Objective. CD14؉,CD16؉ monocytes, identified as a minor population of monocytes in human peripheral blood (PB), have been implicated in several inflammatory diseases. We undertook this study to investigate the relevance of this phenotype to joint inflammation in rheumatoid arthritis (RA).Methods Results. The mean ؎ SD frequency of CD14؉,CD16؉ blood monocytes was significantly increased in RA patients (11.7 ؎ 5.6%; n ‫؍‬ 105) compared with healthy controls (9.5 ؎ 2.2%; n ‫؍‬ 15) (P < 0.01), and the patient group with an increased frequency of CD16؉ monocytes (>13.9%) had active disease, as defined by increased counts of tender and swollen joints, levels of acute-phase reactants, and titers of rheumatoid factor. The response to drug therapy correlated with changes in the frequency of this phenotype. The expression of CD16 on SF monocytes from RA patients was markedly elevated compared with the expression on PB monocytes. CD16 expression on RA blood monocytes was augmented in vitro by IL-10, M-CSF, and TGF␤1. Plasma concentrations of these cytokines and of sCD14 were significantly higher in RA patients with high CD16؉ monocyte frequencies than in those with low CD16؉ monocyte frequencies or in healthy controls. CD14؉,CD16؉ monocytes expressed higher levels of CCR1, CCR5, and ICAM-1 than did regular CD14؉؉,CD16؊ monocytes, particularly in active RA.Conclusion. These results indicate that the maturation of blood monocytes into tissue-infiltrative CD16؉ cells before entry into the joint, induced by cytokine spillover from the inflamed joint, may contribute to the persistent joint inflammation of RA.In human peripheral blood (PB), two monocyte subpopulations with distinct functional properties have been defined by their expression of CD14 and CD16 molecules (1,2). CD14 is the receptor for complexes of lipopolysaccharide (LPS) and LPS-binding protein (3). CD16 is the low-affinity receptor for the Fc region of IgG (Fc␥ receptor type III [Fc␥RIII]) and plays an important role in the clearance of immune complexes (4). Significant expression of CD16 was originally found to be induced on blood monocytes during their differentiation to macrophages in culture, while expression of other Fc␥R decreased (5), but 2-color immunofluorescence analysis with antibodies against CD14 and CD16 has revealed the existence of CD16-expressing monocytes with weak CD14 staining in PB. These CD14ϩ,CD16ϩ cells show typical monocyte morphology with irregular nuclei and account for ϳ10% of circulating monocytes in healthy individuals (1,2,6).

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Objective. To examine the ability of fibroblast-like synoviocytes in rheumatoid arthritis (RA) to produce interleukin-7 (IL-7) and IL-15, and the ability of these cytokines to induce the proliferation of synoviuminfiltrating T cells.Methods. Messenger RNA (mRNA) and protein levels of IL-7 and IL-15 in synovial tissue cells and fibroblast cell lines were determined by reverse transcriptase-polymerase chain reaction and enzymelinked immunosorbent assay, respectively. T cellenriched populations from RA synovial tissues were isolated by deleting adherent cells after a 14-hour incubation in plastic dishes or by expanding T cells during a 14-day incubation of tissue cells with IL-2 alone, and their proliferative responses to IL-7, IL-15, and IL-2 were measured by 3 H-thymidine incorporation. Results. Freshly isolated cells from RA synovial tissues more strongly expressed mRNA for both IL-7 and IL-15 compared with the cells from osteoarthritis tissues, and could spontaneously release greater amounts of these cytokine proteins in culture. Fibroblast cell lines prepared from RA patients were able to produce large amounts of IL-15 and small amounts of IL-7 at both the transcriptional and protein levels, and their cytokine production was significantly elevated when stimulated with IL-1 and tumor necrosis factor ␣. Purified synovial tissue macrophages spontaneously released IL-15 but not IL-7, and synovial T cells did not produce either cytokine. IL-7 and IL-15, similar to IL-2, stimulated the proliferation of synovial tissue T cells from RA patients; IL-7 was less potent than IL-15 or IL-2.Conclusion. These results indicated that fibroblast-like synoviocytes are an important source of the cytokines with IL-2-like activity, IL-15 and IL-7, in RA joints, and that IL-15 may be mainly responsible for local T cell activation and expansion in the presence of deficient IL-2 production by T cells.

Objective Interleukin‐18 (IL‐18) is a proinflammatory cytokine that is involved in immunologically mediated tissue damage, but its bioactivity is regulated in vivo by its soluble decoy receptor, IL‐18 binding protein (IL‐18BP). This study was undertaken to determine levels of IL‐18 and IL‐18 binding inhibition in the blood of patients with adult‐onset Still's disease (ASD). Methods Serum concentrations of IL‐18 in ASD patients were compared by enzyme‐linked immunosorbent assay (ELISA) with those in patients with other systemic rheumatic diseases and healthy controls. The biologically active mature protein of IL‐18 was detected by Western blot analysis with anti–IL‐18 antibody and its induction of interferon‐γ (IFNγ) secretion from IL‐18–responding human myelomonocytic KG‐1 cells. The inhibitory activity on IL‐18 binding to its receptor was determined by 125I–IL‐18 binding inhibition assay using the Chinese hamster ovary cell line transfected with a murine IL‐18 receptor (CHO‐K1/mIL‐18R). Results Concentrations of serum IL‐18 were extremely elevated in patients with active ASD compared with those in patients with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, polymyositis/dermatomyositis, Sjögren's syndrome, or healthy individuals. Levels of IL‐18 were found to correlate with serum ferritin values and disease severity in ASD. Western blot analysis revealed that serum samples from patients with active ASD contained an 18‐kd polypeptide of IL‐18, corresponding in size to the mature form. Accordingly, the samples were able to induce IFNγ secretion from KG‐1 cells, which was largely abolished by neutralizing anti–IL‐18 antibody. However, the level of IL‐18 bioactivity was more than 10‐fold weaker than the concentration of IL‐18 protein measured by ELISA. Serum samples from patients with active ASD showed an inhibitory effect on the binding of 125I–IL‐18 to CHO‐K1/mIL‐18R cells, and this activity was associated with elevation of IL‐18. Conclusion These data indicate that systemic overproduction of IL‐18 may be closely related to the pathogenesis of ASD, despite the restriction on its inflammatory activity by IL‐18 binding inhibitors such as IL‐18BP. The disease activity appears to be determined on the basis of the relative levels of IL‐18 and its specific inhibitors.

S100A8 and S100A9, two Ca 2+ -binding proteins of the S100 family, are secreted as a heterodimeric complex (S100A8/A9) from neutrophils and monocytes/macrophages. Serum and synovial fluid levels of S100A8, S100A9, and S100A8/A9 were all higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA), with the S100A8/A9 heterodimer being prevalent. By two-color immunofluorescence labeling, S100A8/A9 antigens were found to be expressed mainly by infiltrating CD68 + macrophages in RA synovial tissue (ST). Isolated ST cells from patients with RA spontaneously released larger amounts of S100A8/A9 protein than did the cells from patients with OA. S100A8/A9 complexes, as well as S100A9 homodimers, stimulated the production of proinflammatory cytokines, such as tumor necrosis factor alpha, by purified monocytes and in vitro-differentiated macrophages. S100A8/A9-mediated cytokine production was suppressed significantly by p38 mitogen-activated protein kinase (MAPK) inhibitors and almost completely by nuclear factor kappa B (NF-κB) inhibitors. NF-κB activation was induced in S100A8/A9-stimulated monocytes, but this activity was not inhibited by p38 MAPK inhibitors. These results indicate that the S100A8/A9 heterodimer, secreted extracellularly from activated tissue macrophages, may amplify proinflammatory cytokine responses through activation of NF-κB and p38 MAPK pathways in RA.