: Mizuki Hirata, Department of Parasitology, Kurume University School of Medicine, Kurume, Fukuoka 830–0011, Japan (e-mail: email@example.com).
The roles of interleukin (IL)-4 and interferon (IFN)-γ in Schistosoma japonicum egg granuloma formation were investigated in cercariae-infected (infection model) or after implantation of laid parasite eggs (egg implantation model) in cytokine deficient mice. Two weeks after hepatic egg-implantation, a markedly decreased mononuclear cell infiltration and lack of multinuclear cell formation were characteristic features in IL-4 deficient mice. By 4 weeks (late stage), the cellular reactions around the eggs were negligible in the deficient mice. Compared to the controls, there was a drastic reduction in the production of the Th2 cytokines, IL-4, IL-5 and IL-13. MCP-1 levels were also significantly lowered. In mice experimentally infected with cercariae, granuloma cellularity in both the wild-type and IL-4 deficient mice at 45 days and 10 weeks postinfection was analogous to the egg implantation model at 2 and 4 weeks. Overall, the effects of IFN-γ deficiency on granuloma induction differed markedly from the IL-4 results. Two weeks after egg implantation, IFN-γ deficient mice showed suppressed neutrophil response and hepatic necrosis with confluent mononuclear cell infiltration along the outer layer of granulomas. By 4 weeks, there was a decrease in cell infiltration, fibrosis and MCP-1 production while IL-10 production increased. While these early characteristic features for IFN-γ deficiency were common to both the egg implantation (at 2 and 4 weeks) and cercariae infection model (at 45 days), there was a surprising difference, i.e. marked fibrosis was found in the late stages (at 10 weeks postinfection) of cercariae-infected mice, but not in parasite egg implanted mice. Furthermore, while IL-13 levels were unchanged, both MCP-1 and IL-4 production were significantly lower at 10 weeks in comparison with wild-type. The present study clearly demonstrates the importance of both Th1 and Th2 cytokine responses in S. japonicum egg-induced granuloma formation.
Schistosomiasis is a devastating waterborne disease of the tropics. Hypersensitivity reactions in the host to the parasite's eggs lead to the formation of granulomas in the intestine and liver, which is the pathological hallmarks of intestinal schistosomiasis. It has been elucidated that CD4+ T cells mediate granuloma formation and that cytokines produced by a variety of activated immune cells help regulate the pathogenic process (1,2). Among the known cytokines, interleukin (IL)-4 and interferon (IFN)-γ are recognized to play a pivotal role in the regulation of the balance between Th1 and Th2 responses (3,4). The importance of Th2 cytokines has been demonstrated in a number of studies with Schistosoma mansoni. As an example, it has been shown that granuloma development caused by egg deposition is upregulated by IL-4 and/or IL-13 and downregulated by IL-10 (5–10), whereas the roles of IFN-γ are conflicting (11–18).
S. japonicum, a parasite that is prevalent in south-east Asia, and S. mansoni is generally thought to cause similar pathological consequences, yet there are important differences. While studies on the role of IL-4 or IFN-γ in S. japonicum infections are few, there are indications that S. mansoni and S. japonicum respond differently to cytokine regulation, at least in the mouse. Several studies with S. mansoni-infected mice have shown no significant changes in hepatic granuloma formation in either IL-4 deficient mice or IL-4 deficient mice treated with antibody to IL-4, when compared to control mice (8,19,20). However, using mice infected with S. japonicum, Cheever et al. (21) have shown that treatment with anti-IL-4 antibody, which diminished the secretion of Th2 cytokines, surprisingly induced the formation of larger granulomas lacking in fibrotic elements 10 weeks after infection. It has also been shown that treatment with anti-IFN-γ antibody, while reducing the granulomatous reactions, had no effect on hepatic fibrosis (11).
The differences in cytokine responses seen in mice infected with S. mansoni or S. japonicum might provide important clues regarding the nature of the disease. While the use of cytokine deficient animals would help define some of the underlying mechanisms, to the best of our knowledge, experimental data using cytokine deficient animals have not been published. In the present study, we employed IL-4 or IFN-γ deficient mice to clarify the stage specific roles of IL-4 and IFN-γ cytokines in S. japonicum infection. Specifically, we looked for differences of S. japonicum egg induced hepatic granuloma development caused either by infection with cercariae (infection model) or by experimental implantation of laid parasite eggs (egg implantation model) (22) in IL-4 and IFN-γ deficient mice. The results of this study strongly indicate crucial roles for Th1 and Th2 cytokines in both the acute and chronic stage of hepatic granuloma formation, especially in regards to fibrosis and the inflammatory cell types present.
Materials and methods
Animals and parasites
The C57BL/6 wild-type mice use in this study were purchased from Shizuoka Laboratory Animal Center, Japan. C57BL/6 background IL-4 deficient mice (originally constructed by Dr Kopf, Switzerland) were kindly provided by Dr Iwakura, Tokyo University and C57BL/6 background IFN-γ deficient mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). All mice were females between 8 and 15 weeks of age. The Japanese strain of Schistosoma japonicum used in our laboratory has been maintained for 25 years by passage through snails (Oncomelania hupensis nosophora) and rabbits.
Induction of granulomas
Hepatic granulomas were induced either by infection with cercariae (infection model) or by implantation of parasite eggs (egg implantation model). For the infection model, mice were percutaneously exposed to 30 S. japonicum cercariae and killed 45 days and 10 weeks after infection. In the egg implantation model, freshly laid S. japonicum eggs were obtained from mated worms taken from infected rabbits and cultured for 2 days, in RPMI 1640 containing 10% foetal bovine serum (FBS). Eggs were then washed three times with serum-free RPMI 1640 by centrifugation. Fifteen hundred eggs were implanted into the livers of mice through the coecal vein, as previously described (22). Each group of 3–4 mice was sacrificed at different stages of the inflammatory process, usually at 2 weeks (early stage) or 4 weeks (late stage), and most experiments were repeated twice. In some experiments, mice were sacrificed 10 days or 3 weeks after egg implantation.
For routine histological examination, livers were fixed in 10% neutral-buffered formalin, embedded in paraffin wax and stained with haematoxylin and eosin, azan or silver stain. Eosinophils were stained by 0·2% eosin Y (23) and anti-neutrophil antibody (Rb6–8c5) (kindly provided by Dr Sendo, Yamagata University) was used to help differentiate between infiltrating granulocytes from neutrophils. Sections were processed according to Reichert et al. (24) with minor modifications.
In the evaluation of granuloma size, granulomas larger than 100 µm in diameter were selected because disintegrated eggs showed very slight reactions. Diameters (the mean width and length of the lesion) were measured using a micrometer. Routinely, 25 −30 lesions were measured for each specimen and the mean values obtained from 4 to 6 mice were used for the statistical analysis.
Spleens were aseptically removed and single cell suspensions prepared in RPMI 1640 containing 10% FBS. For the assays, 5 × 106 nucleated cells/well were plated in 24-well plates and challenged with 2·5 µg/ml soluble egg antigen that was prepared from eggs isolated from the intestine of infected rabbits (25). After the total final volume was adjusted to 2 ml, the plates were incubated for 48 h at 37°C in a humidified CO2 incubator. Supernatant fluids were harvested after centrifugation, aliquoted and stored at − 80°C until use.
The cytokines or chemokine investigated were IL-5, IL-4, IL-10, IL-13, IFN-γ, TNF-α and MCP-1 and their levels of production were determined by sandwich ELISA assay using commercially available kits. Kits for the determination of IL-5 and MCP-1 were obtained from Genzyme (Cambridge, MA, USA) and BSI (Camarillo, CA, USA), respectively, while reagents for the other cytokines were from R & D Systems (Minneapolis, MN, USA). The samples were diluted 5- or 10-fold when values reached the upper limits of the assay system. For each time point, 4–6 duplicate samples were used. Furthermore, the experiments were repeated to ensure the validity of the results.
The Mann–Whitney's U-test was used for statistical analysis of experimental data. P < 0·05 was considered statistically significant.
Early stages of granuloma formation in implantation model
Both wild-type (Figure 1A) and IL-4 deficient mice (Figure 1B) elicited similar neutrophil responses and coagulative hepatic necrosis around the deposited eggs in the induced granulomas 2 weeks after implantation of S. japonicum eggs. However, differences were observed in mononuclear cell infiltration that appeared at the outer layer of the granulomas. A narrow rim of mononuclear cells was consistently found encircling the granulomas of IL-4 deficient mice (Figure 1B), whereas the mononuclear cellularity was apparently greater in wild-type mice (Figure 1A). In addition, scanty mononuclear cell infiltration was also seen as an advanced feature of granulomas that were characterized by the disappearance of hepatic necrosis (Figure 1C). Interestingly, the lack of multinuclear cell formations was another characteristic feature observed in the IL-4 deficient mice. In contrast, the incidence of multinuclear cells in the wild-type mice, determined in low-power fields, represented 56% of granulomas observed at this early stage. Although not quantitatively measured, the infiltration of eosinophils (determined by eosin Y staining) into the granulomas in the IL-4 deficient mice, in comparison to the wild-type animals, was apparently less.
In the IFN-γ deficient mice, differing from wild-type and IL-4 deficient mice, little or no neutrophil responses were seen 2 weeks after parasite egg implantation (Figure 1D). However, large numbers of eosinophils were found to accumulate around the eggs. Hepatocyte necrosis was also lacking in the IFN-γ deficient mice. These results differed significantly from that seen in either wild-type or IL-4 deficient mice (Figure 1A,B). However, confluent cells, macrophages, fibroblasts and lymphocytes, and eosinophils were found to infiltrate along the outer layer of the induced hepatic granulomas. Thus, these histopathological appearances that represented an early advanced feature of granuloma formation are in striking contrast to those in IL-4 deficient mice (Figure 1B). Since the apparent lack of a neutrophil response in IFN-γ deficiency at 2 weeks was curious, we reexamined this response at an earlier time after egg implantation (10 days). Interestingly, it was found that at this earlier time, the IFN-γ deficient mice were able to elicit apparent neutrophil responses around the eggs as well as induce coagulative necrosis, similar to wild-type and IL-4 deficient mice (data not shown). Thus, this suggests that this response is restricted to the early stage of granuloma formation in IFN-γ deficiency.
Analysis of granuloma size showed that there were no differences between IL-4 deficient and wild-type mice, while granulomas of IFN-γ deficient mice were significantly decreased at 2 weeks of egg implantation (Figure 2).
Late stage of granuloma formation in implantation model
In IL-4 deficient mice, granuloma size was found to diminish by the third and fourth weeks after experimental egg implantation. While, at 4 weeks, cellular infiltration was very apparent in the livers of wild-type mice (Figure 3A), one group of IL-4 deficient mice failed to show any cell infiltration around the parasite eggs. Yet in another experiment, some reactions were seen, albeit strikingly small (Figure 3B). In azan stained sections, the development of fibrosis was not seen inside lesions at 3 weeks, although some fibrosis was seen at the periphery (data not shown). Morphometric analysis confirmed our observation that there is a drastic decrease in granuloma size at 4 weeks in the IL-4 deficient mice (Figure 2).
In IFN-γ deficient mice, three different types of hepatic granuloma could be morphologically distinguished 4 weeks after egg implantation. One type of granuloma was indistinguishable from that frequently seen in wild-type mice, showing well-developed fibrosis accompanied by a moderate extent of mononuclear cell infiltration (Figure 3A). The morphologies of the other two types of granuloma observed were not commonly seen in wild-type mice. The second type of granuloma was predominantly composed of eosinophils (approximately 60–90% in histological examination) (Figure 3C). The third type was characterized by a scanty cellularity as well as an oedema-like appearance (Figure 3D). Since small number of eosinophils were present inside, the third type of granulomas was considered to be an advanced form of the second type of eosinophilic granulomas.
Cytokines and MCP-1 production in egg implanted mice
Cytokines produced by splenic cells were determined. IL-4 deficient mice had drastically reduced levels of the Th2 cytokines, IL-4, IL-5 and IL-13, at 2 and 4 weeks after egg implantation (Figure 4a). No differences were found in the levels of IFN-γ or TNF-α(Figure 4b). Since decreased mononuclear cell infiltration was found to be a characteristic feature in IL-4 deficiency, we further examined MCP-1, a chemokine reported to be a crucial monocyte chemoattractant (26)(Figure 4b). The production of MCP-1 decreased significantly in IL-4 deficient mice at both early and late stages of granuloma formation.
The picture in IFN-γ deficient mice was different. Unlike IL-4 deficient mice, no significant changes in IL-4, IL-13, and IL-5 levels were expressed by splenic cells from IFN-γ deficient mice, compared to wild-type (Figure 4a). Nevertheless, levels of IL-10 did decrease by 2 weeks, then increased significantly by 4 weeks, while TNF-α was shown to decrease at 2 weeks (Figure 4b). MCP-1 production was also observed to decrease (Figure 4b), particularly at 4 weeks after egg implantation, at a time when a markedly decreased cellularity was seen by histology.
In order to confirm the results produced using our egg implantation model, we infected mice with S. japonicum cercariae (infection model) and examined the hepatic lesions induced at 45 days and 10 weeks postinfection. It has been previously reported that S. mansoni-infected IL-4 deficient mice tend to lose weight and succumb to infection (27). However, in the present study, all groups of mice showed similar growth, worm recovery and survival characteristics throughout the course of observation. In addition, vascular dilation that has been observed in the IL-4 deficient mice (27) was not found in intestinal tissues (data not shown).
In both wild-type and IL-4 deficient mice, the cellularity and extent of the hepatic tissue lesions found in our infection model at either 45 days (Figure 5A,C) or 10 weeks (Figure 5B,D) were generally observed to be analogous to the egg implantation model (Figure 1A,B and Figure 3A,B). Compared to wild-type mice, while a smaller number of infiltrating eosinophils were seen at the early stages (45 days), there were no apparent differences observed at 10 weeks. Although there was difficulty in determining quantitatively the magnitude of tissue reactions, IL-4 deficient mice again showed drastically reduced granulomatous reactions at 10 weeks, when compared to wild-type mice.
IFN-γ deficient mice killed at 45 days of infection showed similar granuloma pathology (Figure 5E) to that observed in egg-implanted mice (Figure 1D). But, by 10 weeks after infection, the infiltration of inflammatory cells into the granulomas of IFN-γ deficient mice again appeared to be less (Figure 5F) than those seen in wild-type mice (Figure 5B), a finding comparable to the egg-implanted model. However, the development of intense fibrosis inside the majority of the granulomas seen in the IFN-γ deficient animals was a surprising finding at this chronic stage (Figure 5F), with the results contrasting to that found using egg-implanted mice (Figure 3D). Therefore, while our results indicated differences in the extent of fibrosis between the egg implantation model and infection model, in both models, the suppression of cell infiltrates into the granulomas was a consistent finding.
Cytokines and MCP-1 production in infected mice
We observed decreased fibrosis in IL-4 deficient mice in both models of egg implantation and infection. In comparison, the IFN-γ deficient mice showed different patterns of fibrosis at the later stages of infection between the two models; however, mononuclear cell infiltration similarly decreased. To examine cytokine regulation, we also analysed IL-4, IL-13, IFN-γ and MCP-1 production 10 weeks (late stage) postinfection (Figure 6). In IL-4 deficient mice, the drastic reduction in Th2 cytokines, IL-4 and IL-13, was again seen. No significant changes were found in IFN-γ. However, in contrast to the egg implantation model, in the infection model a decrease in MCP-1 production was not found. Surprisingly, we obtained some unexpected cytokine profiles in the IFN-γ deficient mice. There was a significant reduced production of IL-4, while IL-13 was comparable to wild-type mice. Furthermore, for the infection model, the level of IL-13 production was relatively low in comparison to that seen in the egg-implantation model (Figure 4a). Consistent with the egg implantation model, MCP-1 was significantly reduced in infection.
S. japonicum is known normally to deposit large numbers of aggregated eggs into tissues of the infected host. This is also the major factor that makes dissection of the underlying mechanisms of egg induced granuloma formation experimentally difficult. However, by physically introducing known numbers of schistosome eggs into genetically disrupted (knockout) mice lacking IL-4 or IFN-γ function, we have been able to gain new insights into the stage specific roles of these cytokines in S. japonicum egg-induced granuloma formation.
Decreased mononuclear cell infiltration and fibrosis was a characteristic feature seen in IL-4 deficient mice. With progression to the later stages, when changes into the granulomatous response were more obvious, cellular reactions around eggs decreased to negligible levels. Together with the lack of multinuclear cell and lowered eosinophil number, these results highlight the features of the impaired granuloma development in the deficient mice. Although our results indicate a critical role of IL-4 in granuloma formation, other cytokines are undoubtedly important as reflected by the drastic decreases in Th2 cytokine production observed, particularly IL-5 and IL-13. While previous studies have indicated that IL-5 is not essential for S. mansoni eggs granuloma formation (28), a significantly reduced granuloma reaction has been observed for S. japonicum egg-induced granulomas after anti-IL-5 antibody treatment (11). IL-13 also shares several biological functions with IL-4 (29), and it has been shown that sIL-13Rα2-Fc, an inhibitor of IL-13, can induce more profound effects on granuloma development and inhibit fibrosis more effectively than that observed in S. mansoni -infected IL-4 deficient mice (8,9). The significance of IL-13 has also been demonstrated in IL-13 deficient mice (10). Our finding of a drastically lowered IL-13 response in IL-4 deficient mice is in strong agreement with the data from other studies obtained using either IL-4Rα knock out mice (mice deficient in a receptor for IL-4 and IL-13 (20), or Stat6 knockout mice (30). In these studies, S. mansoni egg-induced granuloma development was also greatly reduced.
The significant decreases in IL-5 or IL-13 have also been reported for S. mansoni in IL-4 deficient or anti-IL-4 antibody treated, infected mice (8,19,20). However, there are important differences in host response to S. mansoni and S. japonicum because, in these same studies, significant reduction was not shown for S. mansoni egg hepatic granuloma formation. Clearly, further studies are required to define the distinct roles of IL-4 and IL-13 in S. japonicum infections.
In IL-4 deficient mice implanted with eggs, there appears to be a close relationship between decreased mononuclear cell infiltration and MCP-1 production. However, the results using cercariae infected mice did not reveal any significant decrease in MCP-1 level even after 10 weeks postinfection, during which time granuloma development was apparently reduced. Successive stimuli from a large number of newly laid eggs and adult worms may be responsible for a high level of MCP-1 production observed during infection. Nevertheless, it is generally thought that the development of fibrosis, which is impaired in IL-4 deficient mice, may be crucial for cell infiltration around egg nidi. This is because connective tissue elements, such as collagen, laminin or fibronectin and others, are known to include multiple adhesion-associated molecules for inflammatory cells,
It is well known that IFN-γ plays a role as an antiproliferative and antifibrogenic agent in a variety of mesenchymal cells. Thus one would anticipate that blockage of IFN-γ signal would augment the granulomatous reaction. However, results in the literature are conflicting. In some murine studies, S. mansoni egg-induced granuloma size has been shown either to decrease in IFN-γ receptor deficient mice (17) or to increase in size as a result of anti-IFN-γ antibody treatment (12–14). Yet, other investigators using either recombinant IFN-γ treatment (16), IFN-γ deficient mice (18), or IFN-γ receptor deficient mice (15) were unable to report any effect on S. mansoni granulomatous reactions. Regarding S. japonicum egg granuloma formation, anti-IFN-γ antibody treatment resulted in decreased granulomatous reactions (11). In this study, we demonstrated striking changes in granulomatous reactions at early and late stages of egg implantation. Significant reduction of granuloma size during early stages is thought to be ascribable to the suppression of neutrophil responses and hepatocyte necrosis. Preliminary studies have indicated that inhibition of the neutrophil response by the administration of antineutrophil antibody (Rb6–8c5) significantly suppressed the development of hapatocyte necrosis in the egg-implanted mice, suggesting that the two phenomena be closely linked.
Evidence on S. japonicum egg-induced hepatic granuloma formation in wild-type or IL-4 deficient mice was generally found to be analogous using either the cercariae infection or egg implantation murine model. However, there were differences observed between the models concerning the IFN-γ deficiency, especially during the later stages of granuloma formation. In the egg implantation model, a marked inhibition in the infiltration of mononuclear cells and fibrosis was a characteristic feature in IFN-γ deficiency. In contrast, cercariae-infected IFN-γ deficient mice showed a marked fibrosis within the granulomas. This observation is similar to that made by Resende et al. (17) who also found a marked decrease of inflammatory cell infiltration and development of fibrosis in S. mansoni-infected, IFN-γ receptor knockout mice. It was initially anticipated that the production of Th2 cytokines, i.e. IL-4 and/or IL-13 that are associated with the development of fibrosis would be higher in cercariae-infected mice than in egg-implanted mice. However, surprisingly, IL-4 production in infected mice was markedly reduced. IL-13 was also apparently produced at lower levels compared to those in egg-implanted mice. Nevertheless, the fact that both the level of IL-13 and the extent of the considerable fibrosis observed are comparable to wild-type mice strongly suggests a crucial role of IL-13 in fibrosis development. While a moderate level of IL-13 may be sufficient for fibrosis development, further study on cytokine production within granulomas appears to be warranted.
Decreased cell infiltration was also another finding consistently seen at late stages in both models regarding IFN-γ deficiency and, in concert with this, we found markedly decreased production of MCP-1 in both models, particularly at the later stages. Therefore, MCP-1 levels seem to correlate well with granuloma cellularity. In addition, in egg-implantation models, it was found that IL-10 production was significantly raised in the IFN-γ deficient mice compared with the controls at the later stages of granuloma formation, when cellularity was drastically decreased, whereas it was significantly lower than controls at early stage. Recent reports indicating that IL-10 suppresses S. mansoni-induced Th2 responses, in addition to downregulating Th1 responses (31,32) lend support to our histological findings.
In conclusion, using IL-4 or IFN-γ deficient mice we have demonstrated that IL-4 or IFN-γ are critical for the development of S. japonicum egg-induced granulomas. Our experimental hepatic egg implantation model revealed that cytokine deficiency had striking effects on granuloma size, cellularity and/or cell composition (monocytes/macrophages, neutrophils and eosinophils). Since the lack of IL-4 or IFN-γ profoundly affected the levels of other important cytokines or chemokine production, other related molecules may be involved directly or indirectly in the pathological process. Nevertheless, our present observations indicate that S. japonicum egg granuloma formation is both a Th1 and Th2 cytokine-associated process.
The authors thank Manami Ohba for the excellent technical assistance. We express our thanks to Dr M. Kopf for kindly providing permission to use the IL-4 deficient mice, to Dr Y. Iwakura for generously supplying the animals, and to Dr F. Sendo for kindly supplying the antineutrophil antibody. We also thank Dr Dennis J. Grab, Kurume University Medical School, for critical reading and editing of the final manuscript. This work was supported by a Grant-in-Aid (10670245) from the Ministry of Education, Science and Culture, Japan, and by Health Sciences Research Grants (Research on Emerging and Re-emerging Infectious Diseases) from Ministry of Health and Welfare, Japan.