Toll-like receptor homolog RP105 modulates the antigen-presenting cell function and regulates the development of collagen-induced arthritis

Abstract

Introduction: RP105 is a Toll-like receptor homolog expressed on B cells, dendritic cells (DCs), and macrophages. We investigated the role of RP105 in the development of collagen-induced arthritis (CIA).

Methods: CIA was induced in RP105-deficient DBA/1 mice and the incidence and arthritis index were analyzed. The cytokine production by spleen cells was determined. The functions of the DCs and regulatory T cells (Tregs) from RP105-deficient or control mice were determined by adding these cells to the lymph node cell culture. Arthritis was also induced by incomplete Freund's adjuvant (IFA) plus collagen or by injecting anti-collagen antibody and lipopolysaccharide.

Results: RP105-deficient mice showed accelerated onset of arthritis and increased severity. Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha production by spleen cells from RP105-deficient mice was increased in comparison with that from wild-type mice. The DCs from RP105-deficient mice induced more IFN-gamma production, whereas Tregs from those mice showed less inhibitory effect against IFN-gamma production. RP105-deficient mice also showed more severe arthritis induced by collagen with IFA.

Conclusions: These results indicate that RP105 regulates the antigen-presenting cell function and Treg development, which induced the attenuation of the cell-mediated immune responses and, as a result, suppressed the development of CIA.

Figure 1

The development of collagen-induced arthritis in RP105-deficient mice. (a-d) RP105^+/+ (n = 26, 13 males and 13 females) and RP105^-/- (n = 27, 13 males and 14 females) mice were immunized with type II collagen (CII), and signs of arthritis were monitored as described in Materials and methods. The incidence of arthritis in RP105^-/- mice was higher than that in RP105^+/+ mice between days 31 and 42 (*P < 0.05 and **P < 0.01 for comparison with RP105^+/+ mice, chi-square test) (a). The disease severity, expressed as the mean arthritis index (and standard error) of the total mice (b) and of the arthritic mice (c), is shown (*P < 0.05 and **P < 0.01 for comparison with RP105^+/+ mice, Mann-Whitney U test). (d) A histological examination was performed in RP105^+/+ (n = 20, 10 males and 10 females) and RP105^-/- (n = 23, 11 males and 12 females) mice at day 35 (*P < 0.05, chi-square test). (e) The anti-CII antibodies of IgG1 and IgG2a classes were measured from RP105^+/+ (n = 18) and RP105^-/- (n = 19) mice on days 28 and 42. Values are mean ± standard error (*P < 0.001, Wilcoxon matched pairs test). AU, arbitrary units.

Figure 2

The cytokine production from spleen cells in response to denatured type II collagen (CII) and ConA in RP105-deficient mice. Mice were immunized with CII with complete Freund's adjuvant on day 0 and with CII with incomplete Freund's adjuvant on day 21. The spleen cells from day-28 mice were harvested and stimulated with CII or ConA (5 μg/mL) for 48 hours. Interferon-gamma (IFN-γ) (a), tumor necrosis factor-alpha (TNF-α) (b), interleukin-4 (IL-4) (c), and interleukin-2 (IL-2) (d) produced in the culture supernatant were measured by cytometric beads assay. The results from two experiments (eight mice per experiment) are shown and expressed as the mean ± standard error (*P < 0.05, **P < 0.01, Mann-Whitney U test).

Figure 3

The functional analysis of dendritic cells (DCs) and regulatory T cells (Tregs) from RP105-deficient mice. (a) The splenic DCs from type II collagen (CII)-immunized RP105^+/+ or RP105^-/- mice were mixed with pooled adherent cells removed lymph node cells (LNCs) from RP105^+/- mice, as described in Materials and methods. The cells were stimulated with denatured CII for 2 days and interferon-gamma (IFN-γ) production was measured. Values are mean ± standard error. The DCs from RP105^-/- mice induced higher IFN-γ production from LNCs than did the DCs from RP105^+/+ at 50 × 10⁴ cells. The summary of five experiments, using three mice per group, is shown (*P < 0.05, Mann-Whitney U test). (b) The Tregs (1 × 10⁵) purified from the spleens of CII-immunized RP105^+/+ and RP105^-/- mice were mixed with pooled LNCs (1 × 10⁶) from RP105^+/- mice, which were immunized with CII 14 days before. The cells were stimulated with denatured CII (20 μg/mL) or anti-CD3 antibody (1 μg/mL) for 2 days and IFN-γ production was measured. The percentage suppression (100 × [IFN-γ without Tregs – IFN-γ with Tregs]/IFN-γ without Tregs) is shown. Values are mean ± standard error of the six experiments, using three mice per group (*P < 0.05, Wilcoxon matched pairs test).

Figure 4

The inflammatory cytokine production from splenic dendritic cells in response to lipopolysaccharide (LPS) in RP105-deficient mice. The dendritic cells (4 × 10⁵) purified from the spleens of RP105^+/+ and RP105^-/- mice were stimulated with LPS at various concentrations for 24 hours. The supernatants were collected and the concentrations of tumor necrosis factor-alpha (TNF-α) (a), interferon-gamma (IFN-γ) (b), interleukin-6 (IL-6) (c), and interleukin-10 (IL-10) (d) were measured. Values are mean ± standard error of five experiments, using three mice per group.

Figure 5

The development of anti-CII antibody and lipopolysaccharide (LPS)-induced arthritis and tumor necrosis factor-alpha production after LPS challenge in RP105-deficient mice. (a-d) RP105^+/+ (n = 9) and RP105^-/- (n = 9) mice were injected with a cocktail of monoclonal anti-CII antibodies intravenously and with 50 μg (a, b) or 10 μg (c, d) of LPS intraperitoneally 2 days later. The incidence of arthritis of paws (arthritic paws/total paws) (a, c) and disease severity, expressed as the mean arthritis index (and standard error) of mice (b, d), is shown. Arthritis index was significantly higher in RP105^-/- mice with 10 μg of LPS (*P < 0.05 for comparison with RP105^+/+ mice, Mann-Whitney U test). (e, f) RP105^+/+ and RP105^-/- mice were challenged intraperitoneally with 50 μg (e) (n = 10 and 11, respectively) or 10 μg (f) (n = 7 and 9, respectively) of LPS. The serum was collected 1 hour later and the tumor necrosis factor-alpha levels were measured. Values are mean ± standard error. **P < 0.01, Mann-Whitney U test. CII, type II collagen.

Figure 6

The development of collagen-induced arthritis induced with type II collagen (CII) and incomplete Freund's adjuvant (IFA) in RP105-deficient mice. RP105^+/+ (n = 18, 11 males and 7 females) and RP105^-/- (n = 22, 11 males and 11 females) mice were immunized with CII and IFA on days 0 and 21, and signs of arthritis were monitored. The arthritis incidence was shown as per body (a) and per paw (100 × [number of the arthritic paws/number of total paws]) (b). The incidence of arthritis in RP105^-/- mice was higher than that in RP105^+/+ mice on days 42, 66, and 73 (a) as was the per-paw incidence after day 38 (b). (*P < 0.05, **P < 0.01, and ***P < 0.001 for comparison with RP105^+/+ mice, chi-square test) (c, d) The disease severity, expressed as the mean arthritis index (and standard error) of total mice (c) and of arthritic mice (d), is shown (*P < 0.05 and **P < 0.01 for comparison with RP105^+/+ mice, Mann-Whitney U test).