Th2-type cytokine pattern dominates the immune response in mice infected with S. mansoni
). Previous IL-4 depletion studies and experiments with IL-4–deficient mice, however, failed to show an indispensable role for this cytokine in the pathogenesis of schistosomiasis (8
). Indeed, whereas a partial reduction in fibrosis was observed in some studies (9
), egg-induced granuloma formation could proceed in the complete absence of IL-4 (8
). In contrast to these observations, granuloma formation and the development of hepatic fibrosis was severely impaired in Stat6-deficient mice (10
), which display a major defect in the production of several Th2-associated cytokines (39
). IL-4 and IL-13 both activate the Stat6 signaling pathway. Therefore, the apparent differences in pathology observed between infected IL-4–deficient and Stat6-deficient mice may be explained by IL-13. Nevertheless, the distinct contributions of IL-4 and IL-13 in disease progression cannot be discerned from studies in Stat6, IL-4Rα, or IL-4–deficient mice alone. In this study, we used a potent inhibitor of IL-13 in infected WT and IL-4–deficient mice, and demonstrate that IL-13 and IL-4 exhibit redundant, as well as unique, roles in the pathogenesis of schistosomiasis.
Several studies have shown that Th2-type cytokine responses can develop in vivo in the absence of IL-4 or the IL-4 receptor (19
), which is consistent with our findings that the levels IL-13, IL-10, and IL-5 were much reduced, but still significantly detectable in MLNs (Figure ) and livers (Figure ) of infected IL-4–deficient mice. Their production was also highly dependent on a CD4+
T-cell response (Figure ), further indicating that a conventional Th2-type response was established. These findings provide evidence that whereas maximal IL-13 expression is dependent on IL-4, the continued production of IL-13 might explain the maintenance of a significant granulomatous response in the absence of IL-4 (8
). Indeed, whereas blocking IL-13 alone had no effect on granuloma size in WT mice, inhibiting the residual IL-13 in IL-4–deficient mice resulted in a marked and highly significant reduction in granuloma volume (Figure a). These findings demonstrate that IL-4 and IL-13 are both sufficient to mediate granuloma development, and formally explain the maintenance of granulomatous inflammation in IL-4–deficient mice (10
). Similar findings were also reported in the pulmonary granuloma model, where the simultaneous depletion of IL-4 and IL-13 reduced granuloma formation to a much greater extent than either treatment alone (29
). Because granulomas serve an important host-protective role by walling off potentially lethal hepatotoxins released by the eggs (40
), the host may have evolved redundant mechanisms for granuloma formation in order to ensure a favorable host-parasite relationship.
Whereas these observations clearly demonstrate that IL-4 and IL-13 actively participate in granuloma formation, unique roles for both cytokines in mast cell recruitment, tissue eosinophilia, and, most importantly, the generation of hepatic fibrosis were revealed. Histological examinations of liver sections from infected mice demonstrated that IL-13 is not required for mast cell (Table ) or eosinophil (Figure b) differentiation and recruitment, because granulomas of sIL-13Rα2-Fc–treated WT mice showed no decrease in either cell type. In fact, eosinophil numbers were increased in the lesions of IL-13–inhibited WT mice (Figure b), suggesting that IL-13 may partially antagonize this effect in the presence of IL-4. In contrast, mast cells were almost completely absent from the lesions in IL-4–deficient mice, and eosinophils were decreased by over 50%. Interestingly, IL-13 appears to mediate the tissue eosinophilia in IL-4–deficient mice, because eosinophils were reduced to below 10% in the IL-4–deficient/sIL-13Rα2-Fc–treated animals. Nevertheless, these data indicate that IL-4 is the dominant cytokine responsible for the development of eosinophil and mast cell populations within granulomas.
Probably the most important advance from this study was the finding that hepatic fibrosis could be blocked by sIL-13Rα2-Fc. Indeed, microscopic (Table ), biochemical (Figure c), and molecular techniques (Figure ) all indicated that IL-13, not IL-4, plays the major role in the development of egg-induced liver fibrosis. Previous studies showed that perturbations in the Th1/Th2 cytokine balance can significantly effect the extent of tissue fibrosis in S. mansoni
–infected mice (13
). Nevertheless, this study suggests that the effects of sIL-13Rα2-Fc were not mediated through a skewing of the Th-cell cytokine response. Blocking IL-13 had no significant effect on the production of IFN-γ, IL-4, IL-5, IL-10, or IL-13 by MLN (Figure ) or spleen cells in vitro, and there was also no change in cytokine mRNA expression in vivo at the site of lesion formation (Figure ). In contrast to these observations, IL-4–deficient mice displayed an increased IFN-γ response in the draining lymph nodes (Figure ) and decreased IL-5 and IL-13 expression in the lymph nodes (Figure ) and liver (Figure ). Thus, the less-pronounced reduction in fibrosis detected in IL-4–deficient mice by hydroxyproline analysis (Figure c) may be attributable to decreased IL-13 production. The fact that IL-4 production was unaffected by IL-13 blockade, yet fibrosis was maximally reduced in these animals, emphasizes the important role played by IL-13. Indeed, sIL-13Rα2-Fc–treated IL-4–deficient mice showed little additional decrease in hydroxyproline levels (Figure c) and no difference in collagen I or collagen III mRNA expression (Figure ) over that observed in similarly treated WT mice. There was also no change in collagen I or collagen III mRNA expression in control-Fc–treated IL-4–deficient mice when compared with WT animals, further de-emphasizing the contribution of IL-4. It is also important to note that whereas the fibrosis scores by histology (Table ) and collagen I and collagen III mRNA data (Figure ) do not agree completely with the sensitive hydroxyproline results reported for the IL-4–deficient mice (Figure ), they are completely consistent with the published and controversial role of this cytokine in schistosomiasis pathogenesis. Indeed, some studies reported a partial role for IL-4 in the generation of egg-induced fibrosis (9
), whereas a related study demonstrated no contribution at all (18
). Therefore, the fact that the IL-13 blockade results were highly significant with all 3 techniques, but IL-4 deficiency achieved significance in only 1 assay (Figure ), and to a lesser degree than IL-13 blockade, emphasizes the potential utility of reducing tissue fibrosis through the inhibition of IL-13.
Moreover, in vitro studies with 3T3 cells may demonstrate for the first time the ability of IL-13 to stimulate collagen production in fibroblasts (Figure ). Thus, the effects of IL-13 on fibrosis are likely direct, and not dependent upon modulations in the Th1/Th2 cytokine response. In support of this conclusion, recent studies identified IL-13 receptors on fibroblasts (22
) and exogenous IL-13 increased adhesion molecule and inflammatory cytokine expression in human lung fibroblasts (41
). Additional studies are needed however, to determine whether granuloma-derived fibroblasts exhibit similar activity. Finally, although IL-13 (Figure ) and IL-4 (42
) are both capable of promoting collagen production in fibroblasts, the fact that cultured lymph node cells produced nearly 100-fold more IL-13 than IL-4 (Figure ) only serves to emphasize the potentially important contribution of IL-13 in this process. Indeed, studies in the pulmonary granuloma model revealed that IL-4 mRNA expression is more tightly regulated at the site of lesion formation, whereas the induction of IL-13 mRNA is much more sustained over time (29
) and, similar to the findings reported here, is observed at greater levels than IL-4 (8
). Nevertheless, we have not examined the kinetics of IL-4 and IL-13 mRNA expression in infected animals, so we cannot say whether a similar pattern holds in the granulomatous livers.
IL-13 was also recently shown to play a central role in murine asthma (30
). Indeed, the targeted pulmonary expression of IL-13 was shown to cause mucus cell metaplasia, airway fibrosis, and nonspecific airway hyperreactivity (43
). Subepithelial fibrosis and airway smooth muscle hypertrophy are common features of chronic severe asthma (44
), and chronic pulmonary fibrosis is associated with the production of collagen I and collagen III. Thus, the link between IL-13 and fibrosis revealed in our study may extend to other important Th2-related human diseases and may lead to more effective modes of treatment of fibrotic disease in general. In summary, our findings provide evidence that IL-13 inhibitors, such as the sIL-13Rα2-Fc, may be of general therapeutic benefit in preventing fibrosis associated with chronic infectious disease, and demonstrate the important and nonredundant role of IL-13 in the pathogenesis of schistosomiasis.