Significant quantities of PGE2 were produced by cercariae of Schistosoma mansoni following incubation with linoleic acid, a free fatty acid found on the surface of the skin. Cyclooxygenase (COX) 2 inhibitors failed to block this PGE2 production, suggesting that a different biochemical pathway may be involved in the production of PGE2 by the parasite. In addition, the parasites were also able to induce PGE2 and IL-10 from human and mouse keratinocytes. Analysis of mouse skin during skin migratory phases of infection confirmed these in vitro observations. COX2 inhibitors blocked the parasite-induced PGE2 and IL-10 from keratinocytes. Further analysis of the parasite secretions showed that the PGE2/IL-10-inducing effect was associated with a fraction <30 kDa molecular size. Addition of this fraction or parasite-stimulated keratinocyte culture supernatant to Con A-stimulated spleen cells resulted in the suppression of cell proliferation. This effect could be blocked by anti-IL-10 treatment. In sharp contrast, attenuation of the parasites with γ-irradiation significantly abrogated their ability to induce PGE2 or IL-10 from skin cells. Significance of IL-10 in host immunoregulation by skin stage schistosomula of S. mansoni was further confirmed by using IL-10-deficient mice. In these mice the normal subdued cutaneous reaction to the parasite was absent. Instead, a prominent cellular reaction occurred around the parasite, and there was considerable delay in parasitic migration through the skin. Thus these results suggest a key role for parasite-induced PGE2 in IL-10-dependent down-regulation of host immune responses in the skin.

Human Schistosomiasis mansoni infections are acquired through the skin (1). Larval stages of the parasite (schistosomula) after gaining entry into the body remain in the skin for about 48–72 h before migrating to the lungs (1, 2, 3). This stay in the skin potentially provides ample opportunity for the host immune system to mount an effective immune response against the migrating parasite. Yet the host fails to elicit any tissue response against the skin-residing schistosomula (3). Conversely, infection with γ-irradiation-attenuated parasites, which are known to confer significant immunity against this infection, stimulated a marked inflammatory response in the skin, resulting in delayed migration of the parasite through the skin (2, 3, 4). This delayed migration correlated with the development of IFN-γ-producing putative effector cells in the skin and draining lymph nodes (5, 6). Interestingly, a challenge infection with normal parasites down-regulated the IFN-γ response in the skin and draining lymph nodes (5, 7). This suggested that a normal infection could potentially hamper the generation of a protective immune response against the parasite in immune individuals. The mechanism of this down-regulation is poorly understood. Cytokine analysis in the skin and draining lymph nodes of mice infected with normal parasites showed predominantly a Th2-type cytokine response (5, 7). Specifically, mRNA for IL-10 and IL-4 were increased in the skin within 16 h after infection (5). It is baffling, especially in immunized animals, how the skin-stage schistosomula are able to induce an overbearing IL-10 response in a milieu predominated by IFN-γ-producing cells (4, 5, 6, 7). IL-10 has been implicated in the down-regulation of various immune responses against S. mansoni in both mice (8, 9) and humans (10, 11). Therefore, it is possible that this early IL-10 response in the skin and draining lymph nodes may play a pivotal role in parasite establishment by modulating the Th1-type responses (11, 12).

Previous studies showed that the infective stages of S. mansoni (cercariae) has the capacity to produce a wide range of eicosanoids, including the arachidonic acid metabolite, PGE2 (13, 14), which is a potent regulator of immune responses (15). Picomolar concentrations of PGE2 can up-regulate IL-10 production and down-regulate IL-12 production from various cell types including skin cells (16, 17). In turn, IL-10 can potentially influence the local and systemic immune responses by modulating the Th1 type cytokine production in the skin (17, 18, 19). PGE2 is also known to be produced by other helminth and protozoan parasites (20). However, the role of PGE2 in parasite-induced immunomodulation has not been fully understood. This study focuses on the role of parasite-induced PGE2 in the host immunoregulation via IL-10.

Collection of cercariae, in vitro transformation, and collection of secretory products from schistosomula were as described before (3). Biomphalaria glabrata snails infected with S. mansoni were obtained from Dr. Fred Lewis (Biomedical Research Institute, Rockville, MD). Normal or irradiated cercariae (exposed to 20 kR γ-irradiation) suspended in MEM (at a concentration of 10,000 cercariae/ml) were transformed into schistosomula by incubating them in sterile MEM for 3–5 h in linoleic acid (2 μg/ml)-coated culture flasks as described previously (21). These culture supernatants will contain both secretions produced at the time of transformation (transformation fluid) and material secreted during the 3- to 5-h incubation period. This preparation is called, collectively, the “secretory products of schistosomula”. Following incubation, the culture supernatant was collected, concentrated, and sterile filtered (0.2 μm; Costar, Cambridge, MA), and protein concentration was estimated using a micro BCA protein assay kit purchased from Pierce (Rockford, IL). In some studies cercariae were mechanically transformed into schistosomula in the absence of linoleic acid by a syringe passage (22). Briefly, cercariae were concentrated after shedding by centrifugation at 600 × g for 15 min at 4°C and resuspended in MEM (at a concentration of 10,000 cercariae/ml). Using a 5-ml syringe, the cercarial suspension was passaged 10–15 times. This procedure induces transformation of cercariae into schistosomula. The mechanically transformed schistosomula were then incubated for 3–5 h in the same medium, and the supernatant was collected, concentrated, and sterile filtered, and protein concentration was estimated as above. This preparation is called “secretory products of mechanically transformed schistosomula”. In some studies PGE2 present in the secretory products was affinity depleted by incubating with PGE2 affinity sorbent beads (mouse anti-PGE2 linked to Sepharose 4B; Cayman Chemicals, Ann Arbor, MI) per manufacturer recommendations and removing the beads by centrifugation (200 × g for 10 min). All culture supernatants were aliquoted and stored at −70°C until use. In some studies the secretory products depleted off PGE2 were separated into four fractions. Fraction I (>100 kDa), fraction II (>50 kDa), fraction III (>30 kDa), and fraction IV (<30 kDa) using Centriprep concentrators (Amicon, Beverly, MA) having membrane with respective m.w. cut-off.

Neonatal human keratinocytes (clone NHEK P131; Clonetics, San Diego, CA) were grown in keratinocyte growth medium (KG2M; Clonetics) and used in their second passage in all experiments. Mice epidermal keratinocytes were separated from C57BL/6 mouse ear skin as described previously (23). Briefly, ear skin was split in half. After removing the s.c. tissue, ears were floated on 0.05% trypsin (diphenyl carbamyl chloride-treated; Sigma, St. Louis, MO) and 160 μg/ml DNase I (Sigma) for 30 min at 37°C. Single cell suspensions were prepared by mechanical disruption, and keratinocytes were isolated by density gradient centrifugation (20 min for 400 × g) using Lympholite-M (Cederlane Laboratories, Ontario, Canada). Sedimented keratinocytes were washed and used in functional studies. Cells (∼5 × 104) were seeded into each well of a six-well plate coated with mouse type IV collagen (Life Technologies, Gaithersburg, MD) and allowed to adhere overnight. Cells were fed with fresh medium every other day until a 70–80% confluence was reached, and then were used in stimulation assays. Keratinocytes were stimulated with either 100 cercariae (normal or irradiated) or with 10 μg/ml of secretory products (from normal or irradiated cercariae) for 72 h at 37°C. At the end of the incubation period, culture supernatants were collected, aliquoted, and stored at −70°C for measuring PGE2 and IL-10. Control cultures were incubated with medium alone.

Levels of PGE2 present in the secretory products of linoleic acid or mechanically transformed schistosomula, culture supernatant of keratinocytes, homogenate of cercariae, and homogenate of adult worms were all quantitated using a competitive enzyme immunoassay kit (catalog no. DE0100; R&D Systems, Minneapolis, MN) per manufacturer instructions. This assay is based on the competitive binding of PGE2 present in the sample to a mouse mAb against PGE2 bound to goat anti-mouse Abs immobilized to a microtiter plate. A fixed amount of alkaline phosphatase-labeled PGE2 was used as the competitor. The intensity of color developed is inversely proportional to the concentration of PGE2 present in the sample. Media controls incubated in the presence of linoleic acid had no PGE2 (data not shown). Indomethacin (Sigma), aspirin (Sigma), or nimesulide (Sigma) was used as inhibitor of PGE2. In blocking studies, the samples were incubated with linoleic acid (0.2 μg/ml in PBS) in the presence or absence of respective inhibitors at three different concentrations (50, 25, and 12.5 μg/ml) for 3 h at 37°C. Following incubation, PGE2 levels in the supernatant were measured as described above. Wells with no linoleic acid served as negative controls. Levels of IL-10 in the culture supernatant of keratinocytes were quantitated using a sandwich ELISA kit purchased from Endogen (Woburn, MA) per manufacturer instructions.

Single cell suspension of spleen cells was prepared from naive C57BL/6 mice in complete RPMI medium containing 10% FCS. A total of 2 × 105 cells was plated in each well of a 96-well plate and stimulated with Con A (0.1 μg/ml; Sigma). Use of animals in this study was approved by the Biological Resources Committee of the University of Illinois at Rockford. Different concentrations of secretory products (1, 5, and 10 μg/ml), their fractions (14–20 μg/ml), or keratinocyte culture supernatant stimulated with normal cercariae (100 μl/well) was then added to these cultures to measure their effect on Con A-induced cell proliferation. After 72 h in culture, cells were pulsed with 1 μCi of [3H]thymidine (Amersham, Arlington Heights, IL) for an additional 18 h. Following incubation, cells were harvested (Nalge Nune, Naperville, IL) and incorporated [3H]thymidine was measured in a β counter (Beckman, Palo Alto, CA). In some experiments, monoclonal rat anti-mouse IL-10 Abs (10 μg/ml, azide free; Endogen) was added to the wells at the start of the culture to neutralize IL-10 in the cultures.

C57BL/6 mice were infected with 500 cercariae via the abdominal skin as described previously (3). On days 2 and 5 after infection, each mouse received 100 μg of monoclonal rat anti-mouse IL-10 (Endogen) or control rat IgG (Pierce) injected i.p. On day 5 after infection, mice were immunized with 2 × 107 BALB/c spleen cells injected s.c. into each flank. Six days later, that is, on day 11 after infection, thickness of hind footpads of each mouse was first measured using a plastic dial caliper (General Hardware, New York, NY) and then challenged with 107 BALB/c cells (in 50 μl) into each hind footpad. At 20–24 h after the challenge, footpad thickness was again measured and values were recorded.

C57BL/10J mice with an IL-10 gene-targeted mutation (Il10tm1Cgn) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/10J wild-type mice served as controls. Mice were infected with 150 normal cercariae of S. mansoni via the abdominal skin, and the skin sites were collected 72 h later for histological analysis as described earlier (3). Migration of schistosomula from the skin to the lungs was evaluated by a compressed organ autoradiography technique using [35S]methionine-labeled cercariae as described previously (3). Briefly, S. mansoni-infected snails were labeled with Tran35S-label (25 μCi of [35S]methionine/milliliter of water; sp. act. 1047 Ci/mmol, catalog no. 51006; ICN Pharmaceuticals, Irvine, CA) and cercariae released 72 h after labeling were collected, counted, and used in infections. To determine the presence of the parasite, skin and lungs were collected from each animal and pressed in a tissue press for 24 h. The pressed tissue was then exposed to x-ray film at −70°C for 5–7 days. Following autoradiographic development, the spots (which represent reduced silver foci on the film) were counted, and the percentage of worm establishment was calculated (number of spots/total number of cercariae used for infection × 100).

All the values are expressed as mean ± SD. Statistical difference between the different groups was determined by one way ANOVA using Student-Newman-Keuls method, with p < 0.05 considered significant.

Significant amounts of PGE2 were present in the secretory products of normal schistosomula of S. mansoni after exposure to linoleic acid-coated plates (Fig. 1). PGE2 was also present in the culture supernatant of irradiated schistosomula. However, on a comparative basis, the levels of PGE2 produced by normal schistosomula were 2.35-fold higher (p < 0.01) than those produced by irradiated schistosomula. In the absence of linoleic acid, no transformation of cercariae to schistosomula occurred and no PGE2 was detected in the culture supernatant. Cercariae were also mechanically transformed into schistosomula by syringe passage in the absence of linoleic acid, and PGE2 levels were measured in the culture supernatant. These studies showed that significant amounts of PGE2 are present in the secretory products of mechanically transformed schistosomula in the absence of linoleic acid (Fig. 1). However, the levels of PGE2 in these samples were significantly lower than those exposed to linoleic acid. Nearly identical levels of PGE2 were present in the secretory products of mechanically transformed normal or irradiated schistosomula. When secretory products of mechanically transformed normal schistosomula were added to linoleic acid-coated plates, significant amounts of PGE2 were present in the culture supernatant, suggesting that these secretions contain activities that can produce PGE2 from linoleic acid.

Although the levels were low, the presence of PGE2 in the secretory products of mechanically transformed schistosomula suggested that PGE2 may be constitutively produced in the tissues of the parasite. Therefore, we quantitated the amount, if any, of PGE2 present in different life cycle stages of the parasite after homogenizing the worm tissues. These studies showed that significant amounts of PGE2 were present in the sporocyst, cercaria, and schistosomula stages of the parasite (Fig. 2 a). On a per gram basis only negligible amounts of PGE2 were present in the tissues of the adult parasites.

Interestingly, when 100 μg of worm homogenate (sporocyst, cercariae, schistosomula, or adult) was incubated with linoleic acid, significant amounts of PGE2 was produced in the culture supernatant (Fig. 2 b), suggesting that these samples contain activities that can produce PGE2 from linoleic acid.

In mammalian system COX enzymes are involved in the production of PGE2 from arachidonic acid (24). Therefore, the above results suggested that worm homogenate may potentially contain COX-like enzymes. To test this, we added COX2 inhibitors such as indomethacin, aspirin, and nimesulide to parasite cultures before incubation with linoleic acid. These studies showed that addition of mammalian COX2 inhibitors had no effect on PGE2 production by normal cercariae/schistosomula (Fig. 3).

We measured PGE2 levels in the skin during the course of infection. These studies showed a significant increase in PGE2 levels in the skin initially following infection; thereafter, the levels dropped as parasites migrated out of the skin, especially 5 days after infection (Fig. 4). A similar analysis after infection with radiation-attenuated cercariae did not show an increase in PGE2 levels in the skin.

Keratinocytes constitute >90% of cells in the epidermal layer of skin where the parasite resides during the initial 2–3 days after infection (25). Among other factors, keratinocytes can produce PGE2 in response to specific stimuli (24). Therefore, we wanted to analyze whether cercariae or their secreted products can induce PGE2 production from human keratinocytes.

Addition of normal cercariae to human or mouse keratinocyte cultures resulted in a significant increase in PGE2 levels in culture supernatant within 24 h (Fig. 5,A). Within a few minutes after culture, the majority of the cercariae were transformed into schistosomula. However, a similar addition of irradiated cercariae to human keratinocyte cultures produced significantly lower amounts of PGE2 in the culture supernatant. Interestingly, there was a slower transformation of irradiated cercariae to schistosomula. Even after 24 h of culture, a substantial population of irradiated cercariae did not transform into schistosomula. Therefore, we repeated the above experiment with equal quantities (10 μg protein/ml) of secretory products depleted of PGE2 from either normal or irradiated parasites (Fig. 5,A). These studies confirmed that normal cercariae (or their secretory products) are potent inducers of PGE2 from human keratinocytes compared with irradiated parasites (or their secretory products). Addition of the COX2 inhibitor indomethacin to the keratinocyte cultures blocked the secretory product-induced PGE2 secretion from human keratinocytes (Fig. 5 A).

The PGE2-inducing activity in the secretory products of normal schistosomula were then narrowed down by separating the secretory products into four different fractions and testing each fraction for its ability to induce PGE2 in human keratinocytes. These studies showed that only the fraction below 30 kDa (fraction IV) retained the ability to induce PGE2 from human keratinocytes (Fig. 5 B).

Significant amounts of IL-10 were present in the culture supernatant of human keratinocytes incubated with normal cercariae or their fraction IV secretory product, whereas incubation of cells with irradiated cercariae or their fraction IV secretory product induced only low levels of IL-10 (Fig. 6). Addition of the COX2 inhibitor indomethacin to these keratinocyte cultures blocked the secretory product-induced IL-10 production by human keratinocytes (Fig. 6). Similar results were obtained when mouse keratinocytes were used (data not shown).

Migration of schistosomula from skin to the lungs was traced in IL-10-deficient C57BL/10 mice using 35S-labeled cercariae. These studies showed that there was a delayed migration of schistosomula from the skin to the lungs of IL-10-deficient mice compared with wild-type mice (Table I). Histological analysis of the skin showed massive accumulation of inflammatory cells around the parasite in the skin of IL-10-deficient mice, whereas fewer inflammatory cells were seen in the skin of wild-type mice (Fig. 7).

Addition of secretory products from normal schistosomula to Con A-stimulated spleen cells resulted in a significant dose-dependent inhibition of cell proliferation (Table II). This activity appeared to be associated with fraction IV of the secretory product. The secretory product-induced inhibition could be reversed by adding neutralizing Abs against murine IL-10 to these cultures. In the above studies, we showed that normal parasites induce IL-10 production from keratinocytes. Therefore, we cocultured mouse keratinocytes with normal cercariae and added the culture supernatant to spleen cell cultures. We observed a similar inhibition of cell proliferation as with the secretory products (Table II). This inhibition could be reversed with a neutralizing Abs against murine IL-10 (Table II).

C57BL/6 mice sensitized and challenged with BALB/c spleen cells showed a significant swelling of the foot pad (DTH response) compared with media controls (Fig. 8). However, when concurrent infection with S. mansoni was present at the time of sensitization, there was a significant reduction in the DTH response (Fig. 8). Injection of neutralizing anti-IL-10 Abs (100 μg/mouse) on days 2 and 5 postinfection reversed the parasite-induced suppression of DTH response (Fig. 8). Injection of a control IgG had no effect on the parasite-induced DTH suppression. When the above experiment was repeated in IL-10-deficient mice, S. mansoni infection had no suppressive effect on DTH reaction (Fig. 8).

Results from this study suggest a plausible role for PGE2- and/or PGE2-dependent IL-10 in the host immunoregulation by skin stage schistosomula of S. mansoni. Larval stages of the parasite S. mansoni are notorious for evading host cellular immune responses in their natural hosts (26, 27). Schistosomula of S. mansoni can down-regulate IFN-γ response and produce an IL-4 and IL-10 response in the skin and draining lymph nodes soon after entry into the host (5, 7). This transient switch in the cytokine pattern from Th1 to a more Th2 type response initially after infection is believed to be an evasive mechanism by the parasite to establish in the host. In this study, we show that the parasite may achieve this by inducing large quantities of PGE2 in the microenvironment, which in turn may be responsible for the predominant IL-10 response.

Previous studies showed that polyunsaturated essential free fatty acids, such as linoleic acid, present on the surface of the skin can act as potent penetration stimuli for the cercariae of S. mansoni (28). Upon exposure to free fatty acids such as 3.0 mM of linoleate, the cercariae transform into schistosomula, and detectable levels of PGE1, PGE2, 5-HETE, and 15-HETE appear in the culture medium (13, 14). This suggested that the skin stage schistosomula of S. mansoni have the ability to synthesize eicosanoids from free fatty acids (29). However, because only a small amount of PGE2 is produced by the parasite, the biological significance of parasite-produced PGE2 may be trivial compared with the substantial amounts of PGE2 induced in the host cells. Nevertheless, significant amounts of PGE2 were present in the culture supernatant of mechanically transformed schistosomula in the absence of any free fatty acids (Fig. 1), suggesting that the parasites can constitutively produce PGE2 in their tissue. Among the various life cycle stages, the tissue migratory stages of the parasite appeared to produce substantial amounts of PGE2.

In the mammalian tissue, biosynthesis of PGE2 is a complex multistep pathway mediated by COX enzymes. There are two COX isoenzymes, COX1 and COX2. COX1 is constitutively expressed in mammalian cells, whereas COX2 is inducible (24). In the presence of COX2, arachidonic acid is metabolized to PGE2 (24). Because PGE2 is produced by several larval stages of S. mansoni (Fig. 2), it is apparent that these parasites may possess enzymes similar to mammalian COX2. However, an immunoblot analysis using a polyclonal Ab against mammalian COX2 failed to detect the presence of COX2 in the parasite tissue or in their secretions (data not shown). This finding was further confirmed by the fact that COX2 inhibitors such as indomethacin, aspirin, and nimesulide failed to block PGE2 production by cercariae or schistosomula. In addition, we also searched the schistosome expressed sequence tag database and found no DNA or amino acid sequence homology to human or mouse COX2 (data not shown). Based on these observations, we hypothesize that S. mansoni lack COX2 enzyme and may possess biochemical pathways different from their mammalian counterpart for their production of PGE2.

Our studies show that in addition to producing substantial quantities of PGE2 from free fatty acids, schistosomula of S. mansoni also has the ability to induce PGE2 production in human keratinocytes. Although the molecular mechanism of this PGE2 induction from host cells is not fully understood at this time, our findings tentatively suggest that the parasite secretory products contain a factor that can potentially induce PGE2 production from human keratinocytes. Preliminary characterization studies suggest that the size of this factor may be <30 kDa and is not PGE2. Further studies are in progress to characterize this molecule. Velupillai and Harn (30) reported the presence of an oligosaccharide Ag (LNFP-III) in the egg of S. mansoni that can induce IL-10 and PGE2 from spleen cells. At this time we do not know whether the factor that we observed in the secretions of schistosomula is similar to LNFP-III.

Thus the skin-residing schistosomulum has the potential to generate substantial quantities of PGE2 around their local milieu (29). This notion was confirmed when we analyzed the PGE2 levels in the skin. A significant correlation was seen between the presence of the parasite in the skin and elevated PGE2 levels. As parasites migrated out of the skin, the levels of PGE2 also substantially decreased, suggesting that the majority of the PGE2 produced in the skin is in response to the parasite. Among other various functions, PGE2 is a potent vasodilator (24). Such a vasodilatation in the skin may help easy passage of the parasite into the circulation (29). Thus, production and/or induction of PGE2 in the skin may be a devious adaptive mechanism developed by the parasite for its survival in the host.

After entry into the host, schistosomula remain in the skin for >3 days before migrating further into the lungs. Analysis of the cytokine response during this skin migratory phase of the parasite shows a significant increase in IL-10 within a few hours after entry of the parasite into the skin (5). Previously we reported that T cells are not a source for this early IL-10 in the skin (5). These studies suggest that the majority of the parasite-induced IL-10 in the skin may be produced by keratinocytes (Fig. 6). It is well established that keratinocytes are a major source of IL-10 in the epidermis (16). Using COX2 blockers we show that this parasite-induced IL-10 by keratinocytes is PGE2 dependent. Previous studies have also shown that PGE2 is a potent stimulator of IL-10 from a variety of cells including keratinocytes (17, 18, 19). Thus our finding potentially revealed an interesting pathway by which the parasites may be inducing a strong IL-10 response in the skin.

Previously we showed that the host mounts very little inflammatory response against the skin-residing parasite, despite the fact that the parasite stays in the skin for >72 h (3). IL-10 and IL-1ra are potent anti-inflammatory molecules that play a key role in orchestrating inflammatory reaction in the skin by limiting the production of a large spectrum of proinflammatory cytokines and mediators (18, 19). Cytokine analysis in the skin following infection with S. mansoni showed a significant reduction in the levels of IL-1α and IL-1β, whereas the levels of IL-1ra were substantially increased (21). IL-10 has been shown to inhibit IL-1α and IL-1β (18, 19) and to up-regulate production of IL-1ra (31). Therefore, PGE2-driven IL-10 production in the skin following infection may play a significant role in the immunomodulation of inflammatory responses in the skin of infected animals. These findings are similar to those reported by Hoffmann et al. (32), who state that a major consequence of schistosome-induced IL-10 production is to down-regulate the immune responses of the host against the parasite. However, this subdued inflammation is seen only so long as live parasites are present in the skin (3), suggesting that the PGE2/IL-10-inducing activity is present only in the secretions of the tissue-migrating parasites.

Previous studies showed that secretions of schistosomula contain an activity that inhibited lymphoproliferation (33). In this study we show that this inhibitory activity is potentially mediated by IL-10 induced in the host cell via a PGE2-dependent mechanism. Because parasite-stimulated keratinocyte supernatant can induce suppression of lymphoproliferation, it is possible that a similar mechanism may be operative in vivo in the skin of infected animals. Such a down-regulatory mechanism may be responsible for the parasite-induced suppression of IFN-γ-producing T cells in the skin (5) and draining lymph nodes (7). A role for IL-10 in the suppression of Th1 cytokine synthesis in murine and human schistosomiasis has already been reported previously (8, 9, 10, 11). Our studies underscore the significance of parasite-induced PGE2 in these IL-10-mediated mechanisms.

Parasite-induced suppression of DTH reaction has been reported previously in murine schistosomiasis (34), and a role for IL-10 in this down modulation has been well documented (35). In this study we show that this IL-10-induced DTH down modulation may be mediated by parasite-induced PGE2, especially during the acute phase of infection (Fig. 8). This finding may be critical for a realistic goal of an antischistosome vaccine using candidate Ags, cautioning that the skin stage parasite can still modulate the immune responses in the host, irrespective of whether the host is immune or not.

Interestingly, exposure to γ-irradiation appears to interfere with the PGE2 production by the parasite and its ability to induce PGE2 production from human keratinocytes (Fig. 1). Because immunization with γ-irradiation-attenuated parasites confers significant immunity against challenge infection (4), it is possible that the initial PGE2 production by the normal parasite is a survival mechanism to down-regulate host inflammatory and immune responses. Cytokine analysis of the skin following infection with radiation-attenuated parasites showed a predominately IFN-γ response, and there was very little IL-10 response in the skin (5). Because the PGE2/IL-10-inducing factor appears to be absent in the secretions of irradiated parasites, it is possible that the harmful effects of irradiation could have damaged the ability of the parasite to produce this host immunomodulatory factor (36). A severe inflammatory reaction surrounds the γ-irradiation-attenuated parasites in the skin and potentially allows immune cells to come in close contact with the parasites (3, 4). This initial host response is believed to be critical for the generation of a protective immune response against this parasite (5, 6, 7).

A pivotal role for IL-10 in the parasite-induced immunoregulation in the skin was further reiterated by our studies using IL-10-deficient mice. Parasite-induced immunosuppression was less prominent in these mice, and the cutaneous responses resembled those in immune mice vaccinated with irradiated cercariae (3). Therefore, these findings complement those reported by Hoffmann et al. (32), who show vigorous expansion of Ag-specific cytokine-producing effector cells in IL-10-deficient mice in response to parasite Ags. In addition, we observed a severe inflammatory reaction around the parasite in the skin that potentially delayed the migration of the parasite through the skin (Fig. 7 and Table I). Similar results were reported by Hoffman et al. (32), who show increases in perivascular inflammatory responses in the lungs of IL-10-deficient mice following infection with S. mansoni.

Our results support the notion that a major consequence of schistosome-induced IL-10 production is to down-regulate host immune responses against the parasite, especially during the early phase of infection. Furthermore, our studies suggest a central role for parasite-induced PGE2 in the IL-10-dependent down-regulation. The study also suggests the presence of a low m.w. PGE2/IL-10-inducing factor in the secretions of normal parasites. Neutralizing the effect of this factor and subverting the initial outburst of PGE2 production in the skin by the parasite may contribute to decreased IL-10 responses, which in turn might prove an effective approach to increase the efficacy of some of the defined vaccine Ags against schistosomiasis.

1

This work was supported by Grant AI39066 from the National Institutes of Health and College Research Board, University of Illinois. Schistosome life cycle stages for this work were supplied through National Institutes of Health-National Institute of Allergy and Infectious Diseases Contract N01-A1-55270.

3

Abbreviations used in this paper: DTH, delayed-type hypersensitivity; COX, cyclooxygenase.

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