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*
Division of Molecular Infection Biology, Research Center Borstel, Borstel, Germany;
Department of Anatomy, Medical University of Lübeck, Lübeck, Germany; and
Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12, or TNF
in TNFRp55-/- mice infected i.v. with
M. avium. Infected TNFRp55-/-
mice treated with a control mAb became moribund between days 26 and 34
postinfection, showing widespread inflammatory cell apoptosis within
disintegrating granulomas. In contrast,
TNFRp55-/- mice depleted of either
CD4+ or CD8+ cells after granuloma initiation
stayed healthy until at least day 38 postinfection and showed no signs
of granuloma destruction. Neutralization of IL-12, but not of IFN-
or TNF, also protected M. avium-infected
TNFRp55-/- mice from granuloma decomposition
and from premature death. Treatment with dexamethasone or with a
specific inhibitor of inducible NO synthase did not prevent granuloma
dissolution or death of TNFRp55-/- mice. In
conclusion, granuloma disintegration in
TNFRp55-/- mice is a lethal event that is
dependent on IL-12 and that is mediated by an excess of T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The integrity of a developing granuloma is crucial in terms of disease
progression and survival. For example, treatment of mice with an
anti-TNF antiserum during the chronic stage of infection with
Mycobacterium bovis bacillus Calmette-Guérin resulted
in granuloma dissolution and extensive bacterial proliferation
(4), and mice deficient in some aspect of organized
mononuclear cell recruitment, such as SCID, IFN--knockout, or
TNF-knockout mice, readily succumbed to infection with
Mycobacterium tuberculosis (5, 6, 7). In
particular, the disintegration of the granuloma structure in the form
of caseous necrosis and rupturing of the cavity into a bronchus is
characteristic of advanced human tuberculosis (2, 3). The
cellular and molecular mechanisms involved in this process have largely
defied definition because of the lack of a mouse model of infection
that adequately reflects the full spectrum of the immunopathology
evident in humans.
Experimental infection in mice with Mycobacterium avium, the causative agent of the most prevalent opportunistic infection in AIDS patients, is particularly well-suited to the study of granuloma induction, maintenance, and necrosis. After i.v. infection with M. avium, mice develop persistent granulomatous lesions in all infected organs (8, 9, 10, 11). Aerogenic infection with highly virulent M. avium strains induces a pulmonary pathology remarkably similar to that found in human tuberculosis (12).
Exploiting the M. avium model for the study of factors
involved in the formation and maintenance of granulomas, we previously
showed that granuloma development was significantly delayed in mice
deficient for the TNFRp55 (13). Although these mice had
similar bacterial counts in infected organs compared with
immunocompetent mice, M. avium-infected
TNFRp55-/- mice all
succumbed to infection. Before death, granulomatous lesions in
TNFRp55-/- mice
acutely disintegrated, showing widespread inflammatory cell apoptosis
and necrosis of both granulomatous and surrounding parenchymal tissues.
Furthermore, significantly increased numbers of
CD3+ cells within disintegrating lesions of
TNFRp55-/- mice were
found, and higher levels of the proinflammatory mediators IFN-,
IL-12p40, and TNF were detected in organ homogenates of M.
avium-infected
TNFRp55-/- mice
(12, 13).
It remained unknown whether T cells and/or the mentioned key inflammatory mediators were causally involved in both the death and the granuloma disintegration occurring in TNFRp55-/- mice in the course of M. avium infection. Therefore, we selectively depleted T cell subpopulations or neutralized proinflammatory mediators in M. avium-infected TNFRp55-/- mice and investigated the effect of these treatments on survival, bacterial containment, and histopathology. Our results show that both granuloma disintegration and death of TNFRp55-/- mice infected with M. avium are dependent on T cells and IL-12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The bcg-susceptible TNFRp55-/- mice used in these studies are fifth generation backcrosses of the original TNFRp55-deficient 129Sv strain (14) onto C57BL/6 mice. TNFRp55-/- and syngeneic C57BL/6 TNFRp55+/+ mice were raised in the animal breeding facilities of Charles River Wiga (Sulzfeld, Germany). TNFRp55-/- mice on a 129Sv background (15) were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and were the kind gift of Dr. U. Steinhoff (Max-Planck-Institute of Infection Biology, Berlin, Germany). All mice were serologically found to be free of Abs to the most common viral and bacterial mouse pathogens. Mice were used when they were 8–12 wk old. During the course of M. avium infection, age- and sex-matched groups of four to five mice per experimental group were housed in isolator cages under barrier conditions in the animal facilities at the Borstel Research Center.
Bacteria
M. avium, strain TMC724 (originally obtained from Dr. F. Collins, Trudeau Institute, Saranac Lake, NY), was passaged in C57BL/6 mice twice and cultured in Middlebrook 7H9 (Difco, Detroit, MI) medium supplemented with OADC (oleic acid, albumin, dextrose, catalase; Becton Dickinson, Heidelberg, Germany) to a mid-logarithmic phase. Aliquots were frozen at -70°C until they were needed. An inoculum of bacteria was prepared by thawing an aliquot and diluting it in PBS. Groups of five mice per experimental variable were infected i.v. via a lateral tail vein with indicated inocula in 0.2 ml PBS. Mice were scored as moribund and sacrificed when weight loss exceeded 25% of the body weight at the beginning of infection. Mice were anaesthetized and killed at indicated time points to determine bacterial CFU in infected organs. Organs were removed aseptically and homogenized in 10 ml distilled water to determine bacterial loads by plating serial 10-fold dilutions of whole organ homogenates on nutrient Middlebrook 7H10 agar (Difco) supplemented with OADC. Bacterial colony numbers (CFU) were determined after 14–21 days incubation at 37°C in humidified air. Data are shown as mean log10 CFU counts ± SD. The natural course of infection and the kinetics of granuloma formation and disintegration in immunocompetent and TNFRp55-/- mice infected with these strains were previously described (11, 13). All animal experiments were approved by the local ethics committee instituted by the Ministry of Nature, Environment and Forestation (Kiel, Germany).
Reagents
The following mAbs (specificities in parentheses) were used for
in vivo studies: GK1.5 (anti-CD4), 2.43 (anti-CD8), 23/7
(irrelevant epitope), XMG1.2 (anti-IFN-, kindly provided by Dr.
A. O’Garra from the DNAX Research Institute, San Diego, CA), and C
15.1 and C 15.6 (both anti-IL-12p40, kindly provided by the Wistar
Institute, Philadelphia, PA). Abs were purified by ammonium sulfate
precipitation and subsequent dialysis of crude ascites produced in nude
mice by Dr. Rui Appelberg (Instituto de Biología Molecular e
Celular, Porto, Portugal). The anti-TNF mAb TN3-19.12 was a
kind gift from Dr. R. Schreiber (Washington University School of
Medicine, St. Louis, MO). The hamster anti-IFN- mAb (H22) was
purchased from Genzyme (Rüsselsheim, Germany). Hamster control
IgG was from Dianova (Hamburg, Germany). For immunohistochemistry, the
mAbs KT174 (anti-CD4) and KT15 (anti-CD8
; BioSource
International, Camarillo, CA) or a rabbit antiserum specific for the
proliferation-associated Ag Ki-67 (16) were used.
Secondary (rabbit anti-rat IgG) and tertiary Abs (goat
anti-rabbit IgG and donkey anti-goat IgG) were from Dianova.
L-N6-imino-ethyl-lysine
(L-NIL)3
was purchased from Alexis (Läufelfingen, Switzerland) and
dissolved in water (adjusted to pH 2.7 to prevent microbial growth).
Dexamethasone (Fortecortin Mono 8) was from Merck (Darmstadt,
Germany).
Treatment protocols
For T cell subset depletion, groups of five mice each were
injected i.p. with 500 µg of the specific mAb in a volume of 500 µl
PBS once weekly. This depletion protocol was previously shown to result
in >95% depletion of the targeted T cell subset when assessed by FACS
analysis of spleen cells (17). For cytokine
neutralization, the following Ab concentrations were used: 2 mg/500
µl anti-IFN- (XMG 1.2), 300 µg/500 µl anti-IFN-
(H22), 250 µg/500 µl anti-TNF, and anti-IL-12p40 (mixture
of 1 mg C15.1 and 1 mg C15.6 in 500 µl). The concentrations of the
anti-cytokine Abs used were previously shown to be neutralizing in
vivo (9, 18, 19). Treatment was by once weekly i.p.
injection of purified Abs and was initiated after granulomas were
established because depletion at the beginning of infection was
previously demonstrated to significantly delay granuloma development
(11, 18). All depletion studies were repeated at least
once and gave qualitatively identical results, although the kinetics of
granuloma disintegration differed slightly between experiments.
Fresh acidified drinking water with or without 5 mM L-NIL was provided to the mice every other day. A toxicity study previously demonstrated that 5 mM L-NIL does not influence water or food intake or impair weight gain in naive mice (20) but that it is fully capable of reducing nitrate/nitrite levels in M. avium- or M. tuberculosis-infected mice to the levels of uninfected controls (21). Dexamethasone was used at 0.4 mg in 100 µl PBS and was injected s.c. at the base of the tail every other day.
Histology
One cranial and one caudal liver lobe per mouse were fixed in 4% formaline-PBS, set in paraffin blocks, sectioned (2–3 µm), and stained using hematoxylin and eosin. In the figure legends, the original magnification of the photographic image is indicated.
Immunohistology
For the detection of CD4+ cells or
CD8+ cells, frozen tissue sections were prepared
using a cryostat (Frigocut E 2800; Leica, Bensheim, Germany).
Four-micrometer sections were air-dried and fixed in acetone before
storage at -70°C. After acetone-chloroform treatment, sections were
blocked with 0.3% superoxide and incubated with mAb KT174
(anti-CD4) or KT15 (anti-CD8). Appropriately diluted rabbit
anti-rat IgG was used as a secondary Ab, and goat anti-rabbit
IgG peroxidase was used as a tertiary Ab. For the detection of
proliferating cells, a rabbit anti-mouse Ki-67 antiserum was used
(16) with goat anti-rabbit IgG and donkey
anti-goat IgG peroxidase as secondary Abs. Development was
performed with 3–3'-diaminobenzidine (Sigma, Deisenhofen, Germany) and
urea superoxide (Sigma), and hemalum was used to counterstain the
slides. The number of granuloma-associated cells positive for a
specific marker was determined by counting them in three randomly
chosen 40x microscopic fields in five nonsequential liver sections per
mouse (four mice per group). Data represent the means of 60
determinations ± SD.
Electron microscopy
Perfusion fixation of the liver was performed at room temperature via the vena portae with 2% glutaraldehyde/0.6% paraformaldehyde in a 0.06 M sodium cacodylate buffer (pH 7.35) for 10 min. Livers were immersed in the fixative for 72 h at 4°C, rinsed in 0.2 M sodium cacodylate buffer (pH 7.35), and postfixed with 1% osmium tetroxide in the same buffer for 2 h at room temperature. After rinsing in 2.4% sodium chloride solution, the samples were washed in 0.2 M sodium acetate buffer (pH 5.0) and block stained with 1% uranyl acetate in 0.2 M sodium acetate buffer in the dark for 30 min. After dehydration in alcohol and embedding in araldite, semithin sections (0.5 µm) were prepared and stained with azure II-methylene blue. Ultrathin sections (60 nm) were stained with lead citrate and examined in a Philips 400 electron microscope.
Cytokine ELISAs and determination of liver enzyme levels
Plasma was obtained after centrifugation of heparinized blood
drawn from the posterior vena cava of anesthetized mice and stored at
-70°C until further use. ELISA measurements of TNF, IL-12p40, and
IFN- levels in the plasma were conducted as stipulated by the
manufacturer (R&D Systems, Wiesbaden, Germany). Levels of alanine
serine aminotransferase (ASAT) and lactate dehydrogenase (LDH) were
measured in the plasma of mice using standard procedures and an
automated sample analyzer in the laboratory of clinical biochemistry of
the Borstel Clinical Center.
Statistics
Quantifiable data are expressed as the means of individual determinations ± SD. Statistical analysis was performed using Student’s t test, or the Welch test in case of unequal variances.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The course of bacterial replication and delayed granuloma
formation in M. avium-infected
TNFRp55-/- mice was
previously described in detail (13).
TNFRp55+/+ mice infected with
106 CFU M. avium TMC724 initiated
granuloma formation between days 10 and 15 postinfection, whereas
similarly infected
TNFRp55-/- mice developed
granulomatous lesions with a delay of 
5–8 days. Mononuclear cell
infiltrations were more diffuse in
TNFRp55-/- mice, had a
higher cellularity in relation to their smaller size, and lacked the
appearance of mature granulomas because they contained fewer
epithelioid macrophages. With an inoculum of 106
CFU M. avium, granuloma disintegration in
TNFRp55-/- mice
reproducibly occurred during the fifth week postinfection, and infected
TNFRp55-/- mice succumbed
to infection before day 35.
When examined by immunohistochemistry at day 21 postinfection, T cell
numbers in incipient lesions of
TNFRp55-/- mice were
similar to those found in the granulomas of
TNFRp55+/+ mice (Ref. 13 and data
not shown). However, when the amount of CD4+ and
CD8+ T cells in disintegrating granulomatous
lesions of TNFRp55-/-
mice were compared with those present in epithelioid granulomas in
TNFRp55+/+ mice at day 33 postinfection (Fig. 1
), a significant increase in both
subsets was evident (59 ± 16 vs 20 ± 7 granuloma-associated
CD4+ cells and 77 ± 11 vs 15 ± 4
granuloma-associated CD8+ cells per 400x
microscopic field; p < 0.01). The staining pattern for
the proliferation-associated Ag Ki-67 showed a similar distribution as
the staining pattern for these lymphocyte markers, and the number of
proliferating Ki-67+ cells was also significantly
increased in lesions of
TNFRp55-/- mice at day 33
postinfection (Fig. 1).
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90–95% of
CD8+ cells (data not shown).
All TNFRp55-/- mice that
had received control mAbs became moribund between days 26 and 30 of
infection (Fig. 2). In contrast,
TNFRp55-/- mice that had
received either anti-CD4 or anti-CD8 mAbs appeared completely
healthy until day 38, when all treated
TNFRp55-/- mice were
sacrificed to compare the lesions of treated surviving mice with the
lesions of control mAb-treated moribund mice.
TNFRp55-/- mice receiving
three injections (on days 24, 31, and 38 postinfection) of both
anti-CD4 and anti-CD8 mAbs remained completely healthy until
day 45, when the experiment was terminated (Fig. 2). Thus, depletion of
T cell subsets significantly prolonged survival of
TNFRp55-/- mice infected
with M. avium.
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). There was no significant difference
between groups in any of the organs tested. Thus, depletion of T cell
subsets, which prolonged survival in
TNFRp55-/- mice, did not
affect bacterial loads in the time frame studied, indicating that death
of TNFRp55-/- mice
did not correlate with bacterial burden.
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Liver histology was examined in moribund
TNFRp55-/- mice treated
with control mAbs and was compared with anti-CD4- or
anti-CD8-treated
TNFRp55-/- mice
sacrificed at day 38 postinfection. Conventional and electron
microscopy was performed (Fig. 4).
Control mAb-treated
TNFRp55-/- mice showed
massive granuloma disintegration with the characteristic hallmarks of
wide-spread apoptotic cell death. Among these apoptotic cells, many
macrophages could be identified by the presence of engulfed
mycobacteria. In contrast, T cell subset-depleted
TNFRp55-/- mice had
almost normal granuloma development, showing incipient epithelioid cell
differentiation and lacking signs of inflammatory cell apoptosis. In
addition, granuloma integrity was fully maintained until day 45 in
those TNFRp55-/- mice
that had been treated with three injections (on days 24, 31, and 38
postinfection) of the combination of anti-CD4 and anti-CD8
mAbs.
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). Thus, prolonged survival of
TNFRp55-/- mice
correlated with granuloma and tissue integrity, and T cells were the
major mediators of host-damaging processes in infected
TNFRp55-/- mice.
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) and developed only small
granulomatous lesions when examined at days 38 and 56 of M.
avium infection. No granuloma destruction was evident in these
mice during the entire observation period (up to 84 days), and ASAT and
LDH levels were comparable to immunocompetent control-infected mice
sacrificed at the same time (Table I). Second, SCID mice deficient in T and B cells were infected with M. avium TMC724 and were treated with a neutralizing anti-TNF mAb starting on day 35 (i.e., subsequent to the delayed granuloma initiation in these mice; Ref. 18) for up to 5 wk. These mice did not show any signs of granuloma dissolution, further substantiating our interpretation that T cells are critically involved in granuloma disintegration.
Effect of cytokine neutralization on the survival of M. avium-infected TNFRp55-/- mice
To analyze the effect of T cell subset depletion on the levels of
proinflammatory cytokines, the amounts of IL-12p40, TNF, and IFN-
were determined in the sera of infected mice (Fig. 5). IL-12p40 levels were somewhat
increased in infected
TNFRp55-/- mice when
compared with infected TNFRp55+/+ mice. However,
T cell subset depletion had no profound effect on IL-12p40 detectable
in the sera of infected
TNFRp55-/- mice. IFN-
and TNF amounts were markedly elevated in infected
TNFRp55-/- mice when
compared with infected TNFRp55+/+ mice. Treatment
of infected TNFRp55-/-
mice with anti-CD4 or anti-CD8 mAbs reduced the levels of
IFN- and TNF to the levels found in M. avium-infected
TNFRp55+/+ mice (Fig. 5).
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Neutralization of TNF or IFN-, as well as treatment with
dexamethasone, failed to increase survival times of infected
TNFRp55-/- mice. All
TNFRp55-/- mice thus
treated died before day 37, which is similar to the results obtained
with control mAb-treated
TNFRp55-/- mice (Fig. 6). In contrast, mice treated for 2 wk
with a combination of two mAbs specific for IL-12p40 survived until day
38 when they were sacrificed to compare their liver histopathology with
that of moribund control mAb-treated
TNFRp55-/- mice.
Infected TNFRp55-/- mice
receiving three injections of the anti-IL-12p40 mAbs (on days 24,
31, and 38 postinfection) remained completely healthy until the
experiment was terminated on day 45 (Fig. 6). Thus, survival of
M. avium-infected
TNFRp55-/- mice was
markedly prolonged by effective neutralization of IL-12.
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). Therefore, survival did not correlate
with different bacterial organ loads in the different mouse groups.
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Histopathological examination revealed that
anti-IL-12p40-treated
TNFRp55-/- mice had
compact granulomas showing epithelioid cell differentiation, whereas
immature granulomatous lesions in
TNFRp55-/- mice treated
with anti-IFN-, anti-TNF, or control mAb had completely
disintegrated and were again characterized by an abundance of apoptotic
cells (Fig. 8). Granulomas in infected,
dexamethasone-treated moribund
TNFRp55-/- mice were
completely necrotic, whereas the surrounding liver tissue appeared less
affected.
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The extent of granuloma disintegration and subsequent tissue necrosis
in the different groups was quantitated using ASAT and LDH levels in
the sera of mice. Only anti-IL-12p40-treated mice had low ASAT and
LDH levels, results similar to those of infected
TNFRp55+/+ controls, whereas infected control
mAb-treated
TNFRp55-/- mice or
infected mice which had received anti-IFN- had high levels of
ASAT and LDH (Table II).
TNFRp55-/- mice treated
with anti-TNF or dexamethasone also showed reduced ASAT and LDH
levels compared with those of untreated
TNFRp55-/- mice;
however, ASAT and LDH levels were still markedly elevated compared with
those of TNFRp55-/- mice
treated with the anti-IL-12p40 mAbs (Table II).
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and was always followed by
premature death. Because the inducible form of NO synthase (iNOS) was previously shown to be up-regulated in M. avium-induced granulomas (13, 21), the possibility was considered that NO might be responsible for the tissue damage in infected TNFRp55-/- mice. Therefore, TNFRp55-/- mice were infected and treated with the selective inhibitor of iNOS, L-NIL. However, L-NIL-treated TNFRp55-/- mice did not survive longer than untreated infected TNFRp55-/- mice, and granulomas of L-NIL-treated mice disintegrated with the characteristic histomorphology also evident in infected TNFRp55-/- mice left untreated. In accordance, ASAT and LDH serum levels were similarly high in both groups of mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pivotal role of both CD4+ and
CD8+ T cells in antimycobacterial protection is
well-established and is thought to involve the enhanced production of
IFN- (6, 9). Regarding M. avium infection,
no role was found for CD8+ cells in this respect
(9, 25). Granuloma formation in response to M.
avium infection proceeds in an accelerated fashion in the presence
of CD4+ cells (11, 18); again,
CD8+ T cells were demonstrated not to be involved
in this process in immunocompetent mice (11, 25).
However, in a disregulated inflammatory response, such as that evident in M. avium-infected TNFRp55-/- mice, both subsets apparently contribute to granuloma disintegration. The cause for the increased presence of both CD4+ and CD8+ T cells in disintegrating granulomatous lesions of TNFRp55-/- mice is not entirely clear. One explanation might be that during the course of infection in TNFRp55+/+ mice, TNFRp55-mediated apoptosis occurs in T cells recruited to or actively dividing in the granuloma. This mechanism would normally regulate the number of T cells present within the lesion, as described in murine autoimmune encephalomyelitis (26). A lack of TNFRp55-mediated T cell apoptosis would result in the enhanced accumulation of T cells, particularly under conditions in which the proliferation of lymphocytes in situ is greatly enhanced, as demonstrated in this study for the lesions in infected TNFRp55-/- mice.
The lethal outcome of M. avium infection in TNFRp55-/- mice investigated in this study is in apparent contrast to another report (10). However, the investigators in that study made use of TNFRp55-/- mice on a 129Sv/BL6 background, which may be assumed to carry the resistant allele of Nramp1. Our own experiments confirmed that genetically resistant TNFRp55-/- mice infected with M. avium do not show granuloma disintegration. Because resistant mice develop only minimal T cell responses to intracellular infections (22, 23), we take this as corroborating evidence that T cells are indeed necessary for the lethal event of granuloma dissolution. Although the inflammatory response in SCID mice is not equivalent to that in immunocompetent mice (18), our experiments in which chronic neutralization of TNF in M. avium-infected SCID mice also did not affect granuloma development tend to further substantiate our interpretation that lethal granuloma disintegration is T cell-dependent.
Most M. avium isolates readily induce IL-12p40 in vitro
(27), and we found high levels in the sera of both
TNFRp55+/+ and
TNFRp55-/- mice.
Neutralization of IL-12 led to prolonged survival of M.
avium-infected
TNFRp55-/- mice, and
granulomatous lesions in these mice contained fewer
CD3+ cells, specifically fewer
CD8+ cells, and did not disintegrate. IL-12 was
previously shown to promote lymphocyte recruitment into developing
granulomatous lesions (28), and this effect may have
critically contributed to lesion development in
TNFRp55-/- mice. On the
other hand, IL-12p70 is also known to activate Th1 cells
(29), and IL-12R interaction in vitro with IL-12p40
homodimer resulted in enhanced Th1 development and increased IFN-
secretion from CD8+ T cells (30).
Moreover, IL-12p40 levels positively correlated with disease activity
in multiple sclerosis (31, 32). In line with these
reports, we hypothesize that IL-12 is involved in both the recruitment
of T cells and their activation within the developing lesion.
The decomposition of granulomas in infected TNFRp55-/- mice always started with conspicuous apoptosis of inflammatory cells within the lesions. Macrophages seemed to be particularly affected, as judged by the frequent presence of mycobacteria within apoptotic cells, although we did not specifically investigate whether other cells, such as T cells or adjacent hepatocytes, also underwent apotosis. However, apoptosis was strikingly absent when T cells were depleted.
Increased numbers of T cells in granulomatous lesions of
TNFRp55-/- mice were
associated with dramatically higher amounts of IFN- in the sera of
these mice, and depletion of T cells resulted in reduced IFN- levels
in the sera of TNFRp55-/-
mice. Therefore, one might argue that T cell-derived IFN- was the
principal inducer of inflammatory cell apoptosis and subsequent tissue
necrosis, as was previously shown in the model of gastrointestinal
infection with Toxoplasma gondii (33, 34).
Although we used, in separate experiments, two different neutralizing
mAbs against IFN-, both of which had previously been shown to be
highly effective in vivo (9, 18), we were unable to
demonstrate a role for IFN- in granuloma disintegration in
infected TNFRp55-/-
mice. This finding was corroborated by the results obtained with
dexamethasone treatment, which reduced serum IFN- in infected
TNFRp55-/- mice
almost to background levels, but it was ineffective at inhibiting
granuloma necrosis. Interestingly, and in contrast to results obtained
in the oral infection model with T. gondii in genetically
susceptible mice (35), inhibition of iNOS also did not
affect the immunopathology observed in
TNFRp55-/- mice infected
with M. avium.
T cells are known to have the potential for autoreactivity resulting in tissue destruction (36, 37). In experimental models of autoimmune diseases, Fas/Fas ligand (FasL) interactions and membrane TNF-TNFRp75 interactions were described as important mechanisms for the induction of apoptosis by CD4+ cells (38, 39, 40). Cytotoxic T cells were shown to destroy infected target cells via either a Fas/FasL interaction or via a granule-dependent mechanism (41). The exact mechanism by which T cells contribute to inflammatory cell apoptosis and tissue necrosis in infected TNFRp55-/- mice remains to be determined. Because TNF neutralization did not affect M. avium-induced lethality in TNFRp55-/- mice, we consider the TNF/TNFRp75 pathway least likely. However, we cannot formally rule out that signaling through the TNFRp75 did occur in our experiments, because membrane-bound TNF may have been suboptimally affected by our anti-TNF treatment. Whether IL-12, in addition to promoting recruitment of lymphocytes into the developing granuloma (28), also directly up-regulates the expression of Fas/FasL or of the perforin/granzyme system is presently not known.
In conclusion, this study shows that lack of TNFRp55 signaling in the course of M. avium infection results in a hyperinflammatory and ultimately fatal response in which IL-12 promotes the recruitment of T cells, which induce inflammatory cell apoptosis leading to granuloma disintegration. Therefore, TNFRp55-mediated signaling appears to be an integral part of a feedback loop between macrophages and T cells that is normally in place to control and regulate the potentially detrimental T cell-mediated inflammatory response to mycobacterial infection.
The integrity of granulomas is a critical determinant of the outcome of mycobacterial diseases, and apoptosis and necrosis are common events in granulomas associated with human tuberculosis (2, 3, 24, 42, 43). The mycobacteria-infected TNFRp55-/- mouse may prove useful in defining suitable molecular targets for therapeutic interventions aimed at reducing tissue-destroying and life-threatening immunopathology caused by an excess of T cells. Certainly, our findings obtained in gene-deficient mice cannot be directly extrapolated to the immunopathology that occurs in immunocompetent individuals with mycobacterial or other intracellular infections. However, recent results from other experimental infections in TNFRp55-/- mice suggest that the observed absence of adequate lesion control is not unique to infections with a particular pathogen because failure to signal through the TNFRp55 also resulted in failure of lesions to regress once Leishmania major or Rhodococcus equi was eliminated (44). The use of TNFRp55-/- mice has thus not only uncovered a unique role of TNF in the adequate maintenance and resolution of inflammatory lesions, but it also highlights the potentially detrimental and pathology-promoting function of T cells that develops in the course of different types of infections and must be effectively harnessed.
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Acknowledgments |
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, anti-IL-12p40, and
nonspecific control mAbs used in this study, Robert Schreiber for his
generous gift of purified anti-TNF mAb, and Johannes Gerdes for his
kind gift of the rabbit anti-mouse Ki-67 antiserum. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefan Ehlers, Division of Molecular Infection Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany.
3 Abbreviations used in this paper: L-NIL, L-N6-imino-ethyl-lysine; LDH, lactate dehydrogenase; iNOS, inducible form of NO synthase; FasL, Fas ligand; ASAT, alanine serine aminotransferase; Nramp1, natural resistance associated macrophage protein.
Received for publication December 22, 1999. Accepted for publication April 13, 2000.
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