Interleukin 10 (IL-10)-mediated Immunosuppression: MARCH-I INDUCTION REGULATES ANTIGEN PRESENTATION BY MACROPHAGES BUT NOT DENDRITIC CELLS

Abstract

Efficient immune responses require regulated antigen presentation to CD4 T cells. IL-10 inhibits the ability of dendritic cells (DCs) and macrophages to stimulate antigen-specific CD4 T cells; however, the mechanisms by which IL-10 suppresses antigen presentation remain poorly understood. We now report that IL-10 stimulates expression of the E3 ubiquitin ligase March-I in activated macrophages, thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells. By contrast, IL-10 does not stimulate March-I expression in DCs, does not suppress MHC-II or CD86 expression on either resting or activated DCs, and does not affect antigen presentation by activated DCs. IL-10 does, however, inhibit the process of DC activation itself, thereby reducing the efficiency of antigen presentation in a March-I-independent manner. Thus, IL-10 suppression of antigen presenting cell function in macrophages is March-I-dependent, whereas in DCs, suppression is March- I-independent.

Keywords: antigen presentation; dendritic cell; immunosuppression; interleukin; macrophage; major histocompatibility complex (MHC); ubiquitin ligase.

FIGURE 1.

IL-10 induces March-I transcription and suppresses MHC-II and CD86 protein expression on activated MΦ. A, MΦ were cultured for 1 day in the absence or presence of IFN-γ and then cultured in the absence or presence of IL-10 for an additional 18 h. CD11b⁺ F4/80⁺ MΦ were analyzed for cell surface expression of MHC-II, CD86, and CD40 by FACS analysis using the indicated antibodies: isotype controls (shaded), untreated (dotted line), IFN-γ and then medium alone (solid line), or IFN-γ and then IL-10 (dashed line). B, cells were cultured in the absence or presence of IFN-γ for 1 day, and then IL-10 (or PBS) was added for an additional 4 h of culture. mRNA expression of the indicated gene product was analyzed by quantitative RT-PCR. Data were normalized for the amount of GAPDH mRNA present in each sample and are shown relative to the expression of each gene product present in cells treated with IFN-γ alone. The data shown are the mean ± S.D. (error bars) from at least three independent experiments. C, MΦ were cultured in the absence or presence of IFN-γ for 1 day, and then IL-10 (or PBS) was added for an additional 4 h of culture. D, MΦ were activated using either LPS or IFN-γ for 1 day, and then IL-10 (or PBS) was added for an additional 4 h of culture. In C and D, mRNA expression of March-I was analyzed by quantitative RT-PCR, and March-I mRNA expression (normalized to GAPDH) was expressed as indicated. The data shown are the mean ± S.D. (error bars) from at least three independent experiments. *, p < 0.05; ns, non-significant differences.

FIGURE 2.

IL-10-mediated down-regulation of MHC-II and CD86 in activated MΦ is March-I-dependent. MΦ were generated from bone marrow cells (A and B) or from the peritoneal cavity (C and D) from either wild-type mice (A and C) or March-I KO mice (B and D). The cells were left untreated or were activated with IFN-γ for 1 day, washed, and recultured in the absence or presence of IL-10 for an additional 18 h. CD11b⁺ F4/80⁺ MΦ were analyzed for cell surface expression of MHC-II, CD86, and CD40 by FACS analysis using the indicated antibodies: isotype controls (shaded), untreated (dotted line), IFN-γ and then medium alone (solid line), or IFN-γ and then IL-10 (dashed line). The geometric geometric mean fluorescence intensity of the indicated protein was expressed relative to that in cells treated with IFN-γ alone. The data shown are the mean ± S.D. (error bars) from three independent experiments. *, p < 0.05; ns, non-significant differences.

FIGURE 3.

IL-10-mediated lysosomal accumulation of MHC-II in MΦ is March-I-dependent. MΦ were generated from bone marrow cells isolated from either wild-type mice or March-I KO mice. The cells were activated by culture for 1 day in the presence of IFN-γ and then cultured in the absence or presence of IL-10 for an additional 18 h. The cells were harvested and fixed/permeabilized for analysis by confocal immunofluorescence microscopy. A, cells were stained with mAb recognizing pMHC-II (green) and the late endosome/lysosome marker LAMP-1 (red). Confocal images of a representative single 0.8-μm-thick optical section taken in the midplane of the cells are shown. B, co-localization of MHC-II with intracellular LAMP-1 was scored by a blinded observer, and the percentage of all cells with significant amounts of lysosomal MHC-II was calculated. The number of cells analyzed in each condition is indicated. Statistical analysis of the data was performed using a Z-test. *, p < 0.05; ns, non-significant differences.

FIGURE 4.

IL-10-mediated suppression of macrophage APC function is March-I-dependent. MΦ generated from bone marrow cells from wild-type or March-I KO mice were activated by treatment with IFN-γ for 1 day in the presence of OVA(323–339) peptide. Activated MΦ were cultured alone (solid line) or with IL-10 (dashed line) for 18 h, washed, and incubated with CFSE-labeled OT-II CD4 T cells for 36 h at an APC:T cell ratio of 1:2. CD4 T cells were isolated from the co-culture after 36 h and cultured alone for an additional 5 days. CFSE dye dilution was measured by FACS analysis. Activated MΦ were unable to stimulate OT-II T cells if the MΦ were not exposed to OVA(323–339) peptide during the culture (shaded). Data shown are the CD4 T cell division index relative to that of activated MΦ cultured in the absence of IL-10. The mean ± S.D. (error bars) from three independent experiments is shown. *, p < 0.05; ns, non-significant differences.

FIGURE 5.

IL-10 does not affect MHC-II or CD86 expression in immature or mature DCs. BMDCs were cultured in medium alone (immature) or activated with LPS for 1 day (mature) and then cultured in the absence or presence of IL-10 for an additional 18 h. Expression of MHC-II, CD86, and CD40 was determined by FACS analysis using the indicated antibodies. Isotype control antibody staining is shown in the shaded histogram. A, immature DCs incubated in the absence (dotted line) or presence (dashed line) of IL-10. B, LPS-matured DCs incubated in the absence (solid line) or presence (dashed line) of IL-10. The geometric mean fluorescence intensity of the indicated protein was expressed relative to that in cells treated with either PBS alone (A) or LPS alone (B). The data shown are the mean ± S.D. (error bars) from three independent experiments. C, immature DCs were incubated with OVA(323–339) peptide for 8 h, washed, activated with LPS for 24 h, and then cultured in the absence or presence of IL-10 for an additional 18 h. The cells were then incubated with CFSE-labeled OT-II CD4 T cells for 72 h at the indicated APC:T cell ratio. CFSE dye dilution was measured by FACS analysis. CFSE-labeled T cells did not proliferate when DCs were not pulsed with OVA(323–339) peptide (shaded histogram). The extent of T cell activation in each condition is shown as the division index relative to that of LPS-treated DCs in the absence of IL-10 at a DC:T of 1:10. The data shown are the mean ± S.D. (error bars) from three independent experiments. ns, non-significant differences.

FIGURE 6.

IL-10 suppression of LPS-induced MHC-II, CD86, and CD40 expression is March-I independent in DCs. DCs generated from bone marrow cells from wild-type mice (A) or March-I KO mice (B) were cultured in medium alone (dotted line) or in medium containing LPS alone (solid line) or LPS and IL-10 together (dashed line). Expression of MHC-II, CD86, and CD40 was determined by FACS analysis using the indicated antibodies. Isotype control antibody staining is shown in the shaded histogram. The geometric mean fluorescence intensity of the indicated protein was expressed relative to that in cells treated with LPS alone. The data shown are the mean ± S.D. (error bars) from three independent experiments. The dashed line represents the baseline expression of each marker in untreated cells. *, p < 0.05. C, spleen DCs isolated from wild-type mice were incubated overnight on ice or at 37 °C in the absence or presence of IL-10 as indicated. Expression of CD86 and CD40 was determined by FACS analysis, and the mean fluorescence intensity of the indicated protein was expressed relative to that in cells incubated at 37 °C in medium alone. The data shown are the mean ± S.D. (error bars) from three independent experiments. *, p < 0.05. D, DCs generated from bone marrow cells from wild-type mice (upper panel) or March-I KO mice (lower panel) were cultured for 4 h in medium alone, with LPS alone, with LPS and IL-10 together, or with IL-10 alone. Expression of mRNA for the indicated DC activation marker was analyzed by quantitative RT-PCR. Data were normalized for the amount of GAPDH mRNA present in each sample and are shown relative to the expression of each gene product present in DCs treated with LPS alone. The data shown are the mean ± S.D. (error bars) from three independent experiments. *, p < 0.05.

FIGURE 7.

IL-10 suppresses activation-induced down-regulation of March-I expression, MHC-II ubiquitination, and activation-induced antigen presentation by DCs. A, bone marrow-derived MΦ were cultured in the absence or presence of IFN-γ for 1 day, and then IL-10 (or PBS) was added for an additional 4 h of culture. Bone marrow-derived DCs were cultured for 2 h in medium alone (None), with IL-10 alone (IL-10), with LPS in the absence or presence of IL-10 (Activating DCs), or with LPS overnight and then cultured for an additional 2 h in the absence or presence of IL-10 (Activated DCs). Spleen DCs were cultured for 1 h on ice or at 37 °C in the absence or presence of IL-10 as indicated. Expression of March-I mRNA was analyzed by quantitative RT-PCR. Data are shown as the 2^ΔCt (ΔCt = GAPDH Ct − March-I Ct) value for each condition. The data shown are the mean ± S.D. (error bars) from three independent experiments. *, p < 0.05; ns, not significant. B, bone marrow-derived DCs were cultured in medium alone, with LPS alone, with LPS and IL-10 together, or with IL-10 alone. After 4 h, cells were harvested and solubilized in Triton X-100 lysis buffer, and pMHC-II was immunoprecipitated using mAb Y3P. The immunoprecipitates were analyzed by immunoblotting with antibodies recognizing ubiquitin or total MHC-II β-chain. A representative anti-ubiquitin blot and total MHC-II β-chain blot are shown. The relative amount of pMHC-II ubiquitination in each condition is expressed as a percentage of that observed in BMDCs cultured in medium alone. The data shown are the mean ± S.D. obtained from three independent experiments. *, p < 0.05; ns, not significant. C and D, DCs generated from bone marrow cells from wild-type or March-I KO mice were incubated with OVA(323–339) peptide for 8 h, washed, and cultured for 18 h in medium alone (dotted line), with LPS alone (solid line), or with LPS and IL-10 together (dashed line). The cells were incubated with CFSE-labeled OT-II CD4 T cells for 72 h at DC:T cell ratios of 1:20 (C) or 1:40 (D). CFSE dye dilution was measured by FACS analysis. DCs were unable to stimulate OT-II T cells if the cells were not exposed to OVA(323–339) peptide during the culture (shaded histogram). Data shown are the CD4 T cell division index relative to that using DCs cultured with LPS alone at a DC:T of 1:20. The mean ± S.D. (error bars) from three independent experiments is shown. *, p < 0.05.