Local application of adipose-derived mesenchymal stromal cells in experimental inflammatory OA results in interleukin-1β-mediated attraction of PMNs and reduced S100A8/A9 release

Abstract

Purpose: Recent studies have demonstrated that mild synovitis in early phases of osteoarthritis (OA) is conducive to development of joint damage. OA synovitis is characterized by elevated levels of pro-inflammatory factors like S100A8, S100A9, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). S100A8/A9 was found to be crucial in mediating joint destruction in inflammatory experimental OA. Previously we found that adipose-derived mesenchymal stromal cells (ASCs) exhibit immunosuppressive characteristics and reduce joint pathology after local application into mouse knee joints with experimental inflammatory OA. This protective effect is only perceived after intra-articular injection in early but not late stage OA, suggesting that the effect may be mediated by an inflammatory environment. Our objective was to examine the working mechanism of ASCs after early injection in experimental OA. Methods: Experimental OA was induced by injection of collagenase into murine knee joints (CiOA). Total knee joints were stained with haematoxylin/eosin and the PMN-specific antibody NIMP-R14. ASCs were isolated from murine adipose tissue and stimulated for 24 hours with IL-1β or S100A8/A9. Gene expression in stimulated cells was analyzed using qPCR. Protein levels of chemokines and cytokines were measured in culture supernatant using Luminex. Migration of MACS isolated bone marrow (BM-) PMNs towards ASC-conditioned medium (CM) was examined using Transwell membranes. ASCs were co-cultured with BM-PMNs and analyzed using histology and Luminex. Results: Injection of ASCs into day 7 CiOA knee joints (when synovitis and IL-1β and S100A8/A9 levels are highest) caused a strong attraction of particularly PMN-like cells and their clustering around ASCs in the synovium shortly after injection (6 hours), which was confirmed by immunohistochemistry. IL-1β stimulation of ASCs in vitro strongly increased gene expression of PMN-attracting chemokines KC, CXCL5, and CXCL7 as well as protein levels of KC, whereas S100A8/A9 did not. The migration of BM-PMNs through Transwell membranes towards CM of IL-1β-stimulated ASCs was significantly increased (from 5% to 10%) when compared to CM of non-stimulated ASCs. Next, ASCs were co-cultured with BM-PMNs in the presence or absence of IL-1β. After 6 hours, a clear clustering of neutrophils around ASCs was observed, with a significant increase in the number of ASCs clustering with PMNs, as well as a significantly elevated number of clustering PMNs per ASC after IL-1β stimulation. Interestingly, association of PMNs with ASCs lead to a significantly diminished release of KC protein by ASCs (69% and 76% lower after 24 h and 48 h respectively), as well as a significantly reduced release of S100A8/A9 protein by the PMNs. This coincided with lowering of S100A8/A9 levels in washouts of inflamed synovium 6 h and 48 h after injection of ASCs in day 7 CiOA knee joints. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in attraction and clustering of PMNs with ASCs in the synovium. This presumably runs via IL-1β-mediated up-regulation of chemokine release by ASCs, and ultimately results in significantly lowered levels of S100A8/A9. This research was supported by the Dutch Arthritis Association.