Endogenous pro- and anti-inflammatory cytokines differentially regulate an in vivo humoral response to Streptococcus pneumoniae

Abstract

Proinflammatory cytokines play a critical role in innate host defense against extracellular bacteria. However, little is known regarding the effects of these cytokines on the adaptive humoral response. Mice injected with a neutralizing anti-tumor necrosis factor alpha (TNF-alpha) monoclonal antibody (MAb) at the time of primary immunization with intact Streptococcus pneumoniae (strain R36A) showed a substantial reduction in both the primary immunoglobulin G (IgG) response specific for the cell wall protein, pneumococcal surface protein A (PspA), as well as in the development of PspA-specific memory. In contrast, anti-TNF-alpha MAb injected only at the time of secondary immunization with R36A failed to alter the boosted anti-PspA response. TNF-alpha was required only within the first 48 to 72 h after primary immunization with R36A and was induced both by non-B and non-T cells and by lymphoid cells, within 2 to 6 h after immunization, with levels returning to normal by 24 h. Thus, the early innate release of TNF-alpha was critical for optimal stimulation of the subsequent adaptive humoral response to R36A. Additional proinflammatory (interleukin 1 [IL-1], IL-6, IL-12, and gamma interferon [IFN-gamma]) as well as anti-inflammatory (IL-4 and IL-10) cytokines were also transiently induced. Mice genetically deficient in IL-6, IFN-gamma, or IL-12 also showed a reduced IgG anti-PspA response of all IgG isotypes. In contrast, IL-4(-/-) and IL-10(-/-) mice immunized with R36A showed a significant elevation in the IgG anti-PspA response, except that there was decreased IgG1 in IL-4(-/-) mice. In this regard, a marked enhancement in the induction of proinflammatory cytokines was observed in the absence of IL-10, relative to controls. Ig isotype titers specific for the phosphorycholine determinant of C-polysaccharide were similarly regulated, but to a much more modest degree. These data suggest that proinflammatory and anti-inflammatory cytokines differentially regulate an in vivo protein- and polysaccharide-specific Ig response to an extracellular bacteria.

FIG. 1.

Endogenous TNF-α is a key positive regulator of the in vivo anti-PspA, anti-PsaA and, to a lesser extent, anti-PC response to R36A. Mice were injected i.p. with 0.5 mg of anti-TNF-α MAb (XT22) or the same amount of control rat IgG1 MAb (GL113) or saline. Twenty-four hours later mice were injected i.p. with R36A (2 × 10⁸ CFU/mouse). Sera were collected 1 week after primary immunization with R36A for determination of anti-PC titers and 2 weeks after immunization for determination of anti-PspA and anti-PsaA titers by ELISA. Values represent arithmetic means ± SEM of eight mice per group. *, P < 0.05. Data are representative of three similar experiments.

FIG. 2.

TNF-α is required for an optimal primary anti-PspA response and the development of PspA-specific memory, but not for elicitation of a secondary response to R36A. (A) Mice were injected i.p. with 0.5 mg of anti-TNF-α MAb or control MAb. Twenty-four hours later mice were injected i.p. with R36A (2 × 10⁸ CFU/mouse). Six weeks after R36A immunization, mice were boosted with R36A alone (2 × 10⁸ CFU/mouse). Sera were collected 2 weeks after primary immunization with R36A (primary) and 1 week after boosting with R36A (secondary) for determination of anti-PspA titers. Values represent arithmetic means ± SEM of eight mice per group. *, P < 0.05. (B) Mice were injected i.p. with R36A alone (2 × 10⁸ CFU/mouse). Two weeks later primed mice were injected i.p. with 0.5 mg of anti-TNF-α MAb and/or 0.5 mg of control MAb, followed 24 h later by boosting with R36A (10⁸ CFU/mouse). Values represent the arithmetic means ± SEM of eight mice per group. *, P < 0.05. One experiment was performed.

FIG. 3.

TNF-α is required within the first 48 to 72 h after R36A immunization for induction of an anti-PspA response. Mice were immunized i.p. with R36A (10⁸ CFU/mouse), and then distinct sets of mice were injected i.p. with 0.5 mg of either anti-TNF-α MAb or control MAb at different times (days 0 to 5). Sera were collected 2 weeks after primary immunization with R36A for the determination of anti-PspA titers. Values represent the arithmetic means ± SEM of five mice per group. *, P < 0.05. Data are representative of two similar experiments.

FIG. 4.

R36A induces a mixed cytokine response in vivo within 2 to 6 h. Real-time RT-PCR analysis of cytokine mRNA expression was performed on freshly harvested splenic non-B, -non-T cells and B + T cells by magnetic sorting at different time points after immunization with R36A (2 × 10⁸ CFU/mouse). Relative levels of cytokine-specific mRNA were standardized based on rRNA levels. Cytokine-specific mRNA levels in sorted spleen cells from unimmunized mice were arbitrarily assigned a value of 1. Data are representative of two similar experiments.

FIG. 5.

Proinflammatory cytokines stimulate the humoral response to R36A. IL-6^−/−, IL-12^−/−, IFN-γ^−/−, and control mice were immunized i.p with R36A (2 × 10⁸ CFU/mouse). Sera were collected on day 7 (primary anti-PC), day 14 (primary anti-PspA), and day 21 (secondary anti-PspA) after immunization. Values are expressed as the arithmetic means ± SEM of eight mice per group. *, P < 0.05. Data are representative of two similar experiments.

FIG. 6.

Role of R36A dose in induction of anti-PspA and anti-PC responses in IL-12^−/− mice. IL-12^−/− and control mice (five per group) were immunized i.p. with three different doses of R36A (10 × 10⁶, 50 × 10⁶, and 200 × 10⁶ CFU/mouse). Sera were collected on day 7 (anti-PC) and day 14 (anti-PspA) after immunization. Values represent the arithmetic means ± SEM. *, P < 0.05. One experiment was performed.

FIG. 7.

Role of anti-inflammatory cytokines in the humoral response to R36A . IL-4^−/−, IL-10^−/−, and control mice were immunized i.p. with R36A (2 × 10⁸ CFU/mouse). Sera were collected on day 7 (primary anti-PC), day 14 (primary anti-PspA), and day 21 after boosting with R36A (secondary anti-PspA). Values represent the arithmetic means ± SEM of seven mice per group. *, P < 0.05. Data are representative of two similar experiments.

FIG. 8.

Role of cytokines in Ig isotype response to S. pneumoniae.

FIG. 9.

Endogenous IL-10 suppresses proinflammatory cytokine induction in response to R36A. Spleen cells from wild-type and IL-10^−/− mice were cultured at 10⁷ cells/ml in the presence of 2 × 10⁸ CFU/ml of R36A for 24 h. In addition, wild-type spleen cells were cultured with anti-IL-10 MAb or control MAb (10 μg/ml). Concentrations of IL-6, IL-10, IL-12, IFN-γ, and TNF-α in the culture supernatants were measured by ELISA. The results are representative of two independent experiments and are shown as means ± SEM of triplicate wells. *, P < 0.05 in Student's t test.