S100A8/A9 is not essential for the development of inflammation and joint pathology in interleukin-1 receptor antagonist knockout mice.

BACKGROUND: Excessive osteoclast activity, which is strongly stimulated by pro-inflammatory mediators, results in bone and cartilage degeneration as central features of many arthritides. Levels of the alarmin S100A8/A9 and interleukin (IL)-1ß are both increased in arthritis patients and correlate with disease activity and progression of tissue erosion. We previously presented S100A8/A9 as a good biomarker for joint inflammation and arthritis pathology under circumstances of high IL-1 signaling in mice that lack the gene encoding IL-1 receptor antagonist (Il1rn-/- mice). Here, we investigated whether S100A8/A9 is also actively involved in the development of joint inflammation and both cartilage and bone pathology under these conditions by comparing Il1rn-/- mice with mice that have an additional deficiency for S100a9 (Il1rn-/-XS100a9-/-). METHODS: Il1rn-/-XS100a9-/- on a BALB/c background were obtained by crossing S100a9-/- mice and Il1rn-/- mice. Arthritis incidence and severity were macroscopically scored. Myeloid cell populations in the bone marrow and spleen were determined using flow cytometry. In vitro osteoclastogenesis of bone marrow cells was evaluated with TRAP staining. Microscopic joint inflammation, cartilage degeneration, and bone destruction were evaluated using histology of ankle joints of 12- and 20-week-old mice. RESULTS: Macroscopically scored arthritis severity was comparable between Il1rn-/- and Il1rn-/-XS100a9-/- mice. Inflammation, cartilage erosion, and bone erosion were clearly present in 12-week-old mice of both strains lacking Il1rn-/-, but not significantly different between Il1rn-/-XS100a9-/- and Il1rn-/-. Moreover, we observed that the numbers of neutrophils and monocytes were increased by the absence of Il1rn, which was affected by the absence of S100a9 only in the spleen but not in the bone marrow. In line with our other findings, the absence of S100a9 did not affect the osteoclastogenic potential of osteoclast precursors in the absence of Il1rn. Finally, in agreement with the findings in early arthritis development in 12-week-old mice, cartilage and bone erosion in 20-week-old mice was significantly higher in both Il1rn-/- strains, but the additional absence of S100a9 did not further affect tissue pathology. CONCLUSION: S100A8/A9 deficiency does not significantly affect inflammation and joint destruction in mice with high IL1ß signaling suggesting that S100A8/A9 is not essential for the development of arthritis under these conditions.

Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis.

BACKGROUND: Osteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA). METHODS: AIA was induced in knee joints of wild-type (WT), FcγRI,II,III-/-, and FcγRI,II,III,IV-/- mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR. RESULTS: FcγRI,II,III,IV-/- mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV-/- mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV-/- and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV-/- mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV-/- mice, AIA induction in knee joints of FcγRI,II,III-/- mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity. CONCLUSIONS: FcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint.

S100A8/A9 increases the mobilization of pro-inflammatory Ly6Chigh monocytes to the synovium during experimental osteoarthritis.

BACKGROUND: Monocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6Chigh and patrolling Ly6Clow monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6Chigh monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6Chigh and Ly6Clow monocytic populations to the inflamed joint in collagenase-induced OA (CiOA). METHOD: S100A8 was injected intra-articularly to investigate monocyte influx. CiOA was induced by injection of collagenase into knee joints of wild-type C57BL/6 (WT), and S100a9-/- mice. Mice were sacrificed together with age-matched saline-injected control mice (n = 6/group), and expression of monocyte markers, pro-inflammatory cytokines, and chemokines was determined in the synovium using ELISA and RT-qPCR. Cells were isolated from the bone marrow (BM), spleen, blood, and synovium and monocytes were identified using FACS. RESULTS: S100A8/A9 was highly expressed during CiOA. Intra-articular injection of S100A8 leads to elevated expression of monocyte markers and the monocyte-attracting chemokines CCL2 and CX3CL1 in the synovium. At day 7 (d7) after CiOA induction in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium compared to saline-injected controls. This coincided with strong upregulation of CCL2, which preferentially attracts Ly6Chigh monocytes. In contrast, S100a9-/- mice showed a significant increase in Ly6Clow monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6Chigh monocytes remained unaffected. In agreement with this finding, the Ly6Clow mobilization marker CX3CL1 was significantly higher within the synovium of S100a9-/- mice. Next, we studied the effect of S100A8/A9 on release of Ly6Chigh monocytes from the BM into the circulation. A 14% decrease in myeloid cells was found in WT BM at CiOA d7. No decrease in myeloid cells in S100a9-/- BM was found, suggesting that S100A8/A9 promotes the release of myeloid populations from the BM. CONCLUSION: Induction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6Chigh monocytes from the BM to the synovium.

S100A8/A9, a potent serum and molecular imaging biomarker for synovial inflammation and joint destruction in seronegative experimental arthritis.

BACKGROUND: Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. METHODS: Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)-/- mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs. RESULTS: Serum levels of S100A8/A9 were significantly increased in IL-1Ra-/- mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra-/- mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. CONCLUSIONS: Expression of S100A8 and S100A9 in IL-1Ra-/- mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.