Background Osteoarthritis (OA) is characterized by cartilage destruction and ectopic bone formation in joints. Several studies have demonstrated that mild synovitis in early phases is conducive to joint damage. This inflammation is reflected by elevated levels of pro-inflammatory factors like S100A8, S100A9, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). Recently we found that adipose derived mesenchymal stem cells (ASCs) exhibit immunosuppressive characteristics (1) and reduce joint pathology after local application into mouse knee joints with experimental OA (2). This anti-inflammatory 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 (1).

Objectives To examine the effect of IL-1β on the immunosuppressive potency of ASCs in early 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 NIMPR14. ASCs were isolated from adipose tissue and stimulated for 24 hours with IL-1β or interferon-gamma (IFN-γ). Gene expression in synovium and stimulated cells were analyzed using qPCR. Protein levels of chemokines and cytokines were measured in the supernatant and washouts using Luminex. ASCs were co-cultured with MACS isolated bone marrow (BM-) PMNs and analyzed using histology, qPCR and Luminex.

Results Injection of ASCs into day 7 CIOA knee joints (when synovitis and IL-1β levels are highest) caused a strong influx of immune cells into the joint cavity shortly after injection (6 hours), which had largely vanished after 24 hours. The attraction of particularly PMN-like cells was confirmed by immunohistochemistry. Synovial gene expression of neutrophil attracting chemokines KC, CXCL5, and CXCL7 were increased. In line with this, IL-1β stimulated ASCs injected in naive knee joints also resulted in a strong influx of PMN-like cells.

IL-1β and IFN-γ (as a positive control) stimulation of ASCs in vitro strongly increased gene expression of KC, CXCL5, and CXCL7 as well as protein levels of KC. Finally, we co-cultured ASCs with BM-PMNs in the presence of IL-1β or IFN-γ. After 3 hours, a clear clustering of neutrophils around ASCs was observed which significantly diminished protein levels of KC (-69% after 24h; -76% after 48h).

Conclusions Local injection of ASCs into a day 7 CIOA knee-joint expressing low levels of IL-1β causes attraction of PMN-like cells. In vitro, IL-1β stimulated ASCs show an increase in chemokine expression, leading to attraction of and clustering with neutrophils which ultimately results in significantly decreased levels of pro-inflammatory factors like KC. The anti-inflammatory effect of locally applied ASCs into OA joints showing synovitis may be triggered by IL-1β and attraction of PMN-like cells.

References

1. ter Huurne M et al. Arthritis Rheum. 64(11):3604-13, 2012.

2. Schelbergen R et al. Osteoarthritis Cartilage. 22(8):1158-66, 2014.

Acknowledgements This research was supported by the Dutch Arthritis Association (RF 12-2-405).

Disclosure of Interest None declared