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Filarial nematodes are arthropod-transmitted parasites of vertebrates, including humans. The human pathogens include Wuchereria bancrofti and Brugia malayi, both of which may cause elephantiasis, and Onchocerca volvulus, infection with which can lead to blindness. An unusual feature of filarial nematodes is that they actively secrete proteins which contain, in covalent association, phosphorylcholine (PC) (reviewed in reference 10). PC is a molecule which has been shown to interact with an as yet undefined receptor on the surface of B lymphocytes (1), and hence it is perhaps not surprising that a number of immunomodulatory properties have been attributed to it (reviewed in reference 9). This has resulted in PC being considered as a contributor to the ability of filarial nematodes to evade the host immune response, and indeed data have been produced revealing PC-containing molecules of the parasites to be able to interfere with lymphocyte activation (reviewed in reference 9). It has also been demonstrated that the PC component of filarial nematode products induces interleukin-10 (IL-10) production in B1 cells of BALB/c mice (21). IL-10 has been indirectly associated with the development of Th2-type immune responses due to its downregulatory effects on Th1 responses (2).

ES-62 is a secreted PC-containing glycoprotein of the rodent filarial nematode Acanthocheilonema viteae (11). Subcutaneous injection of this parasite product into BALB/c mice results in the generation of high levels of immunoglobulin G1 (IgG1) antibody, but virtually no IgG2a, directed against the non-PC epitopes of the molecule (8). Thus, ES-62 is a potent inducer of a Th2 polarized immune response (23), and hence in the present study, we have investigated whether the PC group of ES-62 is involved in promoting this.

ES-62 was purified from spent culture medium of adult A. viteae by ultrafiltration and fast protein liquid chromatography (8). PC-free ES-62 was produced by adding either 1-deoxymannojirimycin (dMM) (13) or hemicholinium-3 (HC-3) (14) to the culture medium as described previously. Groups of four 6- to 8-week-old female BALB/c mice were given a total of four subcutaneous injections of purified parasite material (3 µg per injection), each 1 week apart. Serum samples were collected at day 0 and day 35 and stored at 20°C until assayed by enzyme-linked immunosorbent assay (ELISA) (11).

It was found by ELISA that ES-62 lacking PC had no significant effect on the previously noted (8) IgG1 response, but unlike normal ES-62, was clearly able to induce a substantial IgG2a response (Fig. 1). This implicates a role for PC in blocking the IgG2a response. We thus investigated whether the addition of PC to a proteinfor convenience, we employed bovine serum albumin (BSA)would inhibit any IgG2a antibody response associated with it. PC-BSA was prepared according to the method of Pery and colleagues (22), and BSA subjected to the conjugation procedure, but in the absence of PC ("denatured" BSA), was used as a control. Injection of mice (5 µg per injection), collection of serum samples, and ELISA were undertaken as before, whereupon it was indeed found that although the IgG2a response to BSA was relatively weak, the presence of PC appeared to be inhibiting it (Fig. 2).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Comparison of IgG1 (A) and IgG2a (B) levels in BALB/c mice exposed to either ES-62 or ES-62 synthesized in the presence of dMM. Specific IgG2a titers were significantly higher in mice inoculated with ES-62 synthesized in the presence of dMM (P < 0.05; Mann-Whitney U test). Values represent the mean reciprocal end point dilution ± standard error (n = 4). The same result was obtained when HC-3 rather than dMM was employed to produce PC-free ES-62.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Analysis of IgG2a anti-BSA antibody titers in BALB/c mice exposed to either PC-BSA or BSA. Antibody titers are higher in mice exposed to BSA (P < 0.01; Mann-Whitney U test). Results are expressed as mean reciprocal end point dilutions ± standard error (n = 4).

It is well established that antibody class switching to IgG1 is promoted by IL-4 and that to IgG2a is promoted by gamma interferon (IFN-) (23). Consistent with this, we found by ELISAs, performed as described previously (3), that spleen cells recovered from the mice employed for the antibody studies involving the parasite molecule produced cytokines in response to ES-62 which mirror their antibody status. Thus, there was no statistically significant difference in production of IL-4 in the two "PC-free" groups relevant to the control, but a significant rise in IFN- levels (three- to fourfold) was observed. It is also known that the two cytokines may reciprocally block the activities of each other (23), but clearly the presence of IL-4 did not appear to be inhibiting the IgG2a response of the mice inoculated with PC-free ES-62. We investigated this further by measuring the response to normal ES-62 in IL-4^/ mice. These mice (BALB/c) were generated as described previously (16) and maintained in the University of Strathclyde. Although the IgG1 response was completely eliminated (result not shown), no increase in the specific IgG2a response was observed in these mice (Fig. 3A). Thus, the idea that IL-4 does not influence the IgG2a response was confirmed. We also investigated whether the blocking of the Th1 response could be due to the compensatory effect of IL-13. This cytokine shares many activities with IL-4 as a consequence of employing a common signalling pathway utilizing the IL-4 receptor alpha (IL-4R) subunit (4, 12). We observed however that IL-4R^/ mice behaved in an identical manner to IL-4^/ mice: the IgG1 response was absent (result not shown), but there was no significant increase in the specific IgG2a response (Fig. 3A). IL-4R^/ mice (BALB/c) were generated as described previously (19) and maintained at the University of Strathclyde.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   (A) Comparison of the development of specific IgG2a in IL-4+/+ (wild type), IL-4/, and IL-4R/ mice following 5 weeks of exposure to ES-62. Values represent the mean reciprocal end point dilution ± standard error (n = 4). (B) Comparison of the development of IgG2a in IL-10/ and IL-10+/+ mice following 5 weeks of exposure to ES-62. Specific IgG2a titers were significantly higher in IL-10/ mice (P < 0.025; Mann-Whitney U test). Values represent the mean reciprocal end point dilution ± standard error (n = 4).

It has previously been reported that the PC moiety of filarial nematode proteins can induce IL-10 production in B1 cells (21). IL-10 can downregulate production of IFN-, the cytokine necessary for antibody class switching to IgG2a in mice (2, 23). Thus, to determine whether PC was blocking production of IgG2a antibodies by targeting production of IL-10, the antibody response to normal ES-62 in IL-10^/ mice was investigated. IL-10^/ mice (BALB/c) were a kind gift of Robert Coffman, DNAX, and were maintained in the Department of Pathobiology, the School of Veterinary Medicine, University of Pennsylvania. When ES-62 was injected into these mice, an IgG2a response to the parasite molecule was generated (Fig. 3B). This result therefore implicates IL-10 as playing a role in determining the nature of the IgG subclass response to ES-62.

It is reasonable to speculate that secreted PC-containing molecules (PC-ES) might contribute to the patterns of immune responsiveness observed in humans infected with filarial nematodes. PC-ES are readily detectable in the bloodstream of people harboring Wuchereria bancrofti, particularly if they are microfilaremic (reviewed in reference 10). The latter individuals are also notable in that their peripheral blood mononuclear cells have been shown to be more able to secrete IL-10, either spontaneously or in response to parasite antigens (18). Although this has not been observed in all studies (6), it raises the possibility that PC on ES could be a contributor to the IL-10 production. Elevated spontaneous and parasite antigen-induced IL-10 production has also been detected in onchocerciasis patients relative to healthy subjects in areas of endemicity (17), although the PC-ES status of these individuals was not investigated. Elevated spontaneous levels of IL-10, by downregulating Th1 responses, could contribute to the Th2 phenotype often observed in the specific immune response of microfilaria-positive lymphatic filariasis and onchocerciasis patients (19, 20). Furthermore, there is some evidence that concurrent infection with filarial nematodes can diminish the immune response to unrelated antigens by a mechanism likely to involve IL-10 (5). A logical next step for us therefore will be to investigate whether ES-62 and PC in particular are able to modulate the immune response to other antigens associated with potentiating infections.