Regulation and function of the interleukin 13 receptor alpha 2 during a T helper cell type 2-dominant immune response

Abstract

Highly polarized type 2 cytokine responses can be harmful and even lethal to the host if they are too vigorous or persist too long. Therefore, it is important to elucidate the mechanisms that down-regulate these reactions. Interleukin (IL)-13 has emerged as a central mediator of T helper cell (Th)2-dominant immune responses, exhibiting a diverse array of functional activities including regulation of airway hyperreactivity, resistance to nematode parasites, and tissue remodeling and fibrosis. Here, we show that IL-13 receptor (R)alpha2 is a critical down-regulatory factor of IL-13-mediated tissue fibrosis induced by the parasitic helminth Schistosoma mansoni. IL-13Ralpha2 expression was induced after the onset of the fibrotic response, IL-10, IL-13, and Stat6 dependent, and inhibited by the Th1-inducing adjuvant IL-12. Strikingly, schistosome-infected C57BL/6 and BALB/c IL-13Ralpha2-deficient mice showed a marked exacerbation in hepatic fibrosis, despite displaying no change in granuloma size, tissue eosinophilia, or mastocytosis. Fibrosis increased despite the fact that IL-13 levels decreased significantly in the liver and serum. Importantly, pathology was prevented when IL-13Ralpha2-deficient mice were treated with a soluble IL-13Ralpha2-Fc construct, formally demonstrating that their exacerbated fibrotic response was due to heightened IL-13 activity. Together, these studies illustrate the central role played by the IL-13Ralpha2 in the down-regulation of a chronic and pathogenic Th2-mediated immune response.

Figure 1.

Kinetics of hepatic fibrosis after infection with S. mansoni. Groups of C57BL/6, BALB/c, and C3H/HeN mice were infected with 25–30 S. mansoni cercariae. 5–10 animals per group were killed at the time points specified. Fibrosis was assessed by the amount of hydroxyproline in micromoles detected in the liver per worm pair. Each data point shows the average values per group ± SE.

Figure 2.

Kinetics of IL-4/IL-13 receptor mRNA expression in the liver after infection with S. mansoni. Groups of C57BL/6, BALB/c, and C3H/HeN mice were infected with 25–30 S. mansoni cercariae. 5–10 animals per group were killed at the time points specified. RNA was extracted from the liver of individual mice at different time points and RT-PCR was performed as described in Materials and Methods for IL-4Rα1 (A), IL-13Rα2 (B), γc chain (C), and IL-13Rα1 (D). Average densitometric units from 5 to 10 mice per time point are shown.

Figure 3.

IL-13Rα2 mRNA expression is regulated by the type 1/type 2 cytokine response. (A) All mice (IL-10/IL-12–deficient mice [10/12KO], IL-12–deficient mice [12KO], WT, IL-4–deficient mice [4KO], IL-10–deficient mice [10KO], and IL-4/IL-10–deficient mice [4/10KO]) were infected with 25–30 S. mansoni cercariae. 10 animals per group were killed 9 wk after infection. RNA was extracted from the liver and RT-PCR was performed for IL-13Rα2. Bars, average densitometric units ± SD; *, significant difference when compared with WT values as determined by Student's t test. P < 0.05. (B) C57BL/6 (WT) and IFN-γ–deficient (GKO) mice were sensitized with eggs and IL-12 (egg/IL-12) or not and infected with cercariae as described in Materials and Methods. On week 9, mice were killed and IL-13Rα2 mRNA levels were determined in the liver by real-time PCR. *, significant difference when compared with the respective non–IL-12–treated control group as determined by Student's t test. P < 0.05. (C) Naive C57BL/6 (WT) and IFN-γ–deficient (GKO) mice were injected intravenously with 5,000 schistosome eggs and treated with saline (filled bars) or IL-12 (open bars) as described in Materials and Methods. Mice were killed on days 0, 7, and 11 after challenge and total lung RNA was isolated for the detection of IL-13Rα2 mRNA by real-time PCR. *, significant difference when compared with the respective non–IL-12–treated control group as determined by Student's t test. P < 0.05.

Figure 4.

(A) WT (WT F2) and IL-13–deficient (IL-13KO) mice were infected with 25–30 S. mansoni cercariae and 10 animals per group were killed 9 wk after infection. RNA was extracted from the liver and RT-PCR was performed for IL-13Rα2. *, significant difference when compared with WT values as determined by Student's t test. P < 0.05. (B) Naive C57BL/6 mice were injected with a single 10-μg dose of rIL-13 and liver RNA was prepared at the various time points indicated to determine the level of IL-13Rα2 mRNA expression. The shaded bar denotes the background levels observed in unexposed mice. Average densitometric unit per group ± SD is shown at each time point (n = 5 mice/time point). (C) Immunoprecipitation/Western of sera from schistosome-infected and -uninfected mice. Mice (IL-4/IL-13–deficient mice [IL4/13 KO], Stat6-deficient [B6 Stat6KO], and WT) were infected with 25–30 S. mansoni cercariae and serum was collected 9 wk after infection. All sera were precipitated with biotinylated murine IL-13 and streptavidin agarose beads overnight. The sample was treated with PNGase F, resolved on 12% reducing gel, blotted to polyvinylidene difluoride, and then IL-13Rα2 detected with rabbit anti–mouse A25 and goat anti–rabbit horseradish peroxidase. Comp, competition during the immunoprecipitation step with unlabeled murine IL-13 (note, no IL-13Rα2 [A25] band); arrows, soluble and membrane A25 (IL-13Rα2) have two different mobilities.

Figure 4.

(A) WT (WT F2) and IL-13–deficient (IL-13KO) mice were infected with 25–30 S. mansoni cercariae and 10 animals per group were killed 9 wk after infection. RNA was extracted from the liver and RT-PCR was performed for IL-13Rα2. *, significant difference when compared with WT values as determined by Student's t test. P < 0.05. (B) Naive C57BL/6 mice were injected with a single 10-μg dose of rIL-13 and liver RNA was prepared at the various time points indicated to determine the level of IL-13Rα2 mRNA expression. The shaded bar denotes the background levels observed in unexposed mice. Average densitometric unit per group ± SD is shown at each time point (n = 5 mice/time point). (C) Immunoprecipitation/Western of sera from schistosome-infected and -uninfected mice. Mice (IL-4/IL-13–deficient mice [IL4/13 KO], Stat6-deficient [B6 Stat6KO], and WT) were infected with 25–30 S. mansoni cercariae and serum was collected 9 wk after infection. All sera were precipitated with biotinylated murine IL-13 and streptavidin agarose beads overnight. The sample was treated with PNGase F, resolved on 12% reducing gel, blotted to polyvinylidene difluoride, and then IL-13Rα2 detected with rabbit anti–mouse A25 and goat anti–rabbit horseradish peroxidase. Comp, competition during the immunoprecipitation step with unlabeled murine IL-13 (note, no IL-13Rα2 [A25] band); arrows, soluble and membrane A25 (IL-13Rα2) have two different mobilities.

Figure 5.

Localization of IL-4/IL-13 receptor expression in the livers of infected mice. Liver sections from 9-wk–infected BALB/c mice were fixed in paraformaldehyde and in situ hybridization was performed for IL-4Rα, IL-13Rα2, and IL-13Rα1 mRNA. Anti-sense (A, C, and E) and sense strand (B, D, and F) riboprobes were used in the hybridization reaction using serial tissue sections. The brown areas denote positive staining for each receptor. Each section contains at least one granuloma with a miracidium-containing egg at the center. ×20.

Figure 6.

IL-13Rα2 deficiency does not affect the size or cellular composition of granulomas. C57BL/6 and BALB/c WT and IL-13Rα2–deficient mice were infected with 25–30 cercariae. Granuloma size (A), tissue eosinophilia (B), and mast cell index (C) were assessed 9 wk after infection. Six to eight mice were included in each group and the values for individual animals are shown. Bars denote the means. No significant differences were noted.

Figure 7.

Hepatic fibrosis increases in the absence IL-13Rα2. (A) C57BL/6 and (B) BALB/c WT and IL-13Rα2–deficient mice were infected with 25–30 cercariae and hepatic fibrosis was assessed by hydroxyproline assay 9 wk after infection. Six to eight mice were included in each group and the values for individual animals are shown. Bars, the means; *, significant difference when compared with WT values as determined by ANOVA. P < 0.05.

Figure 8.

IL-4 and IL-13 mRNA expression decreases in IL-13Rα2–deficient mice. (A) WT C57BL/6 (open bars) and IL-13Rα2–deficient (filled bars) mice were infected with 25–30 cercariae. RT-PCR was performed for IL-4, IL-13, and IFN-γ on liver tissues as described in Materials and Methods. *, significant difference when compared with WT values as determined by Student's t test. P < 0.05. (B) RT-PCR for IL-13Rα1, IL-13Rα2, IL-4Rα, and γc chains was performed on the same tissues. Six to eight mice were killed per group and the average arbitrary densitometric units are shown ± SD. No significant differences were found between the WT and IL-13Rα2–deficient mice. Similar results were obtained with tissues from mice on the BALB/c background.

Figure 9.

SEA-specific IgG₁ titers decrease in IL-13Rα2–deficient mice. S. mansoni egg-specific antibody levels were measured in sera of infected WT and IL-13Rα2–deficient mice (C57BL/6) 9 wk after infection as described in Materials and Methods. Bars from bottom to top show the 10th, 25th, 50th, 75th, and 90th percentiles, respectively, of the tested samples. Single outliners are indicated as circles. Data were obtained from groups of mice containing 6–8 mice/group. *, significant difference when compared with WT values as determined by Student's t test. P < 0.05. Note that IgG₁ is the predominant egg-specific Ig isotype detected in serum.

Figure 10.

Exacerbated fibrotic response of IL-13Rα2–deficient mice is prevented by administration of sIL-13Rα2-Fc. WT BALB/c and IL-13Rα2–deficient mice were infected with 25–30 cercariae. Beginning on week 6, IL-13Rα2–deficient mice were treated with sIL-13Rα2-Fc or control Ig (200 μg every other day) for a total of 6 wk. All animals were killed on week 12 and fibrosis (A) and serum levels of IL-13 (B) were determined. The values shown are mean values ± SEM for WT (n = 10), IL-13Rα2 KO (n = 10), and sIL-13Rα2-Fc–treated IL-13Rα2 KO mice (n = 15). The P values, as determined by ANOVA, are shown in A. In B: *, significant difference when compared with the WT group as determined by Student's t test. P < 0.05.