Expression of toll‐like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis

Abstract

Objective CD16 (IgG Fcγ receptor type IIIA [FcγRIIIA])–expressing CD14+ monocytes express high levels of Toll‐like receptor 2 (TLR‐2) and are able to efficiently produce proinflammatory cytokines such as tumor necrosis factor α (TNFα). To understand the role of CD16 and TLR‐2 in monocyte and macrophage activation in rheumatoid arthritis (RA), we investigated the expression of TLR‐2 on CD16+ blood monocytes and synovial tissue macrophages and the effect of CD16 and TLR‐2 activation on cytokine production. Methods The expression of CD14, CD16, TLR‐2, and TLR‐4 on blood monocytes was measured by flow cytometric analysis. CD16 and TLR‐2 expression in RA synovial tissue was detected by 2‐color immunofluorescence labeling. CD16+ mature monocytes were prepared by incubating blood monocytes in plastic plates for 24 hours. These adhered monocytes were stimulated with lipoteichoic acid (LTA), anti‐FcγRIII antibody, and Hsp60 for 5 hours, and culture supernatants were measured for various cytokines by immunoassay. The activation of NF‐κB was detected by electrophoretic mobility shift assay. Results The frequency of CD16+ cells in all blood monocytes was significantly increased in patients with RA compared with healthy controls. TLR‐2 was expressed at higher levels on CD16+ monocytes than on CD16− monocytes, while TLR‐4 was expressed similarly on both monocytes. In RA synovial tissue, CD16+/TLR‐2+ cells were distributed mainly in the lining layer. TLR‐2 expression on monocytes was enhanced by macrophage colony‐stimulating factor (M‐CSF) and interleukin‐10 (IL‐10), but was reduced by transforming growth factor β1, while CD16 expression was inducible by these cytokines. Adhered monocytes (∼50% CD16+) produced TNFα, IL‐1β, IL‐6, IL‐8, IL‐12 p40, IL‐1 receptor antagonist, and IL‐10 after LTA stimulation. This cytokine response was inhibited significantly by anti–TLR‐2 antibody and partly by anti–TLR‐4 antibody. Anti‐FcγRIII antibody stimulation markedly enhanced the LTA‐induced TNFα response. Hsp60 could stimulate TNFα production by adhered monocytes, which was inhibited similarly by anti–TLR‐2 antibody and anti–TLR‐4 antibody. NF‐κB activation in adhered monocytes was induced by LTA, but this NF‐κB activity was not augmented by anti‐FcγRIII antibody stimulation. Conclusion These results suggest that CD16+ monocytes and synovial tissue macrophages with high TLR‐2 expression may be induced by M‐CSF and IL‐10, and their production of TNFα could be simulated by endogenous TLR ligands such as Hsp60 and FcγRIIIA ligation by small immune complexes in RA joints.