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Institut für Klinische Mikrobiologie, Immunologie, und Hygiene and
Nachwuchsgruppe 1 des Interdisziplinären Zentrums für Klinische Forschung, Universität Erlangen-Nürnberg, Erlangen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and inducible
NO synthase. Previous Ab neutralization studies or the use of mice
deficient for both TNF receptors suggested that TNF plays only a
limited role in the control of parasite replication in vivo. In this
study we demonstrate that resistant C57BL/6 (B6.WT) mice
locally infected with L. major rapidly succumb to
progressive visceral leishmaniasis after deletion of the TNF gene by
homologous recombination. A reduction of the parasite inoculum to 3000
promastigotes did not prevent the fatal outcome of the disease. An
influence of the altered morphology of secondary lymphoid organs in
C57BL/6-TNF-/- (B6.TNF-/-) mice on the
course of disease could be excluded by the generation of reciprocal
bone marrow chimeras. Although infected B6.TNF-/- mice
mounted an L. major-specific IFN- response and
expressed IL-12, the onset of the immune reaction was delayed. After in
vitro stimulation, B6.TNF-/- inflammatory macrophages
released 10-fold less NO in response to IFN- than B6.WT cells.
However, in the presence of a costimulus, e.g., L. major
infection or LPS, the production of NO by B6.WT and
B6.TNF-/- macrophages was comparable. In vivo, inducible
NO synthase protein was readily detectable in skin lesions and draining
lymph nodes of B6.TNF-/- mice, but its expression was
more disperse and less focal in the absence of TNF. These are the first
data to demonstrate that TNF is essential for the in vivo control of
L. major. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the induction of inducible NO synthase
(iNOS)3 in macrophages
and the up-regulation of IFN- within 24 h of infection
(3, 4). The subsequent steps of protective immunity are
based on induction and expansion of IFN--producing
CD4+ Th1 cells, which in concert with TNF
activate macrophages to exert NO-mediated leishmanicidal activity
(5, 6, 7, 8). In the resistant C57BL/6 (B6.WT) mouse strain,
healing of the local lesion and lifelong control of L. major
replication are dependent on a permanent presence of iNOS activity in
the tissue. Blocking of this enzyme results in a rapid reactivation of
the disease (9).
In the mouse model of leishmaniasis, the proinflammatory cytokine
TNF has been examined extensively because of its potential effector
function. Treatment with TNF resulted in a reduction of lesion size and
parasitic burden (7, 10, 11). Application of neutralizing
anti-TNF Abs led to a transient aggravation of the disease
(8, 10, 11, 12). In a transgenic model, B6.WT mice that
constantly expressed a neutralizing TNF receptor 1 (p55; TNFR1)-IgG
fusion protein developed a serious, nonhealing lesion at the site of
infection (13). Finally, gene-targeted mice negative for
TNFR1 or both TNFR1 and -2 were able to clear the parasite, although
they continued to exhibit a permanent swelling at the site of
infection. These mice developed a Th1 response and expressed IFN-
and iNOS (14, 15, 16). TNFR2 was not involved in the in vivo
control of the parasite (15). These findings suggest that
TNF acts as a mere cofactor in the development of protective,
anti-leishmanial immunity and is dispensable for the ultimate
control of the infection.
In the present study we have analyzed the anti-leishmanial
immune response of B6.WT mice lacking the TNF gene. Unexpectedly, these
mice developed a visceral form of leishmaniasis. The parasite
disseminated rapidly, and the majority of mice died 6–9 wk after
infection, although the local lesion never reached the size seen in the
highly susceptible BALB/c mouse strain. Fatal disease also occurred
after low dose infections as well as in radiation bone marrow chimeras
lacking TNF in hemopoietically derived cells. Analysis of the
Ag-specific immune response revealed a delay of the induction of Th1
cells by 1 wk and demonstrated substantial deficiencies in the IgG
response. In vitro, the expression of iNOS by thioglycolate-elicited
peritoneal macrophages after stimulation with IFN- was reduced in
the absence of endogenous TNF, but could be induced to wild-type levels
after costimulation with LPS and was also detectable in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred B6.WT, and BALB/c mice were purchased from Charles River (Sulzfeld, Germany). The CD45-congenic strain B6.WT-Ly5.1 was obtained from Dirk Schlüter (Universität Heidelberg/Mannheim, Mannheim, Germany). B6.TNF-/- mice were generated on a genetically pure C57BL/6 background as previously described (17) and were obtained from the Centenary Institute of Cancer Medicine and Cell Biology (Sydney, Australia). The B6.TNF-/- mice were screened by PCR using primers flanking the excised region of the TNF gene (5' sense, GCG TCC AGC TGA CTA AAC ATC CTT C; 3' antisense, ACC ACT AGT TGG TTG TCT TTG AGA T). The conditions of the PCR were 4 min at 94°C and 35 cycles of 94°C (1 min), 60°C (1 min), and 72°C (1 min). All mice were kept at the Institut für Klinische Mikrobiologie, Immunologie, und Hygiene (Erlangen, Germany). Mice 8–16 wk of age were used in all experiments, and experiments were performed according to the animal experimental ethics committee guidelines of the University of Erlangen-Nürnberg.
Generation of radiation bone marrow chimeras
Bone marrow cells were flushed with cold PBS/0.1% BSA from the long bones (femur and tibia) of matched donor mice. A single-cell suspension was prepared by gently passing the cells through 70-µm pore size cell strainer (Becton Dickinson, Franklin Lakes, NJ). The cells were washed and counted. Recipient animals were irradiated with a dose of 5.5 Gy gamma radiation on day -2 and again on day 0. Bone marrow cells (2 x 107 cells/recipient) were injected i.v. on day 0. As a means to track the level of engraftment, the reconstitution was performed reciprocally between C57BL/6 or TNF-negative mice (both B6.Ly5.2) and B6.WT-Ly5.1 mice. Four weeks after engraftment peripheral blood was drawn from recipients. After RBC lysis lymphocytes were analyzed by flow cytometry for the presence or the absence of the C57BL/-Ly5.1 congenic marker as previously described (18). The origin of the Abs used for the analysis was also detailed previously (18). Engraftment was considered to be sufficient when >80% of peripheral leukocytes exhibited the CD45 allele of the donor type.
L. major parasites and the preparation of L. major Ag
L. major promastigotes (MHOM/IL/81/FEBNI) were propagated in vitro in blood agar cultures as previously described (19). The virulence of the isolate was maintained by monthly passage in BALB/c mice. For the preparation of L. major parasites, stationary phase promastigotes were harvested, washed four times, and resuspended in PBS. The parasites were used for infection or subjected to four cycles of rapid freezing and thawing to prepare L. major Ag.
L. major infection and evaluation of the systemic course of disease
Mice were infected s.c. in one or both hind footpads with 3
x 106 stationary phase promastigotes/footpad of
the third to seventh in vitro passage in a final volume of 50 µl. The
increase in lesion size was monitored twice weekly by measuring the
footpad thickness with a metric caliper (Kroeplin Schnelltaster,
Schlüchtern, Germany). When one footpad was infected, the
increase in footpad thickness (percentage) was determined by the
formula: thickness of infected footpad/mean thickness of noninfected
footpads x 100. When both footpads were infected, the increase in
footpad thickness (percentage) was determined by the formula: thickness
of infected footpad/mean thickness of all footpads in the experimental
group before infection x 100. Infection in one or both hind
footpads did not change the course or outcome of the disease. The
number of viable parasites in draining LN and spleen was estimated
using limiting dilution analysis by applying Poisson statistics and the
2 minimization method as described previously
(20).
Serum isotype detection
IgG1 and IgG2a serum Abs specific for L. major were detected by sandwich ELISA. For quantitation of different isotypes, round-bottom 96-well microtiter plates (Dynex Technologies, Denkendorf, Germany) were coated with 4 x 105 lysed L. major promastigotes/well at 4°C overnight. The following steps alternated with washing of the plates with PBS/0.05% Tween 20. Plates were blocked for 2 h at room temperature with 10% FCS in PBS. Serum was added at dilutions of 1/20 and 1/200 in 3% FCS/PBS followed by incubation at 37°C for 2 h. Isotype-specific anti-mouse alkaline phosphatase-coupled mAbs (anti-mouse IgG1, clone A85-1; anti-mouse IgG2a, clone R19-15; PharMingen, San Diego, CA) were incubated for 2 h at 37°C. Alkaline phosphatase activity was visualized using p-nitrophenyl-phosphate substrate (Sigma, Deissenhofen, Germany). The plates were analyzed in a plate reader (Dynex Technologies) at 405 nm absorbency. The results were standardized using a pool of L. major-specific mouse sera on every plate and are expressed as a relative level (ELISA units). The lower detection levels were 7700 and 3800 ELISA units for IgG1 and IgG2a, respectively.
Proliferation assay and detection of cytokines
At the indicated time points, infected mice were sacrificed, and draining popliteal LN were removed. Single-cell suspensions were prepared by grinding the tissue between two sterile slides, then were plated at a concentration of 3 x 105 cells/well in a 96-well flat-bottom microtiter plate containing a total volume of 200 µl of RPMI 1640 supplemented with 2 mM glutamine, 10 mM HEPES, 13 mM NaHCO3, 50 µM 2-ME, 100 µg/ml penicillin, and 100 µg/ml streptomycin (Seromed-Biochrom, Berlin, Germany) plus 10% FCS (Sigma). Cells were stimulated with lysed L. major promastigote Ag at a ratio of five parasites per cell or with Con A (final concentration, 5 µg/ml). Control wells contained medium without additives. The assay was performed in quadruplicate when possible. After 72 h of incubation, 100 µl of supernatants were removed and replaced by medium without additives. For the final 17 h of culture, cells were pulsed with [3H]thymidine (0.5 µCi/well; Amersham, Aylesbury, U.K.). Cells were harvested onto filter strips using a semiautomated cell harvester, and incorporation of radioactivity was measured in a scintillation counter.
Supernatants of proliferation assays were stored at -70°C until the
determination of IL-4 or IFN- concentrations. The cytokines were
detected by capture ELISA. The following pairs of mAb were used:
biotinylated BVD4-1D11 and purified 11B11 in the IL-4 ELISA, and
biotinylated XMG1.2 and purified RA-6A2 in the IFN- ELISA
(PharMingen). Round-bottom 96-well microtiter plates (Dynex
Technologies) were coated with an anti-IFN- or an anti-IL-4
capture Ab at 4°C overnight. The following incubation steps were
performed in a humidified chamber and alternated with washing of the
plates three to five times using PBS/0.05% Tween 20. Supernatants were
diluted 1/5 for IL-4 or 1/20 and 1/200 for IFN- detection and
incubated with the capture Abs for 2 h at 37°C. Lymphokines were
detected with biotinylated detection Abs and visualized with a
streptavidin/alkaline phosphatase complex (Dako, Copenhagen, Denmark)
followed by p-nitrophenyl-phosphate substrate. The plates
were analyzed in a plate reader (Dynex Technologies) at 405
nm.
Delayed-type hypersensitivity (DTH) reaction
Three weeks after L. major infection L. major promastigote Ag (corresponding to 2 x 107 parasites) was injected in a volume of 20 µl in the footpad of the foreleg. As a control, 20 µl of PBS was injected in the opposite foreleg. The footpad thickness was measured with a metric caliper (Kroeplin Schnelltaster) after 24, 48, and 72 h.
RT-PCR
Total RNA was extracted from snap-frozen tissue or cell pellets
with RNeasy columns (Qiagen, Germany) following the manufacturer’s
protocol. RT of 1 µg RNA was performed using Moloney murine leukemia
virus reverse transcriptase (Promega, Mannheim, Germany). Specific
products were amplified with 1 Taq polymerase (Promega).
Both reactions followed a standard protocol. The PCR products were size
fractioned by electrophoresis on 1% agarose gel containing ethidium
bromide. The following primers were used: IFN- forward, 5'-TGA ACG
CTA CAC ACT GCA TCT TGG-3'; IFN- reverse, 5'-CGA CTC CTT TTC CGC TTC
CTG AG-3'; IL-4 forward, 5'-ATG GGT CTC AAC CCC CAG GTA-3'; IL-4
reverse, 5'-CGA GTA ATC CAT TTG CAT GAT-3; IL-12 (p40) forward, 5'-GAC
CCT GCC CAT TGA ACT GGC-3'; and IL-12 (p40) reverse, 5'-CAA CGT TGC ATC
CTA GGA TCG-3.
Macrophages
Thioglycolate-elicited peritoneal macrophages were prepared from the peritoneal cavity of B6.WT or B6.TNF-/- mice 4 days after i.p. injection of 3 ml of 4% Brewer’s thioglycolate broth (Difco, Detroit, MI). RPMI 1640 medium with 2.5% FCS (Sigma) and supplemented as described above was used for tissue culture. The macrophages were seeded into 24-well plates (1 x 106 cells/well in 500 µl) or into eight-well LabTek chamber slides (Permanox; Nunc, Wiesbaden, Germany) and cultured at 37°C. After 90–120 min, nonadherent cells were washed off, and subsequently macrophages were further incubated in fresh medium, stimulated with cytokines, and infected with L. major parasites as indicated.
Determination of nitrite
As an indirect measurement for the production of NO, culture supernatants were analyzed for their content of nitrite (NO2-) using the Griess reaction as previously described (20).
Immunohistology
Tissue blocks from footpad lesions or LN were embedded in optimal cutting temperature compound (Diatec, Hallstadt/Bamberg, Germany) and stored at -70°C. Tissue sections (5–6 µm) were cut, thawed onto slides, coated with Fro-Marker (Sciences Services, Munich, Germany), air-dried, fixed in acetone (for 10 min at -20°C), and washed briefly in PBS/0.05% Tween 20. Nonspecific binding sites were blocked for 30 min with PBS/1% BSA/20% FCS. Rabbit antisera specific for L. major or a mouse C-terminal iNOS peptide have both been described previously (9). Immunoperoxidase staining (with 3-amino-9-ethyl-carbazole as a substrate) and hematoxylin counterstaining were performed as described previously (9).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B6.WT, BALB/c, and B6.TNF-/- mice were
infected with 3 x 106 L. major
promastigotes, and the course of infection was monitored. At the site
of infection, resistant B6.WT mice developed a small skin lesion, but
were able to resolve the lesion and control the infection (Fig. 1
A). Susceptible BALB/c mice,
in contrast, developed ulcerating skin lesions that progressed without
healing (Fig. 1A). In this mouse strain tissue necrosis
developed soon after infection. B6.TNF-/- mice
showed a moderate increase in lesion size, and ulceration was not
observed. After 6 wk the local skin lesion started to resolve, and the
tissue scared (Fig. 1A). However, the disease progressed
systemically, and the majority of mice died 6–9 wk after infection
(Table I). On day 42 after infection,
spleen and liver of B6.TNF-/- mice were
substantially enlarged (spleen: B6.WT mice, 150 ± 30 mg
(n = 5); B6.TNF-/- mice,
360 ± 50 mg (n = 4); BALB/c, 250 ± 10 mg
(n = 3); liver: B6.WT mice, 1580 ± 50 mg
(n = 5); B6.TNF-/- mice,
2030 ± 390 mg (n = 4); BALB/c, 1240 ± 60 mg
(n = 3)).
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and Fig. 1B). BALB/c mice infected with the
same dose showed a more pronounced footpad swelling than
B6.TNF-/- mice (data not shown), but were able
to control the infection and survived (Table I). Course of L. major infection in reciprocal bone marrow chimeras
Reciprocal chimeras
(B6.TNF-/-
B6.WT-Ly5.1,
B6.WT-Ly5.1B6.TNF-/-) were generated using a
lethal irradiation protocol and a subsequent transfer of bone marrow.
B6.WT-Ly5.1B6.WT chimeras served as a control. Four weeks after bone
marrow transfer flow cytometric analysis of a CD45 congenic marker was
used to examine the efficacy of engraftment. At the time of the
infection >80% of recirculation leukocytes (
TCR+, B220+,
Mac-1+) were of the donor type. Eight weeks after
reconstitution the three types of chimeric mice were infected with
3 x 106 parasites in one hind footpad. The
increase in lesion size in B6.WT-Ly5.1B6.WT and
B6.WT-Ly5.1B6.TNF-/- chimeras after
infection mirrored the lesion size in B6.WT (compare Figs. 1A and 2). In contrast,
B6.TNF-/-B6.WT-Ly5.1 displayed a more
pronounced increase similar to B6.TNF-/- mice
(compare Figs. 1A and 2). Only the
B6.TNF-/-B6.WT-Ly5.1 chimeras died after
6–10 wk of infection.
Development of parasitic burden in TNF-deficient mice
Limiting dilution assays were performed with draining popliteal LN
to determine the number of viable L. major parasites in
these lymphoid organs closest to the site of infection. On day 14
postinfection (p.i.), the parasitic burden in B6.WT mice was comparable
to that in B6.TNF-/- mice (Fig. 3A). The number of parasites
in the highly susceptible BALB/c strain was at this time point 10 times
higher (Fig. 3A). On day 42 p.i., the number of
parasites in B6.WT LN was 65-fold lower than that in
B6.TNF-/- LN (Fig. 3A). BALB/c LN
were suppurated and could not be analyzed.
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B). On day 42 p.i., the number of parasites detectable
in the spleens of B6.TNF-/- mice had increased
remarkably. The titer of L. major in
B6.TNF-/- mice exceeded the parasitic burden of
B6.WT mice by >4 orders of magnitude (35,000-fold) and that of BALB/c
mice by 1 order of magnitude (Fig. 3B). On day 35 p.i. the parasitic burden of the bone marrow was analyzed. Also in the bone marrow of the TNF-negative mice, the number of L. major parasites exceeded the burden in B6.WT controls by a factor of 500 and was increased 16-fold compared with that in susceptible BALB/c mice (data not shown).
Analysis of the cellular and humoral immune responses in TNF-deficient mice
Protective immunity against L. major depends on the
genetically determined ability to mount a Th1-type immune response
(1, 2). Therefore, we isolated and analyzed cells from
draining LN at various time points after infection. The relative
proportions of T and B lymphocyte populations in draining LN were
analyzed by FACS analysis and were found to be stable throughout the
early phase of infection (days 0–21 p.i.; data not shown). The
determination of the total number of lymphocytes in the draining LN
showed a reduction in B6.TNF-/- mice on day 3
after infection. However, this retardation of cell influx to the local
LN in B6.TNF-/- mice was transient and had
stopped on day 4 (data not shown). Additionally, the proliferative
response of T cells to L. major Ag of infected B6.WT and
B6.TNF-/- mice was studied. On day 7 p.i.,
a marked L. major-specific proliferation was present in
B6.WT mice, whereas in B6.TNF-/- mice only a
limited proliferative response to L. major could be detected
(Fig. 4A). Based on analysis
of the cellularity of the draining LN, differences in total cell
numbers did not explain the limited proliferative response of
B6.TNF-/- LN cells to L. major Ag on
day 7 after infection. After 18 days of infection, LN cells from
B6.TNF-/- mice showed a stronger response to Ag
than B6.WT LN cells (Fig. 4A). At this time point the titers
of parasites in the LN (mean >500 viable parasites/1000 cells) were so
high that LN cells proliferated in the absence of exogenous Ag (Fig. 4A).
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and IL-12 and low levels of IL-4. Therefore, we analyzed
B6.WT and B6.TNF-/- popliteal LN and spleen
tissue for the presence of IFN-, IL-12 (p40), and IL-4 mRNA by
RT-PCR before the infection occurred (day 0) and 14 days after
infection (day 14). At these time points these cytokines were expressed
at equivalent levels in B6.WT and B6.TNF-/-
mice (data not shown). To study the expression of IFN- and IL-4 by
ELISA on a protein level, LN cells were isolated and stimulated with
L. major Ag. B6.WT LN cells produced a significant amount of
IFN- after antigenic stimulation. Interestingly, the expression of
IFN- was substantially higher in B6.TNF-/-
mice than in B6.WT mice (Fig. 4B). IL-4, as protagonist of
the Th2 response, could not be detected in either supernatant (data not
shown).
Protective immunity to L. major is independent of the
presence of Ag-specific Abs (21). However, Ab production
reflects the ability of the host to respond to Ag efficiently.
Moreover, proportional distribution of serum isotypes is supposed to
mirror a bias toward either a Th1 or Th2 response, whereby IgG2a marks
a Th1 response, and IgG1 is preferentially expressed in Th2-type
responses. Therefore, we investigated the level of Ag-specific Abs of
the IgG1 and IgG2a isotypes on days 28 and 42 after infection. In B6.WT
mice, we detected at these time points no preference of either isotype,
but a substantial production of both IgG isotypes (Fig. 4C).
Interestingly, in B6.TNF-/- mice the production
of Ag-specific IgG1 was below the detection level at both time points,
whereas IgG2a could be detected on day 42 p.i., however, at a
10-fold reduced concentration compared with B6.WT mice (Fig. 4C).
Delayed-type hypersensitivity
The presence of Ag-specific T cells is required to mount a DTH
response. To determine the ability of B6.TNF-/-
mice to mount such a response, we injected L. major Ag in
one footpad of L. major-infected B6.WT,
B6.TNF-/-, and BALB/c mice. After 48 h the
swelling of the footpads in B6.TNF-/- and
BALB/c mice was significantly reduced compared with that in B6.WT mice.
However, B6.TNF-/- mice showed a stronger
reaction than BALB/c mice (Fig. 5).
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A major antimicrobial effector mechanism of the innate immune
system is the IFN-- and TNF-dependent up-regulation of iNOS and the
subsequent production of large amounts of NO (22).
Thioglycolate-elicited peritoneal exudate macrophages from B6.WT and
B6.TNF-/- mice were stimulated with IFN- and
LPS, respectively (Fig. 6), and the
accumulation of NO in the supernatant was determined. Upon stimulation
with IFN- alone B6.WT macrophages showed a strong production of NO
(34.6 ± 11.1 µM NO; mean ± SEM of four experiments; Fig. 6A), whereas NO levels generated by TNF-deficient
macrophages were low (13.3 ± 9.5 µM NO; n = 4;
Fig. 6A). Stimulation of TNF-negative cells with LPS or LPS
plus IFN- resulted in an activation comparable to that in B6.WT
macrophages (Fig. 6B). Thus, IFN--induced, but not LPS-
or LPS- plus IFN--induced, production of NO is dependent on
endogenous TNF.
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, A and B). Later
in the course of the infection (day 40 p.i.), the draining
popliteal LN in B6.WT mice exhibited a defined lymphoid
microarchitecture and distinct focal expression of iNOS in the
paracortical T cell area adjacent to B cell follicles. In contrast, in
the LN of B6.TNF-/- mice, lymphoid structures
were replaced by connective tissue. This can be interpreted as an
indication for an ongoing inflammatory process with tissue destruction
and subsequent scaring. The overall production of iNOS was strong, but
instead of large iNOS-positive foci, much smaller iNOS-positive
clusters or single iNOS-positive cells prevailed (Fig. 7, C
and D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The course of infection observed in B6.TNF-/- mice was different from the form of leishmaniasis observed in the susceptible BALB/c strain. In B6.TNF-/- mice, there was neither ulceration of the lesions nor suppuration of infected tissues or draining LN. Most importantly, B6.TNF-/- mice displayed a higher susceptibility to the pathogen, which was illustrated by a markedly shorter period of survival than that in infected BALB/c mice. The role of TNF in leishmaniasis has been analyzed in a number of previous studies using application of TNF or neutralizing anti-TNF Abs or transgenic models. Ab treatment resulted in a transient aggravation of the course of disease (7, 8, 10, 12), whereas application of therapeutic doses of TNF led to an amelioration of symptoms in susceptible mice (7, 10, 11). Unexpectedly, mice that were negative for both TNFRs, which according to our knowledge should lack any TNF-signaling, did not succumb to the parasite and were able to control the infection, but developed a nonhealing inflammatory lesion (15). The fatal course of infection in the B6.TNF-/- mice suggests that TNF can signal through a third receptor and that this signaling is necessary to confer protection in leishmaniasis. In this context it is worth mentioning that a recent report by Hayder et al. shows that a ligand other than TNF or lymphotoxin can bind to TNFR1 and -2 (23). Taken together, our data are compatible with the view that the one ligand-two receptors model might not be entirely sufficient to describe the more complex situation in vivo. Alternatively, the difference in disease outcome between TNF- and TNFR1/2-negative mice could also be the result of the use of different L. major strains. A comparison of the two strains will answer this question.
TNF-negative mice display changes in the microarchitecture of secondary lymphoid organs such as the absence of distinct B cell follicles and are not able to generate germinal centers after antigenic challenge (17, 24). These alterations are relatively minor, but could influence the efficiency of the anti-L. major immune response. To investigate this, radiation bone marrow chimeras (18) were infected with L. major. This experiment indicates that the sole factor necessary for survival is the ability of hemopoietic cells to produce TNF. TNF-positive bone marrow cells are able to mount a protective immune response in a TNF-negative stromal environment. This also shows that TNF produced by nonhemopoietic cells such as keratinocytes in the skin cannot substitute for leukocytic TNF. Therefore, we conclude that the effects of TNF on hemopoietic cells, rather than those on the formation of the correct lymphoid microarchitecture, are critical for the control of L. major.
Our experiments showed that the clinical course of leishmaniasis in
B6.TNF-/- mice correlated with the parasitic
burden of the host organism. The uncontrolled replication of parasites
in the spleen of B6.TNF-/- mice demonstrated
that as yet undefined mechanisms, which normally control the
replication of L. major, are TNF dependent and cannot not be
compensated for by other control mechanisms. In the mouse model of
Mycobacterium tuberculosis infection, TNF-negative mice were
not able to form granulomas (25). This deficiency had
already been revealed by treatment of infected mice with anti-TNF
Abs (26, 27) and was attributed to a role of TNF in the
priming of the immune response and leukocyte migration in general
(28, 29). An insufficiency in T cell priming or migration
to the site of infection or the lymphoid tissues due to the lack of TNF
should impair the development of a specific cellular or humoral
response to L. major. Indeed, we were able to demonstrate
that B6.TNF-/- mice displayed a delay in
generating a T cell response. Nevertheless, LN cells from
B6.TNF-/- mice produced a substantial amount of
IFN-, which indicates a Th1-type response. This is consistent with
results obtained in studies using TNFR1-negative mice, which also
showed an elevated level of IFN- after L. major infection
(14). Therefore, the T cell response in
B6.TNF-/- mice was of the expected type, but it
was delayed by 1–2 wk. We can exclude that the limitations of
B6.TNF-/- mice in starting the immune response
are due to deficiencies at the level of signal transduction pathways
(30). Thus, we suggest that the delay in generating
specific T and B cells implies a paucity of cell-cell communication or
cell migration. Moreover, infected B6.TNF-/-
mice experience a major deficiency in the DTH reaction to L.
major Ag. After 48 h, at the peak of the reaction, B6.WT mice
showed a marked increase in footpad thickness at the injected site.
Compared with the DTH response of B6.WT mice,
B6.TNF-/- mice only reacted at low level (35%
of the B6.WT swelling 48 h after injection; Fig. 5). This is in
contrast to the outcome of similar experiments in the M.
tuberculosis model (25), where the same strain of
TNF-negative mice displayed a normal DTH response. This is another
strong indication for a defective T cell priming in
B6.TNF-/- mice in the model of experimental
leishmaniasis.
NO has been described as an important effector molecule of an innate
and adaptive response to L. major. Recently, the cascade of
events that results in the expression of iNOS and the production of NO
has in part been defined (31). An infection of macrophages
with L. major causes the production of IFN-. These
cytokines are needed as a necessary second signal and induce in concert
with L. major a rapid expression of iNOS. The enzyme is
readily detectable in the skin within 24 h after infection
(4). This leads to an up-regulation of IFN- production
of NK cells, and in a positive feedback loop to an increased production
of iNOS. TNF has been shown to synergize with IFN- for the
activation of macrophages (21). Furthermore,
neutralization of TNF in the presence of IFN- or the stimulation of
B6.TNF-/- macrophages with IFN- alone
demonstrated a strong dependency of the production of NO on the
presence of endogenous TNF and have proven the importance of this
cytokine (5, 6, 7, 31). In this study we could demonstrate
that in vivo B6.TNF-/- mice, like
TNFR-/- mice (14, 15), do not need
TNF signaling for the expression of iNOS during the infection.
In conclusion, we have determined an unexpected role of TNF in the control of leishmaniasis. Abolishing the signaling through both TNFR1 and 2 can be compensated, whereas absence of the ligand is fatal. Our investigations have not yet revealed which defense mechanism has failed or is absent. However, we found a retarded proliferative response of T cells, a delayed or missing switch to Ag-specific, T cell-dependent serum Igs, and an impaired formation of iNOS-positive cell clusters. These results together with the virtual absence of a DTH reaction point to a deficit in cell recruitment and priming of the immune response that is currently under investigation.
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2 Address correspondence and reprint requests to Dr. Heinrich Körner, Nikolaus Fiebiger Zentrum für Molekulare Medizin, IZKF, Nachwuchsgruppe 1, Glückstrasse 6, D-91054, Erlangen, Germany.
3 Abbreviations used in this paper: iNOS, inducible NO synthase; B6.WT, C57BL/6; B6.TNF-/-, C57BL/6-TNF-/-; LN, lymph node(s); TNFR1, TNF receptor 1 (p55); TNFR2, TNF receptor 2 (p75); p.i., postinfection; DTH, delayed-type hypersensitivity.
Received for publication April 26, 2000. Accepted for publication January 2, 2001.
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) and day 42 p.i. (
) by limiting dilution
analysis. The mean parasitic burden in the organs of four to six mice
is shown. One triangle represents one animal. One of three experiments
is shown.
), B6.TNF-/- (
), and BALB/c (
) mice
were infected in the hind footpads and challenged after 3 wk with
2 x 107 L. major particles in the fore
footpad. The contralateral footpad received an equivalent volume of PBS
as a control. The data shown represent the mean (±SEM) of four animals
(for B6.WT control, three animals). Data represent one of two
experiments. *, p > 0.001; **,
p > 0.01.
;
n = 4) served as controls. The relative increase in
footpad thickness is shown as the mean ± SEM. The curve of the
B6.TNF-/-