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The Journal of Immunology, 2005, 175: 3920-3926.
Copyright © 2005 by The American Association of Immunologists

Severe CD4 T Cell-Mediated Immunopathology in Murine Schistosomiasis Is Dependent on IL-12p40 and Correlates with High Levels of IL-171

Laura I. Rutitzky, Jessica R. Lopes da Rosa and Miguel J. Stadecker2

Department of Pathology, Tufts University School of Medicine, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
C57BL/6 mice infected with the helminth Schistosoma mansoni develop small hepatic granulomas around parasite eggs, but concomitant immunization with soluble schistosome egg Ags (SEA) in CFA (SEA/CFA) causes marked exacerbation of the lesions in a Th1-dominated environment characterized by high levels of IFN-{gamma}. We explored the cause of the severe immunopathology by using IL-12p40–/– and IL-12p35–/– mice. SEA/CFA-immunized IL-12p40–/– mice, incapable of making IL-12 or IL-23, were completely resistant to high pathology, and their SEA-stimulated lymphoid cells failed to secrete significant IFN-{gamma} or IL-17. In contrast, SEA/CFA-immunized IL-12p35–/– mice, able to make IL-23 but not IL-12, developed severe lesions that correlated with high levels of IL-17, low IFN-{gamma}, and an expansion of activated CD4 T cells with a CD44high/CD62Llow memory phenotype. In vivo administration of neutralizing anti-IL-17 mAb markedly inhibited hepatic granulomatous inflammation. Importantly, CBA mice, a naturally high pathology strain, also displayed elevated IL-17 levels comparable to those seen in the SEA/CFA-immunized BL/6 mice, and their lesions were similarly reduced by in vivo treatment with anti-IL-17. Our findings indicate that an IL-17-producing T cell population, likely driven by IL-23, significantly contributes to severe immunopathology in schistosomiasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hepatointestinal perioval granuloma formation, the main pathology in infection with the helminth parasite Schistosoma mansoni, is mediated by CD4 T cells specific for egg Ags (1, 2). Pronounced egg-induced immunopathology in a proinflammatory Th1-type environment, seen in the early stage of patent infection, gradually diminishes in intensity with the changeover to a dominant Th2 response. In contrast, a persistent Th1 response, due to the absence of costimulatory molecules (2, 3, 4, 5) or of anti-inflammatory Th2-type cytokines IL-4 (6) and IL-10 (7), is associated with exacerbated granulomatous pathology, hepatocellular injury, and in instances, death. Genetically determined, high pathology schistosomiasis linked with a Th1 response is also observed in certain inbred mouse strains, such as C3H and CBA (both H-2k), but not in others, such as C57BL/6 (BL/6; H-2b) (8, 9). However, a marked exacerbation of disease in a Th1-biased environment, similar to that seen in C3H and CBA mice, can be readily achieved in the low pathology BL/6 strain by concomitant immunization with soluble schistosome egg Ags (SEA)3 in CFA (SEA/CFA) (10).

The outcome of adaptive CD4 T cell-mediated immune responses critically depends on a number of factors including the nature and amount of Ag, type of APC, costimulation, and cytokine environment. In the case of Th1 differentiation, lineage commitment involves the transcription factor T-bet as well as the signature cytokines IL-12 and IFN-{gamma} (11). However, additional complexity in Th1-type immunity has been revealed with the discovery of the IL-12-like cytokine IL-23 (12, 13). IL-12 and IL-23 are dendritic cell- and macrophage-derived heterodimeric cytokines with distinct functions and structures. They share a common p40 subunit that is bound, respectively, to a distinct p35 vs p19 subunit, with each subunit binding to a specific cell receptor (13, 14). IL-12 preferentially induces T cells to produce IFN-{gamma}, thereby activating phagocytes to kill intracellular pathogens. Conversely, IL-23 induces T cell production of IL-17A and IL-17F, which are the most phylogenetically related proinflammatory members of the IL-17 family of cytokines (15, 16, 17). IL-23 also induces TNF and IL-6 production (18). Importantly, IL-23 promotes chronic inflammatory responses such as those observed in experimental autoimmune encephalomyelitis (EAE) (18, 19) and collagen-induced arthritis (CIA) (20), largely by stimulating T cells to produce IL-17A (17). The designations of Th1 and ThIL-17 have been proposed for these two distinct Th cell populations (21).

To further understand how IL-12 and IL-23 contribute to the development of severe immunopathology in experimental schistosomiasis, we used mice deficient in IL-12p40 (incapable of producing both IL-12 and IL-23), in IL-12p35 (able to produce IL-23 but not IL-12), and in IFN-{gamma}. Because the available mice are on a low pathology (H-2b) background, they were infected in conjunction with the pathology-enhancing regimen afforded by immunization with SEA/CFA. We now report that IL-17, but not IFN-{gamma}, is the CD4 T cell-derived cytokine most directly associated with the severity of hepatic granulomatous inflammation, indicating that an IL-17-producing T cell, likely driven by IL-23, is a major force behind severe pathology in schistosomiasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, infection, and treatments

Female wild type (WT) BL/6 (BL/6), BL/6 IL-12p40–/– (IL-12p40–/–), BL/6 IL-12p35–/– (IL-12p35–/–), BL/6 IFN-{gamma}–/– (IFN-{gamma}–/–), and CBA/J (CBA) mice 6 to 8 wk old were purchased from The Jackson Laboratory and maintained in the Animal Facility at Tufts University School of Medicine (Boston, MA) in accordance with the American Association for the Assessment and Accreditation of Laboratory Animal Care guidelines. Mice were infected by i.p. injection with 80 cercariae of S. mansoni (Puerto Rico strain), which were obtained from infected Biomphalaria glabrata snails, provided to us by the Biomedical Research Institute (Rockville, MD) through National Institutes of Health, National Institute of Allergy and Infectious Diseases (contract NO1-AI-55270). Some mice were immunized by s.c. injection with 50 µg of SEA/CFA, as described (10). Treatment of BL/6 mice with SEA/CFA causes marked exacerbation of their egg-induced immunopathology; either SEA or CFA alone are ineffective (10). SEA from S. mansoni was purchased from the Biomedical Research Institute and prepared as described before (22). Some SEA/CFA-immunized BL/6 and CBA mice were treated in vivo by i.p. injection with 70 µg of neutralizing anti-IL-17 (clone 50104) or control rat IgG2a mAb (R&D Systems) at 2 days, as well as 4, 5, and 6 wk after infection. This anti-IL-17 mAb recognizes IL-17A, although cross-reactivity with IL-17F has not been excluded.

Cell preparations and cytokine determinations

Mesenteric lymph node cells (MLNC) and liver granuloma cells (GC) were isolated from 7-wk infected mice as previously described (10). Cell preparations were >95% viable by trypan blue exclusion. Bulk MLNC and GC suspensions (5 x 106 cells/ml) were incubated in the presence or absence of 20 µg/ml SEA for 36–48 h, and the supernatants were variously assayed for the presence of IFN-{gamma}, IL-17, IL-5, and IL-10 by ELISA. Monoclonal Ab, standard cytokines, and protocols from BD Pharmingen were used for the detection of IFN-{gamma}, IL-5, and IL-10. For IL-17, the ELISA was done with reagents obtained from R&D Systems. The assay involves an mAb (clone 50101) that recognizes IL-17A, although cross-reactivity with IL-17F has not been excluded.

Flow cytometric analysis

GC were stained ex vivo for flow cytometry analysis using PE-conjugated anti-CD4 (clone GK1.5), FITC-conjugated anti-CD69 (clone H1.2F3), CyChrome-conjugated anti-CD44 (clone IM7), and FITC-conjugated anti-CD62L (clone MEL-14) mAb (BD Pharmingen), as described before (23). Data were analyzed using the WinList 5.0 software. Lymphocyte-rich areas (lymphocyte gate), comprising 8–15% of the GC, were defined and gated based on forward and side light scatter.

Assessment of hepatic pathology

Sections from liver samples fixed in 10% buffered formalin and processed by routine histopathologic technique were stained with H&E and examined by optic microscopy for qualitative and quantitative changes. The size of the granulomatous lesions was measured by computer-assisted morphometric analysis, as described (10). An average of 15 granulomas was measured in each liver section. Serum levels of the liver-associated enzyme aspartate transaminase (AST) were measured as an indicator of hepatocellular damage. The enzyme was determined by colorimetric assay in sera from individual mice. Background serum values from normal BL/6 mice were subtracted.

Statistical analysis

ANOVA and Student’s t tests were used to determine the statistical significance of the differences among groups. Results differing with a value p < 0.05 were considered significant. Each individual experiment was conducted with groups of three to six mice.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-12p40, but not IL-12p35, is required for the development of severe hepatic immunopathology

In our model, schistosome-infected, SEA/CFA-immunized BL/6 mice display pronounced hepatic granulomatous inflammation with increased parenchymal mixed mononuclear cell and granulocyte infiltration, hepatocellular damage, elevated Th1-type cytokines, and death occurring at ~7 wk of infection (10); this picture contrasts sharply with the milder pathology in a Th2-type environment observed in control-immunized or unimmunized BL/6 mice (10). To investigate the basis of the Th1-biased high pathology schistosomiasis, we first used mice lacking either the IL-12p40 or the IL-12p35 subunits of IL-12. Strikingly, unlike their BL/6 WT counterparts, the SEA/CFA-immunized IL-12p40–/– mice were completely resistant to developing the larger granulomas (Fig. 1), with survival rates, hepatic inflammation, and serum AST levels equal to those seen in the unimmunized low pathology controls (Fig. 2, A–C). In contrast, the immunized IL-12p35–/– mice displayed increased mortality and significantly more hepatic pathology than the IL-12p40–/– mice, resembling those seen in the BL/6 group (Fig. 2, A–C); no appreciable differences in pathology were detected among the unimmunized mouse groups (Fig. 2, B and C). Cytokine analysis revealed a sharp increase in SEA-specific IFN-{gamma} production by MLNC from SEA/CFA-immunized BL/6 mice, confirming our previous observation (10); however, IFN-{gamma} production was markedly inhibited in both the IL-12p40–/– and IL-12p35–/– counterparts (Fig. 2D). Significantly, IL-17 was also markedly increased in the SEA/CFA-immunized BL/6 mice, but was only inhibited in the IL-12p40–/– mice and not in the IL-12p35–/– group (Fig. 2E). Both IFN-{gamma} and IL-17 were very low to absent in all the control unimmunized mice. IL-5 was generally reduced in all the immunized animal groups (Fig. 2F), whereas IL-10 was also reduced in the immunized BL/6 mice but remained unchanged in the other two groups (Fig. 2G), with all unimmunized mouse groups displaying relatively higher steady levels of both IL-5 and IL-10. Cytokine production by SEA-stimulated GC mirrored the above-described pattern, albeit with somewhat lower levels. The values for IFN-{gamma} and IL-17 are shown in Fig. 2, H and I, respectively.



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  		FIGURE 1. Hepatic granulomatous inflammation in schistosome-infected, SEA/CFA-immunized and unimmunized BL/6 and IL-12p40–/– mice. Egg granulomas after a 7-wk infection, seen here as yellowish-white dots on the surface of the livers, are normally small in BL/6 mice (A), but markedly increase in size in mice immunized with SEA/CFA (B). The IL-12p40–/– mice also develop small granulomas (C), but completely fail to exacerbate the lesions following immunization with SEA/CFA (D). The photograph is from the first experiment comparing BL/6 WT and IL-12p40–/– mice, and is representative of five experiments in which WT mice were compared with either the IL-12p40–/– or IL-12p35–/– mouse groups, or the three mouse groups were examined together. Shown are three livers per group of mice.

 


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  		FIGURE 2. Mortality, quantitative hepatic immunopathology, serum AST levels, and MLNC and GC cytokine production in infected, SEA/CFA-immunized BL/6, IL-12p40–/–, and IL-12p35–/– mice. Mortality (A), hepatic granulomatous inflammation (B), and serum AST levels (C), measured as described in Materials and Methods, are significantly increased in 7-wk infected, SEA/CFA-immunized vs control unimmunized BL/6 and IL-12p35–/– mice (all p < 0.05), but not in the IL-12p40–/– group. Shown are pooled data from four to five separate experiments. D–G, Cytokine levels in 48 h supernatants from SEA-stimulated MLNC measured by ELISA. D, IFN-{gamma} is increased in SEA/CFA-immunized vs unimmunized BL/6 mice (p < 0.01), but not in the IL-12p40–/– and IL-12p35–/– groups. E, IL-17 is increased in SEA/CFA-immunized vs unimmunized BL/6 mice and in the IL-12p35–/– (p < 0.001), but not in the IL-12p40–/– group. F, IL-5 is reduced in all SEA/CFA-immunized groups (p < 0.001). G, IL-10 is reduced in the SEA/CFA-immunized vs unimmunized BL/6 mice (p < 0.01), but unchanged in the other two groups. H and I, Cytokine levels in 36 h supernatants from SEA-stimulated GC measured by ELISA. H, IFN-{gamma} is increased in SEA/CFA-immunized vs unimmunized BL/6 mice (p < 0.01), but not in the IL-12p40–/– and IL-12p35–/– groups. I, IL-17 is increased in SEA/CFA-immunized vs unimmunized BL/6 mice, and in the IL-12p35–/– (p < 0.05), but not in the IL-12p40–/– group. Cytokine levels are expressed as mean ± SE of two to five pooled experiments. In all cases, background levels from unstimulated cultures are subtracted. Ratios in A denote cumulative dead mice per total mice in group.

 
Severe IL-12p40-mediated immunopathology correlates with high IL-17 production and is independent of IFN-{gamma}

The previous experiments revealed an obvious correlation between enhanced pathology and high levels of IL-17, clearly implying a pathogenic role for IL-23. However, they left open the possibility that resistance to severe pathology in the absence of IL-12p40 was also related or influenced by the low levels of IFN-{gamma}. To directly address this question, we examined schistosome-infected, SEA/CFA-immunized IFN-{gamma}–/– mice, and found that they developed severe pathology, similar to the SEA/CFA-immunized BL/6 controls (Fig. 3A), despite the expected total absence of IFN-{gamma} (Fig. 3B). However, in both SEA/CFA-immunized BL/6 and IFN-{gamma}–/– mice there was abundant IL-17 production (Fig. 3C), which correlated well with their severe immunopathology. High IL-17 levels together with exacerbated pathology were also observed in infected, SEA/CFA-immunized mice lacking the receptor for IFN-{gamma} (IFN-{gamma}R–/–, on a 129 [H-2b] background), even though the IFN-{gamma} produced by their SEA-stimulated MLNC was disproportionately elevated (data not shown). However, there were no significant differences in IL-5 or IL-10 production between the IFN-{gamma}–/– or IFN-{gamma}R–/– mice with respect to the BL/6 or 129 control mice, suggesting that these cytokines did not play a significant role in the observed differences in pathology (data not shown).



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  		FIGURE 3. Hepatic immunopathology and MLNC cytokine production in infected, SEA/CFA-immunized BL/6, and IFN-{gamma}–/– mice. A, Hepatic granulomatous inflammation, measured as described in Materials and Methods, is increased in 7-wk infected, SEA/CFA-immunized vs control unimmunized BL/6 and IFN-{gamma}–/– mice (p < 0.05). B and C, Cytokine levels in 48 h supernatants from SEA-stimulated MLNC measured by ELISA. B, IFN-{gamma} is increased in SEA/CFA-immunized vs unimmunized BL/6 mice (p < 0.001) but absent in the IFN-{gamma}–/– mice. C, IL-17 is increased in SEA/CFA-immunized vs unimmunized BL/6 and IFN-{gamma}–/– mice (p < 0.001). Cytokine levels are expressed as the mean of triplicate determinations ± SD; background levels from unstimulated MLNC are subtracted. Results shown are from one experiment representative of two.

 
Severe hepatic immunopathology is associated with an increase in activated CD4 T cells with a memory phenotype

The studies with IFN-{gamma}-deficient mice confirmed that the high pathology phenotype was unrelated to IFN-{gamma} but coincided with increased levels of IL-17. IL-17 (16, 17) is a proinflammatory cytokine derived from a distinct activated CD4 T cell subpopulation that is sensitive to stimulation by IL-23 and characterized by a memory phenotype (12, 21). In our system, the complete absence of IL-17 in supernatants from T lymphocyte-depleted cell populations confirmed its origin in T cells (data not shown). Comparative flow cytometric analysis of GC from SEA/CFA-immunized vs unimmunized mice demonstrated that the immunization caused, in the lesional environment, a marked increase in activated, CD69-expressing CD4 T cells in BL/6 and IL-12p35–/– mice, but not in the IL-12p40–/– group (Fig. 4A). Moreover, the same cell population from the SEA/CFA-immunized BL/6 and IL-12p35–/– mice exhibited a higher proportion of T cells with a CD44high/CD62Llow phenotype, characteristic of memory cells; this trend, however, was reversed in the IL-12p40–/– group (Fig. 4B).



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  		FIGURE 4. Phenotype of CD4 T cells in granulomas from infected, SEA/CFA-immunized BL/6, IL-12p40–/– and IL-12p35–/– mice. Liver GC were isolated and stained ex vivo with anti-CD4 and anti-CD69, or anti-CD4, anti-CD44, and anti-CD62L mAb, as described in Materials and Methods. A, SEA/CFA immunization of infected BL/6 and IL-12p35–/– mice causes an increase in activated CD4+CD69+ T cells shown in the box (inset); this difference is not seen in the same cells from IL-12p40–/– mice. The percentage shown is based on CD4+ cells present in the lymphocyte gate. B, SEA/CFA immunization of infected BL/6 and IL-12p35–/– mice causes an increase in the population of CD4+ T cells expressing the CD44high/CD62Llow memory phenotype, shown in the left box (inset). In contrast, there is a decrease in this cell population in the IL-12p40–/– mouse group. The CD4+ T cells expressing CD44high/CD62Lhigh in the right box are shown for comparison. The results are from one representative experiment of three performed using pooled GC from five to six mice per group.

 
Exogenous administration of neutralizing anti-IL-17 mAb significantly abrogates the severity of immunopathology in SEA/CFA-immunized mice

In analogy with previous observations in EAE (18, 19) and CIA (20), our results linked the development of immunopathology to a population of activated memory T cells with a secretory program that includes IL-17. To more directly ascertain the specific role of IL-17 in the induction of severe pathology, we administered neutralizing anti-IL-17 mAb i.p. to SEA/CFA-immunized BL/6 mice during the course of the schistosome infection. As shown in Fig. 5, anti-IL-17 mAb, but not the control mAb, significantly reduced the severity of the immunopathology, which approached the level exhibited by the unimmunized mice. There were no significant differences in cytokine production by SEA-stimulated MLNC and GC from mAb-treated and untreated SEA/CFA-immunized mice in vitro, after the cells were no longer under the effect of these mAb (data not shown).



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  		FIGURE 5. In vivo treatment with anti-IL-17 mAb inhibits the severe immunopathology in infected SEA/CFA-immunized BL/6 mice. Infected SEA/CFA-immunized BL/6 mice were treated with neutralizing anti-IL-17 mAb or isotype control mAb, as described in Materials and Methods. The egg granulomas in livers from 7-wk infected mice were measured as described. Treatment with anti-IL-17, but not with control mAb, caused a significant reduction in granuloma size (p < 0.05) in the SEA/CFA-immunized mice. Values from unimmunized BL/6 mice are shown for comparison. Results shown are from one experiment representative of two.

 
SEA-stimulated MLNC from CBA mice produce high levels of IL-17, and in vivo treatment with neutralizing IL-17 mAb significantly reduces their hepatic immunopathology

The results described appear to identify IL-17 as a marker of high egg-induced hepatic immunopathology. However, because the granulomatous inflammation in the mice was enhanced by means of immunization with SEA/CFA, we further investigated this correlation in CBA mice, a strain in which an identical infection protocol results in the development of natural high pathology. The results showed that SEA-stimulated MLNC from infected CBA mice produce significantly larger amounts of IL-17 than MLNC from their BL/6 counterparts (Fig. 6A), with levels comparable to those observed in the SEA/CFA-immunized BL/6 mice (Fig. 2E). These findings also link IL-17 with the development of high natural pathology in CBA mice, and validate the observations made in the immunization model. To directly test the contribution of IL-17 to the pathology, CBA mice were treated with neutralizing anti-IL-17 mAb. Treatment with this mAb, but not with the control mAb, resulted in a significant reduction of granuloma size after 7 wk postinfection (Fig. 6B), clearly confirming that IL-17 plays an important role in the development of severe disease.



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  		FIGURE 6. IL-17 is elevated in schistosome-infected CBA mice and its neutralization in vivo reduces immunopathology. A, IL-17 levels in 48 h supernatants from SEA-stimulated MLNC, measured by ELISA, are significantly higher in CBA mice (p < 0.05). IL-17 levels are expressed as mean of triplicate determinations ± SD; background levels from unstimulated MLNC are subtracted. Results shown are from one experiment representative of three. B, Infected CBA mice were treated with neutralizing anti-IL-17 mAb, or isotype control mAb, as described in Materials and Methods. The egg granulomas in livers from 7-wk infected mice were measured as described. Treatment with anti-IL-17, but not with control mAb, caused a significant reduction in granuloma size (p < 0.05). Results shown are from one experiment representative of two.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our analysis using mouse strains deficient in IL-12 subunits demonstrated that the absence of IL-12p40 affords complete protection against the development of severe hepatic granulomatous inflammation induced by immunization with SEA/CFA, whereas the absence of IL-12p35 has virtually no effect on the pronounced immunopathology and mortality. Cytokine analysis of supernatants from SEA-stimulated MLNC and GC indicated that the SEA/CFA-immunized IL-12p40–/– mice displayed low levels of both IFN-{gamma} and IL-17, consistent with their inability to make IL-12 and IL-23. In contrast, the SEA/CFA-immunized IL-12p35–/– mice produced high levels of IL-17 and low IFN-{gamma}, a pattern that reflects the presence and stimulatory activity of IL-23, but not of IL-12. These findings clearly indicate that the development of severe murine schistosomiasis correlates with high levels of IL-17, and suggest that the exacerbated egg-induced immunopathology is largely mediated by a subset of SEA-specific T cells producing IL-17. This powerful pathogenic mechanism is analogous to that recently described in EAE and CIA, two models of murine autoimmune inflammatory disease precipitated by immunization with self-Ag in CFA (19, 20).

In both EAE (18) and CIA (20), it has been shown that accessory cell-derived IL-23 activates a CD69+ subset of CD4 T cells with a CD44high/CD62Llow memory phenotype. Our study similarly found that granulomas from the SEA/CFA-immunized BL/6 and IL-12p35–/– high pathology groups harbor an enriched activated CD69-expressing CD4 T cell population with an increase in the CD44high/CD62Llow memory cell phenotype; this was not the case in IL-12p40–/– low pathology mice. The IL-23-driven T cell subset mediating the immunopathology in EAE and CIA, characterized by the signature cytokine IL-17, has been further demonstrated to produce a variety of additional mediators including TNF, IL-6, GM-CSF, and the chemokines IL-8, MIP-2, and MCP-1 (18, 24), all of which likely contribute to the induction and maintenance of a proinflammatory state.

To further explore the specific role of IL-17 in the development of high pathology, we treated infected SEA/CFA-immunized BL/6 mice with anti-IL-17 mAb recognizing IL-17A. The surprisingly strong reduction of hepatic granulomatous inflammation, owed to this treatment, suggested that IL-17A is able, per se, to mediate a substantial proportion of the immunopathology, although a role for the related IL-17F cannot be excluded. In vivo administration of anti-IL-17 mAb has similarly offered variable protection against EAE (18) and CIA (25); additionally, in a murine model of Lyme disease, this treatment prevented the establishment of severe destructive arthritis (26). Amelioration of severe inflammatory disease following neutralization of IL-17 may at least in part be due to a reduction of proinflammatory molecules responsible for leukocyte recruitment.

In our studies, we used a severe form of murine schistosomiasis precipitated in low pathology BL/6 (H-2b) mice by immunization with SEA/CFA; this model was a necessity because the examined cytokine deficient mice are only available on the H-2b background. The induced high pathology in this model has been previously shown to share morphologic and immunological features with the natural high pathology characteristic of the CBA (H-2k) strain (10). Nonetheless, we validated the relevance of the immunization model by demonstrating that in natural high pathology CBA mice there is also a markedly increased production of IL-17 by SEA-stimulated MLNC, which is similar to that observed in the SEA/CFA-immunized BL/6 mice. Importantly, in vivo administration of anti-IL-17 mAb also caused a significant reduction of pathology in the CBA strain, although the effect of this treatment was slightly less than in the immunization model. This may be due to a less efficient neutralization of the IL-17 given that CBA mice have a relatively greater proportion of CD4 T cells in the granulomas and their T cells are less prone to apoptosis than in the BL/6 strain (23).

Although an unambiguous hallmark of Th1 immunity, the IL-12/IFN-{gamma} axis appears to play a relatively smaller role in the immunopathology associated with the schistosome infection and autoimmune disease (21), as severe inflammation proceeds independently of either of these cytokines. In the schistosome system, unexpectedly, the IL-12p35–/– mice were capable of developing remarkably pronounced hepatic lesions despite the low levels of IFN-{gamma}; this observation was reinforced in IFN-{gamma}–/– mice in which this cytokine is absent. Clearly, lesions of comparable severity develop irrespective of the presence or absence of IFN-{gamma}, which may explain previous conflicting reports variously suggesting that IFN-{gamma} stimulates (27), inhibits (28), or has no significant effect (29) on the schistosome egg-induced immunopathology. Regardless, an elevation of IFN-{gamma} in WT mice remains a faithful indicator of a proinflammatory environment conducive to increased immunopathology (9, 10). These findings notwithstanding, it is still possible that some initial Th1 lineage commitment is necessary to generate IL-12/IL-23 receptor-positive precursor T cells susceptible to IL-23 stimulation and IL-17 production (30).

Although ostensibly of dissimilar pathogenic potential, both Th1 and ThIL-17 cells can produce a number of proinflammatory cytokines in common; in fact, one of these, TNF-{alpha}, has been suggested to play a role in schistosome egg granuloma formation (31, 32). Because granulomatous inflammation is strictly dependent on SEA-specific CD4 T cells and fails to materialize in immunodeficient mice (33, 34, 35), the development of lesions in the absence of IL-23 (i.e., in the IL-12p40–/– mice) clearly demonstrates that products from "classical" Th cells contribute to the background level of immunopathology precipitated by the schistosome eggs (1).

In summary, our study has for the first time documented a pathogenic role for the IL-23-induced, IL-17-producing CD4 T cells in a parasitic helminthic disease, further demonstrating that this subpopulation is capable of driving inflammatory reactions to pathogens and not solely those associated with autoimmune diseases. The discovery of the ThIL-17 cell subset should prompt a thorough re-evaluation of T cell-mediated diseases, both in mice and in humans, as the targeted neutralization of these highly pathogenic cells may represent a powerful new approach to curtailing excessive immune-mediated tissue destruction.


   Acknowledgment
 
The authors thank Dr. Eduardo Finger for taking the photograph in Figure 1.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by U.S. Public Health Service Grants RO1-18919 and RO1-48736. Back

2 Address correspondence and reprint requests to Dr. Miguel J. Stadecker, Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111. E-mail address: miguel.stadecker{at}tufts.edu Back

3 Abbreviations used in this paper: SEA, schistosome egg Ags; GC, granuloma cell; MLNC, mesenteric lymph node cell; EAE, experimental autoimmune encephalomyelitis; CIA, collagen-induced arthritis; AST, aspartate transaminase; WT, wild type. Back

Received for publication April 6, 2005. Accepted for publication July 7, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Pearce, E. J., A. S. MacDonald. 2002. The immunobiology of schistosomiasis. Nat. Rev. Immunol. 2:499.-511. [Medline]
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