Background Rheumatoid arthritis (RA) is marked by the persistent inflammation and osteodestruction, causing progressive disability. 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 and arthritic patients.

Objectives 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 compared with peripheral blood OCPs of healthy controls. In addition, results were correlated with clinical data, including inflammatory indicators and variables describing disease activity and severity.

Methods Mononuclear cells 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 mononuclear cells was determined within peripheral blood and synovial fluid mononuclear cells using flow cytometry for the following surface markers: CD3, CD11b, CD14, CD16, CD19, CD56, CD115, CCR1, CCR2, CCR4. Lymphoid lineage negative (CD3-CD19-CD56-) population expressing myeloid markers (CD11b/CD14) 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 cytochemical staining for tartrate-resistant acid phosphatase. In addition, expression of chemokine receptors was profiled in order to determine the migratory properties of putative osteoclast progenitor subpopulations.

Results We have verified that peripheral blood and synovial fluid samples of RA patients contain an osteoclastogenic population bearing the phenotype CD3-CD19-CD56-CD11b+CD14+. A proportion of cells within this population also express myeloid markers (CD115 and CD16) and different chemokine receptors. This osteoclastogenic population is significantly increased in RA patients, comprising 2.5-5.5% of circulating mononuclear cells compared to approximately 1.5% in healthy controls. The same population was found to comprise 1% of synovial fluid monocytes. Cultures of sorted OCPs from RA patients revealed that both circulatory and synovial progenitor populations 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 Our study revealed that peripheral blood and synovial fluid monocyte subpopulations, including OCPs, are heterogeneous by surface marker expression, size and function, and could be specifically induced in chronic inflammatory joint diseases such as RA.

References

1. Komano et al. Arthritis Research & Therapy 2006, 8:R152.

2. Husheem et al. Calcif Tissue Int (2005) 76:222–230.

Disclosure of Interest None declared

DOI 10.1136/annrheumdis-2014-eular.4681