Modulation of B-cell proliferative response by a soluble extract of Nippostrongylus brasiliensis

Abstract

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.

FIG. 1

AWH inhibits B-cell proliferation in response to LPS stimulation. Purified splenic B cells (from BALB/c nude mice) were activated with LPS (5 μg/ml) alone (filled bar) or in the presence of various concentrations of AWH (open bars). After 72 h of incubation at 37°C, the cultures were pulsed for 18 h with [³H]TdR. Data shown are expressed as the mean dpm of triplicate wells ± the SD. The results are from a single experiment and are representative of 10 separate experiments (∗∗, P < 0.01; ∗∗∗, P < 0.001, NS, not significant; P > 0.05, one-way ANOVA).

FIG. 2

AWH has no inhibitory effect on the proliferative response of spleen cells to various T-cell activators. Spleen cells were activated with ConA (5 μg/ml) or anti-CD3 MAb (final dilution, 1:10) alone or in the presence of AWH. The cultures were pulsed for 18 h with [³H]TdR after initial 72 h of incubation. The data shown are expressed as the mean dpm of triplicate wells ± the SD. The results are from one experiment and are representative of five independent experiments (NS, not significant; P > 0.05, one-way ANOVA).

FIG. 3

Effect of AWH added after LPS stimulation of B cells. B cells were stimulated with LPS (5 mg/ml) 24, 48, or 72 h prior to the addition of AWH (either 40 or 8 μg/ml). Cells were incubated for 72 h from the initial time of LPS stimulation and then were pulsed with [³H]TdR for an additional 18 h. The data are expressed as the mean dpm of triplicate wells ± the SD. The results are from a single experiment and are representative of five separate experiments. White bars, no AWH; black bars, AWH (40 μg/ml); striped bars (8 μg/ml).

FIG. 4

Removal of macrophages from B cells reverses the inhibitory effect of AWH. B cells were activated with LPS (10 μg/ml) in a flask for 24 h. Adherent accessory cells were removed, and the remaining cells were then restimulated with LPS (5 μg/ml) in the presence or absence of AWH. After 72 h of incubation at 37°C, the cells were pulsed with [³H]TdR for an additional 18 h. The data are expressed as the mean dpm of triplicate wells ± the SD (∗, P < 0.05; NS, not significant; P > 0.05, one-way ANOVA). The results are from one experiment and are representative of five separate experiments. The control for the suppressive activity of AWH in this data set can only be the consistent results of AWH in all the other experiments reported here. The same batch of AWH used in these experiments was used in the experiment shown in this figure.

FIG. 5

Inhibitory activity of AWH is not reversed by blocking antibodies to IL-10 and TGF-β and inhibitors of prostaglandins, nitric oxide, and hydrogen peroxide. B cells were stimulated with LPS (5 μg/ml) and cultured in the presence or absence of different concentrations of AWH. The cultures in panel A received either 50 μg of anti-IL-10 MAb or 25 μg of anti-TGF-β MAb per ml. The cultures in panel B received either 10⁻⁶ or 10⁻⁷ M indomethacin (prostaglandins inhibitor). The cultures in panel C received 10⁻⁴ M L-NMMA (nitric oxide inhibitor). The cultures in panel D received 0, 10, 2, or 0.4 U of catalase (hydrogen peroxide inhibitor) per ml at the beginning of the culture. The effectiveness of these inhibitors was tested before use. The culture was incubated for 72 h at 37°C and then pulsed with [³H]TdR for an additional 18 h. The data are expressed as the mean dpm of triplicate wells ± the SD.

FIG. 6

Percent inhibition by AWH of B-cell stimulation by other agents. Purified B cells were stimulated with LPS (5 mg/ml), anti-CD40 MAb (0.5 mg/ml), PWM (5 μg/ml) or SAC (1:2,000 dilution) with or without AWH (40 μg/ml). After 72 h of incubation, the cultures were pulsed for 18 h with [³H]TdR. The data shown are expressed as the mean percent inhibition of proliferation ± the SD. The data shown are representative of four similar experiments.

FIG. 7

AWH inhibitory effect targets signaling pathway downstream of PKC. Purified B cells were activated with PMA (10 ng/ml) in combination with ionomycin (1 μg/ml) in the presence of different concentrations of AWH. After 72 h of incubation at 37°C, the cultures were pulsed for 18 h with [³H]TdR. The data are expressed as the mean dpm of triplicate wells ± the SD. The results are from one experiment and are representative of three separate experiments (∗, P < 0.05; ∗∗∗, P < 0.001; NS, not significant; P > 0.05, one-way ANOVA).

FIG. 8

Heat and proteolytic enzyme treatment reverses the inhibitory effect of AWH on LPS-induced B-cell proliferation. B cells were stimulated with LPS (5 μg/ml) and cultured in the presence of AWH (10 μg/ml) that had either been heat treated by boiling (100°C) for 5 to 10 min (A) or digested with the proteolytic enzyme trypsin (10 U/ml) bound to an insoluble agarose matrix (B). The culture was incubated for 72 h at 37°C and then pulsed with [³H]TdR for additional 18 h. The data are expressed as the mean dpm of triplicate wells ± the SD (∗∗, P < 0.01; ∗∗∗, P < 0.001; NS, not significant; P > 0.05, one-way ANOVA). The results are from a single experiment and are representative of five separate experiments.

FIG. 9

Inhibitory effect of N. brasiliensis extract is specific to the adult stage. B cells were activated with LPS (5 μg/ml) alone or in the presence of various concentrations of extract of the adult stage (AWH) or extract of the larvae stage (LWH). After 72 h of incubation at 37°C, the cultures were pulsed for 18 h with [³H]TdR. The data are expressed as the mean dpm of triplicate wells ± the SD (∗∗∗, P < 0.001; NS, not significant; P > 0.05, one-way ANOVA). The results are from a single experiment and are representative of three separate experiments.