Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis.
OBJECTIVE
CD16 (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.


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-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.


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 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.


CONCLUSIONS
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 TNFalpha could be simulated by endogenous TLR ligands such as Hsp60 and FcgammaRIIIA ligation by small immune complexes in RA joints.
Iwahashi
Mitsuhiro
M
Department of Medicine and Clinical Science, Graduate School of Medicine and Dentistry, Okayama University, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Yamamura
Masahiro
M
Aita
Tetsushi
T
Okamoto
Akira
A
Ueno
Akiko
A
Ogawa
Norio
N
Akashi
Sachiko
S
Miyake
Kensuke
K
Godowski
Paul J
PJ
Makino
Hirofumi
H
D000328:Adult; D000906:Antibodies / Q000494:pharmacology; D001172:Arthritis, Rheumatoid / Q000276:immunology* / Q000378:metabolism; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016753:Interleukin-10 / Q000494:pharmacology; D008024:Ligands; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017452:Receptors, IgG / Q000276:immunology / Q000378:metabolism; D013583:Synovial Membrane / Q000166:cytology* / Q000276:immunology; D013682:Teichoic Acids / Q000494:pharmacology; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000906:Antibodies; D018834:Chaperonin 60; D016207:Cytokines; D008024:Ligands; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; D017452:Receptors, IgG; C508960:TGFB1 protein, human; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D013682:Teichoic Acids; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; C009900:lipoteichoic acid; D016173:Macrophage Colony-Stimulating Factor