Induction of gamma interferon and nitric oxide by truncated pneumolysin that lacks pore-forming activity

Abstract

Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is known to exert various effects on the host immune cells, including cytokine induction, in addition to its known cytolytic activity as a member of the thiol-activated cytolysins. It is of interest to determine whether cytolytic activity is involved in triggering the cytokine production. In this study, we constructed full-length recombinant PLY and noncytolytic truncated PLYs with C-terminal deletions to examine the response of spleen cells to these PLY preparations. When cytolytic activity was blocked by treatment with cholesterol, full-length PLY was capable of inducing gamma interferon (IFN-gamma) production. Truncated PLYs that originally exhibited no cytolytic activity were also active in IFN-gamma induction. Therefore, the IFN-gamma-inducing ability of PLY appeared to be independent of the cytolytic activity. Furthermore, IFN-gamma-inducing preparations were also capable of inducing nitric oxide synthase expression and nitric oxide (NO) production, and the addition of neutralizing antibody to IFN-gamma abolished the NO production. These results clearly demonstrated that PLY is capable of inducing IFN-gamma production in spleen cells by a mechanism different from pore formation and that the induced IFN-gamma stimulates NO production. These findings were discussed with reference to the contribution of PLY to the virulence of S. pneumoniae in vivo.

FIG. 1.

IFN-γ-inducing ability and cytotoxicity of full-length PLY with or without cholesterol treatment. To determine IFN-γ-inducing ability, normal spleen cells were stimulated for 24 h with PLY471 (full-length PLY) that had been treated with or without 10 μ g of cholesterol/ml and the level of IFN-γ in the culture supernatant was assayed by EIA. To determine cytotoxicity for spleen cells, cells were incubated with the same PLY471 preparations for 6 h, and the level of LDH released in the culture supernatant from the cells was measured. Each column represents the level of IFN-γ induced by PLY471 with (open column) or without (closed column) cholesterol, and each point connected by a line represents the cytotoxicity of cholesterol-treated (open squares) or nontreated (closed circles) PLY471. Data are shown as the mean of triplicate determinations ± the standard deviation (SD). The result shown here is the representative of three similar experiments.

FIG. 2.

IFN-γ-inducing ability and cytotoxicity of truncated PLYs with C-terminal deletions. Normal spleen cells were stimulated with PLY437 (A) and PLY426 (B). The levels of IFN-γ (column) and LDH release (points connected by a line) in the culture supernatant were measured after 24 and 6 h, respectively. Data are shown as the mean of triplicate determinations ± the SD. The result shown here is the representative of three similar experiments.

FIG. 3.

Effect of polymyxin B (PMB) on IFN-γ production induced by recombinant PLYs. Normal spleen cells were stimulated with recombinant PLYs or LPS for 24 h in the presence (open column) or absence (closed column) of 5 μ g of polymyxin B/ml. The culture supernatant was collected, and the IFN-γ was measured by EIA. Data are shown as the mean of triplicate determinations ± the SD. The same experiment was repeated twice, and a similar result was obtained.

FIG. 4.

Expression of iNOS mRNA in the spleen cells stimulated with recombinant PLYs. Normal spleen cells were stimulated for 24 h with PLY471 (25 nM), PLY437 (400 nM), or PLY426 (800 nM), at the concentration optimal for IFN-γ induction for each recombinant protein. Total RNA was extracted and subjected to RT-PCR. PCR products obtained by using specific primer sets for iNOS and β-actin were electrophoresed. The predicted sizes of PCR products for iNOS and β-actin are indicated. Bands in the left lane indicate the positions of DNA size markers. The same experiment was repeated twice, and a similar result was obtained.

FIG. 5.

NO production by spleen cells after stimulation with recombinant PLYs. Normal spleen cells were stimulated with recombinant PLYs for 48 h in the presence or absence of anti-mouse IFN-γ antibody. The culture supernatant was collected and nitrite was quantified by Griess reaction. Data are shown as the mean of triplicate determinations ± the SD. The result shown here is representative of three similar experiments.