Prevention of arthritis by interleukin 10-producing B cells

Abstract

In this study we have shown that activation of arthritogenic splenocytes with antigen and agonistic anti-CD40 gives raise to a B cell population that produce high levels of interleukin (IL)-10 and low levels of interferon (IFN)-gamma. Transfer of these B cells into DBA/1-TcR-beta-Tg mice, immunized with bovine collagen (CII) emulsified in complete Freund's adjuvant inhibited T helper type 1 differentiation, prevented arthritis development, and was also effective in ameliorating established disease. IL-10 is essential for the regulatory function of this subset of B cells, as the B cells population isolated from IL-10 knockout mice failed to mediate this protective function. Furthermore, B cells isolated from arthritogenic splenocytes treated in vitro with anti-IL-10/anti-IL-10R were unable to protect recipient mice from developing arthritis. Our results suggest a new role of a subset of B cells in controlling T cell differentiation and autoimmune disorders.

Figure 1.

Transfer of anti-CD40–stimulated splenocytes inhibits arthritis in DBA/1-TcR-β Tg mice. Spleen cells isolated from mice with established arthritis were cultured in vitro for 48 h with CII/isotype control or CII/anti-CD40. 5 × 10⁶ cells were transferred to DBA/1-TcR-β Tg mice on the day of CII/CFA immunization. A group of mice was left untreated. (a) Incidence of arthritis; groups of mice were compared by statistical analysis using the Fisher exact test. (b) Arthritis severity; data are expressed as mean ± SE. Groups of mice were compared by statistical analysis using the nonparametric Mann-Whitney U test. Data are representative of three experiments.

Figure 2.

Are B cells the key players in the antiinflammatory effect mediated by in vitro stimulation with agonistic anti-CD40? B cells or DCs were depleted by positive selection from splenocytes isolated from arthritic DBA/1-TcR-β Tg mice. Undepleted or depleted splenocytes were stimulated with CII/isotype control, CII/anti-CD40. After 48 h incubation, 5 × 10⁶ nondepleted or depleted cells were transferred intraperitoneally, at the time of CII/CFA immunization, to DBA/1-TcR-β Tg mice. (a and b) Incidence of arthritis. Mice groups were compared by statistical analysis using the Fisher exact test. Data are representative of three experiments.

Figure 3.

Generation of B cells with a regulatory phenotype is controlled by endogenous IL-10. Arthritogenic splenocytes were cultured with CII/isotype control, or CII/anti-CD40 for 48 h. B cells were then purified by negative selection and cultured with PMA plus ionomycin in the presence of Brefeldin A for an additional 6 h. (a) The intracellular levels of IFN-γ and IL-10 were measured using PE-conjugated mAb against IFN-γ or IL-10 and the purity of B cells was controlled using anti-CD19 FITC mAb. (b) Supernatant was collected from purified B cells stimulated in vitro with PMA plus ionomycin and the secreted cytokines were measured by ELISA. Data show mean ± SE of triplicate wells, and are representative of four experiments. Data were compared by statistical analysis using the unpaired t test. (c) B cells were purified from arthritogenic splenocytes isolated from mice immunized with CII/CFA or with CFA alone. Negatively purified B cells were cultured with or without CII anti-CD40, anti-CD40 + anti-κ for 72 h. Data are representative of three experiments that gave similar results.

Figure 3.

Generation of B cells with a regulatory phenotype is controlled by endogenous IL-10. Arthritogenic splenocytes were cultured with CII/isotype control, or CII/anti-CD40 for 48 h. B cells were then purified by negative selection and cultured with PMA plus ionomycin in the presence of Brefeldin A for an additional 6 h. (a) The intracellular levels of IFN-γ and IL-10 were measured using PE-conjugated mAb against IFN-γ or IL-10 and the purity of B cells was controlled using anti-CD19 FITC mAb. (b) Supernatant was collected from purified B cells stimulated in vitro with PMA plus ionomycin and the secreted cytokines were measured by ELISA. Data show mean ± SE of triplicate wells, and are representative of four experiments. Data were compared by statistical analysis using the unpaired t test. (c) B cells were purified from arthritogenic splenocytes isolated from mice immunized with CII/CFA or with CFA alone. Negatively purified B cells were cultured with or without CII anti-CD40, anti-CD40 + anti-κ for 72 h. Data are representative of three experiments that gave similar results.

Figure 4.

Transfer of B cells isolated from anti-CD40 activated splenocytes prevents chronic arthritis development. (a–c) DBA/1-TcR-β-Tg splenocytes were stimulated in vitro for 48 h with isotype control/CII; isotype control alone or with anti-CD40/CII or anti-CD40 alone. B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, and 5 × 10⁵ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. Percentage of mice with arthritis. Mice groups were compared by statistical analysis using the Fisher exact test. (b–d) Severity of disease. The data represent the mean of (n) mice ± SE and they are representative of five experiments. Groups of mice were compared by statistical analysis using the nonparametric Mann-Whitney U test. The purity of B cells, isolated from (e) control or (f) anti-CD40–treated splenocytes, was checked before transfer, by staining the negative fraction with FITC-conjugated anti-CD19 and PE-conjugated anti-CD3 mAbs. (c and d) DBA/1-TcR-β-Tg splenocytes were stimulated in vitro for 48 h with isotype control or with anti-CD40 alone. B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, and 5 × 10⁵ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. Percentage of mice with arthritis. (g) B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, from DBA/1-TcR-β-Tg arthritogenic splenocytes. Purified B cells were stimulated in vitro for 48 h with isotype control/CII; or with anti-CD40/CII. 5 × 10⁶ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. A group of mice was left untreated. Percentage of mice with arthritis. Data are representative of four experiments.

Figure 4.

Transfer of B cells isolated from anti-CD40 activated splenocytes prevents chronic arthritis development. (a–c) DBA/1-TcR-β-Tg splenocytes were stimulated in vitro for 48 h with isotype control/CII; isotype control alone or with anti-CD40/CII or anti-CD40 alone. B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, and 5 × 10⁵ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. Percentage of mice with arthritis. Mice groups were compared by statistical analysis using the Fisher exact test. (b–d) Severity of disease. The data represent the mean of (n) mice ± SE and they are representative of five experiments. Groups of mice were compared by statistical analysis using the nonparametric Mann-Whitney U test. The purity of B cells, isolated from (e) control or (f) anti-CD40–treated splenocytes, was checked before transfer, by staining the negative fraction with FITC-conjugated anti-CD19 and PE-conjugated anti-CD3 mAbs. (c and d) DBA/1-TcR-β-Tg splenocytes were stimulated in vitro for 48 h with isotype control or with anti-CD40 alone. B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, and 5 × 10⁵ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. Percentage of mice with arthritis. (g) B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, from DBA/1-TcR-β-Tg arthritogenic splenocytes. Purified B cells were stimulated in vitro for 48 h with isotype control/CII; or with anti-CD40/CII. 5 × 10⁶ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. A group of mice was left untreated. Percentage of mice with arthritis. Data are representative of four experiments.

Figure 4.

Transfer of B cells isolated from anti-CD40 activated splenocytes prevents chronic arthritis development. (a–c) DBA/1-TcR-β-Tg splenocytes were stimulated in vitro for 48 h with isotype control/CII; isotype control alone or with anti-CD40/CII or anti-CD40 alone. B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, and 5 × 10⁵ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. Percentage of mice with arthritis. Mice groups were compared by statistical analysis using the Fisher exact test. (b–d) Severity of disease. The data represent the mean of (n) mice ± SE and they are representative of five experiments. Groups of mice were compared by statistical analysis using the nonparametric Mann-Whitney U test. The purity of B cells, isolated from (e) control or (f) anti-CD40–treated splenocytes, was checked before transfer, by staining the negative fraction with FITC-conjugated anti-CD19 and PE-conjugated anti-CD3 mAbs. (c and d) DBA/1-TcR-β-Tg splenocytes were stimulated in vitro for 48 h with isotype control or with anti-CD40 alone. B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, and 5 × 10⁵ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. Percentage of mice with arthritis. (g) B cells were enriched by negative selection, using an anti-CD43 magnetic beads conjugated antibody, from DBA/1-TcR-β-Tg arthritogenic splenocytes. Purified B cells were stimulated in vitro for 48 h with isotype control/CII; or with anti-CD40/CII. 5 × 10⁶ B cells were transferred intraperitoneally, at the time of CII/CFA immunization, to syngeneic mice. A group of mice was left untreated. Percentage of mice with arthritis. Data are representative of four experiments.

Figure 5.

Treatment with anti-CD40–stimulated B cells, inhibits IFN-γ production, and increases the release of IL-10 in CII immunized DBA/1-TcR-β-Tg mice. Six DBA/1-TcR-β-Tg mice were injected intraperitoneally with B cells isolated from isotype control or anti-CD40 in vitro challenged splenocytes. A group of six mice were left untreated. 20 d after transfer draining lymph nodes were isolated, and cultured in medium alone or in the presence of CII for 72 h. (a) The proliferation of LNCs was measured in medium alone or in response to CII; data show mean ± SE of triplicate wells, and are representative of four experiments. Data were compared by statistical analysis using the Unpaired t test. (b) Splenocytes, were cultured with CII/isotype control, or CII/anti-CD40 for 48 h PMA, ionomycin and Brefeldin A were added for an additional 6 h. The intracellular levels of IFN-γ and IL-10 were measured using PE-conjugated mAb against IFN-γ or IL-10. Splenocytes were stained with FITC-conjugated mAb against CD19, to define the percentage of B cells synthesizing cytokines. Data are representative of four experiments. (c) Lymph nodes were isolated from the same group of mice and cultured with CII/isotype control, or CII/anti-CD40 for 48 h. T cells were purified by positive selection using anti–Thy-1.2 magnetic beads antibodies and cultured for an additional 6 h with PMA, ionomycin, and Brefeldin A. The intracellular levels of IFN-γ and IL-10 were measured using and PE-conjugated mAb against IFN-γ or IL-10.

Figure 6.

In vivo cytokine production by anti-CD40 stimulated B cells reisolated from spleen of treated mice. B cells were purified by negative selection from arthritic splenocytes and labeled with CFSE. The labeled fraction was reconstituted with the CD43⁺-positive fraction and cultured with CII/isotype control or with CII/anti-CD40 for 48 h. 10⁷ CFSE⁺ B cells were then transferred to immunized DBA/1-TcR-β-Tg mice after purification by negative selection. 3 d after the transfer, 5 × 10⁶ splenocytes were incubated in medium alone or with CII. 48 h later CFSE labeled B cells were sorted and stimulated for a 6 h with ionomycin and PMA. The release of (a) IFN-γ and (b) IL-10 was analyzed by ELISA. The data (mean ± SE of triplicate wells) are representative experiments, using six to eight mice per group. Each experiment was repeated twice with similar results. Data were compared by statistical analysis using the unpaired t test.

Figure 7.

B cells isolated from IL-10 KO/H2^q mice fail to differentiate into a “regulatory” phenotype. Spleens were isolated from arthritic IL-10 KO/H2^q mice or from the control IL-10 wild-type mice. Splenocytes were cultured for 48 h with CII/isotype control, CII/anti-CD40. B cells were enriched by negative selection, using an anti-CD43 magnetic beads–conjugated antibody, and 5 × 10⁵ cells were transferred intraperitoneally, at the time of CII/CFA immunization, to IL-10 wild-type mice. Arthritis evolution and severity was measured as described previously. Data shows incidence of arthritis (a) and mean of clinical score ± SE (b), and are representative of three experiments. Groups of mice were compared by statistical analysis using the Fisher exact test.

Figure 8.

(a) Generation of B cells with “protective” phenotype is controlled by endogenous IL-10. Splenocytes isolated from DBA/1-TcR-β-Tg were cultured with CII/isotype control, or CII/anti-CD40 for 48 h alone or in the presence of neutralizing anti–IL-10R mAb (1B1.2) or anti–IL-4 mAb (11B11). 5 × 10⁵ B cells were purified and transferred intraperitoneally to DBA/1-TcR-β-Tg mice at the time of CII/CFA immunization. Data are representative of two experiments. Groups of mice were compared by statistical analysis using the Fisher exact test. (b) Antigen-specific CD4⁺ T cells also play a crucial role in the generation of B cells with “protective” phenotype. CD4⁺ T cells were depleted by positive selection from splenocytes isolated from arthritic DBA/1-TcR-β Tg mice. Undepleted or depleted splenocytes were stimulated with CII/isotype control, CII/anti-CD40. After 48 h incubation, 5 × 10⁶ nondepleted or depleted cells were transferred intraperitoneally, at the time of CII/CFA immunization, to DBA/1-TcR-β Tg mice. Incidence of arthritis. Mice groups were compared by statistical analysis using the Fisher exact test. Data are representative of three experiments.

Figure 9.

Therapeutic effect of anti-CD40–treated B cells on established arthritis. B cells were isolated from CII/anti-CD40 or CII/isotype control stimulated arthritogenic splenocytes and enriched by negative selection. DBA/1-TcR-β-Tg mice were treated (intravenously) at the day of disease onset and every 5 d (three doses in total) with 5 × 10⁵ B cells in the experiment (a) and at day 5 and 10 in experiment (b). The severity of arthritis is reported as clinical score. Mice groups were compared by statistical analysis using two-sided Student's t test.