells, and
some lymphocytes.

Efferent lymphatic vessel. Lymph leaving via the efferent lymphatic vessel
contains newly secreted antibodies, effector lymphocytes (due to clonal expan-
sion of T cells), and mostly naive lymphocytes that migrated from the blood into
the lymph node through the paracortical HEVs.

62
Spleen

The spleen is the largest of the secondary lymphoid organs and is therefore the
major organ in the body in which antibodies are produced.

It is the major site of immune responses to blood-borne antigens; therefore,
splenectomy can lead to an increase in blood-borne microbial infections.

There are two main types of splenic tissue: the white pulp and the red pulp.

The white pulp. The lymphoid tissue of the white pulp is organized around the
central arteriole to form the periarteriolar lymphoid sheath [PALS].

T cells are mostly found around the central arteriole.

B cells are organized into 1
o
and 2
o
follicles. The follicles also contain macro-
phages and follicular dendritic cells [FDCs]. The follicles are anatomically
and functionally the same as in lymph nodes.

The red pulp. Contains macrophages, some activated B cells and plasma cells.
It is a storage site for erythrocytes, platelets, and granulocytes.

Macrophages in the red pulp phagocytose old platelets and RBCs and also
clear the blood of microorganisms and other particles; thus, the spleen is the
major site for the phagocytosis of antibody-coated (opsonized) microbes.

Marginal zone. Separates the white pulp from the red pulp. It contains macro-
phages and some lymphocytes.

63
Mucosal-Associated Lymphoid Tissue (MALT)

The MALT comprises all lymphoid cells present as either solitary or aggregated
nodules in epithelia, lamina propria and submucosa of the gastrointestinal tract,
respiratory tract and genitourinary tract. The main sites of MALT are:

Gut-associated lymphoid tissues (GALT). Lymphoid tissues in the lamina
propria of the intestines, Peyers patches, appendix, and tonsils.

Bronchial-associated lymphoid tissues (BALT); and genitourinary tract.

Cutaneous immune system. Consists of lymphocytes and accessory cells
in the epidermis and dermis that respond to environmental antigens.

Lymphocyte Recirculation

Lymphocyte recirculation is the continuous movement of lymphocytes from one
lymphatic organ or tissue to another via the blood and lymph, and, if activated, to
peripheral inflammatory sites.

Naive lymphocytes specific for any given antigen are very few in number [~
one in 10
5
lymphocytes], thus, lymphocyte recirculation ensures that an
antigen-specific lymphocyte will come in contact with that antigen no matter
where in the body the antigen
is located. A lymphocyte may
recirculate from the blood to
the tissues and lymph and
back again as often as 1-2
times per day.

It also ensures that activated
(effector) lymphocytes are de-
livered to the particular tissue
where they are required for
elimination of the antigen.







Recirculation of Naive T Cells

The predominant lymphocytes in blood are T lymphocytes. Extravasation of na-
ive lymphocytes from the blood into a peripheral lymph node occurs selectively at
64
modified postcapillary venules within the paracortex of the lymph node, called
high endothelial venules (HEVs). HEVs are present in each of the 2
o
lymphoid
organs, [eg, Peyers patches, tonsils, etc] but not in the spleen.

HEVs possess cuboidal (plump) endothelial cells; they are cytokine-activated
cells that express a variety of adhesion molecules not found on the flat resting
endothelial cells of ordinary venules.

The recirculation patterns of na-
ive T lymphocytes is governed
by the combination of adhesion
molecules [eg, L-selectin] and
chemokine receptors [eg,
CCR7] that they express.


Ordinarily, there is continuous lymphocyte movement through the nodes, but
when an antigen enters the nodes, there is a temporary shut down in lympho-
cyte traffic, allowing antigen-specific lymphocytes to be activated and change
into effector cells or memory cells [see Page 87]. I

If a naive T cell does not encounter antigen, it exits through an efferent lym-
phatic vessel, reenters the circulation, and homes to another lymph node.

Recirculation of B lymphocytes

Mature B lymphocytes reside mainly in the follicles of peripheral lymphoid or-
gans; however, they do recirculate, migrating from one peripheral lymphoid or-
gan to the next. Even though B cells recirculate, only a small fraction of blood
lymphocytes are B cells.

Memory B cells, some activated B cells, and some plasma cells, also recir-
culate.
65
THE MAJOR HISTOCOMPATIBILITY COMPLEX
AND ANTIGEN PRESENTATION

The major histocompatibility complex [MHC] is a cluster of genes located in close
proximity that encode the MHC proteins [molecules]. MHC proteins perform a
number of functions in the immune response:

Positive selection of T cells in the thymus [see T Cells, page 82].

Presentation of antigenic peptides to T cells.

The MHC encodes some complement proteins, some cytokines, and proteins
involved in antigen processing.

MHC class I and II proteins are also the most important antigens recognized
in the graft rejection process.

Nomenclature and Location of MHC Genes

All mammals and birds have an MHC complex [the structure and functions are
basically the same]. The nomenclature begins with the initial of the specie, fol-
lowed by LA for leukocyte antigen, since the proteins are readily detected on
blood leukocytes.

HLA [Human MHC complex]; DLA [Dog MHC complex; chromosome 12]; FLA
[Feline MHC complex; chromosome B2]; ELA [Equine MHC complex; chro-
mosome 20]; BoLA [Bovine MHC complex; chromosome 23]; OLA [Ovine
MHC complex; chromosome 20]; CLA [Caprine MHC complex; chromosome
23]; SLA [Swine MHC complex; chromosome 7]; B locus [Chicken MHC com-
plex; chromosome 16].

Human Leukocyte Antigens (HLAs)

Human MHC genes are located on a segment of the short arm of chromosome 6.
The MHC is the most polymorphic gene cluster in the human genome, with large
numbers of alleles [variants of polymorphic genes] at several different loci.










66
MHC polymorphism is usually detected using antibody, hence MHC proteins
are often referred to as major histocompatibility complex antigens. The set of
MHC alleles present on each chromosome is called an MHC haplotype. Wi-
thin a family, there are only 4 HLA haplotypes.

Haplotypes from both parents are inherited and expressed codominantly,
ie, each individual expresses the MHC alleles on both chromosomes that
are inherited from both parents.

MHC proteins are highly specific for each individual species, hence MHC
proteins are considered as species or strain markers.

Class I MHC Molecules (Proteins; Antigens)

Class I MHC molecules consist of two noncovalently linked polypeptide chains
that are constitutively expressed on the cell membranes of most nucleated cells
except primitive cells in early embryonic life, sperms, neurons, and erythrocytes
of most mammals.

Expression of class I molecules on most cell types is enhanced by exposure
to cytokines such as interferons-,-, and -

Alpha (Heavy) Chain (44-47kD)

Each individual expresses 6 different class I molecules on every cell, containing
chains derived from the two alleles of HLA-A, HLA-B, and HLA-C genes that
are inherited from both parents.

The chain consists of three external domains
[1, 2, and 3 domains], a transmembrane
segment and a cytoplasmic tail.

The peptide-binding cleft or groove, located be-
tween domains 1 and 2, binds peptides con-
taining 8-11 amino acids.

A single class I molecule can bind several
different antigenic peptides [but only one at
a time].

The 3 domain contains a loop that serves as
the binding site for T cell coreceptor, CD8.

The polymorphic amino acid residues are con-
fined to the 1 and 2 domains.

67

2
-Microglobulin [
2
m; Light Chain; 12 kD]

2
m is a non-MHC-encoded protein [encoded by a gene on chromosome 15]
that associates noncovalently with the 3 domain of the chain. It is invariant
[nonpolymorphic], ie, all
2
m chains of class I molecules are the same.

It is essential for the expression of all class I molecules at the cell surface.

The assemblying of a class I MHC molecule begins with the folding heavy
chain first interacting with
2
m. The empty dimeric molecular complex is
then stabilized by the binding of a peptide [self peptide or foreign peptide].
The completed trimolecular complex [ chain,
2
m, bound peptide] is then
transported to the cell membrane.

In the absence of
2
m, the class I heavy chain is not expressed on the cell
surface.

Function of MHC Class I Proteins

The physiologic function of class I MHC molecules is the presentation of peptides
derived from proteins in the cytosol, eg, viral proteins, to CD8
+
T cells.

Class II MHC Molecules

Class II molecules consist of two nonidentical protein chains [ and chains],
that are constitutively expressed on the cell membranes of dendritic cells and B
lymphocytes, and on activated macrophages. Expression of class II molecules is
enhanced by interferon-.

Class II molecules are encoded by three
gene regions: HLA-DP, HLA-DQ, and HLA-
DR. Each individual possesses about 10 to
20 class II molecules.

Each of the two chains has two domains, a
transmembrane segment, and a cytoplasmic
tail. The polymorphic amino acid residues
are located in 1 and 1 domains.

The peptide-binding groove, formed by inter-
action of the and domains, binds pep-
tides containing10 to 30 amino acids. A sin-
gle class II molecule can bind several differ-
ent peptides [albeit one peptide at a time].

A loop in the 2 domain is the binding site for the T cell coreceptor, CD4.
68
A nonpolymorphic protein called the invariant chain [Ii] is associated with
newly synthesized class II molecule [plays a role in antigen presentation].

Function of MHC Class II Proteins

Class II molecules present peptides derived from protein antigens degraded in
cellular vesicles [phagosome or endosome] to CD4
+
T cells.

Like class I molecules, a fully assembled class II molecule consists of an
chain, a

chain, and a bound peptide; stable expression of class II molecules
on cell surfaces requires the presence of all 3 components.

Additional Genes within the Class II MHC Locus

Within the class II locus are genes that encode several proteins important in anti-
gen processing.

HLA DM. It facilitates the removal of the invariant chain-derived CLIP protein
and the binding of endosomal peptide to class II MHC molecules.

Proteasome. It is a cytosolic protease complex that cleaves proteins into
small peptide fragments that are subsequently presented by class I MHC mo-
lecules [see Cytosolic Antigen Processing Pathway, page 124].

TAP [transporter associated with antigen processing]. TAP transports pep-
tides from the cytosol into the endoplasmic reticulum, where the peptides as-
sociate with newly synthesized class I molecules.

Comparison of Class I and Class II MHC Molecules
Characteristic Class I MHC Class II MHC
Structure

Distribution


Sites of polymorphic residues

Peptide-binding cleft


Source of peptides

Peptide presented to

Outcome
chain +
2
m

Most nucleated cells


1 and 2 domains

Closed; binds peptides of 8-11
amino acids

Cytosol

CD8
+
T cells

T cell-mediated cytotoxicity
and chains

Professional APCs [B cells,
dendritic cells, macrophages]

1 and 1 domains

Open; binds peptides of 10-30
amino acids

Endosome

CD4
+
T cells

T cell-mediated help

Class III MHC Region

Contains genes that code for several complement components, cytokines such
as tumor necrosis factor, etc.
69
MHC Restriction

This is the concept that a given T cell will recognize antigen only after it is pro-
cessed and the ensuing antigenic peptide is bound to a particular class I or II
MHC molecule. During T cell development in the thymus, a pre-T cell acquires
TCR that is specific for both an antigenic peptide and a self-MHC molecule.

Self-MHC Restriction of Antigen Recognition by T Cells


CD8
+
T cells (cytotoxic T cells) recognize peptide antigen only in association
with particular self-MHC class I molecules.

CD4
+
T cells (helper T cells) recognize peptide antigen associated with par-
ticular self-MHC class II molecules.

MHC Molecules and Disease

Certain diseases such as various allergies, some viral infections, certain auto-
immune diseases, etc, have been associated with the possession of certain MHC
haplotypes. Various hypotheses have been proposed that attempt to explain the
relationship between MHC and disease susceptibility:

The absence of an MHC molecule that can bind and present an antigenic
peptide.

The absence of TCRs that can recognize the complex of antigenic peptide-
MHC molecule.

The MHC allelic gene may encode molecules that are recognized as recep-
tors by bacterial toxins or viruses.
70
Within a species, a reduction in MHC polymorphism may predispose that
species to infectious diseases, eg, cheetahs and the Florida panther.

ANTIGEN PROCESSING AND PRESENTATION

Antigen processing and presentation is defined as the series of events that result
in the conversion of protein antigens to self-MHC-associated peptide fragments
that are then presented to antigen-sensitive T lymphocytes. T lymphocytes res-
pond only to processed protein antigens.

The high degree of polymorphism of MHC class I and II molecules concen-
trated at the peptide-binding region ensures that a particular species has the
potential to effectively respond to and eliminate any antigen it encounters.

However, a single individual expresses a finite number of MHC molecules;
this means that no single individual or animal is capable of responding
vigorously to each and every pathogen requiring a T cell response for eli-
mination.

When a peptide binds to an MHC molecule, it may stay bound for hours to
several days. Thus peptide-MHC complexes persist long enough on the cell
membranes of antigen-presenting cells and target cells to ensure productive
interaction with antigen-specific T cells.

MHC molecules bind to, and present, both self-peptides and foreign peptides
to T cells.

For example, in a normal healthy cell, class I MHC molecules will display
self peptides that are a result of normal turnover of self proteins. If the cell
is infected by a microbe, eg, virus, viral peptides, as well as self peptides,
will be displayed by class I MHC molecules. However, T cells usually
respond only to the foreign peptides.














71
Phases of Naive T cell Responses

Role of Host Cell Compartments in Antigen Presentation



















72
A cell is divided into two compartments: (1) the cytosol and (2) the vesicular
system, which includes the endosome, endoplasmic reticulum, lysosomes, etc.
Microorganisms can replicate in either of the two intracellular compartments.

Cytosolic antigens. Antigenic peptides are presented in association with
class I MHC molecules to CD8
+
T cells.

Endosomal antigens. Antigenic peptides are presented in association with
class II MHC molecules to CD4
+
T cells.

The MHC Class II Processing Pathway (Endosomal Antigens)

The cells that present peptides associated with class II MHC molecules to helper
T cells [T
H
] are called professional antigen-presenting cells [APCs]. These cells
express both class I and class II MHC molecules on their cell membrane. The
best defined APCs are macrophages, B cells, and dendritic cells.

Dendritic Cells (Interdigitating Dendritic Cells).

Dendritic cells arise from hematopoietic stem cells in the bone marrow and are
found in the T cell areas of lymphatic tissues. They possess long thin cytoplasm-
ic processes called dendrites. They constitutively express many molecules as-
sociated with naive T cell activation, eg, class I and II MHC molecules, the co-
stimulatory B7 molecules, and the cell adhesion molecules ICAM-1, ICAM-2,
LFA-1, and LFA-3 [CD58]. Dendritic cells are the most potent stimulators of na-
ive T cell responses.

Dendritic cells also express high levels of the dendritic cell-specific adhesion
molecule DC-SIGN and also secrete a che-
mokine called DC-CK [CCL18] that speci-
fically attracts naive T cells.

Immature dendritic cells called Langerhans
cells [LCs] are found in nonlymphoid tissues
such as the skin and mucous membranes.
They are actively phagocytic, however, they
do not express co-stimulatory molecules.

When a protein antigen invades the skin or
mucosa, LCs bind the antigen to their surfaces, endocytose it into vesicles
and process it. They migrate as veiled cells, via the afferent lymphatics, into
the paracortex of the draining lymph nodes, where they mature and become
interdigitating dendritic cells [IDCs; mature nonphagocytic cells].

The migration of LCs provides a mechanism for transporting antigens from
the skin and mucosa to the T cells located in the lymph nodes.
73























Macrophages

Most of the antigen ingested by macrophages is degraded and eliminated by
exocytosis; however, some of the peptide products form complexes with MHC
class II molecules. The complexes are displayed on the cell surface where they
are presented to T
H
cells.

Macrophages do not express
MHC class II proteins unless
they are activated by phagocyto-
sing microorganisms.

Macrophages are beneficiaries
of T
H
cell effector functions, ie,
cytokines secreted by activated
T
H
cells activate macrophages to
kill phagocytosed microbes.






Role of dendritic cells in antigen cap-
ture, processing, and presentation.
Langerhans cells in the skin engulf
antigens that enter the body through
the epidermis and transport the anti-
gens to regional lymph nodes.
74
B Lymphocytes

B cells bind to antigens using their B cell receptors [BCRs], endocytose them and
present processed peptides from the antigens to T
H
cells. In turn, cytokines pro-
duced by effector T
H
cells stimulate B cells to produce antibodies against protein
antigens.

Antigen Processing and Presentation

Extracellular protein antigens are phagocytosed or endocytosed into vesicles that
fuse with lysosomes containing acidic proteases. Partial degradation of the anti-
gen occurs in the acidic endosomes, leading to the generation of short peptide
fragments of 10 to 20 amino acid residues.


Newly synthesized class II MHC molecules in the endoplasmic reticulum [ER]
associate with an MHC class II-associated trimeric invariant chain [Ii, CD74].

Invariant chain. When Ii chain binds to the class II
molecule, part of its polypeptide chain lies within the
peptide-binding cleft, ensuring that class II molecules
cannot bind and present peptides they encounter in the
ER. Additional functions of Ii:

Promotes the folding and assembly of class II MHC
molecules. In its absence, many class II molecules
remain in the ER as complexes with misfolded proteins.
75
Directs newly formed class II molecules to the specialized endosomal
compartment called MIIC [MHC class II compartment], where peptide
loading takes place.

HLA-DM. This is a specialized MHC class II-like molecule that is found in the
MIIC, where it facilitates the loading of class II molecules with peptides.

In the MIIC, the Ii chain is cleaved by proteases to leave a fragment bound
to the peptide groove called CLIP [class II-associated invariant chain pep-
tide]. HLA-DM catalyzes the release of CLIP. Additional functions:

Stabilizes the empty class II molecules so they do not aggregate; cata-
lyzes the binding of peptides to the empty peptide groove.

Facilitates the release of weakly bound peptides from the peptide grooves
and allows other peptides to replace them. This ensures that the peptide-
MHC II complexes on the cell surface of the APC will survive long enough
to encounter the antigen-specific T
H
cell.

Individuals with mutations in the HLA-DM genes, demonstrate defective
antigen presentation. The class II molecules assemble correctly with the Ii
chain, however, the MHC molecules fail to bind endosome-derived pep-
tides and are displayed at the cell surface still bound to the CLIP peptide.


Class II molecules with bound peptides are transported to the surface of the
APC, where they are displayed for recognition by CD4
+
helper T cells. If an
APC is not processing a foreign antigen, MHC class II molecules are dis-
played at the cell surface with bound self peptides.

Co-stimulation

Presentation of an antigen [signal 1] to a naive helper T cell by an MHC class II
molecule on an APC is, by itself, insufficient to trigger an immune response. The
T cell must also be exposed to co-stimulating molecules [signal 2], such as cyto-
kines and cell membrane proteins, produced by the APC.
76
Co-stimulatory molecules for helper T cells include the cytokine interleukin 1
[IL-1] and the cell membrane proteins B7-1 [CD80] and B7-2 [CD86] (see
Activation of T
H
Cells, page 84).

Signal 1 leads to the induction of various transcription factors, some of which
bind to the promoter regions of the interleukin-2 [IL-2] and interleukin-2 recep-
tor [IL-2R] genes, enhancing their transcription in the T cell. Delivery of the
co-stimulatory signal increases the half-life of IL-2 mRNA, resulting in IL-2
production [T cell autocrine growth factor].

This leads to the proliferation and differentiation of naive T helper cell into
effector T cells and memory T cells.

Signal 2 also induces the synthesis of anti-apoptotic proteins.

In the absence of the co-stimulatory signal, the IL-2 mRNA is rapidly degrad-
ed, preventing the synthesis of IL-2. As a result, the activation process is
terminated and the naive T cell either fails to respond and die by apoptosis or
enters a state of unresponsiveness called anergy.




Activation of nave T helper
cell by both signal 1 [MHC-
peptide-TCR complex] and
signal 2 [co-stimulatory sig-
nal]
77
THYMUS-DERIVED CELLS (T CELLS)

T cell precursors differentiate into immunocompetent T cells within the thymus
before populating the peripheral lymphoid organs and tissues. T cells are rela-
tively long-lived cells that may survive from 6 months to over 20 years.

T cells constitute up to 80% of the recirculating pool of small lymphocytes.

T CELL DEVELOPMENT IN THE THYMUS

In the thymus, a developing T cell acquires antigen-specific receptors and other
functional and phenotypic characteristics that mark it as a mature T cell.

T Cell Surface Molecules

Mature T cells express a variety of cell surface proteins. Among them are pro-
teins involved in antigen recognition, signal transduction, and adhesion to APCs
and target cells. Antigen activated T cells express additional molecules that en-
able them to carry out their effector functions.

A number of the cell surface proteins have
been given a CD (cluster of differentiation)
designation.

Activation of a cell surface receptor, re-
quires binding to its specific ligand, which
may be a counter-receptor on another cell
or a soluble protein, eg, cytokine.







Accessory Molecules of T Cells

Accessory molecules are cell membrane proteins which play critical roles in T
cell response to antigen. Some are adhesion molecules; others deliver signals to
the T cell that function, in concert with signals from the TCR-CD3/,, complex, to
fully activate the T cell.

CD4 and CD8 Molecules

They promote adhesion of T cells to APCs and target cells and also play a role
in signal transduction, thereby potentiating T cell activation.
78
CD4 (TCR coreceptor). It is found on helper T cells [present on monocytes
and macrophages of some species; unknown function]. It binds to the |2
domain of the class II MHC molecule on the surface of professional APCs.

CD8 (TCR coreceptor). It is found on cytotoxic T cells. Its function is the
recognition of the o3 domain of class I MHC molecules, enabling cytotoxic T
cells to bind to target cells.

Lymphocyte Adherence Molecules

They strengthen interactions between helper T cells and professional APCs or
cytotoxic T cells and target cells (eg, virus-infected cells).

CD11aCD18 (Lymphocyte function-associated antigen-1, LFA-1). Binds T
cells to CD54 molecules (ICAM-1) on APCs and target cells. Promotes
binding of T cells to endothelial cells during T cell extravasation.

CD2 (LFA-2). CD2 molecules are found on mature T cells. CD2 binds to a
protein called CD58 (LFA-3) present on APCs and target cells. CD2 functions
both as an intercellular adhesion molecule and as a T cell signal transducer
[contributes to optimal T cell activation].

T Cell Receptor for Co-stimulation

CD28. It is a constitutively expressed membrane receptor for co-stimulatory
signals [signal 2]. Its ligand is B7-1 [CD80] and B7-2 [CD86] cell surface pro-
teins that are expressed on professional APCs.

- Signal 2 functions with signal 1 [delivered by the TCR complex] to acti-
vate naive T cells [see Co-stimulation, page 75].

T Cell Receptor Complex (TCR Complex)

The T cell receptor complex consists of the T cell receptor (TCR), CD3 complex
and , (zeta) proteins.

The TCR is a disulfide-linked heterodimer glycoprotein that enables T cells to
recognize processed antigens presented in association with MHC molecules.
Two types of TCRs have been identified: TCRo| and TCRo.

TCRo|

The o| chains are integral membrane proteins with an extracellular domain, a
transmembrane domain, and a short cytoplasmic tail. The extracellular domains
consist of variable [N-terminal], constant, and hinge regions.

79
Hypervariable regions (Complementarity-determining regions, CDRs). The
CDRs reside within the variable region; they consist of unique amino acid se-
quences that form the antigen-binding site of the TCR. Both chains possess
3 CDRs [CDR1, CDR2, and CDR3] juxtaposed to one another.

- CDR3 is in the center, displays most diversity [a product of junctional di-
versity, see page 81], and directly interacts with the antigenic peptide,
while CDR1 [interacts with both peptide and MHC] and CDR2 [interacts
with MHC] are in the periphery.

- The | chain V domain contains a fourth hypervariable region, which does
not appear to participate in peptide recognition but is the binding site for
microbial products called superantigens [see Bacterial Superantigens,
page 147].

Each T cell possesses 50,000 or more TCRs. These are clonotypic, ie, will
recognize only a single epitope bound to an MHC protein. In humans and
nonruminants, 90-99% of T cells carry o| receptors. Unlike BCR, TCR is not
produced in secreted form, hence it does not perform effector functions on its
own [see Comparison of TCR and BCR, page 95].






TCR o| Repertoire

The repertoire of TCRs available to an animal or individual is carefully regulated,
ie, can respond to as many different foreign antigens as possible but not to self-
antigens.

The phenomenon of allelic exclusion controls the genetic expression of TCRs
[in a heterozygous animal or individual, only one of the allelic forms of a gene
is expressed]. Allelic exclusion occurs when only one of the parental alleles
80
that code for the TCR is functional, rendering each T cell responsive to only a
single epitope. The genes encoding the o chain and | chain are found on se-
parate chromosomes.
Recombinase Enzymes

Rearrangement of TCR gene segments and BCR gene segments [see page 91]
is regulated by several enzymes collectively called recombinases.

Recombination-activating genes I and 2 [RAG-1 and RAG-2]. The genes are
found only in T and B lymphocytes; however, they are active only in develop-
ing lymphocytes, which explains why TCR and BCR rearrangements do not
continue in mature T and B cells that have completed gene rearrangements.

- RAG proteins cleave DNA at recombination sequences which are located
adjacent to the V, D, or J coding sequence.

- Individuals or animals lacking RAG genes fail to produce both BCR and
TCR proteins and manifest severe combined immunodeficiency, SCID
[lack of T and B cells; see Immunodeficiency Diseases, page 240].

DNA repair enzymes [eg, DNA-dependent protein kinase, DNA-PK]. Unlike
RAG proteins, these enzymes are expressed in many cell types.

- Their role in TCR and immunoglobulin [BCR] gene recombination is to
repair the double-stranded breaks introduced by the RAG proteins. Simi-
larly, lack of DNA repair enzymes results in SCID.

- In Arabian foals with SCID, there is a defect in the DNA repair enzyme
that joins the DNA ends.

| chain Gene Rearrangement

The | chain genes rearrange first, followed by the o chain genes. Multiple va-
riable (V
n
) region genes, diversity (D
n
) region genes and joining (J
n
) region genes
occur at the TCR| locus. First, D| gene segments rearrange and join to J | gene
segments, followed by V| to the DJ | gene rearrangement, and then to a C|
gene.



o chain Gene Rearrangement

Multiple variable (V
n
) region genes and joining (J
n
) region genes occur at the
TCRo locus. During the rearrangement of o chain genes, a randomly selected V
81
gene is joined to a J gene and the exon is
transcribed, combined with a constant (Co)
region gene, and translated.

Rearranged TCR Genes

















Diversity of TCR

Accounted for by multiple germ-line genes; gene rearrangements; and Junctional
diversity.
J unctional Diversity

Base deletions. Removal of a few bases from the broken ends of the coding
sequences by endonucleases. Thus, the precise nucleotide at which V, D,
and J genes join can vary, resulting in greater diversity.

N-region nucleotide addition (nongermline nucleotides). Up to 10 nucleo-
tides may be added to the cut ends of V/J [o chain] and V/D/J [| chain] co-
ding sequences by the enzyme terminal deoxynucleotidyl transferase [TdT].

P-nucleotides (nongermline nucleotides). When genes are cleaved, the
ends may be cleaved asymmetrically so that one DNA strand is shorter than
the other. The shorter strand is usually extended with nucleotides comple-
mentary to the longer strand before the ligation of the two segments. The
added nucleotides at the V/D/J and V/J junctions are called P-nucleotides.

Potential TCR Repertoire

Once rearranged in the thymus, the coding sequence of each TCR remains
unchanged and does not display somatic hypermutation as with B cell-derived B
A diagram of rearranged TCR
genes showing the exons that
code for the various regions of
the chains. J unctional diver-
sity [arrows] contribute to the ge-
neration of CDR3.
82
cell receptors [BCRs; see Somatic Mutations, page 100]. It is estimated that the
total TCR repertoire may be in the order of 1 x 10
16
.

CD3 Complex and ,, (Zeta) Proteins

CD3 and ,, proteins are accessory molecules that noncovalently associate with
the T cell receptor. The CD3 complex consists of 4 invariant [ie, the same on all
T cells] polypeptide chains [, o, and two c chains]. The two ,, chains are the
same on all T cells.

When a TCR binds an antigen, a signal must be sent to the T cell nucleus in
order to initiate its response. The cytoplasmic tail
of the TCR is too small to transduce signals,
therefore, the biochemical signals for T cell acti-
vation do not come from the TCR, but, rather,
from the CD3 complex and the ,, chains [collec-
tively referred to as signal transducers].

The CD3 complex also functions in stabilizing cell
surface expression of TCR.

Mutations in the CD3 genes effectively prevent T
cell expression of TCRs.





Positive and Negative Selection of Thymocytes

During T cell development, thymocytes undergo two critical selection processes
that result in self-MHC restriction of TCRs and the elimination of thymocytes
whose TCRs bind strongly to self peptides [so-called forbidden clones].

Thymocyte selection is carried out in the cortex by thymic stromal cells [cor-
tical epithelial cells, macrophages, and dendritic cells]; these cells express
class I and class II MHC molecules.

Positive selection. Thymocytes whose TCRs have moderate affinity for self-
MHC molecules are rescued from programmed cell death. This ensures that
mature T cells are self-MHC restricted, ie, CD4
+
T cells interact with peptide-
MHC II complexes, whereas, CD8
+
T cells interact with peptide-MHC I com-
plexes.

Negative selection. Thymocytes whose TCRs have low affinity or high affi-
nity for self-MHC molecules are eliminated by apoptosis. Death by apoptosis
83
does not elicit an inflammatory response or trigger host defense mechanisms.
Cellular debris resulting from apoptosis is taken up by macrophages.

- In addition, thymocytes
that express TCRs with
high affinity for self-antigen
presented by self-MHC
molecules are eliminated,
contributing to the mainte-
nance of self-tolerance
[see page 225].




























TCRo

TCRo receptors are expressed on a subset of T cells that are found mostly in
epithelial locations, so-called intraepithelial lymphocytes. TCRo recognizes
native antigen rather than peptide-MHC complex and displays limited antigenic
diversity. The role of o TCR-bearing T cells in host immune responses is poorly
understood.
Positive and negative selection
of thymocytes [pre-T cells] in the
thymus.
84
T Cell Subsets

Helper T cell (T
H
; CD4
+
). Helps B cells produce antibodies to protein anti-
gens. In most species, the ratio of T
H
cells to Tc cells is 2:1.

Cytotoxic T cell (Tc; CD8
+
). Causes lysis of antigen-bearing target cells.

T CELL ACTIVATION IN PERIPHERAL LYMPHOID ORGANS

Helper T Cells (CD4
+
T Cells)

T
H
cells play a major role in promoting innate and adaptive immune responses
by releasing soluble helper factors [cytokines] that affect the activities of several
cell types.

Professional APCs (Macrophages, B cells, Dendritic cells)

Activation

Helper T cell
Autocrine
effect
Cytokines



Tc cells B cells Macrophages NK cells Other cells

Activation of Naive Helper T Cells

Helper T cells respond only to processed antigen when it is presented by pro-
fessional APCs in association with self-MHC class II molecules. Interaction with
an APC involves multiple T
H
cell membrane proteins that recognize different
ligands on the APC.

Naive T
H
cell activation requires two signals:
TCR complex and CD4 recognition of peptide-
MHC complexes on the APC constitute the
initial signal [signal 1] and co-stimulation, the
second signal [signal 2].

Of the professional APCs, mature dendritic
cells are the most potent activators of naive
T
H
cells since they constitutively express
MHC molecules and co-stimulators.



85
Co-stimulation

B7 proteins [B7-1, CD80; B7-2, CD86]. The best characterized co-stimula-
tors for naive T
H
cells are the B7 proteins, which are absent or expressed at
low levels on resting APCs.

- Enhanced expression occur when APCs are stimulated by endotoxin
[LPS], interferon-, and binding of T cell CD40L to CD40 on the APC.

CD28. T cell receptor for B7 proteins. Signaling by CD28 enhances several
T
H
cell responses to antigen, including the production of cytokines such as IL-
2 and differentiation of naive T cell into effector cells and memory cells [see
Co-stimulation, pages 75 and 76].

Cytokines. Interleukin-1 is a major co-stimulator of T
H
2 cells [T
H
1 cells lack
IL-1 receptors and do not respond to IL-1]. Interleukin-6 promotes IL-2 and
IL-2 receptor production and T cell proliferation.

Role of CD40 in T cell Activation

Antigen recognition by helper T cell induces the expression of CD40L. CD40L
binds to CD40 on the APC and stimulates the expression of B7 molecules which
bind to CD28 on the helper T cell and the secretion of cytokines that activate the
helper T cell.


Naive Helper T cell Proliferation and Differentiation

Antigen-activated naive T
H
cells synthesize IL-2 and IL-2 receptors (IL-2Rs). If
the IL-2Rs are bound by IL-2 (T cell autocrine growth factor), the cell undergoes
clonal expansion (ie, proliferation of the antigen-specific clone).

Most of the daughter cells differentiate into effector cells; a small fraction of
the daughter cells differentiate to become antigen-specific memory cells.
86
Phases of Naive T helper Cell Responses

Effector T
H
Cells

The terminally differentiated effector T
H
cells enter the circulation and are carried
to peripheral tissues [lymphatic and nonlymphatic]. Here, they are triggered to
effector function by encountering MHC-peptide complexes on APCs.

In contrast to naive T
H
cells that require 2 signals for activation, effector T
H

cells are more sensitive to TCR/CD4 recognition of peptide-MHC complexes
[signal 1] and, therefore, have no need for co-stimulation [signal 2].

- As a result, effector T
H
cells are able to respond to peptide-MHC com-
plexes displayed on APCs that lack the co-stimulatory B7 molecule.

The result of effector T
H
cell activation is the synthesis of various membrane
bound [eg, CD40L] or secreted [eg, cytokines] effector molecules.
87
- CD40L. Binds to CD40 on macrophages and B cells and activates them
[see Role of CD40L in B cell Activation, page 97].

- Cytokines. Various cytokines are secreted by the activated effector T
H

cells that promote and regulate the effector phases of innate and adaptive
immune responses.

Effector cell responses decline following elimination of the antigen. The vast
majority of antigen-activated T cells die by apoptosis within a few days. The
decline in effector response is significant because it returns the immune sys-
tem to a state of rest or homeostasis.

Comparison of Naive and Effector T Cells

The three types of effector T cells are: CD4
+
T
H
1 and T
H
2 cells [see T
H
cell Sub-
sets, page 88] and CD8
+
T cell [see CMI, page 127].

Feature Naive T cells Effector T cells
Costimulatory signal [signal
2; CD28-B7 interaction]

Cell adhesion molecules
[CD2 and LFA-1]


Trafficking patterns

Lifespan

Effector functions
Required for activation


Low



Peripheral lymphoid organs

Long-lived cells

No
Not required for activation


2-4 fold higher [bind more
effectively to APCs and tar-
get cells]

Inflammatory sites, etc

Short-lived cells

CD40L, cytokine secretion,
killing of target cells, etc

Memory T Helper Cells

Memory T
H
cells, unlike effector cells, are long-lived cells. The expanded clone
of antigen-specific memory T
H
cells account for the increased and accelerated
secondary response on subsequent exposure to the same antigen.

Normally, they are functionally quiescent until reexposure to the antigen
[presented by APCs] induces them to undergo clonal expansion, followed by
differentiation of daughter cells into effector cells and memory cells.

Like effector T
H
cells, memory T
H
cells do not need B7-CD28 co-stimulatory
interactions [signal 2] to induce full T cell activation.

Recirculation of Effector and Memory T Cells

The recirculation patterns of effector and memory T cells differ from those of
naive T cells. They preferentially home to inflamed peripheral tissues where they
88
are needed to eliminate antigens in the effector phase of adaptive immune res-
ponses.

Effector T cells down-regulate the expression of
L-selectin and CCR7 and up-regulate the ex-
pression of the chemokine receptor CCR10 and
the adhesion molecules LFA-1 and VLA-4, allow-
ing them to bind to ICAM-1 and VCAM-1, res-
pectively, on peripheral vascular endothelium at
sites of inflammation.

Thus, newly differentiated effector T cells are di-
rected to the site of infection in the peripheral
tissues.


T Helper Subsets

Helper T cells are subdivided into T
H
1 helper cells and T
H
2 helper cells on the
basis of their production of and responses to specific cytokines. T
H
1 and T
H
2
subsets develop from the same naive CD4
+
T lymphocytes [T
H
0], in the peri-
pheral lymphoid organs.

Cytokines produced in the
innate immune response to
microorganisms or early in
adaptive immune responses
influence the differentiation
of naive CD4
+
T cells into
T
H
1 or T
H
2 cells.












Differentiation of naive CD4
+
cells into T
H
1 cells occurs in response to many
pathogens, especially intracellular bacteria and viruses that infect or activate
macrophages and NK cells to produce interleukin-12 or interferon-.

IL-12: promotes the production of
the T
H
1-promoting transcription
factor, T-Bet. IL-4: promotes the
production of the T
H
2-promoting
transcription factor, GATA-3. T
H
1
cytokine and T
H
2 cytokine posi-
tively regulate the subset that pro-
duces it, while negatively regula-
ting the other subset.
89
Interleukin-4, which is produced in response to helminth parasites and other
pathogens, causes proliferating CD4
+
cells to differentiate into T
H
2 cells.

Cytokine Profiles of T
H
1 and T
H
2 Subsets

Cytokine T
H
0 T
H
1 T
H
2
IL-2
IFN-
TGF-|
GM-CSF
IL-3
IL-4
IL-5
IL-10
IL-12
+
+
+
+
+
+
+
+
+
++
++
-
++
++
-
-
-
+
-
-
++
++
++
++
++
++
-
Abbreviations: see Cytokines
























The cytokines produced by the T
H
1 and T
H
2 subsets exhibit cross-regulation, ie,
cytokines from T
H
1 cells can block the production and/or activity of the cytokines
secreted by T
H
2 cells and vice versa [see Immune Regulation, page 135].

Hence each subset amplifies itself and cross-regulates the other subset.
The significance of cross-regulation is that an immune response tends to set-
tle into a T
H
1 type of response or a T
H
2 type of response.

90
BURSA/BONE MARROW-DERIVED CELLS (B CELLS)

In 1956, Dr. Bruce Glick [Poultry Science Department, Mississippi State Univer-
sity], reported that removal of the bursa of Fabricius from chickens during the
early part of its rapid growth would markedly compromise antibody production.

However, up until the mid-1960s, it was believed that the thymus either regu-
lated or was the site of early development of the lymphoid precursors of anti-
body-forming cells. The concept was finally rejected upon demonstration that
lymphoid development proceeds along two separate pathways.

In birds, B cells mature in the BF. In mammals, the early events of B cell
development begin in the bone marrow or Peyers patches. Immature B cells
leave these organs and complete their maturation in the spleen before re-
circulating to seed all the peripheral lymphoid organs.

- In humans, approximately 1 x 10
9
B cells are produced daily. B cell lym-
phopoiesis occurs throughout the lifetime of mammals, though in gradually
decreasing numbers with age.

B cells constitute up to 10-25% of the recirculating pool of lymphocytes; they
are mostly confined to peripheral lymphoid organs and tissues.

Unless mature B cells encounter their antigens and respond to them, they die
in a few days or weeks [short-lived cells]. Memory B cells are long-lived cells.

B Cell Antigen Receptor (BCR) Complex

The B cell antigen receptor complex consists of membrane immunoglobulins
(monomeric IgM and IgD) and two invariant proteins that are disulfide linked to
each other (Igo |CD79a| and Ig| [CD79b]) and which noncovalently associate
with IgM and IgD. Each B cell expresses up to 500,000 BCRs.

All the BCRs on a single B cell have the same
variable region and therefore recognize only a
single antigen [clonotypic]. They are larger
than their secreted counterparts, due to a
stretch of hydrophobic amino acids at the C
terminus of the heavy chain. The additional
amino acids traverse the cell membrane to
anchor the Ig molecule.

The BCR plays two key roles in B cell acti-
vation: [1] delivery of activating signals to the
B cell when two or more receptor molecules
are cross-linked by multivalent antigens and,
91
[2] triggering endocytosis, leading to processing of protein antigens and the
presentation of antigenic peptides in association with MHC class II molecules
to helper T cells.

Like TCRs, the cytoplasmic tails of the membrane Igs are too small to trans-
duce signals to the B cell nucleus, therefore, BCR-mediated signals are trans-
duced by the CD79 molecules. CD79 molecules are also required for the
surface expression of IgM and IgD.

Immunoglobulin (BCR) Gene Rearrangement

The orderly, somatic gene rearrangement [somatic recombination] of Ig genes in
a developing B cell results in Ig diversity and also ensures that only B cells will
produce immunoglobulins. A productive rearrangement of the immunoglobulin
heavy chain [IgH] gene must occur first before rearrangement of the immuno-
globulin light [IgL] chain gene will begin. In humans and domestic animals, Ig
molecules are coded for by three unlinked gene families:

Gene family located on chromosome 14 [humans] codes for Ig heavy chains.
It consists of ~200 V
H
genes, 30 diversity gene segments [D
H
], 6 J
H
genes,
and 9 C
H
genes for each Ig class and subclass.






Gene family located on chromosome 2 [humans] codes for k light chains. It
consists of ~100 different variable gene segments [V
k
], 5 different joining
gene segments [J
k
|, and a single constant gene segment [C
k
].




Gene family located on chromosome 22 [humans] codes for light chains. It
is made up of ~100 V

genes, 6 J

genes, and 4 C

gene segments.





Recombination of IgH Chain Genes

DNA rearrangement occurs first on one chromosome. If this is successful, a
[IgM] heavy chain protein is synthesized. Subsequently, there is irreversible inhi-
bition of DNA rearrangement of the heavy chain gene segments on the other
92
chromosome [allelic exclusion]. This ensures that one B cell will react with only
one antigenic determinant and will produce antibodies to only that antigen.

If the first rearrangement is
nonproductive, for example
due to introduction of a new
stop codon or frame shift,
rearrangement then proceeds
to the second allelic chro-
mosome.

If both alleles undergo non-
productive recombination, the
developing B cell cannot
produce antibodies and un-
dergoes apoptosis.

The production of mature
RNA requires both recom-
bination of DNA segments
and splicing of primary RNA
transcript. Translation of the
heavy chain mRNA leads to
production of the protein.

Recombination of IgL Chain Genes

Production of a chain protein triggers IgL chain gene rearrangement, beginning
with the k light chain. If the rearrangement is successful and functional k light
chains are synthesized, irreversible inhibi-
tion of the gene segments occurs [light
chain isotype exclusion or allelic exclusion].

If the k allele undergoes nonfunctional
rearrangement, DNA rearrangement oc-
curs on the allele. If nonfunctional re-
arrangement occurs with both k and al-
leles, the developing B cell undergoes
apoptosis.

Thus, a single B cell clone can produce
only one of the two types of light chains
during its life.

Similar to the heavy chain protein, the
production of mature RNA requires both
93
recombination of DNA segments and splicing of primary RNA transcript.
Translation of the light chain mRNA leads to production of the light chain
protein.

Diversity of the B cell Repertoire

It is estimated that an individual may possess up to 1 x 10
11
BCRs. Several
mechanisms account for this enormous diversity [see TCR Diversity, page 81].

Somatic recombination of multiple germline gene segments.

J unctional diversity. Base deletion; P-nucleotide and N-nucleotide additions.

- N-nucleotides. TdT is expressed by pro-B cell only during the stage of
IgH gene rearrangement. Therefore, only IgH shows N-region diversity.

Combining of identical IgH chain proteins with different IgL chains yields mul-
tiple specificities.

THE DEVELOPMENT OF B CELLS

Stages in the Life History of B Cells












B Cell Development in the Bone Marrow

The hallmark events of B cell development are: (1) the rearrangement of antigen
receptor genes and the expression of membrane Igs with a single antigenic spe-
cificity by the immature B cell and (2) the selection of the mature B cell repertoire.

Bone marrow stromal cells. Stromal cells in the bone marrow provide the
microenvironment necessary for B cell development.

- Stromal cells interact directly with progenitor B cells and precursor B cells,
providing them with cell-bound survival signals.

- Secretion of various cytokines [eg, IL-7], that support B cell development.
94
- Selection of the B cell repertoire.

Progenitor B cell [Pro-B cell]. Expresses Igo [CD79a] and Ig| [CD79b].
These signaling molecules transmit biochemical signals after the variable
region of the BCR binds to its antigen.

- Expression of the recombinase enzymes RAG-1
and RAG-2 [see T cells, page 80]; and the en-
zyme TdT [see T cells, page 81].

- IgM [] heavy chain gene rearrangement begins. It starts with heavy
chain D
H
to J
H
gene rearrangement, followed by V
H
to D
H
J
H
rearrange-
ment. The cell is now called precursor B cell.

Precursor B cell [Pre-B cell]. Continued expression of RAG-1 and RAG-2
enzymes. Termination of TdT activity.

- Synthesis of IgM heavy chain. The heavy chain pairs with surrogate light
chain. The pair is expressed on the pre-B cell surface in association with
the Igo and Ig| molecules, to form the pre-B cell receptor. Only pre-B
cells that successfully express cell surface heavy chain/surrogate light
chain receptors can proceed to become immature B cells.

- Surrogate light chain. It is made up of two inva-
riant proteins called VpreB and 5.

- Pre-B cell receptor. Signals generated through
the receptor are essential for continued develop-
ment of pre-B cell. Pre-B cell receptor signaling
results in the following events: (1) survival and
proliferation of pre-B cell, (2) inhibition of V
H
to D
H
J
H
rearrangement of the
other heavy chain allele [allelic exclusion, ie, expression of only one of two
inherited alleles], (3) termination of surrogate light chain transcription, (4)
initiation of light chain [k or ] gene rearrangement.

- Following rearrangement of the light chain genes, IgM containing both
heavy and light chains is expressed on the cell membrane. The cell sheds
its pre-B cell receptors and is now called immature B cell.

Immature B cell. Membrane Ig [ heavy chain and
k or light chain] in association with Igo and Ig|.

- Selection of the B cell repertoire. If an imma-
ture B cell that expresses high-affinity BCR for self
antigen binds the antigen in the bone marrow: (1)
it dies by apoptosis [clonal deletion], or [2] it be-
95
comes anergic [long-lasting inactivation], or [3] may undergo receptor
editing.









- Receptor editing. This is a process that attempts to rescue a self-
reactive B cell from undergoing apoptosis. The B cell reactivates the
RAG-1 and RAG-2 genes and begins further rearrangement of the light
chain genes. If it is successful, the new light chain results in a nonself-
reactive BCR, thus the cell escapes negative selection. TCRs do not
undergo receptor editing.

- Migration of immature B cells from the bone marrow to the spleen.

Mature B cell. In the spleen, maturation of B cells requires cytokines and also
biochemical signals that emanate from the B cell receptor. Full maturation of a B
cell is signaled by the coexpression of IgM and IgD on the cell surface.

This involves a change in RNA processing of the heavy chain primary RNA
transcript to allow production of two mRNAs, one that codes for the mIgM and
the other that codes for mIgD.

- Both mIgM and mIgD use the same VDJ exon and associate with identical
light chains, hence both mIgs possess the same antigen specificity.

Following maturation, naive B cells move through the circulation to populate
the peripheral lymphoid organs and tissues.

Comparison of BCR and TCRo|
Feature BCR TCRo|
Valency Bivalent Monovalent
Binding of soluble antigen Yes No
Chemical nature of antigen Protein, polysaccharide, lipid Only protein
Epitope recognized Native epitope Processed epitope
MHC molecule required No Displays processed antigen
Receptor editing Yes No
Isotype switching Yes (IgG, IgA, IgE) No
Secreted form Yes No
Somatic hypermutations Yes (affinity maturation) No
Effector function Yes No

96
B CELL ACTIVATION IN PERIPHERAL LYMPHOID ORGANS

Mature B cells express several cell surface proteins that are responsible for anti-
gen recognition, signal transduction, regulation of B cells, etc.



















The B Cell Coreceptor Complex

The B cell coreceptor complex is a complex of three cell surface molecules, CR2
[CD21], CD19, and CD81. CR2 is a complement receptor that binds complement
component, C3d.

C3d [cleavage product of C3b] generated
during complement activation becomes
covalently attached to microorganisms or
antigen-antibody complexes that initiate
complement activation. This complex of
C3d and antigen binds to B cell, with the
BCR binding to the antigen and CR2
recognizing the bound C3d.

Binding of C3d to CR2 results in phospho-
rylation of CD19, generating intracellular
signals that augment the signal transduced
via the B cell receptor itself. Thus, when the coreceptor is triggered at the
same time as the BCR, a much lower level of antigen [100- to 1000-fold less
antigen] is needed to generate an activating signal and the response of the B
cell is greatly enhanced compared to when antigen interacts only via the
BCR.
97
Response of B Cells to T-Dependent (Protein) Antigens

Humoral immune responses to protein antigens require recognition of the antigen
by helper T cells and cooperation between the antigen-specific B and T cells.

Helper T cell-B cell interactions occur at the junction of the cortex and
paracortex. The interactions involve antigen presentation by B cells to helper
T cells, activation of the helper T cells, expression of CD40L and secretion of
cytokines by the helper T cells that bind to and activate the B cells. Activated
B cells migrate back into the primary follicles.

Antigen recognition by B cells enhances the expression of co-stimulators that
increase the ability of the B lymphocyte to activate helper T cells. B cells also
express receptors for cytokines secreted by the activated helper T cell. Addi-
onally, during antigen processing, B cells alter their cell surface chemokine
receptors; this enables them to follow a chemokine gradient and migrate to-
ward the paracortex.

A single B cell may bind and endocytose a protein and present multiple differ-
ent peptides in association with class II MHC molecules to different helper T
cells but the resultant antibody response remains specific for the previously
rearranged V[D]J variable region gene segments during B cell development.

The net results are induction of B cell clonal expansion, isotype switching, af-
finity maturation, and differentiation into plasma cells or memory B cells.

Role of CD40L and Cytokines in B cell Activation

CD40 [costimulatory receptor for B cells]. Binds to CD40L on T
H
cell.

- Promotes expression of co-stimulatory molecules, growth and differentia-
ion of B cells, and, together with cytokines, isotype switching, affinity ma-
turation, and development of memory cells.
98
IL-2, IL-4, and IL-5. Contribute to B cell proliferation.

IL-6. Plays a key role in the differentiation of progeny B cells into antibody-
secreting plasma cells. Multiple cytokines, including IL-2, IL-4, IL-5, IL-6, IFN-
, and TGF-| stimulate antibody synthesis and secretion by plasma cells.


Isotype (Class) Switching

This is the change in antibody class that occurs during the course of an immune
response to a protein antigen as a result of heavy chain gene switching.

The switching process is not random but is regulated by cytokines secreted
by helper T cells. Different cytokines preferentially induce switching to differ-
ent immunoglobulin isotypes.

Isotype switching occurs via switch recombination, in which the rearranged
VDJ gene segment recombines with a downstream C
H
region and the inter-
vening DNA is deleted.

Initially, most daughter cells express IgM-BCRs. Eventually, a responding
daughter B cell switches to express IgG-BCRs, IgA-BCRs, or IgE-BCRs. The
plasma cell resulting from the differentiation of a daughter B cell is committed
to secreting the immunoglobulin isotype expressed by the daughter B cell.

The effector function of an immunoglobulin molecule is determined by the
isotype of its heavy chain C region [see Immunoglobulins, page 107]. Isotype
switching ensures that the same variable region, and hence the same anti-
gen specificity, can be expressed with different C
H
region isotypes that are
involved in host defense against different types of infectious agents.
99




















Somatic Mutations (Hypermutations; Affinity Maturation)

Affinity maturation is the process that results in an increase in the affinity of
specific antibody for its antigen and is the result of somatic mutations of the pre-
viously rearranged Ig V(D)J genes, followed by selective survival of the B cells
producing the antibodies with the highest affinity.




















100
Affinity maturation occurs only in humoral immune responses to protein anti-
gens, since CD40-CD40L and T helper cell-derived cytokines regulate soma-
tic mutations.

Proliferating germinal center B cells show high rates of point mutations in their
rearranged Ig V(D)J genes. Some of the mutations generate high-affinity
membrane immunoglobulins [BCRs], whereas many of the mutations may
result in a decrease or sometimes in a loss of antigen binding.

Selection of high-affinity B cells by follicular dendritic cells [FDCs]

- FDCs [not of bone marrow origin] are found in the lymphoid follicles of the
lymph nodes, spleen, and MALT. FDCs express Fc receptors [CD32]
and the complement receptors CR1 [CD35], CR2 [CD21] and CR3. FDCs
are nonphagocytic cells and do not present antigens to helper T cells.

- Nondividing small B cells in the germinal center, which are the cells in
which immunoglobulin genes have undergone point mutations, need to
bind to antigen to be rescued from programmed cell death.

The source of antigens is the FDCs in the germinal center that trap
antigens complexed with IgG or complement fragments C3b and C3d.
Biochemical signals generated by antigen binding to surface BCR on
the B cell, blocks a default apoptotic pathway in the B cell.

The net result of this selection process is a population of B cells that become
memory B cells with high affinity BCRs or B cells that become plasma cells
that produce antibodies with significantly higher affinities for antigen than the
antibodies produced by the same clones of B cells earlier in the immune
response.

PLASMA CELLS

Most of the progeny of B cells that proliferate in response to antigens, differen-
tiate into antibody-secreting plasma cells. Plasma cells are defined as terminally
differentiated B cells. Plasma cells are short-lived cells [live only a few days, 3
to 6 days].

However, it has been discovered that some plasma cells may migrate from
the peripheral lymphoid organs to the bone marrow, where they continue to
produce low levels of antibodies for many years.

Plasma cells are larger than naive B cells, ovoid in shape and possess an
eccentric spoke-wheel nucleus. They possess intense basophilic cytoplasm
[rich in ribosomes] with characteristic parallel arrays of rough endoplasmic
reticulum.
101













Few plasma cells are found in the circulation [0.1%]; mostly, they are found
in extrafollicular sites in peripheral lymphoid organs and at inflammatory sites.

- Red pulp areas of the spleen; medulla of lymph nodes; MALT [lamina pro-
pria of intestinal and respiratory tracts]; and bone marrow sinusoids.

More than 40% of the total proteins produced by a plasma cell are antibodies.
A plasma cell is capable of synthesizing over 2,000 molecules of antibodies
per second. The antibodies are transported to the cell surface in secretory
vesicles and released into the extracellular fluid in the peripheral lymphoid
organ.

The mechanism that enables plasma cells to produce secreted Igs and B
cells to produce membrane-bound Igs [BCRs], involves differential processing
of heavy chain 1
o
RNA transcript into messenger RNA, an event regulated by
RNA cleavage and the choice of polyadenylation sites.

- Differentiation of B cells into plasma cells introduces changes in mRNA
processing, such that the heavy chains of Igs produced by plasma cells
lack the transmembrane hydrophobic amino acids [~ 26 in number] and
cytoplasmic segments found in membrane-bound Igs.

The specificity of the Igs produced by a plasma cell is identical to that of the
BCR on the parent B cell. All the antibodies secreted by a single plasma cell
have the same specificity and isotype [see Immunoglobulins, page 111].

Occasionally, the production of immunoglobulins may be so rapid that they
accumulate within cells to form large vesicles known as Russell bodies.

Multiple Myeloma

Multiple myeloma is a tumor of plasma cells arising as a result of a random
mutation in a single B cell. They are almost always first detected as multiple foci
102
in bone marrow [characterized by osteolysis]. They have been reported in hu-
mans, horses, cats, dogs, cows, pigs, etc.

A myeloma cell continues to produce specific antibody. The antibody is re-
ferred to as myeloma protein or M protein. It is indistinguishable from normal
antibody molecules. M proteins may belong to any Ig class, eg, IgG, IgA, etc.

Light-chain disease is a myeloma in which antibody light chains alone are
produced or the production of light chains predominates. The light chains
pass through the glomerulus and are excreted in the urine. They are toxic to
renal tubular cells and may cause renal failure.

- The light chains can be detected by heating or electrophoresis of concen-
trated urine. Light chains precipitate when heated to 50
o
-60
o
C and redis-
solve at 90
o
-100
o
C. Proteins possessing this property are known as
Bence Jones proteins, and their presence in urine suggests a myeloma.

When grown in tissue culture, myelomas can survive indefinitely; this property
of immortality is utilized in the production of monoclonal antibodies [see
Monoclonal Antibodies, page 116].

Memory B Cells

These are small, postmitotic B lymphocytes that are morphologically indistin-
guishable from the parent B cell. Memory cells are functionally quiescent long-
lived cells [months to years].

T cell-derived cytokines and CD40L promote memory cell development and
survival by activating the Bcl-2 antiapoptotic gene. Bcl-2 protein protects
against apoptosis, thus, allowing the B cell to differentiate into a memory cell.

Some memory B cells remain at the site of antigenic stimulation; others exit
germinal centers and recirculate between the blood and lymphoid tissues.

The specificity of the BCR on memory cells remains unchanged from that of
the parent B cell. However, memory B cells express high-affinity Ig receptors
of switched isotypes, ie, IgG [most of them], IgE, or IgA. As a result, the
optimal antigenic doses required for stimulating memory cells are lower than
those for naive B cells.

The large clonal size [100- to 1000 fold] of antigen-specific memory B cells,
compared to the size of the original clone in the naive B cell population,
accounts for the accelerated production of large quantities of isotype-
switched, high-affinity immunoglobulins [predominantly IgG], after 2
o
exposure
to the same antigen [see Secondary Immune Responses, page 123].

103
Comparison of Naive and Memory B Cells
Feature Nave B cell Memory B cell
Membrane Ig isotype

Life span

BCR affinity

Antigenic dose

Effector function
IgM and IgD

Short-lived

Usually low

Usually high

None
IgM, IgG, IgA, or IgE

Long-lived

Usually high

Usually low

None

Response of B Cells to T-Independent Antigens

T-ind antigens [nonprotein antigens] such as polysaccharides, glycolipids, and
nucleic acids, are able to provoke Ig production in the absence of helper T cells.

T-ind antigens bind directly to B cells. Because they
are multivalent, they possess multiple identical epitopes
and so can cross-link several BCRs at one time. As a
result, the effective dose of these epitopes is relatively
large and it provides a sufficient stimulus for the proli-
feration of B cells.

Responses to most T-ind antigens consist mainly of
IgM antibodies of low affinity and do not show signifi-
cant heavy chain class switching, affinity maturation, or
memory.






Comparison of T-dependent and Tindependent Antigens
Feature T-dependent antigen T-independent antigen
Chemical nature


Antigen processing

Isotype switching

Memory response
Proteins


Yes

Yes

Yes
Polysaccharides, nucleic
acids, glycolipids, etc

No

Little or no

Usually not

Clonal Selection Theory

It is the theory of antibody synthesis proposed in the 1950s by F. McFarlane
Burnet to explain why antibodies, which can be induced in response to virtually
104
any antigen, are produced in each individual only to those antigens to which he
or she is exposed. He postulated that:

Lymphoid stem cells differentiate randomly to produce clones of lymphocytes,
each of which is committed to respond to a single antigenic determinant.

Antigen binding to lymphocyte receptors triggers them to proliferate and dif-
ferentiate into antibody-producing effector plasma cells or memory cells.

The specificity of the antibodies produced by a lymphocyte is identical to that
of its antigen receptor.

Lymphocytes bearing receptors specific for self-peptides are deleted [nega-
tive selection] at an early stage in lymphocyte development and are therefore
absent from the repertoire of mature lymphocytes.
























Lymphocyte Mitogens

Lymphocyte mitogens are agents capable of inducing cell division in a high
percentage of T or B cells. Unlike an antigen which only activates lymphocytes
bearing antigen receptors [TCRs or BCRs] specific for that antigen, a mitogen
can activate many clones of T or B cells irrespective of their antigen specificity
[polyclonal activators].

Each B cell expresses antigen-spe-
cific BCRs. An antigen selects the
clones of B cells that can produce
antibodies against it.
105
A number of mitogens are sugar-binding proteins called lectins, which bind
specifically to different cell surface glycoproteins on various cells, including
lymphocytes, triggering cell division.

Lymphocytes normally exist as resting cells in the G
o
phase of the cell cycle.
When stimulated with polyclonal mitogens, they rapidly enter the G
1
phase
and progress through the cell cycle.

Responses to polyclonal mitogens have been instrumental in working out the
mechanisms of lymphocyte growth. They are also used clinically for assess-
ing the ability of lymphocytes from patients with suspected immunodeficien-
cies to proliferate in response to a non-specific stimulus.

B cell mitogens: Lipopolyssacharide [LPS], Protein A [S. aureus], etc.

T cell mitogens: Phytohemagglutinin [PHA, red kidney bean], Concanavalin A
[Con A, J ack bean], etc.

T and B cell mitogens: Pokeweed [PWM].

Differentiation Between T and B Lymphocytes

T and B lymphocytes are morphologically identical. However, they can be identi-
fied by characteristic cell surface proteins using serologic techniques.

Identifying Features of T and B Lymphocytes
Property B cells T cells

Sites of development

Distribution


Antigen receptors

Important surface antigens

Antigens recognized

Progeny cells

Secreted products

Bone marrow, BF

Lymph node cortex
Splenic follicles

BCRImmunoglobulins

Immunoglobulins

Native antigens

Plasma cells, memory cells

Soluble immunoglobulins

Thymus

Lymph node paracortex
Splenic PALS

TCRo/| heterodimer

CD2, CD3, CD4, or CD8

Processed antigens

Effector and memory cell

Cytokines

106
ANTIBODIES (Abs) OR IMMUNOGLOBULINS (Igs)

Antibodies are plasma glycoproteins produced by plasma cells (Ab-producing
cells of B cell lineage) in response to antigen. Antibodies bind to their specific
antigens in both the recognition phase (as membrane Igs [BCRs]) and the
effector phase (as secreted Abs) of humoral immunity.

Secreted Abs are found in the plasma (~20% of total plasma protein), in mu-
cosal secretions, and to some extent, in the interstitial fluid of tissues. Bind-
ing of secreted Abs to their antigens trigger various effector mechanisms that
bring about elimination of the antigens.

Plasma proteins can be analyzed
using electrophoresis. Three types of
globulins, o, |, and , based on their
electrophoretic migration rate, have
been identified. Antibodies are glo-
bulins.

Carbohydrates. The carbohydrate con-
tent of Igs ranges from 3-4% for IgG,
to 10-18% for IgA, IgM, IgD, and IgE.
The oligosaccharide side chains are
usually attached at one or more sites
to the C
H
2 or C
H
3 domains of the Ig
heavy chain. They possess subtermi-
nal galactose and terminal neuraminic acid.

- The half-life of Igs in the circulation depends on the status of the oligo-
saccharide side chains. When Abs in the circulation have the neuraminic
acid removed by the enzyme neuraminidase, the now terminal galactose
binds to a receptor (asialoglycoprotein receptor) on liver cells, triggering
endocytosis of the Ig. The Ig is then degraded by the proteolytic enzymes
in the lysosomes of the cell.

Structure of Antibodies

Although Ig molecules are structurally heterogenous, all Igs consist of four poly-
peptide chains: two identical light (L) chains and two identical heavy (H) chains
held together by noncovalent and covalent inter- and intrachain disulfide bonds.
Each chain consists of a variable (V) and a constant (C) region.

The chains are composed of 3-dimensionally folded, repeating segments
called domains [a loop of about 60 AAs around an intrachain S-S bond]. An
L chain consists of one variable (V
L
) and one constant (C
L
) domain. Most H
chains consist of one variable (V
H
) and 3 or 4 constant (C
H
) domains.
107
- IgA, IgG, and IgD possess 3 C
H
domains: C
H
1, C
H
2, and C
H
3. IgE and
IgM possess 4 C
H
domains: C
H
1, C
H
2, C
H
3, and C
H
4.




















Hinge region. This is the proline-rich region between the C
H
1 and C
H
2 domains.
Confers segmental flexibility on the Ig molecule, allowing the two antigen com-
bining sites (Fabs) to simulta-
neously bind two epitopes se-
parated by varying distances.

IgE and IgM do not possess
hinge regions.






The Bifunctional Nature of Immunoglobulins

The bifunctional nature of Ig molecules, ie, that the antigen recognition functions
and the effector functions of Ig molecules are spatially separated from each
other, was determined following limited proteolytic digestion of rabbit IgG using
papain and pepsin.

Papain. Cleaves IgG above the S-S bond at the hinge region, producing two
antigen-binding fragments ([bivalent]; [Fab: Fragment antigen binding]) and
one crystallizable fragment (Fc: Fragment crystallizable).
108
Pepsin. Cleaves IgG below the S-S bond at the hinge region, hence the
hinge region and interchain disulfide bonds are retained in an F(ab')
2
mole-
cule, but the Fc fragment is degraded.





















Fab and F(ab')
2
can bind to
antigens without activating
Fc-dependent effector func-
tions.

The Fc piece of the antibody
(CH
2
, CH
3
and [CH
4
: IgE and
IgM]) activates complement,
can bind to Fc receptors on
various cells, initiates placen-
tal transfer, etc.

Light (L) Chains (24 kD)

Each Ig molecule possesses two identical light chains; each light chain consists
of 200-250 amino acids linked by S-S bonds to a heavy chain. A light chain con-
sists of a variable region (V
L;
first 100-110 AAs) and a constant region (C
L
).

There are two isotypes [classes] of light chains, kappa [k] and lambda [],
based on the amino acid differences in their C
L
regions. A given Ig molecule
always contains two k or two light chainsnever a mixture of k and .
109
The proportion of k to chains in Ig molecules varies from species to species
(eg, humans: 60% k to 40% ).

The C
L
regions of light chains have no known effector functions. There are no
known differences in function between k-containing Igs and -containing Igs.

Heavy Chains (55-70 kD)

Each Ig molecule consists of two identical heavy chains; each heavy chain con-
sists of 450 to 600 amino acids. An Ig heavy chain consists of a variable region
(V
H
; amino-terminal first 100-110 AAs) and a constant region (C
H
).

Heavy chains are divided into isotypes (classes) and subisotypes (sub-
classes) based on the amino acid sequence of the constant region (C
H
).

Different C
H
isotypes and associated Fc regions (C
H
2, C
H
3) perform distinct
effector functions, eg, complement activation (IgG and IgM), opsonization
(IgG), etc.

Some Mammalian Immunoglobulin Classes and Subclasses
Hypervariable Regions (Paratopes)

Hypervariable regions (3) are sites within the V
L
and V
H
chains where the amino
acid sequences are highly variable. Each hypervariable region is about 5 to10
amino acid residues long. Between the hypervariable regions are framework re-
gions which are relatively constant in amino acid sequence, and provide the
molecular scaffold for V region structure.

The 3 hypervariable regions of a light
chain and the 3 hypervariable regions of
a heavy chain form the antigen-binding
site. Because the hypervariable regions
form a surface that is complementary to
the epitopes of the bound antigen, they
are also referred to as complementarity-
determining regions (CDRs: CDR1, CDR2, and CDR3).
110
The CDR3 of both the V
L
segment and the V
H
segment are the most variable
of the CDRs [a result of junctional diversity]. Thus, the most extensive con-
tact with antigen occurs with CDR3.

Antigen-Antibody Interaction

To bring about the neutralization and/or elimination of an antigen, an antibody
must bind tightly to the antigen. Antigen-antibody interaction is characterized by
noncovalent, reversible binding.

Various noncovalent forces contribute to antigen-antibody binding. They in-
clude ionic bonds [electrostatic interactions], van der Waals forces, hydrogen
bonds, and hydrophobic interactions.

Affinity and Avidity

Affinity. Refers to the strength of the binding between one Fab fragment of Ab
and an epitope of an antigen. Ag-Ab complexes of low affinity dissociate readily.

An increase in the affinity of Abs (due to point mutations in the rearranged V,
J , (D) genes; see affinity maturation) occurs during the course of a humoral
immune response.

Avidity. This is the sum total of
the strength of binding of two
molecules, such as antibody
and antigen. It takes into consi-
deration binding of Fabs to all
the available epitopes, thus,
avidity is much greater than the
affinity of any given antigen-
binding site.






Antibodies as Antigens

Being glycoproteins, Abs can act as antigens when introduced into a foreign
host, inducing the production of anti-antibodies (antiglobulins; anti-species Abs).

Antiglobulins for an Ig class or subclass are commercially available and are
used routinely in clinical [see Coombs Test, page 211] as well as research
analyses of humoral immune responses.
111
Variability of Immunoglobulin Molecules

Variability between Ig molecules can be detected by antibodies produced to
amino acids located in characteristic portions of immunoglobulin molecules.

Isotypes (subisotypes). Refers to the amino acid differences in the C
L
and
C
H
regions that distinguish each immunoglobulin class and subclass. The
variants produced are present in all healthy members of a species.

Allotypes. Characterized by a few amino acid differences of Ig C
L
and C
H

regions. Within a species, the genes that code for the C
L
and C
H
chains are
polymorphic, hence individual members can express different alleles.

- Allotypic differences are used to establish paternity and determine blood
stain origins.

Idiotypes. These are generated by the unique combination of amino acids in
the variable regions of the light and heavy chains. Each idiotype is unique for
the antigen specific antibody produced by a clone of B cells.









Effector Functions of Antibodies

The primary function of an antibody molecule is to bind antigen. In a few in-
stances, this has a direct effect, eg, neutralization of microbial toxins or preven-
tion of viral penetration of cells.

Most of the time, however, the effector functions of antibodies are initiated
only when antibodies bind to their specific antigens and engage other effector
molecules or cells, eg, complement proteins, neutrophils, etc.

Most of the effector functions of immunoglobulins are mediated by the Fc
portion of the heavy chain. Neutralization is the only function of Igs mediated
exclusively by Fab binding of antigen and does not require participation of the
C
H
regions.

- Some bacteria produce proteins [eg, Protein A of S. aureus, see page
148] that either bind to or proteolytically cleave the Fc portion, hence pre-
venting it from carrying out its effector functions.

112
Neutralization of Pathogens and Toxins by Antibodies






















Fc receptors. These are cell-surface receptors that bind the Fc portion of an
antibody molecule. Fc receptors are signaling receptors specific for antibodies of
different classes, eg, FcRs, FcoRs, and FccRs.

Different cells express Fc receptors for immunoglobulins of different isotypes.
An individual cell may express different FcRs for different antibody classes,
eg, eosinophils.

IMMUNOGLOBULIN ISOTYPES (CLASSES)

Immunoglobulin Gamma (IgG)

IgG is the major Ig in serum and is also the major Ig produced in the secondary
[2
o
] immune response.

It is found in blood, tissue spaces and extravascular spaces.

Fc receptors. These are cell-surface receptors that bind the Fc portion of
IgG. There are 3 FcRs: FcRI, FcRII (FcRIIA and FcRIIB), and FcRIII.

- FcRIIB [found on B cells]. Expresses an inhibition motif: when cross-
linked to the BCR, it delivers inhibitory signals to the B cell, blocking B cell
activation [see Regulation of the Immune Response, page 133].
113
Property FcRI (CD64) FcRII (CD32) FcRIII (CD16)
Affinity

Cell distribution



Function
High

Macrophages, Neutro-
phils (low levels),
Eosinophils

Phagocytosis, ADCC
Medium

Macrophages, FDCs
Neutrophils, Platelets
Eosinophils, B cells

Phagocytosis, ADCC,
Feedback inhibition of
B cells.
Low

NK cells, Eosinophils
Macrophages


Phagocytosis, ADCC
ADCC: Antibody-dependent cell-mediated cytotoxicty; FDC: Follicular dendritic cell

Phagocytosis. FcRs on phagocytes interact more efficiently with IgG bound to
antigen because the IgG form multivalent arrays and are bound with much
higher avidity than are free circulating IgG. This binding triggers engulfment and
delivery of signals that enhance the microbicidal activities of the phagocyte, eg,
activation of NADPH oxidase which catalyzes the intracellular generation of reac-
tive oxygen intermediates [see Opsonization, page 48].















Functions of IgG

Opsonization; agglutination of particulate antigens and precipitation of soluble
antigens.

Neutralization of viruses and microbial toxins. Complement activation.

IgG is the only antibody to cross the placenta in some species; only its Fc
portion binds to receptors on the surface of placental cells.

Immunoglobulin Mu (IgM)

First Ig class produced both during the development of B cells and during the
primary immune response. In serum, it is a pentamer with 5 H2L2 units plus one
molecule of J (joining) chain.
114
Because the pentamer has 10 Fabs,
it is the most efficient Ig in agglutina-
ting particulate matter, eg, bacteria,
complement activation, etc.

It has the highest avidity of the Igs;
its interaction with antigen can involve
all 10 of its binding sites. However,
its large size confines it to the blood
vascular system, and is of little impor-
tance in conferring protection in tissue
fluids or body secretions.


Functions

Because the pentamer has 10 Fabs, it is the most efficient Ig in agglutination,
complement activation, etc.

Monomeric IgM: Antigen receptor [BCR] on naive B cells.

Neutralization of viruses and microbial toxins; agglutination; complement acti-
vation.

Opsonization. IgM is not an opsonin because phagocytes do not possess
FcRs; however, IgM is a potent activator of the complement system, gener-
ating the opsonins C3b, iC3b, and C4b.

IgM is the predominant Ig produced by the fetus. An elevated IgM level in the
blood of a newborn is indicative of transplacental infection.

Immunoglobulin Alpha (IgA)

Synthesis of IgA occurs mainly in mucosal lymphoid tissues, especially the gas-
trointestinal and respiratory tracts, hence it is the most abundant Ig in the body.

IgA is present in two forms: Serum IgA and secretory IgA (sIgA) which is pre-
sent in various body secretions. Serum IgA is present mostly as a monomer
whereas sIgA is a dimer plus a joining chain and secretory component.

115
- The secretory component is a polypeptide synthesized by epithelial cells
that helps transport IgA to the mucosal surface. It also protects IgA from
proteolytic degradation [see Immunity in Mucosal Surfaces, page 140].

Functions

Secretory IgA is the major effector Ig at the mucosal level. It is also the pre-
dominant Ig in various secretions, eg, saliva, tears, milk, etc.

Protects mucosal surfaces by preventing the attachment of microorganisms
(eg, bacteria, viruses, etc) and microbial toxins to mucous membranes.

Agglutination of particulate antigens.

Serum IgA. Because of the low concentration of IgA in serum, it is a minor
component of systemic humoral immunity compared with IgG and IgM.

Immunoglobulin Epsilon (IgE)

Similar to IgM, IgE possesses 4 C
H
domains [C
H
1, C
H
2, C
H
3, and C
H
4]. Its Fc
portion binds to FccRs on mast cells, basophils, and eosinophils.

Heat-labile at 56
o
C for 30 mins. Other Ig isotypes are heat-stable at 56
o
C.

Functions

Mediates immediate hypersensitivity [anaphylaxis] by causing release of me-
diators, eg, histamine, from mast cells and basophils upon exposure to anti-
gen [allergen]. Thus, it is referred to as reaginic antibody.

Defense against some helminth infestations by causing release of enzymes
from eosinophils onto the parasite [see Immunity to Parasites, page 153].

Immunoglobulin Delta (IgD)

Its only known function is as an antigen receptor [together with IgM] on nave B
cells. Activation of B cells provokes loss of membrane IgD; daughter cells do not
express IgD. Like IgE, it is heat-labile at 56
o
C for 30 mins.

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

ADCC is the killing of antibody-coated target cells [eg, virus-infected cells] by
nonspecific cells with Fc receptors that recognize the Fc region of the bound Ab.

The bound antibody acts as a marker, enabling the attacking cell to recog-
nize and kill the target cell.
116
Most ADCC is mediated by natural killer [NK] cells that have FcRIII [CD16]
on their surface [see Cell-Mediated Immunity, page 130].

Macrophages and neutrophils express FcRs and kill target cells via IgG-
mediated ADCC.

Eosinophils express FccRs and
kill helminth parasites via IgE-
mediated ADCC [see Immunity
to Parasites, page 151].













Properties of Immunoglobulins
Property IgG IgM sIgA IgE IgD
Mol. Weight

Heavy chain

~percentage of
serum Igs

Serum half-life

Valency

Crosses placenta

Fixes complement

Opsonin

Principal site of ac-
tion

Largely synthe-
sized in

Antibacterial activi-
ty

Antiviral activity
150,000

Gamma []

85


23 days

2 Fabs

Yes

Yes

Yes

Serum and
tissue

Spleen and
lymph nodes

Yes


Yes
900,000

Mu []

5-10


5 days

10 Fabs

No

Yes

No

Serum


Spleen and
lymph nodes

Yes


Yes
400,000

Alpha [o]

5-15


6 days

4 Fabs

No

No

No

Secretions


GI and respi-
ratory tracts

Yes


Yes
190,000

Epsilon [c]

<1


2-3 days

2 Fabs

No

No

No

Mast cell de-
granulation

GI and respi-
ratory tracts

No


No
180,000

Delta [o]

<1


2-3 days

2 Fabs

No

No

No

Receptor
for B cells

BF and
Spleen

No


No

117
Monoclonal Antibodies

When an antigen is injected into an animal, the resulting antibodies are poly-
clonal, ie, consists of various classes, specificities, and affinities for the different
epitopes on the antigen.

Antibodies that arise from a single clone of cells are homogenous and are
directed against only one epitope; such antibodies are called monoclonal anti-
bodies [mAbs].

It is possible to isolate a single, antibody-producing B lymphocyte and grow it
in cell culture. Such a B cell would produce monospecific Abs. However,
normal lymphocytes are not easy to grow and maintain in cell culture.

On the other hand, B lymphocytes can be fused with mouse myeloma cells
[plasma cell tumor] to form B cell lines [combining the immortality of myeloma
cells and the antibody-producing ability of the B cell] that will grow in cell
culture, yet maintain the ability to produce antibodies.

The technique of B cell/myeloma fusion is known as the hybridoma tech-
nique [described by Georges Kohler and Cesar Milstein in 1975].

The Basic Theory of Monoclonal Antibody Production
























118
Method of Production of Monoclonal Antibodies













































119
Procedure

Inject mouse with antigen of interestboost for about two months. Remove
spleen [rich source of B cells].

The B cells are mixed with myeloma cells and exposed to polyethylene glycol,
which causes cell fusion.

The fused cells are grown in a culture medium containing hypoxanthine-
aminopterin-thymidine [HAT]. In the HAT medium, unfused myeloma cells die
[myeloma cells are chosen for their inability to synthesize immunoglobulins
and also their lack of hypoxanthine phosphoribosyl transferase [HPRT] activi-
ty]; unfused B cells die because of their short lifespan.

The clones of hybridoma cells are screened for the production of antibody to
the antigen of interest. Hybridoma cells can be grown in the abdomen of
mice, providing relatively large supplies of antibodies [present in ascitic fluid];
or they can be frozen and stored and reused later.

Applications

Clinical uses

Monoclonal antibodies that carry anticancer agents, eg, radioisotopes, are
being used in the treatment of cancer.

HLA detection. MAbs have been prepared that can distinguish human trans-
plantation antigens, thus improving the specificity of the tissue matching pro-
cess for successful transplantations.

Pregnancy determination. Employs mAbs prepared against specific hor-
mones associated with the pregnant state.

Drugs. MAbs are being used to accelerate removal of drugs from the circu-
lation when they reach toxic levels (eg, digoxin).

Diagnostics (Reduces the problem of cross-reactivity in immunodiagnosis).

Serotyping of bacteria, viruses, etc.

Monoclonal fluorescent antibody technique.

Detection and/or quantitation of antibodies, eg, indirect ELISA, etc.

MAbs directed against specific cell markers, eg, CD4 or CD8 lymphocyte
markers, can be used to separate mixtures of these cells from one another.
120
SPECIFIC IMMUNE RESPONSES
(ADAPTIVE/ACQUIRED IMMUNITY)

The characteristic features of adaptive immunity that enable it to perform its phy-
siologic function of host defense are:

Specificity. Immune responses are specific for individual antigens, as well as
for different structural components of the same antigen, eg, fimbria, capsule, etc.

Diversity. The total number of antigenic specificities of the TCRs [T cells] and
BCRs [B cells] in an individual, constitutes the individuals lymphocyte repertoire.
It is estimated that the mammalian immune system can recognize at least 10
9
-
10
11
distinct antigenic determinants.

Specialization. Humoral immunity protects against extracellular pathogens and
bacterial exotoxins, whereas cell-mediated immunity protects against intracellular
pathogens. Therefore, different classes of microbes elicit variable immune res-
ponses; or the same microbe can elicit different immune responses during differ-
ent stages of its infection [eg, virusesextracellular and intracellular].

Clonal expansion. Enables the few antigen-specific T and B lymphocytes to
increase in number so that they can effectively combat the pathogen that induced
the immune response.

Memory. Exposure of the immune response to a protein antigen enhances its
ability to respond again to that antigen.

Self-limitation. Immune responses usually wane with time after antigenic stimu-
lation [homeostasis], allowing the immune system to respond to new invaders.

Self-tolerance. The lymphocytes in each individual are able to recognize and
respond to many foreign antigens but are normally unresponsive to self-antigens.

Acquired Immunity
(Adaptive; Specific Immunity)


Passive Active


Artificial Maternal Artificial Natural
infection

Immune Transplacental Vaccination
globulin Colostrum

121
Active and Passive Immunity

Passive Immunity. This is the transfer of preformed antibodies or immune
cells [adoptive immunity] to a naive [nonimmune] individual.

Active Immunity. This is immunity acquired actively following natural infec-
tion or immunization by vaccines. Mediated by humoral and cell-mediated
immune responses.

Humoral (Antibody) Immune Responses

The physiologic function of the humoral immune response is defense against
extracellular organisms and microbial toxins, eg, tetanus toxin.

Immunoglobulins against surface antigens on bacteria and viruses may con-
fer protection from disease [see Accessibility of the epitope, pg 14].

Conversely, a humoral immune response to antigens from inside viable bac-
teria and viruses [eg, bacterial ribosomes] are not protective because they are
inaccessible to immunoglobulins.

Immunity is transferable from one individual to another by immune serum.

Passive Humoral (Antibody) Protection

Provides immediate protection; however, because the Igs are decaying steadily
while no new ones are being formed, passive protection is short-term [only a few
weeks]. There is no memory response.

Possible hypersensitivity reactions if Igs from another species are used.
122
Sources. Hyperimmune serum, colostrum, transplacental transfer, egg yolk,
and albumen [egg white].

Uses. Can be used for both prophylactic and therapeutic purposes.

Active Humoral Immunity

Develops slowly over a period of days or weeks; however, it tends to persist,
usually for months or years. Usually demonstrates a memory response.

Used mainly for prophylaxis.

Phases of Antibody Production

The time course and titers reached will depend on the nature of the antigenic
challenge and the physiologic state of the host.

Lag phase. Antibody to the antigen cannot be detected in serum. It includes
the time required for B cells and T cells to recognize the antigen, to undergo
clonal expansion, and to differentiate. Plasma cells must also secrete anti-
body in sufficient quantity so it can be detected in the serum.

Log [exponential] phase. Antibody titer [concentration] rises exponentially.

Plateau [stationary] phase. Antibody titer stabilizes. Secretion and degra-
dation of antibody are balanced.

Decline phase. Antibody titer declines due to natural catabolism of antibo-
dies and clearance of immune complexes [Ag-Ab complexes] from the circu-
ation by the mononuclear phagocytes.















Threshold antibody titer. Minimum antibody titer required for disease pro-
tection; varies from disease to disease.
123
Primary and Secondary Antibody Responses


Feature Primary Response Secondary Response
Synonyms

Responding B cell

Lag phase

Threshold titer

Peak antibody level

Antibody isotypes

Ig affinity (affinity maturation)
None

Naive (virgin) B cell

Usually 5-10 days

3-4 weeks

Low

Mainly IgM >IgG

Lower
Anamnestic, booster, memory

Memory B cell

Usually 1-3 days

1-2 weeks

High [~100-1000x higher]

Mainly IgG >IgM

Higher


The Differences in the Time Course of a T-dep and T-ind Antibody Response
124
CELL-MEDIATED IMMUNITY (CMI)

CMI is an immune response mediated mainly by antigen specific T cells and
other nonspecific accessory cells [NK cells, macrophages] of the immune sys-
tem. CMI can be transferred to naive [nonimmunized] individuals with lympho-
cytes but not with plasma or serum.

Destruction of virus-infected cells, tumor cells, and allografts.
CMI inhibits organisms such as fungi, parasites, and intracellular bacteria.
Mediates delayed-type hypersensitivity [DTH; Type IV hypersensitivity].

Cytotoxic T Cells (Tc Cells)

Cytotoxic T cells [cytolytic T lymphocytes, CTLs] are CD8
+
T cells that kill target
cells expressing peptide epitopes in association with MHC class I molecules.

The interaction of a CD8
+
CTL with a target
cell, involves multiple T cell membrane pro-
teins that recognize different ligands on the
target cell.

LFA-1 [CD11a/CD18]

TCR [/ TCR]

CD3 and proteins: Signal transducers

LFA-3 ([CD58]; target cell)



Cytosolic Antigen Presentation to CTLs

Cytosolic proteins are degraded into peptides that associate with class I MHC
molecules for presentation to CTLs [as a rule of thumb, host cells that present
peptide-MHC I complexes to CTLs are referred to as target cells and not anti-
gen-presenting cells].

Sources of cytosolic proteins

[1] Foreign antigens such as viruses or other intracellular pathogens, [2]
microorganisms internalized into phagosomes [some pathogens produce
enzymes that damage phagosome membranes, creating pores through
which the pathogen and its antigens enter the cytosol, and [3] cellular
proteins such as faulty or damaged proteins, regulatory proteins, etc.

125
The proteins are degraded into small peptides in the proteasome, a multi-
protein cytoplasmic enzyme complex whose function is the degradation of
ubiquitin tagged cytoplasmic proteins [proteins are targeted for proteasomal
degradation by covalent linkage of several copies of ubiquitin, a small in-
variant peptide].

Energy-dependent transport of peptides from the cytosol into the endoplasmic
reticulum [ER] where MHC class I proteins are assembled, is mediated by a
heterodimeric protein located in the ER membrane called TAP [transporter
associated with antigen processing]. In cells that lack functional TAP, Class I
MHC molecules are not loaded with peptides, leading to their degradation in
the ER.

A number of viruses, eg, equine herpesvirus-1, code for proteins that
inhibit TAP. This inhibition blocks peptide delivery to class I MHC mole-
cules, thereby preventing presentation of viral antigen to CTLs.

The TAP protein is linked to newly synthesized class I MHC molecule by the
TAP-associated protein tapasin [a key molecule in the assembly of class I
MHC molecules]. Mutations in the tapasin gene, leads to the expression of
unstable class I MHC molecules on the cell surface.

In the ER, peptides 8-10 amino acids long, bind to the cleft of MHC class I
molecules. Longer peptides that cannot bind to the cleft are trimmed to the
correct size by endoplasmic reticulum aminopeptidase [ERAP] enzyme.

Peptide-MHC I complex is released from tapasin and moves through the
Golgi into exocytic vesicle. Fusion of exocytic vesicle with plasma membrane
and expression of peptide-MHC I complex on cell surface to be recognized by
antigen-specific CTL.

126
Differentiation of Pre-CTLs to Functional (Effector) T Cells

CTLs are not fully differentiated when they exit the thymus. Although they ex-
press functional TCRs that are specific for a particular foreign antigen, they can-
not lyse target cells; hence undifferentiated CD8
+
T cells are called pre-CTLs or
naive CD8
+
T cells. Differentiation to effector CTLs requires two signals: recog-
nition of MHC I-associated peptides [signal 1] and co-stimulators [signal 2].

Pre-CTL is activated by virus-infected professional APC expressing peptide-
MHC class I complex on its surface. The activated pre-CTL produces IL-2
and proliferates in response to it [autocrine effect], eventually differentiating
into armed or effector cytotoxic CD8
+
T cells.

Professional APC can also present peptides from ingested virus-infected
[often apoptotic] cells which move from the phagosome to the cytosol, a pro-
cess called cross-priming or -presentation.










CD4
+
T cell may provide the cytokines that stimulate pre-CTLs. In this ins-
tance, both the pre-CTL and the CD4
+
T cell must recognize antigen on the
surface of the same professional APC.
127
Effector (Differentiated) CTLs

Differentiated CTLs contain membrane-bound cytoplasmic granules that con-
tain proteins used in killing target cells.

CTLs also acquire the ability to secrete cytokines, eg, IFN-, TNF, low level
IL-2, and lymphotoxin, which activate phagocytes and induce inflammation.

Mechanisms of Cell Killing

Activation of the Intrinsic and Extrinsic Cellular Apoptotic Pathways
























Effector CTL cells in circulation encounter target cells that express the same
MHC class I-associated peptides that triggered the proliferation and differentia-
tion of pre-CTLs and bind to them using specific TCRs and accessory molecules.

Binding of TCRs to peptide epitopes results in the clustering of the TCRs, and
generation of biochemical signals that activate the CTL. Co-stimulators and
cytokines, which are required for the differentiation of pre-CTLs into active
CTLs, are not necessary in triggering the effector function of CTLs.

Activation of the CTL results in the release of granule contents onto the target
cell. The most important granule proteins are perforin 1 and granzymes
(granular enzymes [fragmentins]).
128
Perforin. This is a pore-forming protein present as monomers in the gra-
nules of CTLs. Interaction with Ca
2+
ions in the extracellular environment
results in polymerization of the monomers to form tubular pores called
polyperforins.

Polyperforins insert themselves into the target membranes so that they
form transmembrane channels. If a sufficient number of these chan-
nels are present, the target cell will be unable to exclude water and
ions, leading to osmotic swelling and death. High concentrations of
calcium that enters the cell may also trigger apoptosis.

Granzymes. When released via exocytosis by CTLs, they enter target
cells mainly through the perforin-created membrane channels and proteo-
lytically cleave and activate caspases [intracellular proteases], which in
turn cleave several substrates and induce target cell apoptosis.

The nucleases activated in apoptosis can also degrade viral DNA, pre-
venting the assembly of virions and the infection of nearby cells.






Fas ligand (FasL). Fas ligand [CD95L] is a membrane protein expressed by
activated CTLs that binds to Fas protein [CD95] which is expressed on vari-
ous cells. The interaction also results in activation of caspases and apopto-
sis of target cells.

Although CTLs can program target cells to die within minutes [~5 mins], target
cell death may take several hours [2 to 6 hours].

The highly polar release of the effector molecules [ie, CTLs orient their
secretory apparatus to focus it on the point of contact with a target cell]
ensures that the CTL itself and non-antigen expressing nearby cells
[innocent bystander cells] are not killed.
129
As the antigen is eliminated, many of the effector CTLs die by apoptosis, re-
turning the immune system to its basal state of rest. The only surviving cells
are long-lived memory CD8
+
T cells generated during the immune response.

Natural Killer Cells (NK Cells)

NK cells are large granular lymphocytes [LGLs] that originate in bone marrow;
they represent 5% to 10% of lymphocytes found in the peripheral blood and
spleen and are rare in other lymphoid organs. NK cells do not recirculate.

NK cells do not possess antigen receptors; are not subject to MHC restric-
tion; and have no memory.

NK cell proliferation and cytolytic activity are enhanced by IL-2, IL-12, IL-15,
IFN-, IFN-, and IFN-. IL-12, IL-15, IFN- are produced by macrophages in
response to infection.

NK cells express CD16 [FcRIII], CD2, CD11a/CD18, killer-cell immunoglo-
bulin-like receptors [KIRs], and killer-cell activatory receptors [KARs].

Effector Functions

The main functions of NK cells are to recognize and kill certain tumor cells and
virus-infected cells and to secrete IFN- that activates macrophages to destroy
intracellular parasites.
















Target Cell Lysis

NK cells kill target cells that fail to express or express reduced MHC class I pro-
teins. Some viruses produce virokines that can down-regulate MHC class I
expression and some tumor cells often do not express MHC class I molecules.
NK cells are an early component of the
host response to virus infection. IFN-
and IFN- appear first, followed by a
wave of NK cells, which together control
vi-rus replication before antigen-specific
CTLs become fully active.
130
KARs on NK cells recognize
carbohydrate molecules on self
cells. This recognition event trig-
gers NK cells to kill target cells,
using exocytosed perforin 2 and
granzymes.

Interaction of KIRs with MHC
class I molecules expressed by
the target cells inhibits killing by
NK cells in a dominant way.

Granulysin. This is an anti-
microbial peptide present in the
granules of NK cells and CTLs.
When released, it kills a wide
variety of extracellular bacteria,
fungi, and parasites; some pass
through the perforin channels
and kill microbes in the target
cell.



Antibody-dependent cell-mediated cytotoxicity [ADCC]. NK cells also kill
target cells coated with IgG via their CD16 receptor [FcRIII].










Activated Macrophages

Monocytes moving into inflamed tissues are referred to as inflammatory ma-
crophages because of their enhanced activities. Inflammatory macrophages can
be stimulated further to become activated macrophages.

Activation consists of quantitative changes in the expression of various pro-
teins that enable activated macrophages to perform some function(s), eg,
synthesis of nitric oxide [NO] that cannot be performed by resting macro-
phages.
131
Macrophages are activated by CD40L-CD40 interactions and by IFN- pro-
duced by T
H
1 cells and NK cells. Bacterial endotoxin [LPS] is a potent acti-
vator of macrophages [binds to CD14 receptor on macrophages].
















Intracellular Parasites

Various microorganisms such as viruses, protozoa, bacteria, and fungi can repli-
cate within macrophages and kill the macrophages.







Protection against these microbes is afforded by macrophage activation. The
response is nonspecific and activated macrophages are capable of destroy-
ing a wide range of normally resistant intracellular parasites.

Macrophage activation results in increased synthesis of NADPH oxidase,
which catalyzes the generation of reactive oxygen intermediates [ROI], and
the production of inducible NO synthase, which stimulates the synthesis of
nitric oxide [see Inflammation, page 52]. ROI and NO are potent microbicidal
agents.

Activated macrophages also contain increased amounts of lysosomal
enzymes.

T
H
2 cells cross-regulate T
H
1-mediated macrophage activation and inflamma-
tion by secreting cytokines [eg, IL-4, IL-10, and TGF-] that antagonize T
H
1-
derived cytokines.
132
REGULATION OF THE IMMUNE RESPONSE

One of the cardinal features of all immune responses is their self-limitation. The
main reason for this self-limitation is that each immune response eliminates the
antigen that initiated the response and thus eliminates the necessary first signal
for lymphocyte activation. However, the initial response establishes memory for
the antigen, so that subsequent exposure to the same antigen triggers a more
vigorous immune response.

A variety of control mechanisms restore the immune system to a resting state
when responsiveness to a given antigen is no longer required.

Regulation by Antigen

An antigen is the engine that drives the immune response. When the immune
system encounters an antigen, one of two outcomes may develop: (1) immunity
or (2) a state of immunologic unresponsiveness called tolerance.

Antigen concentration. This is the primary regulator of the intensity of an im-
mune response. Optimal antigenic doses vary for different antigens.

Very large doses of antigen often result in tolerance and inhibition of the
immune response [see Immune Paralysis, page 227].

Chemical nature of the antigen

Protein antigens (T-dep antigens). Induce both humoral and cell-mediated
immune responses. Proteins stimulate isotype switching, affinity matura-
tion, and the generation of memory B cells.

Non-protein antigens (T-ind antigens). The immune response to T-ind an-
tigens consists largely of IgM antibodies.

Portal of entry

Protein antigens administered subcutaneously [SC] or intradermally [ID]
are endocytosed by Langerhans cells and carried to regional lymph nodes
where immune responses are initiated.

Antigen given intravenously [IV] or orally may cause tolerance; possibly
due to the rapid elimination of the antigen.

Regulation by Antibody

It has been observed for a long time that an antibody exerts feedback inhibition
on its own further production.
133
If an animal is immunized
with a specific antigen and is
injected with preformed anti-
body to that same antigen
just before or within 5 days
after antigen priming, the im-
mune response to the anti-
gen is reduced up to 100-
fold.


Antibodies eliminate the antigen they were produced against, thereby remov-
ing the initiating stimulus for antigen-reactive B cell clonal expansion.

Regulation by Immune Complexes

Circulating antigen-antibody complexes [immune complexes] can either enhance
or suppress specific immune responses.

Inhibition. Antigen-antibody complexes can simultaneously bind to BCR [via
antigen] and the CD32 [FcRIIB] via the Fc portion of the antibody (IgG).

























134
As a result of this simultaneous cross-linking of receptors, phosphatases as-
sociated with the cytoplasmic tail of the FcRIIB inhibit signaling by the B cell
receptor complex and block naive B cell activation [receptor cross-linking
does not appear to affect memory B cells].

Enhancement. Immune complexes trapped by follicular dendritic cells [FDCs;
via FcRs], are crucial in the selection of B cells that express high-affinity BCRs
during the process of affinity maturation in antibody responses to T-dep antigens
[see page 100]. FDCs are also capable of retaining Ag-Ab complexes for long
periods, thereby prolonging and enhancing the antibody response.

Inactivation and Apoptosis of Activated Lymphocytes

T cells responding to antigens proliferate and differentiate into effector and me-
mory cells. T cell survival is dependent upon antigens, costimulators and cyto-
kines produced during the immune response.

Survival stimuli for lymphocytes induce the formation of antiapoptotic proteins.
However, as the effector response leads to the elimination of the antigen,
effector T cells are deprived of survival stimuli and die by apoptosis.

Therefore, effector T cells have short tissue half-lives [days] and the only cells
surviving following an immune response are the long-lived, functionally quies-
cent memory T cells.


















CTLA-4 (Cytolytic T lymphocyte-associated protein 4; CD152). CTLA-4 is a
physiologic terminator of T cell activation that is expressed only by activated
T cells. It is a high affinity receptor for B7-1 and B7-2 [the same proteins also
bind to CD28].
135
When CTLA-4 interacts with B7
proteins, it sends an inhibitory
signal to the T cell. CD28, the
activating receptor for B7 pro-
teins, is constitutively express-
ed on resting T cells, where it is
needed to initiate the immune
response.

Mutations in CTLA-4 gene re-
sults in uncontrolled T cell proli-
feration and autoimmunity.



Immune Cross-Regulation by T
H
1 and T
H
2 Cytokines


Transforming growth factor- [TGF- ]. It is a potent inhibitor of T cell and B
cell proliferation.

IL-10 inhibits macrophage activation; IL-4 and IL-13 are antagonists of IFN-,
the most potent activator of macrophages. Therefore, these cytokines may
suppress cell-mediated immunity and delayed hypersensitivity reactions [see
Type IV Hypersensitivity, page 218].
136
Stress

Animals or individuals under stress usually show reduced immune functions.

CRF ACTH
Stress Hypothalamus Anterior pituitary Adrenal


Endorphins Glucocorticoids
Enkephalins, etc

CRF: Corticotropin-releasing factor
ACTH: Adrenocorticotropin Immune System

Glucocorticoids. Act directly on immune cells to suppress their activity [see
Corticosteroids, page 250].

If stress occurs before an immune response, antigen may not be able to
activate immune cells that have been suppressed by corticosteroids.

If stress occurs during an immune response, the level of corticosteroids
may be higher and remain elevated for a longer period of time, resulting in
pro-longed immune suppression of effector functions.
137
CUTANEOUS AND MUCOSAL IMMUNE SYSTEM (MIS)

The epithelial surfaces of the body play a major role in the interaction between
the external and internal environment. Invading microbes are first encountered
at these surfaces where they may be largely repelled or destroyed by the MIS.

Mucosal Immune System

The mucosal immune system responds to and protects against microorganisms
that enter the body through mucosal surfaces. The MIS is composed of muco-
sal-associated lymphoid tissues [MALT], a collection of dispersed aggregates of
non-encapsulated lymphoid tissues found especially in the epithelia, lamina pro-
pria and submucosal areas of the gastrointestinal and respiratory tracts.

Gut-associated lymphatic tissue [GALT]. Includes Peyers patches, cecal ton-
sils [avian], tonsils, and appendix [colon]. Tonsils respond to antigens enter-
ing through the nasal and oral epithelial routes.

Bronchus-associated lymphatic tissues [BALT]; genitourinary tract [ureter,
bladder]; mammary gland; conjunctiva; and salivary glands.

It has long been established that the presence of antibody [sIgA; generated
by MALT] in local secretions correlates better with protection against patho-
genic microbes than serum antibody.

Gut-Associated Lymphatic Tissue (GALT)

In the intestinal mucosa, lymphocytes are found in three main regions:

Intraepithelial lymphocytes [IELs]. Found within the epithelium. The majority
of these lymphocytes are CD8
+
T cells that express TCRs, with limited
diversity of antigen receptors.

The lamina propria contains large
numbers of activated B cells, plasma
cells, activated CD4
+
T cells, follicu-
lar dendritic cells [FDCs], and ma-
crophages in loose clusters.

Within the submucosa is an aggre-
gation of solitary nodules consisting
of lymphoid follicles, the Peyers
patches. The lymphoid follicles can
develop into 2
o
follicles with germinal
centers. Peyers patches possess all
the components required to mount
138
an immune response, namely T cells, B cells, FDCs, and macrophages.

Antigen Uptake

There are no afferent lymphatics that carry antigens to Peyers patches. Antigen
transport from the luminal surface to Peyers patches is carried out by specialized
epithelial cells overlying Peyers patches, called M [microfold] cells.

M cells are flattened epithelial cells scattered among the enterocytes. They
lack microvilli but possess deep invaginations of the basolateral plasma mem-
brane which form pockets containing T and B lymphocytes, FDCs, and ma-
crophages.

Luminal antigens and microorganisms are transcytosed into these pockets
and to the organized mucosal lymphoid tissue under the epithelium [M cells
do not function as antigen presenting cells].

M cells are also found associated with lymphoid cell accumulations at other
mucosal sites. In mucous membranes, M cells are located in so-called in-
ductive sites [small regions of a mucous membrane that lie over organized
lymphoid follicles].

Antigens transported across the mucous membrane by M cells at an inductive
site activate B cells in the underlying lymphoid follicles. The activated B cells
differentiate mainly into IgA-producing plasma cells. B cells expressing IgM,
IgG, or IgE BCRs are also present.

The IgA produced are transported across the epithelial cells and released
as secretory IgA into the lumen where they can interact with antigens pre-
sent in the lumen.


139
Interdigitating dendritic cells present in mucosal epithelia also take up
antigens, process them and transport them to regional lymph nodes where
they present them to CD4
+
T cells.

Lymphocyte Circulation within the MIS

Many of the activated B cells and plasma cells migrate via efferent lymphatics to
regional lymph nodes and thoracic duct and into the bloodstream.

The recirculating lymphocytes have an affinity for mucosal surfaces, conse-
quently they lodge throughout the lamina propria of the intestinal tract, the
respiratory tract, urogenital tract, and the mammary gland.

Specific recirculation
within the MIS is
made possible be-
cause the lymphoid
cells express homing
molecules that bind
to vascular addres-
sins expressed on
mucosal high endo-
thelial venules, but
which are absent from lymph node HEVs.

The movement of these IgA-producing cells to the mammary gland is very
important in domestic animals because it provides a route by which cells
stimulated by intestinal pathogens can secrete their IgA into colostrum and
milk and thus protect the intestines of newborn animals.

Role of Immunoglobulins in Mucosal Immunity

IgA

IgA is the predominant Ig synthesized in the MIS. It is found in significant
amounts in intestinal fluid, nasal and tracheal secretions, milk, colostrum, urine,
tears, saliva, etc.

Since the MIS contains up to 75% of all the B cells of the body, IgA accounts
for 60-70% of the daily total output of antibodies. IgA isotype switch is media-
ted by transforming growth factor- [TGF-] and the T
H
2 cytokine IL-5. T
H
2
cells are the predominant helper cells in the MIS.

IgA is produced by plasma cells in the lamina propria in the form of a dimer
that is held together by a J chain. Formation of polymers appears to be criti-
cal to allow IgA to bind to the mucosal transport receptor.
140
The mucosal transport receptor [poly-Ig receptor, pIgR] is a glycoprotein
synthesized by mucosal epithelial cells and expressed on their basal and
lateral surfaces. It is a member of the Ig superfamily.

The dimeric IgA binds to the pIgR and the complex is endocytosed into the
epithelial cell and actively transported in vesicles to the luminal surface. The
extracellular domain of the pIgR, which carries the IgA molecule, is then
cleaved by proteolytic enzymes so that the IgA, with the receptor peptide still
attached [secretory IgA] is released into the intestinal lumen.

The IgA-associated receptor peptide is called secretory component. It
protects the dimeric IgA from proteolytic enzymes in the lumen.

The most important mode of action of sIgA is the prevention of the adherence
of microorganisms and toxins to epithelial surfaces.















IgG

In ruminants, especially bovine, IgG1 is the predominat Ig in colostrum, milk, in-
testinal contents and nasal washings. However, IgA is the predominant Ig on
other body surfaces.

IgG is not as resistant to proteolysis as sIgA, hence its role in defending the
intestinal mucosa is less significant. It is more significant in the respiratory
tract.

IgE

Mainly synthesized by plasma cells in the lamina propria of the respiratory and
intestinal tracts. Thus it is found in the secretions of the nose, eyes, bronchi, and
gut. The production of IgE is associated with immunity to helminths and type I
hypersensitivity.
141
IgM

IgM also binds to pIgR and is carried through the epithelial cell to the lumen.
However, SIgM is susceptible to proteolytic degradation and it is a minor com-
ponent in secretions.

Immunity in the Gastrointestinal Tract

Low pH of the stomach; peristaltic movement; proteolytic enzymes, bile acids,
and pancreatic secretions.

Normal flora. If the natural flora of the intestine is eliminated or its composi-
tion drastically altered, eg, prolonged antibiotic therapy, an overgrowth of po-
tential pathogens may occur.

SIgA. Prevents microbial adherence

IgG. Diffusion from the blood into an area of an inflammatory response.

IgE. Immunity to helminth parasites.

Immunity in the Respiratory Tract

Air that enters the respiratory tract is mostly cleared of suspended particles by
turbulence that directs them onto its mucus-covered walls, where they adhere.
The turbulence filter serves to remove particles as small as 5 m in size before
they reach the alveoli.

Mucociliary escalator. Microorganisms in the lower respiratory tract are
trapped in mucus produced by goblet cells, then propelled upward by the
synchronized beating of cilia. Mucus also contains sIgA and lysozyme.

Alveolar macrophages. Particles <5 m that can bypass the mucociliary
escalator and reach the alveoli are phagocytosed by alveolar macrophages.

Pulmonary intravascular macrophages. Adherent to capillary endothelium of
the lungs. They are found in ruminants, cats, and pigs. They clear microbes
present in blood.

IgA. Synthesized in lymphoid nodules in the walls of the bronchi and lym-
phocytes distributed diffusely throughout the lung and walls of the airways.

IgG. Of major importance during acute inflammation when transudation of
serum protein occurs.

IgE. It mediates type I hypersensitivity reactions in the respiratory tract.
142
Immunity in the Mammary Gland

Teat canal. Between milking it dries out, forming a seal to the teat entrance.

It is lined by keratin that may bind ~1 x 10
6
bacteria. Periodic shedding at
milking removes attached bacteria. Keratin also contains dissolved fatty
acids, making it bactericidal. If the keratin lining of the streak canal is dam-
aged, bacteria readily colonize the canal and infect the mammary gland.

It is normal for the teat canal to be dilated for ~ 2 hours after milking, making
the udder very susceptible to infection. Cows should be fed immediately after
milking so that they remain standing for as long as possible and therefore
avoid udder contact with the ground [the source of pathogens].

Flushing action of milk.

Citrate-lactoferrin ratio. Lactoferrin permanently binds iron, an essential growth
factor for bacteria. Citrate in milk also binds iron, however, bacteria can utilize
iron bound by citrate.

Thus binding of iron to citrate competes with chelation of iron by lactoferrin. A
high molar ratio of citrate to lactoferrin enhances bacterial growth.

Somatic cells. Normally, in the dairy cow, there are 0.5 to 2.0 x 10
5
somatic
cells/ml of milk. Somatic cells are mostly macrophages, epithelial cells, and
some lymphocytes. A low somatic cell count predisposes the udder to infection
by opportunistic pathogens.

In acute mastitis, neutrophil influx into the gland cistern increases the somatic
cell count in milk.

IgA. Locally synthesized in the mammary gland although many of the IgA-
producing cells in the gland originated from precursor cells in the intestinal tract.

IgG. Selectively transferred by an active transport mechanism from the serum.

Immunity in the Urogenital Tract

Flushing action of urine. Urinary stasis may result in urethritis due to the uni-
lateral ascent of pathogenic bacteria.

pH of urine. Influenced by diet and species. Carnivores [acidic pH]; herbi-
vores [neutral to alkaline urine].

Adult female animals. The squamous epithelial cells of the vagina store gly-
cogen. When the cells desquamate, they provide a substrate for lactobacilli
143
[Lactobacillus acidophilus] that, in turn, generate large quantities of lactic
acid, which protects the vagina [pH 3.5-4.5] against invasion.

Glycogen storage in the vaginal epithelial cells is stimulated by estrogens
and thus occurs only in sexually mature animals. Hence, vaginal infec-
tions in humans are most common prior to puberty and after menopause.

IgA. Predominant Ig in cervicovaginal mucus; low amounts in urine.

IgG. Predominant Ig in the uterus.

Cutaneous Immune System

Anatomic barrier to the external environment; secretion of sebum; resident flora;
and epithelial desquamation.

Keratinocytes. Secrete several cytokines, [eg, IL-1, IL-6, IL-8] that may play a
role in local inflammatory reactions.

Langerhans cells [see Antigen Processing and Presentation, page 73].



144

HOST IMMUNE RESPONSES TO BACTERIAL INFECTIONS

Bacterial infectious diseases are complex interactions between bacteria and
host, each seeking survival within one ecosystem. The outcome of the infection
will depend on the following factors:

The resistance of the host
The virulence of the organism
The presence of damaged tissue
The location of the organism within the body

SOME MAJOR ANTIGENS OF BACTERIA

Cell Wall Antigens

Gram-positive bacteria: Peptidoglycan, teichoic acid.

Gram-negative bacteria: Lipopolysaccharide [LPS; T-independent antigen].
Directly activates B cells to produce IgM antibodies. LPS is also a potent
activator of macrophages. Somatic [O] antigen [specific sugars].
Other Bacterial Antigens

Capsular antigen [K antigen]. Like LPS, the polysaccharide-rich capsules of
bacteria directly activate B cells to produce IgM anticapsular antibodies.

Fimbria antigen. Antifimbria antibodies prevent bacterial attachment to mucosal
surfaces.
145

Flagellar antigen [H antigen]. Antibodies against flagella can immobilize motile
bacteria, however, this is not significant in bacterial pathogenesis.











Exotoxins. Proteins secreted by some Gram-positive and Gram-negative bacte-
ria which are responsible for the diseases caused by the organisms, eg, tetano-
spasmin [Clostridium tetani] and botulinal toxin [Clostridium botulinum].

Toxoids derived from exotoxins are highly immunogenic and they induce the
formation of antitoxins in the host [see Toxoid Vaccine, page 172.

Extracellular and Intracellular Sites of Bacterial Growth


IMMUNITY TO EXTRACELLULAR BACTERIA

Extracellular bacteria replicate outside host cells [eg, in the blood, in intestinal
lumen, in connective tissues, etc]. They do not have the capacity to survive for
long periods in phagocytic cells, thus they cause tissue damage while they are
outside phagocytes and other cells.

Extracellular bacteria cause disease by producing toxins [exotoxins, endo-
toxins] or induce inflammation which results in tissue damage at the site of in-
fection.

Innate Immunity to Extracellular Bacteria

Phagocytic cells [eg, neutrophils]. Eliminate bacteria via phagocytosis.
Surface antigens of a
typical bacterial cell.
146

Complement activation on bacterial surfaces.

Complement-mediated lysis of bacteria [Gram-negative bacteria].

Opsonization [eg, C3b, iC3b and C4b] and phagocytosis.

Inflammation. C5a, C3a, and C4a induce local mast cell degranulation and
the release of vasoactive mediators [eg, histamine] that mediate vasodilation
and extravasation of leukocytes into tissues. In addition, C5a is chemotactic
for neutrophils, etc.












Adaptive Immune Responses to Extracellular Bacteria

The humoral immune response constitutes the principal host defense against
extracellular bacteria.

Neutralization of bacterial toxins.
Opsonization [IgG] and Fc receptor-mediated phagocytosis.
Complement activation via the classic pathway [IgG, IgM].
Prevention of bacterial adherence to mucosal surfaces.

IMMUNITY TO INTRACELLULAR BACTERIA

Facultative intracellular bacteria can survive and, in some instances, multiply in
phagocytic cells [mainly macrophages]. Within the macrophage, the bacteria are
protected from extracellular host defense mechanisms.

Antibodies produced during these infections merely indicate exposure to
antigen but does not denote protective immunity.

Innate Immunity to Intracellular Bacteria

Intracellular bacteria stimulate macrophages to secrete IL-12, a potent activator
of NK cells. Activated NK cells, in turn, produce IFN- which activates macro-
phages and promotes the killing of intracellular bacteria [see CMI, page 131].
147

Adaptive Immune Responses to Intracellular Bacteria

Adaptive immunity to intracellular bacteria is principally cell mediated.

CD4
+
helper T cells respond to MHC class II-associated peptide antigens
derived from bacteria in the endosome and IL-12 from the macrophage. The
activated CD4
+
T cell expresses CD40L and secretes IFN-, both of which
activate macrophages, enhancing the killing of the microbes in the phago-
lysosomes.

Peptide antigens from bacteria in the cytosol are presented in association
with MHC class I proteins. CD8
+
cytolytic T cells responding to the MHC-
peptide complex kill the infected macrophage.



























Bacterial Superantigens

These are soluble bacterial proteins, including various exotoxins, which bind
simultaneously to all TCRs bearing certain specific V domains regardless of
their antigenic specificity and to the chain of a class II MHC molecule on
antigen-presenting cells [APCs] without a need for intracellular processing.
148

Cross-linkage of a T cell receptor and class II MHC molecule by a super-
antigen provides an activating signal that induces T cell activation and proli-
feration.

Since superantigens bind outside of the TCR antigen-binding groove, any T
cell expressing a particular V domain will be activated by a particular super-
antigen.

The massive activation following
cross-linkage by a superantigen
results in overproduction of T
H
cell
cytokines [esp. TNF- leading to
systemic toxicity.

Superantigens have so far been
associated with bacteria such as
Staphylococcus aureus [food-
borne illness, scalded-skin syn-
drome, and toxic shock syndrome], streptococci, and mycoplasmas; and
some viruses, eg, rabies virus.

Bacterial Evasion of Host Immune Mechanisms

Killing the phagocyte

S. aureus, Fusobacterium necrophorum, Mannheimia hemolytica, etc: Se-
crete leukocidin which induces lysosomal granule discharge into the cell
cytoplasm.

Inhibition of membrane attachment

Capsule [hydrophilic]. Protects pathogenic bacteria from phagocytosis.
Capsules rich in sialic acids resist C3b opsonization since C3b binds to
factor H with resulting factor I degradation of C3b.

Pathogenic streptococci. M protein alters cell surface membrane.

S. aureus. Protein A binds to the Fc portion of IgG, thereby competing
with Fc receptor on phagocytic cell. Pathogenic staphylococci can secrete
a coagulase enzyme that produces a fibrin coat around the organism,
shielding it from phagocytic cells.

Inhibition of fusion of lysosomal granules and phagosome

Virulent Mycobacterium tuberculosis. Failure of lysosomal fusion due to
sulfatides produced by the bacterium.
149

Resistance to killing and digestion in phagolysosome. The cell wall lipids of
Corynebacterium pseudotuberculosis render it resistant to lysosomal killing.

Escape from the phagosome

Listeria monocytogenes. Produces the enzyme listeriolysin O which me-
diates escape from the phagosome by lysing the phagosome.

Some bacteria [eg, Neisseria gonorrhoeae] evade sIgA response in mucosal
surfaces by secreting proteases that cleave sIgA dimers.

Antigenic variation. Campylobacter fetus subsp. fetus colonizes the male and
female genital tracts of cattle. Organisms emerging following a local immune
response possess epitopes different from those of the original population.

Gram-positive bacteria. Resistance to complement-mediated lysis.

Immunity to Fungal Infections

Innate immunity to fungal infections is mediated by macrophages and neutro-
phils.

Neutrophils may phagocytose fungi for intracellular killing (cationic proteins
[defensins] play a crucial role in intracellular killing) or release their lysosomal
enzymes extracellularly and damage the fungus. Animals or individuals with
neutropenia are extremely susceptible to opportunistic fungal infections.

Facultative intracellular parasites such as Histoplasma capsulatum that live in
macrophages are eliminated following activation of macrophages by IFN-
secreted by NK cells and T cells or lysis of the macrophage by CD8
+
CTLs
[same mechanism as elimination of intracellular bacteria].

Antibodies produced in response to fungal infections are useful in serologic
diagnosis. However, the protective role of humoral immunity in fungal infec-
tions has not been established.
150
IMMUNITY TO PARASITES

Parasites are responsible for a wide variety of diseases in both humans and
domestic animals. It is estimated that ~30% of the world population suffer from
parasitic infestations.

General Features of Parasitic Infections

Parasites generally are host-specific and most of them cause chronic infec-
tions, a consequence of weak innate immunity and the ability of parasites to
evade or resist elimination by adaptive immune responses.

Many parasites have complex life cycles, part of which occurs in vertebrates
[humans or domestic animals] and part of which occurs in intermediate hosts.

Due to their large size, many parasites possess a variety of antigens; also as
a result of their complex life cycles, many parasites express antigens which
are specific to a particular stage of their development.

A consequence of stage-specific antigens is that by the time the immune
system has responded to the infection, the parasite expresses new anti-
gens and is no longer a target for immune elimination.

Chronic and persistent parasitic infestations result in immune complex hyper-
sensitivity [see page 212]. The complexes may be deposited in blood vessels
and kidney glomeruli and produce vasculitis and nephritis, respectively.

Some parasites and their products induce granulomatous responses with
concomitant fibrosis.

The effectiveness of the immune response that occurs is influenced by the
particular parasite and the stage of infection.

Humoral immune responses are most effective against extracellular
stages of the parasite; whereas, during intracellular growth, cell-mediated
reactions are responsible for host defense.

Immunity to Protozoa

Protozoa are microscopic, unicellular eukaryotic organisms. In the host, they are
found in the gut, in macrophages, in the blood, in red blood cells, or in muscle.

Innate Immunity

Phagocytosis by macrophages and neutrophils. However, many protozoa are
resistant to intracellular killing and may multiply in macrophages.
151
Adaptive Immunity

Humoral immunity

IgG and C3b deposited on parasite membrane enhance phagocytosis.

Infected RBCs that carry protozoa antigens on their surfaces are opsonized
and removed by splenic macrophages and/or destroyed by an ADCC res-
ponse.

Splenectomy in animals latently infected with hemoparasites, may result
in reactivation of the infection and clinical disease.

Cell-mediated immunity

Cytotoxic T lymphocytes [CTLs] recognize cytosolic parasite antigens com-
plexed with MHC class I molecules and lyse the infected cells.

Activated macrophages. Destroy intracellular parasites, eg, Toxoplasma ta-
chyzoites.

Immunity to Helminths

Helminths are large, multicellular parasitic roundworms (nematodes) and flat-
worms (tapeworms [cestodes] and flukes [trematodes]) that normally do not mul-
tiply within their hosts; thus the number of worms in an individual or animal in-
creases only through repeated exposure. Helminths are extracellular parasites.

Because an individual or animal carries low numbers of worms, immune sys-
tem exposure to helminth antigens is limited and consequently only a low le-
vel of immunity is induced.

Phagocytic cells such as neutrophils and macrophages can kill some helminth
parasites with their toxic proteins and reactive oxygen intermediates; how-
ever, many helminths have thick cuticles that make them resistant to the cyto-
cidal products of neutrophils and macrophages.

Polymorphonuclear Eosinophils

A major mechanism of defense against many helminth parasites involves T
H
2
cells, IgE antibodies, and eosinophils. Serum levels of eosinophils and specific
IgE increase and remain high until the parasite is cleared from the body.

Eosinophils constitute 1-5% of the total circulating leukocytes. They are dis-
tinguished by the affinity of their coarse granules for the acid dye eosin.

152
Eosinophils are normally present in peripheral tissues, especially in mu-
cosal lining of the respiratory, gastrointestinal, and genitourinary tracts.
They are most abundant at sites of parasitic, allergic, or some inflamma-
tory diseases.

The growth and differentiation of eosinophils is dependent on 3 cytokines:
interleukin-3 [IL-3], IL-5, and granulocyte macrophage-colony stimulating
factor [GM-CSF].

IL-5 is the most significant cytokine [eosinophil differentiation factor]; it
also stimulates release of eosinophils from the bone marrow.

Eosinophil cell membrane receptors include Fc receptors for IgG, IgA, and
IgE and complement receptors.

Eosinophils respond to many of the same stimuli and chemotaxins as neutro-
phils, as well as eosinophil-specific chemotaxins, eg, eotaxins [see CC
chemokines, page 22]. The half-life of eosinophils in tissues is 12 days.

Granular contents include:

Major basic protein. Parasite killing; neutralizes heparin.
Eosinophil cationic protein. Parasite killing.
Arylsulfatase B. Inactivates leukotrienes [LTC
4
, LTD
4
, LTE
4
].
Histaminase (diamine oxidase). Inactivates histamine.
Phospholipase D. Inactivates platelet activating factor [PAF].
Peroxidase, collagenase, elastase, platelet activating factor, etc.

Although eosinophils can generate a respiratory burst, they are weak phago-
cytic cells and are much more important to extracellular destruction of large
parasites eg, helminths, which are often resistant to the lysosomal enzymes
of neutrophils and macrophages.

Though eosinophils suppress inflammation by destroying mast cell factors,
activated eosinophils produce and release lipid mediators such as leuko-
trienes that have proinflammatory properties.

Eosinophil collagenase and elastase degrade extracellular matrix proteins,
thus, if the inflammatory stimulus is persistent, too many eosinophils accu-
mulate in tissue for prolonged period of time, contributing to adverse tis-
sue reactions and tissue damage.

Immune Response Against Helminth Parasites

Helminth parasites have a propensity to stimulate the T
H
2 subset of CD4
+
helper
T cells, resulting in the production of T
H
2 cytokines such as IL-3, IL-4, and IL-5.
153
IL-3 and IL-4. Stimulate mucosal mast cell growth. IL-4 also induces IgE iso-
type switch.

Cross-linking of IgE on mucosal mast cells by parasite antigen results in rapid
mast cell degranulation and release of vasoactive mediators [eg, histamine]
that result in extravasation of inflammatory cells such as neutrophils, macro-
phages and eosinophils. Eosinophil chemotactic factor of anaphylaxis [ECF-
A] present in mast cell granules enhances eosinophil recruitment.

Fc receptors on eosinophils mediate eosinophil binding to IgE-bound para-
site. Subsequent killing of parasite occurs via ADCC. IgG also mediates
eosinophil ADCC.

However, some helminth parasites bound by eosinophils shed their glyco-
calyx coat together with the attached eosinophils.

Major basic protein. Constitutes about 50% of eosinophil granule proteins.
It is strongly cationic [positively charged], therefore it interacts readily with ne-
gatively charged surface of the parasite. It is toxic to helminths.

Eosinophil cationic protein. This is a pore-forming protein that is toxic to
helminths.

The Interaction of Mast Cells, Eosinophils and IgE in the Coordinated Response
To a Helminth Parasite

154
Evasion of Immune Mechanisms by Parasites

Most parasites have evolved various mechanisms for evading their hosts im-
mune responses. Among them are the following:

Camouflaging. This is the ability of parasites to acquire an outer coat of host
proteins [eg, MHC proteins] so that these proteins, as opposed to the parasite
outer membrane proteins, will not be recognized as antigens and, therefore,
not bind antigen-specific IgE antibodies.

Antigenic variation. There are two types of antigenic variation:

Stage-specific antigens. The mature tissue
stages of parasites may express antigens dis-
tinct from those expressed by the infective
stages.

Continuous antigenic variation. African trypano-
somes produce successive waves of progeny
with different surface antigens.

A number of trematodes secrete proteases which cleave Igs, removing the Fc
portion. The resulting Fab and Fc fragments have a shortened half-life in
mucous secretions.

Some parasites may conceal themselves in cysts, eg, Trichinella spiralis.

Protozoan parasites that multiply within macrophages employ various strate-
gies to escape digestion by lysosomal enzymes.
















155
IMMUNITY TO TUMORS

Tumor or neoplastic cells are altered self-cells that fail to respond to normal
growth regulating mechanisms. Certain surface antigens of tumor cells distin-
guish these cells from normal cells of the same type.

Some tumor antigens can elicit immune responses in the tumor-bearing host.
The responses to these antigens can kill tumor cells and eradicate tumors; on
the other hand, some immune responses frequently fail to prevent the growth
of tumors, resulting in the death of the host.

Terminology

Benign tumor. This is a tumor that is not capable of indefinite growth. It remains
confined to its original location, neither invading the surrounding normal tissue
nor spreading to distant body sites.

Malignant tumor (cancer). This is a tumor that continues to grow and becomes
progressively invasive.

The tumor may also be metastatic, ie, small clusters of cancerous cells dis-
lodge from the tumor and spread via lymphatic vessels or bloodstream to
other parts of the body, where they continue to grow. In this way a primary tu-
mor at one site can give rise to a secondary tumor at another site.

Carcinomas. Malignant tumors of epithelial cells.

Sarcomas. Solid tumors of connective tissues, such as bone, muscle, fibrous
tissue, cartilage, and fat.

Leukemia and lymphoma. These are malignant tumors of hematopoietic cells
of the bone marrow; leukemias proliferate as single cells whereas lymphomas
tend to grow as tumor masses.

Tumor Antigens

Tumor cells that are functionally different from their normal precursors may also
be antigenically different, in that they gain or lose cell-membrane antigens. Many
tumor antigens are cellular proteins that give rise to peptides presented with
MHC molecules. Tumor antigens are classified on the basis of their expression
pattern.

Tumor-Specific Antigens (TSAs)

TSAs are unique to tumor cells and do not occur on normal cells in the body.
They are induced by viruses or chemical or physical carcinogens.
156
Virus-induced TSA. Virally induced tumors express tumor antigens shared by
all tumors induced by the same virus, such as FOCMA antigens [feline
oncornavirus membrane-associated antigens] found on the neoplastic lymphoid
cells of cats infected with feline leukemia virus.

The antigens may be found in the nucleus, cytoplasm, or plasma membrane
of the tumor cell.

Chemical carcinogen-induced TSA. Carcinogens induce random mutations in
the genome of affected cells. Consequently, each mutated gene product [anti-
gen] differs, depending on which gene has been affected by the carcinogen.

As a result, when the same chemical carcinogen induces two separate tu-
mors at different sites in the same animal, the tumor antigens are distinct and
the immune response to one tumor does not protect against the other.













Tumor-Associated Antigens (TAAs)

These are tumor antigens that are also expressed on normal cells; however, they
are generally present in higher concentrations in cancer patients.

Mutated gene products. Mutated forms of normal cellular proteins may be
produced by mutated genes.

Carcinogen-induced tumors produce tumor anti-
gens that are peptides derived from mutated
self-proteins. These altered self-proteins are pro-
cessed as cytosolic proteins and presented as
peptides together with class I MHC molecules on
the surface of tumor cells where they are recog-
nized by CTLs as altered self-cells.



157
Reactivated gene products. These are the products of developmental genes
that are programmed to be expressed only during embryonic development and
are usually turned off in adult cells.

Oncofetal tumor antigens. Oncofetal antigens are proteins that are ex-
pressed at high levels on embryonic and fetal tissues and cancer cells but not
adult tissues. It appears that the genes encoding these proteins are silenced
during development and are
derepressed upon malignant
transformation of the cell.






However, current evidence indicates that the expression of oncofetal anti-
gens in adults may not be limited to tumors; increased levels of the pro-
teins have been demonstrated in tissues and in the circulation in various
inflammatory conditions and they are found in trace amounts in some nor-
mal adults.

Oncofetal antigens are poor immunogens and do not provoke protective
immunity. However, their detection is important in tumor diagnosis and
prognosis.

Alpha-fetoprotein (AFP). AFP is the major protein in fetal serum (it is an
immunoregulatory protein). AFP is synthesized by the yolk sac and fetal
liver. Low levels may be present in the serum of some adults.

Elevated AFP levels are found in patients with hepatocellular carci-
noma and, occasionally, gastric and pancreatic cancers.

Carcinoembryonic antigen (CEA). CEA is an integral membrane protein
found on gastrointestinal, pancreatic, and liver cells during the first two tri-
mesters of gestation; low expression is seen in normal adult colonic
mucosa and lactating breast.

CEA expression increases in many carcinomas of the colon, pancreas,
stomach, and breast, and these patients have increased levels of CEA
in their serum. CEA is an intercellular adhesion molecule that func-
tions to promote the binding of tumor cells to one another.

Since CEA and AFP can be found in trace amounts in some normal adults
and in some inflammatory conditions, the presence of these oncofetal anti-
gens is not necessarily diagnostic of tumors.
158
Immune Responses to Tumors

If tumor cells are sufficiently antigenically different from normal, they are regard-
ed as foreign and attacked. Tumor antigens induce both humoral and CMI res-
ponses resulting in the killing of the tumor cells. However, the CMI response ap-
pears to play the major role in the destruction of tumors.

CD8
+
Cytolytic T Lymphocytes (CTLs)

CTLs kill malignant cells that express peptides derived from mutant cellular pro-
teins or oncogenic viral proteins.

Tumor antigens or tumor cells are internalized by host APCs, which process
the antigens. Peptides derived from the antigens are presented in associa-
tion with class I MHC molecules.

The professional APCs express co-stimulators [eg, B7-1], that provide the 2
nd

signals needed for differentiation of CD8
+
T cells into antitumor CTLs. This
process is called cross-priming to indicate that one cell type [the host APC]
may prime T cells specific for the antigens of another cell [the tumor cell].

Once effector CTLs are generated, they are able to recognize and kill the tu-
mor cells without a requirement for co-stimulation.

Natural Killer Cells

NK cells kill tumor cells that have reduced class I MHC expression and can es-
cape lysis by CTLs. In some cases, FcRIII receptors [CD16] on NK cells can
bind to IgG-coated tumor cells leading to ADCC.

Macrophages

IFN- activated macrophages play a significant role in the immune response to
tumors. Macrophages express Fc receptors, enabling them to bind to IgG on
tumor cells and mediate ADCC.
159
Killing of tumor cells by macrophages include the release of lysosomal en-
zymes, reactive oxygen intermediates and nitric oxide. Activated macro-
phages also secrete the cytokine TNF- that has potent antitumor activity
[TNF- injected into tumor-bearing animals induces hemorrhage and necrosis
of the tumor].

Antibodies

Antibodies may kill tumor cells by activating complement or by ADCC in con-
junction with NK cells and macrophages.

Evasion of Immune Responses by Tumors

Malignant neoplasms may evolve mechanisms that enable them to elude the
effector arm of the immune response. Many factors may contribute to the immu-
nologic escape of tumors.

Failure to produce tumor antigen. A malignant cell may not generate peptides
of novel proteins that can be presented by MHC molecules, therefore such a cell
will appear normal to the immune system.

Loss or reduced expression of class I MHC molecules. Malignant trans-
formation of cells is often associated with a reduction [or sometimes a complete
loss] of class I MHC molecules, such that the tumors cannot be recognized by
CD8
+
CTLs. However, such cells can trigger NK cell activity.

Lack of co-stimulation. The poor immunogenicity of many tumor cells may be
due in large part to the lack of co-stimulatory molecules [most tumor cells do not
express co-stimulatory molecules].

As a result, if sufficient numbers
of professional APCs are not
available in the immediate vici-
nity of a tumor to present tumor
antigens and activate tumor-spe-
cific naive T cells, maximal anti-
tumor CTL differentiation may
not occur.

Immunosuppression. It has been
observed that tumor-bearing animals
or individuals are usually immuno-
suppressed. Various products of
tumor cells may suppress antitumor
immune responses.

160
TGF- is secreted in large quantities by many tumors, inhibiting the prolifera-
tion and effector functions of macrophages and lymphocytes.

Apoptosis of CTLs. Some tumor cells express Fas ligand [FasL], which re-
cognizes Fas receptors [death receptor] on CTLs that attempt to attack the
tumor; engagement of Fas by FasL results in apoptotic death of the CTLs.

Antigen masking. Increased expression of cell surface glycocalyx molecules by
the tumor cell can hide or mask the tumor antigens from the immune system.

Tumor Immunotherapy

Immunotherapy for tumors is aimed at augmenting or supplementing the weak
host immune response to the tumors [active immunity] or at administering tumor-
specific antibodies or T cells [passive immunity].

Cytokine Therapy. Recombinant cytokines, individually or in combination, have
been used to augment the immune responses to tumors.

The cytokines stimulate the proliferation and activation of T lymphocytes and
NK cells.

The cytokines that have been tried in cancer immunotherapy include IL-1, IL-
2, IL-12, GM-CSF, TNF, and the interferons.

Tumor Necrosis Factors

TNF- and TNF- exhibit direct antitumor activity, killing some tumor cells and re-
ducing the rate of proliferation of others while sparing normal cells. In the pre-
sence of TNF- or TNF-, a tumor undergoes visible hemorrhagic necrosis and
tumor regression.

TNF- has also been shown to inhibit tumor-induced vascularization [angio-
genesis] by damaging the vascular endothelial cells in the vicinity of a tumor,
thereby decreasing the flow of blood and oxygen that is necessary for pro-
gressive tumor growth.

Interferons

Purified recombinant preparations of IFN-, IFN-, and IFN- have shown some
promise in the treatment of certain cancers. However, most of the clinical trials
have involved IFN-.

Interferon-mediated antitumor activity involves various mechanisms. All three
types of interferon increase class I MHC expression on tumor cells, thereby
increasing CTL activity against the tumors; IFN- also increases class II MHC
161
expression on macrophages, enhancing tumor antigen presentation to CD4
+

T cells.

IFN- inhibits cell division of both normal and tumor cells, therefore the anti-
tumor effects of the interferons may also be related to this ability to directly
inhibit tumor cell proliferation.

Adoptive Cellular Therapy. This is the transfer of cultured autologous lympho-
cytes that have antitumor activity into a patient.

Lymphokine-activated killer (LAK) cells. LAK cells are generated by removing
peripheral blood leukocytes from tumor patients, culturing the cells in high
concentrations of recombinant IL-2, and injecting the LAK cells back into the
patient together with in vivo administration of IL-2. LAK cells are derived
mainly from NK cells.

Various undesirable side effects are associated with the high levels of IL-2
required for LAK-cell activity in vivo. They include fever, chills, weight gain
and, more importantly, vascular leak syndrome, which involves emigra-
tion of lymphoid cells and
plasma from the peripheral
blood into the tissues, leading
to massive pulmonary edema
and shock.




Tumor-infiltrating lymphocytes
(TILs). TIL cells are lymphocytes
[tumor-specific CTLs and activat-
ed NK cells] obtained from biopsy samples of tumors and expanded in vitro
with IL-2. When reinjected into the patient, TILs have a wide range of anti-
tumor activity; however, the tumor-specific CTLs recognize and infiltrate only
the tumors they come from.

When TILs are injected into the donor patients together with IL-2, they pro-
duce very high remission rates, especially in patients with melanomas and
colorectal and kidney cancer. TIL cells require 100-fold lower levels of IL-
2 for their in vivo activity than do LAK cells.

Monoclonal Antibodies

Monoclonal antibodies specific for tumor antigen can be infused into the patient,
either directly or with a toxin, drug, or radioisotope conjugated to the antibody.
The antibody directs the conjugate exclusively to the cancer cells.
162




















Tumor Vaccines

Viral vaccines. Successful vaccines against viral tumors have been used in
feline leukemia in cats and Mareks disease in chickens.
163
IMMUNITY IN THE FETUS AND NEONATE

In domestic animals, all components of the innate and adaptive immune systems
develop in utero and are functional at birth. However, they are generally less
efficient in responding to antigens than in the adult.

Development of the Prenatal Immune System

The immune system of all domestic species begins development fairly early in
gestation. In general, the shorter the gestation period, the less developed the
immune system is at birth.

The thymus is the first lymphoid organ to develop, followed by the peripheral
lymphoid organs [eg, spleen, lymph nodes, etc]. Ig-containing cells develop
soon after the appearance of the spleen and lymph nodes; however, serum
immunoglobulins are not usually found until late in fetal life, if at all.

A steady increase in peripheral lymphocytes occurs throughout gestation.
The majority of these circulating fetal lymphocytes are T cells. Development
and expansion of other white blood cell populations occurs at the same time
that lymphocyte development is going on in the fetus.

Intrauterine Infections

If a fetus is exposed to a foreign antigen, whether an immunologic response
occurs depends on the stage of development of the fetus and the nature of the
particular antigen. The outcomes of fetal infections may be as follows:

Death of the fetus. Occurs if the immature defense mechanisms allow infec-
ting organisms to replicate freely and destroy fetal tissues. This is seen most-
ly during early gestation.

Persistently infected fetuses that remain infected into neonatal or adult life.
Various viruses can induce immunologic tolerance or hyporesponsiveness,
resulting in little or no antibody production [see Tolerance, page 224], eg,
bovine viral diarrhea infection of cattle.

If fetal lymphocytes have matured to the point at which they can recognize
antigenic determinants of the invading organism, the immune response may
be successful in terminating the infection.

The Neonatal Immune System

Although neonates are capable of mounting an immune response, they are re-
ferred to as being immunonaive. This inability to initiate a successful immune
response stems from the physiologic immaturity of their immune systems.
164
The initial immune response mounted to an antigen is a 1
o
immune response
with a prolonged lag period and low concentrations of immunoglobulins being
produced. Therefore, unless adequate maternal immunologic assistance is
provided, neonates have an increased likelihood of succumbing to infections
that are innocuous to adult animals.

The immunologic assistance that neonates receive is through immunoglo-
bulins and other factors present in colostrum [natural passive immunity].

Natural Passive Immunity

Natural passive immunity is important for two main reasons: (1) it is essential for
the protection of the newborn, during the first weeks or months of life, from the
microorganisms present in its environment and (2) maternal antibodies interfere
with active immunization of the newborn, therefore this must be taken into consi-
deration when designing vaccination schedules [see Vaccines and Vaccination,
page 178].

Maternal antibodies may be acquired transplacentally, via colostrum and/or
milk, and via the egg yolk and egg white.

Transfer of Maternal Antibodies

In domestic animals, maternal antibodies may or may not reach the fetus depen-
ding on the nature of the placenta. Placental classification by microscopic ap-
pearance is based on the number of placental layers [six altogether] that sep-
arate the fetal blood from the maternal blood:

Maternal capillary endothelium, (2) uterine connective tissue, (3) endometrial
epithelium, (4) chorionic epithelium [trophoblast], (5) fetal connective tissue
and (6) fetal capillary endothelium.

Epitheliochorial type: All six layers are intact, thereby preventing antibody
[IgG] transfer. It is found in ruminants, sows, and equine. The presence of
antibodies in the precolostral serum of a newborn, suggests intrauterine infec-
tion.

Endotheliochorial type: There is complete erosion of the endometrial epi-
thelium and underlying interstitium, thus maternal capillaries are directly ex-
posed to chorionic epithelium [ie, 4 intervening layers]. Less than 10% of ma-
ternal IgG cross the placenta. Present in queens and bitches.

Hemochorial type: The chorionic epithelium is in direct apposition to mater-
nal pools of blood [ie, 3 intervening layers]. IgG readily crosses the placenta.
Present in humans, primates, and rodents.
165
Colostrum

Colostrum consists of the accumulated secretions of the mammary gland over
the last few weeks of pregnancy together with proteins transferred from the ma-
ternal circulation under the influence of estrogen and progesterone.

Colostral and Milk Immunoglobulin Levels (mg/dL) in Domestic Animals and Humans


Species


IgG
Colostrum

IgA


IgM


IgG
Milk

IgA


IgM
Horse

Cow

Ewe

Sow

Bitch

Queen

Human
1500-5000

2400-8000

4000-6000

3000-7000

120-300

3250-4400

30
500-1500

100-700

100-700

950-1050

500-2200

150-340

12,000
100-350

300-1300

400-1200

250-320

14-57

47-58

120
20-50

50-750

60-100

100-300

1-3

100-440

10
50-100

10-50

5-12

300-700

110-620

240-620

150
5-10

10-20

0-7

30-90

10-54

0

1

IgG. This is the 1
o
colostral Ab in most domestic species except in ruminants,
in which IgG1 is the 1
o
Ab. IgG is produced systemically and is selectively
transferred by an Fc-receptor-mediated process into the mammary gland. As
IgG accumulates in the mammary gland to 5 to 50 times the concentration in
plasma, systemic concentrations of IgG fall by 50%.

IgM Most colostral IgM [~85%] are produced systemically; about 15% of
IgM are produced locally in the mammary gland.
166
IgA. About 60% of IgA are produced locally in the mammary gland; ~40% of
IgA are of serum origin.

Colostrum is generally nursed out by 48 hours after parturition and is replaced
by milk.

Absorption of Colostral Antibodies

In neonates, the level of proteolytic activity in the digestive tract is low soon after
birth, therefore colostral antibodies are not degraded but reach the small intestine
intact. Colostral Igs are also protected from enzymatic digestion by the presence
of trypsin inhibitors in colostrum. Ig absorption is most efficient in the jejunum
and ileum.

Ig in colostrum binds to an Fc receptor [called FcRn] on the specialized
epithelial cells present in the small intestine; are actively pinocytosed; and
eventually reaching the neonatal circulatory system in an undegraded form.

Horses and pigs. IgG and IgM are preferentially absorbed, whereas IgA
mainly remains in the intestines. In ruminants, all Ig classes are absorbed,
however, IgA is gradually reexcreted into the intestinal lumen.

Translocation cutoff time [gut closure]. The ability of the neonates intestine
to absorb Igs is time dependent [~48 hours]. Feeding colostrum tends to
hasten gut closure while delaying feeding results in a slight delay of closure.
Gut closure time is different for each species and for each Ig class. It is a
result of the sloughing of the specialized enterocytes and their replacement
by more mature epithelial cells incapable of protein absorption.

Permeability is highest immediately after birth and declines after about 6-8
hours. However, the absorption of all Ig isotypes drops to low levels after
about 24 hours. Colostrum administered after Ig absorption has stopped
has significant effects only in neutralizing enteric pathogens.

In neonates, peak serum antibody levels are normally reached between 12
and 24 hours after birth.

Half-life of maternal antibodies. After absorption ceases, maternal antibodies
in the bloodstream of the neonate begin to decrease rapidly due to normal
catabolic processes. Half-life of maternal antibodies varies among species:

Horses and cattle: 21 days; dogs and cats: 8-10 days.

However, protection in a neonate against a specific pathogen may extend
beyond the normal range of Ig half-life if the initial antibody titer against
that pathogen is high.
167
Failure of Passive Transfer (FPT)

Failure of passive transfer is the absence of adequate concentrations of plasma
IgG with the potential consequence of a diseased neonate [septicemia and
diarrhea]. Colostral transfer is a function of quality [titer of specific antibodies to
specific pathogens], quantity of the colostrum, and the timing of colostral intake.

FPT is the most common immunodeficiency disease of domestic animals.
Between 10-40% of dairy calves and 20% of foals fail to receive adequate
levels of maternal Igs. The risk of death for a calf with FPT is 3-10x greater
than a calf absorbing adequate amount of colostral antibodies.

Immunoglobulin content of colostrum may be quantitated with a colostro-
meter, a hydrometer that measures the specific gravity of colostrum. Specific
gravity >1.050 indicates adequate globulin content [ 5000 mg/dL].

The causes of FPT include the following:

birth of weak or deformed animals
delay to first suckle
failure of intestinal absorption
poor colostrum production and/or premature lactation
poor maternal instinct especially in primiparous dams
low antibody levels in maternal serum and thus in colostrum
cows with poor udder conformation [eg, dropped teats]
too many in the litter
bullying of the weak by the strong
death or disability of the dam

Diagnosis of Failure of Passive Transfer

The diagnosis of FPT is based on measurement of serum IgG concentrations in
the neonate, preferably between 18 to 48 hours of age. Numerous test kits are
available to measure IgG concentrations.

Single radial immunodiffusion test [Mancini test; gold standard]
Latex agglutination test [whole blood or serum]
ELISA test [whole blood or serum]
Sodium sulfite precipitation test [serum]
Glutaraldehyde coagulation test [whole blood]
Refractometry [serum or plasma]
Zinc sulfate turbidity test [serum].

FPT in calves and lambs. Serum IgG concentration of <1000 mg/dL.


168
FPT in Foals

Threshold concentration of serum IgG for prophylaxis in foals: 800 mg/dL.
Levels between 400 and 800 mg/dL should be considered partial FPTs, and may
require treatment depending upon circumstances [eg, cleanliness of environ-
ment, etc]; levels of 400 mg/dL are FPTs and should definitely be treated.

Foals < 15 hours old. A total of 2 to 3 L of colostrum divided into 3 or 4
doses at hourly intervals should be provided. It may be given by bottle or
nasogastric tube.

Foals > 15 hours old. 2 to 4 L of normal plasma [IgG concentration between
1000 and 2000 mg/dL] are required to achieve desired concentrations of Ig in
the foal. It can be administered IV or intraperitoneally. The plasma must be
checked for anti-erythrocyte Abs [hemolytic disease of the newborn; see Type
II hypersensitivity] and must be free of bacterial contamination.

Alternatives to Fresh Colostrum

Stored frozen colostrum. Colostrum can be stored at 15
o
to 20
o
C for up to
one year. Colostrum may be thawed with little loss of immunoglobulin.

Colostrum substitutes. Many of the commercial products do not have the pro-
tective qualities of fresh colostrum in establishing adequate passive immunity.
Some products provide significant Ig and appear to provide protection from
disease.

Bovine colostrum has been used as an alternate source of colostrum for
some neonates, eg, foals. The following must be taken into consideration: (1)
the half-life of heterologous Igs is much shorter, (2) antibody specificity is not
ideal due to different vaccination schedules, and (3) specie differences in
susceptibility to various pathogens.

Cell-Mediated Immunity

For all mammalian species, colostrum contains >1 x 10
6
cells per ml. The cells
are mostly lymphocytes and macrophages and a few neutrophils.

Some lymphocytes are absorbed through the gut wall and reach the circula-
tory system. However, it is not clear if the passively transferred lymphocytes
confer significant cell-mediated immunity on the offspring.

Passive Immunity in Chickens

Passively transferred Igs from the hen provide temporary protection for the chick
during the period after hatching when it is least immunocompetent.
169
IgG. In birds, IgG is selectively transferred from the maternal circulation into
the yolk. The level of IgG in the yolk is 250 mg/dL [maternal circulation con-
tains ~600 mg/dL].

IgG enters the vitelline circulation and hence that of the chick from day 12
of incubation. J ust before hatching, the yolk sac with the remaining mater-
nal IgG is completely taken into the abdominal cavity and incorporated into
the wall of the small intestine.

IgA and IgM. Maternal IgA and IgM are added to the developing egg via the
albumen [albumin] as the yolk reaches the magnum of the oviduct. At mid-
incubation, IgA and IgM diffuse into the amniotic fluid and are swallowed by
the embryo. Thus, when the chick hatches, it has IgG in its serum and IgM
and IgA in its intestines.


170
VACCINES AND VACCINATION

The goal of active immunization of an individual or animal is to elicit specific
protective immunity and immunologic memory [anamnestic response]. This
anamnestic response is the protective principle on which vaccination is based.

The role of memory cells in immunity depends, in part, on the incu-
bation period [IP] of the pathogen. Effective protection against dis-
eases with short IP depends on maintaining high levels of neutra-
lizing antibody by repeated immunization. Conversely, for patho-
gens with a longer IP, demonstrable neutralizing antibody at the
time of infection is not necessary

Vaccines are almost always intended for use only on healthy animals.

Vaccines are most effective against poorly infectious agents whose antigens
are relatively invariant.

Vaccination does not protect against infection nor does it eliminate or prevent
the development of carrier states. The correct use of vaccines reduces the
consequence of the particular infection on the animal or individual.

The nonspecific immune system is not directly affected by vaccination.

Terminology

Variolation. In the eleventh century the Chinese vaccinated their children
against smallpox using vesicular fluid containing live smallpox virus [Variola vi-
rus]; reduced the case fatality rate from 25% to 1%.

Vaccination (vaccinuspertaining to cows). The administration of an antigen
[vaccine] to an animal or individual with the intention of stimulating a protective
immune response.

In 1796, Edward Jenner introduced vaccination to Europe when he used live
cowpox virus to protect individuals against smallpox.

Vaccine. A preparation of antigenic material used to induce immunity against
pathogenic organisms.

Homologous vaccine. A vaccine made from the causative agent responsible
for the disease in question.

Heterologous vaccine. A vaccine from an agent other than the one responsi-
ble for the disease in question, such as protecting puppies against canine
distemper using measles vaccine.

171
Bacterin. Killed bacterial vaccine. Bacterins are generally less effective than
viral vaccines and provide short-lived, partial immunity.

Autogenous vaccine. This is prepared from the particular strain of pathogen
causing disease in that specific group of animals.

General Principles of Vaccination

There must be absolute identification of the causal organism.

It must be established that an immune response can in fact protect against
the disease in question.

The disease should be serious enough to justify vaccination.

The risks of vaccination should not exceed the chance of contracting the dis-
ease itself. Thus, a vaccine, even if not completely safe, should be safer than
exposure to the disease.

Requirements for an Effective Vaccine

The vaccine must induce a humoral or CMI response directed against an anti-
gen involved in pathogenesis.

Vaccine should be safe, with minimal and acceptable side effects.

Stability. Vaccines are stable for 1 year when maintained at a temperature of
4
o
C; they can deteriorate in 2-3 days at a temperature of 37
o
C.

Be reasonably cheap and adaptable to mass vaccination.

Types of Immunization Procedures

Artificial Passive Immunization

This involves the production of antibodies in one animal by active immunization,
followed by transfer of these preformed antibodies to susceptible animals to
confer immediate protection. Passive immunization is routinely administered to
animals or individuals exposed to tetanus, botulism, rabies, snake bites, black
widow spider bites, etc.

The antisera [immune globulins] are usually produced in young horses by a
series of immunizing inoculations.

Sometimes, the recipient may produce anti-specie antibodies to the isotypic
antigenic determinants of the foreign antibody.
172
If the recipient produces IgG or IgM specific for the foreign antibodies, this
may result in immune complex hypersensitivity [see Type III Hypersensiti-
vity, page 212].

To reduce their antigenicity for other species, immune globulins of horse
origin are usually treated with pepsin to degrade the Fc regions [see Im-
munoglobulins, page 108].

Artificial Active Immunization

This involves the administration of antigen to an animal so that it can respond by
mounting a protective immune response.

Stimulates the development of effector cells and memory cells.

TYPES OF VACCINES

Nonliving Vaccines

Nonliving vaccines cannot replicate and are unable to cause infectious disease.
To induce a protective immune response, nonliving vaccines require a large anti-
genic dose, multiple immunizations, and usually the addition of adjuvants.

Toxoid vaccine (Nontoxic derivative of a toxin). Immunity to toxigenic bacteria
is based on circulating antibodies [antitoxins] to bacterial exotoxins. Purified exo-
toxin is chemically modified by formaldehyde treatment so that it retains its anti-
genicity but loses its toxicity. The toxoid preparation is combined with an adju-
vant, eg, aluminum hydroxide gel, before administration.

Vaccination with the toxoid triggers the production of antitoxin antibodies,
which can bind to the toxin and neutralize its toxic effects.

Subunit vaccine. A subunit vaccine contains only one or more of the antigens
of the pathogen necessary to induce protective immunity, rather than the entire
organism. The efficacy of a subunit vaccine is highly dependent upon the use of
an effective adjuvant.

The advantages of subunit vaccines include the reduction in risk of disease,
abortion or allergic reaction to nonessential components of the organism.

M protein vaccine against strangles in horses [Streptococcus equi ss. equi].

Subunit feline leukemia [FeLV] vaccine. This recombinant vaccine protects
cats from feline leukemia. The antigen is derived from the envelope glyco-
protein of FeLV subgroup A, expressed in E. coli [see page 175].

173
Plasmid DNA vaccine. Plasmid DNA encoding a protein antigen involved in
pathogenesis, is injected directly into the muscle of the recipient. The DNA is
injected in saline solution or adsorbed to microscopic gold beads and fired
into muscle cells with a gene gun.

Because the DNA-encoded proteins are synthesized in the cytosol of trans-
fected cells, DNA vaccines elicit strong and long-lived humoral and cell-me-
diated [cytolytic T lymphocyte] immune responses to the antigen even when
administered without adjuvants.

The potential advantages of DNA vaccines include:

(1) the ease of manipulating DNAs to express multiple viral and bacterial
antigens, (2) stability [DNA vaccines can be stored without refrigeration],
(3) low cost, (4) long-lasting protection, and (5) the ability to coexpress
other proteins that may enhance immune responses [eg, cytokines and
co-stimulators].

A potential problem is that continuous antigenic stimulus may lead to tole-
rance or autoimmunity.

Inactivated (killed) vaccines. Inactivated vaccines cannot replicate and are un-
able to cause infectious disease. It is essential to maintain the structure of epi-
topes on surface antigens during inactivation.

In general, inactivated vaccines produce weaker immune responses with a
shorter duration than the immune responses produced by attenuated vacc-
ines.

Heat inactivation. Not recommended because heat causes extensive protein
denaturation and lipid oxidation.

Chemical inactivation. Chemical inactivation with formaldehyde, ethylene
oxide, -propiolactone, etc, result in little or no change in antigenicity.

Live Vaccines

Virulent vaccine. Administered by an unnatural route or at a safe age, eg, ovine
parapoxvirus [contagious ecthyma] applied to a scarified area of the inguinal
region in sheep; Anaplasma marginale vaccine [infected blood] administered to
young calves.

Attenuated vaccines. Consist of a naturally occurring avirulent organism or a
virulent organism is grown under unusual conditions (eg, passages in cell culture
or embryonated eggs, growth in embryonic tissue, or at low temperatures) that
reduces its ability to cause disease.
174
The process of attenuation selects mutants that are better suited to growth in
the abnormal culture conditions and are therefore less capable of growth in
the original host.

Because attenuated vaccine organisms replicate in the host, they more close-
ly resemble virulent infection and generally produce a stronger and more dur-
able protective immune response than do nonliving vaccines.

Underattenuation. Some attenuated live-virus vaccines have been associa-
ted with significant disease in some recipients.

Overattenuation. Can result in a decline in the capacity of the organism to
replicate in the host, with a corresponding loss of immunogenicity.

Temperature-sensitive mutants (Ts mutants). Ts mutants grow optimally at
low temperatures, but poorly at normal body temperatures.

Advantages and Disadvantages of Attenuated Live-Virus and Inactivated Vaccines
Parameter Attenuated Vaccine Inactivated Vaccine
Antigen per dose

Cost

Number of doses needed

Need for adjuvant

Duration of immunity

Type of immunity induced

Induction of interferon

Relative stability

Interference

Dangerous in pregnant animals

Reversion to virulence
Low

Low

Usually a single dose

No

Many years

Humoral and CMI

Yes

Less stable

Occasional

Some

Possible
High

High

Multiple doses

Yes

Months or years

Mainly humoral

No

More stable

No

No

No


Genetic Engineered Vaccines

Recombinant antigen vaccines. The gene encoding an immunogenic protein
is isolated and cloned in bacterial, yeast, or mammalian cells using recombinant
DNA technology. When the gene is expressed, the resultant proteins are har-
vested, purified, and used for vaccine development.

Since the protein antigens are processed in endosomal vesicles [see MIIC, page
75], they primarily induce humoral immunity.

175

















Recombinant vector vaccines. Genes encoding major antigens of pathogens
may be cloned directly into attenuated viruses [eg, vaccinia virus, canarypox vi-
rus] or bacteria [eg, attenuated strain of Salmonella]. The attenuated organism
serves as a vector, replicating within the host and expressing the gene product of
the pathogen.

A vaccinia virus recombinant containing the gene for the rabies virus enve-
lope glycoprotein [G-protein], administered in a bait, has been successfully
used to protect wild carnivores against rabies.

Deletion mutants. It is possible to modify the genes of an organism by deleting
a sequence of nucleotides from the gene so that the organism becomes irrever-
sibly attenuated.

A pseudorabies vaccine in which the thymidine kinase [TK] gene has been
deleted is currently available for use against pseudorabies in pigs. TK is re-
quired by herpesviruses to replicate in nondividing cells, eg, neurons. Viruses
from which the TK gene has been removed are able to infect nerve cells but
cannot replicate and cause disease.

Adjuvants (from Latin adjuvare, to help)

Adjuvants are substances that, when mixed with an antigen and injected with it,
enhance the immune response to that antigen. In conventional vaccines, adju-
vants are used to elicit an early, strong, and long-lasting immune response.
They are usually combined with nonliving antigens.

An adjuvant allows the use of reduced antigen concentration to achieve the
desired immune response, thus reducing vaccine production costs.
The production of a recombinant viral
protein for use in a vaccine
176
Mode of Action

The formation of a depot of antigen at the site of inoculation, resulting in slow
release of antigen. Antigenic stimulus is prolonged over a period of weeks,
creating the effect of multiple, microbooster exposure.

Stimulate antigen-presenting cells to express more co-stimulatory molecules,
[eg, B7 molecules] and increased production of cytokines.

Stimulate granuloma formation. Because macrophages in a granuloma are
activated macrophages, this mechanism also enhances T
H
cell activation.

Mode of Action of Some Commonly Used Adjuvants
Type Adjuvant Mode of Action
Aluminum salts



Calcium salts

Water-in-oil emulsion



Surface-active agents
Aluminum phosphate
Aluminum hydroxide
Aluminum potassium sulfate

Calcium phosphate gel

Freunds incomplete adjuvant
Freunds complete adjuvant


Saponin
Slow-release antigen depot;
induce granuloma formation


Slow-release antigen depot

Slow-release antigen depot;
induce granuloma formation;
enhance co-stimulation

Stimulates antigen processing
Freunds complete adjuvant: Water-in-oil emulsion plus heat-killed Mycobacteria

Side effects. Some adjuvants may induce severe local inflammatory reac-
tions, resulting in abscess formation and severe granulomas.

Polyvalent (Multiple-Antigen) Vaccines

Several disease syndromes may be caused by more than one organism, thus it
is now commonplace to incorporate several antigens in one vaccine. In addition,
a single vaccine may contain several antigens against unrelated diseases.

Interference. When different antigens in a mixture are inoculated simulta-
neously, competition may occur between antigens. This may result in less
than an optimum immune response to a number of the antigens. Manu-
facturers take this into account and modify their mixtures accordingly.

Administration of Vaccines

Whether parenteral or local immunization is necessary for protection depends on
the pathogenesis of the disease.

Systemic immunity. Subcutaneous [SC], transdermal or intramuscular [IM]
administration.

Local immunity. Intranasal, ocular, or oral routes.
177
Mass vaccination. Aerosolization [spray method], feed, or drinking water [not
tap water].

Vaccination Schedules

Vaccination schedules vary among the domestic species and even from area to
area. When formulating a vaccination protocol, the following questions must be
considered:

Is it likely that sufficient immunity to protect the animal against a given dis-
ease is already present?

What is the nutritional status of the animal?

Inadequate nutrition, including deficiencies of protein and certain micro-
nutrients [eg, copper and zinc], is known to restrict immune responses.

What is the health status of the animal?

Does the animal have possible immunosuppression attributable to physiologic
stressors or pharmacologic intervention?

Animals under stress are poor subjects for immunization because the
immune response may be diminished by various mechanisms, including
immunosuppression by endogenous corticosteroids. Glucocorticoid thera-
py should be stopped a week before and two weeks after 1
o
vaccina-
tion, if possible.

What is the level of exposure that the animal is likely to encounter for the dis-
ease[s] being considered for vaccination?

Disease prevalence, population density, exposure to other animals, etc.,
are important factors in formulating a vaccination protocol.

Neonates

Maternal antibody inhibits the immune response of the newborn to vaccines.
Ideally vaccination should be delayed until the titer of maternal antibody in the
young animal has declined to near zero. However, this may introduce a window
of susceptibility in the newborn animal.

For kittens and puppies, a series of vaccinations are given at 3- to 4-week
intervals between 6 and 16 weeks of age.

Vaccination can break through maternal antibody interference by 12
weeks of age in most puppies and kittens.
178
Comparison of response to sequential
(2-week interval) vaccination in neo-
nates with (top) and without (bottom)
maternal antibody protection. The
presence of maternal antibody delays
the neonates ability to mount a suc-
cessful active immunization.













Calves, lambs, kids, foals, and piglets can be vaccinated early [3-4 months]
and a second injection given about 6 months of age.

Vaccination of the Dam

Vaccination of the pregnant animal or laying hen such that high levels of mater-
nal antibodies transferred in the colostrum and milk or in the egg, ensures that
the offsprings have a protective level of antibodies during the critical early days of
their lives.

Guidelines for Proper Handling of Vaccines
All vaccines should be maintained at the recommended temperature from the time
they leave the manufacturer until they are administered to the animal.

All vaccines should be protected against exposure to ultraviolet light.

The diluent provided by the manufacturer has been pH-adjusted for a particular
organism. Therefore, practitioners should never use a diluent intended for a dif-
ferent vaccine to rehydrate a lyophilized producteven a diluent from the same
manufacturer.

Because different vaccines require different pH values and may have various acti-
vating agents or adjuvants that can affect the pH, practitioners should never mix
vaccine products unless it is recommended by the manufacturer.

Attenuated vaccines should be used soon after they have been reconstituted.
Therefore, practitioners should ensure that the vaccines are not stored for use at a
later date.

Chemically sterilized syringes should not be used because traces of disinfectant
can inactivate attenuated vaccines.
179
The normal distribution of protective
immune responses in a population
of vaccinated animals
Revaccination

Viral diseases for which long-term, protective immune responses are developed
after vaccination [eg, canine distemper and canine parvovirus vaccines] do not
require annual revaccinations once the animal reaches adulthood.

Herd Immunity

Refers to the immune status of a population, rather than an individual animal.

If a significant fraction of the population is immune to an infectious agent, the
probability of a susceptible animal contacting an infected animal is so low that
the susceptible animal is not likely to become infected.

The percentage of immune animals for herd immunity to work will vary be-
tween vaccines, and on the nature of the disease, but is generally between 80
to 95%.

Failures in Vaccination

No vaccine is 100% effective; therefore, the immune response never confers
100% protection and is never equal in all members of a vaccinated population.
The immune response usually follows a normal distribution pattern.


















Causes of Vaccination Failures

Apparent failure due to animal incubating disease.

Apparent failure due to poorly distributed vaccine in aerosols or feed.
180
Animal genetically incapable of responding to antigen.

An immune response is overwhelmed by extreme exposure to a particular
pathogen.

Failure in the vaccine itself

Death of live vaccine; inappropriate strain of organism or wrong organism
employed; and insufficient dose of vaccine.

Failure in the immune response

Due to passive immunity [maternal antibodies].

The animal produces a humoral response to the vaccination, but a cell-
mediated response is necessary for protection.

Stress, fatigue, malnutrition, heavy parasite load [disturbances in protein
metabolism].

Complications and Side Effects of Vaccines

Virulent infectious material in vaccine

Incomplete inactivation in case of killed vaccines.

Some attenuated vaccines may have residual virulence.

Allergic effects. Observed more frequently with killed vaccine [multiple dosing
and/or high individual doses of antigen].

Puppies. Vaccination with whole killed leptospiral vaccine induces allergy
in about 11% of puppies vaccinateddyspnea, urticaria, facial swelling,
and death.

Corneal edema (blue eye). May occur in 1% of puppies vaccinated with
modified live infectious canine hepatitis vaccine.

Nonmicrobial antigens in vaccines can cause allergic reactions, eg, egg
proteins or proteins derived from tissue culture.

Harmful effects on the developing fetus. Abortogenic or teratogenic effects.

181
HYPERSENSITIVITY REACTIONS

Immunity is a double-edged sword. When an immune system acts against a for-
eign antigen, the result is protection; when it acts against the hosts own cells,
the result is the disruption of body systems.

Hypersensitivity is an inappropriate or exaggerated immune response that is
harmful to the host.

Hypersensitive reactions may develop in the course of either humoral [initiat-
ed by antibody or antigen-antibody complexes] or cell-mediated [initiated by T
cells] responses.

In 1839, Magendie reported on the sudden death of dogs receiving repeated
injections of egg albumin. However, his observation was not followed up.

In 1902, Paul Portier and Charles Richet were developing an antitoxin to
protect swimmers from attacks by sea anemones and the Portuguese man-
of-war jellyfish. After immunizing dogs with a sublethal extract of sea
anemone tentacles, they observed that a 2
o
challenge several weeks later
with the same sublethal extract resulted in a reaction characterized by
vomiting, bloody diarrhea, suffocation, unconsciousness, and death. They
called the phenomenon anaphylaxis (Gk ana, against; phylaxis, protection).
Richet received a Noble Prize in medicine in 1913 for his work on
anaphylaxis.


Terminology

Allergen. An antigen that causes IgE-mediated hypersensitivity reaction.

Allergy. A harmful antigen-antibody reaction, usually caused by a foreign anti-
gen in food, pollen or chemicals.

Atopy (Unusual). Hereditary tendency to develop IgE-mediated allergic reac-
tions to allergens that do not cause a reaction in the normal individual or animal.

Anaphylaxis. An immediate-type hypersensitivity reaction that results from sen-
sitization of tissue-fixed mast cells by IgE following exposure to allergen. Usually
systemic and severe.

Anaphylactoid reactions. Anaphylaxis that is not of IgE origin. May occur via
activation of the complement cascade by either the classic or alternate pathways,
resulting in generation of the anaphylatoxins C3a, C4a, and C5a. They can
cause degranulation of mast cells resulting in release of vasoactive mediators.

Sensitization. Allergic reactions require prior immunization by the allergen that
elicits the acute response. Allergic reactions only occur in sensitized individuals.
182
Gell and Coombs Classification

The classification scheme is based on the immune mechanisms involved and the
time lag between antigen exposure and appearance of clinical signs.

Immediate hypersensitivity reactions

Acute. Mediated by IgE; begins and ends within 30 minutes. Type I.

Subacute. Mediated by IgG and/or IgM; begins after about 2 hours and its
persistence depends on antigen-antibody concentration. Types II and III.

Delayed hypersensitivity reactions (cell-mediated)

Mediated by T cells and macrophages and is a delayed reaction; begins
within 1-3 days and persists for several days to a few weeks. Type IV.


183
TYPE I (IMMEDIATE) HYPERSENSITIVITY

This is an exaggerated immune response which develops within minutes follow-
ing exposure of a sensitized individual or animal to antigen. It is dependent on
activation of mast cells and basophils by IgE and the release of mediators of in-
flammation.

It is characterized by rapid increase in vascular permeability, vasodilation,
bronchial and visceral smooth muscle contraction, and local inflammation. In
the most extreme systemic form of the reaction [anaphylaxis], death may re-
sult from asphyxiation and cardiovascular collapse.

Manifested in bronchial asthma, hay fever [allergic rhinitis], bee stings, rag-
weed allergy, etc.

Experimental Induction of Type I Hypersensitivity

Systemic anaphylaxis can be readily demonstrated in guinea pigs. Sensitization
is induced by a single injection of a foreign protein (eg, egg albumin).

After a waiting period of 2 to 3 weeks, the guinea pig is challenged with an in-
travenous injection of the same protein. Within 1 minute the guinea pig be-
comes restless, shows labored breathing, and its blood pressure drops. Con-
traction of the smooth muscles of the gastrointestinal tract and bladder results
in defecation and urination. Bronchiole constriction results in death by asphy-
xiation within 2-4 min of the injection.

The Mechanism of Type I Hypersensitivity Reaction
184
Allergens

The allergens that elicit IgE responses usually enter the body by inhalation, in-
gestion or through the skin. They are all noninfectious [nonparasitic] proteins
or chemicals bound to proteins to which the allergic individual or animal is con-
tinuously exposed.

Most allergens are low-molecular-weight, highly soluble protein molecules
bound to dessicated particles such as pollen grains or mite feces. On contact
with the mucosa, the soluble allergens are eluted from the delivery particles
and diffuse into the mucosa.

The allergens are taken up by mucosal dendritic cells [ie, Langerhans cells]
which carry them to T cell areas of regional lymph nodes. The allergenic pep-
tides are presented to naive T
H
cells, leading to their activation and the provi-
sion of T
H
2 cell help for B cell IgE production.

Several allergens such as ragweed pollen are multiallergenic systems con-
taining a number of allergenic components that elicit IgE responses and non-
allergenic components that elicit IgM or IgG responses.

Since type I hypersensitivity reactions are dependent on T
H
2 cells, T cell-inde-
pendent antigens such as polysaccharides cannot elicit such reactions unless
they become attached to proteins.

Common Antigens Associated with Type I Hypersensitivity Reactions
Proteins

Foreign serum
Vaccines
Plant Pollens

Ragweed, rye grass, birch,
timothy grass
Insect Products
Bee venom, ant venom,
wasp venom, dust mite
feces
Foods
Shellfish, milk, eggs, fish,
wheat, nuts, peas, beans
Drugs
Sulfonamides, penicillin,
local anesthetics, etc

Mold spores, animal hair
and dander

Mast Cells

Mast cells originate from hematopoietic precursors in the bone marrow. Normally,
mature mast cells are not found in the circulation. The precursor cells migrate to
the peripheral tissues where they undergo differentiation to become mature mast
cells. The life span of mast cells varies from weeks to months.

Maturation of mast cells in the tissues is regulated by stem cell factor [SCF],
a cytokine derived from fibroblasts and bone marrow stromal cells and the T
cell cytokines, IL-3, IL-4, IL-9, and IL-10. The cytokines also regulate the
proliferation of mast cells in tissues.

Mature mast cells are found throughout the body, predominantly located near
blood vessels and nerves and beneath epithelia. The greatest concentration
185
of mast cells are in the skin and respiratory and gastrointestinal tracts. They
are also present in the lymphoid organs.

Mast cells are large, round cells
with cytoplasm containing mem-
brane-bound granules and lipid
bodies. The granules contain
pharmacologically active media-
tors that are released following
activation of the mast cell.

Mast cell populations in different
anatomic sites exhibit significant
variation in morphology and bio-
chemical and functional charac-
teristics.
Mast cells also secrete a variety of cytokines including IL-1, IL-3, IL-4, IL-
5, IL-6, GM-CSF, TGF-, and TNF-.

Polymorphonuclear Basophils

Basophils are terminally differentiated inflammatory granulocytes that develop
from myeloid progenitor cells in the bone marrow. They constitute about 0.5% of
blood leukocytes. Basophils have a short life span [days].

Basophils are not normally found in extravascular tissues, however, they may
be recruited to some inflammatory sites, usually with eosinophils. Basophil
granules contain pharmacologically active mediators similar to those found in
mast cells.

Features of Mast Cells and Basophils
Feature Mast cells Basophils
Site of maturation

Major cell in blood

Recruited into tissues
from blood

Resident cells in
connective tissue

Ability to proliferate

Life span

Expression of FcRI

Major granule contents
Connective tissue

No

No


Yes


Yes

Weeks to months

High levels

Histamine, chondroitin sul-
fate, heparin, proteases
Bone marrow

Yes

Yes


No


No

Days

High levels

Histamine, chondroitin sul-
fate, protease

186
IgE (Reaginic Antibody)

IgE is usually present in serum in very small quantities [eg, 9 to 700 g/ml in
dogs and 0.1 to 0.4 g/ml in humans]. Most animals and humans mount signi-
ficant IgE responses only as a defense against helminth parasites.

T
H
2 cells. Synthesis of IgE by B cells is dependent on cytokines produced by
activated T
H
2 cells, such as IL-4, IL-5, and IL-13. IL-4 is required for isotype
switching to IgE.

The IL-4 response is inhibited by T
H
1 cytokines such as IFN- and IL-12.
IFN- can inhibit isotype switching to IgE because IFN- antagonizes the
actions of IL-4 [see Cytokines, page 24].

Heredity. The proclivity to produce IgE is influenced by the inheritance of
several genes. Abnormally high levels of IgE synthesis and associated atopy
run in families.

Atopic individuals or animals produce high levels of IgE in response to
certain antigens, whereas normal individuals generally synthesize other
immunoglobulin isotypes such as IgG and IgM, and only small amounts of
IgE.

History of antigen exposure. In general, repeated exposure to a particular
antigen is necessary to develop an atopic reaction to that antigen because
IgE isotype switching and binding to mast cells and basophils must occur
before an allergic reaction to an antigen can take place.

IgE mediates mast cell and basophil activation and type I hypersensitivity
by binding to Fc receptors on these cells.

IgE-Binding Fc Receptors

IgE mediates type I hypersensitivity by binding to a receptor on mast cells and
basophils specific for the Fc region of the heavy chain. Two FcRs have been
identified, designated as FcRI and FcRII.

FcRI. Mast cells and basophils constitutively express FcRI, which binds to IgE
with a high affinity. Up to 90,000 FcRI molecules have been shown to be pre-
sent on a human basophil.

Each FcRI molecule is composed of three separate transmembrane sub-
units; one chain that interacts with the heavy chain, one chain and a
dimer of identical disulfide-linked chains which contribute to signal trans-
duction and subsequent mast cell degranulation.
187
Although the serum concentration of
IgE is normally very low compared to
other Ig isotypes, the high affinity of the
FcRI for IgE overcomes this draw-
back. FcRI bound IgE functions as an
antigen receptor on the surface of mast
cells and basophils.

In serum, the half-life of IgE is only 2 to
3 days. Once IgE is bound to its re-
ceptor on mast cells and basophils, it is
more stable in the bound state and
has a half-life of 11 to 12 days. This
helps explain prolonged sensitization,
which may occur after an initial exposure to an allergen.

In addition to mast cells and basophils, FcRI is expressed on activated eosi-
nophils where it mediates IgE-directed ADCC.

Activation of Mast Cells and Basophils

The binding of IgE to FcRI does not result in mast cell or basophil activation.
Mast cells and basophils are activated by cross-linking of FcRI molecules by
binding of multivalent antigen to the fixed IgE molecules. Maximum activation
occurs when 10% or more of the FcRI receptors are aggregated.

In an individual or animal allergic to a particular antigen, a significant pro-
portion of the IgE bound to mast cells is specific for that antigen. Exposure
to the antigen will cross-link sufficient IgE molecules to trigger mast cell acti-
vation. On the other hand, in nonatopic individuals, the mast cell-associated
IgE is specific for many different antigens [all of which may have induced low
levels of IgE production]. Therefore, no single antigen will cross-link enough
of the IgE molecules to cause mast cell activation.

The bridging of IgE molecules by allergen results in methylation of various
plasma membrane phospholipids. This event results in an increase in the
fluidity of the plasma membrane and the formation of Ca
2+
channels.

Increase in intracellular Ca
2+
. Due to recruitment of Ca
2+
from intracellular
stores in the ER and increased uptake of extracellular Ca
2+
. The Ca
2+
influx
leads to two parallel and interdependent events:

Degranulation [occurs in seconds to minutes]. An increase in the activity
of the membrane-bound enzyme adenylate cyclase, leads to a transient
increase in cAMP. As a result, cAMP-dependent protein kinases, which
phosphorylate granule membrane proteins, are activated. Phosphory-
lation of the proteins increases the permeability of the granules to water
188
and Ca
2+
. The granules swell, facilitating their later fusion with the plasma
membrane. For degranulation to proceed, however, there must be a drop
in cAMP levels [also mediated by protein kinase]. Degranulated mast
cells do not die but remain viable and synthesize new granules.

The de novo synthesis and release of secondary mediators.

Eicosanoids [occurs in minutes]. The cytosolic enzyme phospholi-
pase A
2
hydrolyzes membrane phospholipids to release arachidonic
acid, which is then converted by enzyme cascades to leukotrienes
[lipoxygenase pathway] and prostaglandins [cyclooxygenase pathway].

Cytokines [occurs in hours]. A consequence of newly induced cyto-
kine gene transcription.

Both mast cells and basophils can be directly activated by a number of other
stimuli independent of allergen-mediated cross-linkage of FcRI. These in-
clude substances that may be present at inflammatory sites, such as chemo-
kines, the anaphylatoxins C3a, C4a, and C5a; various drugs such as syn-
thetic ACTH, morphine and codeine; cold, heat, exercise, etc.


























189
Mediators derived from Mast Cells and Basophils

The clinical manifestations of type I hypersensitivity reactions are related to the
biological effects of the soluble mediators released during mast cell or basophil
degranulation. The mediators may be classified as either primary or secondary.

Primary (Preformed) mediators. These are produced before degranulation
and are stored in the granules.

Secondary (Newly synthesized) mediators. These are either synthesized af-
ter target-cell activation or are released by the breakdown of membrane phos-
pholipids during the degranulation process.

Primary (Preformed) Mediators

Histamine (Biogenic amine)

Histamine is formed by the decarboxylation of the amino acid histidine. It is pre-
sent in very high concentrations in mast cells. Histamine acts by binding to hista-
mine receptors [eg, H
1
, H
2
, H
3
] on target cells. Different cell types express dif-
ferent histamine receptors.

Most of the biologic effects of histamine in type I hypersensitivity reactions are
mediated by the binding of histamine to H
1
receptors.

Causes increased vascular permeability by inducing retraction of endothelial
cells; results in edema and swelling of local tissue.

Stimulates endothelial cells to synthesize vascular smooth muscle cell relax-
ants, such as prostacyclin [PGI
2
] and nitric oxide, which cause vasodilation;
may lead to a drop in blood pressure.

Causes constriction of intestinal and bronchial smooth muscle, resulting in
increased peristaltic movements and bronchospasm.

Potent stimulator of exocrine secretions, eg, salivation, lacrimation, and in-
creased secretion by nasal and bronchial mucous glands. Stimulates nerve
endings, resulting in itching and pain in skin.

The actions of histamine are short-lived because histamine is rapidly removed
from the extracellular milieu by amine-specific transport systems.

This explains why antihistamines are ineffective in individuals with asthma.
Bronchoconstriction in asthma is more prolonged than the effects of hista-
mine, indicating that other mast cell-derived mediators are more important
than histamine in asthma.
190
Antihistamines. Act as H
1
and H
2
histamine receptor antagonists. Antihista-
mines are effective in the immediate-phase response only.

Serine proteases

Mast cell granules contain abundant amounts of neutral serine proteases, repre-
sented primarily by the proteases tryptase and chymase.

The proteases activate complement leading to the formation of the anaphyla-
toxins C3a and C5a; C3a and C5a initiate mast cell degranulation, resulting in
smooth muscle contraction and increased vascular permeability. The pro-
teases can also trigger the generation of bradykinin from serum kininogen.

Chymase. Stimulates bronchial mucus secretion.

Tryptase. Activates collagenase, thereby contributing to degradation of extra-
cellular matrix.

Proteoglycans (Heparin and chondroitin sulfate)

Heparin and chondroitin sulfate help prevent mast cell proteases from being self-
destructive by stabilizing and altering their biological activity, thereby limiting the
extent of tissue damage.

Heparin also functions extracellularly as a potent anticoagulant.

Eosinophil chemotactic factor (ECF-A)

Recruitment of eosinophils to the site of inflammation.

Secondary (Newly Synthesized) Mediators

Leukotrienes (Slow-reacting substances of anaphylaxis, SRS-A). Leukotrienes
are lipoxygenase metabolites of arachidonic acid. LTC
4
, LTD
4
, and LTE
4
are a
1000 times more potent than histamine.

They are associated with prolonged bronchoconstriction, increased vascular
permeability and mucus production. They are the major contributors to the
prolonged bronchospasm and mucus buildup in individuals with asthma.

LTB
4
. Chemotactic for neutrophils and eosinophils.

Prostaglandins. Prostaglandins are cyclooxygenase metabolites of arachidonic
acid. The most important is PGD
2
. PGD
2
causes prolonged bronchoconstriction,
vasodilation, and mucus production.

191
PGD
2
synthesis can be prevented by inhibitors of cyclooxygenase, such as
aspirin and nonsteroidal anti-inflammatory agents [NSAIDS]. These drugs
exacerbate asthmatic bronchoconstriction by shunting arachidonic acid into
the 5-lipoxygenase pathway, resulting in increased leukotriene production.

Platelet-activating factor (PAF)

In mast cells and basophils, PAF is synthesized from membrane phospholipids.
PAF causes bronchoconstriction and increased vascular permeability. It is also
associated with neutrophil and eosinophil chemotaxis and activation.

PAF is rapidly destroyed by a plasma enzyme called PAF hydrolase, which
limits its biologic functions.

Cytokines

Mast cells and basophils produce a variety of cytokines that promote late- phase
inflammatory events.

Late-phase reaction is observed within 4 to 6 hours following mast cell and
basophil degranulation and persists for 1 to 2 days. It is characterized by a
cellular infiltrate made up of eosinophils, neutrophils, macrophages, lympho-
cytes and other inflammatory cells, which secrete a variety of toxic products.

Much of the tissue damage that is associated with chronic allergic reac-
tions is thought to occur in this way. The late-phase reaction is best inhibi-
ted by corticosteroids.

Eosinophils

Eosinophils play a principal role during the late-phase reaction, making up about
30% of the cellular infiltrate. The recruitment of eosinophils as well as their acti-
vation, are mediated by mast cell and T
H
2 cytokines and chemoattractants, such
as IL-3, IL-5, GM-CSF, PAF, eotaxin, LTB
4,
and the complement protein C5a.

Tissue damage in allergic disease results from release of toxic granular con-
tents such as lysosomal hydrolases, major basic protein, etc

Activated eosinophils also secrete many proinflammatory mediators, including
PAF, LTC
4
, LTD
4
, and LTE
4
that contribute to vasodilation, bronchoconstric-
tion, and mucus production.

Clinical Manifestations of Type I Hypersensitivity

The clinical and pathologic features of the allergic reaction depends on a number
of variables:
192
The concentration of IgE antibody present, (2) the route of allergen entry [de-
termines the organs or tissues that are involved], and (3) the dose of the aller-
gen.

Systemic Anaphylaxis

If the allergen is given systemically, disseminated mast cell activation results in
widespread vasodilation leading to a severe drop in blood pressure, constriction
of the airways, and swelling of the epiglottis that often leads to suffocation, a
syndrome called anaphylactic shock. Anaphylactic shock is rapidly fatal but
can be controlled by immediate injection of epinephrine.

The allergen may be introduced by injection, an insect sting, or absorption
across an epithelial surface such as the skin or gut mucosa.

The signs and symptoms of anaphylaxis vary among species, reflecting the
differences in the distribution of mast cells and in the sensitivity of their
smooth muscles to the biologically active contents of their mast cell granules.

Anaphylactic Shock Organs in Humans and Domestic Animals
Species Shock Organ(s) Primary Clinical Signs Major Mediators
Humans

Ruminants


Swine

Horse

Cat


Dog


Chicken
Respiratory tract

Respiratory tract


Lungs and intestines

Lungs and intestines

Lungs and intestines


Liver (occlusion of hepa-
tic veins)

Respiratory tract
Dyspnea, urticaria

Coughing, dyspnea
(pulmonary edema)

Dyspnea, cyanosis

Dyspnea, diarrhea

Dyspnea, vomiting, diar-
rhea

Vomiting, collapse, diar-
rhea

Dyspnea, convulsions
Histamine, leukotrienes

Serotonin, leukotrienes,
kinins, dopamine

Histamine

Histamine

Histamine, leukotrienes


Histamine, leukotrienes,
prostaglandins

Histamine, leukotrienes

Milk Allergy

Milk allergy is sometimes observed in high-producing Jersey and Holstein cattle.
The allergic reaction is in response to the milk protein -casein. If milking is
delayed, the increased intramammary pressure sometimes forces milk protein,
including -casein, into the circulatory system where it is recognized as a for-
eign protein and stimulates an IgE response.

If there is another incidence of delayed milking, -casein may be forced into
the circulation again and precipitate an allergic response with signs ranging
from localized urticarial skin lesions to acute systemic anaphylaxis and death.
193
The condition can be readily treated by prompt milking.

Drug Hypersensitivity

Some non-protein drugs, such as penicillin and its derivatives sometimes elicit
strong IgE responses. Penicillin has a highly reactive -lactam ring [critical for its
antibiotic activity] that reacts with amino groups in self-proteins to form hapten-
carrier conjugates.

When penicillin is ingested or injected, it is degraded to several compounds,
the most important of which contains a penicilloyl group. The penicilloyl
group can bind to host proteins [eg, albumin], altering them sufficiently to form
hapten-carrier conjugates.

The conjugates are internalized by
hapten-specific B cells, processed and
presented to helper T cells. The
helper T cells in turn activate penicillin-
binding B cells to produce antibody to
the hapten. For unknown reasons, the
antibodies produced are usually IgE
antibodies.

Sensitized animals and individuals.

Oral administration: Severe diarrhea
Parenteral administration: Acute systemic anaphylaxis

Bee Stings

Bee sting in an atopic individual can result in acute anaphylactic shock. Sys-
temic reactions occur because venom diffuses into circulating blood, which then
carries it to sensitized mast cells located throughout the body. The allergens in
bee venom are the enzymes phospholipase A
2
and hyaluronidase.

One or more of the following symptoms may be observed:

Severe drop in blood pressure may result in dizziness, seizures, cardiac
arrhythmias and heart attacks.

Swelling of the lips and tongue and of the larynx. Constriction of the lower
airways.

Skin eruptions such as urticarial lesions [hives; urticaria Latin for stinging
nettle] accompanied by intense pruritus.

194
Localized Anaphylaxis

Introduction of allergen via the respiratory tract [most common route for allergen
entry], intestinal tract, or the skin results in a more localized release of mediators
from activated mast cells.

The inflammation stimulated by these mediators is typically biphasic with an
immediate reaction occurring within minutes after mast cell degranulation fol-
lowed by a late-phase reaction.

Localized anaphylactic reactions include allergic rhinitis [hay fever], asthma,
atopic dermatitis [eczema], food allergies, etc.

Allergic Inhalant Dermatitis

In dogs and cats, inhalant allergy presents primarily as an atopic dermatitis [AD]
characterized by intense pruritus. These allergies were originally associated with
changing seasons, however, they are now a year-round problem. It has been es-
timated that ~10% of the canine population and an unknown number of cats suf-
fer from inhalant allergies.

AD is one of the most common causes of chronic pruritus in dogs and may
affect dogs of any breed but occurs more commonly in certain breeds or fami-
lies of dogs such as Cairn terriers, West Highland white terriers, English and
Irish setters, Dalmatians, boxers, and Labrador retrievers.

Allergens. Allergens are mostly inhaled, but they may be absorbed through the
skin and possibly the GI tract.

The major allergens implicated include tree, weed, and grass pollens; molds;
house dust mites; animal dander; etc. Dogs with AD are frequently hyper-
sensitive to more than one allergen.

Clinical features. AD occurs primarily as a pruritic dermatitisface rubbing, ax-
illary pruritus, and foot licking [the so-called allergic triad].

Acute erythema and edema to chronic 2
o
changes including crusting, scaling,
hyperpigmentation, and pyoderma.

Otitis externa or conjunctivitis may be observed in some animals.

Diagnosis

History

Is the condition seasonal or nonseasonal?
195
What is the age of onset? Most animals develop allergies to inhalant aller-
gens at age 1 to 2 years [food allergiesup to 1 year of age].

Does the condition usually worsen when the animal is in the house, rather
than outdoors?

Identification of allergens by intradermal skin tests.

Detection of allergen-specific serum IgE using ELISA or RAST tests.

Treatment

Avoidance of the offending allergens.

Corticosteroids or immunotherapy [desensitization/hyposensitization therapy].

Chronic Obstructive Pulmonary Disease (COPD, Heaves)

COPD is usually observed in horses housed in dusty conditions. It is charac-
terized by an insidious onset of exercise intolerance, chronic coughing, nasal dis-
charge, and dyspnea caused by mucopurulent bronchiolitis.

Allergens implicated in the pathogenesis of COPD include grass, weed and
tree pollens, and molds and actinomycetes that grow in damp hay or straw.

Type III hypersensitivity is also thought to be involved in COPD.

Food Allergies in Dogs

Food allergy is defined as an immune-mediated hypersensitivity to proteins in
various foods. In dogs, there appears to be no breed predilection. Most food
allergies begin at <12 months of age.

Food intolerance. This is a general term that describes any adverse reaction
to food that does not have an immunologic basis, including food poisoning.

Foods involved. Protein-rich foods have been incriminated; they include beef
[single most common allergen], chicken, milk, eggs, corn, wheat meal, and soy.

Clinical features. Mast cell degranulation along the gut can increase the perme-
ability of mucous membranes, so that the allergen enters the bloodstream and
incites systemic reactions.

The most common clinical sign of food allergy is nonseasonal pruritus, which
is usually generalized. However, pruritus may be localized on the feet, ears,
or perianal area.
196
About 10-15% of the dogs show gastrointestinal problems.

Mild: Irregularity in the consistency of the feces.

Severe: Vomiting, cramps, and violent, sometimes hemorrhagic diarrhea.

In some cases, otitis externa may be the only presenting sign.

Lesions. The most common 1
o
lesions are papules and erythema. Common
2
o
lesions include pyotraumatic dermatitis [hot spots], hyperpigmentation,
and seborrhea.

Diagnosis. Intradermal skin tests and serologic testing have proved unreliable.

Intestinal biopsy. An intestinal biopsy with an eosinophilic inflammatory
infiltrate will substantiate the diagnosis of food allergy.

Elimination (Hypoallergenic) Diet

The diet should be balanced and nutritionally complete and must contain only
one protein to which the animal has never been exposed. The protein must be
highly digestible. Large, intact proteins elicit stronger immune responses than
small, digestible proteins.

Other than fresh water, nothing else should be fed to dogs during the elimi-
nation diet trial.

The diet is fed for a minimum of 8 to 12 weeks. If there is complete resolution
or marked improvement in clinical signs, a presumptive diagnosis of food
allergy can be made.

To confirm the diagnosis of food allergy, the dog is challenged with its regular
diet. Recurrence of clinical signs is usually noted between an hour and 14
days, the average being 3 days. Once a food allergy is confirmed, the elimi-
nation diet should be reinstituted until clinical signs resolve, which may take
up to 14 days.

At this stage, previously fed individual ingredients should be added to the eli-
mination diet for a period of up to 14 days. If pruritus increases, the indivi-
dual ingredient is considered positive for having a role in the food allergy.

Treatment

Elimination of the suspect food. Feed a hypoallergenic diet. It utilizes protein
sources that are not found in most pet foods.

197
Insect Hypersensitivity

Insect hypersensitivity affects mostly young to middle-aged animals. No breed or
sex predilection has been shown. In many insect hypersensitive reactions, the
severity of clinical signs is often disproportionate to the number of parasites
found on the animal.

Both types I and IV hypersensitivities play an important part in the hypersensi-
tive reactions.

Antigen source

Stinging insects often introduce antigens into the skin by injection. Biting in-
sects may introduce the antigens as they feed on the host. Inhalant exposure
should be considered for nonstinging and nonbiting insects.

The antigens are usually insect proteins, especially salivary components.

Clinical signs

The major clinical signs associated with insect hypersensitivities are urticaria
accompanied by intense pruritus and subsequent self-trauma.

Flea-Bite Hypersensitivity

Flea-bite hypersensitivity is probably the single most important allergic skin dis-
ease in dogs and cats. There is no breed or gender predisposition.

Both type I and IV hypersensitivities are thought to be involved in the patho-
genesis of flea-bite allergic dermatitis.

Clinical features. Dogs and cats appear to fall into one of two categories:

Flea-tolerant animals can harbor large numbers of fleas without showing cli-
nical signs. Dogs continually exposed to fleas fail to develop flea-bite hyper-
sensitivity or only develop mild clinical signs.

In flea-allergic animals or animals exposed to fleas on an intermittent basis
[as infrequently as every 14 days], clinical signs are more severe and they
tend to persist.

Lesions include wheals, papules, pruritus, excoriations, alopecia, lichenifica-
tion, crusting, and hyperpigmentation.

Diagnosis. Intradermal skin test. Most positive animals usually respond within a
few minutes.
198
Treatment. Total flea control.

Hyposensitization therapy is of questionable value.

Diagnosis of Type I Hypersensitivity

Intradermal Allergy Tests

Skin tests detect the presence of allergen-specific IgE bound to mast cells in the
dermis; it also assesses the complete mast cell response to a specific allergen.

An allergen solution is injected intradermally or placed on the skin. A light
scratch is made with a needle to allow the allergen to penetrate the skin.

If an animal or individual is allergic
to the allergen, vasoactive media-
tors are released by local mast
cells, resulting in vasodilation and
increased vascular permeability.
These changes are observed as
redness and local swelling [a
wheal]. Subsequent dilation of
blood vessels on the edge of the
swelling produces the appearance
of a red rim [the flare].

The response reaches maximal in-
tensity within 30 min and then
fades and disappears within a few
hours. In a few animals, a 2
o
in-
flammatory reaction [late-phase re-
action] sometimes occurs 4-6 h
after testing and may last for up to
24 h.

Advantages

A skin test is relatively inexpen-
sive to perform and allows screening of a large number of allergens simulta-
neously.

The test assesses tissue-fixed IgE, the IgE that actually mediates the skin
disease in question.

It is considered the best method for selecting allergens for hyposensitization
therapy.
199
Disadvantages

Animals must be sedated or properly restrained. The test cannot be perform-
ed on dogs with generalized dermatitis.

Animals should be withdrawn from antihistamines for 10 to 14 days and from
all steroid-containing medications for at least 4 weeks prior to testing, or false-
negative results may occur.

False-positive reactions and false-negative reactions may occur.

The dose of antigen in commercial skin testing solutions may be too low.

Some dogs strongly suspected to have atopy have had negative intrader-
mal test results; dogs may be up to 10 times less sensitive to intradermal
allergens, such as pollen, fungi, or dander than humans.

Serologic Allergy Testing

In vitro allergy tests measure serum concentrations of allergen-specific IgE. In
vitro tests commercially available include the radioallergosorbent test [RAST],
Western blotting, and the ELISA.

Advantages

Generally, patients do not need to be withdrawn from medication that would
interfere with traditional intradermal allergy testing.

Because in vitro tests require only a serum sample, they are readily available
to the clinician; the tests can be performed on animals with generalized der-
matitis.

Results are quantitative rather than based on subjective evaluation.

Disadvantages

They are subject to a high level of false-positive results [low specificity], which
may lead to inappropriate hyposensitization.

There may not be a direct correlation between antigen-specific IgE in tissue
and that in blood; circulating IgE levels may drop very rapidly in the absence
of allergen exposure.

Radioallergosorbent Test (RAST)

The allergen is bound to a solid-phase substrate, such as beads or paper disks.
200
The allergen-bound substrate is incubated with the patients serum. Serum
antibody that is specific for the allergen being tested will bind to the fixed
allergen.

Unbound antibody is washed away and the amount of specific IgE bound to
the solid-phase allergen is measured by adding
125
I-labeled rabbit anti-IgE.

After repeated washings, a gamma counter determines the radioactivity of the
sample. The number of counts per minute is proportional to the amount of
allergen-specific IgE that was present in the patients serum.

Therapy for Type I Hypersensitivity

The first priority is identification of the allergen and its avoidance. Therapeutic
approaches to atopic disorders include blocking the production or release of mast
cell mediators or reversing the action of mediators on target cells.

Alpha and Beta Adrenergic Receptors

Two major types of adrenergic receptors, alpha [] and beta [] receptors, have
been identified. The receptors are divided into
1
and
2
and
1
and
2
recep-
tors.

Some alpha functions are excitatory while others are inhibitory. Similarly, cer-
tain beta functions are excitatory and others are inhibitory. Hence, and
receptors are not necessarily associated with excitation or inhibition but rather
with the affinity of the drug or hormone for the receptors in the specific effect-
tor organ.

Type Response to Catecholamines Mechanism of Action

1
NE >E >ISO Ca
2+
Increase

2
NE >E >ISO AC Inhibited

1
ISO >E >NE AC Stimulated

2
ISO >E >NE AC Stimulated
NE, norepinephrine; E, epinephrine; ISO, isoproterenol (synthetic
catecholamine); AC, adenylate cyclase.
201
Effects of Stimulating and Adrenergic Receptors
System Stimulation of receptor Stimulation of receptor
Mast cells

Smooth muscle

Blood vessels
Enhances degranulation

Contraction

Vasoconstriction
Suppresses degranulation

Relaxation

Vasodilation

Mast Cell Degranulation

Stimulation of receptor. Results in inhibition of adenylate cyclase and de-
creased intracellular cAMP, enhancing mast cell degranulation. Drugs stimu-
lating receptors include norepinephrine.

Stimulation of receptor. Results in stimulation of adenylate cyclase and in-
creased intracellular cAMP, thus decreasing mast cell degranulation [although
cAMP rises transiently during mast cell activation, degranulation is prevented
if cAMP levels remain high]. Stimulators of receptors include epinephrine,
isoproterenol, and salbutanol.

Cromolyn sodium [Disodium cromoglycate]. Blocks Ca
2+
influx into mast
cell thus antagonizing IgE-induced release of mediators.

Theophylline prolongs high cAMP levels in mast cells by inhibiting phospho-
diesterase, which cleaves cAMP to 5-AMP.

Antihistamines

Most antihistamines bind to H
1
receptors on target cells but have the side effect
of causing sedation as a result of the antihistamine crossing the blood-brain bar-
rier.

The second generation antihistamines bind to H
2
receptors and are less prone
to cross the blood-brain barrier and therefore do not cause sedation.

Antihistamines are very useful in some immediate-phase type I reactions.
They are not of much value in patients with asthma because the prolonged
bronchospasm is mediated by leukotrienes.

Epinephrine (Adrenalin)

Stimulates cAMP production by binding to receptors on mast cells.

Promotes vasoconstriction in the skin and viscera, thereby reducing vascular
permeability and raising blood pressure [increase in cardiac output].

Promotes relaxation of bronchial smooth muscles, causing bronchodilation
202
Corticosteroids

Late-phase type I reactions and chronic allergic diseases associated with antigen
persistence are treated with topical or systemic corticosteroids, which are power-
ful anti-inflammatory drugs.

Decrease histamine levels by blocking conversion of histidine to histamine.

Stimulates mast cell production of cAMP.

Immunotherapy (Hyposensitization/Desensitization)

The objective of desensitization is to cause the production of IgG while simulta-
neously reducing the quantity of IgE present in the individual or animal.

Allergens are usually chosen based on a diagnosis of hypersensitivity, patient
history, and results from a skin or blood allergy test.

Small but increasing doses of aller-
gen are carefully injected SC over a
period of weeks or months. Such
repeated introduction of allergen by
the SC route appears to cause a
shift towards IgG production.

The IgG antibody is referred to as
blocking antibody because it com-
petes for the allergen, binds to it,
and forms a complex that is removed
by phagocytosis; as a result, the
allergen is not available to cross-link
the fixed IgE on the mast cell membrane and allergic signs decrease.

Allergy shots may also induce T cell-mediated suppression, possibly via a
shift to the T
H
1 subset and IFN- production. IFN- is an antagonist of IL-4
and it inhibits IgE isotype switching.

Desensitization is not a routinely successful procedure. In humans, it is ef-
fective in 65-75% of individuals suffering from inhalant allergies, and in a
reported 97% for insect venom.

In domestic animals, a high success rate has also been reported in dogs
with insect hypersensitivity. The success rate in other allergic conditions
in dogs varies between 50-85%.
203
TYPE II (CYTOTOXIC) HYPERSENSITIVITY

Type II hypersensitivity reactions involve antibody [IgG and/or IgM]-mediated ly-
sis of target cells. IgG or IgM reacts with antigens present on the surface of cells
or other tissue elements.

The antigens may be normal cell surface molecules that act like antigens [see
Pemphigus, page 231] or exogenous antigens, such as drug metabolites, vi-
ruses, or bacterial products that adhere to tissues or to the cell membrane.

Destruction of Target Cell or Tissue

Complement-mediated cytotoxicity.

Opsonization [C3b/IgG] and phagocytosis of target cell by macrophages loca-
ted in the spleen and liver.

Antibody-dependent cell-mediated cytotoxicity ([ADCC]; see Immunoglo-
bulins, page 115). ADCC is usually involved in the destruction of targets that
are too large to be phagocytosed, such as parasites and tumor cells.

Interference with Normal Cellular Functions

A variation of type II hypersensitivity involves those diseases mediated exclusive-
ly by antibodies, without involvement of complement or cytotoxic cells. The dis-
eases involve other mechanisms leading to functional alterations.

Antireceptor antibody diseases. In these diseases, antibodies are directed
against specific receptors, and the binding of the antibody either increases or
decreases receptor functions, eg, myasthenia gravis [see page 229].

TRANSFUSION REACTIONS

Red blood cells possess cell-surface antigens which are integral components of
the cell membrane. Most of these antigens are either glycoproteins or glyco-
lipids. RBC antigens are called blood group antigens. They vary in their anti-
genicity; some are more potent in stimulating an immune response than others.

RBC antigens affect blood transfusion and influence graft rejection [grafts in-
compatible in the major blood groups are rapidly rejected].

ABO Blood Group Antigens in Humans

Two antigens, type A and type B, occur on the surfaces of the RBCs in a large
proportion of the population. It is these antigens [called agglutinogens because
they often cause blood cell agglutination] that cause blood transfusion reactions.
204
Natural antibodies [called alloantibodies or isohemagglutinins] to the A and B
antigens are produced in response to microorganisms [eg, bacteria, pro-
tozoa], that have antigenic determinants very similar to blood group antigens.
These antibodies are usually [but not always] of the IgM isotype.

An individual with blood type
A, for example, recognizes B-
like epitopes on intestinal bac-
teria and produces alloanti-
bodies to the B-like epitopes.














Genotype
Blood group
phenotype
Antigens on RBCs
(agglutinogens)
Serum antibodies
(isohemagglutinins)
AA or AO
BB or BO
AB
OO
A (41%)
B (9%)
AB (3%)
O (47%)
A
B
A and B
None
Anti-B
Anti-A
None
Anti-A and B

Incompatible Transfusion

Transfusion of blood into an individual possessing alloantibodies to one of the
blood group antigens can result in an immediate transfusion reaction.

In the absence of natural isohemagglutinins, the transfused RBCs stimulate
an immune response in the recipient. The transfused cells then circulate for a
period of time before antibody production takes place and immune elimination
occurs within 5-21 days [delayed hemolytic transfusion reaction].

The clinical manifestations of transfusion reactions result from: (1) Massive
intravascular hemolysis of the transfused RBCs by IgM/IgG and complement,
(2) agglutination of erythrocytes, and (3) opsonization and clearance of ery-
throcytes by macrophages in the spleen and liver [extravascular hemolysis].

Fever, salivation, tachycardia, tremors, paresis, hemoglobinemia, hemo-
globinuria, hypotension, DIC, paresis, dyspnea, convulsions, etc.
A group O pig produces anti-A anti-
bodies to cross-reacting A epitope
of bacteria. Transfusion with A
blood triggers an immediate trans-
fusion reaction
205
Extravascular hemolysis: Hyperbilirubinemia and bilirubinuria.

Treatment. Immediate termination of the transfusion and maintenance of
urine flow with a diuretic since accumulation of hemoglobin within the kidney
may result in acute tubular necrosis. IV fluids to combat hypotension and
shock. Recovery follows elimination of all the transfused RBCs.

Prevention. Prior testing [crossmatching] of the recipients serum for alloanti-
bodies against donor erythrocytes.

Mix recipients serum with donor RBCs and incubate at 37
o
C for 30 min.

If donor RBCs are lysed or agglutinated by the recipients serum, then
transfusion should not be attempted.

The Rhesus (Rh) System

The Rh system [Rh antigens were first identified on the RBCs of rhesus mon-
keys] is a major cause of hemolytic disease of the newborn. There are 6 Rh
antigens: RhC or c, RhD or d, and RhE or e. Each is called an Rh factor:

RhD is the most important clinically due to its high antigenicity. It is also
widespread in the population, therefore any individual who has the RhD anti-
gen is said to be Rh positive [Rh
+
], whereas a person who does not have type
D antigen is said to be Rh negative [Rh
-
].

In Rh
-
individuals, the RhD gene locus is missing completely, however,
they do express the RhC or c and RhE or e antigens. About 85% of Cau-
casians are Rh
+
and 15% are Rh
-
; 95% of blacks are Rh
+
and 5% are Rh
-
.

Unlike the ABO system in which natural alloantibodies to blood group anti-
gens develop spontaneously, alloantibodies to Rh antigens develop only in
response to transfusion of blood or by a mother having a baby who has the
RhD antigen.

Transfusion of Rh
+
blood into an Rh
-
individual does not result in an imme-
diate transfusion reaction. However, in some individuals, anti-Rh anti-
bodies develop in sufficient quantities within 2-4 weeks to cause agglutina-
tion of the transfused cells that are still in circulation. The cells are phago-
cytosed and hemolysed by macrophages in the liver and spleen. Thus, a
delayed transfusion reaction occurs although it is usually mild.

On subsequent transfusion of Rh
+
blood into the same person, who is now
immunized against the Rh factor, the transfusion reaction is greatly en-
hanced and can be as severe as the transfusion reactions caused by type
A or type B blood.
206
Important Blood Groups of Domestic Animals
Species Blood Group Systems Features
Cattle



Sheep


Pig



Horse


Cat
A, B, C, F, J , L, M,
R, S, Z, T


A, B, C, D, M, R, X


A, B, C, D, E, F, G, H,
I, J , K, L, M, N, O, P


A, C, D, K, P, Q, U


AB
11 total; B and J are most important; J anti-
gen is a serum lipid that passively adsorbs
onto erythrocytes

7 total; B and R important; R like bovine J
group

16 total; A system most important and con-
trols the expression of two antigens, A and
O

7 total; Aa and Qa are most important in
neonatal isoerythrolysis [NI]

Only one system: A completely dominant
over B; up to 95% of cats are A positive

Horses. Blood groups with transfusion significance are Aa, and Qa, especially
neonatal isoerythrolysis [NI]. The incidence of Aa and Qa is breed-dependent.
Other blood groups can occasionally give NI reactions.

All horses lack a unique RBC antigen found on donkey RBCs, so they will
produce antibodies and NI when exposed to donkey blood [such as in mule
pregnancies].

Natural alloantibodies exist in horses, especially to Ca antigens, which cause
weak agglutination and hemolytic crossmatch reactions, however, the anti-
bodies to Ca do not appear to produce a significant hemolytic reaction in vivo.

Cats. One blood group system, the AB system. In this system, there are 3 blood
types, A, B, and AB [rare]. The frequency of feline A and B blood types varies
geographically and between breeds. Naturally-occurring alloantibodies have
been reported in cats. Type AB cats have no alloantibodies.

In all type B cats, the anti-A antibodies are strong hemolysins and hemagglu-
tinins [titer >1:32] and are responsible for serious transfusion reactions and
NI. In contrast, anti-B antibodies in type A cats are weaker hemagglutinins
and hemolysins [titer 1:2]; they shorten the half-life of transfused B cells in
type A cats, but have not been associated with NI. All cats should be typed or
crossmatched before their first blood transfusion.

The half-life of transfused RBCs in matched transfusions [ie, type A blood
to a type A cat or type B blood to a type B cat] is 29 to 30 days.

The half-life of type A RBCs in type B cats is ~ 1.3 hours; transfusion of
type B blood into type A cats produces milder clinical signs and the trans-
fused erythrocytes have a half-life of ~ 2.1 days.
207
Pigs. Natural anti-A antibodies may occur in A-negative pigs; transfusion of A-
positive blood into the pig may result in transient collapse and hemoglobinuria.

Cattle. J -negative cattle may possess natural anti-J antibodies.

Canine Blood Groups





















There are 8 major blood groups in the dog, DEA [dog erythrocyte antigen] 1 to 8.
DEA 1.1 and DEA 1.2 are the most antigenic. Dogs can be positive for either
DEA 1.1 or DEA 1.2 [not both] or be negative for both.

Acute transfusion reactions only occur in DEA 1.1 and DEA 1.2 negative
dogs. Since there are no naturally occurring antibodies to these blood group
antigens, a reaction will only be seen after prior exposure to DEA 1.1 and
DEA 1.2 positive blood. Thus, an initial crossmatch of a dog that has not
previously been transfused should be compatible.

It should be noted that a compatible crossmatch in a dog does not prevent
sensitization against donor cells within 1 to 2 weeks. Thus, a dog that
was given a compatible transfusion from a donor earlier may turn out to be
incompatible with the same donor 1-2 weeks later. Therefore, all dogs
should be crossmatched before a second blood transfusion.

The life span of compatible transfused RBCs in dogs is ~ 21 days. In an
acute hemolytic transfusion reaction, the life span of incompatible RBCs is
~a few minutes to 12 hours.
208
The naturally occurring antibodies in DEA 3, 5, and 7 negative dogs do not
induce severe hemolytic reactions, instead, incompatible blood is hemolysed
more rapidly [within 4 to 5 days] than compatible blood [ie, delayed hemolytic
reaction].

HEMOLYTIC DISEASE OF THE NEWBORN (HDNB)

HDNB may result from incompatible blood transfusions or as a result of leakage
of fetal red cells into the maternal circulation through the placenta during parturi-
tion. HDNB has been reported in humans and domestic animals.

Erythroblastosis Fetalis in Humans

Erythroblastosis fetalis [immune hydrops fetalis] is a disease of the fetus and
neonate characterized by the destruction of fetal or neonatal RBCs. In most
instances, the mother is Rh
-
and the father is Rh
+
.

During a first incompatible pregnancy [ie, Rh
+
fetus], some fetal RBCs can
cross the placenta and enter the mothers circulation, however, they are not
enough to sensitize her. During parturition, or after a miscarriage or abortion,
large amounts of fetal umbilical cord blood enter the mothers circulation and
are recognized by the maternal immune system, resulting in the production of
IgM antibodies and memory cells. The secreted IgM antibody clears the Rh
+

fetal RBCs from the mothers circulation.
209
In a 2
nd
incompatible pregnancy, activation of the memory cells by fetal RBCs
results in the formation of IgG anti-Rh antibodies. The antibodies cross the
placenta and destroy the fetal RBCs.

About 3% of second Rh
+
babies exhibit some signs of erythroblastosis fe-
talis; about 10% of the third babies exhibit the disease; and the incidence
rises progressively with subsequent pregnancies.

Clinical features. Mild to severe anemia and jaundice [bilirubinemia]. Perma-
nent brain damage results from the accumulation of the lipid soluble bilirubin in
neuronal cells and their subsequent destruction.

Diagnosis

Amniocentesis (15 to 20 weeks): Detects bile pigments and possibly anti-
bodies in the amniotic fluid.

Testing of maternal serum at intervals during pregnancy for antibodies to the
Rh antigen. A rise in the titer of these antibodies as pregnancy progresses
indicates that the mother has been exposed to Rh antigens and is producing
increasing amounts of antibody.

Detection of maternal IgG on the surface of fetal red blood cells using
Coombs test.

Treatment. Started during the last trimester.

Intrauterine exchange transfusion. The fetus is transfused with Rh
-
blood
every 10-21 days until parturition. In less severe cases, exchange transfusion
is not performed until after birth, mainly to remove bilirubin.

The neonate is also exposed to low levels of UV light which breaks down the
bilirubin and prevent any brain damage.

Immune prophylaxis. Administration of anti-Rh antibodies [IgG], eg, Rhogam
D, to the mother within 24-48 hours after delivery.

The antibodies prevent sensitization of the mother by binding to fetal RBCs
that enter the mothers circulation at the time of delivery and facilitate their
clearance. The maternal RBCs are Rh
-
and are unaffected by the anti-Rh
serum.

Neonatal Isoerythrolysis (NI) in Domestic Animals

NI is a special category of transfusion-like reaction which occurs when a female
of one blood type is mated to a male of another type. Neonates that inherit the
210
blood group of the sire develop hemolytic anemia when they ingest colostrum
containing antibodies against their erythrocytes.

NI is fairly common in foals; its been occasionally reported in dogs, cats,
piglets, and calves.

Clinical signs include weakness, anemia, hemoglobinuria, and death.

Dogs. This occurs in dams that have been previously sensitized to DEA
1.1 and 1.2.

Cats. It occurs following the mating of type B queens to type A or AB toms.
All kittens bearing the type A antigen will suffer a hemolytic anemia at birth
after ingestion of colostrum containing naturally occurring anti-type A anti-
bodies.

Neonatal Isoerythrolysis in Foals

NI in horses is most often associated with Aa negative or Qa negative mares.
The other blood groups may occasionally cause NI. In mules, about 8-10% of
foals may be affected; in thoroughbreds and standardbreds, 0.05-2% of foals
may be affected.

Because of the epitheliochorial type of placenta, it is unclear as to how fetal
RBCs get into the dams circulation during pregnancy. It is believed that dur-
ing parturition, the breakdown of placental blood vessels facilitates entry of
fetal RBCs into the mare.

The level of maternal sensitization is weak following a first pregnancy, thus
the problem is seen only in mares that have had several pregnancies or mul-
tiple blood transfusions.

Foals acquire maternal anti-
RBC antibodies following in-
gestion of colostrum, thus
foals are born healthy but
become sick several hours
after ingesting colostrum.

Horses can be screened for
NI by testing the mares se-
rum against the sires RBCs just before foaling.

Clinical signs. Weakness and depression, pale mucous membranes, icterus,
and hemoglobinuria.

211
Diagnosis. Clinical signs.

Coombs test.

Hemolysis of the foals RBCs on addition of mares serum and fresh normal
rabbit serum [source of complement components].

Treatment. Acute cases.

Exchange transfusion requiring at least 5 liters of blood.

Transfusion of washed erythrocytes from the mare. 3-4 liters of blood are
collected in sodium citrate and centrifuged; the plasma is discarded. The
RBCs are washed once in saline and transfused slowly into the foal. The
blood is given in divided doses about 6 hours apart.

Prevention. Strip mare of colostrum. Foal should not be allowed to suckle for
24-36 h. When suckling is allowed, the foal should only be allowed to take small
quantities at first.

Coombs Test (Antiglobulin Test)

The Coombs test is used to detect the presence of maternal IgG on the neo-
natess RBCs. If antibody is present, the RBCs can be agglutinated by anti-spe-
cie antibody; if no antibody is present on the RBCs, they are not agglutinated by
anti-specie antibody.

Drug Hypersensitivity

A number of drugs [eg, penicillin, sulfonamides, phenylbutazone, cephalosporin,
chloramphenicol, etc], can bind nonspecifically to proteins on the membranes of
RBCs, platelets, and leukocytes, forming a complex similar to hapten-carrier
conjugates.

In some individuals, the drug-protein complexes induce formation of anti-
bodies, which then bind to the adsorbed drug on the cell, inducing comple-
ment-mediated lysis and anemia, thrombocytopenia, neutropenia, etc.
212
TYPE III (IMMUNE COMPLEX) HYPERSENSITIVITY

The occurrence of diseases due to immune complexes [Ag-Ab complexes] was
first observed by the physician Clemens von Pirquet in 1911 when he observed
signs of serum sickness in diphtheria patients being treated with antiserum from
horses immunized with diphtheria toxoid.

Immune complex-mediated reactions are initiated by antigen-antibody com-
plexes that either are formed locally at the site of tissue damage or are depo-
sited there from the circulation.

The clinical signs of immune complex disorders are dependent on the location
of immune complex deposition and can include arthritis, vasculitis, glomerulo-
nephritis, or skin lesions.

Pathogenesis of Immune Complex Disorders

IgG or IgM combine with antigen to form immune complexes; these com-
plexes activate complement with the release of C3a and C5a [anaphylato-
xins]. C3a and C5a increase vascular permeability by directly activating ba-
sophils, causing the release of vasoactive amines [eg, histamine] and plate-
let-activating factor [PAF].

PAF is a potent platelet agonist that triggers the further release of vaso-
active mediators.

Neutrophils attracted to the
site of immune complex de-
position by C5a attempt to
phagocytose the immune
complex. Because the com-
plex is attached to the base-
ment membrane surface,
phagocytosis is impeded, al-
lowing lytic enzymes to be
released during the unsuc-
cessful attempts of the neu-
trophil to ingest the adhering
immune complex [frustrated
phagocytosis].

The membrane attack com-
plex [MAC] also formed as a
result of complement acti-
vation, may cause cell membrane damage, contributing to tissue injury.

213
Platelets. PAF induces platelet aggregation and microthrombus formation
and release of vasoactive amines.

Platelets also release tissue cell growth factors which may be responsible
for the cellular proliferation found in certain immune complex diseases,
such as lupus glomerulonephritis.

Nature of Antigens Causing Immune Complex Diseases

Virtually any antigen that induces a detectable antibody response can induce im-
mune complex hypersensitivity.

Self-antigens (autoimmunity)

The continued production of autoantibody to a self-antigen leads to pro-
longed immune complex formation and deposition in various tissues, eg, sys-
temic lupus erythematosus [SLE].

Repeatedly inhaled antigens

The antigens interact with antibodies in the respiratory tract and form immune
complexes that are deposited in bronchiolar and alveolar walls resulting in
hypersensitivity pneumonitis.

Farmers lung occurs in farmers chronically exposed to dust from moldy
hay containing the spores of Micropolyspora faeni; librarians lung results
from inhalation of antigens from old books; extrinsic allergic alveolitis of
cattle; and the lung lesion of COPD in horses.

Persistent infection (microbial antigens)

The combined effects of a low-grade persistent infection and fairly low level of
antibody production results in immune complex formation, deposition, and
subsequent tissue injury.

Localized Type III Reactions

Immune complexes are deposited locally within tissues. The prototype of local-
ized type III hypersensitivity is the Arthus reaction, described by Maurice Arthus
in 1903.

Arthus Reaction

It is induced by the injection of an antigen intradermally or subcutaneously into
an animal with high levels of circulating antibody specific for that antigen. The
antigen diffuses through the tissue and localizes in the walls of small arteries at
214
the injection site, where it forms immune complexes with the circulating antigen-
specific antibodies.

Microscopic examination of the tissue reveals neutrophils adhering to the vas-
cular endothelium and then migrating into the tissues at the site of immune
complex deposition.

The resulting inflammation manifests as edema and erythema and even-
tually, if the reaction is severe enough, hemorrhage and necrosis of the skin
at the site of antigen injection. The Arthus reaction reaches peak intensity in 4
to 8 h and then wanes and is usually markedly decreased by 24 to 48 h.

Generalized Type III Hypersensitivity

When large amounts of antigen enter the circulation and bind to antibody, cir-
culating immune complexes can form. The pathogenicity of the circulating im-
mune complexes is determined primarily by the ratio of antigen to antibody and
the class of antibody involved.

Size of circulating immune complexes

Complexes formed in antibody excess tend to be large and are rapidly re-
moved from the circulation by cells of the mononuclear phagocyte system.

Immune complexes formed in slight antigen excess are small to intermediate
in size and are the most pathogenic because they are not easily cleared by
the phagocytic cells and therefore circulate longer.

Immunoglobulin isotype

Since IgM and IgG are the only Igs that can activate complement via the clas-
sic pathway, they are the ones usually involved in immune complex reactions.

Charge of the immune complexes [anionic vs cationic]

Complexes that contain cationic Ags bind avidly to negatively charged com-
ponents of the basement membranes of blood vessels and kidney glomeruli.

Clearance of Circulating Immune Complexes

Removal of circulating immune complexes is determined by the functional inte-
grity of the mononuclear phagocyte system and the binding of complement pro-
teins, which enhance the clearance of the complexes.

Complement protein C3b binds to immune complexes and attach them to
complement receptors [CR1, CD35] on erythrocytes [primates] or platelets
215
[non-primates]. The coated RBCs or platelets circulate to the spleen and li-
ver where the immune complexes, released by Factor I, are phagocytized by
splenic macrophages and Kupffer cells [the RBCs and platelets are recycled].

In the absence of complement components, significant accumulation of
immune complexes occur in tissues.

Immune Complex Deposition

The sites of deposition of immune complexes are determined by the anatomic
and physiological character of the tissue rather than by an immunological rela-
tionship of the inducing antigen or antibody to the tissue.

Increase in vascular permeability

Immune complex deposition in blood vessels and tissues is preceded by a
local increase in vascular permeability.

Anatomic and hemodynamic processes

Immune complex deposition is more probable in locations that have a filtering
function, such as the glomeruli [in the glomerular capillaries the blood pres-
sure is about 4X that of most other capillaries], blood vessels, the joint where
plasma is ultrafiltered to form synovial fluid, and the choroid plexus.

Serum Sickness

This syndrome follows the injection of foreign serum [usually equine hyperim-
mune serum] into humans. Injections of hyperimmune sera were the primary
treatment for persons with diphtheria [Corynebacterium diphtheriae], tetanus, or
exposed to rabies virus, etc.

The person receiving the equine
hyperimmune serum develops
antibodies specific for the equine
proteins; these antibodies then
form circulating immune com-
plexes with the equine proteins
that can be deposited in joints,
lymph nodes, skin, kidneys, etc.

Symptoms of acute serum sick-
ness are usually observed within
8 to 10 days following injection
of antiserum and include fever,
malaise, arthritis, lymphadenopa-
216
thy, glomerulonephritis, urticarial and erythematous skin rashes, etc. In some
cases serum sickness can prove fatal.

The symptoms usually subside within 1 to 2 weeks, following removal of the
immune complexes and the remaining antigen [characterized by return of
complement levels to normal and free antibody in the serum].

Immune-Complex Glomerulonephritis

The function of the glomerulus as an arterial capillary filter, together with a fenes-
trated endothelial lining, makes the glomerulus a particularly susceptible site for
immune complex accumulation.

Immune complex-mediated glomerulonephritis is also known as mesangio-
proliferative glomerulonephritis [MPGN] since it is characterized by proli-
feration of glomerular cells, especially mesangial cells.

The hallmark of MPGN is its granular [lumpy-bumpy] appearance as detected
by fluorescent antibody test using tagged antibody specific for Ig or comple-
ment [differentiates MPGN
from autoimmune or idiopa-
thic injury to the glomerular
basement membrane, in
which a smooth linear pat-
tern of Ig deposition is ob-
served. The nature of MP-
GN varies with the size of
immune complexes formed.

Small complexes [slight
antigen excess]. The
complexes can penetrate
both the vascular endothelium and the basement membrane, ending up
on the subepithelial surface where they stimulate epithelial cell prolifera-
tion and thickening of the basement membrane [form the so-called wire
loop lesion or membranous glomerulopathy]

Large complexes [antibody excess]. They are unable to cross the base-
ment membrane and largely accumulate between the endothelium and the
basement membrane, where they stimulate endothelial cell swelling and
proliferation.

Glomerulonephritis is characterized by increased water consumption [poly-
dipsia] and increased frequency of urination [polyuria]; hematuria; proteinuria
[albumin is the major protein lost] in combination with an elevated blood urea
nitrogen, etc.
217
Immune Complex Diseases of Domestic Animals

Immune complex disease usually occurs in an animal that produces only mo-
derate amounts of antibody, resulting in the formation of large quantities of im-
mune complexes of the appropriate size for tissue deposition.

On the contrary, an animal producing very high or very low levels of antibody
fails to produce sufficient depositable immune complexes of the right size;
thus animals determine their own fate by how much antibody they produce.

Species Disease Major Lesions
Dog




Cat


Equine



Swine


Bovine
Infectious canine hepatitis
Systemic lupus erythematosus
Lyme disease
Dirofilaria immitis [Heartworm]

Feline leukemia
Feline infectious peritonitis

Equine infectious anemia
Strangles [S.equi ss. equi]
Equine viral arteritis

Erysipelothrix rhusiopathiae
Hog cholera

Mycobacterium johnei
Uveitis, glomerulonephritis
Glomerulonephritis, arthritis, dermatitis
Glomerulonephritis
Glomerulonephritis

Glomerulonephritis
Glomerulonephritis, peritonitis, uveitis

Glomerulonephritis
Purpura hemorrhagica
Arteritis

Arthritis
Glomerulonephritis

Enteritis

Detection of Immune Complexes

C1q solid-phase binding assay

C1q is linked to an inert solid
phase support, such as a poly-
styrene tube. Serum to be
tested is added and the com-
plexes bind to the solid phase
C1q via the array of Fc regions
presented to C1q.

After incubation, the sample is
washed out of the tube. The
amount of complex bound to
C1q is measured using a radio-
labelled antibody to IgG and the
radioactivity measured in a
gamma counter.
218
TYPE IV (DELAYED-TYPE, DTH) HYPERSENSITIVITY

DTH is a T cell-mediated hypersensitivity reaction occurring more than 24 hours
after contact with the antigen [delayed hypersensitivity]. The reaction is charac-
terized by an influx of nonspecific inflammatory cells, predominantly macro-
phages, into tissues.

A DTH response is not always detrimental; although in some cases the res-
ponse causes extensive tissue damage and is, in itself, pathologic, in many
other cases tissue damage is minimal, and the response plays an important
role in host defense against intracellular pathogens and contact antigens.

Antigens that stimulate DTH reactions are often infectious agents that escape
elimination by the immune system, survive, and multiply in macrophages, eg,
Mycobacterium tuberculosis and other Mycobacterial spp. Many other bacte-
ria, fungi, protozoa, and noninfectious agents [eg, chemicals] may also cause
type IV hypersensitivity reactions.

Type IV hypersensitivity differs from the other hypersensitivities due to lack of
antibody involvement; hence the reaction cannot be transferred from sensi-
tized to normal animals by serum but only by T lymphocytes.

A variety of DTH reactions have been described, including the tuberculin re-
action, granulomatous hypersensitivity, allergic contact dermatitis, and cuta-
neous basophil hypersensitivity.

The Tuberculin Reaction

The tuberculin reaction was first demonstrated by Robert Koch in 1890. In
attempting to develop a vaccine for tuberculosis, he discovered that a killed
tuberculin preparation was not effective in preventing tuberculosis in previously
unexposed individuals but could be used in a diagnostic test.

Individuals injected ID or SC with the tuberculin preparation developed fever
and a localized skin reaction [hardening and swelling] if they had been pre-
viously exposed to M. tuberculosis whether or not they were symptomatic.

The sensitization phase occurs within 1 to 2 weeks following primary contact with
the antigen. When Mycobacterium tuberculosis invades a host, the organisms
are ingested by macrophages, which present peptide epitopes in association with
MHC class II proteins to CD4
+
T cells, resulting in a cell-mediated response and
the generation of memory T
H
1 CD4
+
cells [also called T
DTH
cells].

ID injection of mycobacterial PPD [purified protein derivative], results in the
activation of memory T
H
1 cells by APCs [macrophages and Langerhans cells]
and the initiation of the delayed hypersensitivity reaction.

219
The activated T
DTH
cells secrete a variety of cytokines that result in vasodila-
tion, increased vascular permeability, and the recruitment and activation of
macrophages and other inflammatory cells.

Neutrophils appear early in the reaction, peaking by about 6-12 hours and
then declining in numbers. Blood monocytes and T cells [mainly CD4
+
T
cells and some CD8
+
T cells] arrive between 24 and 48 hours following
antigen exposure.

Cytokines. Several cytokines produced by T cells and macrophages play
crucial roles in DTH reactions.

IL-1, TNF-, TNF-. Act on nearby endothelial cells, enhancing expres-
sion of adhesion molecules.

IFN-. Potent activator of macrophages; enhanced expression of endothe-
lial cell adhesion molecules.

Macrophage-chemotactic factor (MCF). Recruitment of macrophages.

Migration-inhibition factor (MIF). Inhibits macrophage migration and there-
by preventing the macrophages from migrating beyond the site of a DTH
reaction.

Lesion. Tissue swelling and induration at the injection site result from ede-
ma, fibrin deposition, and infiltrating cells and reach a maximum in 24 to 72
hours. The lesion usually resolves within 5-7 days, but if there is persistence
of antigen in the tissues it may develop into a granulomatous reaction.

Tissue damage results from the release of hydrolytic enzymes and reac-
tive oxygen intermediates from macrophages.


220
Granulomatous Hypersensitivity

Granulomatous hypersensitivity is clinically the most important form of DTH,
causing many of the pathological effects in diseases which involve cell-mediated
immunity. Chronic granulomatous inflammation occurs with some frequency in
some bacterial, fungal, and protozoa infections.

Granulomas may arise from: (1) persistence within macrophages of intra-
cellular organisms or other particles that the macrophage is unable to kill, (2)
persistent immune complexes, and (3) chronic irritants in tissues, such as as-
bestos particles, silica, etc [such non-immunological granulomas may be dis-
tinguished by the absence of lymphocytes in the lesion].

The lesion consists of a pali-
sade of macrophages, epi-
thelioid cells, and multinucle-
ated giant cells that surround
the infectious agent

Macrophages, epithelioid
cells, and giant cells are,
in turn, surrounded by a
cuff of lymphocytes [most-
ly nonantigen-specific T
cells]. Collagenous cap-
sules may also develop a-
round some pathogens
due to proliferation of fi-
broblasts and increased collagen synthesis.

Extensive tissue damage is due to the release of lytic enzymes from
activated macrophages.

Allergic Contact Dermatitis (ACD)

ACD is a T cell-mediated [T
H
1 cells and T
C
cells] eczematous disease. It is cha-
racterized by a 48 to 72 hour delayed eczematous response to the epicutaneous
application of the antigen. ACD has been reported in humans, horses, dogs, and
rarely cats.

Sensitization phase. Sensitization usually takes ~10-14 days.

The sensitizing chemicals are haptens that form complexes with skin pro-
teins. The modified self-proteins are internalized by epidermal Langerhans
cells, which leave the epidermis and migrate as veiled cells, to regional lymph
nodes.
221
In the lymph nodes, they present processed hapten-protein complexes to-
gether with class II MHC molecules to CD4
+
T cells, producing a popula-
tion of memory CD4
+
T
H
1 cells [T
DTH
cells].

The sensitizing chemicals include some insectides, dermatological drugs [eg,
neomycin], formaldehyde, salts of metals such as cobalt and nickel, cosme-
tics and hair dyes, oils from poison ivy and poison oak, aniline dyes, chemi-
cals in latex gloves, etc.

Elicitation phase

A subsequent exposure to the hapten elicits activation of T
DTH
cells and cyto-
kine production. Within 48-72 hours after this secondary exposure, the cyto-
kines cause macrophages to accumulate at the site. Activation of the macro-
phages and the release of their lytic enzymes result in the eczematous reac-
tion.

CD8
+
T cells contribute to tissue damage mainly by cell-mediated cytotoxicity.

Some chemicals are lipid soluble and can therefore cross the cell mem-
brane and modify intracellular proteins. These modified proteins generate
modified peptides within the cytosol, which are presented on the cell sur-
face together with class I MHC molecules.

CD8
+
T cells can cause tissue damage either by killing the eliciting cell or
by secreting cytokines such as IFN-, a potent activator of macrophages.












Lesion. This is an eczematous reaction observed in the hairless areas, usually
ventral abdomen, scrotum, foot pads, nose, neck, and ears.

Acute form. Erythema [redness], edema, vesiculation, and pruritus. In ani-
mals, because of the intense pruritus, self-trauma, excoriation, ulceration and
2
o
pyoderma may mask the true nature of the lesion.

Chronic form. Hyperkeratosis, acanthosis, and dermal fibrosis.
222
Diagnosis. History [must be exhaustive] and distribution of lesions.

Patch testing

Suspected chemicals are used to impregnate gauze swabs, which are
then attached to the shaved skin with tape. After 48 to 72 hours the
dressing may be removed and the areas in contact with the swabs exa-
mined.

A positive reaction is indicated by local erythema and vesiculation.

Dogs and cats. A solution of the suspected allergen is applied to shaved
normal skin and the area examined daily for up to 5 days.

Treatment. Identification and elimination of allergen from the animals environ-
ment.

Corticosteroids and antibiotics to control 2
o
bacterial infections.

Poison Ivy and Poison Oak (leaves of three, let it be)

Prior exposure is necessary to sensitize the individual. A subsequent exposure
will result in ACD if the oil in the leaves [urushiol] remains in contact with the skin
for as little as 10 to 15 minutes. The resulting rash begins after a 12 to 48 hour
delay and persists for about 2-4 weeks or longer.

Oxidation of urushiol results in formation of intermediates, such as pentade-
cacatechol, a lipid soluble hapten.

Pentadecacatechol may cross cell membranes and bind to intracellular
proteins, yielding peptide-MHC class I conjugates that are expressed on
the cell membrane, activating CD8
+
T cells.

Pentadecacatechol may also bind extracellularly to skin proteins to form
hapten-protein complexes which are internalized in endosomes and pro-
cessed and presented in association with class II MHC proteins to sensi-
tized T
DTH
cells.

Lesions. Result from lysis of altered epidermal cells by CD8
+
T cells and lytic
enzymes released from activated macrophages. Characterized by redness,
papules, vesicles [blisters], etc.

Treatment. Wash your skin with rubbing alcohol [the resin is soluble in rub-
bing alcohol]; should lesions develop, coat the rash with Calamine lotion to re-
lieve the itch. Application of topical corticosteroids.

223
































Comparison of Atopic Dermatitis and Allergic Contact Dermatitis
Atopic Dermatitis Allergic Contact Dermatitis
Pathogenesis
Clinical signs
Distribution
Major allergens
Diagnosis
Pathology
Treatment
Type I hypersensitivity
Hyperemia, urticaria, pruritus
Face, nose, eyes, feet, perineum
Foods and pollen, fleas, etc
Intradermal testingimmediate
Eosinophilic infiltration, edema
Steroids, antihistamines, desensiti-
zation
Type IV hypersensitivity
Hyperemia, vesiculation, erythema
Hairless areas, eg, feet
Reactive chemicals
Patch testdelayed response
Mononuclear cell infiltration
Steroids

Poison oak dermatitis. Cyto-
kines such as IFN-, MCF,
and MIF released from sensi-
tized T
DTH
cells mediate the
reaction. Tissue damage re-
sults from lytic enzymes re-
leased from activated macro-
phages. MCF, macrophage-
chemotactic factor; MIF, mi-
gration-inhibition factor.
224
AUTOIMMUNITY

Since 1900, the central dogma of immunology has been that the immune system
does not normally react to self [self-tolerance]. Autoimmunity is a state in which
the natural unresponsiveness to self-antigen terminates, ie, antibodies or T cells
react with self components, thereby causing disease.

Tolerance. Failure of the adaptive immune system to respond to an antigen.
Tolerance is antigen specific, ie, does not result in general immunosuppression.

Tolerogens. Antigens that induce tolerance. However, the same substance can
be both an immunogen and a tolerogen, depending upon how it encounters host
immune cells.

Central tolerance. This is tolerance that is established in immature lymphocytes
developing in central lymphoid organs.

Central tolerance ensures that the repertoire of mature lymphocytes cannot
recognize self-antigens that are present in the central lymphoid organs.

Peripheral tolerance. This is tolerance that renders self-reactive mature lym-
phocytes in the peripheral tissues inactive or anergic.

Peripheral tolerance ensures immunologic unresponsiveness to self-antigens
that are expressed in peripheral tissues and not in the 1
o
lymphoid organs.

Acquired or induced tolerance. Immunologic unresponsiveness to an exoge-
nous antigen, eg, in utero viral infection leading to persistently infected fetuses,
etc.

Factors Favoring Induction of Tolerance

The structure and dose of the antigen. A very simple molecule induces tole-
rance more readily than a complex molecule. Complex molecules are readily
processed and presented by APCs, hence are highly immunogenic.

All antigens have an optimal immunogenic dose range. Very high and
very low doses of antigen can elicit tolerance. Moderate doses tend to be
immunogenic.

Persistence of antigen. Tolerance is maintained best if the antigen to which
the immune system is tolerant continues to be present.

Antigen persistence results in continuous engagement of TCRs of specific
T cells; without activating signals provided by APCs, the T cells become
anergic or undergo activation-induced apoptosis.
225
Portal of entry and location. Antigens administered SC or ID tend to be im-
munogenic; whereas the same antigens administered IV or orally tend to be
tolerogenic.

Oral tolerance. Lack of a humoral or cell-mediated immune response to
an oral antigen. It is mediated by T cells [anergy of antigen-specific T
cells] or the production of immunosuppressive cytokines such as TGF-.

This may be a means for preventing immune responses to food anti-
gens and to the normal flora that are present as commensals in the in-
testinal lumen.

Antigen sequestration. This is the compartmentation of antigens, such that
under normal circumstances, they do not encounter self-reactive lympho-
cytes.

However, if an antigen is sequestered within an organ or tissue, thus pre-
cluding contact with developing T cells in the thymus, self-tolerance to the
antigen will not be induced.

Self-Tolerance

Self-tolerance is the ability of an individuals immune system to distinguish be-
tween self and non-self antigens.

Burnet and Fenner. The existence of immune tolerance was postulated by
Burnet and Fenner in 1948 in the self-marker aspect of their clonal selection
theory on antibody formation.

They hypothesized that immune tolerance to self-antigens is subserved by a
mechanism that causes fetal immunocytes to be deleted by contact with their
specific autoantigen. Thus, loss of self-tolerance leading to autoimmunity
would occur by mutation in adult life, producing new clones of anti-self lym-
phocytes.

Evidence for the hypothesis was provided by chimeric blood groups in cattle.
When a cow has dizygotic [nonidentical] twin calves, vascular anastomosis
may occur resulting in an interchange of hematopoietic stem cells.

When the calves are born, each possesses a mixture of RBCs [some ori-
ginating from the other calf]. Despite being genetically different, the for-
eign cells persist indefinitely.

Thus, each calf is a chimera, tolerating the presence of foreign RBCs.
Cells from an unrelated calf would be rejected if administered after birth.

226

















Medawar, Billingham, and Brent produced an experimental tolerance by
inoculating the spleen cells from mice of one inbred strain [A] into the new-
borns of a second inbred strain [CBA]. When the CBA mice grew to maturity,
they would accept skin grafts from strain A mice but not from unrelated
strains.

This was further demonstrated when they succeeded in having a skin graft
from a black mouse grow on a white one. This experiment ably supported
Burnet and Fenners hypothesis.

Although both T cells and B cells participate in tolerance, it is T cell tolerance
that plays the primary role. Tolerance is more readily induced and lasts lon-
ger in T cells than in B cells.

T cells B cells

Induction 24 hours 10 days

Duration 100 days 50 days





T Cell Tolerance

Clonal deletion [central tolerance]. The main factors that determine whether or
not a particular self-antigen will induce negative selection of self-reactive pre-T
cells are (1) the concentration of that antigen in the thymus and (2) affinity of the
pre-T cell TCRs [/ or /] that recognize the antigen.
Fusion of the placentas of dizygotic
twin calves resulting in the develop-
ment of chimeric calves. Each calf is
tolerant to the cells of the other twin
and will accept a skin graft from its
twin but not from an unrelated calf.
227
Self-proteins are processed and presented in association with MHC mole-
cules on thymic APCs [see The Lymphoid System]. Those pre-T cells carry-
ing TCRs for self antigens and so potentially are able to mount autoimmune
responses damaging to the host are eliminated.

Clonal deletion affects both class I and class II MHC-restricted T cells and is
therefore significant in the induction of self-tolerance in both CD4
+
and CD8
+

lymphocyte populations.

Clonal anergy (peripheral tolerance). Because self-tolerance is crucial to sur-
vival, any T cells recognizing self-antigens that are not present in the thymus and
therefore avoid elimination, can be functionally inactivated after they leave the
thymus.

Full activation of naive T cells requires the
recognition of MHC-peptide complexes as
well as co-stimulators [mainly B7-1/B7-2].

However, since most tissue cells lack the
ability to supply the co-stimulatory signal,
these antigens do not elicit an immune res-
ponse. Instead, the T cells responding to
them are rendered anergic and will not res-
pond to the antigen even if it is later pre-
sented by competent APCs.















Anergic T cells fail to produce the growth factor IL-2 and to proliferate in res-
ponse to antigen. Once induced, the anergic state can last for several weeks.

Immune paralysis. Very high doses of antigen induce a form of clonal aner-
gy known as immune paralysis. The high doses of antigen bypass APCs and
reach T
H
cells directly, rendering them anergic.
228













B Cell Tolerance

Clonal deletion [central tolerance]. When an immature B cell [pre-B cell ex-
pressing IgM BCRs only] binds self-antigens in the bone marrow with high affi-
nity, the BCR transmits a signal to the immature cell that arrests its development
and triggers apoptosis.

Receptor editing [Central tolerance; see Receptor editing, page 92]. Self-reac-
tive immature B cells in the bone marrow may be induced to undergo secondary
rearrangement of the light chain variable region gene segments. If receptor ed-
iting is successful, it results in a BCR with a new specificity and reduced affinity
for self antigen.

Clonal anergy [peripheral tolerance]. Mature B cells that recognize self-anti-
gens in peripheral tissues in the absence of co-stimulation by specific helper T
cells may be rendered functionally unresponsive [functional deletion].


Autoimmunity

In certain circumstances, self-tolerance may be lost and immunologic reaction to
host antigens develops. The most important step in the production of autoim-
mune disease is the activation of self-reactive T
H
1 or T
H
2 cells, which can induce
either cell-mediated or antibody-mediated autoimmune responses, respectively.

Many autoimmune diseases exhibit a marked familial incidence, which sug-
gests a genetic predisposition to these disorders.

Autoimmune response. An individual makes auto-antibodies or activates
self-reactive T cells to a self-antigen.

Autoimmune disease. Pathologic condition arising from an autoimmune
response.
229
Mechanisms of Autoimmune Diseases

Release of sequestered antigens

Tissue damage caused by trauma, inflammation, viral or bacterial infection,
may disrupt cell or tissue barriers and release the sequestered antigens into
the circulation, providing an opportunity for auto-antibody formation.

Examples: (1) formation of antisperm antibodies in some vasectomized men,
(2) milk allergy in J ersey cows, (3) posttraumatic uveitis that can result from
rupture of the lens capsule releasing previously sequestered lens proteins, (4)
release of heart muscle antigens after myocardial infarcts, etc.

Molecular mimicry. Various viruses and bacteria possess antigenic determi-
nants that are identical or similar to normal host cell components. Such cross-
reacting epitopes trigger the activation of autoreactive T cells or B cells.

In porcine enzootic pneumonia, antibodies to Mycoplasma hyopneumoniae
cross-react with pig lung tissue.

In humans, antibodies produced in response to infections with some strepto-
coccal species cross-react with kidney, joint, and heart antigens to produce
rheumatic fever.

Altered antigen or neoantigen. Development of neoantigens or altered anti-
gens that were unavailable when fetal tolerance was induced.

Polyclonal B cell activation. Polyclonal activators are agents that are capable
of activating many clones of lymphocytes, regardless of their antigen specificity.

A number of viruses and bacteria such as Epstein-Barr virus [EBV] and gram-
negative bacteria, can induce the proliferation of numerous clones of B cells
that express IgM in the absence of T
H
cells. If autoreactive B cells are activa-
ted by this mechanism, autoantibodies can appear.

Organ-Specific Autoimmune Diseases

In organ-specific autoimmune diseases, the self-antigens and lesions are essen-
tially localized to a given organ. The target organ may be subject to direct cellu-
lar damage by humoral or cell-mediated mechanisms; alternatively, the function
of a target organ may be stimulated or blocked by auto-anti-bodies.

Myasthenia Gravis (Type II Hypersensitivity)

In myasthenia gravis, autoantibodies are produced to the acetylcholine receptors
on the motor end-plates of muscles. Binding of antibody to the receptors pre-
230
vents binding by acetylcholine and inhibition of muscle contraction. The anti-
bodies also induce complement-mediated degradation of the receptor.

Myasthenia gravis is characterized by progressive weakening of the skeletal
muscles. It is seen in humans, dogs, and rarely, goats and cats














Graves Disease (Type II Hypersensitivity)

In Graves disease, autoantibodies are produced to the TSH receptor on thyroid
epithelial cells. Binding of the autoanti-
bodies to the receptor mimics the nor-
mal action of TSH, stimulating the se-
cretion of excess thyroid hormone.

Unlike TSH, the autoantibodies are
not regulated, and hence they
overstimulate the thyroid gland, re-
sulting in hyperthyroidism. Graves
disease has been reported in hu-
mans, dogs, and cats





Autoimmune Hemolytic Anemia (Type II Hypersensitivity)

Auto-antibodies to self-antigens
on RBCs results in erythrocyte
destruction by macrophages in
the spleen or complement-me-
diated cytolysis.

231
Autoimmune hemolytic anemia has been reported in humans and in most do-
mestic animals.

Autoimmune Thrombocytopenia (Type II Hypersensitivity)

Platelets coated with autoantibody are either destroyed in the spleen or lysed by
the membrane attack complex of complement, resulting in thrombocytopenic pur-
pura [TP].

The most frequent clinical signs are hemorrhages of the skin and mucous
membranes. Other features include epistaxis, melena, and hematuria which
may cause severe anemia.

Autoimmune thrombo-
cytopenia has been re-
ported in most domes-
tic animals, especially
dogs.

Pemphigus (Type II Hypersensitivity)

Auto-antibodies are produced to cell surface proteins of dermal and mucosal ke-
ratinocytes or the epidermal basement membrane.

Binding of antibody to keratinocytes leads to
loss of cell-cell adhesion and the develop-
ment of bullae [blisters or vesicles; pemphi-
gus], whereas the anti-basement membrane
antibodies cause a loss of cell-extracellular
matrix adhesion [bullous pemphigoid].

The pemphigus complex has been reported
in humans, dogs, cats, and horses.

Equine Recurrent Uveitis (ERU)

Equine recurrent uveitis [periodic ophthalmia, moon blindness, immune-mediated
uveitis or iridocyclitis] is the leading cause of blindness in horses worldwide. It is
a devastating ocular inflammation that can result in blindness in otherwise
healthy, young horses.

Various etiologies have been proposed as the cause of equine uveitis, in-
cluding infection with the nematode Onchocerca cervicalis, however, it is of-
ten associated with systemic Leptospira interrogans infection, especially sero-
var pomona. Affected horses usually possess antibodies to L. interrogans.

232
It appears the disease may be due to an autoimmune attack on ocular tissues
as a result of cross-reactivity [molecular mimicry] with L. interrogans.

In acute cases horses show blepharospasm, pain, lacrimation, and photopho-
bia. The eye[s] is/are also cloudy.

Administration of anti-inflammatory or immunosuppressive drugs may reduce
the inflammation, however, each subsequent episode leads to increased loss
of vision.

Systemic Autoimmune Diseases

In systemic autoimmune diseases, chronic IgG antibody production directed at
self-antigens causes immune complex deposition in a number of organs and tis-
sues, such as the kidneys, joints, small blood vessels, and the skin.

Systemic Lupus Erythematosus (SLE)

SLE is an episodic multisystemic disease that is seen in humans, equine, canine,
and feline. In humans, the female:male ratio is10:1.

SLE is characterized by fever, weakness, arthritis, an erythematous rash, kid-
ney dysfunction, and pleurisy.

Affected animals and individuals produce multiple autoantibodies against di-
verse tissue antigens, such as DNA, histones, RBCs, platelets, and lympho-
cytes. The most dominant antibody is antinuclear antibody [ANA], which is
nonspecific. Antibodies to dsDNA are specific for SLE.

Interaction of the autoantibodies with their specific antigens produces various
symptoms. Autoantibody specific for RBCs and platelets can lead to comple-
ment-mediated lysis, resulting in hemolytic anemia and thrombocytopenia.

Systemic deposition of immune complexes of autoantibodies and various nu-
clear antigens results in vasculitis, glomerulonephritis, arthritis, etc.
233
IMMUNODEFICIENCY DISEASES

Immunodeficiency diseases occur when one or more components of the immune
system is defective. The deficiency may result from a primary congenital defect
or may be acquired from a secondary cause, such as viral infections.

Immunodeficiency diseases may result from congenital or acquired defects in
hematopoietic stem cells, T cells, B cells, phagocytic cells, and the comple-
ment system.

The Points in the Immune System where Developmental Blocks
may lead to Immunodeficiencies



















The basic clinical manifestations of immunodeficiency are frequent, prolong-
ed, severe infections, which are often caused by unusual infectious agents,
especially opportunistic pathogens.

The nature of the infection in a particular animal depends on the degree of
deficiency and the component of the immune system that is defective.

Congenital (Inherited/Primary) Immunodeficiencies

Most of the gene defects that cause inherited immunodeficiencies are recessive,
and several are caused by mutations of genes on the X chromosome. Recessive
defects cause disease only when both chromosomes are defective.

Because males have only one X chromosome, all males who inherit an X
chromosome carrying a defective gene will manifest disease, whereas female
carriers, having two X chromosomes, are perfectly healthy.
234
Phagocytic Deficiencies

Phagocytosis of microbes by phagocytic cells involves a series of processes that
include adherence of phagocytes to vascular endothelial cells, emigration across
the vascular endothelium, chemotaxis to the site of inflammation, attachment to
the microorganisms, phagocytosis, and subsequent killing and digestion.

A dysfunction in any one of these processes may severely limit the effect-
tiveness of the phagocytic defense system. The hallmarks of phagocytic defi-
ciencies are recurrent bacterial or fungal infections; the clinical manifestations
range from mild skin infections to severe systemic infections.

Extrinsic Factors

Deficiencies of opsonins secondary to deficiencies of antibody and comple-
ment.

Suppression of the total number of phagocytic cells by immunosuppressive
agents, eg, corticosteroids and radiation therapy.

Neutropenia due to destruction of neutrophils by autoantibodies, eg, systemic
lupus erythematosus.

Abnormal neutrophil chemotaxis secondary to complement deficiencies.

Intrinsic Factors

Leukocyte-Adhesion Deficiency (LAD)

LAD is an autosomal recessive defect that occurs in Irish setters, Holstein cattle,
and humans. Included among the deficiencies is the inability of neutrophils,
monocytes, and lymphocytes to adhere to vascular endothelial cells, thus pre-
venting extravasation of these
cells into the extravascular tis-
sue spaces.

Also impaired is the ability of
CTLs and NK cells to ad-
here to their target cells and
T
H
cell/B cell interaction.

The biochemical basis of
LAD is a deficiency in the
biosynthesis of the -chain
component [CD18] of the
2
-
integrin subfamily. The inte-
235
grin molecules affected by this defect include CD11a/CD18, CD11b/CD18
[CR3/Mac-1], and CD11c/CD18 [CR4].

Consequently, the neutrophilic response to infection is impaired because cir-
culating neutrophils cannot bind to the intercellular adhesion molecules ex-
pressed on the surface of activated endothelial cells and therefore are unable
to leave the bloodstream [neutrophilia].

Chdiak-Higashi Syndrome (CHS)

CHS is a potentially fatal autosomal recessive disorder characterized by recur-
rent infections by pyogenic bacteria, partial oculocutaneous albinism, and a mild
bleeding tendency. CHS has been reported in humans, Persian cats, foxes, cat-
tle [Hereford, Brangus, J apanese black], white tigers, etc.

CHS is due to mutations in the LYST [lysosomal trafficking regulator] gene.
The gene encodes a protein called lysosomal trafficking regulator. This regu-
lator protein plays a critical role in determining the size, structure, contents,
movement, and normal functioning of lysosomes and related structures found
in cells throughout the body.

The neutrophils, monocytes and eosinophils of af-
fected animals contain giant lysosomes. There is re-
duced phagolysosome formation leading to impaired
intracellular killing of microbes. Neutrophils and ma-
crophages also demonstrate decreased migration and
chemotactic responses.

In pigment cells [melanocytes], melanin is trapped within giant melanosomes
[related to lysosomes], resulting in partial oculocutaneous albinism.

Enlarged lysosomes in platelets results in abnormal platelet function, pre-
disposing affected animals to bruising and bleeding. Death from acute he-
morrhage may occur in these animals.

In the nervous system, abnormal lysosomes in nerve cells account for the
neurological problems that accompany CHS.

The secretion of granule proteins [granzymes/perforins] by CTLs and NK cells
is affected, resulting in impaired target cell killing. Affected animals may show
increased susceptibility to neoplasms.

Diagnosis. Examination of stained blood smears and demonstration of gross-
ly enlarged lysosomes in leukocytes.

Examination of hair shafts for enlarged melanosomes.
236
Complement Deficiencies

Inherited deficiencies have been reported for each of the complement compo-
nents, including the inhibitors and inactivators that regulate the activation cas-
cade, with the exception of Factor B.

Complement deficiencies result in recurrent infections, defective humoral
functions [some complement components regulate antigen-mediated signals
received by B cells], and persistence of immune complexes.

Physiologic depletion: Gram-negative sepsis and immune complex diseases.

Classical Pathway

Deficiencies in any of the early components of the classical pathway [C1q, C1r,
C1s, C4, and C2] manifest similar clinical presentations, notably a marked in-
crease in immune complex diseases, such as SLE, glomerulonephritis, and vas-
culitis and recurrent infections by pyogenic [pus-producing; encapsulated] bac-
teria, such as staphylococci.

These deficiencies highlight the important role of the early complement reac-
tions in generating C3b, which is critical for the clearance of immune com-
plexes and as an opsonin.

Alternative Pathway

Deficiencies in components of the alternative pathway, including properdin and
Factor D, result in increased susceptibility to infection with pyogenic bacteria but
not with immune complex disease.

C3 Deficiency

C3 deficiencies have the most severe consequences, reflecting the central role of
C3 in activation of C5 and the formation of the MAC.

The majority of patients with C3 deficiency have recurrent bacterial infections
and manifest immune complex diseases.

Terminal Pathway

Deficiencies in the terminal complement components, including C5, C6, C7, C8,
and C9, have also been reported. Terminal pathway deficiencies predispose to
infections with Gram-negative bacteria.

A deficiency in C9 results in no clinical signs, suggesting that in some cases
the entire MAC is not necessary for complement-mediated lysis to occur.
237
Complement Regulatory Proteins

Deficiencies in complement regulatory proteins are associated with abnormal
complement activation and a variety of related clinical abnormalities.

Humoral (B cell) Deficiencies

Any interruption in B cell maturation and differentiation from the level of the stem
cells in the bone marrow to the mature functioning plasma cells of peripheral
lymphoid organs will affect immunoglobulin synthesis.

Deficient humoral immunity usually results in increased susceptibility to infec-
tion by extracellular microbes, especially pyogenic bacteria [these bacteria
possess polysaccharide capsules that make them resistant to phagocytosis in
the absence of opsonins]; immunity to most viral and fungal infections may be
normal.

B cell immunodeficiencies are diagnosed by reduced levels of serum immu-
noglobulin, defective antibody responses to vaccination, and in some cases,
reduced numbers of B cells in the circulation or lymphoid tissues or absence
of plasma cells in tissues.

X-Linked Agammaglobulinemia

This disorder is caused by a defect in the maturation of pre-B cells to mature B
cells. Affected individuals and animals have low or undetectable serum immuno-
globulin levels.

Fluorescent antibody staining reveals a complete absence of recirculating
mature B cells and lack of cells in the B cell-dependent areas of the peri-
pheral lymphoid organs and tissues.

Selective Immunoglobulin Deficiencies

Selective Ig deficiencies involving a deficiency in a single immunoglobulin class
or subclass, have been observed in humans and in several animal species.

Selective IgA deficiency is the most common Ig deficiency in humans, affect-
ing about 1 in 700 individuals. The heavy chain genes and the expression
of membrane IgA are normal; the defect appears to be in the differentiation of
these cells into IgA-secreting plasma cells.

Many patients suffer from recurrent respiratory and gastrointestinal infec-
tions.


238
Cell-Mediated Immunodeficiencies

Abnormalities in T lymphocyte maturation and function lead to deficient cell-me-
diated immunity and increased incidence of infection with intracellular microbes,
such as viruses, protozoa, and fungi.

Due to the central role of T
H
cells in the immune system, a T cell deficiency
can also result in deficient antibody production.

T cell immunodeficiencies are diagnosed by reduced numbers of peripheral
blood T cells, ie, lymphopenia; deficient cutaneous delayed-type hypersensi-
tivity [DTH] reactions to specific antigens, eg, purified protein derivative [PPD]
of Mycobacteria spp; and low proliferative responses of blood lymphocytes to
T cell mitogens, such as Con A and PHA [see Mitogens, page105].

Severe Combined Immunodeficiency (SCID)

Immunodeficiencies that affect both T and B cells, with resultant loss in both T
and B lymphocyte functions, are called severe combined immunodeficiencies.

Animals or individuals with SCID have an extraordinary high incidence of
infectious disease and malignancy associated with an abbreviated life span.

X-Linked SCID

The X-chromosome inherited defect is due to mutations in the gene encoding the
common chain [
c
] shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15.
X-Linked SCID has been reported in humans and dogs.

The disease is characterized by impaired T cell maturation and NK cell de-
velopment [IL-15 is an important growth factor for the development of NK
cells].

T cell deficiency is a result of the inability of the lymphopoietic cytokine
IL-7, whose receptor uses the same
c
for signal

transduction, to stimulate
the growth of immature thymocytes. B cell numbers are normal, however,
B cell responses to most antigens are impaired.

Reticular Dysgenesis SCID

Reticular dysgenesis is a fatal congenital disease characterized by the absence
of T and B lymphocytes and myeloid cells, such as phagocytic cells of the mono-
cyte and granulocyte series.

The defect is suspected to be a failure of the lymphoid and myeloid stem cells
to differentiate during hematopoiesis.
239
Therapeutic Approaches for Congenital Immunodeficiencies

Passive immunization with pooled gamma globulin helps to minimize and
control infections.

Bone marrow transplantation.

Gene Therapy (Somatic Gene Therapy)

This is a procedure that aims to correct a genetic defect by the introduction of a
normal gene into bone marrow cells or other cell types.

The first gene therapy was performed in 1990 on two young girls with adeno-
sine deaminase [ADA]-deficiency SCID. ADA deficiency affects purine degra-
dation, resulting in an accumulation of nucleotide metabolites that are toxic to
T and B cells.

The procedure involved the use of a retrovirus in which key retroviral genes
had been replaced with the ADA gene.

The patients bone marrow cells were removed and infected in vitro with
the engineered retrovirus; the genetically altered bone marrow cells then
were reinfused back into the girls. Years later, researchers were success-
ful in introducing the ADA gene into self-renewing pluripotent stem cells.

Acquired or Secondary Immunodeficiencies

Acquired immunodeficiencies are a result of malnutrition, infection of cells of the
immune system, or exposure to drugs [eg, treatment with immunosuppressive
drugs] or environmental toxins.

Malnutrition

Malnutrition is the most common cause of immune deficiency diseases world-
wide. Malnutrition includes deficiencies and excesses or imbalances of individual
nutrients.

Virus-Induced Immune Deficiencies

Viruses may either multiply in and destroy primary and/or secondary lymphoid
organs or may cause lymphoid neoplasia.

Some Viruses that Destroy Lymphoid Tissues

Feline immunodeficiency virus [Feline lentivirus]
Human immunodeficiency virus [Human lentivirus]
240
Infectious bursal disease virus in chickens [Avibirnavirus]
Feline leukemia virus [Gammaretrovirus]
Feline panleukopenia virus [Feline parvovirus]
Canine distemper virus [Canine morbillivirus]
Bovine virus diarrhea virus [Bovine pestivirus]

Viruses that cause Lymphoid Neoplasia

Mareks disease virus [Gallid herpesvirus 2]
Feline leukemia virus [Gammaretrovirus]
Bovine leukemia virus [Deltaretrovirus]
Avian leukosis virus [Alpharetrovirus]

Immunodeficiencies of Domestic Animals

Equine

SCID. This is an autosomal recessive disorder that occurs in both male and fe-
male Arabian foals. Affected foals are clinically normal at birth and remain so
until1 to 3 months of age when colostral antibodies decline.

The foals are very susceptible to infectious agents that primarily infect the
respiratory or gastrointestinal tracts.

Equine adenovirus is the most significant pathogen of SCID foals;
Cryptosporidium parvum and Rhodococcus equi are also involved. Foals
infected with any of these organisms usually die by 5 months of age.

The biochemical basis of SCID in Arabian foals is a defect in the enzyme
DNA-dependent protein kinase [DNA-PK], which is required for receptor gene
rearrangements. Without functional DNA-PK, receptor gene rearrangements
in pre-B and pre-T cells are impaired, resulting in the absence of mature,
functional T and B cells.

Agammaglobulinemia. This has been reported in male horses [Thoroughbred,
Quarter Horse, and Standardbred] that lack mature B cells and plasma cells and
do not produce antibodies after exposure to antigen. Affected foals die within
17-18 months due to recurrent pyogenic bacterial infections.

Transient hypogammaglobulinemia. Between 2 to 3 months of age, some
foals experience a transient hypogammaglobulinemia due to a delayed onset of
immunoglobulin synthesis. Lymphocyte numbers remain normal.

Selective IgM deficiency in foals. Immune functions appear to be normal in all
respects except for very low levels of IgM.

241
Dogs

X-linked SCID. Has been reported in Bassett hounds and a Cardigan Welsh
corgi.

Selective IgA deficiency. Has been reported in German shepherds, beagles,
and shar pei.

Canine leukocyte-adhesion deficiency [Canine granulocytopathy syndrome].
Reported in Irish setters.

Increased susceptibility to bacterial and fungal infections. Although there is
evidence of severe persistent leukocytosis, there is impaired pus formation at
sites of bacterial infection. There is delayed wound healing in affected dogs.

Affected dogs may be kept alive with long-term bactericidal antibiotics.

Diagnosis. Mutation-specific DNA testing; demonstration of a lack of neutro-
phil adherence to glass or plastic; DNA PCR to detect altered gene, etc.

Bovine

Bovine leukocyte-adhesion deficiency (BLAD). BLAD is a defect inherited as
an autosomal recessive trait in Holstein-Friesian cattle.

It is characterized by persistent neutrophilia, recurrent infections associated
with poor growth performance, poor wound healing, and a deficiency or ab-
sence of pus in inflammatory sites despite a striking blood neutrophilia. Af-
fected calves usually die before 1 year of age.

Selective IgG2 deficiency has been reported in 1-2% of Red Danish cattle.

Cats

Hypotrichosis with thymic aplasia. This is an autosomal recessive disease
characterized by lack of a thymus, no body hair, and depletion of lymphocytes in
the T cell areas of the lymph nodes, spleen, and Peyers patches. The condition
has been reported in Birman kittens.
242
TRANSPLANTATION IMMUNOLOGY

Transplantation is the process of transferring cells, tissues, or organs, called a
graft, from one site to another. It has long been known that an animal or indivi-
dual will accept a graft of its own tissue, eg, skin grafts, but not that of another of
the same species except an identical twin.

Donor. The individual who provides the graft.

Recipient (host). The individual who receives the graft.

Terminology

Histocompatible. Refers to individuals whose major histocompatibility antigens
are identical. Grafts between such individuals are readily accepted.

Histoincompatible. Individuals that display significant antigenic differences in
their major histocompatibility antigens.

Autograft. Self-tissue transferred from one site to another within the same in-
dividual [esp. patients with severe burns]. It takes permanently.

Isograft. Tissue transferred from one individual to a genetically identical indivi-
dual. In humans an isograft can be performed between genetically identical [mo-
nozygotic] twins.

Allograft. Tissue transferred between genetically different members of the same
species. It is rejected by the homograft reaction.

Xenograft. Tissue transferred from one specie to another. Xenografts exhibit the
greatest genetic disparity and therefore induce the most vigorous graft rejection.

The Laws of Transplantation

The law states that the vigor and speed of rejection is related to the genetic dis-
parity between the donor and the recipient [ie, the degree of foreignness].

Donor Recipient Outcome
Mouse A
Mouse B
Mouse B
Mouse (A x B)F
1

Mouse A
Mouse A
Mouse (A x B)F
1

Mouse B


Transplantation Antigens

Rejection of grafts is antigenically specific and is determined primarily by alloge-
neic differences in the histocompatibility antigens.
243
In humans, the various cell membrane antigens [proteins] that determine his-
tocompatibility are encoded by >40 different loci; however, the major histo-
compatibility complex encoded proteins [MHC I and II] are responsible for the
most vigorous allograft rejection reactions [see Major Histocompatibility Com-
plex, page 65].

Minor histocompatibility antigens. These are peptides derived from poly-
morphic cellular proteins of the donor that are bound to MHC molecules. The
peptides are encoded by genes outside the major histocompatibility complex.

Minor histocompatibility antigens induce weak or slower [more gradual] re-
jection reactions. However, because the combined response to several
minor histocompatibility antigens can be quite vigorous, transplantation
even between MHC I and II identical individuals requires some degree of
immune suppression.

Specificity and Memory of the Allograft Reaction

Skin grafts have been used extensively to study the mechanisms of the allograft
reaction because of certain technical advantages: (1) they are easy to prepare,
(2) their rejection is readily detected, and (3) their median survival times provide
an estimate of the intensity of the hosts immune response.

First-Set Reaction

Following a skin graft, there is an initial vascularization and cell proliferation. The
vascularized transplant becomes infiltrated with lymphocytes, monocytes, and
other inflammatory cells; there is decreased vascularization of the transplanted
tissue by 6-9 days, visible necrosis by 10 days, and complete rejection by 14
days.

Second-Set Reaction

A second skin graft from the same donor to the same recipient is rejected
much more rapidly than the first graft, usually in 5-6 days.

The accelerated rejection is specific for a particular donor. If another donor,
antigenically different from the first, provides skin grafts to the same recipient,
first- and second-set reactions to successive grafts are again observed.

Lymphoid cells from a sensitized animal transferred to a first graft recipient
will accelerate rejection of the graft.

Thus the capacity to reject an allograft is specific and has a memory res-
ponse.
244
Pathology of Allograft Rejection

Rejection of a graft begins following vascularization of the graft which allows in-
flammatory cells to penetrate the graft.

There is an initial transient neutrophil accumulation around the blood vessels
at the base of the graft, followed by mononuclear cells, especially cytotoxic
lymphocytes, that cause progressive damage to the endothelial cells lining
small blood vessels.

Capillary endothelial cell destruction results in thrombosis, vascular stasis,
hemorrhage, and infarction of the transplanted organ or tissue.

Mechanisms Involved in Allograft Rejection

Graft rejection is mostly a result of cell-mediated [delayed hypersensitivity] res-
ponse to alloantigens expressed on cells of the graft. CD8
+
T cells [CTLs] and
macrophages [activated by CD4
+
T cells] mediate most rejection reactions.

Nude mice (athymic mice) which lack functional T cells do not reject foreign
skin grafts; indeed the mice will even accept xenografts.

Neonatally bursectomized chickens, which totally lack B cells, are still able to
reject allogeneic graft. Antibodies are most significant in hyperacute rejection
reactions.

The process of graft rejection is divided into two phases: (1) a sensitization
phase in which antigen-reactive lymphocytes of the recipient proliferate in res-
ponse to alloantigens on the graft and (2) an effector phase in which immune
destruction of the allograft takes place.

Sensitization Phase

T cells respond to allogeneic MHC molecules in two ways:
245
Direct alloantigen presentation. Organ transplants carry with them a popula-
tion of donor APCs called passenger leukocytes. Migration of the donor APCs
from the grafted organ to regional lymph nodes occurs via the lymph.

Because passenger cells express the allogeneic MHC antigens of the donor,
they are recognized as foreign by host T cells that possess the corresponding
TCRs. The resulting alloreactive effector T cells are then carried from the
lymph nodes to the grafted organ, which they attack.

If the organ is depleted of donor APCs by careful flushing with warm physiolo-
gical saline or by treatment with antibodies, rejection of the allograft occurs
only after a prolonged time period.








Indirect alloantigen presentation. Occurs when allogeneic MHC molecules
from graft cells are taken up and processed by recipient APCs, and peptide frag-
ments of the allogeneic MHC molecules containing polymorphic amino acid resi-
dues are bound and presented by recipient [self] MHC molecules.











Effector Phase

Allograft rejection reactions have various time courses depending upon the type
of tissue or organ grafted and the immune response involved.

Hyperacute Rejection

This type of rejection reaction may occur when the donor and recipient have not
been matched for ABO blood group antigens or when the recipient has pre-
formed alloantibodies to other donor antigens such as HLA antigens as a result
of multiple transfusions. Rejection occurs in minutes to hours.
246
As soon as the vascular supplies between the recipient and donor organs are
linked, the alloantibodies bind to endothelium and activate complement [ABO
antigens are present on endothelial cells in the graft]. Complement activation
leads to endothelial cell injury, platelet activation and coagulation.

These processes contribute to thrombosis and vascular occlusion, and the
organ suffers irreversible ischemic damage.

Acute Rejection

This type of reaction occurs 10 to 30 days after transplantation. It is character-
ized by necrosis of parenchymal cells. Mediated by T cells and macrophages.

Cytokines [eg, IL-2, IFN-] released by activated CD4
+
T cells activate CD8
+
T
cells and macrophages.

CD8
+
T cells bind to the graft antigens via specific receptors and release ef-
fector molecules, such as perforins, granzymes, which mediate cellular injury.

Macrophage activation by IFN-, results in increased lysosomal activity, pha-
gocytosis, respiratory burst, and the release of TNF-; these events result in
direct cytotoxicity of graft cells.

Chronic Rejection

A slow loss of tissue function occurs over a period of months or years after
acute rejection reactions have subsided. It is characterized by fibrosis with loss
of normal organ structures.

The mechanisms of chronic rejection include both humoral and cell-mediated
responses. The antigens that evoke chronic rejection may be weak antigens
of the HLA system or antigens in minor histocompatibility loci.

Graft-Versus-Host Disease (GVHD)

GVHD occurs when immunocompetent lymphoid cells [T cells] are transferred to
a histoincompatible recipient who is immunocompromised and therefore unable
to reject the allogeneic cells in the graft [ie, host-versus-graft reaction does not
take place].

The donor T cells mount a cell-mediated immune response against the allo-
antigens on the host cells and attempt to reject them. This usually occurs in
bone marrow transplantation performed as a treatment protocol in patients
with certain leukemias or other blood diseases such as aplastic anemia.


247
The activation and proliferation of the T cells and the subsequent production
of cytokines generate inflammatory reactions in the skin, gastrointestinal tract,
and liver.

If it is severe, GVHD can result in generalized skin rashes, gastrointestinal
hemorrhage, and liver failure [seen as jaundice].

Tissue Typing

ABO blood typing. It is essential that the ABO blood groups of the donor and
recipient be compatible. The A, B, and O antigens are expressed on donor
RBCs, epithelial cells, and endothelial cells.

HLA typing. HLA typing of potential donors and a recipient can be accom-
plished with a lymphocyte microcytotoxicity test. More than 200 specific anti-HLA
alloantibodies are available.







Donor 1 shares antigens 1, 4, and
7 with the recipient, whereas do-
nor 2 has no antigens in common
with the recipient.
248
Mixed leukocyte reaction (MLR; blastogenesis assay). This assay is a mi-
totic response of T cells to HLA. It uses DNA synthesis to detect the incom-
patibility of class II antigens. Over 60 antigens are encoded by HLA-D region,
and all can be detected by MLRs.

Cross-matching. The recipients serum may have preformed alloantibodies
against the donors HLA antigens as a result of multiple blood transfusions, an
earlier transplantation, or multiple pregnancies.

May be performed by complement-dependent lymphocytotoxicity using the
donors lymphocytes as targets for the recipients preformed antibodies.

Plasmapheresis. Levels of preformed antibodies can be reduced by plasma-
pheresis.

In this process, plasma is removed from a patients blood by continuous-
flow centrifugation. The RBCs are then resuspended in a suitable medium
such as saline, and returned to the patient.

Immunologically Privileged Sites

Immunologically privileged sites tolerate allografts without sensitization of the re-
cipient. They include the anterior chamber of the eye, the cornea, the thymus,
the testes, the uterus, and the brain.

These sites are characterized by an absence of lymphatic vessels and some-
times an absence of blood vessels. They fail to induce an immune response
because the alloantigens of the graft are not generally able to sensitize the
recipients lymphocytes, and the graft shows an increased likelihood of accep-
tance, even when HLA antigens are not matched.
249
IMMUNOMODULATION

Immunomodulation refers to the means by which the immune response can be
either enhanced or inhibited. An understanding of the cellular and molecular
mechanisms by which the immune system is regulated, has enabled immunolo-
gists to come up with agents that can influence the immune response.

IMMUNOPOTENTIATION

In several situations it may be desirable to enhance the immune response.
These include the potentiation of the normal immune response in order to en-
hance protection and the treatment of immunosuppressive conditions.

Vaccination. The antigen specificity and memory of the immune system have
been used clinically to enhance the immune response to infectious agents by
prior exposure to the microbe or its product[s].

Passive Immunization. This is the administration of immune cells or antibodies
that are already reactive with a specific pathogen.

Immunostimulants [Immunomodulators]. These are substances that induce a
short-term, nonantigen-specific enhancement of the immune response. Unlike
adjuvants, immunostimulants need not be administered together with antigen to
enhance the immune response.

Immunostimulants act primarily by activating macrophages or other antigen-
presenting cells and/or induction of cytokine synthesis. They have been used
prophylactically to control infectious agents or as an adjunct to antimicrobial
therapy. In chronic diseases, they may aid in the clearance of persistent
microbes.

Inactivated Propionebacterium acnes (EqStim; ImmunoRegulin). This is a
gram-positive bacterium that is part of the normal flora of the skin. P. acnes
stimulates macrophages to release various cytokines and interferons and en-
hances T cell and NK cell activity.

Dogs and cats. Treatment of certain tumors [eg, malignant melanoma],
chronic recurrent canine pyoderma, feline leukemia, etc.

Horses. Treatment of chronic infectious respiratory disease, papillomato-
sis [warts], sarcoid skin tumors, etc.

Mycobacteria and Mycobacterial Cell Wall Preparations (Equimune; Settle;
Regressin-V). Muramyl dipeptide [MDP; glycopeptide constituent of cell
wall peptidoglycan] derived from the fractionation of mycobacteria, activates
macrophages and also enhances antibody production and NK cell activity.
250
Horses. Treatment of respiratory infections, sarcoid skin tumors, endo-
metritis, etc.

Dogs and cats. Treatment of canine mammary tumors, lymphomas, and
sarcomas.

Acemannan (Carrisyn). Acemannan is a water-soluble, complex carbohy-
drate [mannan] obtained from the aloe vera plant. It stimulates release of
cytokines and promotes cell-mediated immune responses.

Cats: Treatment of feline leukemia-positive and feline immunodeficiency-
positive cats; and vaccine-induced fibrosarcomas [intralesional injection].

IMMUNOSUPPRESSION

These are measures that are used to reduce the immune response. Such
measures are appropriate in the following circumstances: allergic conditions,
autoimmune diseases, and prolongation of graft survival.

Immunosuppressive therapy is best directed at cells in the process of proli-
feration, therefore it is most effective just before or at the time of antigen
exposure.

NONSPECIFIC IMMUNOSUPPRESSIVE THERAPY

Nonspecific immunosuppressive therapy usually involves the inhibition of cell
division in general, thereby reducing the response of T and B cells to antigens.
There are a number of disadvantages associated with such approach:

[1] increased risk of infection in the recipient and;

[2] because any rapidly dividing non-immune cells [eg, epithelial cells of the gut
or bone marrow hematopoietic stem cells] are also affected, serious or even life-
threatening complications can occur.

Ionizing Radiation

Ionizing radiation damages DNA, resulting in a permanent mutation of a gene.
The injurious effects are greatest on cells that are under-going cell division. High
doses can effectively eliminate all lymphocytes, includ-ing bone marrow stem
cells.

Corticosteroids

Corticosteroids are produced by the adrenal cortex in response to the release of
corticotropin [adrenocorticotropic hormone] by the pituitary gland or to angioten-
251
sin. Response to corticosteroids varies among species; humans are much more
sensitive to the immunosuppressive effects of corticosteroids than domestic ani-
mals.

Corticosteroid induces cellular synthesis of proteins that mediate its effects.
The corticosteroids are lipophilic and thus can cross the cell membrane and
bind to receptors in the cytoplasm. The resulting corticosteroid-receptor com-
plexes are then transported to the nucleus where they bind to specific regula-
tory DNA sequences, either up-regulating or down-regulating transcription.

Steroid-induced proteins called annexins [lipocortin or lipomodulin] block the
activities of cell membrane phospholipase A
2
and thus prevent the produc-
tion of arachidonic acid and synthesis of the leukotrienes and prostaglandins.

Effects on leukocyte circulation and platelets: neutrophilia, monocytosis, lym-
phopenia, eosinopenia, and thrombocytopenia.

Anti-inflammatory activities.

Inhibits increases in vascular permeability and vasodilation, thus prevent-
ing edema formation and fibrin deposition.

Inhibits expression of adhesion molecules, resulting in decreased adher-
ence of leukocytes to vascular endothelium and reduced migration to in-
flamed tissue.

Depresses chemotaxis of both neutrophils and macrophages.










Stabilizes lysosomal membrane so that decreased levels of lysosomal
enzymes are released at the site of inflammation.

Corticosteroids inhibit capillary and fibroblast proliferation and enhance
collagen breakdown, resulting in delayed wound and fracture healing.

If corticosteroids are used in bacterial infections, bactericidal antibacterials
may be preferred to bacteriostatic agents because of corticosteroid-in-
duced dysfunction of phagocytes.
252
Effect on immune cells and functions.

Macrophages. Depresses microbicidal activity, antigen processing, and
cytokine production, eg, IL-1 and TNF.

Corticosteroids inhibit cytokine production by blocking transcription fac-
tors, thereby preventing the mRNA for these cytokines from being syn-
thesized.

Animals suffering from infectious diseases controlled primarily by CMI
[eg, systemic mycoses] should not receive corticosteroids [unless clini-
cal judgment deems them absolutely necessary and if appropriate anti-
fungal therapy is instituted].

Lymphocytes. Corticosteroid treatment causes a decrease in the num-
ber of circulating lymphocytes as the result either of steroid-induced lysis
of lymphocytes or of alterations in lymphocyte-circulation patterns.

Lympholysis [esp, immature cortical thymocytes]. Corticosteroids pro-
mote the release of cellular endonucleases, resulting in the induction of
lymphocyte apoptosis.

Depresses lymphocyte proliferation, the release or effect of various
lymphokines, such as IL-2, TNF, etc.

Autoimmune hemolytic anemia. Corticosteroids suppress erythrophagocy-
tosis and antibody binding.

Long-term corticosteroid therapy causes weakening of bone and connective
tissues and atrophy of the adrenal glands due to inhibition of the release of
adrenocorticotropic hormone [ACTH] from the pituitary gland.

Cytotoxic Drugs (Mitotic Inhibitors)

These drugs inhibit cell division in general, hence they serve to reduce multipli-
cation of antigen-sensitive cells upon encountering antigens.

Alkylating agents. These drugs insert into the DNA helix and become cross-
linked, thereby preventing their separation and thus inhibiting template formation.

Cyclophosphamide. It is toxic for resting and dividing cells, especially for ra-
pidly dividing immunocompetent cells; thus it is sometimes given at the time
of transplantation to block T cell proliferation.

It has been of some benefit in the treatment of lymphomas, sarcomas,
mammary adenocarcinoma and immune-mediated skin diseases.
253
DNA synthesis inhibitors. These drugs inhibit the synthesis of nucleic acids,
hence they affect only proliferating lymphocytes.

Azathioprine [Imuran]. Azathioprine acts on cells in the S
phase of the cell cycle to block synthesis of inosinic acid, a
precursor used for the synthesis of the purines adenylic and
guanylic acid. Both T cell and B cell proliferation is dimi-
nished in the presence of azathioprine.

Before the discovery of cyclosporin A, transplant patients were given a
combination of azathioprine and corticosteroids just before and after trans-
plantation to diminish T cell proliferation in response to the alloantigens of
the graft. With this therapy, the survival rate of unrelated donor grafts was
50-60% at 1 year.

It is used by many clinicians for the treatment of immune-mediated skin
diseases and lymphoplasmacytic enteritis in dogs.

Folic acid antagonists. These drugs act as folic acid antagonists to block pu-
rine biosynthesis.

Methotrexate. It is used to treat lymphoma, Sertoli cell tumor, osteosarcoma,
and transmissible venereal tumor.

SPECIFIC IMMUNOSUPPRESSIVE THERAPY

Specific immunosuppression involves selective elimination of T and B cells.

Specific T Cell Immunosuppression

Cyclosporin A [CsA]. CsA is the single most important immunosuppressive
agent in clinical transplantation, increasing the survival rate of heart, kidney, and
liver transplants for over 5 years [increases the survival rate of unrelated donor
grafts to ~80%]. It is a cyclic polypeptide derived from fungi.

CsA blocks activation of resting T cells by inhibiting the transcription of IL-2
and the high affinity IL-2 receptor [IL-2R], which are essential for activation.

CsA binds with high affinity to a cytoplasmic protein called cyclophilin
forming a complex that blocks the phosphatase activity of calcineurin, a
254
protein required in the activation of the genes for IL-2, IL-2R, and other
cytokines.

A second mechanism of CsA is that it induces the synthesis of the immuno-
suppressive cytokine TGF-.

The major side effects of CsA are renal toxicity and, to a lesser degree, he-
patic toxicity.

Cyclosporin is also used as an adjunct therapy for feline ulcerative gingivo-
stomatitis and conjunctivitis sicca.

Tacrolimus [FK506]. It is a macrolide antibiotic obtained from the filamentous
bacterium Streptomyces tsukabaensis. FK506 and its binding protein [called
FKBP] bind calcineurin and inhibit its activity.

FK506 is 10-100 times more potent than CsA and therefore can be adminis-
tered at lower doses than CsA. FK506 also produces renal toxicity, but it ap-
pears to provide a wider therapeutic window between effective immunosup-
pressive levels and those that cause toxicity. It can reduce transplant rejec-
tion by as much as 75-90%.

FK506 has been used most frequently in liver transplant recipients and in
cases of kidney allograft rejection that are not adequately controlled by CsA.

Anti-CD3 monoclonal antibody (OKT3). Depletes T cells by binding to CD3
and promoting phagocytosis or complement-mediated lysis.

Anti-IL-2R monoclonal antibody. Prevents T cell activation and proliferation by
blocking IL-2 binding to activated T cells and also depletes activated T cells that
express IL-2 receptors.

255
SEROLOGY

Serology is the study of antigen-antibody [Ag-Ab] interactions in vitro. The prin-
ciple of all antigen-antibody reactions lies in the specific combination of epitopes
on the antigen with the hypervariable regions or complementarity-determining re-
gions of the antibody molecule.

Serologic assays differ in their sensitivity, specificity, and speed; some are
strictly qualitative; others may be qualitative and/or quantitative.

Serologic Applications

Serologic assays can be used for the detection and measurement of anti-
bodies or antigens.

Disease diagnosis

Detection of antibody titer rise: 4-fold increase between acute and con-
valescent serum samples collected 2-4 weeks apart against an etiologic
agent. Acute samples should be frozen and submitted at the same time
as the convalescent sample to avoid differences due to laboratory techni-
ques.

The detection of circulating antigen in certain infections, eg, hepatitis B
surface antigen by serum of known antibody; the matching of red blood
cells against serum antibodies of a recipient.

Monitoring the level of the humoral [antibody] immune response.

Seroepidemiology. A population survey using a serologic test to screen for
exposure and immunity to an infectious disease.

Collection and Handling of Serologic Samples

Virtually all serologic tests require serum or plasma as the sample. The most
practical method of collection is the Vacutainer System [Becton-Dickinson, Ru-
therford, NJ ].

A red-top vacuum tube is used when serum is required and a lavender-top
tube is used when plasma is required.

Serum. This is the fluid portion of blood remaining after the blood cells and ma-
terials responsible for clotting are removed. When collecting serum, allow the
blood sample to clot for 20 to 30 minutes at room temperature and then cen-
trifuge for 10 minutes at a speed not to exceed 1500 rpm.

256
After centrifugation, a small pipette is used to aspirate the serum off the
packed erythrocytes. Place the aspirate into a transfer tube or other sealable
tube and label clearly. The serum may be submitted immediately or frozen or
refrigerated for later use.








Normal serum. Serum devoid of antibodies.
Antiserum. Serum containing antibodies.
Hyperimmune serum. Serum containing high concentrations of antibodies.

Plasma. This is the noncellular portion of blood. It is obtained by collecting
blood in an anticoagulant, followed by centrifugation.









Terminology

Agglutinin. Antibody that aggregates or clumps particulate antigen.

Antiglobulin (antispecie antibody). Antibody made against an immunoglobulin,
usually by injecting immunoglobulin into an animal of another species.

Antitoxin. Antibody that neutralizes a toxin.

Lysin. Antibody that causes lysis of cell membrane.

Neutralizing antibody. Antibody that renders the microbe [commonly viruses]
noninfectious.

Opsonin. Antibody that combines with surface components of microbial and
other particles so that they are more readily engulfed by phagocytic cells.

Precipitin. Antibody that forms precipitates with soluble antigen.

257
Seroconversion. The appearance of antibody in the blood in response to infec-
tion, disease, or immunization, ie, seronegative seropositive.

Seronegative: Animals with no detectable antibodies.

Seropositive. Animals with detectable antibodies. However, classification of
an animal as seropositive is usually based on the titer being above a certain
threshold level [cut-off point]. For example, animals with titers >1:4 may be
classified as positive, whereas animals with titers 1:4 may be classified as
negative.

Serotype. The type of a microorganism as determined by its distinct antigenic
properties, eg, E. coli O157:H7.












Subtype. Variation within a serotype, eg, Canine parvovirus 2a and 2b.

Serogroup. A group of bacteria containing a common antigen or a group of viral
species that are closely related antigenically.

Sensitivity. The minimum concentration of a substance that can be reliably
measured by a serologic test. Measurement of false-negative.

Relative Sensitivity of Tests Measuring Antibody
Test ~Detectable Amount (g/ml)
ELISA
Radioimmunoassay (RIA)
Precipitation reaction in fluids
Double diffusion in agar gel
Complement fixation
Bacterial agglutination
Passive agglutination
Hemagglutination inhibition
Virus neutralization
Antitoxin neutralization
0.0005
0.00005
20.0
3-20
0.05
0.05
0.001-0.01
0.005
0.00005
0.06

Specificity. The specificity of an antibody refers to its capacity to discriminate
between ligands of similar structure by combining with them at different extents.
Surface antigens of a typical
bacterial cell
258
The greater the differences in affinity for two closely related structures, the more
specific the antibody.

Although Ag-Ab reactions are highly specific, in some cases antibody elicited
by one antigen can cross-react with an unrelated antigen. Such cross-reacti-
vity occurs if two different antigens share an identical epitope or if antibodies
specific for one epitope also binds to an unrelated epitope possessing similar
chemical properties. Measurement of false-positive.

Titer. The reciprocal of the highest dilution [lowest concentration] of serum [anti-
body] that produces a test reaction.

Therefore, if the highest dilution that produces a test reaction is 1 in16, then
the titer is 1/16. This means that the undiluted serum contains 16 times the
antibody for the reaction.

Two-Fold Serial Dilution of Serum (Antibodies)

In certain situations it is desirable to determine the concentration [titer] of anti-
body present in a serum sample, eg, following vaccination. Titration is accom-
plished by serially diluting the serum in a series of decreasing concentrations in a
test tube.

Antibody is detected using a suitable fixed amount of antigen.

259
PRIMARY BINDING ASSAYS

Antigens and specific antibodies combine reversibly to form immune complexes.
In general, primary binding tests are performed by allowing the reactants to com-
bine and then measuring the amount of immune complex formed. It is usual to
use fluorescent dye or enzyme-labeling in order to identify one of the reactants.

Tests Involving Enzyme Labeling

The most important of these techniques are the enzyme-linked immunosorbent
assays [ELISAs]. In these tests, an enzyme covalently conjugated to an antibody
reacts with a colorless substrate [chromogen] to generate a colored reaction pro-
duct. ELISA assays possess both high specificity and high sensitivity.

Enzymes employed in ELISA tests include horseradish peroxidase, and al-
kaline phosphatase.

Direct ELISA (Sandwich ELISA)

The sandwich ELISA can be used to detect or quantitate an antigen. Monoclonal
antibody is bound to the walls of wells in a microtiter plate, to a membrane, or to
a plastic wand.

Add sample [that may contain antigen] to the antibody-coated wells; incubate
for 1 hour at room temperature. If antigen is present in the sample it will be
trapped by the antigen-binding sites on the fixed antibody.

After washing unbound materials
away, a secondary [labeled] anti-
body containing a conjugated en-
zyme [horseradish peroxidase] is
added; incubate for 1 hour at room
temperature. The 2
o
antibody is al-
so specific for the antigen so it will
bind to any remaining exposed an-
tigenic determinants.

Following a wash the enzyme acti-
vity of the bound material in each
microtiter well is determined by ad-
ding the substrate of the enzyme
[hydrogen peroxide] and a chromo-
gen [in the presence of oxygen it
changes from colorless to a colored compound].

260
The color formed can be read in a spectrophoto-
meter and is directly proportional to the amount of
antigen originally present.



Canine Parvovirus Antigen Test Kit (Snap Test)





















Indirect ELISA

An indirect ELISA is used to detect or quantitate antibody. Serum or some other
sample containing 1
o
antibody is added to an antigen-coated microtiter well and
allowed to react with the bound anti-
gen.

After any free primary antibody is
washed away, the presence of an-
tibody bound to the antigen is de-
tected by adding an enzyme-conju-
gated 2
o
anti-isotype antibody [anti-
species antibody], which binds to
the 1
o
antibody.

Any free 2
o
antibody is then
washed away, and a substrate and
chromogen mixture is added.
261
The intensity of the color is proportional to the amount
of enzyme-linked 2
o
antibody that is bound, which in
turn is proportional to the amount of 1
o
antibody pre-
sent in the sample under test.


Immunofluorescence

The technique of immunofluorescence was introduced in 1941 by Coons, who
employed -anthracine, a blue fluorescing compound, coupled to pneumococcus
antiserum to detect bacterial antigens in tissue sections.

Fluorochromes are chemical substances that are capable of absorbing a
short wavelength of light and instantaneously emitting a longer wavelength
light. The emitted light is generally viewed with a fluorescence microscope,
which is equipped with a UV light source and excitation filters.

The most commonly used fluorescent dyes are fluorescein isothiocyanate
[FITC] and rhodamine. Both dyes can be conjugated to the Fc region of an
antibody molecule without affecting the specificity of the antibody.

FITC absorbs blue light [490 nm] and emits an intense yellow-green fluo-
rescence [517 nm].

Rhodamine absorbs in the yellow-green range [515 nm] and emits a deep
red fluorescence [546 nm].

Fluorochrome-labeled antibodies may be employed in various techniques, the
most important of which are the direct and indirect fluorescent antibody tests.

Direct Fluorescent Antibody Test (FAT)

Direct FAT is used to detect the presence of antigen, eg, bacterial cultures and
viral or other antigens, in a smear or tissue.

The antibody directed against the antigen
is labeled with FITC.

A tissue section or smear containing the
organism is fixed with acetone and incu-
bated with labeled antibody, and then
washed to remove unbound antibody.

When examined with a fluorescence mi-
croscope, the antigenic particles bound to
the labeled antibodies are seen to fluo-
262
resce.

Indirect Fluorescent Antibody Test (IFAT)

The IFA test may be used for the detection and quantitation of antibodies in
serum or for the demonstration and identification of antigens in tissues or cell
cultures. Three reactants are needed:

Antigen A, unlabeled1
o
antibody A, and a fluorochrome-labeled anti-isotype 2
o
antibody.

The IFA test has two advantages over direct staining:

The 1
o
antibody does not need to be conjugated with label; this eliminates
the need to purify and individually conjugate each serum sample.

Indirect methods increase the sensitivity of staining because multiple fluo-
rochrome anti-isotype antibodies can bind to each 1
o
antibody molecule.

Detection of Bacterial Surface Antigens Using IFA Test

Testing for Antibody

Known antigen is employed as a tissue smear, section, or cell culture on a slide
or coverslip. This is incubated in a serum suspected of containing antibodies to
that antigen, and the serum is then washed off, leaving only specific antibodies
bound to the antigen.

The bound antibodies may be visualized by incubating the smear in FITC-
labeled antiglobulin serum. When the slide is washed [to remove unbound
antiglobulin] and examined, fluorescence indicates that antibody was present
in the test serum.
274
IDENTIFICATION OF PROTEINS

A number of techniques are available that can be used to identify and character-
ize specific proteins in a complex mixture of proteins. One such technique,
Western blotting, is widely used to determine the presence and size of a protein
in a biologic sample.

Southern is the last name of the scientist who first blotted
DNA from a separating gel to a membrane, a technique
since called Southern blotting. By analogy, Northern
blotting was applied to the technique of transferring RNA
from a gel to a membrane, and Western blotting was
applied to protein transfer.

Western Blotting

In Western blotting a protein mixture is electrophoretically separated on a poly-
acrylamide slab gel in the presence of sodium dodecyl sulfate [SDS], a dissocia-
ting agent. The final positions of different proteins in the gel are a function of
their molecular size.



The protein bands are transferred from the separating SDS-PAGE to a sup-
port membrane [eg, nitrocellulose membrane] by capillary action [blotting] or
by electrophoresis, such that the membrane acquires a replica of the array of
separated proteins present in the gel. SDS is displaced from the protein
during the transfer process, and native antigenic determinants are often re-
gained as the protein refolds.

The individual protein bands are identified by flooding the nitrocellulose mem-
brane with radiolabeled polyclonal or monoclonal antibody specific for the pro-
tein of interest.

275
The Ag-Ab complexes that form are visualized by autoradiography. Antibody
probes labeled with enzymes that generate chemiluminescent signals and
leave images on photographic film have been used.

Clinical Uses

The Western blot is used to determine whether a positive result in a screening
immunologic test is a true-positive or a false-positive result. For example the test
is used to confirm a positive ELISA test for HIV infection or Lyme disease; to
detect feline immunodeficiency virus antibodies, antibodies to Sarcocystis neu-
rona [equine protozoal myeloencephalitis, EPM], etc.

The HIV virus is dissociated into its constituent proteins by treatment with
SDS and the proteins separated by SDS-PAGE.

The separated proteins are transferred to a nitrocellulose sheet and reacted
with the serum from the patient. Anti-HIV antibodies in the serum bind to the
various HIV proteins [primarily gp41 and p24] and are detected using horse-
radish peroxidase-linked anti-human immunoglobulin, which produces a visi-
ble color change when the enzyme substrate is added.


263
SECONDARY BINDING ASSAYS

Secondary binding assays are two-stage processes: (1) the first stage is the in-
teraction between antigen and antibody, and (2) the second stage is determined
by the physical state of the antigen.

If antibodies combine with soluble antigens in solution under appropriate con-
ditions, the immune complexes precipitate. If the antigens are particulate, eg,
bacteria, erythrocytes, then they agglutinate.

Precipitation Reactions

The interaction between an antibody and a soluble antigen in aqueous solution
forms a lattice that eventually develops into a visible precipitate. Formation of an
Ag-Ab lattice depends on the valency of both the antibody and antigen:

The antibody must be bivalent; a precipitate will not form with monovalent
Fab fragments.

The antigen must either be bivalent, ie, it must have two copies of the same
epitope, or polyvalent, ie, have different epitopes that react with different an-
tibodies present in polyclonal antisera.

Precipitation is a consequence of the growth of Ag-Ab aggregates in such a
way that each one Ag molecule is linked to more than one Ab molecule and
each Ab molecule is linked to more than one Ag molecule.

The precipitate develops as neighboring antibody molecules within the lat-
tice form ionic bonds with each other, causing the lattice to lose its charge
and thus become insoluble.

Precipitation Reactions in Solution

A simple way to demonstrate the presence of antibody against an antigen in so-
lution is to layer a small volume of one over the other in a test tube. At the inter-
face, precipitation will occur, forming a ring. This gives a qualitative evidence of
an Ag-Ab reaction.

A quantitative precipitation reaction can be performed by placing a constant
amount of antibody in a series of tubes and adding increasing amounts of
antigen to the tubes.

After the precipitate forms, each tube is centrifuged to pellet the precipi-
tate, the supernatant is poured off, and the amount of precipitate is mea-
sured. Plotting the amount of precipitate against increasing antigen con-
centrations yields a precipitin curve.
264
When the supernatants are examined (each supernatant is divided into
aliquots, to one is added a small amount of fresh antigen [to detect excess
antibody], and to the other a small amount of fresh antiserum [to detect
excess antigen]), it is observed that excess of either antibody or antigen
interferes with maximal precipitation.


Prozone. Uncombined antibody is present in the supernatant.
Due to the presence of excess antibody, each antigen mole-
cule is covered with antibody, preventing cross-linkage and
thus precipitation.

Equivalence zone. Both antigen and antibody
are completely precipitated and no uncombined
Ag or Ab is present in the supernatant. The ratio
of Ab to Ag is optimal such that extensive cross-
linking and lattice formation occurs. As this lattice
grows in size it becomes insoluble and eventually
precipitates.





Zone of antigen excess (postzone). Uncombined Ag is present in the super-
natant. In this zone, precipitation is partly or completely inhibited because solu-
ble Ag-Ab complexes form in the presence of excess Ag.

Where Ag is in excess, each Ab molecule is bound to a pair
of Ag molecules. Further cross-linkage is impossible since
these complexes are small and soluble, hence no precipitation occurs.
265
Agar Gel Immunodiffusion Reactions

When Ag and Ab diffuse toward one another in an agar matrix or when antibody
is incorporated into the agar and antigen diffuses into the antibody-containing
matrix, a visible line of precipitation will form. As in a precipitation reaction in
solution, visible precipitation occurs in the region of equivalence, whereas no
visible precipitate forms in regions of Ab or Ag excess.

Single Radial Immunodiffusion (Mancini Method)

In 1965, Mancini introduced a novel technique employing single diffusion for ac-
curate quantitative determinations of antigens. The technique involves incorpo-
ration of specific antibody into the agar plate.

The principle of the test is that a quantitative relationship exists between the
amount of Ag placed in a well cut in the agar-antibody plate and the resulting
ring of precipitation.

Specific antiserum is incorporated in agar and wells are cut in the agar. Ag is
placed in the wells and allowed to diffuse into the agar.

A ring of precipitate indicating the zone of optimal proportions will form
around the well. The area of this ring is directly related to the amount of
antigen added to the well.

266
The Mancini method is routinely used to quantitate serum levels of IgM, IgG,
and IgA by incorporating class-specific anti-isotype antibody into the agar.
The assay is also used to determine the concentrations of complement com-
ponents in serum.

Ouchterlony Test (Double Diffusion Test)

First described by Elek and Ouchterlony in 1948, the test is based on the prin-
ciple that Ag and Ab diffuse through a semisolid medium [eg, agar] and form sta-
ble immune complexes which then can be analyzed visually.

Molten agar is poured on a clean glass slide or into Petri dish and allowed to
harden. Small wells, about 5 mm in diameter and 1 cm apart, are punched
out of the agar.

Samples containing Ag and Ab are placed in opposing wells and allowed to
diffuse toward one another in a moist chamber for 18-24 hours. The resultant
precipitation lines that represent Ag-Ab complexes are analyzed visually in
indirect light with the aid of a magnifying lens.


Applications

Identifying the presence of either soluble Ag or Ab in body fluids, eg,
Coggins test [detects presence of Abs against equine infectious anemia
virus in horses].

Comparing antigens. The pattern of the precipitin lines that form when
two different antigen preparations are placed in adjacent wells, indicates
whether or not they share epitopes.

Identity occurs when two Ags share identical epitopes. The antiserum
forms a single precipitin line with each Ag that grow toward each other
and fuse to give a single curved line of identity.

Nonidentity occurs when two Ags are unrelated [ie, share no common
epitopes]. The antiserum forms independent precipitin lines that cross.
267
Partial identity occurs when two Ags share some epitopes but one or
the other has a unique epitope[s]. The antiserum forms a line of iden-
tity with the common epitope[s] and a curved spur with the unique epi-
tope[s].











Agglutination Reactions

Whereas the Ag is in solution in precipitation reactions, it is particulate in agglu-
tination reactions. The agglutination [visible clumping] of either insoluble native
antigens [eg, bacteria, fungi] or antigen-coated particles can be simply assessed
visually with or without the aid of a microscope.

The main advantages of aggluti-
nation reactions are their high de-
gree of sensitivity and the great
variety of substances detectable
through use of antigen- or anti-
body-coated particles.

Prozone effect. J ust as in preci-
pitation reactions, an excess of
Ab inhibits agglutination reac-
tions. A number of mechanisms
can cause the prozone effect:

Excess antibody increase the likelihood that a single antibody molecule
will bind to two or more epitopes on a single particulate antigen rather than
cross-linking epitopes on two or more particulate antigens.

Incomplete antibodies. When high concentrations of incomplete anti-
bodies bind to the antigen but do not induce agglutination. Incomplete
antibodies are often of the IgG class; the lack of agglutinating activity may
be due to restricted flexibility in the hinge region, making it difficult for the
antibody to assume the required angle for optimal cross-linking of epitopes
on two or more particulate antigens. For this reason, pentameric IgM is
the most efficient agglutinin.
268
No agglutination









Bacterial Agglutination

Serotyping. Agglutination reactions provide a way to type bacteria, eg, different
species of the bacterium Salmonella can be distinguished by agglutination reac-
tions with a panel of typing antisera.

Disease diagnosis. The agglutinin titer of an antiserum can be determined
using the tube agglutination test and used to diagnose a bacterial infection. The
tube agglutination test can also be used to assess response to bacterial vac-
cines.

Serum from the patient is serially
titrated in 2-fold dilutions and a
suitable fixed amount of Ag is
added to each tube. After tho-
rough shaking, the tubes are in-
cubated in a waterbath at 37
o
C
for 1-2 hours. The result is deter-
mined by looking for sedimented
clumps and clear supernatant.

The titer of the serum is the reci-
procal of the highest dilution with
clearly visible agglutination.


Slide, card, or plate agglutination
test. Bacteria can be directly agglu-
tinated by mixing a loopful of anti-
serum with a suspension of the or-
ganism on a slide, card, or porcelain
plate and inspected for agglutination.

Indirect (Passive) Agglutination

The sensitivity and simplicity of agglutination reactions can be extended to solu-
ble antigens by the technique of passive agglutination. Soluble antigens or anti-
269
bodies can be coupled to the surface of artificial or biological carriers, such as
latex particles, colloidal charcoal, or erythrocytes and then detected by agglutina-
tion reactions.

Latex Agglutination

In latex agglutination, antigen or antibody is adsorbed to the surface of latex po-
lystyrene beads and suspended in water. The addition of specific Ab or Ag re-
sults in the formation of Ag-Ab complexes, causing agglutination [clumping].

The reaction results in changes in the appearance of the latex suspension
from smooth and milky to clumpy because the latex particles have clustered
together.

If no reaction occurs, the mixture remains evenly dispersed.

Detection of Bacteria in Cerebrospinal Fluid Using Latex Agglutination Test












Commercial latex agglutination test kits are available for the detection of
rheumatoid arthritis factor in canine serum, K99+ [F5] E. coli in stools of
diarrhetic calves, S. aureus and Streptococcus agalactiae in bovine mastitis,
etc.

270
Hemagglutination (HA)/Hemagglutination Inhibition (HI) Tests

A number of viruses are capable of binding and agglutinating mammalian and
avian erythrocytes. Antibodies directed against these viruses may inhibit this he-
magglutination by blocking their attachment sites.

Virus-induced hemagglutination may be used as a preliminary test when at-
tempting to identify a virus, eg, canine parvovirus.

Inhibition of hemagglutination by specific antibody may be used to identify a
specific virus or to measure antibody levels in serum.

Preparation of 10% RBC Suspension

Collect blood using sodium citrate as anticoagulant: I part of 5% sodium citrate
to 5 parts of blood. Mix the blood-citrate solution by repeated inversions of the
stoppered tube used to collect the blood.

Wash the cells 3 times in 0.85% saline; gently mix the citrate-blood suspen-
sion with ~5 volumes of the saline, sediment the suspension at 1500 rpm for
10 min, pour off the supernatant, add fresh saline, and repeat twice more.

Drain off the last wash; resuspend the washed cells in saline to give a 10%
stock solution. This may be kept at 4
o
C for up to 5 or 6 days. For the HA/HI
tests, 0.5% [1:200] RBC suspension is used.

Schematic Representation of Viral Hemagglutination Test









Schematic Representation of Viral Hemagglutination Inhibition Test











271
Hemagglutination Inhibition Antibody Titer Determination















Neutralization Tests

Neutralization tests are used to estimate the ability of antibody to neutralize the
biological activity of viable viruses in vitro.

Virus Neutralization Test

These are widely employed in the quantitation of antiviral antibodies, or virus
identification. They utilize the ability of antibody-containing sera to block the in-
fectivity of viral agent upon inoculation of the mixture into susceptible hosts or
cell cultures.

Virus identification. Incubate virus with antiserum at room temperature for 1
hour. Control virus is incubated with normal serum.

Inoculate mixtures
into indicator sys-
tem: embryonated
eggs, monolayer
cell culture, or lab-
oratory animals.
Observe for lack
of mortality or cy-
topathic effect in
test sample.





272
Complete inhibition of CPE at a challenge dose of 10 or 100 TCID
50
by a
known antiserum type is considered a positive serum neutralization test and
indicates the identity of the virus.

Neutralizing antibody titer

Serially dilute serum [2-fold] and add constant virus dilution [10 TCID
50
] to
each serial dilution. Incubate mixture at room temperature for 1 hour.

Inoculate serum-virus mixture into indicator system and determine Ab titer.

Complement-Fixation Test (CFT)

The CFT is one of the most widely applicable of all serologic tests. Once the
required reagents (antigen, complement, sheep RBCs, and antibody against the
erythrocytes [hemolysin]) are prepared and standardized, the CFT may be used
to measure serum antibody levels and/or diagnosis of viral, bacterial, and fungal
diseases.

Titration of Complement

Sensitized sheep RBCs [antibody-coated sheep RBCs] are added to varying
amounts of the complement source in microtiter wells.

After a suitable incubation period at 37
o
C, the wells containing the various
complement dilutions are examined for hemolysis after centrifuging the micro-
titer plate.

The amount of complement needed for the complement-fixation test is the
dilution of complement necessary to achieve lysis of half the RBCs added, ie,
the CH
50
dilution.



273
First, antigen and the serum under test [deprived of its complement by heat-
ing at 56
o
C] are incubated in the presence of normal guinea pig serum,
which provides a source of complement.

After allowing the Ag-Ab-complement mixture to react for a short period, the
amount of free complement remaining in the mixture is measured by adding
an indicator system (antibody-coated sheep RBCs [sensitized RBCs]).

Negative result. Lysis of sensitized RBCs [seen as the development of a
transparent red solution].

Positive result. Absence of lysis [seen as a cloudy red cell suspension].