IL-18 stimulates IL-13-mediated IFN-gamma-sensitive host resistance in vivo

Abstract

IL-4 and IL-13 are up-regulated during in vivo responses to many nematode parasites, but increasing evidence suggests that increases in IL-13 can also occur independently of the IL-4-dominant Th2 response. Blocking B7 after Trichuris muris inoculation inhibits resistance and IL-4 elevations, instead resulting in an IFN-gamma-dominant response associated with susceptibility. However, blocking IFN-gamma under these conditions restores IL-13-dependent resistance. In this study, we examined the mechanism of IL-13 up-regulation and associated protection during this in vivo immune response. CD4+ T cells and DX5+ TCR- cells were identified as the major producers of IL-13, and the DX5+ TCR- cells were phenotyped as NK cells, since they expressed CD11b, IL-2Rbeta and Ly49C but not c-kit or Fc epsilonRI. NK cell-derived IL-13 elevations were T cell-dependent, as CD4+ T cell depletion blocked IL-13 production by mesenteric lymph node cells and induced susceptibility. IL-13 expression was increased independently of IL-12; however, blocking IL-18 function inhibited IL-13 production and increased susceptibility. These results indicate that CD4+ T cells and NK cells are the major sources of IL-13 during the in vivo Th1 response induced by B7 blockade and that under these conditions, IL-18 is specifically required for the in vivo up-regulation of IL-13 production and associated host protection.

Figure 1

CD4⁺ and DX5⁺ cells are the major producers of IL-13 following B7 blockade in T. muris-immunized mice. CTLA4-Ig or L6 (500 μg) was administered at days 0, 1 and 12 after oral inoculation of BALB/c mice (five mice/group) with T. muris (Tm) eggs. At day 21, CD4⁺, CD4⁻CD8⁺ and CD4⁻CD8⁻DX5⁺ cell populations were purified from pooled MLN cells using anti-CD4, anti-CD8 or anti-DX5 magnetic beads. IL-13, IL-4 and IFN-γ mRNA expression were detected by quantitative fluorogenic real-time PCR. Data were individually normalized to rRNA, and data for treatment groups are expressed relative to the uninfected controls. Similar results were obtained in two additional experiments.

Figure 2

NK cells are a major non-T cell source of IL-13. BALB/c mice were inoculated orally with T. muris (Tm) eggs and treated with CTLA4-Ig as previously described. At day 21 after inoculation (a) MLN and spleens were assayed for IL-13 gene expression as described in Fig. 1, and (b) spleen cells from CTLA4-Ig-treated infected mice were depleted of CD4⁺ T cells by magnetic bead cell sorting and then stained with PE-labeled anti-mouse TCRαβ and FITC-labeled anti-mouse DX5 mAb. DX5⁺TCRαβ⁺ cells (gate P1) and DX5⁺TCRαβ⁻ (gate P2) cells were purified by electronic cell sorting. (c) Isolated DX5⁺TCRαβ⁺ and DX5⁺TCRαβ⁻ cells were analyzed for IL-13 gene expression. RNA from total spleen cells in untreated mice was used as a control. (d) DX5⁺TCRαβ⁻ cells from spleens were analyzed using multi-color FACS staining. Single-color histograms represent the expression of c-kit, CD11b, IL-2Rβ, Ly49C and FcεRI on gated DX5⁺TCRαβ⁻ cells (solid line). Corresponding isotype controls are shown in gray. Asterisk indicates a statistically significant difference (*p<0.05) between the indicated groups.

Figure 3

IL-4-independent IL-13 up-regulation and the associated protection against T. muris after blockade of both B7 and IFN-γ is CD4⁺ cell-dependent. BALB/c mice were administered CTLA4-Ig or L6 after inoculation with T. muris (Tm) eggs as previously described. At days 0, 7 and 14, 1 mg anti-IFN-γ mAb or the isotype control (GL113) and anti-CD4 mAb were administered to specific treatment groups. At day 21, (a) susceptibility was determined by assessment of worm burden. (b) Serum IgE levels were determined by ELISA. The means and SE from five individual BALB/c mice are shown for each group (ND: not detectable). (c, d) At day 21, CD4⁺, CD4⁻CD8⁺ and CD4⁻CD8⁻NK⁺ cells were purified from pooled MLN cells. RNA was isolated from these cell populations. Expression of IL-13 (c) and IFN-γ (d) mRNA was detected by real-time PCR. Data were individually normalized to rRNA, and treatment groups are expressed relative to the uninfected control. Similar results were obtained in two independent experiments. Asterisks indicate a statistically significant difference (**p<0.01) between the indicated groups.

Figure 4

IL-13-mediated protection to T. muris after blockade of both B7 and IFN-γ is not dependent on IL-12. IL-12p40^–/– and WT BALB/c mice (five mice/group) were administered CTLA4-Ig or L6 after T. muris (Tm) inoculation. Anti-IFN-γ mAb was used to block IFN-γ function in vivo. At day 21 post-immunization, (a) worm burdens were assessed by counting total numbers of adult worms in the gut, and (b) MLN cells were collected and IFN-γ-producing cells detected by ELISPOT. The means and SE from five individual mice are shown for each group. Similar results were obtained in two additional experiments. Asterisks indicate a statistically significant difference (**p<0.01) between the indicated groups.

Figure 5

Increases in IL-13 are comparable in T. muris-inoculated IL-12p40^–/– and WT mice following B7 and IFN-γ blockade. IL-12p40^–/– and WT BALB/c mice (five mice/group) were immunized with T. muris (Tm) and treated with CTLA4-Ig or L6 and anti-IFN-γ or GL113 (isotype control). At day 21 post-inoculation, (a) MLN cells were restimulated with anti-CD3 (0.625 μg/mL) for 72 h and IL-13 supernatant levels detected by ELISA; (b, c) IL-13 and IL-4 gene expression were determined by quantitative fluorogenic real-time PCR; and (d) total serum IgE levels were determined by ELISA. The means and SE from five individual BALB/c mice are shown for each group. Similar results were obtained in two additional experiments. Asterisks indicate a statistically significant difference (*p<0.05, **p<0.01) between the indicated groups.

Figure 6

IL-18BP-Fc treatment abrogates IL-13 production and associated protection in T. muris-inoculated WT mice after B7 and IFN-γ blockade. BALB/c mice (five mice/group) were immunized with T. muris (Tm) and treated with CTLA4-Ig and anti-IFN-γ. IL- 18BP-Fc was administered weekly by i.v. injection to appropriate treatment groups. At day 21, (a) worm burden was determined. (b) MLN cells were restimulated with anti-CD3, and IL-13 production in the supernatants was determined by ELISA. (c, d) IL-13 and IL-4 mRNA expression were determined by real-time quantitative PCR. (e) Sera were collected from the mice and total serum IgE levels determined by ELISA. The means and SE from five individual BALB/c mice are shown for each group. Asterisks indicate a statistically significant difference (*p<0.05, **p<0.01) between the indicated groups. Similar results were obtained in two experiments.