Background The early but not the late phase of experimental inflammatory osteoarthritis (CiOA) is characterised by strongly elevated levels of S100A8/A91 and interleukin-1 beta (IL-1β).1,2 Adipose-derived mesenchymal stromal cells (ASCs) exhibit anti-inflammatory characteristics and reduce development of joint pathology after intra-articular injection into murine CiOA joints.3,4 This protective effect is only perceived when ASCs are applied in the early but not in the late stage of CiOA, suggesting that the inflammatory environment mediates this effect. Our objective was to examine the working mechanism of ASCs after injection into mouse knee joints with early CiOA.
Methods CiOA was induced by intra-articular injection of collagenase. Knee joint sections were stained with haematoxylin/eosin and the PMN-specific antibody NIMP-R14. Murine ASCs were stimulated for 24 hour with IL-1β or S100A8/A9. Protein levels of chemokines were measured in culture supernatant using Luminex. Migration of MACS-isolated bone marrow PMNs towards ASC-conditioned medium (ASC-CM) was examined using Transwell membranes. ASC-PMN co-cultures were analysed using histology and Luminex.
Results ASC injection into day 7 CiOA knee joints (when IL-1β and S100A8/A9 levels are highest) strongly attracted particularly PMNs which clustered around ASCs in the synovium 6 hour after injection, as visualised by immunohistochemistry. IL-1β-stimulation of ASCs in vitro strongly increased protein release of PMN-attracting chemokine KC (13-fold increase), whereas S100A8/A9 did not. Migration of PMNs through Transwell membranes towards ASC-CM of IL-1β-stimulated ASCs was significantly enhanced (two-fold increase) when compared to ASC-CM of non-stimulated ASCs. After 6 hour co-culturing ASCs with PMNs, both the number of ASCs clustered with PMNs and clustered PMNs per ASC were significantly increased after IL-1β-stimulation. Interestingly, association of PMNs with ASCs significantly diminished the release of KC protein by ASCs (69% lower after 24 hour), and also significantly reduced the release of S100A8/A9 protein by the PMNs. This coincided with reduced S100A8/A9 levels in synovial washouts 6 hour and 48 hour after ASC injection in day 7 CiOA joints4.
Conclusions Local application of ASCs in inflamed CiOA joints results in attraction and clustering of PMNs with ASCs in the synovium, which is likely mediated by IL-1β-induced up-regulation of KC release by ASCs, resulting in significantly lowered S100A8/A9 levels.
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Ter Huurne M, et al. Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis. Arthritis Rheum. 64(11):3604-13, 2012.
Schelbergen R, et al. Treatment efficacy of adipose-derived stem cells in experimental osteoarthritis is driven by high synovial activation and reflected by S100A8/A9 serum levels. Osteoarthritis Cartilage. 22(8):1158-66, 2014.
Acknowledgements This research was supported by the Dutch Arthritis Association.