engleski

Phenotypic and functional characterization of peripheral blood osteoclast progenitor population in patients with rheumatoid arthritis

Introduction. Rheumatoid arthritis (RA) is the most severe chronic joint disease marked by the persistent inflammation and osteodestruction, causing progressive disability and crippling. The mechanisms leading to joint destruction involve differentiation and activation of osteoclasts, multinucleated cells derived from monocyte/macrophage lineage. Human osteoclast progenitors (OCPs) represent a subpopulation of peripheral blood monocytes and have been shown to be present at low frequency in the peripheral blood of healthy subjects. Tumor necrosis factor-α (TNFα) increases the peripheral blood OCP numbers in arthritic patients, implying that circulating OCPs may have an important role in the pathogenesis of RA. The aim of our study was to analyze the phenotype, frequency and osteoclastogenic potential of OCPs in the peripheral blood and synovial fluid of RA patients in relation to anti-TNF therapy. Methods. Monocytes were isolated from peripheral blood of healthy controls and RA patients, after obtaining approval from the Ethical Committee and informed consent from patients. A subset of peripheral blood and synovial fluid samples were collected from RA patients prior and in the follow-up of TNF-blockage treatment. The phenotype of isolated monocytes was determined within peripheral blood mononuclear cells using flow cytometry for the following surface markers: CD3, CD11b, CD11c, CD14, CD16, CD19, CD56, CD115 and CCR2. Lymphoid lineage negative (CD3-CD19-CD56-) population was then sorted and cultured with the addition of macrophage colony-stimulating factor (M-CSF ; 30 ng/mL) and receptor activator of nuclear factor-kappa-B ligand (RANKL ; 60 ng/mL) for two weeks to stimulate osteoclast differentiation. Osteoclasts were detected by the cytochemical staining for tartrate-resistant acid phosphatase (TRAP). Results were correlated with clinical data, including inflammatory indicators and variables describing disease activity and severity. Results. We have verified human peripheral blood OCPs as an osteoclastogenic population bearing the phenotype CD3-CD19-CD56-CD11b+CD115+. This monocyte subpopulation is significantly increased in RA patients, comprising 2.5-5.5% of circulating monocytes compared to 1.5% in healthy controls. The same population was found to comprise approximately 1% of synovial fluid monocytes. A portion of cells within this population express other monocyte markers, including CD14 and CD16. Cultures of sorted lymphoid lineage negative population from RA patients revealed that both circulatory and synovial monocyte progenitors exhibit in vitro osteoclastogenic potential. Anti TNF-treatment was not able to significantly decrease the proportion of OCPs, and only transiently suppressed their differentiation in cultures stimulated by RANKL and M-CSF . Conclusions. Peripheral blood monocyte subpopulations, including OCPs, are heterogeneous by surface marker expression, size, and function, and could be specifically induced during chronic inflammatory diseases. Many studies attempted to define source of OCPs specifically associated with arthritis as well as mechanisms of their activation, in order to develop efficient therapeutic approaches. Our study revealed that anti-TNF treatment only transiently suppressed osteoclastogenic potential of peripheral OCPs, indicating that additional therapeutic modalities, besides TNF-blocking agents, should be considered for sustained antiresorptive effect.

reumatoidni artritis; osteoklastni progenitori; periferna krv; sinovijalna tekućina; koštana razgradnja

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