Role of interleukin-4 (IL-4) and IL-10 in serum immunoglobulin G antibody responses following mucosal or systemic reovirus infection

Abstract

Mucosal and parenteral immunizations elicit qualitatively distinct immune responses, and there is evidence that mucosal immunization can skew the balance of T helper 1 and T helper 2 responses. However, a clear picture of the effect of the route of infection on the balance of the T helper responses has not yet emerged. Our laboratory previously demonstrated that oral reovirus infection elicits specific serum immunoglobulin G2a (IgG2a), while parenteral reovirus infection elicits the mixed production of specific serum IgG2a and IgG1 in mice of the H-2(d) haplotype. Knowing that IgG2a production is indicative of a T helper 1 response and IgG1 production is indicative of a T helper 2 response, we hypothesized that the route of infection influences the development of T helper 1 and T helper 2 responses. Using quantitative reverse transcription-PCR, we found that mRNA for the T helper 1 cytokines gamma interferon and interleukin-12 (IL-12) were expressed in draining lymphoid tissues following both oral and parenteral infections. However, we observed that mRNA for the T helper 2 cytokine IL-10 was suppressed in the Peyer's patches and mesenteric lymph nodes and IL-4 mRNA was suppressed in the mesenteric lymph nodes compared to noninfected controls, following oral infection. Using recombinant cytokines and cytokine knockout mice, we confirmed that IL-4 plays a major role in mediating the route-of-infection-dependent differences in serum IgG subclass responses. Therefore, the route of infection needs to be taken into consideration when developing vaccines and adjuvant therapies.

FIG. 1.

Reovirus-specific IgG2a (closed bars) and IgG1 (open bars) serum antibody concentrations following mucosal or parenteral infections. BALB/c mice were infected with reovirus orally, nasally, or in the FP with 3 × 10⁶ PFU/mouse, and serum antibody responses were determined 10 days after infection. Bars indicate the standard errors among groups of three mice. The dashed line indicates the limit of detection. Data shown are from one of three similar experiments.

FIG. 2.

IL-12 (A and B) and IFN-γ (C) mRNA expression following oral or FP reovirus infections. Each dot in panels A and C represents an individual mouse at the represented time postinfection, with three to four mice per group. Values in panel B are the fold induction of mRNA expression relative to noninfected (NI) controls. Onefold induction in panel B indicates endogenous levels. The dashed line in panel C indicates the limit of detection. Because the control IFN-γ levels were below the limit of detection, fold change levels were not calculated for IFN-γ. Asterisks indicate a significant difference from NI controls as determined by one-way ANOVA and Dunnett's test (P < 0.05). The data are representative of two similar experiments.

FIG. 3.

IL-4 (A and B) and IL-10 (C and D) mRNA expression following oral or FP reovirus infection. Each dot in panels A and C represents an individual mouse at the represented time postinfection, with three to four mice per group. In panels B and D values are expressed as the fold induction of mRNA expression relative to noninfected (NI) controls. The dashed lines in panels B and D at 1-fold induction indicate endogenous levels. Asterisks indicate a significant difference from NI controls by one-way ANOVA and post-hoc tests (P < 0.05). The data are representative of two similar experiments.

FIG. 4.

Reduction of IgG2a/IgG1 ratios by rIL-4 treatment in orally infected mice. BALB/c mice were infected either orally or in the FP, and some orally infected mice received rIL-4 at 3 μg/dose (with or without 30 μg of anti-IL-4 MAb carrier/dose as described in the text) on day −1 and on day 2 of infection. The serum IgG subclass responses were determined by ELISA 10 days following infection. Data are represented as the ratio of the geometric means of IgG2a and IgG1 titers. Bars indicate the standard errors among groups of 5 to 11 mice from two independent experiments. Asterisks indicate significant differences from orally infected mice as determined by a one-way ANOVA and Dunn's post-hoc test (P < 0.05).

FIG. 5.

Increase in the IgG2a/IgG1 ratio in FP-infected IL-4^−/− mice. IL-4^−/− mice or BALB/c controls (IL-4^+/+) were infected either orally or in the FP with 10⁷ PFU of reovirus T1/L per mouse. Serum IgG subclass responses were determined by ELISA 10 days following infection. Data are represented as the ratio of the geometric mean titers of IgG2a and IgG1. Bars indicate the standard errors among groups of 4 to 10 mice from two independent experiments. The asterisk indicates a significant difference from FP-infected BALB/c control mice as determined by a one-way ANOVA and Dunn's post-hoc test (P < 0.05).

FIG. 6.

Enhancement of reovirus-specific IgA production by in vivo rIL-4 treatment. BALB/c mice (solid bars) or IL-4^−/− mice (hatched bars) were infected orally with 10⁷ PFU of reovirus T1/L per mouse. Groups of mice received rIL-4 at 3 μg/dose (with or without 30 μg of a carrier/dose as noted) on day −1 and on day 2 of infection. The fragment culture supernatants were collected, and IgA concentrations were determined by reovirus-specific ELISA. Bars indicate the standard errors among groups of 4 to 10 mice from two independent experiments. The asterisk indicates a significant difference from nontreated orally infected mice as determined by one-way ANOVA and Dunn's post-hoc test (P < 0.05).

FIG. 7.

Partial effect of IL-10 treatment on the IgG2a/IgG1 ratio in orally infected mice. BALB/c mice were infected either orally or in the FP with 3 × 10⁷ PFU of reovirus/mouse. Orally infected mice received rIL-10 at 100 ng/dose (or PBS as vehicle where noted) every 12 h for the first 5 days postinfection, and serum antibody responses were determined 10 days following infection. Bars indicate the standard errors among groups of four mice. Representative data are from one of three independent experiments.

FIG. 8.

Failure of anti-IL-10R treatment to alter the IgG2a/IgG1 ratios in FP-infected mice. BALB/c mice were infected either orally or in the FP. FP-infected mice received anti-IL-10R MAb at 1.0 mg/mouse (or rat IgG1 as isotype control [IC] where noted) the day before infection and 0.5 mg/mouse every other day for 10 days. Data are represented as the ratio of the geometric means of IgG2a and IgG1 titers. Bars indicate the standard errors among groups of six to eight mice from one of two independent experiments.

FIG. 9.

Reovirus serum IgG subclass ratio was not affected following mucosal or parenteral infections. BALB/c mice were infected with reovirus orally or in the FP with the indicated reovirus dose ranging from 10⁴ to 10⁹ PFU per mouse, and serum antibody responses were determined 10 days after infection. Bars indicate the standard errors among groups of four mice. Representative data are from two similar experiments.