• Mitsuhiro Iwahashi, Masahiro Yamamura, Tetsushi Aita, Akira Okamoto, Akiko Ueno, Norio Ogawa, Sachiko Akashi, Kensuke Miyake, Paul J Godowski, and Hirofumi Makino.
• Department of Medicine and Clinical Science, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
• Arthritis Rheum. 2004 May 1; 50 (5): 1457-67.

ObjectiveCD16 (IgG Fcgamma receptor type IIIA [FcgammaRIIIA])-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 alpha (TNFalpha). 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.MethodsThe 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-FcgammaRIII antibody, and Hsp60 for 5 hours, and culture supernatants were measured for various cytokines by immunoassay. The activation of NF-kappaB was detected by electrophoretic mobility shift assay.ResultsThe 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 beta1, while CD16 expression was inducible by these cytokines. Adhered monocytes ( approximately 50% CD16+) produced TNFalpha, IL-1beta, 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-FcgammaRIII antibody stimulation markedly enhanced the LTA-induced TNFalpha response. Hsp60 could stimulate TNFalpha production by adhered monocytes, which was inhibited similarly by anti-TLR-2 antibody and anti-TLR-4 antibody. NF-kappaB activation in adhered monocytes was induced by LTA, but this NF-kappaB activity was not augmented by anti-FcgammaRIII antibody stimulation.ConclusionThese 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 TNFalpha could be simulated by endogenous TLR ligands such as Hsp60 and FcgammaRIIIA ligation by small immune complexes in RA joints.

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