Th2 lymphocytes have been postulated to play a major role in the immunopathology induced by Schistosoma mansoni infection. Nevertheless, infected IL-4 knockout (KO) and wild-type (wt) mice develop egg granulomas comparable in size. To further investigate the function of the Th2 response in egg pathology we studied IL-4Rα-deficient mice, which are nonresponsive to both IL-4 and IL-13. In striking contrast to IL-4 KO animals, infected IL-4Rα KO mice developed only minimal hepatic granulomas and fibrosis despite the presence of CD3+ T cells in the residual egg lesions. Moreover, liver lymphokine mRNA levels in these animals and IL-4 KO mice were equivalent. In addition, infected IL-4Rα-deficient, IL-4-deficient, and wt animals developed similar egg Ag-specific IgG Ab titers, arguing that CD4-dependent Th activity is intact in KO mice. As expected, IFN-γ secretion was strongly up-regulated in mesenteric lymph node cultures from both groups of deficient animals, a change reflected in increased serum IgG2a and IgG2b Ab levels. Surprisingly, Th2 cytokine production in infected IL-4Rα KO mice was not abolished but was only reduced and resembled that previously documented in IL-4 KO animals. This residual Th2 response is likely to explain the ability of IL-4 KO mice to generate egg granulomas, which cannot be formed in IL-4Rα-deficient animals because of their lack of responsiveness to the same cytokine ligands. Taken together, these findings argue that tissue pathology in schistosomiasis requires, in addition to egg-specific CD4+ lymphocytes, a previously unrecognized IL-4Rα+ non-T cell effector population.

CD4+ T cells of the Th2 subset, while helpful in host defense and in the regulation of Th1-mediated immunopathology, can themselves trigger detrimental host responses in the form of allergy and inflammation (1). Th2 differentiation is known to be selectively driven by exogenous IL-4 (2, 3, 4). Indeed, in most in vitro systems IL-4 appears to be an obligatory signal for Th2 development. Therefore, one would predict that IL-4 is critical in the pathogenesis of Th2-mediated disease. Nevertheless, recent studies indicate that mice genetically deficient in IL-4 may retain the ability to mount Th2-dependent immunopathologic responses evident in IL-4-sufficient host (5, 6).

A pertinent example of this paradox is the granulomatous inflammation in murine schistosomiasis. Humans and experimental animals infected with the helminth parasite Schistosoma mansoni react to eggs produced by the female worms by forming granulomas in the liver and other organs. This host inflammatory response leads to tissue fibrosis and circulatory impairment. Both granuloma formation and fibrosis have been shown to be CD4+ lymphocyte dependent (7). Since Th2 cytokines dominate during the peak of the granulomatous response, it has been proposed that schistosome egg pathology depends primarily on the activity of Ag-specific Th2 cells (8). However, it has been difficult to formally demonstrate a requirement for Th2 cells in this process, since anti-IL-4-treated mice show only a minor reduction in granuloma volume (9), and IL-4 KO4 mice infected with S. mansoni display either no (10) or only a small reduction (11) in granuloma size. Nevertheless, mice deficient in the Th2-specific signaling molecule STAT-6 recently have been shown to develop markedly reduced granulomas (12). The latter observation suggests that although IL-4 itself may not be essential for egg pathology, the IL-4R signaling pathway is nevertheless involved.

In the present study we directly addressed the role of the IL-4R in egg pathology in S. mansoni-infected mice by comparing granuloma formation and fibrosis in wild-type (wt), IL-4 KO, and IL-4Rα-deficient animals. The latter mice lack the α subunit of the IL-4R and as a result are unresponsive to both IL-4 and IL-13 (13) and thus in contrast to IL-4 KO animals permit an analysis of the function of signaling by both cytokines. Our results demonstrate that S. mansoni-infected IL-4Rα-deficient mice are distinct from both IL-4 KO and wt animals in showing near complete ablation of egg pathology while retaining the same lymphokine profile as IL-4 KO mice.

BALB/c IL-4 KO and BALB/c IL-4Rα KO mice were generated by homologous recombination in BALB/c I embryonic stem cells as previously described (13, 14). Double IL-4/IL-4Rα KO animals, obtained by an intercross between the KO mice, were also analyzed. The mice were bred and maintained at the American Association for Accreditation of Laboratory Animal Care facility of the National Institute of Allergy and Infectious Diseases. Control BALB/cJ and BALB/c RAG-2 KO mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and Taconic Farms (Germantown, NY), respectively. Age-matched (8–12 wk) and sex-matched mice were used in each experiment. Mice were percutaneously infected with 35 S. mansoni cercariae (NMRI strain) as previously described (15). A soluble extract of schistosome eggs (SEA) was prepared according to published procedures (16).

Liver granuloma diameters were measured in histological sections (17), and the presence of eosinophils (percentage) and mast cells (arbitrary units from 0–7, where a score of 7 indicates approximately 10 mast cells/lesion) in the same granulomas were estimated by microscopic examination. Tissue egg counts were performed on liver digested in 4% KOH, and liver collagen levels were determined by measurement of hydroxyproline (18). The statistical significance of differences in granuloma volumes and cell composition between groups was evaluated by Student’s two-tailed t test, and differences in fibrosis were evaluated by analysis of covariance.

Liver sections were stained with a polyclonal rabbit anti-human CD3 Ab cross-reactive with mouse CD3 (Dako, Carpinteria, CA), and the reaction was developed using an ABC Vectastain Elite kit (Vector Laboratories, Burlingame, CA) (19).

Mesenteric lymph nodes were pooled from mice (n = 6/group) at 8 wk after infection, and single-cell suspensions (3 × 106/ml) were stimulated in vitro with medium alone or SEA (20 μg/ml) for 72 h (17). When indicated, anti-CD4 mAb (GK1.5, rat IgG2b) (20) and anti-CD8 mAb (2.43, rat IgG2b) (21) were added to the cultures 2 h before addition of SEA. IFN-γ, IL-5, and IL-10 were measured in culture supernatants by ELISA as previously described (22, 23), while IL-4 and IL-13 were detected using commercial kits from Endogen (Woburn, MA) and R&D Systems (Minneapolis, MN), respectively.

Liver tissue from infected animals was homogenized in RNA STAT-60 (Tel-Test, Friendswood, TX) using a tissue Polytron (Omni, Waterbury, CT), and total RNA was isolated as recommended by the manufacturer. A RT-PCR (24) was used to determine relative amounts of mRNA for IL-4 (34 cycles), IL-5 (35 cycles), IL-13 (33 cycles), and hypoxanthine phosphoribosyltransferase (HPRT) (23 cycles). The sequences of the primers and probes have been previously published (24, 25).

Blood was collected from individual mice at the time of sacrifice. Levels of egg-specific Ab were determined by ELISA (26) in pools obtained by mixing equal volumes of serum from each animal within a group. The assay employed Immunolon 2 plates (Dynex Laboratories, Chantilly, VA) coated overnight at 4°C with SEA (0.5 μg/well). Peroxidase-conjugated rabbit Ab specific for mouse IgG or specific IgG isotypes (Zymed, San Francisco, CA) were used to develop the reactions. Total serum IgE was measured by a specific ELISA (PharMingen, San Diego, CA).

To formally address the requirement for IL-4 signaling in egg-induced granuloma formation and fibrosis, we infected groups of BALB/c IL-4 KO, IL-4Rα KO, and double IL-4/IL-4Rα-deficient as well as wt animals by percutaneous exposure to cercariae. At 8 wk postinfection no significant differences in worm or tissue egg burdens were observed among the four groups of mice (data not shown), confirming the lack of involvement of IL-4 in parasite development (10, 11). In contrast, the same animal groups displayed major differences in egg-induced pathology. As observed with mice on other genetic backgrounds (10, 11), BALB/c IL-4 KO animals failed to display significant alterations in granuloma size, and only a small difference in hepatic fibrosis measured by tissue hydroxyproline. In striking contrast, granuloma volumes in both IL-4Rα and double IL-4/IL-4Rα KO mice were markedly diminished to the level seen in T cell-deficient RAG-2 KO mice (Fig. 1,A). Hepatic fibrosis was also dramatically reduced in IL-4Rα-deficient animals but remained significantly higher than that observed in RAG-2 KO mice (Fig. 1,B). Similarly, the percentage of eosinophils in granulomas from IL-4Rα-deficient animals was significantly lower than that in lesions from wt or IL-4 KO animals but, nevertheless, was higher than that in RAG-2 KO granulomas (Fig. 1,C). As described previously (10, 11) granulomas in IL-4 KO mice were only marginally reduced in their eosinophil composition with respect to lesions in wt animals. In contrast, mice deficient in either IL-4 or IL-4Rα developed granulomas almost totally devoid of mast cells (Fig. 1 D), consistent with the known involvement of IL-4 in mastocytosis (27). In general, the residual lesions in IL-4Rα KO animals were found to be primarily mononuclear in composition and, with the exception of the reduced numbers of eosinophils and mast cells and the absence of concentric fibrosis, failed to display detectable cellular differences when compared with granulomas in wt mice.

FIGURE 1.

IL-4R signaling, but not IL-4, is required for schistosome egg pathology. A, Hepatic granuloma sizes (mean ± SE) in 8-wk infected BALB/c wt (n = 26), IL-4 KO (n = 17), IL-4Rα KO (n = 14), IL-4/IL-4Rα KO (n = 10), and RAG-2 KO (n = 9) mice. B, Hepatic fibrosis as measured by hydroxyproline in the same animals. Values were normalized to the number of eggs per liver. C and D, Eosinophil and mast cell composition of granulomas measured in A. ∗, Mean values statistically different (p < 0.001) from those in both wt and IL-4KO mice; §, values in IL-4 KO mice statistically different (p < 0.001) from those in wt animals. Values statistically different from those in RAG-2 KO mice are indicated directly.

FIGURE 1.

IL-4R signaling, but not IL-4, is required for schistosome egg pathology. A, Hepatic granuloma sizes (mean ± SE) in 8-wk infected BALB/c wt (n = 26), IL-4 KO (n = 17), IL-4Rα KO (n = 14), IL-4/IL-4Rα KO (n = 10), and RAG-2 KO (n = 9) mice. B, Hepatic fibrosis as measured by hydroxyproline in the same animals. Values were normalized to the number of eggs per liver. C and D, Eosinophil and mast cell composition of granulomas measured in A. ∗, Mean values statistically different (p < 0.001) from those in both wt and IL-4KO mice; §, values in IL-4 KO mice statistically different (p < 0.001) from those in wt animals. Values statistically different from those in RAG-2 KO mice are indicated directly.

Close modal

Immunohistochemistry was performed on liver sections from 8-wk infected wt and KO animals to determine whether the decreased pathology in schistosome-infected IL-4Rα KO mice is due to defective migration of effector T cells into egg-induced lesions. The granulomas in IL-4Rα KO mice, although reduced in size, nevertheless contained large numbers of CD3+ lymphocytes, while no staining was observed in the equivalent lesions in RAG-2 KO mice (Fig. 2). Accurate quantitation of the number of CD3+ cells present was not feasible, although they appeared to be in higher density in granulomas from IL-4Rα KO livers.

FIGURE 2.

Normal infiltration of T cells in residual granulomas in infected IL-4Rα KO mice. Immunohistochemical staining of liver sections from wt (A), IL-4 KO (B), RAG-2 KO (C), and IL-4Rα KO (D) animals with anti-CD3 Ab.

FIGURE 2.

Normal infiltration of T cells in residual granulomas in infected IL-4Rα KO mice. Immunohistochemical staining of liver sections from wt (A), IL-4 KO (B), RAG-2 KO (C), and IL-4Rα KO (D) animals with anti-CD3 Ab.

Close modal

To confirm that the infiltrating T cells are functional, liver tissue from infected wt and KO animals was tested for expression of Th2 lymphokines by RT-PCR. Although IL-4, IL-5, and IL-13 liver mRNAs were diminished in IL-4Rα KO mice compared with those in wt animals, they were not significantly different from the levels in IL-4 KO mice, which display unimpaired egg pathology (Fig. 3). Since previous studies have determined that the CD8+ cells in S. mansoni- infected mice are Tc1 rather than Tc2 in phenotype (28), the presence of comparable levels of Th2 cytokine mRNAs in livers of IL-4Rα KO and IL-4 KO animals argue that the CD3+ cells within the residual lesions are likely to be CD4+ rather than CD8+ lymphocytes.

FIGURE 3.

Th2 lymphokine mRNA expression in livers of schistosome-infected wt, IL-4 KO, and IL-4Rα KO mice. Cytokine as well as hypoxanthine phosphoribosyltransferase (HPRT) mRNA levels were measured by RT-PCR and expressed as arbitrary OD units. The ratio (cytokine/HPRT) was then calculated for each mouse and the mean and SE for each group (n = 5–10 animals) determined. Uninfected mice gave values <0.1.

FIGURE 3.

Th2 lymphokine mRNA expression in livers of schistosome-infected wt, IL-4 KO, and IL-4Rα KO mice. Cytokine as well as hypoxanthine phosphoribosyltransferase (HPRT) mRNA levels were measured by RT-PCR and expressed as arbitrary OD units. The ratio (cytokine/HPRT) was then calculated for each mouse and the mean and SE for each group (n = 5–10 animals) determined. Uninfected mice gave values <0.1.

Close modal

To further characterize the T cell response in infected IL-4Rα KO mice, mesenteric LN cells were stimulated in vitro with SEA, and lymphokine secretion was assayed by ELISA. As previously shown (29), wt mice mount marginal IFN-γ responses to SEA at 8 wk postinfection. In contrast, simultaneously infected IL-4, IL-4Rα or IL-4/IL-4Rα KO animals display markedly increased and comparable IFN-γ levels (Fig. 4). Thus, the absence of either IL-4 and/or IL-4R signaling allows the development of Th1 responses normally suppressed in infected IL-4 intact mice.

FIGURE 4.

In vitro lymphokine responses of mesenteric LN cells from S. mansoni-infected wt, IL-4 KO, IL-4Rα KO, and IL-4/IL-4Rα KO mice in response to SEA. Mesenteric LN cells were stimulated with SEA for 72 h, and lymphokine levels were measured in supernatants by ELISA. Results shown are means of duplicate values (±SD) for one of five experiments performed.

FIGURE 4.

In vitro lymphokine responses of mesenteric LN cells from S. mansoni-infected wt, IL-4 KO, IL-4Rα KO, and IL-4/IL-4Rα KO mice in response to SEA. Mesenteric LN cells were stimulated with SEA for 72 h, and lymphokine levels were measured in supernatants by ELISA. Results shown are means of duplicate values (±SD) for one of five experiments performed.

Close modal

When Th2 cytokine production was assessed in the same animals, a more complex series of effects was observed. In agreement with previous studies (10, 11) IL-4 KO mice showed diminished, but significant, IL-5 and IL-10 production. In addition, IL-13 production was retained at substantial levels (Fig. 4). Infected IL-4Rα and IL-4/IL-4Rα KO mice showed nearly the same levels of IL-5, IL-10, and IL-13 as IL-4 KO animals, indicating that Th2 development is reduced, but not ablated, in the absence of either IL-4 or IL-4R signaling. In infected IL-4Rα KO mice, in which it was possible to assay the synthesis of IL-4, production of this Th2 lymphokine was also only partially diminished relative to that in wt animals (Fig. 4). Nevertheless, the lack of IL-4 consumption in vitro in the absence of IL-4Rα is likely to account for part of the cytokine accumulation observed. In vitro treatment with anti-CD4- or anti-CD8-specific mAb confirmed that the observed Th2 as well as Th1 cytokine production by mesenteric LN cells from IL-4Rα KO mice are primarily dependent on CD4+ cells (data not shown).

Isotype-specific anti-SEA Ab were also measured as a parameter of Th activity. Infected wt, IL-4 KO, and IL-4Rα KO mice displayed indistinguishable levels of total serum anti-SEA IgG (Fig. 5). However, titers of IgG2a and IgG2b Ab in IL-4 KO and IL-4Rα KO animals were significantly increased with respect to those in wt mice, consistent with the augmented Th1 cytokine production observed in the former animals. Titers of SEA-specific IgG1 Ab were only partially affected by IL-4/IL-4Rα deficiency (Fig. 5), and in some experiments no difference in IgG1 levels between wt and KO mice was detected. Nevertheless, total IgE was reduced to undetectable levels in both groups of KO animals (data not shown).

FIGURE 5.

Anti-SEA Ab levels in pooled sera of infected wt, IL-4 KO, and IL-4Rα KO mice. SEA-specific total IgG, IgG1, IgG2a, and IgG2b Ab were determined by ELISA. Sera from uninfected mice gave values <0.1. The experiment shown is representative of two performed.

FIGURE 5.

Anti-SEA Ab levels in pooled sera of infected wt, IL-4 KO, and IL-4Rα KO mice. SEA-specific total IgG, IgG1, IgG2a, and IgG2b Ab were determined by ELISA. Sera from uninfected mice gave values <0.1. The experiment shown is representative of two performed.

Close modal

While the schistosome egg granuloma has many features of a Th2 response, it has been difficult to reconcile the near normal development of granulomas and fibrosis in S. mansoni-infected IL-4 KO mice with a Th2 model of pathogenesis. We have now partially resolved this paradox by demonstrating that infected IL-4Rα KO animals show a near complete impairment of both granulomatous inflammation and fibrosis. Thus, while IL-4 itself is not essential for pathogenesis, signaling through IL-4R by another cytokine ligand is clearly required. IL-13, a Th2 cytokine structurally related to IL-4, is known to signal through the IL-4Rα/IL-13R complex and therefore is the likely mediator involved. The latter hypothesis is strongly supported by recent experiments in the i.v. egg injection model, in which pulmonary granuloma formation was shown to be partially inhibited in wt mice and ablated in IL-4 KO mice after in vivo administration of soluble IL-13R/Fc fusion protein (30). Our findings also extend previous observations demonstrating impaired hepatic pathology in schistosome-infected mice deficient in the Th2 signaling component STAT-6 (12) by implicating both the receptor and the cytokine involved.

A key question raised by the requirement for IL-4R triggering in egg pathology concerns the identity of the IL-4R+ effector cell necessary for granulomatous inflammation. The most simple explanation is that CD4+ lymphocytes or their helper functions are defective in infected IL-4Rα, but not in IL-4 KO mice. Since total IgG SEA-specific Ab levels are equivalent in wt, IL-4-deficient, and IL-4Rα-deficient animals, overall CD4-dependent helper function appears to be intact (Fig. 5). Nevertheless, at a more specific level it is conceivable that the CD4+ Th2 cells that mediate pathology are somehow dependent on IL-4R, but not on IL-4, for their generation. For example, one might postulate that although T cells lack IL-13R (31), IL-13 indirectly influences Th2 development in this model as proposed in another experimental system (32). However, it is clear that S. mansoni-infected IL-4 and IL-4Rα KO animals display indistinguishable in situ (Fig. 3) and in vitro (Fig. 4) lymphokine as well as qualitatively similar Ab isotype (Fig. 5) profiles and therefore do not appear to differ in their Th1/Th2 response patterns. Thus, the critical IL-4Rα+ effector cell is unlikely to be a CD4+ lymphocyte. Since CD8 cells do not appear to contribute to the observed cytokine production in either wt or IL-4Rα-deficient animals, and CD8-deficient mice show unimpaired acute granuloma formation (33), it is equally improbable that IL-4R+ CD8 cells are the target of the defect. Instead, the cell involved most probably is a non-T lymphocyte that receives the Th2 signal and responds by performing a critical, but as yet undefined, function in granuloma formation and fibrosis.

IL-4Rα is widely distributed on numerous cell types (34), including eosinophils and mast cells, which are normal constituents of schistosome egg granulomas. While both of these cell populations are reduced in the granulomas arising in IL-4Rα KO mice (Fig. 1), their loss is unlikely to be the underlying explanation of the ablated tissue pathology. Thus, previous studies have indicated that eosinophil-depleted animals develop egg granulomas that are nearly normal in size (35). Similarly, as illustrated by the data presented here (Fig. 1), the egg lesions developing in IL-4 KO mice are of comparable volume to those occurring in wt animals despite the notable absence of mast cells. Alternative candidates include macrophages, endothelial cells, and/or fibroblasts, all of which are IL-4Rα+ and, in contrast to T lymphocytes, are responsive to IL-13 as well as to IL-4 (36, 37, 38).

Taken together the results presented in this paper argue that egg pathology requires both a Th2 response (which occurs residually in IL-4 KO mice) as well as the IL-4Rα+ non-T cell population discussed above acting as a primary effector of the granulomatous response. The latter component of the disease process may offer a novel target for immunological intervention. On this note, it is of interest that mice vaccinated with IL-12 plus egg Ags develop reduced granulomatous pathology and display a Th1/Th2 profile closely resembling that seen in infected IL-4Rα KO animals, raising the possibility that the IL-12 immunization protocol functions by reducing IL-4Rα expression. While this mechanism has not been formally examined, a more likely explanation of the effects of the IL-12 vaccination procedure is the near complete suppression of the synthesis of the relevant IL-4R ligands, IL-4 and IL-13, observed (39).

Unexpectedly, S. mansoni-infected IL-4Rα KO mice produced significant levels of Th2 cytokines despite their inability to receive IL-4 differentiation signals. Nevertheless, these animals, like infected IL-4 KO mice, showed markedly enhanced Th1 development, indicating that, as expected, cross-regulation is impaired. The above observations argue for the existence of alternative mechanisms for generating Th2 responses independent of IL-4 signaling. Studies of the CD4+ T lymphocyte populations arising in schistosome-infected IL-4Rα as well as STAT-6-deficient mice are currently in progress to further investigate this issue.

We thank Drs. David Sacks and George Yap for their critical comments, Dr. Hugues Charest for assistance with RT-PCR assays, and Ricardo Dreyfuss for help with the photomicrographs.

1

This work was supported in part by grants from the Swedish Medical Research Council and the Swedish Institute (to M.C.K.).

4

Abbreviations used in this paper: KO, knockout; wt, wild type; SEA, soluble egg Ag; RAG, recombinase-activating gene.

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