Biochemistry of

Imunology - Hematology

1,2
MarhaenHardjo
1
Head of Biochemistry Department, Medical Faculty of Hasanuddin University
2
Director of Stem Cell Center Hasanuddin University Hospital

DEPARTMENT OF BIOCHEMISTRYY
MEDICAL FACULTY OF
HASANUDDIN UNIVERSITY
Topics:
Membrane & Metabolism of Erythrocyte
Structure & Function of Haemoglobine
Metabolism of Leucocyte
 The erythrocyte membrane

Structure of RBC membrane:

Lipid bilayer(40%)
Membrane proteins (52%)
Carbohydrate (8%)

Planes of design:
Vertical interaction
Stabilise the lipid bilayer membrane
Horizontal interaction
Support structural integrity of RBC.
Structure of RBC membrane
 Membrane Lipids

Asymmetric phospholipid distribution

Unesterified, free cholesterol between layers.
Cholinecontaining, uncharged phospholipids,
outer layer:
Phosphatidylcholine(PC) (30%)
Sphingomyelin(SM) (25%)
Charged phospholipids, inner layer:
Phosphatidylethanolamine (PE) (28%)
Phosphatidylserine (PS) (14%)
 Membrane Lipids

Asymmetric phospholipid distribution is

maintained by:
Differential rate of diffusion through membrane
bilayerof choline containing phospholipids (PC and
SM diffuse slowly)
Charged phospholipids interaction with
membrane skeletal protein.
Active transport of aminophospholipids(PC and
PE) from outer to inner layer.
 Membrane Lipids
Unesterified cholesterol lies between the two
layers of the lipid bilayer.
Mature RBC cannot synthesis lipids in vivo:
renewal through exchange between plamsaand
membrane lipids.
PS on the outer layer may be recognised by
macrophages as a signal for attachment and
phagocytosis.
May occur following RBC damage (eg b
thalassoc RBC scrambling due to oxidative
damage).
 Membrane Lipids

Integral proteins
Embedded in membrane via hydrophobic
interactions with lipids.
Peripheral proteins
Located on cytoplasmic surface of lipid
bilayer, constitute membrane skeleton.
Anchored via integral proteins
Responsible for membrane elasticity and
stability.
 Integral Proteins
Band 3
Glycophorin
Aquaporin
 Band 3
Constitutes 25% of total membrane protein
911 Amino Acid protein
Comprises 3 distinct proteins:
Cytoplasmic domain:
Hydrophilic, interacts with proteins of skeleton
Transmembrane domain
Contains multiple membrane spanning domains
Forms the anion transporter
C-terminal domain
?binds carbonic anhydrase
Single oligosaccharide possesingblood group antigen I and
i.
 Band 3
Functions:
Anion transport
Exchanges bicarbonate

for chloride
Structural:
Linkage of lipid bilayerto underlying
membrane skeleton.
Interaction with ankyrin and protein
4.2, secondarily through binding to
protein 4.1.
Important for prevention of surface
loss.
Chromosome 17
Spectrin: the most prominent component (two isoforms ,; a tetramer; a meshwork )
fixed to the membrane- ankyrin
binding sites for several other proteins (glycophorin C, actin, band 4.1,
adducin)
This organization keeps the erythrocyte shape.
Haemoglobin
4 protein chains + 4 haem groups
Haem
Movements of the heme and the F helix during the T R transition in
hemoglobin:
Hemoglobin saturation curves:
Hemoglobin autooxidation

O2 binds Fe2+ - an intermediate structure - an electron is delocalized between

the iron ion and the O2
the side effect - every so often a molecule of oxyhaemoglobin undergoes
decomposition and release superoxide

Hem - Fe2+- O2 Hem - Fe3+ - O2-

3% of the haemoglobin undergoes oxidation every day

Methemoglobin (Fe3+) is unable to bind O2 (methaemoglobin reductase)

 Erythrocyte exceptions

They lack organelles

no ATP production in oxidative phosphorylation
no ability to replace damaged lipids and proteins (low metabolic activities,
with no ability to synthesize new proteins or lipids)

Free radicals exposure

haemoglobin autoxidation (O2- release)
a cell membrane rich in polyunsaturated fatty acids (susceptible to lipid
peroxidation)
deformation in tiny capillaries; catalytic ions leakage (cause of lipid
peroxidation)
 Erythrocyte metabolism

Glucose as a source of energy

Glycolysis generates ATP and 2,3-bisphosphoglycerate

The pentose phosphate pathway produces NADPH

Glutathione synthesis- the antioxidant defence system

 Glucose- source of energy

Glucose transporter:
integral membrane protein (12 membrane-spanning helices)
a channel for the glucose transport
insulin-independent transporter

Glycolysis in erythrocytes

1. Source of ATP
Lactate- the end product
Cover energy requirement

2. Generate 2,3-bisphosphoglycerate (2,3-BPG)

a major reaction pathway for the consumption of glucose in erythrocytes
the specific binding of 2,3-BPG to deoxyhemoglobin decreases the oxygen
affinity of hemoglobin and facilites oxygen release in tissues
 2,3-bisphosphoglycerate

Allosteric effector of haemoglobin:

binds to deoxyhaemoglobin (a central cavity capable of binding 2,3-
BPG)
decreases haemoglobins O2 affinity

Clinical aspects:
In people with high-altitude adaptation or smokers the concentration of
2,3-BPG in the blood is increased (low oxygen supply)

Fetal haemoglobin has low BPG affinity - the higher O2 affinity -

facilitates the transfer of O2 to the fetus via the placenta
Glutathione synthesis in erythrocytes
 Glutathione
Elimination of H2O2 and organic hydroperoxides
1. Cofactor for the glutathione peroxidase (removes H2O2 formed in
erythrocytes)
2. Involved in ascorbic acid metabolism
3. Prevents protein SH groups from oxidizing and cross-linking

Glutathione peroxidase
Gly Gly Gly
+ R-O-O-H

Cys SH Cys S S Cys + H 2O

+ NADPH
Glu Glu Glu
Glutathione reductase

Reduced form of glutathione Oxidized form of glutathione

(monomer) (dimer, disulphide)
 The pentose phosphate pathway in
erythrocytes

Generates NADPH - reduction of glutathione (eliminates H2O2 formed in

erythrocytes)

Clinical apect:
Glucose-6-phosphate dehydrogenase deficiency
Causes hemolytic anemia (decreased production of NADPH - reduced
protection against oxidative stress - haemoglobin oxidation and Heinz
bodies formation, membrane lipid peroxidation and hemolysis)
Hemolytic crises are evocated by drugs (primaquine, sulphonamide
antibiotics) and foods (broad beans)

The most common enzyme deficiency disease in the world (100 million
people)
 Oxyhaemoglobin
O2

Superoxide dismutase
Haemoglobin Superoxide H2O2

Catalase

Methaemoglobin reductase
Methaemoglobin
O2+H2O
Pentose phosphate GSH
pathway NADP+

Glutathione reductase Glutathione peroxidase

NADPH GSSG
H2O

GSH-reduced form; GSSG-oxidized form of glutathione

 Haemoglobinopathy
abnormal structure of the haemoglobin (mutation)
large number of haemoglobin mutations, a fraction has deleterious
effects
sickling, change in O2 affinity, heme loss or dissociation of tetramer
haemoglobin M and S, and thalassemias

Haemoglobin M
replacement of the histidine (E8 or F7) in or -chain by the tyrosine
the iron in the heme group is in the Fe3+ state (methaemoglobin)
stabilized by the tyrosine
methaemoglobin can not bind oxygen

Thalassemias
genetic defects- or -chains are not produced ( or -thalassemia)
 Haemoglobin S (sickle-cell)

Causes a sickle-cell anemia

Erythrocytes adopt an elongated sickle shape due to the
aggregation of the haemoglobin S
 Replacing Glu with the less polar amino acid Val - forming an
adhesive region of the chain
Hg proteins aggregate into a long rodlike helical fiber

Cross section
 Red blood cells adopt a sickle shape in a consequence of the forming haemoglobin S
fibers
The high incidence of sickle-cell disease coincides with a high incidence of malaria
Individuals heterozygous in haemoglobin S have a higher resistance to malaria; the
malarial parasite spends a portion of its life cycle in red cells, and the increased
fragility of the sickled cells tends to interrupt this cycle

Scanning electron micrograph of a sickled erythrocyte.

The haemoglobin S fibers can be seen within the
distorted cell. The cell has ruptured and haemoglobin
fibers are spilling out.
Hemoglobin switching:
 Glycosylated haemoglobin (HbA1)

formed by hemoglobin's exposure to high plasma levels of glucose

non-enzymatic glycolysation (glycation)- sugar bonding to a protein
normal level HbA1- 5%; a buildup of HbA1- increased glucose concentration
the HbA1 level is proportional to average blood glucose concentration over
previous weeks; in individuals with poorly controlled diabetes, increases in
the quantities of these glycated hemoglobins are noted (patients
monitoring)

Sugar CHO + NH2 CH2 Protein

Sugar CH N CH2 Protein

Schiff base
Amadori reaction

Sugar CH2 NH CH2 Protein

Glycosylated protein
Summary

1) The erythrocyte membrane

2) Haemoglobin O2 binding
3) Haemoglobin allosteric efectors
4) Free radicals in erythrocytes
5) Metabolic pathways in erythrocytes
6) Haemoglobinopathy
7) Glycosylated haemoglobin
 Hemoglobin
synthesis, structure & function
 Introduction

The hemoglobin are red globular proteins which

have a molecular weight of about 64,500 and
comprise almost one third of the weight of a red
cell. Their primary function is the carriage of
oxygen from the lungs to the tissues.
Over 500 different haemoglobin variants have
been described but all share the same basic
structure of four globin polypeptide chains each
with haem group. Functional haemoglobin
composed of two pairs of dissimilar globins.
 Haemoglobin synthesis

Although haem & globin synthesis

separately within developing red cell
precursors their rate of synthesis are
carefully coordinated to ensure optimal
efficiency of haemoglobin assembly.
1 : Haem synthesis
st
The first step in
haem synthesis
is the
combination of
succinyl CoA
and glycin to
produce
aminolaevulinic
acid ( ALA).
This reaction is
energy
dependent and
so occurs in the
mitochondria.
 Haem synthesis
Its catalyzed by the enzyme ALA synthetase.
This step is a first-limiting step for the whole process of
haem synthesis.
It is stimulated by the presence of globin chains and
inhibited by the presence of free haem groups.
This represents an important control mechanism of the rate
of haem synthesis and its coordination with globin
synthesis.
Several factors are required for this step, including vitamin
B6, free ferrous and copper ions.
Synthesis of the enzyme ALA synthetase is inhibited by
the presence of free haem.
This represents a further feedback mechanism for haem
synthesis.
Mitochondrial -aminolevulinic acid (ALA) is transported
to the cytoplasm, where ALA dehydratase (also called
porphobilinogen synthase) dimerizes 2 molecules of
ALA to produce the pyrrole ring compound
porphobilinogen (PBG).
 Haem synthesis
The next step requires the synthesis of porphyrin
ring.
The reactions involved are extremely complex
but can be summarized as the condensation of
four PBG molecules to form the asymmetric
cyclic uroporphyrinogen III (UPGIII).
Synthesis of UPGIII requires the presence of two
enzymes (uroporphyrinogen I synthetase and
uroporphyrinogen III cosynthetase) and involves
the formation of several short-lived
intermediates.
 UPG III is converted to coproporphyrinogen III
(CPGIII) by decarboxylation of the acetate side
chains under the influence of the enzyme
uroporphyrinogen decarboxylase.
 CPGIII enters the mitochondria where it converted to
protoporphyrinogen IX (PPG IX) by an unknown
mechanism. This reaction is catalyzed by the enzyme
coproporhyrinogen oxidase.
PPG IX is further converted within the mitochondria to
protoporphrin IX.
 It only remains for the central ferrous ion to be inserted
to complete the synthesis of haem. This reaction is
catalyzed by the enzyme ferrochelatase and requires the
presence of reducing agents.
 2 : Globin synthesis
nd

Humans normally carry 8 functional globin

genes, arranged in two duplicate gene
clusters:
The -like cluster on the short arm of
chromosome 11.
The -like cluster on the short arm of
chromosome 16.
These genes code for 6 different types of
globin chains: ,,,,,, globin.
 Ontogeny of globin synthesis
Time Region Type of Globin Type of Hb
Gene

weeks of 3 Yolk Sac & Hb Gawer1

Gestation ) )2

5 weeks of Yolk Sac & Hb Portland( )2

Gestation Hb GawerII ()2

weeksof 6-30 Liver & spleen & & Hb F ( )2

Gestation

weeks of 30 Liver Hb A2 ( 2(
Gestation

At Birth B.M ___ HbA( )2

 Adult haemoblobin
Hb A Hb A2 Hb F

structure 22 22 22

%Normal % 96-98 % 1.5-3.2 % 0.5-0.8

2 : Globin synthesis
nd
 Haemoglobin Structure
Primary structure of globin

The primary structure of globin refers to the amino acid

sequence of the various chain types. Numbering
from the N-terminal end identifies the position of
individual amino acids. The identity and position of
these amino acids cannot be changed without
causing gross impairment to molecular function.
Secondary Structure of globin :

The secondary structure of all globin chain types

comprises nine non-helical sections joined by
eight helical sections.

The helical sections are identified by the letters

A-H while the non helical are identified by a pair
of letters corresponding to the adjacent helices
e.g. NA (N-terminal end to the start of A helix), AB
(joins the A helix to the B helix) etc.
Tertiary Structure of globin:

The tertiary folding of each globin chain

forms an approximate sphere.

The intra-molecular bonds which give rise

to the helical parts of the impart
considerable structure rigidity, causing
chain folding to occur in the non-helical
parts.
 Tertiary folding gives rise to at least 3 functionally
important characteristics of the hemoglobin molecule :
1- Polar or charged side chains tend to be directed to the
outside surface of the subunit and, conversely, non-polar
structures tend to be directed inwards. The effect of this is to
make the surface of the molecule hydrophilic and the interior
hydrophobic.

2- An open-toped cleft in the surface of the subunit known as

haem pocket is created. This hydrophobic cleft protects
the ferrous ion from oxidation.

3- The amino acids, which form the inter-subunit bonds

responsible for maintaining the quaternary structure, and
thus the function, of the haemoglobin molecule are
brought into the correct orientation to permit these bonds
to form.
 Quaternary structure of Haemoglobin

The quaternary structure of haemoglobin has four

subunits arranged tetrahedrally. In adult
haemoglobin- (HbA), there are different contact
areas:

11 and 2 2 which confirms stability of the

molecule.
1 2 and 2 1 which confirms solubility of
the molecule.
1 2 and 1 2 which are weak bonds to
permit oxygenation and deoxygenation.
 Functions of Haemoglobin
Oxygen delivery to the tissues
Reaction of Hb & oxygen

Oxygenation not oxidation

One Hb can bind to four O2 molecules
Less than .01 sec required for oxygenation
chain move closer when oxygenated
When oxygenated 2,3-DPG is pushed out
chains are pulled apart when O2 is unloaded,
permitting entry of 2,3-DPG resulting in lower affinity
of O2
Oxy & deoxyhaemoglobin
Normal Hemoglobin Function
When fully saturated, each gram of hemoglobin binds
1.34 ml of oxygen.
The degree of saturation is related to the oxygen tension
(pO2), which normally ranges from 100 mm Hg in arterial
blood to about 35 mm Hg in veins.
The relation between oxygen tension and hemoglobin
oxygen saturation is described by the oxygen-
dissociation curve of hemoglobin.
The characteristics of this curve are related in part to
properties of hemoglobin itself and in part to the
environment within the erythrocyte, including pH,
temperature, ionic strength, and concentration of
phosphorylated compounds, especially 2,3-
diphosphoglycerate (2,3-DPG).
Hb-oxygen dissociation
curve
Normal Hemoglobin Function
Oxygen affinity of hemoglobin is generally expressed in
terms of the oxygen tension at which 50% saturation
occurs.
When measured in whole erythrocytes, this value
averages 27.1 mm Hg in normal, nonsmoking males and
27.5 mm Hg in normal, nonsmoking females.
When oxygen affinity is increased, the dissociation
curve is shifted Leftward, and the value is reduced.
Conversely, with decreased oxygen affinity, the curve is
shifted to the right.
 Hb-oxygen dissociation
curve
The
normal position of curve
depends on

Concentration of 2,3-DPG
H+ ion concentration (pH)
CO2 in red blood cells
Structure of Hb
 Hb-oxygen dissociation
curve
Right shift (easy oxygen delivery)

High 2,3-DPG
High H+
High CO2
HbS

Left shift (give up oxygen less readily)

Low 2,3-DPG
HbF
 Bohr Effect
The change in oxygen affinity with pH is known
as the Bohr effect.
Hemoglobin oxygen affinity is reduced as
the acidity increases.
Since the tissues are relatively rich in carbon
dioxide, the pH is lower than in arterial blood;
therefore, the Bohr effect facilitates transfer of
oxygen.
The Bohr effect is a manifestation of the acid-
base equilibrium of hemoglobin.
 2,3-diphosphoglycerate
(2,3-DPG)
This compound is synthesized from glycolytic intermediates by
means of a pathway known as the Rapoport-Luebering shunt.
In the erythrocyte, 2-3-DPG constitutes the predominant
phosphorylated compound, accounting for about two thirds of the
red cell phosphorus.
The proportion of 1,3-DPG pathway appears to be related largely to
cellular ADP and ATP levels; when ATP falls and ADP rises, a
greater proportion of 1,3-DPG is converted through the ATP-
producing step.
This mechanism serves to assure a supply of ATP to meet cellular
needs.
In the deoxygenated state, hemoglobin A can bind 2,3-DPG in a
molar ratio of 1:1, a reaction leading to reduced oxygen affinity
and improved oxygen delivery to tissues.
 When oxygen is unloaded by the hemoglobin
molecule and 2,3 DPG is bound, the molecule
undergoes a conformational change becoming what is
known as the ""Tense" or "T" form.
The resultant molecule has a lower affinity for
oxygen.
As the partial pressure of oxygen increases, the 2,3,
DPG is expelled, and the hemoglobin resumes its
original state, known as the "relaxed" or "R" form, this
form having a higher oxygen affinity.
These conformational changes are known as
"respiratory movement".
The increased oxygen affinity of fetal hemoglobin
appears to be related to its lessened ability to bind
2,3-DPG.
The increased oxygen affinity of stored blood is
accounted for by reduced levels of 2,3-DPG.
 Changes in 2,3-DPG levels play an important
role in adaptation to hypoxia. In a number of
situations associated with hypoxemia, 2,3-DPG
levels in red cells increase, oxygen affinity is
reduced, and delivery of oxygen to tissues is
facilitated.
Such situations include abrupt exposure to high
altitude, anoxia due to pulmonary or cardiac
disease, blood loss, and anemia.
Increased 2,3-DPG also plays a role in
adaptation to exercise. However, the compound
is not essential to life; an individual who lacked
the enzymes necessary for 2,3-DPG synthesis
was perfectly well except for mild polycythemia
 Carbon Dioxide
(CO2)
Transport of carbon dioxide by red
cells, unlike that of oxygen, does not
occur by direct binding to heme.
In aqueous solutions, carbon dioxide
undergoes a pair of reactions:
1. CO2 + H2O H2CO3

2. H2CO3 H+ + HCO3
 (CO2)
Carbon dioxide diffuses freely into the red cell where the
presence of the enzyme carbonic anhydrase facilitates
reaction 1.
The H+ liberated in reaction 2 is accepted by deoxygenated
hemoglobin, a process facilitated by the Bohr effect.
The bicarbonate formed in this sequence of reactions diffuses
freely across the red cell membrane and a portion is
exchanged with plasma Cl-, a phenomenon called the
"chloride shift." the bicarbonate is carried in plasma to the
lungs where ventilation keeps the pCO2 low, resulting in
reversal of the above reactions and excretion of CO2 in the
expired air.
About 70% of tissue carbon dioxide is processed in this way.
Of the remaining 30%, 5% is carried in simple solution and
25% is bound to the N-terminal amino groups of
deoxygenated hemoglobin, forming carbaminohemoglobin.
 Methemoglobinemia
In order to bind oxygen reversibly, the iron in the
heme moiety of hemoglobin must be maintained
in the reduced (ferrous) state despite exposure
to a variety of endogenous and exogenous
oxidizing agents.

The red cell maintains several metabolic

pathways to prevent the action of these oxidizing
agents and to reduce the hemoglobin iron if it
becomes oxidized. Under certain
circumstances, these mechanisms fail and
hemoglobin becomes nonfunctional.
 Methemoglobinemia
At times, hemolytic anemia supervenes as
well. These abnormalities are particularly
likely to occur
(1) if the red cell is exposed to certain oxidant drugs

or toxins
(2) if the intrinsic protective mechanisms of the cell
are defective or
(3) if there are genetic abnormalities of the
hemoglobin molecule affecting globin stability or
the heme crevice.
Pictures used in the presentation:
Marks Basic Medical Biochemistry, A Clinical Approach, third edition, 2009 (M.
Lieberman, A.D. Marks)
Principles of Biochemistry, 2008, (Voet D, Voet J.G., and Pratt C.W)
Color Atlas of Biochemistry, second edition, 2005 (J. Koolman and K.H. Roehm)
Metabolism of leukocytes
and platelets
Differentiation of the bone marrow stem cells
stem cell
myeloid
progenitor
lymphoid
megakaryocyte progenitor
erythroblast

erythrocyte

monocyte

platelets
neutrophil eosinophil basophil

dendritic cell macrophage

plasma
mature cell
lymphocytes
 A) Phagocytic cells:

Neutrophils most abundant

Eosinophils
Monocytes
Macrophages rise by differentiation of monocytes in tissues
 Degradation of the ingested particle:

1) Activation of NADPH oxidase

2) Production of NO by nitric oxide synthase

3) Fusion of phagosome with lysosomes of the phagocytic cell that

contain bactericidal substances and hydrolytic enzymes (often with
acidic pHopt)
 1) NADPH-oxidase

Protein complex of neutrophils, eosinophils, monocytes, macrophages

NADPH + 2 O2 NADP+ + H+ + 2 O2- superoxide anion

2 O2- + 2 H+ O2 + H2O2

H2O2 can damage bacteria directly or after conversion to OH :

H2O2 + M+ OH + OH- + M2+ (M; metal)

 Activation: by association of the components localized in cytosol with
cytochrome b558 in the membrane; electrons from cytosolic NADPH
are via FAD and cytochrome transferred to oxygen

cytochrome b558

active NADPH-oxidase
plasma
membrane

fusion
with
lysosomes
phagosome
 Myeloperoxidase
Present in granules of neutrophils and monocytes, but not macrophages!

Significant portion of H2O2 (produced by dismutation of O2- generated by

NADPH oxidase) is used by myeloperoxidase to oxidize Cl- to HClO

HClO is highly reactive, able to oxidize biomolecules; it also provides

toxic chlorine gas:
HClO + H+ + Cl- Cl2 + H2O

HClO also reacts with O2- yielding OH:

HClO + O2- O2 + OH + Cl-
 Chronic granulomatous disease

Caused by a deficiency of one of the NADPH oxidase subunits

Superoxide and the other reactive oxygen species are not produced

Severe infections that are very hard to treat e.g.:

Burkholdaria cepacea causes pneumonia
Aspergillus causes intractable pneumonia, septicaemia; can lead
to death

Treatment: antibiotics, antifungal agents

 2) Nitric oxide production
Mainly by inducible nitric oxide synthase (iNOS) of macrophages which is
induced by cytokines (INF-, TNF) or bacterial lipopolysaccharide:

Arg citrulline

NO can kill bacteria directly (e.g. by inhibition of the respiratory chain) or

indirectly: by reaction with O2-, generating peroxynitrite ONOO- which attacks
Fe-S proteins and essential SH groups, inactivates enzymes
 NADPH oxidase is effective mainly in degradation of extracellular
pathogens (Salmonella, Staphylococcus, Streptococcus pyogenes)
neutrophils

NO serves mainly to kill the intracellular parasites (Listeria, Brucella,

Candida albicans)macrophages
 3) Granules (lysosomes) of neutrophils
Contain bactericidal substances and hydrolases that, after fusion with
phagosome, destroy the engulfed particles:
myeloperoxidase
lysozyme cleaves glycosidic bonds in peptidoglycan of the
bacterial (primarily G+) cell walls
defensins cationic peptides (Arg) with Mr of 3,5-6 kDa; interact with
anionic lipids of bacterial membrane and make pores in it; can also
inhibit synthesis of DNA and proteins
hydrolases, e.g. elastase serine protease: can damage bacteria
and cleave virulence factors, but also cause harm to host tissues
(cleaves the proteins of extracellular matrix, too)
 Eosinophils
Main task: defence against multicellular parasites
Display all the above-mentioned mechanisms with slight differences:
ROS production
peroxidase of eosinophils similar to myeloperoxidase, but prefers
Br- as a substrate (instead of Cl-), thus generating HBrO (instead of
HClO)
basic protein of eosinophils disrupting the parasite cell membranes
 B) Basophils and mast cells
Activated by antigens / allergens interacting with IgE bound to the surface IgE receptors of basophils (mast cells)
Upon activation, content of their granules is released substances that are harmful to parasite and induce reactions that should lead to its removal; however, they
can also be responsible for allergic symptoms:
hydrolases
histamine
heparin

Synthesis of eicosanoids is activated; leukotrienes are potent bronchoconstrictors, stimulate chemotaxis and leukocyte activation

cytoplasmic granules
 Histamine

Produced by histidine decarboxylation:

Causes vasodilation and bronchoconstriction helps to eliminate

parasites (cough, peristalsis, enhanced production of mucus)
 Atopy
IgE recognizing allergens (from pollen, food) are produced and bind to
IgE receptors of basophils (mast cells). Next exposure to the allergen
can lead to release of histamine and heparin and synthesis of
eicosanoids

Local symptoms occur: allergic rhinitis, asthma, conjunctivitis

If the allergen enters bloodstream, it can cause a massive degranulation

of basophils (mast cells) increase in vascular permeability, decrease in
blood pressure pulmonary oedema, ischemia anaphylactic shock

Treatment: antihistamines block histamine receptors

 C) Lymphocytes
Have specific receptors recognizing one particular antigen: B cell
receptors (BCR) and T cell receptors (TCR), respectively

BRC is a membrane-bound immunoglobulin, TCR is very similar to Ig

B cells (after proliferation and differentiation into plasma cells) secrete

large amounts of antibodies (soluble immunoglobulins)
 Soluble immunoglobulins
2 heavy chains (H) interconnected by
disulfide bonds
VH 2 light chains (L), each connected to
VL one of the H chains (by disulfide bond)
CH1 H chain: 4-5 domains, 50-75 kDa
Fab
CL L chain: 2 domains, 25 kDa
N-terminal domains of H- and L-chains
are variable (VH resp. VL), the others
CH2 are constant (CH resp. CL), i.e. the
Fc same in one type of Ig
Variable domains of H a L chains form
CH3
the antigen-binding site
 Types of immunoglobulins (Ig)
155 kDa
There are 2 isotypes of L: ,
There are 5 isotypes of H:
, , , ,
According to these isotypes of H,
5 types of immunoglobulins can
be distinguished:
IgA (2 subtypes)
IgG (4 subtypes)
IgD
IgE
IgM
IgM can form pentamer, IgA can
(similar: IgD, IgE)
900 kDa form dimer or trimer
 D) Platelets
No nucleus many of their metabolites come from megakaryocytes
Form blood clots, act as vasoconstrictors
Participate in defence against infections, e.g.: they suppress the growth
of Plasmodium falciparum (infectious agent that causes malaria)
Generate O2- and H2O2 that may synergize with pro-aggregatory stimuli
Contain thromboxan A synthase that catalyzes conversion of prosta-
glandin H2 to thromboxan A2:

TXA2 promotes platelet aggre-

gation and vasoconstriction
 Platelets also release two very important factors that can influence
not only platelets but also other cell types:

Platelet-Activating Factor (PAF)

Platelet-Derived Growth Factor (PDGF)
 Platelet-Activating Factor

phospholipid

Mainly juxtacrine and paracrine signalling via GPCR

Promotes platelet aggregation
Induces activation of leukocytes, adhesion, chemotaxis, cytokine
production, causes vasodilation and bronchoconstriction
Mediates interplay between thrombotic and inflammatory cascades
BUT: it is also suspected of contributing to allergy, anaphylactic shock
It is produced also by endothelial cells, monocytes, granulocytes
 Platelet-Derived Growth Factor
Dimeric protein, 3 isoforms
Receptors: tyrosine kinases expressed on fibroblasts, glia, smooth
muscle cells, leukocytes.
Effects:
proliferation
chemotaxis
cytoskeletal rearrangements
differentiation of certain types of cells (e.g. in CNS)
participates in wound healing, capillary formation, embryonic and
postnatal development!
BUT: probably also plays a role in pathogenesis (some tumours)
 Cytokines
Proteins secreted by leukocytes and other cells (but there are also
membrane cytokines) that influence (via receptors) the cells of the
immune system

Cytokine signalling:
autocrine a cytokine influences the same cell that produces it
paracrine a cytokine influences the nearby cells
endocrine a cytokine influences distant cells (after transport by the
bloodstream)
 Types of cytokines
Interleukins e.g. IL-6: produced by macrophages, neutrophils, stimu-
lates lymphocytes, secretion of Ig, synthesis of acute phase reactants

Chemokines induce chemotaxis

Interferons e.g. INF-: produced by lymphocytes, monocytes, and

macrophages, participates in antiviral defense (induces synthesis of
enzymes that block viral replication)

Transforming growth factors e.g. TGF-: produced by T-lymphocytes,

macrophages, and platelets, displays anti-inflammatory effects

Tumor necrosis factors e.g. TNF-: able to induce apoptosis

 Leukocyte infiltration into tissues
= diapedesis (extravasation):

Taken from:
Halliwell, Gutteridge,
Oxford University Press, 1999

Leukocytes are slowed down by the interaction of their mucins with selectines on
the surface of endothelial cells (EC)
Cytokines on the surface of EC interact with the receptors of leukocytes
A strong adhesion mediated by the interaction of integrins with molecules on the
surface of EC migration of leukocytes into the tissue directed by cytokins
released by inflammatory cells or EC
 Regulation
Many functions of leukocytes are regulated by monomeric GTP-binding
proteins, e.g. Rac, Rho:
activation of NADHP oxidase
chemotaxis
phagocytosis
fusion of phagosome with granules

Rho and Rac are able to modulate the assembly of actin filaments,
which plays a role in the processes listed above

http://uk.video.search.yahoo.com/video/play?ei=UTF-8&fr=yfp-t-702&p=chemotaxis&vid=0001539076618&dt=&l=77&turl=http%3A%2F%2Fyts.video.search.yahoo.com%2
Fimage%2F2021a80a1&rurl=http%3A%2F%2Fwww.youtube.com%2Fv%2FZUUfdP87Ssg%26hl%3Den%26fs%3D1&tit=Neutrophil
THANK YOU