A. Sloetjes

Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis

Objective: To study the active involvement of Myeloid-related proteins S100A8 and S100A9 in joint inflammation and cartilage destruction during antigen-induced arthritis (AIA). Methods: Joint inflammation and cartilage destruction was measured with 99mTc uptake and histology. The role of S100A8/A9 was investigated by inducing AIA in S100A9–/– mice that also lack S100A8 at protein level, or after intra-articular injection of rS100A8 in mouse knee joints. Cartilage destruction was measured using immunolocalisation of the neoepitope VDIPEN or NITEGE. mRNA levels of matrix metalloproteinases (MMPs) and cytokines were measured using reverse transcriptase (RT)-PCR. Results: Immunisation of S100A9–/– mice with the antigen mBSA induced normal cellular and humoral responses, not different from wild type (WT) controls. However, joint swelling measured at day 3 and 7 after AIA induction was significantly lower (36 and 70%, respectively). Histologically, at day 7 AIA, cellular mass was much lower (63–80%) and proteoglycan depletion from cartilage layers was significantly reduced (between 50–95%). Cartilage destruction mediated by MMPs was absent in S100A9–/– mice but clearly present in controls. MMP3, 9 and 13 mRNA levels were significantly lowered in arthritic synovia of S100A9–/–. In vitro stimulation of macrophages by the heterodimer S100A8/A9 or S100A8 elevated mRNA levels of MMP3, 9 and in particular MMP13. Intra-articular injection of S100A8 caused prominent joint inflammation and depletion of proteoglycans at day 1. Significant upregulation of mRNA levels of S100A8/A9, cytokines (interleukin 1 (IL1)), MMPs (MMP3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4) was found in the synovium and correlated with strong upregulation of NITEGE neoepitopes within the cartilage layers. Conclusions: S100A8/A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis.

S100A8 enhances osteoclastic bone resorption in vitro through activation of Toll-like receptor 4: implications for bone destruction in murine antigen-induced arthritis

OBJECTIVEnRheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA).nnnMETHODSnBone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed.nnnRESULTSnBone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors.nnnCONCLUSIONnThese results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.

Toll-like receptor 4 induced FcγR expression potentiates early onset of joint inflammation and cartilage destruction during immune complex arthritis: Toll-like receptor 4 largely regulates FcγR expression by interleukin 10

Objective: To study the role of Toll-like receptor (TLR)2 and 4 in the onset of joint inflammation and cartilage destruction during immune complex-mediated arthritis (ICA), and its relationship with FcγR expression. Materials and methods: ICA was induced in knee joints of TLR2−/− and TLR4−/− mice and their wild-type controls. Joint inflammation and cartilage destruction were measured in the knee joint using histology. mRNA levels were determined in synovial specimens and macrophages using quantitative polymerase chain reaction and cytokine protein levels in synovial washouts using Bioplex. Results: Joint inflammation and cartilage destruction were not different in arthritic TLR2−/− and wild-type mice. By contrast, at day 1 after ICA induction, joint swelling and proteoglycan depletion in knee joints of TLR4−/− mice were considerably lower (inflammation 68–79% and proteoglycan depletion 27–76%) when compared with wild-type controls. Cytokine production at this time point was markedly reduced in TLR4−/− mice (interleukin (IL)1, IL6, macrophage inflammatory chemokine (MIP)-1α and keratinocyte-derived chemokine 49%, 72%, 68% and 84%, respectively). In arthritic synovia of TLR4−/− mice, and also after injection of the antigen poly-l-lysine (PLL) lysozyme alone, mRNA levels of FcγR, and the FcγR regulating cytokine IL10 were considerably lower. Stimulation of peritoneal macrophages with PLL lysozyme up regulated mRNA levels of FcγR and IL10, whereas neutralisation by anti-IL10 antibodies largely blocked FcγR up regulation. At day 4, joint inflammation and cartilage destruction were comparable in TLR4−/− mice and wild-type controls. Conclusion: TLR4 regulates early onset of joint inflammation and cartilage destruction during ICA arthritis by up regulation of FcγR expression and enhanced cytokine production. TLR4-mediated up regulation of FcγR is largely mediated by IL10.

Alarmins S100A8/S100A9 aggravate osteophyte formation in experimental osteoarthritis and predict osteophyte progression in early human symptomatic osteoarthritis

Objective Alarmins S100A8 and S100A9 are major products of activated macrophages regulating cartilage damage and synovial activation during murine and human osteoarthritis (OA). In the current study, we investigated whether S100A8 and S100A9 are involved in osteophyte formation during experimental OA and whether S100A8/A9 predicts osteophyte progression in early human OA. Methods OA was elicited in S100A9−/− mice in two experimental models that differ in degree of synovial activation. Osteophyte size, S100A8, S100A9 and VDIPEN neoepitope was measured histologically. Chondrogenesis was induced in murine mesenchymal stem cells in the presence of S100A8. Levels of S100A8/A9 were determined in plasma of early symptomatic OA participants of the Cohort Hip and Cohort Knee (CHECK) cohort study and osteophytes measured after 2 and 5u2005years. Results Osteophyte size was drastically reduced in S100A9−/− mice in ligaments and at medial femur and tibia on days 21 and 42 of collagenase-induced OA, in which synovial activation is high. In contrast, osteophyte size was not reduced in S100A9−/− mice during destabilised medial meniscus OA, in which synovial activation is scant. S100A8 increased expression and activation of matrix metalloproteinases during micromass chondrogenesis, thereby possibly increasing cartilage matrix remodelling allowing for larger osteophytes. Interestingly, early symptomatic OA participants of the CHECK study with osteophyte progression after 2 and 5u2005years had elevated S100A8/A9 plasma levels at baseline, while C-reactive protein, erythrocyte sedimentation rate and cartilage oligomeric matrix protein were not elevated at baseline. Conclusions S100A8/A9 aggravate osteophyte formation in experimental OA with high synovial activation and may be used to predict osteophyte progression in early symptomatic human OA.

Induction of Canonical Wnt Signaling by Synovial Overexpression of Selected Wnts Leads to Protease Activity and Early Osteoarthritis-Like Cartilage Damage

Proteins from the Wnt signaling pathway are very important for joint development. Curiously, osteoarthritis (OA) is thought to be a recapitulation of developmental processes. Various members of the Wnt signaling pathway are overexpressed in the synovium during experimental OA. Here, we investigated the potency of specific Wnt proteins, when expressed in the synovium, to induce OA pathology. We overexpressed Wnt5a, Wnt8a, Wnt16, and WISP1 in the synovium using adenoviral vectors. We determined whether overexpression resulted in OA pathology by histology, and we measured whether Wnt signaling led to increased protease activity in the joint. Synovial overexpression of Wnt8a and Wnt16 led to canonical Wnt signaling in the cartilage, whereas overexpression of Wnt5a did not. Canonical Wnt signaling increased protease activity and induced cartilage damage shortly after overexpression. Specific blocking of the canonical Wnt signaling pathway with Dickkopf-1 reduced the Wnt-signaling-induced cartilage damage. By contrast, the noncanonical signaling Wnt5a did not cause cartilage lesions. Overexpression of WISP1, a downstream protein of canonical Wnt signaling, resulted in increased cartilage damage. In conclusion, our data show that canonical Wnts and WISP1, which we found overexpressed in the synovium during experimental OA, may conduce to OA pathology.

High LDL levels lead to increased synovial inflammation and accelerated ectopic bone formation during experimental osteoarthritis

OBJECTIVEnA relation between osteoarthritis (OA) and increased cholesterol levels is apparent. In the present study we investigate OA pathology in apolipoprotein E (ApoE)(-)(/-) mice with and without a cholesterol-rich diet, a model for high systemic low density lipoprotein (LDL) cholesterol levels independent of weight.nnnMETHODnWild type (WT), Apoe(-)(/-), S100a9(-/-) and Apoe(-)(/-)S100a9(-/-) mice (C57BL/6 background) received a standard or cholesterol-rich diet. Experimental OA was induced by intra-articular injection of collagenase and animals were sacrificed at day 10 and day 36.nnnRESULTSnAlthough minimal differences in cartilage damage were found between the WT and ApoE(-)(/-) mice, increased synovial thickening was found in the latter. Thirty-six days after OA-induction, ApoE(-)(/-) mice on a standard diet showed increased ectopic bone formation, particularly at the medial collateral ligament, compared with OA in WT mice. Furthermore, a significant increase in synovial gene expression of both S100a8 and S100a9 and S100A8/S100A9 protein levels was found in ApoE(-)(/-) mice, suggesting an activated inflammatory status of synovial cells. In both ApoE(-)(/-) and WT mice, addition of a cholesterol-rich diet resulted in excessive bone formation in the medial collateral ligament at late-time-point OA. Interestingly, at the early time point, proteoglycan deposition was already significantly increased in ApoE(-)(/-) mice compared with WT mice. Mice deficient for both ApoE and S100a9 also showed increased ectopic bone formation, but not synovial activation, suggesting a role for S100-proteins in cholesterol-mediated synovial activation.nnnCONCLUSIONSnIncreased cholesterol levels strongly elevate synovial activation and ectopic bone formation in early-stage collagenase-induced OA.

Scavenger receptor class A type I/II determines matrix metalloproteinase-mediated cartilage destruction and chondrocyte death in antigen-induced arthritis

OBJECTIVEnScavenger receptor class A type I (SR-AI) and SR-AII are expressed by macrophages in particular and bind and internalize a broad range of molecules (including endotoxins, apoptotic bodies, and oxidized low-density lipoprotein). This study was undertaken to investigate the role of SR-AI/II in mediating severe cartilage destruction in antigen-induced arthritis (AIA).nnnMETHODSnAIA was induced in the knee joints of SR-AI/II(-/-) mice and wild-type (WT) controls. Joint inflammation and cartilage destruction (chondrocyte death) were measured by examining the histology of total knee joints. Matrix metalloproteinase (MMP)-mediated neoepitopes were measured by immunolocalization using anti-VDIPEN antibodies and chondrocyte activation with anti-S100A8 antibodies. Messenger RNA (mRNA) levels were determined in inflamed synovium using microarray analysis and quantitative reverse transcriptase-polymerase chain reaction. In synovial washouts, cytokines (interleukin-1beta [IL-1beta], IL-10, and tumor necrosis factor alpha) and S100A8/S100A9 were measured using Luminex and enzyme-linked immunosorbent assay.nnnRESULTSnLevels of SR-AI/II mRNA were strongly elevated in inflamed synovium in AIA. On days 2, 8, and 14 after AIA induction, joint inflammation (exudates/infiltrate) was similar between the 2 groups. In WT mice, severe cartilage destruction was found in multiple cartilage surfaces of the inflamed knee joint on day 14 after AIA induction. MMP-mediated matrix destruction ranged between 40% and 60%, and chondrocyte death was prominent in 40-75% of the cartilage surfaces. In striking contrast, in SR-AI/II(-/-) mice, despite comparable joint inflammation, pronounced cartilage destruction was almost completely absent. Levels of IL-1beta and S100A8/S100A9 were significantly lower on days 7 and 14 after AIA induction, but levels of mRNA for various MMPs (MMP-2, MMP-3, MMP-9, and MMP-13) were comparable.nnnCONCLUSIONnOur findings indicate that SR-AI and SR-AII are crucial receptors involved in mediating severe cartilage destruction in AIA.

Induction of Canonical Wnt Signaling by the Alarmins S100A8/A9 in Murine Knee Joints: Implications for Osteoarthritis.

Both alarmins S100A8/A9 and canonical Wnt signaling have been found to play active roles in the development of experimental osteoarthritis (OA). However, what activates canonical Wnt signaling remains unknown. This study was undertaken to investigate whether S100A8 induces canonical Wnt signaling and whether S100 proteins exert their effects via activation of Wnt signaling.

Interleukin-1 is not involved in synovial inflammation and cartilage destruction in collagenase-induced osteoarthritis

OBJECTIVEnInterleukin-1 (IL-1) is an alleged important cytokine in osteoarthritis (OA), although the exact contribution of IL-1 to joint destruction remains unclear. Here we investigated the involvement of IL-1α and IL-1β in joint pathology during collagenase-induced OA (CiOA).nnnMETHODSnCiOA was induced in wild type (WT) and IL-1αβ-/- mice. Additionally, IL-1 signaling was inhibited in WT mice with CiOA using osmotic pumps containing IL-1RA. Joint pathology was assessed using histology. Activity of cartilage-degrading enzymes was determined using antibodies against aggrecan neo-epitopes VDIPEN and NITEGE. Synovial gene expression was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Serum protein levels were measured with Luminex or enzyme-linked immunosorbent assay (ELISA).nnnRESULTSnSynovial IL-1β expression was strongly elevated 7 days after induction of CiOA in WT mice but decreased afterwards, whereas S100A8/A9, previously described to aggravate OA, remained elevated for 21 days. Remarkably, synovial inflammation was comparable between WT and IL-1αβ-/- mice on day 7 of CiOA. In line, synovial mRNA expression of genes involved in IL-1 signaling and inflammatory mediators was comparable between WT and IL-1αβ-/- mice, and serum levels for Keratinocyte Chemoattractant (KC)/IL-6/S100A8/S100A9/IL-10 were equal. Synovial matrix metalloproteinase (MMP)/aggrecanase expression and activity in cartilage was not different in WT and IL-1αβ-/- mice on day 7 of CiOA. Cartilage destruction on day 42 was not different between WT and IL-1αβ-/- mice, which was supported by our finding that IL-1RA treatment in WT mice with CiOA did not alter joint destruction.nnnCONCLUSIONSnIL-1α and IL-1β are not involved in synovial inflammation and cartilage destruction during CiOA, implicating that other mediators are responsible for the joint damage.

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

BackgroundSeronegative 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.MethodsSerum 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.ResultsSerum 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.ConclusionsExpression 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.