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* Centenary Institute of Cancer Medicine and Cell Biology, Sydney, Australia;
Department of Medicine, University of Sydney, Sydney, Australia;
Laboratoire d’Ingenierie des Anticorps, Institut Pasteur, Paris, France; and
Department of Veterinary Anatomy and Pathology, University of Sydney, Sydney, Australia
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting
CD4+ and CD8+ T cells
(2, 3). Granuloma formation, however, is a complex process
that requires not only the activation of the lymphocytes, but also
their recruitment with monocytes to the site of the infection,
migration into the tissues, and juxtaposition around
mycobacteria-infected macrophages (4). This colocation
facilitates the activation of bactericidal mechanisms in infected
macrophages by T cell-derived cytokines (1). Some
mycobacteria, however, survive within macrophages, and persistent
antigenic stimulation perpetuates the process leading to chronic
granuloma formation characterized by dense accumulations of infected
macrophages, epithelioid cells, and T lymphocytes (5).
These granulomas contain the mycobacterial infection and prevent
dissemination to other organs, but they are also responsible for lung
immunopathology, as the granulomas displace and destroy parenchymal
tissue (6). The cytokine and chemokine signals that
regulate granuloma formation and persistence are poorly understood,
although signaling through TNF receptor I plays an essential role
(7, 8).
TNF (previously known as TNF-
) is a highly potent proinflammatory
cytokine with a wide range of activities in both inflammatory and
immune responses (9). TNF (8, 10) and the
related cytokine lymphotoxin-
(LT)4
(11) are essential for host resistance against infection
with Mycobacterium tuberculosis and other mycobacteria.
TNF-deficient (TNF-/-) mice infected by aerosol
with M. tuberculosis develop normal T cell responses to
mycobacterial Ags (10), but are profoundly susceptible to
the infection, succumbing with extensive necrosis in the lungs and
infected organs. A major defect is the failure of granuloma formation
in the infected organs of TNF-/- mice.
Dissecting the effects of TNF deficiency on the sequential steps
involved in granuloma formation in the lung is hampered in M.
tuberculosis infection by the rapidly progressive necrosis
associated with an influx of neutrophils observed in these mice. By
comparison, infection with less virulent mycobacteria allows analysis
of the effects of TNF deficiency on the induction of chemokines and
recruitment of leukocytes into granulomas.
Mycobacterium smegmatis is a rapidly growing mycobacterium that is usually nonpathogenic in immunocompetent subjects. A major component of mycobacterial cell wall is lipoarabinomannan (LAM), which is a complex polysaccharide composed of arabinan and mannan linked to the cell membrane by phosphatidylinositol (12). In the case of M. smegmatis and the avirulent H37Ra strain of M. tuberculosis, LAM is characterized by extensive arabinan side chains (araLAM), whereas in LAM from more virulent mycobacteria (M. tuberculosis H37RV and Mycobacterium bovis) the arabinan side chains are masked by mannan caps (manLAM) (13, 14). These forms of LAM differ markedly in their ability to stimulate TNF production from human and mouse macrophages (15, 16), with purified araLAM inducing more TNF secretion than purified manLAM. Therefore, it was proposed that mycobacterial virulence may be related in part to this differential induction of TNF by the two forms of LAM, with avirulent mycobacteria stimulating increased TNF production resulting in enhanced macrophage bactericidal activities and early clearance of the organisms (15). In this report, we have compared infection with M. smegmatis and M. tuberculosis infection in TNF-deficient (TNF-/-) and normal mice (wild type (WT)). We found that the initial clearance of M. smegmatis during the first week of infection was independent of TNF, but subsequently TNF deficiency resulted in delayed expression of chemokines and reduced cellular recruitment associated with delayed clearance of M. smegmatis. The emergence of an enhanced Th1-like T cell response was associated with the late induction of chemokines and control of the infection. By comparison, infection of TNF-/- mice with virulent M. tuberculosis was associated with an initial delay in chemokine induction and cellular infiltrate into the liver. Then, despite excessive chemokine production, there was a failure to form functional granulomas, resulting in fatal progressive infection. Therefore, although TNF is not essential for chemokine expression per se, it is required both for the early induction of chemokines that initiates timely cell recruitment and for establishing and maintaining the microenvironment of protective granulomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Control WT mice were 6- to 8 wk-old C57BL/6 mice obtained from the Animal Resource Center (Perth, Australia). TNF gene knockout mice (TNF-/-) prepared on a C57BL/6 background have been previously described (17). All mice were housed under specific pathogen-free conditions in the Centenary Institute animal facility until infection, when they were transferred and maintained in a level 2 (M. smegmatis) or level 3 (M. tuberculosis) physical containment facility.
Bacteria and experimental infections
M. smegmatis (mc2155) was grown in Middlebrook 7H9 liquid medium (Difco, Detroit, MI) supplemented with ADC for 7 days at 37°C. Mycobacterium bovis (bacille Calmette-Guérin (BCG)) was derived from the Glaxo strain; it was obtained from CSL Biosciences (Melbourne, Australia) and was cultured as described for M. smegmatis. The M. tuberculosis H37Rv (ATCC 27294; American Type Culture Collection, Manassas, VA) strain was cultured from a low passage seed lot in Proskauer-Beck liquid medium to midlog phase, aliquoted, and frozen at -70°C. TNF-/- and WT mice were infected with 5 x 107 CFU M. smegmatis or 1 x 104 CFU M. tuberculosis via a lateral tail vein. The numbers of viable bacteria in target organs were followed over time by plating serial dilutions of whole organ homogenates on supplemented Middlebrook 7H11 agar (Difco) and counting bacterial colony formation after 3 days (M. smegmatis) or 21 days (M. tuberculosis) of culture. M. smegmatis sonicate was prepared by sonication in PBS as previously described (18) and was stored at -70°C.
Macrophage infections
Bone marrow-derived macrophages were cultured from murine bone
marrow as previously described (11). After 7 days of
culture, these cells were either prestimulated with 100 U/ml IFN-
(Genzyme, Cambridge, MA) or medium alone for 18 h and then
infected with M. smegmatis or M. bovis (BCG) at a
multiplicity of infection of 10:1 for 6 h. Cells were washed to
remove extracellular bacteria and were cultured for 4 days. The
concentration of TNF in the culture supernatants was measured using the
WEHI 164 bioassay, as previously described (11).
Liver leukocyte preparations
Animals were sacrificed by carbon dioxide narcosis, and the liver was perfused with saline through the portal vein to remove blood-borne leukocytes. A single-cell suspension was prepared by sieving a liver lobe through a 200-µm pore size mesh. Liver leukocytes were obtained by spinning the suspension over an isotonic Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). The number of leukocytes in each preparation was counted using a Sysmex KX-21 hemocytometer (TOA Medical Electronics, Kobe, Japan). Flow cytometric analysis of each cell preparation was performed as previously described (3) to determine the number of CD11b+ (M1/70.15; Caltag Laboratories, San Francisco, CA) and CD4+ (CT-CD4; Caltag Laboratories) T cells in the sample.
Analysis of chemokine mRNA
Expression of chemokine mRNA in the livers of infected mice was
measured using the RiboQuant RNase protection assay system (BD
PharMingen, San Diego, CA) according to the manufacturer’s
instructions. Briefly, a 32P-labeled multiprobe
template set specific for lymphotactin, RANTES, eotaxin,
macrophage-inflammatory protein- (MIP-1), MIP-1
, MIP-2,
monocyte chemoattractant protein-1 (MCP-1), and T cell
activation-3 and 2 constitutively expressed genes GADPH and L32
was generated. Total liver RNA from uninfected and M.
smegmatis-infected or M. tuberculosis-infected WT and
TNF-/- mice was prepared using RNAzol B
(Cinna/Tel-Test, Friendswood, TX) according to the manufacturer’s
instructions, with an additional phenol-chloroform extraction before
RNA precipitation. Samples of total RNA from the liver (50 µg) of
uninfected and infected mice were incubated with 1.6 x
106 cpm of probe and incubated overnight at
56°C. Single-stranded RNA was digested with RNase, and the protected
probes were analyzed by PAGE. Gels were dried and exposed to x-ray film
or GEL-DOC 1000 detection screens (Bio-Rad, Hercules, CA). The
migration distance of bands of known sizes on each gel allowed
identification of the band representing each chemokine. The intensity
of each band on the phosphorimager was calculated using Molecular
Analyst software (Bio-Rad). Differences in the amounts of RNA between
samples were corrected for by expressing band intensity as a ratio of
the gene of interest to the constitutively expressed gene,
L32.
T cell responses to mycobacterial Ags
Spleens from M. smegmatis-infected
TNF-/- and WT mice were removed, and
single-cell suspensions were prepared. Erythrocytes were lysed in a
hypotonic ammonium chloride lysis buffer, and the remaining cells were
washed, counted, and suspended in complete RPMI 1640 medium
(Cytosystem, Sydney, Australia) with 10% FCS (Trace, Sydney,
Australia), 2 mM L-glutamine (Sigma-Aldrich, St.
Louis, MO), 10 mM HEPES (Sigma-Aldrich), 10 mM
Na2CO3, 0.5 µM 2-ME
(Sigma-Aldrich), 100 U/ml penicillin (Trace), and 100 µg/ml
streptomycin (CSL). To measure Ag-specific T cell responses,
splenocytes from M. smegmatis-infected cells were cultured
in the presence of M. smegmatis sonicate (10 µg/ml) or in
medium alone. Lymphocyte proliferation and cytokine assays for IFN-
were performed as described previously (19). For
proliferative responses, the cells were pulsed with 1 µCi of
[3H]thymidine (NEN Life Sciences, Boston, MA)
for the final 6 h of culture and then harvested onto glass fiber
filters. The incorporated [3H]thymidine was
determined by liquid scintillation spectroscopy (Pharmacia/Wallace Oy,
Turku, Finland). Specific [3H]thymidine
incorporation was calculated by subtracting the mean counts per minute
in unstimulated wells from the mean counts per minute of test samples.
The concentration of IFN- in culture supernatants was determined
with a capture ELISA using a mAb capture assay with the Abs R4-6A2 and
XMG1.2-biotin (Endogen, Woburn, MA) following the manufacturer’s
instructions. Avidin-alkaline phosphatase (Sigma-Aldrich) and
n-nitro-phenyl-phosphate (1 mg/ml in 10 mM
NaHCO3 and 0.1 mM MgCO3, pH
6.3) were used as the colorimetric reagents, and absorbance was
measured at 405 nm. Proliferation and IFN- release from uninfected
WT and TNF-/- mice were insignificant. To
determine the frequency of IFN--producing cells, splenocytes were
cultured in multiscreen 96-well filtration plates (Millipore, Bedford,
MA) for 16 h in the presence of M. smegmatis sonicate
or medium alone. The ELISPOTs were then developed as previously
described (19).
Histology
Liver tissue samples were fixed in 10% neutral buffered Formalin, processed into paraffin blocks, and sectioned at 5 µm. Sections were stained with H&E, coded, and analyzed in a blinded fashion to assess the number and type of infiltrating leukocytes in the livers of WT and TNF-/- mice. A focus of inflammatory cells (foci) was defined as a collection of 10 or more macrophages and lymphocytes in a cluster.
Serum nitrite measurements
Serum nitrite was assayed by a modification of the nitrate kit for food analysis (Roche, Mannheim, Germany). Briefly, serum nitrate was reduced to nitrite using nitrate reductase. Nitrite levels in the samples were determined using the Greiss reagent (3% phosphoric acid, 1% p-amino-benzene-sulfonamide, and 1% n-1-napthylenediamide (Sigma-Aldrich)). Samples were incubated for 5 min at room temperature, and absorbance was measured at 540 nm.
Statistical analysis
Where appropriate, values were tested for statistical significance by unpaired Student’s t test using StatView (SAS Institute, Cary, NC). Colony-forming unit values were subjected to log10 transformation before analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether M. smegmatis as an avirulent
mycobacteria was more potent in inducing TNF than a more virulent
species, bone marrow-derived macrophages were infected with either
M. smegmatis or M. bovis (BCG), and the
production of TNF was monitored over time. Macrophages infected with
M. smegmatis produced significantly larger amounts of TNF
than those infected with M. bovis BCG over 72 h of
culture (Fig. 1
A;
p < 0.05). Prestimulation of the macrophages for
16 h with IFN- (100 U/ml) abrogated this difference, such that
macrophages infected with either M. smegmatis or M.
bovis (BCG) produced comparable amounts of TNF (Fig. 1B).
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TNF-/- and WT mice were infected with
M. smegmatis, and the clearance of bacteria from the primary
sites of infection, the liver and spleen, was monitored over time. Both
WT and TNF-/- mice controlled the infection in
the liver (Fig. 2A) and spleen
(Fig. 2B) to an equivalent extent over the first 7 days
following infection. The rapid clearance of bacteria continued over the
following 14 days in WT mice. In contrast, bacterial clearance
significantly slowed after 7 days in TNF-/-
mice. There were significantly more bacteria in the liver and spleen of
TNF-/- mice at 14 and 21 days postinfection
compared with WT mice (p < 0.05). After day 21
the clearance rate in TNF-/- increased, so that
by 28 days postinfection bacteria were cleared from both WT and
TNF-/- mice.
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To determine whether the delay in clearance of bacteria was
associated with differences in the recruitment of leukocytes to the
sites of infection and subsequent granuloma formation, the histological
appearance and cellular infiltrate in the liver were examined over the
course of infection in both WT and TNF-/- mice.
In WT mice, a florid monocytic infiltrate was
generated. The number of foci of
inflammatory cells (Fig. 3A and Fig. 4, A and C) and the
total number of leukocytes recruited into the liver (Fig. 3B) peaked on day 14 postinfection in WT mice and then
declined by day 28 postinfection. By contrast, recruitment of
leukocytes into the liver and subsequent formation of foci of
inflammatory cells were significantly delayed in
TNF-/- mice following infection with M.
smegmatis (Fig. 3, A and B, and Fig. 4, B and D). In the absence of TNF, liver leukocyte
numbers did not increase until day 21 postinfection, and by day 28 the
cellular infiltrate of lymphocytes and macrophages was still only
loosely clustered. Flow cytometric analysis of the isolated liver
leukocytes from the WT mice demonstrated that the influx of macrophages
(CD11b+; Fig. 3C) and
CD4+ T cells (Fig. 3D) peaked on days
7 and 14, respectively. In the TNF-/- mice, the
recruitment of CD11b+ cells and
CD4+ T cells was markedly delayed (Fig. 3, C and D), although the absolute rise in numbers
of CD4+ T cells was greater in
TNF-/- than in WT controls.
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To determine the parameters of chemokine induction after M.
smegmatis infection, the relative expression of mRNA for several
chemokines in the liver was measured by RNase protection assays. Both
the kinetics and pattern of chemokine induced differed between WT and
TNF-/- mice (Fig. 5). In WT mice, increased transcription
for RANTES and MCP-1 was evident on day 7 postinfection. mRNA for
RANTES, MIP-1, MIP-1, MIP-2, MCP-1, and eotoxin were up-regulated
in the liver (Fig. 5), with expression peaking on day 14 postinfection
(Fig. 6). In contrast, induction of
chemokine mRNA in TNF-/- was delayed over the
first 2 wk of infection, but from day 21 chemokine expression developed
independently of TNF (Fig. 5). Induction of MIP-2 mRNA was first
evident in TNF-/- mice on day 21 postinfection
(Fig. 5). By day 28 MIP-1 and MIP-1 expression was comparable to
levels observed in WT mice on day 14, while relatively low levels of
RANTES, MIP-2, and MCP-1 were expressed (Fig. 6)
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To determine whether the delayed up-regulation of chemokines and
subsequent clearance of bacteria in TNF-/- mice
were related to differences in the T cell responses, T cell function
was analyzed in WT and TNF-/- mice over the
course of infection. Ag-specific T cell responses were observed in both
WT and TNF-/- mice from day 14 of infection.
Comparable production of IFN- and proliferative responses from
splenic cultures were observed on days 14 and 21 postinfection in both
WT and TNF-/- mice (Fig. 7). The responses in WT mice then
declined, but continued to increase in
TNF-/- mice, so that by day 28 postinfection a
significantly enhanced T cell response was evident in
TNF-/- mice (p <
0.05). This was confirmed by the observation that the frequency of
Ag-specific IFN--producing cells was significantly higher in
TNF-/- mice on day 28 postinfection
(p < 0.05; Fig. 7C).
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To monitor the production of RNI, the major bactericidal effector
species for mycobacterial killing in mice, the concentration of serum
nitrates were measured. In WT mice the serum nitrate concentration
peaked on day 14 postinfection (Table I).
In contrast, serum nitrate concentrations in
TNF-/- mice did not peak until day 28
postinfection. The delayed production of RNI in
TNF-/- mice was temporally associated with the
increased T cell production of IFN- on day 28 and the final
clearance of bacteria in these mice.
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To compare the regulatory actions of TNF on the induction of
chemokines and the recruitment of leukocytes in response to virulent
mycobacterial infection, WT and TNF-/- mice
were infected with 1 x 104 CFU M.
tuberculosis H37Rv i.v., and the course of infection was followed
over time. As observed with aerosol M. tuberculosis
infection, TNF-/- (10) mice
displayed marked susceptibility to infection and succumbed to infection
after 
28 days (Fig. 8A),
with significantly increased bacterial loads in both liver (Fig. 8B) and spleen (data not shown). In contrast, all WT mice
survived during the first 16 wk of infection (data not shown).
Leukocyte infiltrate was evident in the liver of WT mice from 7 days
postinfection, and by day 14 numerous well-defined granulomas were
present in WT mice (Figs. 4
E and 8C). By
contrast, cellular recruitment in TNF-/- mice
was significantly delayed, with very few cellular foci in the liver on
day 14 (Fig. 8C). By day 21 cellular foci were present in
livers of TNF-/- mice, and by day 28 these were
significantly more numerous in TNF-/- than in
WT controls (Fig. 8C). These cellular foci, however,
differed from those in WT mice. Although they contained macrophages and
lymphocytes, they did not form tight clusters typical of WT foci (Fig. 4, G and H). Furthermore, in WT mice most
granulomas on day 28 contained epithelioid macrophages with a
lymphocyte cuff, whereas the granulomas in
TNF-/- mice lacked these differentiated cells.
Interestingly, the large infiltration of neutrophils present in the
lungs of TNF-/- mice during M.
tuberculosis infection (10) did not occur in the
livers of these mice, with only occasional neutrophils present.
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In WT mice chemokine expression was evident at 7 and 14 days
postinfection with induction of mRNA for RANTES, MIP-1, MIP-2, and
MCP-1 (Fig. 9). In
TNF-/- mice the induction of these chemokines
was delayed, with significantly less chemokine mRNA expression in the
first 2 wk of infection. However, the expression of chemokine mRNA was
detected from day 21 in TNF-/- mice, with
significantly increased levels of mRNA for eotaxin, MIP-1, MIP-2,
and MCP-1 compared with WT infected mice (Fig. 9). The increased levels
of chemokine mRNA persisted and were higher on day 28 postinfection
even though the mice were succumbing to the infection at this
time.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
(8, 10). This allowed the identification of three phases
in the cellular control of the infection. First, the rapid clearance of
M. smegmatis during the first week of the infection occurs
independently of TNF (Fig. 2). Therefore, although the araLAM-rich
M. smegmatis is a more potent stimulant of TNF production
than slow-growing M. bovis (BCG) in vitro (Fig. 1), TNF is
not required for the initial killing of the organisms in liver and
spleen, the major sites of infection. The next phase during wk 2 is
characterized in normal mice by a marked influx of macrophages and
lymphocytes into the liver that peak on day 14 (Fig. 3) and a
progressive decline in bacterial load. This is associated with the
induction of both C-C and C-X-C chemokines (Figs. 5 and 6). This phase
is dependent on TNF, and in its absence the cellular infiltrate is
delayed by 2 wk (Fig. 3). This results in a significant reduction in
the rate of clearance of the mycobacteria (Fig. 2). During the third
phase in wk 3 and 4, normal mice show progressive clearance of the
organisms, with a fall in chemokine mRNA levels and a reduction in the
cellular infiltrate. The TNF-/- mice, however,
demonstrated further differences during this phase. Transcripts for
MIP-2, MCP-1, and MIP-1 appeared by day 21 and had significantly
increased by day 28. This was associated with the recruitment of both
macrophages and T cells (Fig. 3, C and D, and
Fig. 4) and reduction in the bacterial loads to the levels in WT mice
(Fig. 2). In addition, the TNF-/- mice showed
significantly greater mycobacteria-specific Th1-like T cell responses
by day 28 (Fig. 7), with the concurrent rise of serum nitrate as
evidence of the late activation of iNOS (Table I).
These results highlight different aspects of TNF’s role in the in vivo
control of mycobacterial infection. First, TNF is essential for the
early induction of chemokines and subsequent leukocyte recruitment to
infected organs, particularly macrophages, which, following activation,
produce RNI essential for the clearance of infection in mice. Infection
of macrophages with M. tuberculosis in vitro leads to the
rapid secretion of MIP-1, MIP-2, and MCP-1 within 12 h
(21). However, during low dose aerosol infection with
M. tuberculosis, transcripts for these chemokines only
appeared in the lungs after day 30 (21). By contrast there
were small, but significant, increases in chemokine mRNA on days 7–14
in the livers of WT mice following i.v. infection with M.
tuberculosis, and these levels peaked on day 21 (Fig. 9). In vitro
studies with neutralizing anti-TNF Ab showed that the MIP-1
response to M. avium infection (22) and the
chemokine response in rat lung injury (23) were also
dependent on TNF.
Recently, neutralization of TNF in vivo was found to reduce the rapid
response of some, but not all, chemokines in an acute model of
pulmonary Th1-like granulomas generated by the
injection of purified protein derivative-coated beads
(24). Of the 24 chemokines studied, eight, including
MIP-2, MIP-1, and MIP-1, increased more in PPD-induced than in
schistosomal Ag-induced granulomas, with transcript levels peaking at
1–2 days. A further five, including MCP-1, were elevated in both types
of granulomas, while eotaxin and three others were elevated in response
to schistosomal Ag-induced granulomas. Interestingly, inhibition of TNF
reduced by 30–50% the early rise in mRNA for five of the nine
chemokines tested, including MIP-1 and MCP-1, but had no effect on
the MIP-2 response (24). By contrast, during in vivo
infection with M. smegmatis, the peak chemokine mRNA in the
liver occurred on day 14 (Fig. 5), and only the constitutively
expressed RANTES showed a significant rise on day 7 (Fig. 6). Also,
there was less apparent selectivity in the pattern of chemokine
observed following mycobacterial infection, with induction of eotaxin
occurring in both M. smegmatis- and M.
tuberculosis-infected mice (Figs. 6 and 9). Further, all six
chemokines examined, including MIP-2, were markedly reduced in
TNF-deficient mice, with no response evident on day 14 when the
inflammation was peaking in WT mice in response to M.
smegmatis (Fig. 3). Subsequently, there was delayed induction of
chemokine transcription within the liver of
TNF-/- mice (Fig. 6), indicating that the
chemokine response to persistent infection was not completely dependent
on TNF.
This TNF-independent component of the chemokine response was more
evident during infection with the more virulent M.
tuberculosis. There were lower chemokine transcript levels in
TNF-/- mice compared with WT mice early in
M. tuberculosis infection, but by day 21 there were
significant increases in hepatic mRNA for both the C-C and C-X-C
chemokines, to levels higher than those in WT mice (Fig. 8). These
continued to rise during the last week of the fatal infection. M.
bovis (BCG) infection of TNF-deficient mice was also associated
with comparable increases in chemokine mRNA expression
(25) and in MCP-1 and MIP-1 protein levels compared
with WT mice (26). Furthermore, delayed expression of
MIP-1 and MIP-1 mRNA was observed in
TNF/LT-/- double-knockout mice
(25). This late excessive chemokine response to M.
tuberculosis or M. bovis in
TNF-/- mice was associated with a delayed or
aberrant inflammatory response with failure to control the infection
(10, 25, 26). Therefore, in addition to its role in the
initial cellular recruitment, TNF plays an essential role in regulation
of the inflammatory response and, in particular, the juxtaposition of
macrophages and lymphocytes to form granulomas. This was evident in
both M. smegmatis and M. tuberculosis infection,
where, despite the influx of leukocytes, there is failure to form the
tight foci of leukocytes evident in the liver of WT mice (Fig. 4). The
inadequate migration of lymphocytes into M. tuberculosis
infection in TNF deficiency may relate to the binding of TNF to the
extracellular matrix to direct migration leukocytes through tissues
(27). TNF may also contribute to the tight apposition of
macrophages and lymphocytes within granulomas, either as secreted or
membrane-bound forms. TNF is highly expressed within granulomas in
human tuberculosis (28), leprosy (29), and
murine (7) mycobacterial infections and is essential for
the differentiation of macrophages into epithelioid cells.
Neutralization of TNF activity in established mycobacterial infections
leads to the disruption of granulomas, with recrudescence and
dissemination of the M. tuberculosis infection
(30, 31, 32).
A further effect of TNF deficiency on M. smegmatis infection
was the enhanced T cell response later in the infection (Fig. 7).
Previously, normal T cell responses have been demonstrated in
TNF-/- mice to keyhole limpet hemocyanin and
alloantigens (38), mycobacterial Ags (8, 10), and autoantigens (33). The enhanced T cell
response during M. smegmatis infection (Fig. 7) was probably
due to the increased bacterial load seen in
TNF-/- from wk 2 to 3 of infection (Fig. 2).
Previously, we have expressed M. tuberculosis genes in
M. smegmatis (34), but found the recombinant
M. smegmatis relatively poor at inducing T cell responses to
the exogenous Ag in comparison to recombinant M. bovis (BCG)
(18) (P. W. Roche, unpublished observations),
presumably due to the rapid clearance of M. smegmatis in
normal mice. In the absence of TNF, the enhanced T cell response with
increased IFN- production on day 28 may have contributed to the
eventual clearance of the organisms. T cells are a potent source of
LT (35) that may also activate macrophages through TNF
receptor I, resulting in iNOS induction (36) and increased
killing of the bacterium. Recent studies with
LT-/- chimeras (11) or
LT/TNF-/- mice reconstituted with TNF-
transgenes (25) have confirmed that LT is essential to
control M. tuberculosis and BCG infection as well as
infection with L. monocytogenes infection (D. R. Roach,
unpublished observation). Nevertheless, LT is unable to
complement for the effects of TNF deficiency in
TNF-/- mice infected with M.
tuberculosis.
In summary, TNF was essential for the early induction of chemokines and leukocyte recruitment following M. smegmatis infection in the liver, but this defect was compensated by a later chemokine response with enhanced Th1-like T cell response that controlled the infection. With the more virulent M. tuberculosis infection, after an initial delay in chemokine response there was a marked TNF-independent rise in chemokines, but the inflammatory response was dysregulated, with failure to form effective tight granulomas. The importance of TNF-dependent granuloma formation in the control of latent M. tuberculosis infection in humans is graphically illustrated by the rapid reactivation of clinical tuberculosis in patients undergoing treatment for rheumatoid arthritis and Crohn’s disease with a humanized mAb to TNF (37).
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Acknowledgments |
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Footnotes |
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2 Current address: CSIRO Livestock Industries, Geelong, Victoria 3220, Australia.
3 Address correspondence and reprint requests to Prof. Warwick J. Britton, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag 6, Newtown, 2042 New South Wales, Australia. E-mail address: wbritton{at}medicine.usyd.edu.au
4 Abbreviations used in this paper: LT, lymphotoxin-; BCG, bacille Calmette-Guérin; araLAM, arabinan side chains of lipoarabinomannan; iNOS, inducible NO synthase; LAM, lipoarabinomannan; manLAM, mannan caps of lipoarabinomannan; MIP-1, macrophage-inflammatory protein-; MCP-1, macrophage chemoattractive protein-1; RNI, reactive nitrogen intermediate; WT, wild type.
Received for publication December 17, 2001. Accepted for publication February 28, 2002.
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) or M. bovis (BCG; multiplicity of infection,
10:1; 
) or were uninfected (
). TNF production was measured by the
WEHI 164 bioassay. A, No prestimulation;
B, prestimulated with 100 U/ml IFN-
) and TNF-/-
(
