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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.

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.

Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1β levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1β or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASC -CM) or IL-1β-stimulated ASCs (ASC -CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1β-stimulation of ASCs strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1β-stimulated ASCs. Also migration of PMNs toward ASC -CM was significantly enhanced (287%) when compared to ASC -CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASC -CM. Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1β-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.

BackgroundSynovitis-associated pain is an important aspect of arthritis pathology. Inflammatory mediators released by the synovium have been implicated in the regulation of pain, including S100A8 and S100A9, which may act via stimulation of TLR4 on the nerve endings in the synovium. In this study we investigated the role of S100A9 in the pain response after induction of an acute synovitis using streptococcal cell walls (SCW) as a trigger, comparing S100A9-/- mice and their WT controls.Materials and methodsAcute synovitis was induced by a single i.a. SCW injection in the knee joint of C57Bl6 (WT) and S100A9-/- mice. Serum S100A8/A9 levels were investigated by ELISA and S100A8 and S100A9 expression in synovium by immunohistochemistry. Joint swelling and cell influx was assessed by 99mTc accumulation and histology, respectively. Pain response was investigated by Incapacitance Tester (weight bearing), Catwalk (gait analysis) and von Frey’s filaments (mechanical allodynia). Gene expression of neuron activation markers in dorsal root ganglia (DRG) were determined by q-PCR.ResultsA single i.a. SCW injection resulted in increased synovial expression of S100A8 and S100A9 as well as significant increased serum S100A8/A9 levels (2.6-fold, p<0.001) 1 day p.i.. These increased levels however, did not contribute to the development of inflammation since joint swelling and cell influx were similar in both mice strains. WT mice showed a significant decrease in percentage of weight bearing on the SCW hindpaw (28%, p<0.001) 1 day p.i., while S100A9-/- mice showed no reduction. In line with that, gait analysis showed that the stand-phase of the unaffected paws was significantly increased in WT mice 1 day p.i., but not in S100A9-/- mice. No difference in mechanical allodynia was observed, both mouse strains showed a similar reduction of paw withdrawal threshold. Gene expression of neuron activation markers NAV1.7, ATF3 and GAP43 in DRG were significantly increased in SCW injected WT mice 1 day p.i (p=0.022, 0.004 and 0.030, respectively) but not in S100A9-/- mice.ConclusionsThese findings show that S100A9, which is released from the synovium upon inflammation, is an important mediator of pain response in the knee during the acute phase of inflammation.

Osteoarthritis (OA) is a destructive disease of the joint with age and obesity being its most important risk factors. Around 50% of OA patients suffer from inflammation of the synovial joint capsule, which is characterized by increased abundance and activation of synovial macrophages that produce reactive oxygen species (ROS) via NADPH-oxidase 2 (NOX2). Both ROS and high blood levels of low-density lipoprotein (LDL) are implicated in OA pathophysiology, which may interact to form oxidized LDL (oxLDL) and thereby promote disease. Therefore, targeting NOX2 could be a viable treatment strategy for OA. Collagenase-induced OA (CiOA) was used to compare pathology between wild-type (WT) and Nox2 knockout (Nox2−/−) C57Bl/6 mice. Mice were either fed a standard diet or Western diet (WD) to study a possible interaction between NOX2-derived ROS and LDL. Synovial inflammation, cartilage damage and ectopic bone size were assessed on histology. Extracellular ROS production by macrophages was measured in vitro using the Amplex Red assay. Nox2−/− macrophages produced basal levels of ROS but were unable to increase ROS production in response to the alarmin S100A8 or the phorbol ester PMA. Interestingly, Nox2 deficiency reduced cartilage damage, synovial lining thickness and ectopic bone size, whereas these disease parameters were not affected by WD-feeding. These results suggest that NOX2-derived ROS are involved in CiOA development.

BackgroundSynovitis-associated pain is an important aspect of arthritis pathology. Inflammatory mediators released by the synovium have been implicated in the regulation of pain, including S100A8 and S100A9, which may act via stimulation of TLR4 on the nerve endings in the synovium. In this study we investigated the role of S100A9 in the pain response after induction of an acute synovitis using streptococcal cell walls (SCW) as a trigger, comparing S100A9-/- mice and their WT controls.Materials and methodsAcute synovitis was induced by a single i.a. SCW injection in the knee joint of C57Bl6 (WT) and S100A9-/- mice. Serum S100A8/A9 levels were investigated by ELISA and S100A8 and S100A9 expression in synovium by immunohistochemistry. Joint swelling and cell influx was assessed by 99mTc accumulation and histology, respectively. Pain response was investigated by Incapacitance Tester (weight bearing), Catwalk (gait analysis) and von Frey's filaments (mechanical allodynia). Gene expression of neuron activation markers in dorsal root ganglia (DRG) were determined by q-PCR.ResultsA single i.a. SCW injection resulted in increased synovial expression of S100A8 and S100A9 as well as significant increased serum S100A8/A9 levels (2.6-fold, p<0.001) 1 day p.i.. These increased levels however, did not contribute to the development of inflammation since joint swelling and cell influx were similar in both mice strains. WT mice showed a significant decrease in percentage of weight bearing on the SCW hindpaw (28%, p<0.001) 1 day p.i., while S100A9-/- mice showed no reduction. In line with that, gait analysis showed that the stand-phase of the unaffected paws was significantly increased in WT mice 1 day p.i., but not in S100A9-/- mice. No difference in mechanical allodynia was observed, both mouse strains showed a similar reduction of paw withdrawal threshold. Gene expression of neuron activation markers NAV1.7, ATF3 and GAP43 in DRG were significantly increased in SCW injected WT mice 1 day p.i (p=0.022, 0.004 and 0.030, respectively) but not in S100A9-/- mice.ConclusionsThese findings show that S100A9, which is released from the synovium upon inflammation, is an important mediator of pain response in the knee during the acute phase of inflammation.

Introduction In a previous study, we showed that LDL accumulation by LDL receptor deficient mice resulted in increased ectopic bone formation during experimental osteoarthritis (OA). Furthermore, we found that S100A8/A9 proteins are crucial in mediating joint pathology during experimental OA. In the present study we investigate OA pathology and its correlation with S100A8/9 in ApoE-/- mice, which is a different model for studying effects of systemically high LDL cholesterol levels. Material and Methods Wild type (WT) and ApoE deficient (ApoE-/-) mice received a normal or cholesterol-rich diet for 54 days. At day 18, experimental OA was induced by intra-articular injection of collagenase and animals were sacrificed at day 28 and 54. Results ApoE-/- mice on a normal diet showed remarkably higher LDL levels than WT mice (8.90 mmol/L and 0.40 mmol/L, respectively; p<0.0001). Experimental OA in ApoE-/- mice showed no increase in synovial thickening and ectopic bone formation, but a significant increase of cartilage damage was found in ApoE-/- mice compared to WT mice at the lateral side of the femoral chondyle (OARSI score 13.7 and 6.8, respectively; p<0.05). Synovial gene expression of both S100A8 and S100A9 was significantly increased in ApoE-/- mice compared to WT mice (fold increase 1.8 and 1.4, respectively; p<0.05). Furthermore, S100A8/S100A9 protein levels of synovial wash-outs was increased in ApoE-/- mice at day 28 (fold increase 5.8; p<0.05), which was confirmed by immunohistochemical staining for S100A8. In addition, we investigated whether a cholesterol-rich diet could increase joint pathology after induction of OA. This diet increased differences in LDL levels even more (18.4 mmol/L in ApoE-/- mice versus 1.2 mmol/L in WT mice; p<0.0001) and already at day 28, histological differences between the two groups were observed. Synovial thickening was increased by 400% in ApoE-/- mice compared to WT mice (p<0.001) and also ectopic bone formation in the medial collateral ligament was strongly increased at this early time point (fold increase 2.7; p<0.01). Cartilage damage, however, was comparable to damage observed in mice on a normal diet. Again, S100A8 and S100A9 levels were strongly increased in ApoE-/- mice, both on protein and gene expression level and significantly correlated with ectopic bone formation. Conclusion LDL cholesterol accumulation by ApoE deficiency results in increased S100A8 and S100A9 production by synovial cells. A cholesterol-rich diet further increases this production which correlates with increased synovial thickening and ectopic bone formation in experimental OA. This suggests a important role for LDL cholesterol in developing OA joint pathology.

Background Monocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6C high and patrolling Ly6C low monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6C high monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6C high and Ly6C low 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 Ly6C high , but not Ly6C low 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 Ly6C high monocytes. In contrast, S100a9 -/- mice showed a significant increase in Ly6C low monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6C high monocytes remained unaffected. In agreement with this finding, the Ly6C low mobilization marker CX3CL1 was significantly higher within the synovium of S100a9 -/- mice. Next, we studied the effect of S100A8/A9 on release of Ly6C high 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 Ly6C high monocytes from the BM to the synovium.

IgG-containing immune complexes, which are found in most RA joints, communicate with hematopoietic cells using three classes of Fc receptors(FcγRI, -II, -III). In a previous study we found that if a chronic T-cell-mediated antigen-induced arthritis (AIA) was elicited in knee joints of FcR γ-chain-deficient mice that lack functional FcγRI and FcγRIII, joint inflammation was comparable but severe cartilage destruction was absent. We now examined the individual role of the stimulatory FcγRI and FcγRIII and inhibitory FcγRII in inflammation and functional cartilage damage in knee joints with AIA using FcγRI-, FcγRII-, and FcγRIII-deficient mice. Three weeks after immunization with the antigen-methylated bovine serum albumin (BSA), cellular (T-cell responses as measured by lymphocyte proliferation) immunity raised against mBSA was comparable in all groups examined. Humoral (total IgG, IgG1, IgG2a, and IgG2b levels) immunity against mBSA was comparable in FcγRI−/− and FcγRIII−/− but higher in FcγRII−/− if compared to controls. Joint swelling as measured by 99mTc uptake at days 1, 3, and 7 was similar in FcγRI−/− and FcγRIII−/− mice and significantly higher in FcγRII−/−. Chronic inflammation and cartilage damage (depletion of proteoglycans, metalloproteinase (MMP)-induced neoepitopes, and matrix erosion) was studied histologically in total knee joint sections stained with hematoxylin or safranin-O. Histologically, at day 7 after AIA induction, exudate and infiltrate in the knee joint was similar in FcγRI−/− and FcγRIII−/− and significantly higher (230% and 340%) in FcγRII−/− mice if compared to controls. Aggrecan breakdown in cartilage caused by MMPs and, which is related to severe irreversible cartilage erosion, was further studied by immunolocalization of MMP-mediated neoepitopes (VDIPEN) and image analysis. MMP-induced neoepitopes determined in various cartilage layers (tibia and femur) were primarily inhibited in FcγRI−/− (79 to 87% and 87 to 88%, respectively) and comparable in FcγRIII−/−. VDIPEN neoepitopes were much higher (82 to 122% and 200 to 250%, respectively) in FcγRII−/− mice. Initial depletion of proteoglycans was similar (60 to 100%) in all groups. In the chronic phase, cartilage matrix erosion in the lateral and medial tibia was significantly elevated in FcγRII−/− (222% and 186%, respectively) but not in FcγRI−/− or FcγRIII−/− mice. These results suggest that during T-cell-mediated AIA, FcγRI and FcγRIII act in concert in acute and chronic inflammation whereas FcγRI is the dominant FcR involved in severe cartilage destruction. FcγRII is a crucial inhibiting factor in acute and chronic inflammation and cartilage erosion.

Monocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6C and patrolling Ly6C monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6C monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6C and Ly6C monocytic populations to the inflamed joint in collagenase-induced OA (CiOA). 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. 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 Ly6C , but not Ly6C 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 Ly6C monocytes. In contrast, S100a9 mice showed a significant increase in Ly6C monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6C monocytes remained unaffected. In agreement with this finding, the Ly6C mobilization marker CX3CL1 was significantly higher within the synovium of S100a9 mice. Next, we studied the effect of S100A8/A9 on release of Ly6C 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. Induction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6C monocytes from the BM to the synovium.