Background/Purpose

Macrophages and synovial fibroblasts (SF) are two major cells implicated in the pathogenesis of rheumatoid arthritis (RA). They can interact in the synovial micro-environment to drive inflammation and bone destruction. The aim of our work was to investigate the effects of SF on monocyte viability and differentiation and to determine which factors were implicated in these effects.

Methods

SF were isolated from synovial tissue of 9 RA patients and CD14+ cells were magnetically isolated from healthy donors by MACS technology. SF conditioned media were collected after 24 hours of culture with or without TNF-α or IL-1β. After 3 days of culture with RA SF conditioned media, monocyte survival was assessed using a WST-1 viability test. To study the involvement of M-CSF, IL-34 and GM-CSF, their expression was quantified in RA SF by qPCR, in RA synovial fluids by ELISA assay, and specific blocking antibodies were used in monocyte cultures. Macrophages polarization after culture with RA SF conditioned media was studied by flow cytometry. The cell surface markers analyzed were CD14, CD16, CD64 (M1), CD200R (M2a) and CD163 (M2c). A non-parametric test (Kruskall Wallis) was used to perform statistical analyzes.

Results

SF conditioned media significantly increased monocyte viability compared to cells cultured in medium alone (p<0.001). This effect was stronger when using conditioned media from IL-1β or TNF-α pre-stimulated SF. Monocyte viability was significantly increased compared to M-CSF (+29% (p=0.05) and +52% (p=0.004) for TNF-α and IL-1-β media respectively). M-CSF and IL-34 blocking antibodies, alone and in combination, had no effect on monocyte viability induced by SF conditioned media. In contrast, blocking GM-CSF resulted in a significant decrease in monocyte viability by 30% when added to the stimulated SF conditioned media (p<0.001). The expression of GM-CSF by RA SF was stimulated by TNF-α and IL-1β and GM-CSF levels in the synovial fluids were correlated with those of these two cytokines. Finally, studying the cell surface markers, we found no regulation of M1/M2 polarization when monocytes were differentiated in the presence of SF conditioned media.

Conclusion

SF conditioned media increased monocyte viability with a greater effect observed when SF were pre-stimulated with IL-1β or TNF-α and this effect was GM-CSF-dependent. However, cells cultured in the presence of conditioned media did not express specific M1 or M2 markers.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-1%ce%b2-and-tnf-%ce%b1-promote-monocyte-viability-through-the-induction-of-gm-csf-expression-by-rheumatoid-arthritis-synovial-fibroblasts/