Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G_i-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G_i-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-α, IL-1β, and TGF-β1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.

Increased bone resorption mediated by osteoclasts causes various diseases such as osteoporosis and bone erosion in rheumatoid arthritis (RA). Osteoclasts are derived from the monocyte/macrophage lineage, but the precise origin remains unclear. In the present study, we show that the purified CD16^- human peripheral blood monocyte subset, but not the CD16⁺ monocyte subset, differentiates into osteoclast by stimulation with receptor activator of NF-κB ligand (RANKL) in combination with macrophage colony-stimulating factor (M-CSF). Integrin-β3 mRNA and the integrin-αvβ3 heterodimer were only expressed on CD16^- monocytes, when they were stimulated with RANKL + M-CSF. Downregulation of β3-subunit expression by small interfering RNA targeting β3 abrogated osteoclastogenesis from the CD16^- monocyte subset. In contrast, the CD16⁺ monocyte subset expressed larger amounts of tumor necrosis factor alpha and IL-6 than the CD16^- subset, which was further enhanced by RANKL stimulation. Examination of RA synovial tissue showed accumulation of both CD16⁺ and CD16^- macrophages. Our results suggest that peripheral blood monocytes consist of two functionally heterogeneous subsets with distinct responses to RANKL. Osteoclasts seem to originate from CD16^- monocytes, and integrin β3 is necessary for osteoclastogenesis. Blockade of accumulation and activation of CD16^- monocytes could therefore be a beneficial approach as an anti-bone resorptive therapy, especially for RA.

Objective: Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive T cell infiltration into the synovium. The accumulated T cells express type 1 cytokines, such as interferon‐γ (IFNγ) and tumor necrosis factor α, and activated markers of inflammation, such as CD154 and inducible costimulator (ICOS). It is thought that chemokines contribute to T cell accumulation in the synovium. In this study, we examined the role of CXCL16 and CXCR6 in T cell migration and stimulation in RA synovium.Methods: Expression of CXCL16 and CXCR6 was analyzed by immunohistochemistry, reverse transcription–polymerase chain reaction, Western blotting, and/or flow cytometry. Migration activity was assessed using a chemotaxis chamber. IFNγ production was analyzed by enzyme‐linked immunosorbent assay. The effect of anti‐CXCL16 monoclonal antibody on murine collagen‐induced arthritis (CIA) was evaluated.Results: CXCL16 was expressed in RA synovium. CXCR6 was expressed more frequently on synovial T cells than in peripheral blood. Moreover, CXCR6‐positive synovial T cells more frequently expressed CD154 and ICOS than did CXCR6‐negative T cells. Stimulation with interleukin‐15 (IL‐15) up‐regulated the expression of CXCR6 on peripheral blood T cells, and then stimulation with CXCL16 induced migration of IL‐15–stimulated T cells and enhanced IFNγ production. Furthermore, anti‐CXCL16 monoclonal antibody significantly reduced the clinical arthritis score and reduced infiltration of inflammatory cells and bone destruction in the synovium of mice with CIA.Conclusion: Our results indicate that CXCL16 plays an important role in T cell accumulation and stimulation in RA synovium and suggest that CXCL16 could be a target molecule in new therapies for RA.

Objective: Rheumatoid arthritis (RA) is characterized by chronic inflammation of multiple joints. Large numbers of T cells, which produce type 1 cytokines, infiltrate into RA synovium. Chemokines and chemokine receptors are considered to contribute to the T cell infiltration. In this study, we examined the role of CX3CL1/fractalkine and its receptor CX3C chemokine receptor 1 (CX3CR1) in the T cell migration into RA synovium.Methods: Using flow cytometry, immunohistochemistry, and reverse transcription–polymerase chain reaction, we analyzed CX3CR1 expression by peripheral blood and synovial T cells, and CX3CL1 expression in synovium from patients with RA. Cytokine and cytotoxic molecule expression by CX3CR1‐positive T cells was analyzed by flow cytometry.Results: CX3CR1 expression by peripheral CD4+ and CD8+ T cells was up‐regulated in RA patients. The peripheral CD4+ and CD8+ T cells expressing CX3CR1 predominantly produced interferon‐γ and tumor necrosis factor α, and expressed cytotoxic molecules such as granzyme A and perforin. Furthermore, CX3CR1+,CD3+ T cells infiltrated into RA synovium. CX3CL1, the unique ligand of CX3CR1, was expressed by endothelial cells and synoviocytes in RA synovium, but not in osteoarthritis synovium.Conclusion: Our findings suggest that the interactions of CX3CL1 and CX3CR1 might contribute to the accumulation of CX3CR1+ T cells expressing type 1 cytokines and possessing cytotoxic granules in RA synovium.

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive infiltration of inflammatory cells in the synovium of multiple joints. We and others have shown that fractalkine (FKN/CX3CL1), a chemokine expressed on fibroblast-like synoviocytes and endothelial cells in RA synovium, may contribute to the accumulation of T cells, macrophages, and dendritic cells, which express CX3CR1, the receptor for FKN. This interaction might be involved in adhesion of the inflammatory cells to endothelial cells, migration into the synovium, and cytokine production. In this study, we examined the effect of FKN inhibition on murine collagen-induced arthritis. Anti-FKN mAb significantly lowered clinical arthritis score compared with control Ab, and reduced infiltration of inflammatory cells and bone erosion in the synovium. However, anti-FKN mAb did not affect the production of either serum anti-collagen type II (CII) IgG or IFN-γ by CII-stimulated splenic T cells. Furthermore, treatment with anti-FKN mAb inhibited migration of adoptively transferred splenic macrophages into the inflamed synovium. Our results suggest that anti-FKN mAb ameliorates arthritis by inhibiting infiltration of inflammatory cells into the synovium. Thus, FKN can be a new target molecule for the treatment of RA.

Introduction: Disturbances in peripheral blood memory B cell subpopulations have been observed in various autoimmune diseases, but have not been fully delineated in rheumatoid arthritis (RA). Additionally, the possible role of tumour necrosis factor (TNF) in regulating changes in specific peripheral blood memory B cell subsets in RA is still unclear. Methods: The frequency and distribution of B cell subsets in the peripheral blood and synovial membrane of active RA patients with long-standing disease have been analysed. Additionally, the possible role of TNF in causing disturbances in memory B cell subsets in RA patients was assessed in a clinical trial with the specific TNF-neutralising antibody, infliximab. Results: RA patients, independent of disease duration, have a significantly lower frequency of peripheral blood pre-switch IgD⁺CD27⁺ memory B cells than healthy individuals, whereas post-switch IgD^-CD27⁺ accumulate with increased disease duration. Notably, both pre-switch IgD⁺CD27⁺ and post-switch IgD^-CD27⁺ memory B cells accumulate in the synovial membrane of RA patients. Finally, anti-TNF therapy increased the frequency of pre-switch IgD⁺CD27 memory B cells in the peripheral blood. Conclusions: The data suggest that decreases in peripheral blood IgD⁺CD27⁺ pre-switch memory B cells in RA reflect their accumulation in the synovial tissue. Moreover, the significant increase in the peripheral blood pre-switch memory B cells in patients who underwent specific TNF-blockade with infliximab indicates that trafficking of memory B cells into inflamed tissue in RA patients is regulated by TNF and can be corrected by neutralising TNF.