Plem van Lent

A1.22 Interleukin-1 does not aggravate joint inflammation and cartilage destruction in experimental osteoarthritis

Background and objectives Osteoarthritis (OA) is a degenerative joint disease characterised by severe cartilage destruction, with a putative role for synovial macrophages. Recently it has become clear that over 50% of the patients also show low grade joint inflammation reflected by a thickened synovial lining and elevated production of cytokines like interleukin-1 (IL-1)by different cell types in the synovium. IL-1 mediates cartilage destruction by degrading existing proteoglycans and inhibiting new formation ofproteoglycans by stimulating degradative enzyme production. However, treatment of OA patients with IL-1 inhibitors has so far been disappointing. In this study we investigated the role of IL-1α/β in synovitis and cartilage destruction during experimental collagenase-induced OA (CiOA). Materials and methods Experimental OA was induced by intra-articular injection of collagenase into WT and IL-1αβ-/- mice. At day 7 after CiOA induction, total knee joints were stained with haematoxylin/eosin (H&E) to score synovial activation (arbitrary score from 0–3). Cartilage destruction at day 7 and 42 after induction of CiOA was determined in knee sections stained with safranin O/fast green using a modified Pritzker score. Gene expression in inflamed synovium was analysed using qPCR with primers for several pro- and anti-inflammatory cytokines. Results Synovitis in IL-1αβ-/- mice at day 7 of CiOAwas not different from WT controls (2.9 ± 0.2 and 2.7 ± 0.4 respectively). Absence of IL-1α/β had no effect on synovial mRNA levels of pro-inflammatory mediators KC, S100A8 and S100A9. IL-6 mRNA levels, however, were significantly down-regulated in the synovium of IL-1αβ-/- mice (3-fold reduction). The lack of IL-1α/β also did not affect gene expression of anti-inflammatory cytokines IL-10, TGFβ and iNOS. Cartilage destruction in CiOA clearly aggravated over time. Cartilage lesions in IL-1αβ-/- mice were not significantly different from WT controls at day 7. Also no difference in cartilage damage was measured at day 42 of CiOA when compared to WT (at the medial femur (95.3 ± 42.0 vs. 65.5 ± 54.5), medial tibia (84.5 ± 36.6 vs. 55.1 ± 38.9), lateral femur (69.6 ± 43.9 vs. 60.8 ± 35.8), and lateral tibia (49.8 ± 26.3 vs. 55.4 ± 17.1)). Conclusions IL-1β does not affect synovial inflammation nor cartilage destruction during collagenase-induced osteoarthritis, implicating that other macrophage-derived mediators are responsible for the joint damage.

A4.6 TGF-β is a potent inducer of nerve growth factor in articular cartilage via the ALK5-SMAD2/3 pathway. Potential role in OA related pain?

Background and objectives Pain is the main problem for patients with OA. Pain is linked to inflammation, but in OA a subset of patients suffer from pain without inflammation, indicating an alternative source of pain. NGF inhibition very efficiently blocks pain during OA, but the exact source of NGF is unclear. NGF is elevated in animal models of OA with cartilage damage without inflammation. TGF-beta is released from cartilage upon damage. We investigated whether TGF-beta could contribute to NGF expression in the joint and which signalling route is involved. Materials and methods Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-β1 and/or IL-1β as a positive control for NGF induction. We analysed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1 (oxozeaenol), Smad2/3 (SB-505124) or Smad1/5/8 (LDN-193189) signalling to identify the TGF-β pathway inducing NGF. Results TGF-beta consistently induced NGF expression at higher levels than IL-1 in all of our experimental models: murine, bovine and human origin, in cell lines, primary chondrocytes and explant cultures. We initially expected TAK1 to be dominant and pivotal in NGF induction due to involvement in both IL-1 and TGF-beta signalling. TAK1 inhibition consistently reduced (but not blocked) TGF-β-induced NGF whereas it fully blocked IL-1β-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-β-induced NGF expression. Blocking the Smad1/5/8 pathway did not affect NGF expression. Human OA primary chondrocytes had a large variation in basal NGF levels (mRNA and histology). TGF-β consistently induced a higher level of NGF induction than IL-1 in these chondrocytes despite variable basal levels of NGF when stimulating human OA cartilage explants with TGF-beta, NGF expression increased, which could be fully blocked by inhibiting ALK5-Smad2/3 signalling. Conclusions This is the first time that it is shown that TGF-β induces NGF expression in (primary) chondrocytes. Moreover, we found that TGF-beta consistently induced NGF at higher levels than IL-1. In addition, IL-1-induced NGF could be fully abrogated by TAK1 inhibition, but this only reduced TGF-beta-induced NGF expression. In contrast blocking signalling via ALK5-Smad2/3 fully abrogated the TGF-beta-induced NGF expression. Clearly, our data show that there are sources of NGF other than inflammation in the joint and that this should be taken into account when studying pain in OA.

Activating FCγ RECEPTORS mediate immune complex-induced inhibition of osteoclastogenesis

Background Rheumatoid arthritis is characterised by osteoclast-mediated bone loss. Co-stimulatory signalling via ITAM- and ITIM-coupled receptors is essential for osteoclast formation and function. The ITAM- and ITIM-coupled Fcγ receptors (FcγR) play a crucial role in mediating inflammation and cartilage destruction in experimental arthritis, but their role in osteoclast-mediated bone loss is unknown. Objectives To investigate the role of FcγRs in osteoclastogenesis and osteoclast function. Materials and methods Bone destruction was analysed in arthritic knee joints of FcγRIIB-deficient, FcRγ-chain−/− (lacking expression of activating FcγRs), and wild type mice. Bone marrow-derived osteoclast precursors were differentiated in vitro towards osteoclasts in the absence or presence of immune complexes (ICs) and stimulated thereafter for 24 h with or without TNFα or LPS. Additionally, mature osteoclasts were stimulated with ICs. Experiments were analysed for osteoclast formation, bone resorption, and the expression of FcγRs and osteoclast markers. Results Bone erosions and cathepsin K-positive osteoclast numbers were significantly increased during antigen-induced arthritis in the knee joints of FcγRIIB-deficient mice. All FcγR classes were highly expressed on bone marrow-derived osteoclast progenitors. On mature osteoclasts, as compared to macrophages, expression of the inhibitory FcγRIIB was similar, whereas expression of activating FcγRs was significantly lower. IC stimulation of mature osteoclasts neither affected their number nor their bone resorptive capacity. Differentiation of bone marrow-derived precursors in the presence of ICs significantly inhibited osteoclast formation, bone resorption, and expression of the osteoclast markers cathepsin K, CTR, DC-STAMP and NFATc1. In the presence of ICs, osteoclastogenesis of FcγRIIB−/− precursors and bone resorption remained inhibited. In contrast, ICs could not inhibit osteoclast formation or bone resorption of FcRγ-chain−/− precursors. When IC-inhibited osteoclastogenesis was followed by stimulation with TNFα or LPS, the inhibitory effects of ICs were overruled. Conclusions Activating FcγRs, but not the inhibitory FcγRIIB, mediate IC-induced inhibition of osteoclastogenesis. In the presence of pro-inflammatory mediators like TNFα and LPS the inhibitory effect might be overruled. This suggests that the balance of FcγR-mediated induction of inflammation, through pro-inflammatory cytokine production, as well as the direct inhibitory effect of ICs on osteoclastogenesis determines the net effect on bone loss.

A1.12 Local experimental osteoarthritis induces systemic changes in monocyte populations regulated by S100A8/A9

Inflammation is increasingly recognised to be involved in osteoarthritis (OA) pathology. In response to pro-inflammatory cytokines, monocytes can be recruited from the bone marrow (BM) where monocyte chemoattractant protein-1 (MCP-1) is a key molecule that drives monocyte efflux via binding with the C-C chemokine receptor type 2 (CCR2). In mice, two functionally distinct monocyte populations are described: pro-inflammatory Ly6C-high monocytes (CCR2high) and patrolling Ly6C-low monocytes (CCR2low). The objectives of our study are to investigate the systemic effects of locally induced OA on BM monocyte populations and their recruitment to the OA joint in collagenase induced OA (CiOA), and the role of the alarmins S100A8/A9 in that. CiOA was induced in C57BL/6 mice by unilateral-articular collagenase-injection and were sacrificed at day 7, 21 and 42, together with age-matched naive mice (n = 6/group), and synovial mRNA expression of several pro-inflammatory cytokines were measured. During CiOA, the absolute amount of cells in the BM per femur was measured and the mRNA expression of BM MCP-1 and CCR2 was determined. Cells from BM, blood and synovial tissue were isolated and analysed by FACS. Monocyte subsets were identified as (B220/CD90/CD49b/NK1.1/Ly6G)lowCD11bhigh(F4/80/MHCII/CD11c)low and further distinguished by their Ly6C expression. In addition, we investigated synovitis in S100A9-/-mice during CiOA. Synovial expression of IL-1β, IL-6, TNF-α, S100A8 and S100A9 was increased at day 7, but only expression of S100A8 and S100A9 remained high until day 21. Local induction of CiOA resulted in systemic effects within the BM showing decreased cell numbers at day 7 and 21 (15% and 14% respectively). Concurrently, the expression of MCP1 at day7 in BM was increased 3.3-fold, suggesting increased BM-efflux. Relative number of BM-Ly6C-high monocytes was increased at day 7 (164%), being reflected by increased CCR2 expression (2.8-fold). This suggests a specific regulation of BM Ly6C-high monocytosis and recruitment during CiOA. During the course of CiOA increased number of Ly6C-high monocytes were observed in the synovium: 398% at day 7 and 510% at day 42, compared to naïve mice. These effects may be caused by the sustained S100A8/A9 levels, we therefore investigated synovitis in S100A9-/- mice during CiOA. The number of cell layers and cell influx in the synovium of S100A9-/- mice was decreased compared to C57BL/6 mice Local induction of OA induces systemic release of BM-derived cells and increased Ly6C-high monocyte populations systemically and locally in the synovium, a process that may be regulated by the sustained release of S100A8/A9 from the synovium. Disclosure of Interest None declared.

A1.09 S100-damps in IL-1RA-/-mice, a serum biomarker andin vivoimaging tool to assess joint inflammation and destruction in experimental seronegative arthritis

Background and objectives Seronegative joint diseases are characterised by the lack of autoantibodies and rheumatoid factor, potent biomarkers for disease activity in seropositive arthritis. Promising alternative biomarkers in seronegative arthritis are the Damage Associated Molecular Patterns (DAMPs), S100A8 and S100A9, proteins specifically expressed by infiltrating phagocytes. In this study we explore the biomarker potential of S100A8/S100A9 to asses joint inflammation and damage in IL-1Ra-/- mice, a mouse model for seronegative arthritis in which serum autoantibodies are not correlated to disease activity. Materials and methods Serum of IL-1Ra-/- and BALB/c mice was collected every two weeks and swelling in the hind paws was macroscopically scored. Serum levels of IL-1β, IL-6 and TNFα were measured by Luminex technology and S100A8/A9 complex levels by ELISA. Hind paws were isolated and histologically scored for cell influx, cartilage damage and bone erosion, sections were also stained for S100A9 expression. Synovial S100A8 was imaged by injection of anti-S100A8-Cy7 antibody and 24 h p.i. fluorescent images were acquired in the IVIS Lumina optical imaging system. Results Starting at week 8 up to week 16, serum levels of S100A8/A9 in IL-1Ra-/- mice were significantly increased (1640 ± 1008 ng/ml) compared to BALB/c mice (429 ± 191 ng/ml, P = 0.005) and strongly correlated to joint swelling (r = 0.740, P < 0.0001) and microscopic parameters for joint pathology (r = 0.672–0.770, P < 0.0001), in contrast to levels of IL-1β, IL-6, and TNFα. Serum S100A8/A9 levels already correlated to joint swelling (r = 0.410, P = 0.047) as early as week 8 and high serum levels at week 10 were predictive for increased joint swelling at week 16 (r = 0.563, P = 0.004). Local expression of S100A9 within the synovium correlated to joint damage and serum S100A8/A9 levels and imaging of S100A8 using specific anti-S100A8-Cy7 antibody showed a specific targeting in inflamed joints of IL-1Ra-/- mice when compared to a control isotype (P = 0.044). Conclusions High levels of serum S100A8/A9 correlate to joint inflammation and destruction in experimental seronegative arthritis and local synovial expression of S100-DAMPs can be monitored in vivo by molecular imaging. These findings underline the potential of S100-DAMPs as a serum and imaging biomarker for disease severity in seronegative arthritis.

A1.14 Synovial macrophages promote TGF-β signalling but protect against influx of S100A8/S100A9-producing cells after intra-articular injections of oxidised low-density lipoproteins

Background and objectives LDL in inflamed synovium is oxidised and taken-up by macrophages, leading to an activated macrophage phenotype. In this study, we investigate whether injection of oxLDL directly into a murine knee joint induces joint pathology and elucidate the role of synovial macrophages in that process. Materials and Methods Synovium was obtained from end-stage OA patients. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (PBS). This procedure was repeated in mice depleted of synovial lining macrophages by intraarticular injection of clodronate liposomes seven days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production. Results Synovial macrophages and fibroblast of OA patients showed extensive accumulation apolipoprotein B, the main protein present in LDL and oxLDL. Multiple injections of oxLDL in murine knee joints significantly increased TGF-β activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of specifically oxLDL in macrophage-depleted knee joints led to increased synovial thickening. Furthermore, protein and RNA levels of CCL2 and CCL3 were significantly upregulated in macrophage-depleted joints after oxLDL injections and FCM-analyses revealed increased presence of monocytes and neutrophils in the synovium, which was confirmed by immunohistochemistry. Also protein levels of S100A8/A9 were significantly increased in synovial wash-outs of oxLDL-injected joints, as was expression of aggrecanase-induced neo-epitopes. Interestingly, no raise in active TGF-β was measured in macrophage-depleted joints. Conclusions Synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity in combination with increased influx of monocytes and neutrophils.

A10.08 Detailed analysis of the effect of cryopreservation on the viability and cytokine release of human synovial tissue

Background and objectives Human synovial tissue derived from joint surgery of osteoarthritis (OA) and rheumatoid arthritis (RA) patients is valuable material for studying fundamental research questions. It would be ideal to store and collect all this valuable tissue for later use. In this study cryopreservation solutions are validated for their capability to preserve the tissue as freshly obtained material. Materials and methods Human synovial OA tissue was derived (n = 4), with informed consent, from joint surgery and frozen as 3 mm biopsies. Cryosections were made to prove synovial origin. Biopsies were cryopreserved by slow freezing using several cryoprotectant (CPA) solutions (Cryostor CS2, Cryostor CS10, CryoSFM, Biofreeze and standard freezing medium containing 10% DMSO and 10% FCS). For slow freezing an isopropanol container was used assuring a temperature decrease of 1°C/min. Frozen tissues were stored for 7 days in liquid nitrogen. Thawing was performed at 37°C, followed by quick removal of the CPA solution. Results To asses general viability after thawing the tissue was cultured overnight. Both the ATP and XTT assays showed viability rates up to 90% of non frozen tissue. However, decreased viability (60%) was observed with higher concentrations of DMSO (CS10 and standard media, XTT) and the complete absence of DMSO (Biofreeze, ATP). Next, the RNA integrity number (RIN) was determined 24 h after thawing to ensure the RNA is suitable for gene array experiments. A high RIN (˜8) was found in all conditions. Stress genes (CASP3, HSPA1A, HSP27, MCL1, BAX, CD95, p53) described to be up/downregulated after cryopreservation did not show deviations from the non frozen tissue in the four donors tested. However, studying OA characteristic gene expression (TIMP3, CCL18, MMP9) showed that the Biofreeze medium had most changes (up/down) compared to non frozen tissue. Furthermore, we showed that the tissue was able to respond to inflammatory stimuli (Pam3Cys/LPS) as cytokine secretion (IL1b, TNFa, IL6, IL8) was highly upregulated. Conclusion Evaluation by two viability assays, stress – and disease characteristic gene expression, and cytokine secretion showed that during a short-time culture of human synovial tissue the disease characteristic phenotype was unchanged after cryopreservation.

A8.1 Alarmins S100A8/A9 Cause Osteophyte Formation in Experimental Osteoarthritis with High Synovial Involvement

Background and Objectives Osteophyte formation is an important hallmark of osteoarthritis (OA) causing limited joint movement and pain. There is increasing belief that synovial activation contributes to OA pathology. As shown recently in our lab, alarmins S100A8 and S100A9 (major products of synovial macrophages) are involved in cartilage degradation and synovial activation during human and murine OA. In the current study, we explored the involvement of S100A8/A9 in osteophyte formation in experimental OA. Methods Experimental OA was elicited in C57Bl/6 (WT) mice and S100A9-/- mice, which also lack functional S100A8. Collagenase induced OA (CIOA) was induced by two times intra-articular injection of 1U collagenase, DMM was induced by transsection of the medial anterior meniscotibial ligament leading to destabilisation of the medial meniscus (DMM). Osteophyte size was assessed by a blind observer using Leica Application Suite (LAS) imaging software. Chondrogenesis was induced by bringing human foetal mesenchymal stem cells (MSCs) in pellet and stimulating for 5 days with BMP-2 and TGFβ1, with or without human recombinant S100A8. Proteoglycan content was quantified using the LAS imaging software on SafO stained sections. Results First, we measured osteophyte size in S100A9-/- mice at day 42 of CIOA. Synovial activation is high in CIOA and this is significantly reduced in S100A9-/- mice. Osteophyte size was dramatically reduced in the S100A9-/- compared to WT in the medial collateral ligament (92.5% reduction) but also significantly at the medial side of both tibia and femur (68.2% and 64.6% reduction) (n = 10). One explanation for the reduced osteophyte size in S100A9-/- mice may be a direct effect of S100-proteins on chondrogenesis. To investigate this, we stimulated MSCs in pellet culture with BMP-2 and TGFβ1, supplemented with 1 and 5 µg/ml S100A8. Proteoglycan deposition as measured by redness in SafO staining was increased 27% and 71% respectively, indicating that S100A8 stimulates chondrogenesis. Finally, we determined osteophyte size in the DMM model, in which synovial involvement is very low. At day 56, we observed no significant differences in osteophyte size between the S100A9-/- and WT at the medial femur and tibia (105% and 136% of WT, n = 8). This confirms the importance of the synovium in the S100-effect on osteophyte development. Conclusions S100A8/S100A9 play a crucial role in osteophyte formation in an OA model that shows clear synovial involvement, probably by stimulating chondrogenesis. Considering also the deleterious effect of S100A8/A9 on joint destruction in OA, targeting these alarmins during OA may be very promising.

S100A8 and S100A9 induce a catabolic shift in chondrocytes from human osteoarthritis patients

Background and objective S100A8 and S100A9 are damage associated molecular patterns that are associated with inflammation and cartilage and bone erosion during human rheumatoid arthritis. They are produced in large amounts by monocytes and activated macrophages and can signal via Toll-like receptor 4. Recent studies in our lab show that S100A8 and S100A9 are also associated with cartilage degradation in experimental osteoarthritis (OA). In the present study, the authors investigated whether S100A8, S100A9 and/or S100A8/S100A9 complex could activate human chondrocytes and skew them towards a cartilage breakdown phenotype. Moreover, the authors wondered whether this catabolic shift was specific for chondrocytes from OA patients. Methods S100A8 and S100A9 protein detection was performed in human cartilage from OA and non-OA donors using immunohistochemistry. In adjoining slides specific matrix metalloproteinase (MMP) and aggrecanases cartilage breakdown epitopes, VDIPEN and NITEGE, were detected with immunohistochemistry. Human chondrocytes from OA and non-OA donors were isolated and stimulated with recombinant S100A8, S100A9 and S100A8/A9 heterodimer. mRNA levels of MMP, cytokines and cartilage matrix molecules were determined with qPCR and protein levels using Luminex. Results S100A8 and S100A9 proteins were abundantly present in and around chondrocytes derived from human cartilage as shown by immunolocalisation, suggesting that these small sized monomeric molecules (12 kD) are able to penetrate the cartilage matrix. Moreover, S100A8 and S100A9 were correlated with VDIPEN and NITEGE cartilage breakdown epitopes. In addition, S100A8 and S100A9, but not the S100A8/A9 heterodimer, upregulated expression of MMP1, −3, −9 and −13 (3- to 6-fold) and cytokines interleukin (IL)-6, IL-8 and MCP-1 (3- to 24-fold) in human OA chondrocytes, on both mRNA and protein level. Furthermore, S100A8 and S100A9 downregulated mRNA expression of matrix molecules aggrecan and collagen type II (2- to 3-fold). Finally, the authors compared the effects of S100A8 and/or S100A9 on OA chondrocytes with those on non-OA chondrocytes. The catabolic effect of S100A8 and S100A9 on MMP1, MMP3 and aggrecan was significantly higher in OA chondrocytes compared to non-OA. Conclusions S100A8 and S100A9 are found in and around human chondrocytes. S100A8 and S100A9, but not the heterodimeric S100A8/S100A9 complex, can skew human chondrocytes towards a catabolic phenotype promoting cartilage breakdown. This skewing seems to be specific for chondrocytes from OA patients. S100A8 and/or S100A9 may prove crucial markers for measuring cartilage destruction.

Activating FcγRIII determines cartilage destruction during immune complex arthritis but not in the presence of T-cell immunity

We have recently shown that activating Fcγ receptors determine metalloproteinase (MMP)-induced cartilage destruction, seen in various murine models of arthritis mediated by immune complexes (IC). In the mouse, two activating FcγR (FcγRI and FcγRIII) which bind IC have been described. In this study, we investigated the role of activating FcγRIII in MMP-mediated cartilage destruction in two different models of experimental arthritis, one induced only by ICs and the second by ICs and T cells.