Immunology and Cell Biology (2007) 85, 103–111

& 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00
www.nature.com/icb

REVIEW

Life and death in the granuloma: immunopathology

of tuberculosis
Bernadette M Saunders1,2 and Warwick J Britton1,2

During tuberculosis (TB) infection, the granuloma provides the microenvironment in which antigen-specific T cells colocate
with and activate infected macrophages to inhibit the growth of Mycobacterium tuberculosis. Although the granuloma is the
site for mycobacterial killing, virulent mycobacteria have developed a variety of mechanisms to resist this macrophage-mediated
killing. These surviving mycobacteria become dormant, however, if host cellular immunity or the signals maintaining granuloma
structure wane, or if mycobacteria resume replication, leading to reactivation of TB. This balance of life and death applies
not only to the mycobacterium but also to the host macrophages that may undergo apoptosis or necrosis, leading to the
characteristic caseous necrosis within the granuloma, and the potential spread of TB infection. The immunological factors
controlling the development and maintenance of the granuloma will be reviewed.
Immunology and Cell Biology (2007) 85, 103–111. doi:10.1038/sj.icb.7100027; published online 9 January 2007

Keywords: granuloma; tuberculosis; reactivation; cytokines; chemokines

The formation and maintenance of granulomas are essential for the
control of mycobacterial infections but, paradoxically, granulomas are
also responsible for the typical immunopathology caused by these
infections. There are over 70 species of Mycobacteria, but Mycobacter-
ium tuberculosis and Mycobacterium leprae, the causative agents of
tuberculosis (TB) and leprosy, are the major human pathogens. These
slow-growing mycobacteria have adapted to survival within the
macrophage, and this capacity for persistence of mycobacteria in the
face of a potent cellular response underlies the chronic inflammatory
reaction of the host. Mycobacterial infection of dendritic cells (DCs)
stimulates CD4 and CD8 T cells, which on recruitment to the sites
of infection activate infected macrophages. M. tuberculosis, however,
blocks phago-lysosomal fusion and acidification of infected phago-
somes, and also partially inhibits the activation of infected macro-
phages by interferon (IFN)-g, the major effector cytokine released by
Th1-like CD4 T cells. As a result, some mycobacteria persist in the
infected lung, leading to chronic antigenic stimulation and T-cell
accumulation around macrophages. In the face of chronic cytokine
stimulation, macrophages differentiate into epithelioid cells and fuse
to form typical giant cells. Within the resulting granuloma, there is a
balance between mycobacterial killing and survival. The local archi-
tecture results in the close apposition of lymphocytes and macro-
phages, and this is necessary for the activation of macrophages to kill
M. tuberculosis. But the survival of some tuberculous bacilli leads to
latent TB infection (LTBI), which is contained by the granulomatous
process. Following acute M. tuberculosis infection, this process is
adequate to control the infection in 95% of subjects, while the
remainder progress to primary TB disease. There are currently two
billion humans with LTBI, and reactivation of the infection occurs
in 5–7%1 of these subjects without, and in up to 50% with, human
immunodeficiency virus (HIV) co-infection.
Although granulomas can develop during other infections such as
schistosomiasis and in non-infectious conditions, including sarcoido-
sis and Crohn’s disease, this brief review will focus on recent deve-
lopments in our understanding of mycobacterial granulomas. In
particular, it will review the regulation of the adaptive CD4 T-cell
response essential for the formation of granulomas, the role of tumor
necrosis factor (TNF) family members and chemokines, and the
processes leading to sustained latency or reactivation of infection
in humans.
ADAPTIVE IMMUNITY AND GRANULOMA FORMATION
Cellular requirements for granuloma formation
The activation of ab T-cell receptor (TcR) expressing CD4 T cells is
essential for the formation of granulomas during mycobacterial
infections.2 Additional types of T cells contribute to the immune
response during M. tuberculosis infection in mice and humans,
including ab TcR expressing CD8 T cells, gd TcR expressing T cells
and CD1-restricted CD4/CD8 double negative T cells; however,
granulomas develop during experimental M. tuberculosis infection
of mice deficient in these cells (Table 1).3,4 For example, the initial
control of infection and inflammatory response is normal in CD8
T-cell deficient mice, although later in infection there is increased
bacterial load and lymphocyte accumulation in the lungs.3 This

1Mycobacterial Research Programme, Centenary Institute, Newtown, New South Wales, Australia and 2Discipline of Medicine, Central Clinical School, University of Sydney,

Sydney, Australia
Correspondence: Dr B Saunders, Mycobacterial Research Programme, Centenary Institute, Locked Bag No 6, Newtown, 2042 New South Wales, Australia.
E-mail: b.saunders@centenary.usyd.edu.au
Received 3 November 2006; revised 7 November 2006; accepted 8 November 2006; published online 9 January 2007
 Life and death in the granuloma
BM Saunders and WJ Britton
104
Table 1 Contribution of T-cell subsets for normal granuloma formation following aerosol M. tuberculosis infection
KO strain Survival Granuloma formation
ab T cell 48 days Extensive inflammatory infiltration, necrotic lesions dominated by neutrophils.
CD4 120 days Delayed inflammation with few lymphocytes, predominantly macrophages localized as perivascular cuffing accompanied by
increased neutrophils.
CD8 4150 days Normal granuloma development initially apart from increased lymphocytes. During chronic infection, there was increased
neutrophils and necrosis.
b2m 40 days Increased inflammatory infiltrate dominated by macrophages and neutrophils with caseating necrosis. Lymphocytes were
restricted to the adjacent perivascular cuff.
CD1 Normal Normal granuloma development.
gd T cell Normal Increased neutrophils within granulomas, otherwise normal.
contrasts with the marked susceptibility to M. tuberculosis of b2-
microglobulin / mice, which lack classical and non-classical major
histocompatibility complex (MHC) class I molecules and therefore
both CD8- and CD1-restricted T cells.5 The increased iron stores
in these mice, which is due to the lack of the b2-microglobulin-
associated iron transport molecule, HFE, also contribute to the failure
to control intracellular mycobacterial infection.6
The CD4 T cells are stimulated by M. tuberculosis-infected DCs in
the lymph nodes draining the lung,7 and differentiate into Th1-like
CD4 T cells secreting interleukin (IL)-2, IFN-g and lymphotoxin-a
(LTa). Infection of DCs with M. tuberculosis or the vaccine strain
Mycobacterium bovis bacille Calmette Guerin (BCG) results in upregu-
lation of MHC class II and the costimulatory molecules, CD80 and
CD86, in a TLR2-dependent fashion8,9 and the secretion of IL-12 and
the pro-inflammatory cytokines. Multiple components of myco-
bacteria can stimulate TLR1/2, TLR2 and TLR9 in vitro (reviewed in
Krutzik and Modlin10), and mice deficient in both TLR2 and TLR9 fail
to develop normal CD4 T-cell responses and are profoundly suscep-
tible to M. tuberculosis infection.11 DCs, rather than macrophages, are
the primary source of both IL-12 and IL-23 during mycobacterial
infection,12 and their secretion is enhanced by further activation of the
infected DCs by IFN-g and CD40 ligand-mediated interaction from
CD4 T cells.13 This IL-12 response is downregulated by endogenous
IL-10, which is also released by mycobacteria-infected DCs.8
Cytokine requirements
Dissection of the genetic basis for Mendelian susceptibility to myco-
bacterial infections (MSMD) in humans, with marked susceptibility
to BCG and non-tuberculous mycobacteria such as Mycobacterium
avium, along with studies in gene-deficient mice, have helped define
the cytokine requirements for the activation and function of CD4
T cells against mycobacteria. Variants in five genes have been asso-
ciated with MSMD, IFN-g receptor chains 1 (R1) and IFN-g R2, Stat1
in the IFNgR signalling pathway, IL-12 p40 and IL-12 receptor Rb1.14
As observed in IFN-g-deficient mice, subjects with defects in the IFN-g
signalling pathway are profoundly susceptible to mycobacterial infec-
tions, and these present at an early age and are difficult to treat.14
These subjects fail to develop granulomas in tissues infected with
mycobacteria, demonstrating the absolute requirement of IFN-g
signalling for the activation of macrophages and granuloma forma-
tion. Mice that lack IFN-g15 also fail to develop granulomas following
aerosol M. tuberculosis infection, and instead of discrete granulomas
composed of activated macrophages and lymphocytes, their lungs are
infiltrated by neutrophils leading to necrosis and death necrosis.16 The
differentiation of Th1 T cells is dependent on the transcription factor
T-bet,17 and mice deficient in T-bet have reduced IFN-g responses and
Immunology and Cell Biology
Reference
4
2
60
113
3
114
increased susceptibility following M. tuberculosis infection.18 Interest-
ingly, the lungs of M. tuberculosis T-bet / mice develop a striking
infiltrate of eosinophilic macrophages and multinucleate giant cells
with neutrophils rather than well-formed granulomas. Within the
granuloma, IFN-g stimulates macrophages to kill mycobacteria
through a variety of pathways, including activation of phagocyte
oxidase and inducible nitric synthase,19 to produce reactive oxygen
and nitrogen metabolites, and the upregulation of the GTPase, LRG47,
which stimulates phago-lysosomal fusion.20
The development of mycobacteria-specific IFN-g secreting effector
T cells is also dependent on the cytokine milieu during their activation
by infected DCs. IL-12 and IL-23 are critical cytokines for the
development of this Th1-like pattern of cellular immunity. They are
heterodimeric cytokines composed of a shared IL-12p40 chain with
IL-12p35 or IL-23p19 chains for IL-12 and IL-23, respectively, and
their receptors also share a common IL-12Rb1 chain.21 Humans with
mutations in either IL-12p40 or IL-12Rb1 are deficient in both IL-12
and IL-23 signalling and have reduced IFN-g T-cell responses.22 These
subjects were identified because of increased susceptibility to myco-
bacterial MSMD and Salmonella infections, but there are significant
differences to those with IFN-g signalling deficiency. The infectious
syndromes are not as severe and may present later in life. Following
successful therapy, they do not recur as readily, suggesting that partial
immunity has developed despite IL-12/IL-23 deficiency.23 Moreover,
these subjects do develop granulomas with aggregations of lympho-
cytes and macrophages in infected organs, rather than the sheets
of heavily infected macrophages present in IFN-g-deficient subjects.
This has led to the suggestion that the IL-12/IL-23 axis is not as
important in humans23 as in mice, where IL-12p40 is essential for the
development of competent antimycobacterial T-cell responses.24 Some
IL-12Rb1-deficient individuals show evidence of an IL-12-indepen-
dent pathway of IFN-g production,25 and this may be sufficient for
macrophage activation and containment of mycobacterial infection
within granulomas in some individuals. In addition, there are func-
tional differences between different IL-12Rb1 alleles so that the degree
of signalling impairment, subsequent IFN-g deficiency and clinical
phenotype varies between individuals.26
IL-23 is also important in the differentiation of a separate effector
T-cell population secreting pro-inflammatory cytokines of the IL-17
family and TNF. These IL-17-secreting T cells are implicated in the
pathogenesis of autoimmune inflammatory processes in mice,27 and
the major role for IL-23 was suggested to be the development of
these pro-inflammatory TH17 cells, whereas IL-12 is the critical
regulatory cytokine for immunity against intracellular pathogens.
Indeed, IL-12 can compensate for the absence of IL-23 in the control
of M. tuberculosis infection;28 however, this does not exclude IL-23

playing a role during mycobacterial infections. Plasmid IL-23 is as
effective as plasmid IL-12, an adjuvant to enhance the protective
effects of DNA vaccines against pulmonary TB,29 and plasmid IL-23
can complement IL-12p40 deficiency in the induction of protective
IFN-g T-cell responses against TB.30 In addition, it is now clear that
the induction of IL-17-producing T-cell responses are not dependent
on IL-23 in vivo, but rather they develop in the absence of the
inhibitory effects of IFN-g. Thus, BCG immunization of IFN-g or
IL-12p40-deficient mice leads to the emergence of mycobacteria-
specific T cells secreting IL-17 and TNF.30 The contribution of these
pro-inflammatory T cells to granuloma formation and their role
in protective immunity remains to be clarified.
Regulation of inflammatory responses
Although granulomas are essential for the containment of mycobac-
teria, this intense inflammatory response in the presence of persisting
mycobacteria has the propensity for tissue damage. Therefore, regula-
tion of the granulomatous process is essential. This occurs in part
through mechanisms for regulating the expansion of activated T cells
common to other infections; however, additional processes for
regulating the granulomatous response have been defined. First,
IFN-g itself, while essential for granuloma formation, may also
limit the uncontrolled expansion of mycobacteria-reactive T cells.
In the M. avium model of pulmonary infection, IFN-g / mice
accumulate large lymphocyte infiltrates in the lungs, which undergo
necrosis and form cavities consistent with unrestrained expansion of
the T cells.31 Second, endogenous IL-10, produced during mycobac-
terial infections, can reduce IL-12 production from mycobacteria-
infected DCs, migration of DCs to draining lymph nodes and the
expansion of IFN-g-secreting T cells.32 In addition, IL-10 ‘de-activates’
macrophages and reduces the impact of IFN-g on macrophage
activation and resultant mycobacterial killing. This effect is most
apparent during infection with less virulent mycobacteria such as
M. avium; however, IL-10-deficient mice, infected with M. tuberculosis,
show a transient increase in IFN-g-secreting T cells and decrease in
bacterial burden, consistent with a partial inhibitory effect of IL-10.33
An additional characteristic of T-bet / mice during M. tuberculosis
infection was the increased production of IL-10 in the lungs as well as
reduced IFN-g production, and this IL-10 may have contributed to the
ineffectual macrophage responses in these mice.18 Overexpression of
huIL-10 in antigen-presenting cells in the lungs of M. avium-infected
mice resulted in markedly increased bacterial burdens with a marked
increase in macrophages in the granulomas.34 Macrophage apoptosis
as well as the production of TNF, IL-12p40 and nitric oxide were
reduced, indicating that IL-10 from macrophages and DCs exerts an
inhibitory effect on the control of mycobacterial infection by suppres-
sing macrophage effector mechanisms.
More recently, IL-27 has been found to provide another regulatory
network for controlling inflammatory responses to mycobacteria. IL-
27, which is a heterodimeric cytokine produced by DCs and macro-
phages, was first characterized as an immunoregulatory cytokine that
increased the production of IFN-g from naı̈ve CD4 T cells.35 In vitro
studies demonstrated that signalling through IL-27R on CD4 T cells
activated Stat1 and T-bet, suggesting IL-27 sensitized naı̈ve CD4
T cells to the Th1 polarizing effects of IL-12.36 Subsequently,
M. tuberculosis infection of IL-27R (WSX-1)-deficient mice revealed
that IL-27 played an inhibitory role in the protective immune response
during the first 3 months of infection, when the IL-27R / mice
showed reduced bacterial burden in the lungs.37 Late accumulation
of IFN-g-secreting cells in the IL-27R / mice caused severe lung
pathology. This is consistent with the lethal CD4 T-cell-mediated
Life and death in the granuloma
BM Saunders and WJ Britton
105
immunopathology that developed in IL-27 / mice infected with
Toxoplasma gondii.38 This downregulation of the adaptive CD4 T-cell
response to infection by IL-27 occurs through a variety of mechan-
isms, including the IL-27-mediated inhibition of IL-2 production
by CD4 T cells and the increased expression of SOCS-3.38 IL-27
also inhibits IL-17 production, independent of the IFN-g-mediated
suppression of IL-17.39
Finally, T regulatory cells (Tregs) may also inhibit the CD4 T-cell
response and limit the granulomatous inflammatory response to
M. tuberculosis. Urdahl et al.40 have recently shown that Foxp3-
expressing Tregs are recruited to the lung during experimental TB
infection in mice along with effector T cells. Depletion of Tregs before
infection resulted in increased clearance of the bacteria from the lung,
indicating that the Tregs were inhibiting the T-cell-effector mechan-
isms. The mechanisms of this effect of Tregs during mouse TB and
their possible role in human TB remain to be elucidated.
MICROARCHITECTURE OF THE GRANULOMA
The presence of granulomas in infected tissue is a hallmark of
mycobacterial disease.
This requires the coordinated recruitment of macrophages and
lymphocytes across the endothelium, their migration through infected
tissues and aggregation to form granulomas. This permits the close
interaction of antigen-specific T cells with infected macrophages,
which act to contain the bacilli and prevent further spread. The
cellular and chemical factors controlling granuloma formation are
considerable and incompletely understood. It is clear, however, that
members of the TNF family, most notably TNF and LTa, are involved
in this process.
TNF family members and granuloma development
The TNF superfamily currently consists of 19 ligands and 29 recep-
tors.41 TNF ligands are type II transmembrane proteins, that assemble
as biologically active trimers. TNF receptors are type I transmembrane
proteins characterized by the presence of one to six cysteine-rich
domains.41 TNF family members have wide-ranging involvement in
immune homeostasis, activation of both macrophages and T cells, and
inflammation. To date the function of at least 20 family members
has been examined during host responses mycobacterial infections
(Figure 1). Five of these molecules (soluble TNF, membrane-bound
TNF (MemTNF), LTa, TNFRI and CD40) are essential for normal
granuloma formation and survival following M. tuberculosis infec-
tion.42–46 For example, in the absence of TNF, there was a delay in the
initial recruitment of monocytes into infected tissue.42,43 Despite
normal activation of antigen-specific T cells,42,43 this was followed
by a dysregulated inflammatory response, with the influx of neutro-
phils, formation of necrotic lesions, uncontrolled bacterial growth
and the rapid demise of infected animals. Interestingly, mice lacking
LTa, which binds to the TNFRI molecule in addition to TNFRII and
HVEM, also exhibit marked susceptibility to TB infection, with a
similar dysregulated inflammation and overwhelming bacterial growth
despite the production of normal levels of TNF.45 This demonstrated
separate, non-compensatory functions for TNF and LTa, and this has
subsequently been confirmed during malaria and Listeria mono-
cytogenes infections.47,48
Both TNF and LTa bind TNFRI and TNFRII; however, signalling
for protective effects occurs primarily through TNFRI, as TNFRII /
mice display only minor increased susceptibility to mycobacterial
infection.49 LTa also binds the HVEM receptor, but the importance
of this interaction in antibacterial immunity or inflammation has not
been established. Interestingly, TNF- and LTa-deficient mice display
Immunology and Cell Biology
 Life and death in the granuloma
BM Saunders and WJ Britton
106
Response to mycobacterial infection
CD95L Reported
CD40L
Unreported
CD30L CD27L SolubleMem
OX40L 4-1BBL TNF TNF LTα LTαβ LIGHT
OX40 CD27 TNFRI TNFRII LTβR
CD40 CD30 4-1BB
CD95
Figure 1 TNF superfamily involvement in granuloma formation and resistance to mycobacterial infection. Reported: Dark gray ligands and receptors are
essential for normal granuloma formation and sustained resistance to mycobacterial infection. Pale gray ligands and receptors are required for optimal
protective immunity to mycobacterial infection. Unfilled ligands and receptors were not required for normal granuloma formation and expression of protective
immunity to mycobacterial infection. Unreported: Ligands and receptors whose function in granuloma formation and resistance to mycobacterial infection
has not yet been reported.
marked differences in their response to Mycobacterium leprae infec-
tion.50 While loss of either cytokine augments growth of M. leprae,
leukocyte infiltration in TNF / mice is more extensive with some
lymphocyte infiltration, while LTa / mice exhibited less inflamma-
tion with minimal lymphocyte infiltration. Further, while the chemo-
kine responses in infected wild type (WT) and TNF / mice were
similar, LTa mice demonstrated five- to 25-fold lower levels of mRNA
expression for the chemokines tested. Therefore, the inflammatory
response of TNF- and LTa-deficient mice are not identical and are
influenced by the virulence of the mycobacterial pathogen.
Soluble and membrane-bound forms of TNF and LT make different
contributions to granuloma formation and protective immunity.
Deletion of LTa removes both soluble and membrane-bound forms
of LT, whereas deletion of LTb leaves the soluble LTa trimer intact.
We have previously shown that mice lacking LTa, but not LTb, are
more susceptible to TB infection,45 indicating that it is the soluble LTa
that coordinates granuloma formation. TNF also has membrane-
bound and soluble forms. In this instance, both memTNF and soluble
TNF are required for optimal resistance to mycobacterial infec-
tion.44,51 MemTNF alone was sufficient for the establishment of
normal granulomatous lesions and containment of bacterial growth
for three months.44 Further, these mice developed a normal antigen-
specific T-cell response, but despite this, increased numbers of
activated T cells accumulated in the lung during the chronic phase
of infection. This resulted in progressive inflammation in the lung,
and the memTNF mice succumbed after day 120 despite control of
bacterial growth.44,51 Therefore, memTNF is sufficient for the initial
formation of granulomas, but soluble TNF is required for long-term
optimal control of mycobacterial infection.
Investigation of other members of the TNF superfamily (Figure 1)
has demonstrated that TNFRII, CD40L, CD95, LIGHT and CD30 are
required for optimal immunity to mycobacterial infection. Mice
lacking these TNF family molecules showed transient or partial
susceptibility to mycobacterial infection. A common feature of these
molecules is their expression during the immunopathological response
to TB infection. For instance, CD40L is expressed on alveolar macro-
phages of tuberculoid granulomas, and increased mRNA for CD40L is
present in M. tuberculosis-infected macrophages.52,53 CD40–CD40L
interaction induces a cascade of events, including the production of
Immunology and Cell Biology
BTLA
GITRL
TWEAK EDA
TRAIL RANKL BAFF APRIL
DcR3 DR4 OPG
BAFFR BCMA
HVEM DR5 RANK TACI Fn14 EDAR
XEDAR
DcR1 GITR
DcR2 HVEM
cytokines and chemokines. The absence of CD40L impaired granu-
loma formation after BCG and M. avium infection, leading to
increased necrosis and impaired control of bacterial growth; however,
CD40L / mice survived M. tuberculosis infection despite impaired
granuloma formation.46,54–56 Interestingly, CD40 / mice were more
susceptible to M. tuberculosis infection than CD40L / mice, exhibit-
ing marked inflammation with increased bacterial growth.46 Human
studies identified variants in the CD40L gene, but none of these
was associated with increased susceptibility to TB.57
LIGHT, which shares common receptors with LT, is required for the
optimal activation of both CD4 and CD8 T cells in vitro.58 Never-
theless, LIGHT / mice infected with M. tuberculosis displayed only
a transient exacerbation of bacterial load at 4 weeks post infection,
with no change in the granulomatous response (Saunders, unpub-
lished observations, Ehlers et al.59). FAS/FASL signalling is critical for
T-cell homeostasis and activation of macrophage apoptosis. Mice
deficient in FAS/FASL signalling controlled acute TB infection, but
displayed increased pyogenic inflammation and bacterial growth
during the chronic phase of infection.60 Therefore, signalling through
both TNFRI and FAS is required for optimal immunity to mycobac-
terial infection and for regulation of the inflammatory response.
Finally, antibody neutralization of the TNF family members, CD27,
CD30, IBB1 and OX40L, indicated that only CD30 was required to
control M. avium infection.61 Further, CD30 / mice displayed
reduced T-cell activation and diminished lymphocyte cuffing around
granulomas, confirming its requirement for optimal granuloma form-
ation. A common feature of TNF family members, in particular TNF
and LTa, in immunity to TB infection, is their regulation of chemo-
kine and chemokine receptor expression, that controls the recruitment
of inflammatory cells.
Chemokine requirements for granuloma formation
The differential regulation of chemokines and their receptors during
M. tuberculosis infection in humans is increasingly recognized
(Table 2); however, the function and contribution of individual
chemokines or receptors to this response is still uncertain. The
establishment of chemokine gradients is crucial for the recruitment
of inflammatory cells and their aggregation to form granulomas.
Multiple studies have demonstrated elevated levels of chemokines,
 Life and death in the granuloma
BM Saunders and WJ Britton
107

Table 2 Chemokine expression in tissues from M. tuberculosis-infected individuals

In vivo/ex
vivo sample Chemokines Response Reference

Pleura MIP-1a, MIP-1b Increased expression in pleura fluid from TB patients 64,115–117

Mig, RANTES MIP-1a, Mig, RANTES and IP-10 were expressed on pleural mononuclear cells and stroma cells,
IP-10, MCP-1 whereas CXCR3 and CCR5 were expressed on T cells in pleura
MCP-1, MIP-1a, MIP-1b Compared to TB infection alone, MCP-1 expression increased in HIV-TB co-infected individuals,
whereas MIP-1a, MIP-1b and RANTES expression was decreased
BALF IP-10, IL-8 Increased in TB patients 62,65,66,118,119

MCP-1, MCP-3, MCP-4

RANTES
MIP-1a Not increased over healthy controls
Exotaxin
Lung MCP-1, MCP-3, MCP-4, IP- Increased in TB patients 66,120

10 TGF-b, IFN-g and IL-12 were also increased in TB granulomas, whereas IL-4 and IL-10 were
decreased
Eotaxin Not increased
Alveolar CCR5 Upregulated following M. tuberculosis infection in PBMC and alveolar macrophages 65,74,121

macropha- RANTES
ges MIP-1a
MCP-1
Plasma IL-8, IP-10 Elevated in TB patients and in those with concurrent HIV infection 73,117,122–124

MCP-1, RANTES IP-10 and MCP-1 even higher in HIV-co-infected patients than TB infected individuals alone
MIP-1a, MIP-1b
RANTES
MCP-1 Not increased in all TB patients, higher in patients with extrapulmonary TB
PBMC MIP-1a Increased in M. tuberculosis-infected PBMC from TB patients. Levels were lower in HIV co-infected 65,125

RANTES individuals 126

127

MCP-1 Increased mRNA and protein in monocytes from TB patients

IL-8 Increased following M. tuberculosis infection
Cerebral MCP-1, IL-8, MIP-1a Elevated in CSF from patients with pyogenic and tuberculous meningitis 128

spinal fluid

including MCP-1, MIP-1a, RANTES, IL-8 and IP-10 in the serum and
bronchial alveolar lavage of TB patients, compared to healthy con-
trols.62–66 Human monocytes, macrophages and DCs infected with
M. tuberculosis produce elevated levels of multiple chemokines,
including MIP-1a, MIP-1b, MCP-1, MCP-3, RANTES, IP-10 and
IL-8.67–71 There is also growing evidence that the expression of
chemokines and the chemokine receptors CCR5 and CXCR4 are
perturbed in HIV co-infected individuals, with increased and
decreased expression depending upon the study and chemokine or
receptor examined. This altered expression may be one mechanism
leading to enhanced disease progression in HIV M. tuberculosis-
co-infected individuals.72,73
The exact requirement for individual chemokines and their recep-
tors in generating protective granuloma formation during TB infec-
tion remains uncertain. Studies utilizing gene-deficient mice have
generally indicated a high degree of redundancy in this system
(Table 3). Indeed, the absence of individual chemokines is adequately
compensated during murine M. tuberculosis infection. As most che-
mokine receptors are bound by multiple ligands, these may be more
critical to coordinate cellular inflammation and granuloma formation.
For instance, CCR1 binds several ligands, including MIP-1a, MIP-1b
RANTES and MCP-3, whose expression is upregulated during myco-
bacterial infection.74 Nevertheless, CCR1 / mice control M. tubercu-
losis infection normally, with no abnormality in granuloma size and
structure (Saunders, unpublished observations). Indeed, of the che-
mokine and chemokine receptor knockout mice challenged with
M. tuberculosis (Table 3),75–79 only CCR2 was essential to control high
dose M. tuberculosis infection,76,78 with aberrant granuloma formation
a distinguishing feature of this enhanced susceptibility.
Despite this redundancy in the effect of chemokines in mice,
individual chemokines may be important in the resistance of humans
to TB. For example, genetic analysis revealed that a single-nucleotide
polymorphism in the MCP-1 gene was associated with increased risk
of developing pulmonary TB.80 WT individuals and homozygotes
for this mutation show an inverse relationship between MCP-1 and
IL-12p40 levels. Interestingly, MCP-1 / mice show equivalent control
of M. tuberculosis infection to WT mice,81 suggesting that there may
be differences in the requirements for individual chemokines and
their receptors between humans and mice.
WHEN GRANULOMAS FAIL
Granulomas fail to form in the absence of either adaptive IFN-g-
secreting CD4 T-cell responses or the co-locating signals provided by
TNF and its family members. This is evident in gene-deficient strains
of mice and subjects lacking functional IFN-g receptor/STAT1 signal-
ling.14 But the majority of humans infected with M. tuberculosis
control the initial infection through the formation of granulomas,
which provide the microenvironment where specific T cells activate
macrophages to contain the infection. Despite optimal activation,
human macrophages fail to eradicate M. tuberculosis infection, and the
dormant bacilli may persist for decades. In a minority of infected
subjects, the granuloma fails for a variety of reasons, leading to

Immunology and Cell Biology

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BM Saunders and WJ Britton
108

Table 3 Response of chemokine ligand and receptor-deficient mice to mycobacterial infection

Chemokine or chemokine
receptor Infection (strain, route) Results Reference

CCR1 M. tuberculosis No effect on bacterial growth or granuloma formation Saunders, unpublished observations
Aerosol
CCR2 M. tuberculosis Low dose, aerosol: normal immunity 76,129

Aerosol, IV High dose, IV: increased susceptibility

CCR5 M. tuberculosis No difference in bacterial growth; normal granuloma formation 75,130

Aerosol Increased lymphocyte infiltration

CXCR2 M. avium No difference in bacterial growth or recruitment of neutrophils to 77

Aerosol the lungs

CXCR3 M. tuberculosis No difference in bacterial growth or survival 79

Aerosol Initial granuloma formation diminished, but effect was lost by

day 60. Similar effect in anti-MIG (CXCR3 ligand)-treated mice
MCP-1(CCL2) M. tuberculosis Transient, increased bacterial growth 81,129

Aerosol and IV

the reactivation of infection and clinical TB.1 Understanding the
process of reactivation is important to devise new strategies to
control TB.
Differences in mycobacterial granulomas between species
The mouse model has provided important information on the
cytokine and cellular requirements for granuloma formation, but
there are significant differences in the granulomatous response to
M. tuberculosis in the lung between mice and humans.82 In the mouse,
there is co-location of CD4 T cells and macrophages, some of which
differentiate into epithelioid cells, but multinucleate giant cells are
absent and the central caseous necrosis, typical of human tuberculous
granulomas, fails to develop.83 With time there is increasing accumu-
lation of CD4 T cells, which form dense wedges in the lungs, whereas
CD8 T cells tend to be scattered at the edge of the granulomas.82 There
is also progressive fibrin deposition around the pulmonary granulo-
mas.83 By contrast, the granulomas that develop about M. tuberculosis-
infected macrophages in the human lung are characterized by a central
region of large CD68+ epithelioid cells, surrounded by a mixture of
macrophages and predominantly CD4 T cells, with a smaller number
of CD8 T cells and multinucleate giant Langhans cells.84 Epithelioid-
like cells develop from M. tuberculosis-infected macrophages in the
presence of continuous cytokine stimulation, and in the presence of
IL-4 and GM-CSF in vitro, these cells fuse to form giant cells.85 In the
highly organized granulomas typical of tuberculoid leprosy, the CD8
T cells form a peripheral cuff around the granulomas.86 Aggregates of
lymphocytes, both T cells and B cells, form adjacent to granulomas
in the human lung, and some B cell aggregates also occur in murine
tuberculosis.82,87
The characteristic feature of human granulomas is the development
of a central, acellular eosinophilic region of caseous necrosis. Both
host and mycobacterial factors contribute to caseation. Under intense
cytokine and direct cell–cell activation, macrophages and epithelioid
cells undergo necrosis and/or apoptosis to form this material.88
Signalling through TNFRI, Fas and the perimetric receptor P2X7
leads to apoptosis of infected human macrophages.89–91 Caseating
granulomas contain apoptotic macrophages and granulocytes,88
whereas T-cell-derived perforin and granulysin and macrophage-
derived reactive oxygen and nitrogen metabolites may also contribute
to the necrosis of the cells.92–94 In addition, macrophages release
proteolytic enzymes such as metalloproteinase 9, which may lead to
cellular breakdown.95 Mycobacterial products such as ESAT 6 may also
cause lysis of lung cells and facilitate bacterial spread.96 Mutants with
deletions of some regulatory mycobacterial genes such as SigH cause
less immunopathology even though the mycobacteria persist in the
lung, suggesting that some mycobacterial products contribute to
necrosis.97 The caseous material may proceed to calcify, and the
granuloma is surrounded by fibrosis to form the chronic Ghon
focus marking past TB infection.
Caseation is central to the ‘life cycle’ of human TB infection as it
carries the potential for the granuloma to erode into air passages to
form a cavity, and into the blood vessels to permit dissemination. This
provides access for tuberculous bacilli to the respiratory tree and to
spread through coughing to other subjects, thus maintaining infection
in the population. The central region of the human granuloma
becomes devoid of blood vessels and hypoxic, and this may further
increase the propensity for necrosis.82,84 This is another significant
difference between the human and mouse granulomatous response to
M. tuberculosis. Studies with hypoxia-sensitive chemicals and direct
oxygen measurements have revealed that the granulomatous regions in
M. tuberculosis-infected lung are not hypoxic in the mouse.82,98 This
limits the suitability of the mouse model for the assessment of
anaerobic anti-microbials for use in human TB.
The wall of the tuberculous cavity demonstrates a gradient from
relatively effective to ineffective granulomatous responses to
M. tuberculosis. Immunohistochemical analysis of human tuberculous
cavities following surgical removal revealed that CD68+ macrophages
accumulated at the luminal surface of the cavity and that these
contained abundant bacilli.82,84,99 Previously, it was inferred that the
mycobacteria replicated in the extracellular environment in the cavity
because it is oxygen rich through its communication with the airways,
but it appears that mycobacteria rapidly replicated within macro-
phages on the surface of the cavity.100,101 Immediately below the
surface was the layer of acellular, caseous material containing few
mycobacteria. The next layer of granulomatous material contains an
aggregation of CD4 and CD8 T cells, epithelioid macrophages and
Langhans cells. These macrophages contained far few mycobacteria,
suggesting that their growth in this region was limited by T cell-
derived signals. This cellular region was surrounded by a fibrous layer,
that separated the cavity from the normal lung. There was increased
expression of mRNA for IFN-g and iNOS in these samples, indicating
that T cell responses were retained in the walls and areas adjacent to
the cavity; however, these had been insufficient to prevent reactivation
of the infection.100

Immunology and Cell Biology


Mechanisms of granuloma failure
Granulomas may fail for two broad reasons. First, the M. tuberculosis-
specific adaptive T cell response may wane or be perturbed for genetic
or environmental reasons. Racial variation in the susceptibility to TB
and twin studies strongly suggest that genetic factors are important
in the reactivation of TB, and polymorphisms in at least 14 genes have
been associated with reactivation disease.102 Variants in a gene may
interact with environmental factors to increase the risk of TB, as is the
case with polymorphisms in the gene for vitamin D receptor and
vitamin D deficiency.103 HIV co-infection is the most dramatic of the
multiple environmental factors associated with failure of the T cells to
control M. tuberculosis infection.104 The lifelong risk of active TB is
50% in subjects co-infected with M. tuberculosis and HIV compared to
10% in non-HIV-infected subjects, and this risk is directly related to
the degree of loss of CD4 T cells. During HIV co-infection, granulo-
mas fail to form in the absence of T cell-derived macrophage
activating cytokines, and infected organs are infiltrated with disorga-
nized sheets of infected macrophages.
The critical role of CD4 T cells in granuloma formation is high-
lighted by the impact of highly active antiretroviral therapy (HAART)
in HIV+ subjects co-infected with M. tuberculosis or members of the
Mycobacterium avium complex.105 The reconstitution of adaptive
immunity and rise in CD4 T cell count is associated with cytokine
release, activation of mycobacteria-infected macrophages and a gran-
ulomatous infiltrate of lung or lymph nodes. This immune recon-
stitution inflammatory syndrome can cause significant pathological
damage and require suppression with corticosteroids.106 The capacity
of granulomas to cause tissue damage is dramatically illustrated by the
effects of HAART in subjects with latent infection with M. leprae. The
resurgent CD4 T cell response results in the formation of granulomas
around infected macrophages and Schwann cells in the skin and
nerves, leading to a severe, reversal or type I leprosy reaction.107
The second mechanism underlying the breakdown of granulomas is
the failure of co-location signals for macrophages and T cells, despite
the presence of otherwise effective anti-mycobacterial T cell responses.
These signals are essential for the maintenance, as well as the
formation, of granulomas in both human and murine TB.108 This is
illustrated by the increased rate of reactivation of clinical TB in
subjects with LTBI receiving anti-TNF therapy for rheumatoid arthri-
tis or Crohn’s disease.109,110 Sustained TNF signalling is required for
the maintenance of the local chemokine gradients necessary to hold
macrophages and lymphocytes in close apposition within the granu-
loma and so permit macrophage activation. Reactivation of TB can
occur rapidly within three months of anti-TNF mAb (Infliximab)
therapy, indicating that the granuloma is a dynamic microenviron-
ment and that continuous activation of infected macrophages is
necessary to restrain replication of mycobacteria. The risk of reactiva-
tion of LTBI is greater following therapy with Infliximab than soluble
TNFRII, and this may be related to the fact that Infliximab inhibits
both soluble and membrane TNF, while soluble TNFRII only inhibits
soluble TNF.111 This is consistent with the capacity of memTNF alone
to provide partial control M. tuberculosis and other intracellular
pathogens in mice.44,112 Other cytokines, chemokines and adhesion
molecules contribute to the cellular migration and cell–cell interaction
necessary for granuloma formations. Although there may be consider-
able redundancy in this process, biological inhibitors to these mole-
cules, such as IL-1b, ICAM and LFA-I which are in use or clinical
trials, may also lead to disaggregation of the granuloma and reactiva-
tion of latent infection.
In summary, granulomas are the essential microenvironment in
which host T cell responses restrain the growth of mycobacteria in
Life and death in the granuloma
BM Saunders and WJ Britton
109
macrophages. At the same time, granulomas have the potential for
pathological damage through caseous necrosis and fibrosis. Erosion
by the granuloma into the airways provides the route of transmission
for M. tuberculosis and maintenance of TB infection within the
population.
ACKNOWLEDGEMENTS
This work in our laboratory is supported by the NHMRC of Australia and the
NSW Department of Health through its infrastructure grant to the Centenary
Institute.
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