Cadherin-11 regulates fibroblast inflammation

Abstract

Fibroblasts are important participants in inflammation. Although not leukocytes, their capacity to produce cytokines, chemokines, and other inflammatory factors locally in tissues suggests that they can contribute to inflammatory diseases. For example, fibroblasts in a rheumatoid arthritis (RA) joint are a dominant source of IL-6 and RANKL in the synovium, both of which are therapeutic targets for inflammation and bone erosion. Previously, we found that fibroblasts can be targeted by mAb directed against cadherin-11 (cad-11), a mesenchymal cadherin that fibroblasts selectively express. Targeting cad-11 significantly reduced inflammation as assessed by joint swelling and clinical inflammation scores. However, the mechanism by which anti-cad-11 reduced inflammation was not known. Here, we show that cad-11 engagement induces synovial fibroblasts to secret proinflammatory cytokines including IL-6. Cad-11 engagement strongly synergized with TNF-α and IL-1β in the induction of IL-6. Importantly, cad-11 activated MAP kinases and NF-κB for IL-6 induction. IL-6 levels in ankles of inflamed joints were reduced in cad-11 mutant mice compared to wild-type mice with inflammatory arthritis. Thus, we suggest that cad-11 modulates synovial fibroblasts to evoke inflammatory factors that may contribute to the inflammatory process in RA.

Fig. 1.

Cad-11 engagement induces synovial fibroblasts to produce inflammatory factors. (A) Primary synovial fibroblasts were incubated with or without 5 μg/mL hIgG1 or hCad-11-Fc for 2 d in 1% FBS/SF medium. Cell culture supernatants were analyzed for IL-6 by ELISA. The results shown were accumulated from more than five independent experiments. (B) Synovial fibroblasts were cultured with or without 20 μg/mL hIgG1 or the indicated concentrations (μg/mL) of hCad-11-Fc for 2 d in 1% FBS/SF medium, and then the supernatants were analyzed for IL-6 by ELISA. Data shown represent more than three independent experiments. (C) Synovial fibroblasts were stimulated with or without 10 μg/mL hIgG1 or hCad-11-Fc in a 3D micromass organ culture for 24 h. Culture supernatants were analyzed for IL-6 by ELISA. Data shown represent three independent experiments. (D and E) After culturing synovial fibroblasts with 10 μg/mL hIgG1, hCad-11-Fc, or 1 ng/mL TNF-α for 24 h, cell-free supernatants were examined by a cytokine antibody array. Each spot was quantified by a densitometer, and the graph shows fold induction of each molecule in the (D) hCad-11-Fc– or (E) TNF-α–treated group relative to the hIgG1-treated group. The factors shown in D and E were at least more than 1.5-fold induction. (F) Synovial fibroblasts were treated the same way as in A. MCP-1 was measured by ELISA. The results were accumulated from more than four independent experiments. Compared with hIgG1, *P = 0.0012 and **P = 0.0073. pos, positive control.

Fig. 2.

Specificity of cad-11–mediated IL-6 production. (A) Synovial fibroblasts were stimulated with wild-type or mutant hCad-11-Fc at various concentrations (μg/mL) in 1% FBS/SF medium. IL-6 secreted into the supernatants was quantified by ELISA. The representative synovial fibroblasts (Left: less responsive cells; Right: more responsive cells) from three independent experiments are shown. (B) After silencing cad-11 by shRNA (cad11-H2 or cad11-H3) in synovial fibroblasts, surface cad-11 was detected by FACS analysis (filled histograms: isotype control; open histograms: cad-11). No virus and control shRNA were used as controls. Data shown represent five independent experiments. (C) Cad-11 shRNA or control shRNA-transduced synovial fibroblasts were stimulated with 5 μg/mL of hIgG1 or hCad-11-Fc in 1% FBS/SF medium for 2 d. Cell-free supernatants were analyzed by ELISA for IL-6. The results shown were accumulated from four independent experiments. When cad11-H2 and cad11-H3 were compared with control shRNA, *P = 0.0018 and **P = 0.0020, respectively. (D) Cad-11–coated beads induce cad-11 clustering on the cell surface. Protein A-precoated polystyrene beads (5.5 μm) were bound with hIgG1 or hCad-11-Fc and added to L cells expressing GFP-cad-11. After fixation, cells were analyzed by confocal microscopy to detect GFP signals clustered around the beads. Digital image correlation was also taken to confirm the locations of beads on the cell surface. Arrowheads indicate the beads on L cells. (Scale bars, 10 μm.)

Fig. 3.

Cad-11 engagement strongly activates JNK and ERK in synovial fibroblasts. (A) After synovial fibroblasts were serum-starved overnight and stimulated with or without hCad-11-Fc (20 μg/mL) or TNF-α (10 ng/mL) for the indicated times in 1% FBS/SF medium, total cell lysates were analyzed for pJNK, pERK1/2, or pp38 by immunoblotting. Total p38 and β-actin were used as loading controls. Molecular markers (kDa) are shown. (B) Synovial fibroblasts were pretreated with or without DMSO (0.16%) or inhibitors for JNK (sp600125, Left) or ERK (U0126, Right) at the indicated concentrations for 30 min, and then cells were stimulated with 5 μg/mL hIgG1 or hCad-11-Fc overnight in 1% FBS/SF medium. IL-6 was measured by ELISA. (C) Synovial fibroblasts were treated the same way as in B except for concentrations of inhibitors, 10 μM of sp600125 (Left), and 20 μM of U0126 (Right). Data were accumulated from more than eight independent experiments. When hCad-11-Fc is compared with hIgG1, *P < 0.01. When sp600125 and U0126 were compared with DMSO control in the hCad-11-Fc–stimulated group, **P = 0.0292 and ***P = 0.0349, respectively.

Fig. 4.

Cad-11 stimulates NF-κB activation in synovial fibroblasts. Serum-starved synovial fibroblasts were stimulated with hCad-11-Fc (20 μg/mL) or TNF-α (10 ng/mL) for the indicated times. (A) Total cell lysates were examined for p-p65 and total p65 by immunoblotting (Upper). The band density of p-p65 at each time point was measured by a densitometer. Relative units were calculated by dividing the band density at each time point by the band density at 0 min (Lower). (B) Synovial fibroblasts were stimulated the same way as in A for 15 min and for 30 min. p-p65 was measured by direct ELISA in total cell lysates (Upper). One representative of three independent experiments is shown. The level of p-p65 was confirmed by immunoblotting in the same cell lysates, and p65 was used as a control (Lower). IB, immunoblotting.

Fig. 5.

Cad-11 synergizes with TNF-α or IL-1β for IL-6 production. (A) Synovial fibroblasts were stimulated with or without 0.1 ng/mL TNF-α (Left) or 0.01 ng/mL IL-1β (Right) in the combination of 5 μg/mL hIgG1 or hCad-11-Fc for 2 d. Cell culture supernatants were analyzed for IL-6 by ELISA. Data represent more than three independent experiments. (B) Serum-starved synovial fibroblasts were stimulated with or without hCad-11-Fc (20 μg/mL) in the presence or absence of the indicated concentrations of TNF-α for 15 min, and total cell lysates were determined for phosphorylation of ERK1/2 and JNK by immunoblotting.

Fig. 6.

Cad-11 regulates IL-6 production in vivo. WT and cad-11–deficient mice (six mice per group) were induced with arthritis by injecting K/BxN serum. Total RNA was isolated from hind ankle joints at day 4. Mouse IL-6 (A), TNF-α (B), and IL-1β (C) were analyzed by quantitative real-time PCR. The expression levels of each cytokine were normalized to GAPDH. When compared the levels of IL-6 and TNF-α in wild-type and cad-11–deficient mice, *P = 0.0472 and **P = 0.0144, respectively. n.s., not significant.