Synovial macrophages promote TGF-Β signaling but protect against influx of S100A8/S100A9-producing cells after intra-articular injections of oxidized low-density lipoproteins

Abstract

Purpose: In previous studies we found that synovial macrophages regulate joint pathology during experimental osteoarthritis (OA) and, more recently, we found that high systemic levels of LDL aggravate joint pathology during experimental OA with synovitis. LDL in inflamed synovium is oxidized and taken-up by macrophages via scavenger receptors, leading to an activated macrophage phenotype. In this study, we investigate whether direct injection of oxLDL into a normal murine knee joint induces joint pathology and elucidate the role of synovial macrophages in that process. Methods: Knee joints of C57BL/6 mice were injected at five consecutive days with 6 μL (1.2 mg/mL) oxLDL, LDL, or an equal volume of vehicle (PBS). This same procedure was done in mice which were depleted of synovial macrophages by intra-articular injection of clodronate liposomes seven days prior to the (ox)LDL or vehicle injections. Joint pathology was investigated by immunohistochemistry and FACS analysis, and RNA expression and protein production by synovium were determined using RT-PCR and luminex, respectively. Active TGF-β was measured using a functional CAGA-luciferase assay. Data are depicted as mean±standard deviation. Results: LDL and oxLDL injection in naïve knee joints did not increase synovial thickening, or production of pro-inflammatory factors (IL-1β, IL-6 and S100A8/9) compared to vehicle injection. Levels of active TGF-β in synovial wash-outs was, however, significantly increased by 33% (from 84.7 ng/mL/g synovium±14.4 to 113.0 ng/mL/g synovium±33.3; p<0.05). Immunohistochemistry of total knee joints showed that oxLDL injection decreased formation of aggrecanase-induced neo-epitopes (NITEGE) compared with vehicle injections (3.6 times; p<0.05). In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to a 3.1 fold increase of synovial thickening, compared with injection of vehicle (p<0.01), while LDL injections did not alter synovial thickening. Protein and RNA levels of chemokines CCL2 (Mcp-1) and CCL3 (Mip-1α) were significantly upregulated in macrophage-depleted joints after oxLDL injections (6.7 fold and 4.6 fold, respectively; p<0.01). FACS-analyses revealed increased presence of monocytes and polymorphonuclear granulocytes (PMN) in the synovium, which was confirmed with immunohistochemical staining for NIMP.R14. Also protein levels of S100A8/A9, markers for inflammation, were significantly increased in synovial wash-outs of oxLDL injected joints, compared with LDL (fold increase 5.6; p<0.05) or vehicle (fold increase 8.3; p<0.01) injection, as was NITEGE expression (fold increase 1.92; p<0.05). Interestingly, no raise in active TGF-β was measured in these macrophage-depleted joints. Conclusions: Synovial macrophages promote anabolic effects after oxLDL injections in knee joints, supporting earlier studies which show increased ectopic bone formation during LDL-rich conditions in experimental osteoarthritis. In absence of synovial macrophages, however, oxLDL induces monocyte and PMN influx, production of pro-inflammatory mediators and aggrecanase activity, thereby increasing catabolic activity.