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Infection and Immunity, July 1999, p. 3571-3579, Vol. 67, No. 7
Division of Molecular Infection Biology,
Received 19 October 1998/Returned for modification 25 January
1999/Accepted 14 April 1999
The pathogenesis of mycobacterial infections is associated with the
formation of granulomas in which both antibacterial protection and
tissue damage take place concomitantly. We used murine
Mycobacterium avium infection to compare the development of
granulomatous lesions in intravenously infected tumor necrosis factor
receptor p55 (TNFRp55) gene-deficient (p55 Granulomas are focal mononuclear
cell infiltrations that appear in response to chronic inflammatory
stimuli and are therefore a hallmark of mycobacterial infections.
Granulomas are an essential component of a coordinated
antimycobacterial defense, because it is within granulomas that
T-cell-macrophage cooperation can take place, allowing the macrophage
to display effective bacteriostatic or bacteriocidal activities
(9, 24). On the other hand, granulomas displace and destroy
adjacent tissues, leading to inflammation-dependent organ damage or
failure (9, 23).
The inflammatory and protective components of the host immune response
against mycobacteria coincide temporally and may be mediated by the
same molecules. A case in point, tumor necrosis factor (TNF) has been
shown with in vitro and in vivo studies to induce antibacterial
mechanisms in macrophages against Mycobacterium tuberculosis
and Mycobacterium avium (2, 14, 17). In addition, TNF plays a pivotal role in mononuclear cell recruitment, because a
polyclonal antiserum against TNF reduced granuloma induction and
accelerated granuloma resolution in mice infected with
Mycobacterium bovis BCG (25). Similarly, in SCID
mice infected with M. avium, T-cell-independent granuloma
formation was completely abrogated by treatment with a monoclonal
antibody against TNF (38).
When mice genetically deficient for the major signalling component of
the TNF receptor p55 (TNFRp55) (p55 M. avium is the cause of the most prevalent opportunistic
infection among AIDS patients (21). Disseminated M. avium infection in these patients leads to hepatosplenomegaly with
variable granuloma formation, anemia, fever, and weight loss (22,
26). Studies using chemotherapy prophylaxes have shown that
quality of life and life expectancy are both significantly reduced by
M. avium infection in these patients (20, 22).
In mice, all strains of M. avium are less virulent than
M. tuberculosis, and murine M. avium infection is
more chronic by nature (6, 33). In addition, mice tolerate
much higher bacterial numbers (up to 1010 CFU) in organs
during M. avium infection than during M. tuberculosis infection (1, 19). M. avium
infection therefore allows for a more detailed kinetic analysis of
granuloma induction and maintenance than infections with highly
virulent organisms like M. tuberculosis or Listeria
monocytogenes, which may cause rapid tissue destruction. In
addition, in vivo studies using neutralizing antibodies against TNF
demonstrated only a marginal, if any, effect on M. avium
replication during the early phase of infection (1).
We therefore used M. avium infection to examine the
contribution of TNFRp55-mediated signals in maintaining long-term
granuloma integrity in response to a persistent replicating stimulus of
low intrinsic toxicity.
Mice.
TNFRp55 Bacteria.
M. avium TMC724 (originally obtained from F. Collins, Trudeau Institute, Saranac Lake, N.Y.) and M. avium
SE01 (an isolate from the blood culture of an AIDS patient) were
passaged twice in C57BL/6 mice and cultured in Middlebrook 7H9 (Difco,
Detroit, Mich.) medium supplemented with OADC (oleic acid, albumin,
dextrose, catalase; Becton Dickinson, Heidelberg, Germany) to
mid-logarithmic phase. Aliquots of the cultured organisms were frozen
at Histology.
One cranial and one caudal liver lobe per mouse
were fixed in 4% formaline-PBS, set in paraffin blocks, sectioned (2- to 3-µm sections), and stained using hematoxylin and eosin (HE), or,
for easier visualization of apopotic cells, with toluidine blue.
Granuloma numbers were determined by counting focal mononuclear
infiltrations in five nonsequential sections per animal (four mice per
group) in a superimposed 0.25-cm2 grid. For the purpose of
quantitation, a granuloma was defined as the focal accumulation of more
than nine mononuclear cells. Data represent the means of 20 determinations ± standard deviations (SD).
Immunohistology.
Tissue sections were deparaffinated and
placed in 10 mM sodium citrate buffer (pH 6) and pressure-cooked for
exactly 1 min (5). After blocking for 20 min in 1%
H2O2 solution, slides were incubated with
appropriately diluted polyclonal rabbit anti-mouse-isoform of
nitric-oxide synthase (iNOS) (Genzyme-Virotech, Rüsselsheim, Germany) in Tris-buffered saline-10% fetal calf serum for 30 min in a
humid chamber. Appropriately diluted goat-anti-rabbit immunoglobulin G
(IgG) peroxidase (Dianova, Hamburg, Germany) was used as a bridging antibody and diluted rabbit anti-goat IgG peroxidase (Dianova) was used
as a tertiary antibody in sequential incubations of 30 min each. For
the detection of CD3+ cells, a rat anti-mammalian CD3
monoclonal antibody (clone CD3-1C; Biotrend, Cologne, Germany) was used
as a primary antibody, diluted rabbit anti-rat IgG (Dianova) as a
secondary antibody, and goat anti-rabbit IgG peroxidase as a tertiary
antibody. Development was performed with 3-3'-diaminobenzidine (Sigma,
Deisenhofen, Germany) and urea superoxide (Sigma), and hemalum was used
to counterstain the slides.
RT-PCR.
A detailed protocol of the procedure employed for
semiquantitative reverse transcription (RT)-PCR has been published
elsewhere (13, 19). Briefly, weighed liver samples
(approximately 150 mg each) were homogenized in 5 ml of 4 M
guanidinium-isothiocynanate buffer, diluted to obtain equalized amounts
of liver in buffer, and after acid phenol extraction of total RNA, cDNA
was obtained by using Moloney murine leukemia virus-RT (Gibco-BRL,
Eggenstein, Germany) and deoxyribosylthymdine (12- to 18-mer; Sigma) as
a primer. After amplification (denaturation at 94°C for 30 s,
annealing at 60°C for 30 s, and extension at 72°C for 30 s) and electrophoresis on a 2% agarose gel, amplicons were blotted
onto a nylon membrane (Hybond N+; Amersham-Pharmacia, Freiburg,
Germany), and hybridization was performed at 42°C with specific
internal oligonucleotide probes (followed by two washes at room
temperature) and 45°C under increasingly stringent salt conditions.
Chemiluminescent labelling and detection of hybridized oligonucleotides
were performed by using the Amersham ECL kit and autoradiography for
approximately 90 min on Hyperfilm-ECL (Amersham-Pharmacia). The
following specific cytokine primers and probes and PCR cycle numbers
were used: Cytokine ELISAs, nitrite-nitrate measurements, and determination
of liver enzyme levels.
Roughly similar-sized pieces of spleen
were weighed (each piece was approximately 300 to 400 mg, except for
controls, which weighed 70 to 80 mg) and homogenized in 2 ml of PBS
containing 10 µg of chymostatin per ml, 5 µg of leupeptin per ml,
and 10 µg of aprotinin (Boehringer, Mannheim, Germany) per ml.
Homogenates were diluted with PBS to equalize the concentrations of
spleen in the samples. Samples were centrifuged at 6,500 rpm for 15 min in an Eppendorf microcentrifuge, after which the supernatant was stored
at Statistics.
Quantifiable data are expressed as the means of
individual determinations ± SD. Statistical analysis was performed by
using Student's t test or Welch analysis of variance in
cases of unequal variances.
Course of M. avium infection in p55+/+ and
p55
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Fatal Granuloma Necrosis without Exacerbated Mycobacterial Growth
in Tumor Necrosis Factor Receptor p55 Gene-Deficient Mice Intravenously
Infected with Mycobacterium avium
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
/) mice to
the development of granulomatous lesions in M. avium-infected syngeneic C57BL/6 (p55+/+) mice. Up to
5 weeks after infection with either the highly virulent M. avium strain TMC724 or the intermediately virulent M. avium strain SE01, bacterial counts in the liver, spleen, and
lung of p55/ mice were identical to or lower than those
in infected p55+/+ mice. However, the formation of
mononuclear cell foci in the liver was delayed by approximately 2 to 3 weeks in p55/ mice compared to the results obtained for
infected p55+/+ mice. Despite comparable bacterial loads,
granulomas in p55/ mice underwent progressive necrosis,
causing damage to the surrounding liver tissue. The appearance of
necrotizing granulomas was associated with the death of all infected
p55/ mice, regardless of the virulence of the M. avium strain used for infection. Granulomatous lesions in the
liver contained three times as many CD3+ cells in
p55/ mice yet appeared more diffuse than in
p55+/+ mice. Semiquantitative reverse transcription-PCR
studies revealed that prior to mouse death, interleukin-12 (IL-12) and
gamma interferon mRNA levels were up regulated in the livers of
infected p55/ mice, while mRNA levels for tumor
necrosis factor, the inducible isoform of nitric-oxide synthase (iNOS),
and IL-10 were similar to those found in infected p55+/+
mice. In response to persistent mycobacterial infection, the absence of
TNFRp55 causes the disregulation of T-cell-macrophage interactions and
results in fatal granuloma necrosis even when adequate antibacterial
functions are maintained.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice) were
infected with live M. bovis BCG or killed
Corynebacterium parvum, they developed fewer and smaller
granulomas than wild-type control mice (37). However, when
M. tuberculosis was used to infect p55/
mice, granuloma formation was found to proceed in an almost unaltered fashion (17). In the latter experiments, and in studies
using transgenic mice expressing soluble TNFRp55 fusion protein, which effectively neutralizes the activity of soluble TNF, mutant mice exhibited greatly increased bacterial organ loads with concomitant tissue necrosis and prematurely succumbed to infection (17, 18). With these models, it has been impossible to distinguish between direct mycobacteriostatic effects and the granulomagenic properties of TNF, and the effects of the lack of TNFRp55-mediated signalling on granuloma maintenance in chronic mycobacterial
persistence could not be studied.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice were obtained as
described (34). The mice used in the studies presented here
are fifth generation backcrosses onto a C57BL/6J strain. The murine
TNFRp55 gene (Tnfrsfla) maps to mouse chromosome 6 at 57.10 centimorgans (cM) (4, 32). Neighboring genes that are linked
within 0.5 cM upstream and downstream encode Bphs (Bordetella
pertussis-induced histamine sensitization), CD9, lymphotoxin-
receptor, nucleolar protein 1, CD27, and Idd19 (insulin-dependent
diabetes mellitus 19). No information about allelic variants within
these loci between 129/Sv and C57BL/6 has been reported so far. The
p55/ and syngeneic C57BL/6 p55+/+ mice were
raised in the animal breeding facilities of the GSF-National Research
Center for Environment and Health (Oberschleissheim, Germany) and (for
repeat experiments) by Charles River Wiga (Sulzfeld, Germany). For the
course of M. avium infection, age- and sex-matched groups of
p55+/+ and p55/ mice were housed in
isolator cages under barrier conditions at the Institute for Medical
Microbiology, Munich, or in the animal facilities at the Borstel
Research Center.
70°C until needed. An inoculum of bacteria was prepared by
thawing an aliquot and diluting it in phosphate-buffered saline (PBS). Mice were infected intravenously via a lateral tail vein with indicated
inocula in 0.2 ml of PBS. Mice were anesthetized and killed at
indicated time points during the course of infection. Organs were
removed aseptically and homogenized in 10 ml of 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 were determined after
14 to 21 days of incubation at 37°C in humidified air. The natural
course of infection and the kinetics of granuloma formation in mice
infected with these strains were previously described (19).
2-microglobulin (14 cycles), sense
5'-TGACCGGCTTGTATGCTATC-3' and antisense
5'-CAGTGTGAGCCAGGATATAG-3' with probe
5'-GAAGCCGAACATACTGAACTGCTAC-3'; interleukin-10 (IL-10) (26 cycles), sense 5'-CGGGAAGACAATAACTG-3' and antisense
5'-CATTTCCGATAAGGCTTGG-3' with probe
5'-GGACTGGCTTCAGCCAGGTGAAGAC-3'; IL-12 p40 (26 cycles), sense 5'-CGTGCTCATGGCTGGTGCAAAG-3' and antisense
5'-CTTCATCTGCAAGTTCTTGGGC-3' with probe
5'-TCTGTCTGCAGAGAAGGTCACA-3'; gamma interferon (IFN-
) (24 cycles), sense 5'-AACGCTACACACTGCATCTTGG-3' and antisense 5'-GACTTCAAAGAGTCTGAGG-3' with probe
5'-GGAGGAACTGGCAAAAGGA-3'; TNF (22 cycles), sense
5'-GATCTCAAAGACAACCAACTAGTG-3' and antisense 5'-CTCCAGCTGGAAGACTCCTCCCAG-3' with probe
5'-CCCGACTACGTGCTCCTCACC-3'; and iNOS (26 cycles), sense
5'-CTGGAGGAGCTCCTGCCTCATG-3' and antisense 5'-GCAGCATCCCCTCTGATGGTG-3' with probe
5'-CTGGATGAGCTCATCTTTGCC-3'.
2-microglobulin was performed, and all samples were equalized (if
necessary) for 2-microglobulin cDNA content prior to analysis of
cytokine cDNA. Quantitation was performed after scanning hyperfilm
radiographs with a Studiostar scanner (Agfa, Cologne, Germany), and the
number of black pixels over background film was measured by using
Photoshop software (Adobe, Edinburgh, United Kingdom) in a preset frame
of 3,750 total pixels. Statistical analysis was carried out with pixel
values obtained from four mice per experimental group. Fold increase
over background was calculated from the titration of the standard cDNA,
assigning an arbitrary value of 1 to the titer with background pixel
levels. Fold differences between experimental groups were calculated by comparing fold increases of p55+/+ mice to levels of
p55/ mice infected with the same strain.
70°C. Plasma was obtained after centrifugation of heparinized blood drawn from the inferior vena cava of anesthetized mice and stored
at 70°C until further use. Enzyme-linked immunosorbent assay
(ELISA) measurements of TNF and IFN- levels in the plasma and spleen
homogenates were carried out as stipulated by the manufacturer (Genzyme-Virotech, Rüsselsheim, Germany), and concentrations were
calculated per input organ weight. As an indicator of nitric oxide
production, the concentrations of the final reaction products nitrate
and nitrite were determined in the plasma by a colorimetric method
(Boehringer), appropriately scaled down to microtiter plate format to
accommodate 50-µl samples. For use in this assay, plasma samples were
diluted twofold, added to a microconcentrator (Amicon, Beverly, Mass.)
(cutoff, 10,000 Da), and centrifuged at 6,000 rpm for 15 min followed
by 8,000 rpm for 30 min in an Eppendorf microcentrifuge. Levels of
aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and
lactatedehydrogenase (LDH) were measured in the plasma of mice by using
standard procedures and an automated sample analyzer in the laboratory
of clinical biochemistry of the Borstel Clinical Center.
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice.
During the first 5 weeks following
intravenous infection with either 105 or 106
CFU of the virulent TMC724 strain, the bacterial counts in the liver
and lungs of p55+/+ and p55/ mice were
almost identical (Fig. 1A and E).
Infection with either 1 × 105 or 8 × 106 CFU of the less-virulent SE01 strain resulted in
similar growth curves in the liver and lungs of p55+/+ and
p55/ mice (Fig. 1B and F). The plateau of bacterial
growth reached after 2 weeks of infection with this strain in the
livers of p55+/+ mice was also evident in
p55/ mice, showing early mycobacteriostatic effector
mechanisms to be intact in these mice. In one of three experiments,
bacterial counts in the lungs of p55/ mice infected
with SE01 were significantly higher than in p55+/+ mice at
5 weeks postinfection (P < 0.05). Following infection with either strain, p55/ mice always had significantly
lower bacterial loads in their spleen at early time points of infection
(P < 0.01) (Fig. 1C and D). This could be attributed
to the smaller size of the spleens of p55/ mice, as
bacterial loads were identical between p55+/+ and
p55/ mice when CFU counts were expressed as the log of
CFU per gram of tissue (data not shown).
View larger version (18K):
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FIG. 1.
Course of M. avium infection in
p55+/+ and p55 / C57BL/6 mice. Mice were
intravenously infected with either 1 × 106 CFU of
M. avium TMC724 (A, C, and E) or 8 × 106
CFU of M. avium SE01 (B, D, and F), and bacterial counts
were determined in the liver (A and B), spleen (C and D), and lungs (E
and F) at indicated time points. Each point reflects the means and SD
(error bars) of four mice per group. Triangles, p55+/+
mice; circles, p55/ mice; *, P < 0.05;
**, P < 0.005.
Kinetics and morphology of granuloma formation in response to
M. avium infection in p55+/+ and
p55/ mice.
During early infection of
p55/ mice with 105 or 106 CFU
of M. avium (TMC724 or SE01) there were numerous Kupffer
cells filled with acid-fast material that were not surrounded by
inflammatory cells, while this was very rarely observed in control
infected mice. In p55/ mice, these foci of infection
without an inflammatory response persisted throughout the course of
infection, while p55+/+ mice increasingly developed
inflammatory cell infiltrations.
/ mice were delayed by approximately
2 weeks in mice infected with TMC724 and by 3 weeks in mice infected
with SE01 compared to p55+/+ mice (Fig.
2). Mononuclear cell infiltrations were
more diffuse in p55/ mice and lacked the appearance of
mature granulomas because they contained fewer macrophages showing the
typical light-microscopic signs of epithelioid differentiation
(elongation of nuclei and enlargement of cytoplasm) (Fig. 3A and
B). Immunohistological staining of
material reactive with a specific rabbit polyclonal anti-iNOS antiserum
within the granulomas was equally intense in p55/ and
p55+/+ mice (Fig. 3C and D).
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/ mice were accelerated compared to
the lower-dose infection but still proceeded in a delayed and less well
organized fashion compared to high-dose infection in p55+/+ mice.
Loss of granuloma integrity during chronic M. avium
infection in p55/ mice.
Within 6 to 8 weeks of
receiving 105 CFU of TMC724 or SE01, or 3 to 4 weeks after
receiving 1 × 106 CFU of TMC724 or 8 × 106 CFU of SE01, p55/ mice began to look
sick, and all M. avium-infected gene-deficient mice
subsequently died within approximately 10 days. Autopsies performed
within this time frame revealed that cellular integrity was severely
impaired in all granulomas of infected p55/ mice, while
infected p55+/+ mice showed typical, well-differentiated
monocytic lesions and control uninfected p55/ mice had
normal liver and spleen histologies at that time. Prior to 5 weeks
postinfection, infected p55/ mice showed no evidence of
granuloma necrosis. Early disintegrating granulomas in
p55/ mice in the 6th week of infection showed typical
signs of apoptosis, like chromatin condensation and the appearance of
pyknotic nuclei, although this was a rare occurrence in intact
granulomas of p55+/+ mice (Fig. 4A and
B). Ziehl-Neelsen staining revealed that
all of these lesions in p55/ mice contained
extracellular acid-fast bacilli, showing that these lesions originated
from lysed macrophages in granulomatous foci of infection, while in
p55+/+ mice, acid-fast mycobacteria were always found
within macrophages (data not shown).
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/ mice was followed
by extensive granuloma necrosis in the liver and damage to adjacent liver tissue regardless of the isolate used for infection (Fig. 5B and
C). Granuloma necrosis and hepatocyte
damage appeared to be progressive, since mice randomly selected for
histology after the 5th week of infection showed necrotizing granulomas
at different stages of development. In particular, mice that appeared
healthy had only a few necrotic granulomas, while mice that appeared
very sick showed abundant granuloma necrosis and widespread hepatocyte damage.
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) (HE; magnification, ×128);
(C) low-power view of multiple foci of granuloma necrosis with
incipient damage (arrows) to surrounding liver tissue (HE;
magnification, ×64).
/ mice had significantly
increased serum levels of liver enzymes compared to infected
p55+/+ mice (levels in p55+/+ mice were
[ASAT] 72 ± 28 U/ml, [ALAT] 55 ± 19 U/ml, and [LDH] 222 ± 67 U/ml; levels in p55/ mice were [ASAT]
983 ± 340 U/ml, [ALAT] 381 ± 144 U/ml, and [LDH] 7,442 ± 2,413 U/ml; and levels in uninfected p55/
mice were [ASAT] 15 ± 5 U/ml, [ALAT] 5 ± 4 U/ml,
[LDH] 70 ± 20 U/ml). In two repeat experiments performed with
5 × 106 to 8 × 106 CFU of the
less-virulent SE01 strain, 20 of 20 p55/ mice died
between 5 and 6 weeks after infection, showing signs of granuloma
necrosis, while all syngeneic p55+/+ infected mice were
healthy for the length of the experiment (4 months) and showed no
structural impairment of granulomas (19). Moribund infected
p55/ mice also had significantly higher bacterial
counts in the kidney, brain, and blood, but there was no evidence of
necrosis in lungs, spleens, kidneys, or brains at the time of death of
these mice.
Evidence for immune disregulation in p55/ mice
infected with M. avium.
In an attempt to elucidate what
precipitated the death of infected p55/ mice, we
investigated the cellular composition and mRNA expression of regulatory
cytokines at the site of infection. Immunohistology of the liver at 5 weeks postinfection revealed that there were approximately three times
the number of CD3+ cells present in the inflammatory
lesions of TMC724-infected p55/ mice compared to the
number present in p55+/+ mice (120 ± 21 versus
44 ± 6 granuloma-associated CD3+ cells per 400×
microscopic field) (an example is shown in Fig. 6). Similar changes in CD3+
cell numbers were found in SE01-infected p55/ mice. The
increase in CD3+ cells was accounted for by an increase in
both the CD4+ and the CD8+ subpopulations of T
cells (data not shown). In p55+/+ mice, CD3+
cells were always confined to granulomas, whereas they were also found
scattered throughout the liver sinusoids in p55/ mice.
To rule out the possibility that there might be a kinetic, rather than
an absolute, difference in the number of CD3+ cells
recruited to granulomatous lesions in p55/ versus
p55+/+ mice, granulomas were also evaluated for their
CD3+ cell content at 3 weeks postinfection with M. avium. Although incipient granulomas of p55/ mice
were rather small when compared to established lesions in p55+/+ mice at this time point, similar numbers of
CD3+ cells were detected (Fig. 6A and B). At no time did
p55+/+ mouse lesions display the high numbers of
CD3+ cells evident in p55/ mouse lesions at
5 weeks postinfection.
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/ and immunocompetent mice,
levels of mRNA for IFN-, TNF, and iNOS were significantly lower in
p55/ mice (twofold, fourfold, and fourfold decreases,
respectively) (Fig. 7, columns A),
reflecting the decreased number of infiltrating inflammatory cells in
these mice at this early point of infection. Semiquantitative RT-PCR of
liver tissue taken from TMC724-infected p55/ mice 1 week prior to death, however, demonstrated that levels of mRNA for
IFN- and IL-12 p40 (8-fold and 10-fold increases, respectively) were
higher than in infected p55+/+ mice, while IL-10, TNF, and
iNOS mRNA levels were comparable to those found in immunocompetent mice
(Fig. 7, columns B). IL-4 mRNA levels did not significantly differ from
uninfected controls at any investigated point of infection. RT-PCR
studies of SE01-infected mice gave similar results (data not shown).
These findings were corroborated by ELISA measurements of cytokine
protein levels, which also showed that in moribund p55/
mice, IFN- and IL-12 levels were consistently higher in both spleen
homogenates and plasma regardless of the M. avium isolate used for infection (Table 1). Plasma TNF
levels of SE01-infected p55/ mice at time of death were
also significantly higher than in p55+/+ mice (Table 1).
Mice that were clinically worse always had the highest TNF and IFN-
plasma levels. In agreement with RT-PCR results on iNOS expression,
nitrite and nitrate levels (as an estimate of nitric oxide production)
in the sera of M. avium-infected mice determined after 5 to
6 weeks of infection were similar, with levels slightly lower in
p55/ mice than in p55+/+ mice (254 ± 95 µM versus 365 ± 39 µM, respectively). Taken together, these
data show that there was a hyperinflammatory response with increased
numbers of CD3+ cells in the livers of M. avium
infected p55/ mice just prior to death and
significantly higher amounts of IL-12 and IFN- present in tissue
lesions.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The major finding from our studies is that, despite comparable
M. avium counts in p55+/+ and
p55/ mice, developing granulomas in
p55/ mice become necrotic and cause tissue damage.
Granuloma necrosis in p55/ mice is associated with
increased infiltration of CD3+ cells and higher IFN- and
IL-12 tissue levels, and it is invariably followed by the death of all
infected p55/ mice, regardless of the virulence of the
M. avium strain used for infection.
M. avium infection is known to progress in two phases in susceptible mice: after an initial phase of almost unrestrained growth with little inflammatory cell accumulation, CD4+ T-cell-mediated effector mechanisms become apparent, resulting in granuloma formation (19) and growth reduction of strains of intermediate virulence (1). Our findings imply that TNFRp55 is not involved in antibacterial mechanisms operating during the early T-cell-independent and T-cell-dependent phases of infection. This interpretation is in agreement with a recent study showing that mice deficient for both components of TNFR (p55 and p75) were similar to littermate controls in restricting the growth of an M. avium strain of intermediate virulence (10).
Since during the first 5 weeks of infection levels of M. avium growth were identical in the livers of p55+/+
and p55/ mice, we were in the position to assess the
inflammatory response in infected livers uninfluenced by an
exacerbation of infection known to occur in other experimental systems
in these mice (e.g., during M. tuberculosis infection
[17]). We found granuloma formation to be delayed in
p55/ mice compared to the formation rate in
p55+/+ mice. Granulomas in p55/ mice had a
more immature and malorganized appearance, but macrophages showed signs
of activation, as evidenced by strong staining for iNOS protein.
Signalling via TNFRp55 was previously shown not to be necessary for the
expression and function of iNOS (14, 17), although we did
find mRNA expression for iNOS to be delayed in p55/
mice as a consequence of delayed mononuclear-cell recruitment (Fig. 7).
In this context, it is important to remember that, in contrast to
M. tuberculosis, most strains of M. avium,
including the ones used here, are resistant to any direct antibacterial effects of nitric oxide (11).
These findings concerning the kinetics, quantity, and quality of
granuloma formation reconcile a number of conflicting reports on the
role of TNF during granuloma initiation (17, 25, 37, 38).
Our kinetic investigations indicate that the speed and magnitude of
granuloma formation and differentiation in the absence of TNFRp55 are
determined by the virulence and inoculum size of the mycobacterium
used. Therefore, when early points of infection are examined, little or
no granuloma formation is evident in p55/ mice whenever
mycobacteria of low virulence, such as M. bovis BCG or
M. avium, are used for infection (37). At later
points of infection with these strain, or during infection with highly virulent M. tuberculosis, there is little difference in the
granuloma number in p55/ mice compared to the number in
p55+/+ mice, and epithelioid differentiation and macrophage
activation does occur to a significant extent (17).
Flynn et al. also noted granuloma necrosis in p55/ mice
infected with M. tuberculosis (17). In those
experiments, however, it was difficult to ascertain the cause of tissue
destruction, because M. tuberculosis proliferated
extensively due to the absence of TNF-mediated antimycobacterial
effects. In that situation, macrophages of p55/ mice
rendered incapable of controlling growth of M. tuberculosis may have been lysed after reaching a threshold load, causing necrotic destruction of granulomatous and parenchymal tissue. In fact, an
identical histopathology was described in M. tuberculosis-infected mice deficient for 2-microglobulin, the
-T-cell receptor, IFN-, or iNOS (7, 16, 28, 29),
implying that unrestricted growth of M. tuberculosis is the
common determining cause of tissue necrosis, rather than a specific
effect due to the absence of TNFRp55.
In contrast, our experiments demonstrate that long-term macrophage integrity and granuloma maintenance during chronic M. avium infection are dependent on TNFRp55-mediated functions distinct from those known to induce antibacterial effector mechanisms. While both this function of regulating granuloma stability and the known antimycobacterial effects induced by TNF may be important in containing infection with M. tuberculosis, only the former is critical in determining the outcome of M. avium infection.
The view that p55/ mice died not as a consequence of
the exacerbation of mycobacterial proliferation or dissemination, but rather because of the pathology (i.e., necrosis) caused by granuloma disintegration, is supported by the fact that mice are capable of
tolerating much higher numbers of M. avium than M. tuberculosis in infected organs. For instance, infection with
M. avium TMC724 in immunocompetent mice leads to bacterial
organ loads in excess of 1010 CFU and is accompanied by
heavy dissemination into the kidneys and the brain without any apparent
impairment of vital organ functions (12a, 19). Although
during this scenario granuloma macrophages are completely filled with
mycobacteria, we have never observed signs of cell death or granuloma
necrosis resembling those in p55/ mice, where they
occur at a much lower mycobacterial load and even during infection with
a strain of lower virulence.
Disintegration of the granulomatous lesion initially involved apoptosis
of mononuclear cells followed by a necrotic decomposition of the entire
granuloma structure. Hepatocyte necrosis with concomitantly increased
levels of hepatic transaminases in serum ensued, and this might have
constituted the direct cause of death in intravenously infected
p55/ mice. In view of the disproportionately increased
levels of LDH, it is also possible that necrosis in tissues other than
the liver (e.g., the lung) may have occurred. We have been unable to
find histopathologic signs of granuloma necrosis in other organs after intravenous infection. However, in a study focusing on the long-term immunopathological sequelae of aerogenic exposure to M. avium in immunocompetent and -deficient mice, we observed that
p55/ mice died at approximately the same rate as their
intravenously infected littermates yet showed few signs of hepatocyte
necrosis, while they had necrotizing granulomatous infiltrations in the lungs (3). Therefore, granuloma breakdown, as such, rather than isolated hepatocyte necrosis, seems to be a prerequisite for the
death of p55/ mice resulting from M. avium
infection. Granuloma necrosis was always followed by increased growth
of M. avium, primarily in less-affected organs, such as the
kidney and brain. This suggests dissemination of mycobacteria from
disintegrating primary granulomas and substantiates the generally held
belief that a major function of granulomas is to physically prevent
bacterial spreading by isolating infectious foci.
Traditionally, TNF has been thought to act as a major inducer of tissue
pathology, particularly in tuberculous lesions (9, 36).
However, p55/ mice infected with Leishmania
major were previously found to have larger inflammatory lesions
than p55+/+ mice, although they remained fully capable of
eliminating parasites from the lesions (39). Moreover,
contact hypersensitivity reactions to dinitrofluorobenzene were
enhanced in p55/ mice compared to syngeneic
p55+/+ mice (27). In these situations,
TNFRp55-mediated signalling would seem to be involved in restricting or
resolving, rather than exacerbating, the inflammatory response.
It seems possible that TNF regulates the inflammatory response by maintaining the viability of activated macrophages at the site of infection. M. avium may have a direct or indirect toxic effect on macrophages, and TNFRp55-generated signals may be needed to antagonize any such effect. In this respect, in vitro cell culture systems demonstrated that mycobacterial infection may lead to macrophage apoptosis, particularly when high numbers of mycobacteria are used (35), and that TNF may, in part, counteract apoptosis, increasing the survival of infected macrophages (12, 30).
The concept of a regulatory role for TNF in chronic inflammation is
further supported by results recently obtained in TNF gene-deficient
mice experimentally challenged with heat-killed C. parvum
(38). These mice also developed poorly organized
hyperinflammatory lesions in their livers and spleens and showed
disseminated foci of necrosis shortly before death (31).
Evidence for an unbalanced, hyperinflammatory response in the absence
of TNF signalling was also obtained in the present studies of
persistent mycobacterial infection. When we examined the lesions in
p55/ mice by immunohistology, there was a threefold
increase in tissue-infiltrating CD3+ cells in the livers of
infected p55/ mice, and mRNA and protein levels for
IFN- and IL-12 (and inconsistently for TNF) were significantly
higher in the liver, spleen, and plasma of these p55/
mice than in p55+/+ mice. It is therefore possible that a
disregulated T-cell-mediated and/or IFN--dependent process led to
granuloma necrosis.
It is unknown whether, during certain stages of granuloma development in human mycobacterial infections, expression of TNF or its receptors is down modulated. It is clear from histological studies that apoptosis and necrosis frequently occur in mycobacterial granulomas (8, 9, 23). In view of the evidence presented here, it seems possible that necrotic breakdown of tuberculous lesions may also be a consequence of a decrease, rather than an increase, in TNF signalling and of a concomitant disregulation of the T-cell response within the lesion.
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ACKNOWLEDGMENTS |
|---|
This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Eh 101/4-1).
We acknowledge the excellent technical assistance of Claudia Hahn.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Molecular Infection Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. Phone: 49-4537-188481. Fax: 49-4537-188686. E-mail: sehlers{at}fz-borstel.de.
Editor: S. H. E. Kaufmann
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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