RESUMO

We and others have previously shown that nematodes or nematode products can stimulate or inhibit the generation of lymphocyte responses, suggesting that nematodes exert diverse effects on the developing immune responses of their host. In this study we examined the immunomodulatory effect of a soluble extract of Nippostrongylus brasiliensis (adult worm homogenate [AWH]) on B-cell responsiveness. We found that the extract inhibited the proliferation of B cells to lipopolysaccharide (LPS) stimulation in a dose-dependent manner. This effect was specific to B cells, since the extract did not inhibit T-cell proliferation to concanavalin A or anti-CD3 stimulation. The data presented here confirm that the extract is not toxic to B cells. We present evidence that the active factor is proteinaceous in nature and that the inhibitory activity is restricted to the adult stage of Nb. The extract does not appear to interfere with early activation events since it can be added up to 48 h after LPS stimulation, and it inhibited responses to phorbol myristate acetate and ionomycin. Furthermore, the proliferation of B cells to other activators was also inhibited by AWH. This observation shows that the inhibitory activity of AWH is not restricted to LPS-mediated B-cell proliferation. We present evidence that, in the absence of accessory cells, the inhibitory effect of the extract was ablated. This observation shows that the activity of AWH is not mediated directly on B cells but is mediated via the production of negative signals from accessory cells (macrophages), which affect a downstream pathway required by all B-cell activators tested. These effects on B-cell and accessory cell function are likely to have a significant effect on the outcome of infections experienced concurrently.

Assuntos

RESUMO

Infection with the nematode parasite Nippostrongylus brasiliensis induces a pronounced type-2 T-cell response that is associated with marked polyclonal immunoglobulin E (IgE) and IgG1 production in mice. To examine the differential roles of the infection and products produced by nematodes, we investigated a soluble extract of N. brasiliensis for the ability to mediate this type-2 response. We found that the extract induced a marked increase in IgE and IgG1 levels, similar to that induced by the infection. The extract did not affect the level of IgG2a in serum, showing that the effect was specific to IgE and IgG1 (type-2-associated immunoglobulin) rather than inducing a nonspecific increase in all immunoglobulin isotypes. This response was also associated with increased interleukin-4 production in vitro. These results confirm that the extract, like infection, is a strong inducer of polyclonal type-2 responses and a reliable model for investigating the regulation of nematode-induced responses. The extract induced the production of IgG1 when added to in vitro cultures of lipopolysaccharide-stimulated B cells. This provides evidence for the induction of class switch. It did not induce upregulation of IgG1 in naive (unstimulated) B cells or expand B cells in in vitro cultures. Analysis of DNA from the spleens of mice treated with the extract by digestion-circularization PCR demonstrated a marked increase in the occurrence of gamma1 switch region gene recombination in the cells in vivo. These results provide strong evidence that soluble worm products are able to mediate the marked polyclonal gamma1/epsilon response and that infection is not required to mediate this response. Furthermore, these data provide evidence that the soluble nematode extract induces this effect by causing de novo class switch of B cells and not by an expansion of IgG1 B cells or an increase in antibody production by IgG1 plasma cells.

Assuntos

RESUMO

BACKGROUND: We have demonstrated that infection with Nippostrongylus brasiliensis (Nb), which induces strong type 2 responses, prolongs kidney allograft survival in rats. Here, we confirm that this effect is not species-specific and address immune modulation in allospecific T-cell responses mediated by nematode infection. METHODS: C57BL/6 mice were injected with Nb or phosphate-buffered saline. Four days later, mice were transplanted with BALB/c hearts and graft survival was assessed. In other experiments, Nb-infected mice were immunized with BALB/c spleen cells and allospecific T-cell responses were determined in vitro. RESULTS: In this study, we show that Nb prolongs cardiac allograft survival in mice. Further, spleen T cells from Nb-infected, allo-immunized mice exhibit reduced allospecific cytotoxic T-lymphocyte activity. In contrast, allospecific proliferation of T cells in the mixed lymphocyte reaction was not reduced by Nb, ruling out immunosuppression as the mechanism of Nb-induced allograft survival. Nb infection induced IL-4 and IL-6 and inhibited IFN-gamma production by T cells in response to allo-antigen. Furthermore, anti-IL-4 treatment reduced allospecific T-cell proliferation from Nb-infected but not control mice, indicating that type 2 allospecific T cells develop in the presence of Nb. We also double-stained T cells for CD8 and IL-4 and showed that Nb induces an 8-fold increase in Tc2 cell numbers. CONCLUSIONS: These results are consistent with a hypothesis that Nb mediates prolongation of allograft survival through induction of type 2 immunity, including the development of regulatory Tc2 cells, and subsequent inhibition of allospecific cytotoxic T-lymphocyte activity.

Assuntos

Assuntos

RESUMO

RNA has been isolated from adult Nippostrongylus brasiliensis (NB) homogenized in guanidium isothiocyanate. The mRNA has been translated in vitro utilizing a rabbit reticulocyte system and translated products immunoprecipitated with rabbit anti-adult NB antiserum and rat infection serum.

Assuntos

RESUMO

Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.

Assuntos

RESUMO

We have examined the biochemical and functional characteristics of mast cells grown in tissue culture from the mesenteric lymph node (MLN) of rats infected with the nematode Nippostrongylus brasiliensis and compared them with mast cells isolated from the small intestinal mucosa and peritoneal cavity of infected animals. Cultured mast cells (MC) and isolated intestinal mucosal mast cells (MMC) had a similar histamine content, and both contained type II protease (RMCP II) which was absent from peritoneal mast cells (PMC). PMC, MMC and cultured MC each responded to immunologically induced histamine secretion, but MMC and cultured MC were hyporesponsive to calcium ionophores and unresponsive to widely used PMC secretagogues including compound 48/80 and bee venom peptide 401. MMC and cultured MC also differed from PMC in their lack of responsiveness to the anti-allergic agent disodium cromoglycate. Thus, MC cultured from the MLN are distinct from PMC but have a biochemical and functional phenotype similar to that of intestinal MMC.

Assuntos

RESUMO

The HLA-A and HLA-B phenotypes of 329 unrelated Haitian adults were determined and gene and haplotype frequencies were estimated. A summary of the data is presented together with a brief comparison with American Black, American White, and African Black populations. HLA gene and haplotype frequencies of Haitians are quite similar to those for American Blacks. The frequency of Aw34 appears to be smaller in Haitians than in American and African Blacks. No statistically significant differences were found in the frequencies of B-locus antigens.

Assuntos