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*
Institut für Neuropathologie, Universitätskliniken Bonn, Bonn, Germany;
Department CNS, F. Hoffmann-La Roche, Basel, Switzerland; and
Institut für Medizinische Mikrobiologie und Hygiene, Universität Heidelberg, Fakultät für Klinische Medizin, Mannheim, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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exerts its biologic activity through two distinct
receptors, TNF receptor type 1 (TNFR1, p55) and TNF receptor type 2
(TNFR2, p75). To analyze their function in toxoplasmosis, we orally
infected mice genetically deficient for TNFR1
(TNFR10/0), TNFR2 (TNFR20/0), or both TNF
receptors (TNFR1/20/0), as well as wild-type (wt) mice with
a low-virulent strain of Toxoplasma gondii.
TNFR1/20/0 and TNFR10/0 mice succumbed to
toxoplasmosis within 17 and 27 days, respectively, whereas
TNFR20/0 and wt mice were equally resistant to acute
toxoplasmosis. Histopathology attributed death of
TNFR1/20/0 and TNFR10/0 mice to a fulminant
necrotizing encephalitis. In addition, pneumonia contributed to the
fatal outcome. The poor prognosis of TNFR1/20/0 and
TNFR10/0 mice was reflected by a significantly increased
parasitic load in the brain and lung as compared with
TNFR20/0 and wt mice. Immunohistochemistry demonstrated a
remarkable reduction of inducible nitric oxide synthase protein in
brain and lung of TNFR1/20/0 and TNFR10/0 as
compared with TNFR20/0 and wt mice. Reverse-transcribed PCR
showed that in contrast to TNFR20/0 and wt mice,
TNFR10/0 mice were unable to up-regulate inducible nitric
oxide synthase mRNA transcripts in the course of infection, whereas
intracerebral levels of IFN-
, TNF-, and IL-1ß mRNA transcripts,
recruitment of immune cells to the brain, and the amount of apoptotic
cells in inflammatory foci did not differ significantly among the
various experimental groups. These results illustrate that in
Toxoplasma encephalitis, TNF--mediated immune responses
are of crucial importance and that signaling through TNFR1, but not
TNFR2, provides the stimulus required for the induction of protective
nitric oxide. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous experimental studies have underlined the key importance of
cytokine production for an effective antiparasitic immune response. In
particular, IFN-, which is produced by NK cells as well as
CD4+ and CD8+ T cells in murine toxoplasmosis
(7, 8, 9), has gained special attention. The fundamental role of IFN-
in toxoplasmosis is illustrated in IFN-- and IFN- receptor
(IFN-R)-deficient mice, which rapidly succumb to fulminant acute
toxoplasmosis (10, 11). In addition, neutralization of IFN- results
in a lethal reactivation of chronic TE (7). There is increasing
evidence that IFN- mediates its protection in part through the
induction of other protective effector molecules. In both IFN-- and
IFN-R-deficient mice, as well as in mice with chronic TE, which
received an IFN- neutralizing Ab, the lethal course of disease was
characterized by a dramatic reduction of TNF- and inducible nitric
oxide synthase (iNOS) production (10, 11, 12).
The importance of iNOS for the antiparasitic immune response in vivo
has recently been demonstrated in iNOS-deficient mice, which succumbed
to toxoplasmosis within 4 wk (13). In addition, neutralization of
nitric oxide (NO) in acute murine toxoplasmosis results in an
exacerbation of TE (14). With respect to TNF-, which is produced by
CD4+ and CD8+ T cells as well as
macrophages/microglial cells in murine TE (9), it has also been shown
that its application in acute toxoplasmosis has a protective effect
(15), whereas neutralization of this cytokine in chronic TE results in
a lethal exacerbation of the disease (12). However, the precise
biologic role of TNF- in toxoplasmosis has not yet been defined.
TNF- exerts its biologic activity via two receptors, the TNF
receptor type 1 (TNFR1, p55) and the TNF receptor type 2 (TNFR2, p75),
which are expressed in virtually all cell types (16). These two
receptors have been implicated in a variety of biologic functions,
including the development of lymphatic organs (17), mediation of
apoptosis (18, 19, 20), regulation of cell adhesion molecule expression
(21), and activation of glial cells (22), as well as immune reactions
to infectious organisms (23, 24). All of these aspects may play a
significant role in the immune response to T. gondii, and it
still remains to be clarified which TNFR(s) mediates the protective
effects of TNF- in toxoplasmosis.
In the present study, we took advantage of the recent development of
TNFR1 (TNFR10/0)- and TNFR2 (TNRF20/0)-, as
well as TNFR1/2 (TNFR1/20/0)-deficient mice (23, 24, 25), which
allow the analysis of the role of TNF- as well as its receptors
under exactly defined genetic conditions. Our experiments show that
TNF--mediated immune responses are of critical importance in
toxoplasmosis and that signaling through TNFR1, but not through TNFR2,
is necessary to mediate protection in this parasitic infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female 129/Sv x C57BL/6 TNFR10/0, TNFR20/0, TNFR1/20/0, and 129/Sv x C57BL/6 wild-type (wt) mice at the age of 8 to 16 wk were used for the experiments. Female C57BL/6 mice (6–8 wk old) were obtained from Harlan-Winkelmann (Borchen, Germany). The animals were kept in an isolation facility before and throughout the studies.
Infectious organisms
Parasites were harvested from the brains of mice chronically infected with a low-virulent strain of T. gondii (DX strain). Brain tissue of these animals was dispersed in PBS. The final concentration of the infectious agents was adjusted to a dose of 10 cysts/0.5 ml, which was administered orally to the experimental animals by gavage.
Experimental procedure and tissue processing
On days 10 and 25 p.i., infected wt, TNFR10/0, TNFR20/0, and TNFR1/20/0 mice were perfused intracardially with 0.9% saline while they were under deep Metofane (Janssen, Neuss, Germany) anesthesia. Uninfected mice of all strains served as controls.
For immunohistochemistry on cryostat sections, liver, spleen, small and large intestine, heart, lung, and brain were dissected. Blocks of tissue were mounted on thick filter paper with Tissue Tek OTC compound (Miles Scientific, Naperville, IL), snap-frozen in isopentane (Fluka, Neu-Ulm, Germany) on dry ice, and stored at -80°C until use for immunohistochemistry.
For immunohistochemistry on paraffin sections and conventional histology, mice were perfused with 4% buffered paraformaldehyde, pH 7.4. Liver, spleen, small and large intestine, heart, and lung were dissected, fixed in 4% buffered paraformaldehyde for 24 h, and embedded in paraffin. Sections (4 µm) were cut from the organs. For conventional histology, sections were stained with hematoxylin and eosin, periodic acid-Schiff stain, or Giemsa solution (Merck, Darmstadt, Germany).
Brain-derived leukocytes were isolated by passing brain tissue through a 100-mesh stainless steel sieve, followed by collagenase/DNase digestion and Percoll (Pharmacia, Freiburg, Germany) gradient separation, as described previously (26). Isolated cells were analyzed by flow cytometry. In control experiments with spleen cells, the concentration and incubation time of enzymes were optimized to minimize a reduction of Ag-specific staining.
For reverse transcription (RT)-PCR determination of cytokine mRNA and Western blot analysis, brain tissues were snap-frozen and stored at -80°C.
Monoclonal and polyclonal Abs
The following anti-mouse mAb-producing hybridomas were obtained from the American Type Culture Collection (Rockville, MD) and kept under standard hybridoma conditions in DMEM with 10% FCS (Life Technologies, Eggenstein, Germany): anti-CD4 (clone G.K.1.5., rat IgG2b), anti-CD8 (clone 2.43, rat IgG2b), CD45 (LCA, clone M1/9.3.4.HL.2, rat IgG2a), CD45R (B220, clone RA3–3A1/6.1, rat IgM), anti-CD54 (ICAM-1, clone YN1/1.7.4, rat IgG2b), and anti-VCAM (clone M/K-2.7, rat IgG1).
For flow cytometry, CD45 was conjugated with biotin by standard labeling procedures.
Rat anti-mouse Ly6-G (Gr-1) was a kind gift of Dr. R. Coffman (DNAX
Institute, Palo Alto, CA). Rat anti-mouse TNF- (clone MP6-XT22),
CD4-FITC, CD8-FITC, and CD45R (B220)-FITC were purchased from
PharMingen (Hamburg, Germany). A polyclonal rabbit
anti-T. gondii antiserum was obtained from Biogenex
(Duiven, The Netherlands). Polyclonal rabbit anti-mouse iNOS,
alkaline phosphatase-conjugated donkey anti-rabbit IgG
F(ab')2 fragments, peroxidase-conjugated goat
anti-rabbit IgG F(ab')2 fragments, Texas Red-conjugated
goat anti-rabbit IgG F(ab')2 fragments, and
biotinylated mouse serum-preadsorbed mouse anti-rat IgG
F(ab')2 fragments were from Dianova (Hamburg, Germany).
FITC-conjugated F4/80 was purchased from Serotec-Camon (Wiesbaden,
Germany). Peroxidase-linked sheep anti-rat IgG F(ab')2
fragments were obtained from Amersham-Buchler (Braunschweig, Germany),
and peroxidase-linked streptavidin-biotin complex and alkaline
phosphatase-linked streptavidin-biotin complex were purchased from
Dakopatts (Hamburg, Germany). FITC-conjugated avidin and rabbit
anti-cow glial fibrillary acid protein (GFAP) were from Sigma (St.
Louis, MO). Mouse serum protein-absorbed PE-conjugated goat
anti-rat IgG and avidin PE/Cy5 were obtained from Southern
Biotechnology Associates-Biozol (Freising, Germany).
Immunohistochemistry
Immunohistochemistry was performed on acetone-fixed 10-µm
cryostat sections according to a protocol previously described in
detail (2). In brief, for demonstration of CD4, CD8, CD45 (LCA), CD45R
(B220), Ly6-G, and TNF-, as well as for visualization of T.
gondii and iNOS, an indirect method using peroxidase-conjugated
sheep anti-rat IgG F(ab')2 fragments or
peroxidase-conjugated goat anti-rabbit F(ab')2
fragments, respectively, as secondary Abs was used. The avidin-biotin
complex technique was employed for demonstration of CD54 (ICAM-1) and
VCAM. In addition, T. gondii Ag was demonstrated in paraffin
sections in a peroxidase-anti-peroxidase protocol, as described
previously (2). The peroxidase reaction products were visualized using
3,3'-diaminobenzidine (Sigma) and H2O2.
Sections were, in part, lightly counterstained with hemalum
(Merck).
To demonstrate TNF- with GFAP on cryostat sections, a
double-labeling immunofluorescence technique was employed. The
incubation steps were 1) rat anti-mouse TNF-, 2) biotinylated
mouse anti-rat IgG F(ab')2 fragments, 3)
FITC-conjugated avidin, 4) rabbit anti-cow GFAP antiserum, and 5)
Texas Red-conjugated goat anti-rabbit IgG F(ab')2
fragments.
To control for nonspecific reactions, incubations either with irrelevant species-specific IgG Abs instead of the primary Ab or with omission of the primary Ab were performed.
Apoptosis was assessed on 1% glutaraldehyde-fixed cryostat sections by use of a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) kit (Boehringer, Mannheim, Germany) according to the manufacturer’s instructions.
Flow cytometry of brain-derived leukocytes
Brain-derived leukocytes were analyzed by double or triple immunofluorescence staining followed by flow cytometry. Murine macrophages and microglial cells were identified by staining with CD45-biotin and F4/80-FITC followed by avidin-PE/Cy5. As reported previously (26), macrophages and microglial cells were differentiated by their different expression of CD45 (LCA). Microglial cells are F4/80+CD45low, whereas macrophages are F4/80+CD45high. For identification of B lymphocytes, double staining with CD45R (B220)-FITC and CD45 (LCA)-biotin followed by avidin-PE/Cy5 was employed. CD4+ and CD8+ T lymphocytes were identified by staining with rat anti-mouse CD8 and goat anti-rat PE followed by CD4-FITC. Granulocytes were identified by triple staining with rat anti-mouse Ly6-G and goat anti-rat PE followed by F4/80-FITC, CD45-biotin, and avidin-PE/Cy5. Granulocytes were defined as Ly6G+F4/80-CD45high.
Control staining was performed with fluorochrome-labeled or unlabeled isotype-matched control Abs. For flow cytometry, a FACScan (Becton Dickinson, Heidelberg, Germany) was used.
Detection of cytokine mRNA by RT-PCR
IFN-, TNF-, iNOS, IL-1ß, and
hydroxyphosphoribosyltransferase (HPRT) mRNA transcripts were analyzed
in brain tissue homogenates according to a protocol previously
described in detail (27). Primer sequences and oligonucleotide probes
for IFN-, TNF-, and HPRT were identical with those described
before. iNOS mRNA transcripts were analyzed by use of the following
pair of primers: 5'-TCA CGC TTG GGT CTT GTT CAC T-3' (sense), 5'-TTG
TCT CTG GGT CCT CTG GTC A-3' (antisense) as described by Stenger et al.
(28), and as the oligonucleotide probe, 5'-TGA CCC TAA GAG TCA CCA
AA-3' was employed.
In brief, mRNA was extracted from the brains of uninfected and T. gondii-infected TNFR1/20/0, TNFR10/0, TNFR20/0, and wt mice by use of an mRNA extraction kit (Pharmacia). After RT of mRNA using the Superscript RT kit (Life Technologies), PCR reactions were conducted in a volume of 50 µl. PCR reaction conditions were optimized for each set of primers to ensure that amplification occurred in the linear range. PCR products were subjected to electrophoresis through an agarose gel, and the DNA was transferred to a nylon membrane (Boehringer). Blots were hybridized using specific oligonucleotide probes, which were 3'-end labeled with digoxigenin by use of a DIG Oligonucleotide 3'-End Labeling Kit (Boehringer). A DIG Luminescent Detection Kit (Boehringer) was used to visualize the hybridization products.
Western blot analysis
For the analysis of iNOS production by Western blot, brain tissue of each experimental group was lysed in ice-cold lysis buffer (10 mM NaH2PO4, pH 8.0, 140 mM NaCl, 3 mM MgCl2, 0.5% IGEPAL, 1 µM DTT, 17.4 µg PMSF/ml, 100 µg aprotinin/ml; all reagents from Sigma).
In addition to brain tissues, IFN- and LPS-stimulated resident
peritoneal cells were used as a positive control as described by Green
et al. (29). In brief, peritoneal cells were isolated from a C57BL/6
mouse and incubated at a concentration of 5 x 105
cells in 500 µl in DMEM with 5% FCS (Life Technologies) in snap-cap
tubes (Becton Dickinson). For the induction of iNOS, cells were treated
with 10 U IFN-/ml (R&D Systems, Wiesbaden, Germany) and 2 ng LPS/ml
(Sigma) for 48 h. Negative controls remained unstimulated. For
Western blot analysis, cells of six snap-cap tubes were pooled after
48 h of incubation, and the cellular proteins were isolated as
described for brain tissues.
The protein content of brain tissue and peritoneal cell lysates was determined by the Bradford assay (Bio-Rad, Munich, Germany). For Western blot analysis, 20 µg of protein was separated on 7.5% reducing SDS-PAGE and transferred in 25 mM Tris, 192 mM glycine, pH 8.3, and 20% methanol to a 0.2 µm-pore size nitrocellulose membrane (Bio-Rad). Detection of iNOS on nitrocellose membrane was performed according to a protocol published recently by Bogdan et al. (30). In brief, membranes were blocked with 1% BSA in TTBS (25 mM Tris, pH 7.5, and 150 mM NaCl with 0.2% Tween-20) for 1 h and, thereafter, incubated with rabbit anti-mouse iNOS diluted 1:2000 in TTBS with 1% BSA at 4°C overnight. After intensive washing with Tris buffer, the membranes were incubated with alkaline phosphatase-conjugated donkey anti-rabbit IgG F(ab')2 fragments (Dianova) at a dilution of 1:10,000 in TBBS for 45 min at RT. After washing in Tris, the membranes were equilibrated in alkaline phosphatase buffer (100 mM Tris, pH 9.5, 100 mM NaCl, and 5 mM MgCl2). Thereafter, they were developed in a solution of nitroblue tetrazolium (330 µg/ml, Sigma) and 5-bromo-4-chloro-3-indolyl phosphate (165 µg/ml, Sigma).
Specificity controls included either incubation with irrelevant rabbit anti-mouse Ab instead of the primary Ab or omission of the primary Ab.
Statistical evaluation
For statistical evaluation of the parasitic load of brain and
lung, the number of parasites was determined on anti-T.
gondii-immunostained sections in TNFR1/20/0,
TNFR10/0, TNFR20/0, and wt mice. High power
fields (n = 100–400) were analyzed per section
in four animals from each group, and the statistical significance of
the differences was evaluated by using the Wilcoxon test. Differences
in the mortality rates among TNFR1/20/0,
TNFR10/0, TNFR20/0, and wt mice were analyzed
using the 
2 test. A p value of <0.05 was
accepted as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After oral infection with 10 T. gondii cysts of the
low-virulent DX strain, all mice developed toxoplasmosis. There were
significant differences in the susceptibility to the parasite among the
various groups of mice as reflected by the mortality rates (Fig. 1
). Inactivation of both TNFRs rendered
mice highly susceptible to T. gondii, and all
TNFR1/20/0 mice died of acute toxoplasmosis up to day
17 p.i. In addition, TNFR10/0 mice also exhibited a
significantly increased susceptibility to the infectious organism, and
death occurred up to day 27 p.i. In contrast, TNFR20/0
mice were as resistant as wt animals, and TNFR20/0 mice
only in rare cases succumbed to acute toxoplasmosis (Fig. 1).
TNFR20/0 mice and wt mice developed a chronic toxoplasmosis
and survived for at least 3 mo.
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To assess the cause of death in TNFR1/20/0 and TNFR10/0 animals, a detailed histopathologic study was performed. TNFR1/20/0, TNFR10/0, TNFR20/0, and wt mice developed acute toxoplasmosis with the same pattern of organs involved. Brain and lung were most severely affected, and major differences among TNFR1/20/0, TNFR10/0, TNFR20/0, and wt animals were detected in these organs. In contrast, in all groups, liver, heart, and small and large intestine were only mildly diseased, and pathologic findings in these organs regressed up to day 25 p.i.
Histopathology attributed death of TNFR1/20/0 mice to a
progressive encephalitis, which was characterized by unrestricted
intracerebral multiplication of T. gondii. Interestingly, at
day 10 p.i., these animals had also developed a ventriculitis with
large numbers of parasites in the lumen and the wall of the lateral
ventricle, which is normally not infected by T. gondii
except in cases of prenatal toxoplasmosis (31), as well as in the
periventricular brain parenchyma (Fig. 2g). In addition, a
severe pneumonia contributed to the fatal outcome of
TNFR1/20/0 mice. In these animals, the parasitic load of
the brain and the lung significantly exceeded the number of parasites
in all other experimental groups (Table I).
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). As observed for TNFR1/20/0
mice, the parasite also infected the lateral ventricle (Fig. 2c). Up to day 25 p.i., the parasite replicated
massively in the brain (Table I) and caused widespread, large areas of
tissue necrosis with trophozoites as well as T. gondii cysts
located at the border (Fig. 2d). Furthermore,
TNFR10/0 mice developed toxoplasmic pneumonia, which was
characterized by numerous toxoplasms in association with large numbers
of inflammatory leukocytes in the alveolar spaces and lung tissue. At
day 10 p.i., many of the alveoli were occluded by a serous
exudate. However, in contrast to the brain, pneumonia was regressive
from day 10 to 25 p.i., and the amount of parasites declined
significantly during this period (Table I).
The increased resistance of TNFR20/0 mice to T.
gondii was reflected by a significantly lower parasitic load in
brain and lung as compared with TNFR1/20/0 and
TNFR10/0 mice (Table I). Although exceptional
TNFR20/0 mice died of acute pneumonia up to day 17
p.i., the vast majority of TNFR20/0 mice were able to
significantly reduce the amount of T. gondii in the lung
from day 10 to 25 p.i. and to effectively control the parasite in
the brain. In contrast to TNFR1/20/0 and
TNFR10/0 mice, TNFR20/0 mice did not differ
significantly from wt mice in the number of parasites in brain and lung
(Table I) or in the histopathology of toxoplasmic pneumonia and
encephalitis. Both TNFR20/0 and wt mice had only single
T. gondii Ag-positive cells in the brain at day 10 p.i.
(Fig. 2, a and e). At day 25 p.i.,
either single cysts or small groups of cysts were scattered throughout
the brain (Fig. 2, b and f).
Cellular immune reactions in the CNS
In the normal brain of TNFR1/20/0,
TNFR10/0, TNFR20/0, and wt mice, only low
numbers of CD4+, CD8+ T cells, B cells,
macrophages, and granulocytes were present, as revealed by flow
cytometry and immunohistochemistry. Infection with T. gondii
induced an increase of these populations of immune cells in the brain
up to day 10 p.i., with a further prominent increase up to day
25 p.i. (Fig. 3).
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). The amount of the various populations of
intracerebral immune cells did not differ significantly among
TNFR10/0, TNFR20/0, and wt mice at days 10 and
25 p.i. These data show that mutation of neither TNFR1 nor TNFR2
interferes with the recruitment of immune cells to the T.
gondii-infected brain. These findings are extended by in situ
immunohistochemical analysis of cell adhesion molecule expression. In
all experimental groups, ICAM-1 and VCAM were strongly up-regulated on
endothelial cells of cerebral blood vessels. In addition, macrophages
and CD4+ and CD8+ T cells were recruited
efficiently to infectious organisms in the brain parenchyma in every
strain. Thus, in all experimental groups, the formation of
inflammatory infiltrates was unimpaired. Since TNFRs have also been shown to be involved in programmed cell death (18), we studied apoptosis in the brains of the various groups by the TUNEL technique. Whereas in the brains of all uninfected animals apoptotic cells were not present, T. gondii-infected mice from all experimental groups exhibited a similar small number of apoptotic cells in leptomeningeal infiltrates, in perivascular cuffs, and in T. gondii-associated infiltrates. Thus, these studies demonstrate that in murine TE, apoptosis of intracerebral immune cells is independent of TNFR1 and TNFR2 expression (data not shown).
Production of intracerebral cytokines in normal and T. gondii-infected TNFR1/20/0, TNFR10/0, TNFR20/0, and wt mice
In uninfected TNFR1/20/0, TNFR10/0,
TNFR20/0, and wt mice, low levels of TNF- and IL-1ß
mRNA transcripts were occasionally observed. In contrast, IFN- and
iNOS mRNA transcripts were not detectable (Fig. 4).
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mRNA levels in all groups (Fig. 4). In addition, in
all experimental groups, intracerebral IL-1ß and TNF- mRNA
transcripts increased in response to T. gondii infection
(Fig. 4). An analysis of at least three mice per group revealed small
variations in the expression of IL-1ß and TNF- mRNA transcripts
within each group, and there were no significant differences among the
various experimental groups. Since TNF- expression is also regulated
posttranslationally, additional studies were performed at the protein
level. Immunohistochemistry confirmed elevated TNF- production in TE
and revealed numerous TNF-+ leukocytes within
inflammatory foci, as well as TNF-+ astrocytes without
obvious differences in their number and topographical distribution in
the various experimental groups (data not shown).
In contrast, differences were observed in the expression of iNOS among
TNFR1/20/0, TNFR10/0, TNFR20/0, and
wt mice. Immunohistochemistry revealed that in the brains of wt and
TNFR20/0 mice, iNOS+ leukocytes were already
present in significant numbers at day 10 p.i., with a further
strong increase toward day 25 p.i. (Fig. 5, a and e).
In these animals, iNOS+ cells were intimately associated
with T. gondii (Fig. 5b). In contrast to
wt and TNFR20/0 mice, T. gondii-associated
leukocytes in TNFR1/20/0 and TNFR10/0 mice did
not express, or only exceptionally single leukocytes expressed, iNOS,
and large numbers of infectious organisms were devoid of accompanying
iNOS+ cells (Fig. 5g). In addition, the
increase in the number of iNOS+ cells, which was observed
in wt and TNFR20/0 animals, was absent in
TNFR10/0 mice at day 25 p.i. (Fig. 5d).
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Moreover, iNOS mRNA transcripts were analyzed. RT-PCR of brain tissue
homogenates performed within the linear range of amplification detected
very low levels of iNOS mRNA transcripts in wt and TNFR20/0
mice at day 10 p.i. Up to day 25 p.i., these strains had a
prominent increase in intracerebral iNOS mRNA levels. In contrast, only
single TNFR10/0 and TNFR1/20/0 mice had faint
signals of iNOS mRNA transcripts at day 10 p.i., and, more
importantly, TNFR10/0 mice failed to increase intracerebral
iNOS mRNA transcripts up to day 25 p.i. (Fig. 4).
With respect to intracerebral iNOS production in the various experimental groups, it is of note that we made the same histopathologic observation with respect to the expression of iNOS in the lung: In contrast to wt and TNFR20/0 mice, which exhibited abundant iNOS+ leukocytes, iNOS expression on pulmonary leukocytes was significantly reduced or even absent in TNFR1/20/0 and TNFR10/0 mice (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated immune
responses play a crucial role in murine toxoplasmosis and, furthermore,
that the protective effects of TNF- are mediated through TNFR1, but
not TNFR2. The observation that TNFR1/20/0 mice
succumbed significantly earlier to the infection with a dramatically
increased parasitic load of brain and lung as compared with
TNFR10/0 mice provides evidence that in the absence of
TNFR1, a part of the function of this receptor is taken over by TNFR2.
This is in keeping with the observation that soluble TNF preferentially
signals through TNFR1 and much less through TNFR2 (32). It also argues
that membrane-bound TNF, the prime activating signal for TNFR2 (33), is
not crucially involved in anti-T. gondii defense.
Histopathology revealed that in all experimental groups, the same
pattern of organs was affected by T. gondii, with the brain
being the most strongly diseased organ followed in severity by the
lung. Thus, although TNFR1/20/0 and TNFR10/0
mice have a disturbed intestinal lymphatic architecture (17, 34) at the
entry site of the parasite into the host, they were able to effectively
clear T. gondii from this organ. Interestingly,
IFN-R-deficient mice on a 129/SvEv background, which are at least as
resistant to T. gondii as are mice on the 129/Sv x
C57BL/6 background used in this study, failed to control T.
gondii already in the intestine (10). Consequently,
IFN-R-deficient mice died of a necrotizing hepatitis and intestinal
lymphadenitis within 10 days. The remarkable differences in the
survival times between IFN-R-deficient and TNFR1/20/0
mice strongly indicate that in murine toxoplasmosis, IFN--mediated
immune responses are crucial for the control of the infection just from
an early phase of infection and TNF-induced processes at a later stage
in the course of infection.
TNFR10/0 mice had a significantly increased parasitic load in the lung and brain as compared with TNFR20/0 and wt mice at days 10 and 25 p.i. However, in contrast to the brain, TNFR10/0 mice achieved a significant reduction of the parasitic burden in the lung from day 10 to 25 p.i. These observations indicate that systemic immune responses differ significantly from intracerebral immune reactions and that TNFR1 is especially important in the brain, where the parasite persists. The CNS differs immunologically from other organs in several aspects. In toxoplasmosis, it seems particularly important that T. gondii infects neurons, which fail to express MHC class I Ags (35). Thus, the well-documented cytotoxic activity of CD8+ T cells against T. gondii (36, 37) is fully active in systemic organs, where all cell populations express MHC class I molecules, but ineffective in the brain. These findings indicate that in the brain, noncytotoxic immune reactions are even more important than in other organs. This may explain why, in our study, TNFR10/0 mice, which have a normal cytotoxic activity of CD8+ T cells (23), could compensate their genetic defect by other immune mechanisms in peripheral organs but not in the brain.
An analysis of the effect of TNFR deficiency on the ensuing immune response detected a significantly reduced production of iNOS mRNA and protein in the brains of TNFR1/20/0 and TNFR10/0 mice. The recent observation of Scharton-Kersten et al. (13) that iNOS-deficient mice succumbed to a progressive TE within 4 wk strongly indicates that in our study, the impaired iNOS production of TNFR1/20/0 and TNFR10/0 mice significantly contributed to the poor outcome of these mice. In addition, it has been shown that neutralization of NO in acute murine toxoplasmosis leads to an exacerbation of TE (14). Interestingly, we observed a reduced iNOS production in the brain as well as in the lung of TNFR1/20/0 and TNFR10/0 mice, which indicates that the increased parasitic load of the lung also results from an insufficient production of NO. However, the significant reduction of the parasitic load in the lungs of TNFR10/0 mice from day 10 to day 25 p.i. despite the persistent low production of iNOS in the lung further argues for a significant role of other immune mechanisms independent of TNFR1 and iNOS in systemic toxoplasmosis (38).
Previous studies have established that in TE, resident brain
parenchymal cell populations including astrocytes and microglial cells
are involved in the antiparasitic immune response (9, 39).
Interestingly, in vitro, neither unstimulated nor IFN- or
TNF--stimulated murine astrocytes are able to control the growth of
T. gondii (40). In addition, recent studies in our
laboratory revealed that IFN-- and/or TNF--stimulated murine
cerebellar granule neurons cannot control the growth of T.
gondii and, furthermore, that constitutive NO synthase expression
of neurons is insufficient to inhibit the growth of the parasite (D.
Schlüter, unpublished observations). In contrast, several in
vitro studies have shown that IFN--stimulated microglial cells and
macrophages exert a toxoplasmastatic activity, which is dependent on
TNF--induced NO production (41). Furthermore, a strong IFN-- (10)
and TNF--dependent (Ref. 12 and this study) iNOS production is
observed in murine toxoplasmosis. In addition, we recently identified
macrophages/microglial cells as iNOS-producing cell types in murine TE
(9). From these data, the question arises as to how NO produced by
cells of the macrophage lineage can control a parasite persisting in an
NO-negative cell type. Recently, Stenger and coworkers described the
control of Leishmania persisting in iNOS-negative cells in
the lymph nodes by neighboring iNOS-expressing cells (42, 43). This
mechanism may also be operative in murine TE. In line with this
assumption is the development of a fatal TE in iNOS-deficient mice and
an exacerbation of TE in mice with neutralized NO (13, 14).
Interestingly, studies of TNFR10/0 mice have also revealed
a crucial role of this receptor in listeriosis and tuberculosis (23, 24, 44). As observed in our study, in murine tuberculosis TNFR1
deficiency was linked to a reduced production of NO in the early stage
of the infection (44). In contrast, Leishmania-infected
TNFR10/0 mice exhibited a normal production of NO, although
parasite clearance was delayed (45). The divergent findings on the
relative role of TNFR1 in NO induction among the various studies may be
explained by different IFN- levels at the site of infection (44, 45). Additionally, TNFR10/0 macrophages, infected with
Leishmania major, were shown to compensate for the lack of
TNF/TNFR1 signaling and to effectively induce NO synthesis in the
presence of the parasite (45). It is noteworthy that T.
gondii-infected normal macrophages stimulated with suboptimal
doses of IFN- require TNF- for an effective induction of NO as
well as an inhibition of proliferation of T. gondii (41).
Furthermore, neutralization of TNF- in chronic murine TE resulted in
a reduced production of NO and an increased proliferation of T.
gondii leading to death in the mice (12). These findings indicate
that in murine TE, endogenous TNF-, which signals preferentially
through TNFR1 (32), acts in synergy with limited concentrations of
IFN- for an optimal NO production and parasite control.
TNF- and TNFR are also involved in a variety of immune reactions
that are independent of NO production. Studies in TNFR10/0
and TNFR20/0 mice have revealed that in cerebral malaria,
TNFR2 mediates the immunopathologic hyperinduction of ICAM-1 on
cerebral blood vessel endothelia leading to occlusion of cerebral blood
vessels by sequestering leukocytes (21). In our model, none of the
TNFR-mutant mice showed an increased expression of ICAM-1 or VCAM on
cerebral blood vessels as compared with wt mice. This difference
between cerebral malaria and TE is explained by the observation that in
murine TE, IFN- is the major mediator inducing an up-regulation of
cell adhesion molecules at the blood-brain barrier (M.
Deckert-Schlüter, unpublished observation), whereas in cerebral
malaria, TNFR2-mediated immunopathologic up-regulation of ICAM-1 is of
key importance (21). Moreover, in our study, the recruitment of immune
cells to the brain as well as the rate of apoptosis of intracerebral
immune cells were as normal in TNFR-mutant mice as in wt mice. Thus,
the number of intracerebral immune cells, especially of
CD4+ and CD8+ T cells, which are of major
importance in toxoplasmosis (2, 7, 46), was equal in the various
experimental groups. Furthermore, the normal rate of apoptosis in
TNFR-mutant mice indicates that CD95 (Fas) and not TNFR regulates
apoptosis in TE (19). Furthermore, intracerebral production of
cytokines with a documented anti-T. gondii activity,
i.e., IFN-, TNF-, and IL-1ß (7, 12, 15, 47, 48), did not differ
between TNFR-mutant and wt mice.
Although these data point to the reduced iNOS production as a crucial
factor responsible for the high susceptibility of
TNFR1/20/0 and TNFR10/0 mice, other yet
undefined factors may also contribute to the insufficient control of
T. gondii in these strains. In particular, the in vivo
regulation of intraneuronal and astrocytic anti-T.
gondii immune responses by IFN- and TNF- is still unknown,
and one may speculate that TNFR1 deficiency directly impairs the immune
response of neurons and astrocytes to T. gondii. In this
context, it is of note that in TNFR1/20/0 and
TNFR10/0 mice, intracerebral cysts developed regularly,
indicating that in these animals the immunologic control of cysts, but
not the induction and formation of cysts, is impaired.
In conclusion, the present study demonstrates an essential function of
TNF-- and TNFR1-mediated immune responses in murine toxoplasmosis,
which cannot be compensated for by other immune mechanisms. The strong
inverse correlation of susceptibility and iNOS levels strongly suggests
a critical role of TNFR1-mediated iNOS and NO production in murine
toxoplasmosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martina Deckert-Schlüter, Institut für Neuropathologie, Universitätskliniken Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany. E-mail address:
3 Abbreviations used in this paper: CNS, central nervous system; TE, Toxoplasma encephalitis; IFN-R, interferon- receptor; iNOS, inducible nitric oxide synthase; NO, nitric oxide; TNFR, tumor necrosis factor receptor; wt, wild type; RT, reverse transcription; LCA, leukocyte common Ag; GFAP, glial fibrillary acid protein; PE, phycoerythrin; p.i., postinfection; HPRT, hydroxyphosphoribosyltransferase; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
Received for publication August 11, 1997. Accepted for publication December 4, 1997.
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