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, IL-12, and TNF-
Are Required to Maintain Reduced Liver Pathology in Mice Vaccinated with Schistosoma mansoni Eggs and IL-121
*
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Biomedical Research Institute, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12, or TNF-
at
the time of egg laying developed granulomas that were similar to the
non-IL-12-treated control group. Although all three
anti-cytokine-treated groups exhibited a dominant type 1 response
in lymph node cells restimulated ex vivo, the expression of type 2
cytokine mRNA was markedly restored at the site of granuloma formation,
which suggests that all three cytokines are required to maintain the
suppressed type 2 pattern. Moreover, egg/IL-12-sensitized mice depleted
of IFN- or IL-12 displayed a partial reduction in IFN-
production, suggesting that multiple type 1 cytokines were required to
maintain polarized type 1 responses to chronic type 2-inducing stimuli.
Together, these data reveal key roles for IFN-, IL-12, and TNF-
in the protective effects mediated by this IL-12-based vaccine to
prevent pathology. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2). Over the
past several years, it has become apparent that IL-12 is essential for
promoting protective immune responses against infectious organisms
including intracellular bacteria (3) and protozoan parasites (4, 5, 6, 7), as
well as assisting in immune-mediated antitumor and antimetastatic
activities (8). The prophylactic ability of IL-12 was additionally
realized by Afonso et al. (9), who demonstrated that BALB/c mice
immunized with IL-12 plus soluble leishmanial Ags developed
leishmanial-specific CD4+ Th1 cells that completely
rendered the normally susceptible animals resistant to Leishmania
major challenge infection. Thus, all of these studies
suggest that IL-12 may aid in the immunotherapeutic or
immunoprophylactic treatments of clinically diverse diseases. Schistosomiasis, a disease that currently affects 200 million people worldwide, is often effectively treated by chemotherapies. In spite of these chemotherapeutic successes, many world health agencies agree that the development of an antischistosomiasis vaccine should be aggressively pursued. This is due, in part, to the recent identification of possible praziquantel-resistant Schistosoma mansoni field isolates (10, 11). Additionally, evidence suggestive of age-dependent immunity (12) indicates that the development of an antischistosome vaccine is feasible and could lead to reduced morbidity and mortality. One current vaccine strategy is to suppress the overall pathology associated with egg deposition in the bladder, liver, or intestines. We have recently shown that IL-12 is a highly efficacious adjuvant for this "antipathology" vaccine strategy (13).
Initially, we demonstrated that IL-12 could be used prophylactically in
combination with egg Ags to reduce subsequent egg-induced lung
pathology (14). More importantly, a similar IL-12-based antipathology
vaccine reduced both granuloma formation and fibrosis in the livers of
infected animals (13). The reduction in fibrosis was accompanied by a
switch in the normal Th2 response to a limited Th1 response. We
observed marked increases in type 1 cytokines including IFN-, IL-12,
and TNF-, and corresponding decreases in type 2 cytokines such as
IL-4, IL-5, and IL-13. We hypothesized that the reduction in
fibrosis/granuloma formation in egg/IL-12-sensitized mice was possibly
due to both the enhanced production of cytokines involved in
suppressing collagen synthesis (IFN-) (15) and the repressed
production of collagen-inducing cytokines (IL-4) (16).
In the current study, we wanted to elucidate the mechanism and describe
the key components of this IL-12-based antipathology vaccine.
Specifically, we examined whether the reduction in hepatic pathology
induced by the egg/IL-12 sensitization protocol was dependent upon the
increased expression of IFN-, IL-12, or TNF-. To address their
contribution to the pathology-reducing effect, egg/IL-12-sensitized
mice were infected and then treated with neutralizing Abs to IFN-,
IL-12, and TNF- at the time of schistosome egg laying. We examined
the effects on granuloma formation, recruitment of eosinophils,
development of hepatic fibrosis, and the evolving cytokine response
both in vitro and in vivo. The results from this study demonstrate that
all three type 1 cytokines are important for the antipathology effect
induced by Ag/IL-12 sensitization. In addition, these data provide
evidence that type 1 responses generated against strong type 2 inducing
stimuli are potentially unstable and remain highly dependent upon
endogenous IFN-, IL-12, and TNF- for their maintenance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female 42-day-old C57BL/6 mice (10 per group) were obtained from Charles River Laboratories (Raleigh, NC). All mice were housed in an National Institutes of Health American Association for the Accreditation of Laboratory Animal Care-approved animal facility. Cercariae of a Puerto Rican strain of S. mansoni (Biomedical Research Institute, Rockville, MD) were obtained from infected Biomphalaria glabrata snails (Biomedical Research Institute). Soluble egg Ag (SEA)3 and soluble worm Ag preparations (SWAP) were derived from homogenized eggs and adult parasites as previously described (13).
Immunizations and infections
The sensitization of mice to S. mansoni egg Ags in
the presence of rIL-12 (generously provided by Genetics Institute,
Cambridge, MA) was performed essentially as previously described (13).
Briefly, S. mansoni eggs were isolated from the livers of
infected mice (Biomedical Research Institute), and 5000 eggs/animal
were injected i.p. on three occasions separated by 2-wk intervals.
Animals were also injected i.p. with rIL-12 (0.25 µg per dose) on
days 0, 1, 2, 3, and 5 after each egg exposure. Mice were infected 4 wk
after the last egg/IL-12 exposure by percutaneous challenge of tail
skin for 40 min in water containing between 20 and 25 cercariae. At the
onset of egg production (week 5), mice were treated twice weekly with
neutralizing mAbs against IFN- (mAb XMG1.6), TNF- (mAb XT22.11),
IL-12 (mAb C17.8.20), or isotype control ß galactosidase (ßGal)
(mAb GL113) at 1 mg/injection. Sera were collected from mice before
they were sacrificed by i.p. administration of sodium pentobarbital (18
mg/mouse, Sigma, St. Louis, MO) on week 8. Tissues were then collected
for the remaining studies. Control mouse groups used in these studies
were either uninfected, egg-sensitized without rIL-12, or infected
without treatment. Previous studies on mice pretreated with rIL-12
alone have demonstrated no significant effects on pathologic parameters
after infection (13).
Histopathology and fibrosis measurement
The collagen content of the liver, determined as hydroxyproline, was measured as described previously (17). Approximately half of the liver was fixed in Bouin-Hollande solution and histologic sections were processed and stained with Giemsa (Histo-Path of America, Clinton, MD). The diameters and eosinophil content of granulomas (30/mouse) surrounding single, mature, and viable eggs were measured by using an ocular micrometer (Leica, Columbia, MD), and the volume of each granuloma was calculated assuming a spherical shape.
Lymphocyte culture and cytokine assays
For in vitro cytokine measurements, mesenteric lymph nodes were
removed aseptically at week 8 after infection, and single-cell
suspensions were prepared. Mesenteric nodes were pooled from three
animals per group, and cells were plated in 24-well tissue culture
plates at a final concentration of 3 x 106 cells per
ml in RPMI 1640 supplemented with 2 mM glutamine, 25 mM HEPES,
10% FCS, 50 µM 2-ME, penicillin, and streptomycin. Cultures were
incubated at 37°C in an atmosphere of 5% CO2. Cells were
stimulated with SEA at 20 µg/ml, SWAP at 50 µg/ml, or medium alone.
Supernatant fluids were harvested at 72 h and assayed for cytokine
activity. IFN-, IL-5, IL-12, and IL-10 were measured by specific
two-site ELISA as previously described (13). Cytokine levels were
calculated using standard curves constructed using recombinant murine
cytokines.
RT-PCR detection of cytokine mRNAs
Two 25-mg portions of each liver were combined and homogenized
in 1 ml RNA STAT-60 using a tissue polytron (Omni International,
Waterbury, CT), and total RNA was isolated as recommended by the
manufacturer. The RNA was resuspended in diethylpyrocarbonate-treated
water and quantitated spectrophotometrically. A RT-PCR procedure was
performed as described (18) to determine relative quantities of mRNA
for IFN-, IL-4, IL-5, TNF-, IL-12p40, and hypoxanthine
phosphoribosyltransferase (HPRT). The primers and probes for all genes
were previously published (18, 19). The PCR conditions and cycle number
were strictly defined for each cytokine primer pair such that a linear
relationship between input RNA and final PCR product was obtained.
Positive and negative controls were included in each assay to confirm
that only cDNA PCR products were detected and that none of the reagents
were contaminated with cDNA or extraneous PCR products. The amplified
DNA was analyzed by electrophoresis, Southern blotting, and
hybridization with cytokine-specific probes. The chemiluminescent
signals were quantified using a 600 ZS scanner (Microtek International,
Torrance, CA). The amount of PCR product was determined by comparing
the ratio of cytokine-specific signal density to that of HPRT-specific
signal density for individual samples (five mice per group). Arbitrary
densitometric units for individual samples were subsequently multiplied
by a factor of 100 and compared with control mice (uninfected mouse
liver). Amplification of HPRT served as an internal control for the
amount of RNA and cDNA from each sample.
Measurement of SEA-specific Ab responses
For assesment of serum Ig, serum was collected at time of sacrifice (week 8 postchallenge). Immulon 4 (Dynatech Laboratories, Chantilly, VA) microtiter plates were coated overnight at 4°C with SEA (1 µg in 50 µl/well) diluted in PBS. Plates were blocked with 200 µl 5% nonfat dry milk/PBS for 2 h at 37°C. The blocking solution was aspirated and the wells washed six times with PBS/0.05% Tween-20 (Sigma). Individual mouse serum was serially diluted 1/100 to 1/102500 in 1% BSA/PBS, and 50 µl was added to appropriate wells. Plates were incubated at 37°C for 90 min and then washed six times with PBS/0.05% Tween-20. Fifty microliters of isotype-specific horseradish peroxidase-conjugated rabbit anti-mouse Abs in 1% BSA/PBS diluted at 1/1000 (measurement of IgG1, IgG2a, IgG2b, and total IgG/A/M, Zymed, San Francisco, CA) were added to the wells and incubated at 37°C for 2 h. Wells were again washed six times with PBS/0.05% Tween-20 and 100 µl of (2,2'-azino-di(3-ethyl-benzthiazoline sulfonate)) (ABTS:H2O2 substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD)) was added and the reactions developed in the dark at room temperature for 20 to 30 min. Absorbance at 405 nm was determined using a Vmax Kinetic Microplate Reader (Molecular Devices, Palo Alto, CA). Specific SEA-isotype titers were calculated by the product of absorbance and the reciprocal of the sera dilution from a point in the linear portion of the dilution curve.
Total serum IgE Abs were quantitated by ELISA using a protocol provided by PharMingen (San Diego, CA). Briefly, plates were coated with anti-mouse IgE capture mAb from clone R35-72 in 0.1 M NaHCO3, pH 8.2, overnight at 4°C. The secondary mAb was a biotinylated anti-mouse IgE from clone R35-92, and the streptavidin-peroxidase reagent was diluted 1/1000 in 1% BSA/PBS. A purified mouse IgE from clone IgE-2 (PharMingen) was used as the control standard.
Parasitologic data
Worm number and the number of eggs in the tissue and feces were determined as previously described (17).
Statistics
Values for secreted cytokine proteins, semiquantitative RT-PCR, serum ELISA data, granuloma volume, and the percentage of eosinophils in granulomas were compared using Student’s two-tailed t test. Hepatic fibrosis was compared by analysis of covariance using the log of total liver eggs as the covariate and the log of hydroxyproline per egg. Values of p < 0.05 were regarded as significant. Two separate experiments were performed for all data presented.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-, or IL-12 restores granuloma
formation in infected egg/IL-12-sensitized mice
Our previous studies have shown that sensitizing mice to egg Ags
in combination with IL-12 before infection led to increased IFN-,
IL-12, and TNF- expression in infected mice (13). To assess whether
the reduced hepatic pathology in egg/IL-12-sensitized animals is
directly influenced by these cytokines, C57BL/6 mice were presensitized
i.p. with eggs/IL-12, infected, and depleted of IFN-, TNF-, or
IL-12 from the start of egg laying up to the time of sacrifice (Fig. 1
). Hepatic pathology in the various
cytokine-depleted mice was compared with three different infected
control groups as well as with uninfected animals. The infected control
groups included mice that were sensitized with only saline, eggs alone,
or with eggs and IL-12 followed by a control mAb (anti-ßGal). As
expected, control mAb-treated egg/IL-12-sensitized mice developed
smaller hepatic granulomas (Fig. 2A) that contained
significantly fewer eosinophils (Fig. 2B) and displayed
marked decreases in fibrosis, as assessed by hydroxyproline levels
(Fig. 2C) when compared with the saline-treated or
egg-sensitized controls. Mice sensitized with eggs alone also showed
reduced levels of fibrosis; however, the changes were less marked than
in the egg/IL-12-sensitized group, which was consistent with previous
findings (13). Interestingly, granuloma development was restored to
control levels in egg/IL-12-sensitized mice by depletion of endogenous
IFN-, IL-12, or TNF- (Fig. 2A). The granuloma
eosinophil composition was also significantly restored in IFN-- or
IL-12-depleted mice, while depletion of TNF- had less of an effect
(Fig. 2B). Moreover, the level of hepatic fibrosis was also
significantly increased in all three cytokine-depleted groups (Fig. 2C). In all cases however, anti-IFN- mAb treatment
had the largest effect.
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Cytokine depletion had no effect on the number of eggs/worm pairs in the tissues or in the number of eggs/worm pairs passed in the feces during the 24 h before sacrifice in any of the examined groups (data not shown). Additionally, the egg- or egg/IL-12-sensitized groups did not differ from the infected untreated mice in these parasitologic criteria (data not shown). Analysis was performed by one-way ANOVA and by Student’s t test between appropriate groups.
Neutralizing endogenous IFN- or IL-12 decreases the type 1
cytokine response in egg/IL-12-sensitized mice but fails to
significantly augment the type 2 response in the lymph nodes or spleen
To determine whether the changes in egg-induced pathology after
neutralization of IFN-, IL-12, or TNF- were accompanied by
alterations in the type 1/type 2 cytokine profile, mesenteric lymph
nodes and spleens were isolated from the animals at the time of
sacrifice and analyzed in vitro for their cytokine-producing potential.
Separate pools of mesenteric lymph nodes and individual spleens were
processed at week 8, and the cells were restimulated in vitro with
either SEA or SWAP. Seventy-two-hour culture supernatants were then
assayed by ELISA for IL-5 and IL-10 as markers of a type 2 response
(Fig. 3) and IFN- and IL-12 (Fig. 4) as indicators of a type 1 cytokine
pattern. Mesenteric lymph node cultures from both saline-treated and
egg-sensitized mice produced predominantly IL-5 and IL-10 in response
SEA or SWAP (Figs. 3A and 3B). Little or no
IFN- was detected in these cultures, confirming that a highly
polarized type 2 response was established in both control-infected
groups (Fig. 3B). In contrast to these data, the
egg/IL-12-sensitized-control mAb-treated group (anti-ßGal)
displayed a highly polarized type 1 cytokine pattern (Fig. 4), which
was consistent with previous findings (13). Similar data were obtained
with splenocytes (data not shown). Despite displaying a marked increase
in pathology after neutralization of IFN-, IL-12, or TNF-, a
statistically significant increase in IL-5 or IL-10 by mesenteric lymph
node cells was not observed (Fig. 3). There was, however, a consistent
trend in both SEA- and SWAP-restimulated cultures toward slightly
elevated type 2-associated responses in anti-IFN--treated mice.
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and IL-12 were observed after neutralization of endogenous
IFN- or IL-12. While all four groups of egg/IL-12-sensitized mice
showed highly polarized type 1 responses (Fig. 4
), IFN- production
by both SEA- and SWAP-stimulated cultures were significantly lower in
animals that were treated in vivo with anti-IFN- or
anti-IL-12 (Fig. 4B). Animals treated with
anti-TNF- consistently produced the largest amount of IFN-
when compared with the other mAb-treated groups (Fig. 4B).
IL-12 production followed a similar pattern as IFN-, although the
highest levels were observed in cultures that were not restimulated
with parasite Ags (Fig. 4A). In all of our studies, SEA or
SWAP restimulation reduced IL-12 expression in lymphocytes obtained
from infected mice. Nevertheless, similar to the results for IFN-,
IL-12 levels were higher in the anti-ßGal-treated
egg/IL-12-sensitized group when compared with both infected control
groups. Neutralization of TNF- or IL-12 had little effect on the
ability to produce IL-12, while mice treated with anti-IFN-
displayed significantly reduced IL-12 levels in SEA, SWAP, and
nonstimulated cultures from both the mesenteric lymph nodes (Fig. 4A) and spleen (data not shown).
Type 2 cytokine mRNAs are restored in the livers of infected
egg/IL-12-sensitized mice after depletion of IFN-, TNF-, or IL-12
Although significant quantitative differences were detected among
the groups, the in vitro cytokine data discussed above suggest that a
prominent type 1 response is maintained in the draining lymph nodes and
spleens of all egg/IL-12-sensitized mice, even after treatment with
neutralizing mAbs to IFN-, TNF-, or IL-12. Thus, these data
suggest that the increased pathology in anti-IFN--,
anti-TNF--, and anti-IL-12-treated mice may not result from
enhanced type 2 responses. The above findings might argue for a direct
anti-inflammatory role for these cytokines in the granulomatous
response. Nevertheless, in vitro cytokine data may not directly reflect
the ongoing cytokine response at the site of granuloma formation. To
more thoroughly examine the cytokine response in situ, liver biopsies
from individual mice were analyzed by semiquantitative RT-PCR for
changes in cytokine mRNA expression. Surprisingly, in contrast to the
in vitro results, these assays revealed that a marked type 2 cytokine
response is induced in the egg/IL-12-sensitized animals after treatment
with anti-IFN-, anti-TNF-, or anti-IL-12 mAbs (Fig. 5, A and B). In the
livers of anti-IFN--treated mice, type 2 cytokine mRNA levels
approached that of the egg-sensitized/infected controls (positive
control for type 2 cytokines). These data thus correlate with the
marked changes in pathology (Fig. 2) and in vitro IFN- expression
(Fig. 4) observed in animals depleted of IFN-.
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protein secretion from
draining lymph node cells in vitro (Fig. 4), mRNA for this type 1
cytokine was abundant in livers of mice that had been sensitized with
egg/IL-12 and/or subsequently treated with anti-IFN-,
anti-TNF-, or anti-IL-12 Abs (Fig. 6A). However, as reported
previously, the increases in liver IFN- mRNA were not significantly
different from the infected controls (13). Neutralization of IFN- or
TNF- had little effect on IFN- mRNA expression, although IL-12
depletion modestly down-regulated IFN- mRNA transcript levels when
compared with the anti-ßGal-treated egg/IL-12-sensitized animals.
Thus, in contrast to the marked decreases in IFN- expression
observed in Ag-stimulated lymph node cultures (Fig. 4B),
neutralization of IFN- did not affect the level of IFN- mRNA
transcripts in the liver. Nevertheless, the overall dominance of type 1
vs type 2 (IFN- vs IL-5) associated cytokines was shifting in the
granulomatous tissues. Neutralization of IFN- (and to a lesser
extent, anti-IL-12 and anti-TNF- treatment) restored the
expression of IL-4 and IL-5 mRNA (Fig. 5), but did so without
significantly modifying IFN- mRNA expression.
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increased TNF- mRNA transcripts
and partially up-regulated p40 IL-12 mRNA levels (Fig. 6, B
and C). By contrast, mice depleted of IFN- demonstrated
the greatest abrogation in TNF- and p40 IL-12 mRNA transcript levels
when compared with mice treated with anti-ßGal, anti-TNF-,
or anti-IL-12 Abs (Fig. 6, B and C).
Anti-IL-12 Ab treatment had little effect on p40 IL-12 or TNF- mRNA
expression in the egg/IL-12-sensitized mice (Fig. 6, B and
C).
SWAP-specific IgG2a titers are decreased in the sera of infected
egg/IL-12-sensitized mice after depletion of endogenous IFN- or
IL-12
The RT-PCR data demonstrated that type 2 cytokine responses were
up-regulated after treatment with anti-IFN-, anti-TNF-,
or anti-IL-12, at least in the livers of egg/IL-12-sensitized mice.
These results likely explain the marked pathologic changes observed in
the cytokine-depleted groups. To determine whether other type
2-regulated responses were similarly effected by depleting these
cytokines, serum IgE and IgG2a titers were examined. For these
experiments, serum was collected from individual animals at the time of
sacrifice and SEA-specific IgG2a (type 1-associated) and total IgE Ab
titers (type 2-associated) were measured by ELISA. As shown in Figure 7, neutralization of IFN- or IL-12
significantly decreased SEA-specific IgG2a Ab titers when compared with
the anti-ßGal-treated controls (Fig. 7A). There were
no significant changes observed in the TNF--depleted mice.
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B). Although animals treated with
anti-IFN-, anti-TNF-, or anti-IL-12 all had more
total IgE than animals treated with anti-ßGal, only those animals
treated with anti-TNF- displayed statistically significant
(p < 0.05) increases in total IgE titers when
compared with the egg/IL-12-anti-ßGal-treated group.
Nevertheless, these levels were still much lower than either of the
infected control groups. Interestingly, the egg/saline-sensitized
animals developed the highest IgE titers. There were no differences in
SEA-specific IgG1 or IgG2b Ab titers between any of the groups (data
not shown) although all egg/IL-12-sensitized animals had the highest
titers of total anti-SEA Abs (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12, and IFN- in regulating many
aspects of this IL-12-based antipathology vaccine.
In a pulmonary model of granuloma formation, IFN- was shown to play
an anti-inflammatory role in egg-induced lesion formation (27).
Neutralization of endogenous IFN- increased granuloma size in the
lungs of i.v. egg-challenged mice and simultaneously up-regulated type
2 and decreased type 1 cytokine responses (14). Thus, maximal pulmonary
granulomatous inflammation correlated with polarized type 2 cytokine
responses. These data suggested that a major role of the type
1-associated IFN- response was to act as a down-regulator of type 2
cytokine production. IL-12 was shown to play a similar role as IFN-
because exogenous treatment with the cytokine significantly decreased
granuloma development and type 2 cytokine expression (14). It was
demonstrated that sensitizing mice to egg Ags in the presence of IL-12,
before egg challenge, could effectively reduce subsequent pulmonary
granulomatous responses. These findings were extended to the livers of
infected animals where fibrosis, as well as granuloma size, were
decreased by prior egg/IL-12 sensitization (13). The livers of
egg/IL-12-sensitized animals displayed marked increases in IFN-,
IL-12 p40, and TNF- mRNA expression when compared with
nonsensitized-infected mice. The cytokine depletion experiments
performed here demonstrate that elevated production of all three
cytokines at the time of egg laying in infected mice is critical for
the suppression of egg-induced pathology (Fig. 2). The increased
pathology in all three anti-cytokine-treated groups correlated with
at least a partial restoration in IL-4 and IL-5 mRNA expression at the
site of granuloma formation (Fig. 5), thus likely explaining the
increased fibrosis and granuloma size, as well as tissue eosinophilia
(Fig. 2).
Although the local type 2/type 1 cytokine balance was altered by all
three cytokine depletions, the production of type 2 cytokines was not
significantly increased in the mesenteric lymph nodes (Fig. 3) or
spleens (data not shown) after in vitro restimulation. Only the
IFN--depleted mice showed a very slight increase in IL-5 production
when compared with the egg/IL-12-sensitized control group.
Nevertheless, this increase was minimal when compared with the control
and egg/saline-infected animals. Indeed, all three groups continued to
display highly significant Ag-specific type 1 responses in the
periphery (Fig. 4). Interestingly, while type 2 responses were not
up-regulated in the draining lymph nodes, IFN- expression was
significantly decreased in both IFN-- and IL-12-depleted mice (Fig. 4B). A similar decrease in the type 1 to type 2 cytokine
ratio was also observed at the site of granuloma formation. However,
the cytokine shift in the liver was a result of increased type 2
cytokine-associated mRNA expression rather than a result of decreased
IFN- mRNA expression. This finding suggests that IFN- responses
may be more stable in the liver as opposed to the draining lymph nodes.
The pathologic changes observed in the liver additionally suggest that
an important functional change occurred as a result of this shift in
the cytokine balance (Fig. 2). These data are somewhat surprising
because studies with protozoan parasites have suggested that while both
IFN- and IL-12 are required to generate Th1 responses (4, 5, 28)
only IFN- is necessary to maintain an established Th1 response (29, 30). The results presented here indicate that IL-12, as well as
IFN-, may be required to maintain strong polarized type 1 responses
to schistosome eggs. Moreover, the in vitro ELISA data, when combined
with the liver RT-PCR results, suggest that the maintenance of
polarized Th1-type responses to Th2-inducing stimuli, particularly in
the local milieu (Fig. 5), remains highly dependent upon endogenously
produced IFN-, IL-12, and, to a lesser extent, TNF-. These
findings may therefore be consistent with in vitro studies that found
some Th1 populations are readily converted into IL-4 producers while
Th2 cells are relatively fixed in their cytokine-producing pattern (31, 32). Nevertheless, we have not formally demonstrated that the changes
in cytokine expression are due solely to changes in cytokine production
by CD4+ T cells. Indeed, cell types other than
CD4+ T cells, such as eosinophils and basophils, clearly
contribute to the overall cytokine response during schistosomiasis (33)
and therefore may also be contributing to the changes reported here.
To begin to understand the connection between IFN-, IL-12, and
TNF- and the mechanism for limiting egg-induced pathology, we
examined the regulation of these cytokines following the various Ab
treatments. Not unexpectedly, the production of all three cytokines was
closely linked. IFN--depletion resulted in the greatest restoration
in liver pathology (Fig. 2) and was associated with the most
significant increase in the local type 2 cytokine response (Figs. 3 and 5). Interestingly, anti-IFN--treated mice also showed marked
decreases in IL-12 (Figs. 4A and 6C) and TNF-
expression (Fig. 6B) when compared with the
anti-ßGal-treated mice. These findings thus confirm that IL-12
and TNF- are up-regulated by IFN- (1, 34, 35) and demonstrate
that anti-IFN- treatment actually yields a triple cytokine
depletion. Anti-IL-12-treated egg/IL-12-sensitized mice also showed
reduced IFN- responses (Fig. 4B), while TNF-
expression was not significantly affected (Fig. 6B). Again,
these animals displayed significant increases in granuloma size,
fibrosis, and tissue eosinophilia, although the changes were less
marked than in the anti-IFN--treated mice (Fig. 2), which may be
explained by the unaffected TNF- response in the anti-IL-12- vs
anti-IFN--treated mice (Fig. 6B).
Anti-TNF--treated mice, while also showing increased IL-4 and IL-5
mRNA expression in the liver (Fig. 5), displayed little change in their
type 1 response when compared with the anti-ßGal-treated controls
(Figs. 4 and 6). In fact, SEA-specific IFN- production was slightly
increased in the anti-TNF--treated animals (Fig. 4B).
TNF- mRNA expression was also increased in the livers (Fig. 6B). The fact that IFN- (Fig. 4B) and IL-12
(Fig. 4A) expression were not affected correlates with the
failure to signficantly restore the tissue eosinophilia in
anti-TNF--treated animals (Fig. 2B). Moreover, these
results suggest that the effects of TNF- on granuloma formation and
fibrosis may be more direct and downstream from IFN- or IL-12
because the latter cytokine-depleted animals manifested multiple
Th1-associated deficiencies while the anti-TNF--treated group
showed only modest changes in their type 1/type 2 cytokine profile.
The results obtained from anti-TNF--treated mice in particular
are surprising given previously published information on the role of
TNF- in schistosome egg-induced granuloma formation. Indeed numerous
studies have suggested that TNF- plays a proinflammatory rather than
anti-inflammatory role in lesion development as demonstrated here.
Amiri et al. (36) were the first to demonstrate a role for the cytokine
in schistosomiasis by showing that TNF- could partially restore
granuloma formation in SCID mice. In related studies, Joseph and Boros
(37) showed that peak liver granuloma size was reduced in infected mice
when the animals were treated with neutralizing Abs to TNF-. In
addition, Adewusi et al. (38) reported increased levels of TNF- in
CBA/J mice that developed a more serious hepatosplenic-like form of the
disease. In contrast to these findings, our studies in
egg/IL-12-sensitized mice suggest that TNF- is playing a
host-protective role (Fig. 2). Indeed, in all of our studies examining
the role of IL-12, the most dramatic reductions in either pulmonary
(14, 27) or hepatic (13) pathology correlated with marked increases in
TNF- expression. These data suggest that TNF- may play distinct
roles in granuloma formation and hepatic fibrosis depending on the
particular cytokine milieu in which it is expressed. Indeed, in a
tuberculosis model (39), TNF- was shown to exhibit unique activities
in a pure type 1 vs a mixed type 1/type 2 dominated immune response. In
type 1 responses, TNF- behaved in a manner similar to IFN-,
acting as a key macrophage-activating cytokine, while in mixed type
1/type 2 or type 0 responses, the cytokine induced tissue damage. Thus
in schistosome-infected mice, where type 2 responses dominate (21),
TNF- appears to play a proinflammatory role (36, 37) leading to
hepatic pathology, while in egg/IL-12-sensitized mice, TNF- may
contribute to the collagen-suppressing activities exhibited by IFN-
(15). Future studies aimed at elucidating the role of this important
immunoregulatory cytokine in type 1- vs type 2-dominated granulomatous
responses could lead to more effective strategies for limiting hepatic
pathology induced by schistosome infection.
A recent study demonstrated that IgE-deficient mice (C
knockout)
develop smaller liver granulomas when compared with WT control-infected
animals (40). These data were the first to suggest a possible
proinflammatory role for IgE in egg-induced granuloma formation. Our
data from egg/IL-12-sensitized mice correlate with these observations
because IgE levels were dramatically reduced in these animals when
compared with infected, non-IL-12-treated controls (Fig. 7); granuloma
size was similarly decreased in these animals as well (Fig. 1A). Nevertheless, the anti-cytokine-treated
egg/IL-12-sensitized mice developed granulomas that were nearly
identical to those in both infected non-IL-12-treated control groups
(Fig. 1A) but showed little restoration of their serum IgE
response (Fig. 7). In addition, infected egg/saline-sensitized mice
showed almost no increase in their acute stage granulomatous response
when compared with the nonsensitized controls, yet displayed
significantly elevated IgE Ab titers. Together, these data suggest that
vaccine-induced IgE responses play little or no role in the regulation
of acute stage granuloma formation. Nevertheless, a role for B cells
(41), Ab (42), and possibly Fc receptor (41, 43) signaling in the
regulation of granuloma formation, particularly in the chronic stage of
infection, has recently been established.
Previous studies showed that IFN- could suppress hepatic fibrosis in
schistosome-infected mice (15). The data presented here demonstrate
that the reduction in granuloma formation, collagen synthesis, and
tissue eosinophilia conferred by prior egg/IL-12 sensitization relies
upon the combined increased expression of multiple cytokines, including
IFN-, IL-12, and TNF-. Nevertheless, IFN- emerged as a central
mediator because depletion of this cytokine alone exerted the most
dramatic effects on pathology while simultaneously decreasing the
expression of the other key cytokines. A more surprising finding of
this study was the relative plasticity of the egg/IL-12-induced type 1
response in infected animals. Most striking was the marked increase in
type 2 cytokines in the local granuloma environment after
neutralization of endogenous IFN-, IL-12, or TNF-. In addition,
the fact that type 1 responses were significantly decreased in the
periphery after depletion of IFN- or IL-12 suggests that
vaccine-induced type 1 responses generated against potent type
2-inducing stimuli such as schistosome eggs may be continuously
dependent on multiple type 1-inducing cytokines for their maintenance.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas A. Wynn, National Institutes of Health, Bldg. 4, Room 126, 9000 Rockville Pike, Bethesda, MD 20892-0425. E-mail address:
3 Abbreviations used in this paper: SEA, soluble egg Ag; SWAP, soluble worm Ag preparation; ßGal, ß galactosidase; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication March 16, 1998. Accepted for publication June 22, 1998.
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References |
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