IL-21-induced Bepsilon cell apoptosis mediated by natural killer T cells suppresses IgE responses - PubMed (original) (raw)

. 2006 Dec 25;203(13):2929-37.

doi: 10.1084/jem.20062206. Epub 2006 Dec 18.

Kumiko Magara-Koyanagi, Hiroshi Watarai, Yuko Nagata, Yasuyuki Ishii, Satoshi Kojo, Shigetoshi Horiguchi, Yoshitaka Okamoto, Toshinori Nakayama, Nobutaka Suzuki, Wen-Chen Yeh, Shizuo Akira, Hiroshi Kitamura, Osamu Ohara, Ken-ichiro Seino, Masaru Taniguchi

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IL-21-induced Bepsilon cell apoptosis mediated by natural killer T cells suppresses IgE responses

Michishige Harada et al. J Exp Med. 2006.

Abstract

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Valpha14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bepsilon cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2-modifying factor, resulting in Bepsilon cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell-dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.

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Figures

Figure 1.

Figure 1.

Requirement of Vα14 NKT cells in BCG-mediated IgE suppression. (A) FACS profiles of liver MNCs. The liver MNCs obtained 1 wk after the last immunization were stained with α-GalCer/CD1d tetramer and anti-TCRβ mAb. Three mice per each group were analyzed and representative data are shown. (B and C) Effects of BCG on antibody responses in WT and Vα14 NKT KO mice. Total and OVA-specific serum IgE (B), IgG1, and IgG2a (C) were assayed by ELISA. Five mice were used in each group. Values are expressed as mean ± SD. The asterisks (*) indicate that the amount of IgE was below the detection level for anti-OVA IgE (<31.2 U/ml), anti-OVA IgG1 (<0.002 U/ml), or anti-OVA IgG2a (<1.25 U/ml). N.S., not significant. All experiments were repeated three times with similar results.

Figure 2.

Figure 2.

Activation of DCs by BCG. IL-12 production (A) and NF-κB activation (B). (A) Intracellular staining of BM-DCs with anti–IL-12p40/p70 and anti-CD11c mAbs with or without in vitro BCG (50 μg/ml) treatment for 12 h. BCG-treated BM-DCs (10,000 cells) were analyzed by FACS, and the number in each panel indicates the percentage of total cells. (B) NF-κB activation. 2 × 105 BM-DCs were stimulated with or without 50 μg/ml BCG or 1 μM CpG in vitro. NF-κB activity was determined by EMSA. (C and D) No requirement of TLR2 and TLR4 in BCG-mediated IL-12 production. 2 × 105 BM-DCs derived from WT (C) or TLR2/4 double KO (D) mice were stimulated in vitro with or without 10 μg/ml LPS, 10 μg/ml PGN, or 150 μg/ml BCG for 48 h, and IL-12p70 levels were measured by ELISA. (E and F) Requirement of IRAK-4 for IL-12 production. 2 × 105 BM-DCs were assayed for IL-12p70 by ELISA after stimulation with 0, 50, or 150 μg/ml BCG or 1 μM CpG (E), and for IL-6 with 10 ng/ml TNF-α stimulation for 48 h (F). In C–F, values are expressed as mean ± SD of triplicate cultures. The asterisks (*) indicate that the levels were below the detection limits for IL-12p70 (<62.5 pg/ml) and IL-6 (<15.6 pg/ml). N.S., not significant. All experiments were repeated twice with similar results.

Figure 3.

Figure 3.

IL-21 expression. (A) Vα14 NKT cell–dependent IL-21 production. Liver MNCs were obtained after BCG injection (500 μg/mouse) and examined for IL-21 mRNA expression. (B) Identification of the source of IL-21. Vα14 NKT and conventional T cells were sorted from liver MNCs and examined for IL-21 mRNA expression. (C) Requirement of DCs for BCG-induced IL-21 expression by Vα14 NKT cells. Liver TCRβ+ cells were cultivated in the presence of 50 μg/ml BCG with (top) or without (bottom) BM-DCs for 24 h and analyzed for IL-21 mRNA expression. Liver TCRβ+ cells stimulated with 10 μg/ml anti-CD3 mAb were used as a positive (Pos.) control. (D) Requirement of IL-12– and CD1d-mediated signals for IL-21 mRNA expression upon BCG stimulation. An isotype control, anti–IL-12p40/p70, or anti-CD1d mAb (20 μg/ml) was added to the cultures of liver TCRβ+ cells and BM-DCs as described in C. All experiments were repeated twice with similar results.

Figure 4.

Figure 4.

IL-21–mediated Bɛ cell apoptosis. (A) RT-PCR analysis. Expression of IgE (Cɛ), IL-21R, and γc was investigated in naive B (left) and Bɛ (right) cells. (B) Suppression of IgE production in naive B cell cultures. Naive B cells and Vα14 NKT cells (105 each) were cocultured in the presence of sCD40L and IL-4. (C) Suppression of IgE production in the Bɛ cell culture. 105 Vα14 NKT cells were added to the Bɛ cell (105) cultures. In B and C, 20 μg/ml anti–IL-21 mAb or isotype control mAb was added at the same time as the Vα14 NKT cells. The concentration of total IgE was measured by ELISA in triplicate. Values are expressed as mean ± SD. N.S., not significant. The experiments were repeated three times with similar results. (D) IL-21–mediated Bɛ cell apoptosis. 2 × 105 Bɛ and Bγ cells were generated and then further cultured with or without 30 ng/ml IL-21 for 30 h. Annexin V staining was then performed. The numbers represent percentage of the gated cells. Annexin V+ cells among Bɛ and Bγ cells just before IL-21 treatment was 25.7 and 29.2%, respectively (not depicted). The experiments were repeated three times with similar results.

Figure 5.

Figure 5.

Bmf-mediated Bɛ cell apoptosis. (A) RT-PCR. RNA from Bɛ and Bγ cells was analyzed for its expression of the indicated genes by RT-PCR. Note that no significant differences in Bcl-2 and IL-21R expression between Bɛ and Bγ cells were observed. (B) Western blotting. Bɛ cells were stimulated with IL-21 at 37°C for 30 min, and their cell lysates (6 × 106) were subjected to immunoprecipitation with anti–Bcl-2 mAb and immunoblotting with anti-Bmf antibody (top) or anti–Bcl-2 mAb (bottom). All experiments were repeated three times with similar results.

Figure 6.

Figure 6.

IL-21 mRNA expression and IL-21–induced IgE suppression in humans. (A) IL-21 mRNA expression in PBMCs. 106 human PBMCs were stimulated with 100 ng/ml α-GalCer or 50 μg/ml BCG and examined for IL-21 expression by quantitative real-time PCR with Taqman probes. The data are representative of five donors. (B) IL-12 and CD1d are required for IL-21 expression. 106 PBMCs were stimulated in vitro with 50 μg/ml BCG in the presence of 10 μg/ml anti-CD1d and/or anti–IL-12p40/p70 mAb. Representative data from five donors are shown. (C) IL-21 mRNA expression in PBMCs. Healthy volunteers were inoculated intradermally with BCG (two drops of 26.7 mg/ml of BCG emulsion per person). In A–C, the data for IL-21 expression were normalized to 18S ribosomal RNA expression, and relative expression levels are shown. Statistical analysis was performed using a matched pairs t test in C. (D) Suppression of IgE production. Left, suppression of IgE production by IL-21. 2 × 105 human B cells were cultured with sCD40L and IL-4 in the presence of human IL-21 for 14 d. Right, suppression of IgE production by BCG-activated human PBMCs. 105 Bɛ cells were cocultured with 105 PBMCs, sCD40L, and IL-4 in the presence of 50 μg/ml BCG for 14 d. Total IgE was measured by ELISA. Values are expressed as mean ± SD of triplicate cultures. The asterisks (*) indicate that the IgE levels are below the detection limit for total IgE (<0.014 IU/ml). Data shown are representative of three donors. Results were expressed as a fold difference in human IL-21 gene expression relative to a control sample (vehicle) after being normalized with 18S ribosomal RNA expressions in each sample.

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