J. Bleil

In Situ Analysis of Interleukin–23– and Interleukin‐12–Positive Cells in the Spine of Patients With Ankylosing Spondylitis

OBJECTIVE The interleukin-12 (IL-12) family of cytokines has been suggested to play a critical role in inflammatory autoimmune diseases, and recent studies analyzing peripheral blood and synovial fluid from patients with spondyloarthritides suggest that IL-23 might be a proinflammatory factor in these disorders. This study was undertaken to investigate the presence and source of IL-23 in the spines of patients with ankylosing spondylitis (AS). METHODS The frequency of IL-23-positive and IL-12-positive cells within the subchondral bone marrow and within fibrous tissue replacing normal bone marrow in facet joints of patients with AS was analyzed by immunohistochemistry. The origin of IL-23-positive cells was determined by double staining of CD163+ macrophages, CD68+ macrophages, CD1a+ dendritic cells, tryptase-positive mast cells, myeloperoxidase-positive cells, CD20+ B cells, and CD3+ T cells. Findings in 28 facet joints from 22 AS patients were compared with those in 20 facet joints from 13 patients with osteoarthritis (OA) and 10 normal control specimens. RESULTS The frequency of IL-23-positive cells in subchondral bone marrow from the joints of AS patients (mean ± SD 42.50 ± 32.81/high-power field [hpf]) was significantly increased compared to that in subchondral bone marrow from OA patients (OA 15.63 ± 29.90/hpf) (P = 0.0017) or controls (19.36 ± 16.8/hpf) (P = 0.03). Myeloperoxidase-positive cells and, to a lesser extent, macrophages and dendritic cells were found to be the origin of IL-23 in the bone marrow. In AS and OA patients, the frequency of IL-23-positive cells was significantly higher than that of IL-12-positive cells (P < 0.001 in both patient groups). Within fibrous tissue from AS and OA facet joints, IL-23 was predominantly produced by CD163+ macrophages (mean ± SD 0.64 ± 0.59/hpf and 4.36 ± 3.4/hpf, respectively) and CD68+ macrophages (2.3 ± 0.65/hpf and 6.54 ± 4.1/hpf, respectively). CONCLUSION IL-23 is expressed in the subchondral bone marrow and in fibrous tissue replacing bone marrow in facet joints of patients with AS. It might have a role in inflammatory processes and in chronic changes in AS joints, which makes it an interesting potential therapeutic target in this disease.

Histomorphologic and Histomorphometric Characteristics of Zygapophyseal Joint Remodeling in Ankylosing Spondylitis

To unravel the mechanisms that control bony ankylosis in ankylosing spondylitis (AS).

Cartilage in facet joints of patients with ankylosing spondylitis (AS) shows signs of cartilage degeneration rather than chondrocyte hypertrophy: implications for joint remodeling in AS

IntroductionIn ankylosing spondylitis (AS), joint remodeling leading to joint ankylosis involves cartilage fusion. Here, we analyzed whether chondrocyte hypertrophy is involved in cartilage fusion and subsequent joint remodeling in AS.MethodsWe assessed the expression of chondrocyte hypertrophy markers runt-related transcription factor 2 (Runx2), type X collagen (COL10), matrix metalloproteinase 13 (MMP13), osteocalcin and beta-catenin and the expression of positive bone morphogenic proteins (BMPs) and negative regulators (dickkopf-1 (DKK-1)), sclerostin, (wingless inhibitory factor 1 (wif-1)) of chondrocyte hypertrophy in the cartilage of facet joints from patients with AS or osteoarthritis (OA) and from autopsy controls (CO) by immunohistochemistry. Sex determining region Y (SRY)-box 9 (Sox9) and type II collagen (COL2) expression was assessed as indicators of chondrocyte integrity and function.ResultsThe percentage of hypertrophic chondrocytes expressing Runx2, COL10, MMP13, osteocalcin or beta-catenin was significantly increased in OA but not in AS joints compared to CO joints. Frequencies of sclerostin-positive and DKK-1-positive chondrocytes were similar in AS and CO. In contrast, wif-1- but also BMP-2- and BMP-7-expressing and Sox9-expressing chondrocytes were drastically reduced in AS joints compared to CO as well as OA joints whereas the percentage of COL2-expressing chondrocytes was significantly higher in AS joints compared to CO joints.ConclusionsWe found no evidence for chondrocyte hypertrophy within hyaline cartilage of AS joints even in the presence of reduced expression of the wnt inhibitor wif-1 suggesting that chondrocyte hypertrophy is not a predominant pathway involved in joint fusion and remodeling in AS. In contrast, the reduced expression of Sox9, BMP-2 and BMP-7 concomitantly with induced COL2 expression rather point to disturbed cartilage homeostasis promoting cartilage degeneration in AS.

Granulation Tissue Eroding the Subchondral Bone Also Promotes New Bone Formation in Ankylosing Spondylitis.

We previously suggested that fibroblast‐rich granulation tissue eroding the subchondral bone is instrumental in the joint remodeling that occurs in ankylosing spondylitis (AS). The purpose of this study was to determine if this granulation tissue also carries bone‐forming capabilities, which we approached by searching for bone‐forming cells (hypertrophic chondrocytes, osteoblasts) in its vicinity. We also assessed adipogenic tissue transformation, which has been suggested to be an intermediate feature in AS bone formation based on imaging studies.

In situ analysis of interleukin-6 expression at different sites of zygapophyseal joints from patients with ankylosing spondylitis in comparison to controls.

Objectives: Analysis of interleukin (IL)-6 serum levels in patients with ankylosing spondylitis (AS) has indicated that IL-6 might be a pro-inflammatory cytokine involved in AS. However, two placebo-controlled trials with monoclonal antibodies directed against the IL-6 receptor have failed to demonstrate the efficacy of the monoclonal humanized anti-human IL-6 receptor antibody over placebo for the treatment of symptoms of AS. In this study we conducted an in situ analysis of IL-6 expression at different sites of inflammation in zygapophyseal joints of patients with AS in comparison to osteoarthritis autopsy controls (CO). Method: Our immunohistochemical analysis involved 14 patients with AS, 12 autopsy controls (CO), and 11 patients with osteoarthritis (OA). Immunohistochemistry was performed to detect IL-6+ cells at five different sites: within subchondral bone marrow, fibrous tissue replacing subchondral bone marrow, hyaline cartilage, and the subchondral bone plate, and at entheseal sites. Results: Apart from changes in subchondral bone marrow, no significant differences were observed at the sites analysed when comparing AS patients and controls. A significantly lower frequency of IL-6+ cells was evident in AS patients compared to controls (p = 0.0043). In addition, AS patients tended to have even lower percentages of IL-6+ cells than controls at subchondral bone plates and entheseal sites. A significantly lower number of IL-6 expressing cells was also seen within the fibrous tissue of AS compared to OA patients (p = 0.0237). Conclusions: This in situ analysis confirms that IL-6 is not a key player in the pathogenesis of inflammatory processes in spondyloarthritides (SpA). The relevance of pro-inflammatory agents in axial SpA might be studied better in situ in bony specimens at the primary site of inflammation.

Anti-RANKL treatment inhibits erosive joint destruction and lowers inflammation but has no effect on bone formation in the delayed-type hypersensitivity arthritis (DTHA) model.

BackgroundThe aims of the present study were to determine the relationship between bone destruction and bone formation in the delayed-type hypersensitivity arthritis (DTHA) model and to evaluate the effect of receptor activator of nuclear factor κB ligand (RANKL) blockade on severity of arthritis, bone destruction, and bone formation.MethodsDTHA was induced in C57BL/6 mice. Inflammation, erosive joint damage, and new bone formation were semiquantitatively scored by histology. Osteoclast activity was assessed in vivo, and messenger RNA (mRNA) expression of mediators of bone destruction and bone formation were analyzed by mRNA deep sequencing. Serum concentrations of tartrate-resistant acid phosphatase 5b, carboxy-terminal telopeptide I (CTX-I), matrix metalloproteinase 3 (MMP3), and serum amyloid P component (SAP) were determined by enzyme-linked immunosorbent assay. Anti-RANKL monoclonal antibody treatment was initiated at the time of immunization.ResultsBone destruction (MMP3 serum levels, cathepsin B activity, and RANKL mRNA) peaked at day 3 after arthritis induction, followed by a peak in cartilage destruction and bone erosion on day 5 after arthritis induction. Periarticular bone formation was observed from day 10. Induction of new bone formation indicated by enhanced Runx2, collagen X, osteocalcin, MMP2, MMP9, and MMP13 mRNA expression was observed only between days 8 and 11. Anti-RANKL treatment resulted in a modest reduction in paw and ankle swelling and a reduction of serum levels of SAP, MMP3, and CTX-I. Destruction of the subchondral bone was significantly reduced, while no effect on bone formation was seen.ConclusionsAnti-RANKL treatment prevents joint destruction but does not prevent new bone formation in the DTHA model. Thus, although occurring sequentially during the course of DTHA, bone destruction and bone formation are apparently not linked in this model.

FRI0425 Analysis of cellular composition and cytokine expression in the subchondral bone marrow in ankylosing spondylitis

Background Ankylosing spondylitis (AS) is characterized by inflammation within the sacroiliac joints and at the spine including vertebral bodies and facet joints. In AS, bone destruction is followed by new bone formation leading to ankylosis of joints and syndesmophyte development. Histological studies of bone material from AS patients showed subchondral bone marrow changes, namely the transition of the bone marrow into granulation tissue which facilitates subchondral bone destruction but also promotes local bone formation [1, 2]. Currently, it is unclear what promotes the transition of the bone marrow into the granulation tissue. Objectives The aim of this study was to look for changes in the subchondral bone marrow in joints from AS patients that may precede and promote transformation into granulation tissue. Therefore, we analyzed the cellular composition of the bone marrow and determined local cytokine expression at subchondral regions of AS facet joints. Methods Facet joints were acquired from AS patients undergoing polysegmental correction surgery and compared to joints from autopsy controls. We performed immunohistochemical stainings to determine the number of T cells (CD3) and B cells (CD20), macrophages (CD68, CD163), monocytes (CD14), dendritic cells (DC; CD1a, DCsign) and myelopoietic cells (MPO). To correct for putative differences in fat cell content, software assisted image analysis was used to calculate the area of bone marrow covered by fat cells and the number of nucleated cells (DAPI) as well as myeloid cells. Cytokine expression was determined by mRNA in situ hybridization (TNF, IFNg) or immunohistochemistry (TGFβ, IL-10). Results We observed no difference in the number of adaptive immune cells, i.e. CD3+ T and CD20+ B cells, between AS and control joints and found no difference in T/B cell aggregates. Furthermore, no difference in the number of macrophages (CD163+, CD68+) and DCs (CD1a+, DCsign+) was found. The only difference in cellular composition appeared to be a slight trend towards a higher percentage of myelopoietic MPO+ cells/DAPI+ cells in AS joints compared to control joints. The overall number of DAPI+ cells as well as the fat content of the bone marrow was not different between AS and control joints. Concerning cytokines, TNF mRNA expression was in general low but significantly increased (p<0.05) in AS versus control joints. No difference was found for IFNg. The number of TGFβ expressing cells was similar in AS and control joints while the number of IL-10 positive cells was decreased (p<0.01) in AS joints. Conclusions The results show a shift from pro-inflammatory to anti-inflammatory cytokine expression within subchondral bone marrow sites in AS joints while no major change in the cellular distribution of the major leukocyte subsets is found. The shift in cytokine milieu may contribute to transformation of the bone marrow into granulation tissue. It would be interesting to determine the cellular source of local cytokines, such as of IL-10. References Cruickshank B. Lesions of cartilaginous joints in ankylosing spondylitis. The Journal of pathology and bacteriology 1956;71(1):73–84. Bleil J, Maier R, Hempfing A, Sieper J, Appel H, Syrbe U. Granulation Tissue Eroding the Subchondral Bone Also Promotes New Bone Formation in Ankylosing Spondylitis. Arthritis Rheumatol 2016;68(10):2456–65. Disclosure of Interest None declared

AB0110 Anti-Rankl Treatment Inhibits Erosive Joint Destruction But Has No Effect on Bone Formation in the DTH Arthritis Model

Background The delayed-type hypersensitivity arthritis (DTHA) model combines collagen type II antibody (anti-CII) treatment with local DTH induction in the footpad [1] which induces a strong arthritis accompanied by bone destruction and excessive peri-articular bone formation. Objectives Here we analyzed the impact of osteoclasts on inflammation, joint damage and bone formation in the DTHA model. Methods C57BL/6 mice were immunized with methylated BSA (mBSA) in CFA, given 1000 μg anti-CII four days later [1] and challenged seven days after immunization with 200 μg mBSA in PBS in one hind foot pad and with 20 μl PBS only in the other foot pad (control). Osteoclast activity was determined in vivo by fluorescence imaging using the Cat K 680 FAST probe. Receptor-activator of nuclear factor kappa B (RANKL), TRAP and Cathespin K (CatK) mRNA expression was analyzed in arthritic paw tissue by mRNA deep sequencing. TRAP5b, CTX-I and CTX-II serum levels were determined by ELISA. In situ expression of tartrate-resistant acid phosphatase (TRAP), collagen 10 (COL10) and osteocalcin was detected by immunohistochemical staining. Inflammation, erosive joint damage and peri-articular bone formation were semi-quantitatively scored. Results In vivo imaging of osteoclast activity showed increased CatK activity on days 3 and 8 after arthritis induction in the arthritic paw. RANKL mRNA expression peaked on d3 while TRAP and CatK mRNA expression peaked on d8. Erosive joint damage was driven from the subchrondal bone marrow side. Here, TRAP-positive osteoclasts were located at the borders of a granulation tissue which eroded the subchondral bone plate. Peri-articular bone formation involved COL10 expressing hypertrophic chondrocytes as well as osteocalcin–expressing osteoblasts. Treatment with anti-RANKL antibody did not affect footpad swelling (p>0.05) but reduced the serum levels of TRAP5b (p<0.001) and CTX-I (p<0.001) and prevented the destruction of the subchondral bone plate (p<0.001) and development of the invasive granulation tissue (p<0.001) at subchondral sites. Scores of intra- and peri-articular inflammation and scores of peri-articular bone formation did not change (p>0.05). Conclusions Inhibition of osteoclast differentiation prevents joint destruction in the DTHA model driven by the subchondral granulation tissue while leaving intra-articular and peri-articular inflammation and bone formation unaffected. Osteoclast activity might be targeted to prevent erosive damage driven from subchondral sites as for instance in sacroiliacal joints of patients with spondyloarthritis. Disclosure of Interest None declared

FRI0193 Osteoblasts Promote New Bone Formation at Sites of Cartilage and Bone Erosions While Fat Marrow is Found Within the Subchondral Bone Marrow in Ankylosing Spondylitis

Background Ankylosing spondylitis (AS) is characterized by erosive bone damage followed by bone formation. A recent MRI study suggested that ankylosis of sacroiliac joints develops following repair of erosions with fat metaplasia and backfill as intermediary steps in this pathway [1]. Objectives In this study we used histological analysis of facet joints of AS patients to elucidate the mechanism of bone formation and the potential role of adipogenic tissue transformation. Methods Sections of facet joints which can also undergo ankylosis in AS were used from patients with AS, patients with osteoarthritis (OA) and from controls (CO). In safranin O stained tissue sections, the subchondral bone marrow area and sites of erosions were evaluated with regard to replacement by fibrous tissue, a pathological feature described before, and adipogenic tissue transformation. To identify putative bone forming cells we analyzed the expression of Runx2 a shared marker of chondrocytes and osteoblasts, and collagen type X and MMP13 as specific markers of hypertrophic chondrocytes as well as CD56 and collagen 1 as markers of osteoblasts within eroded areas of the subchondral endplate and the articular cartilage. Results Within the subchondral bone marrow adipogenic tissue transformation as well as fibrous transformation was found: 10/16 (62.5%) of CO joints, 18/29 (62%) of AS joints and 16/17 (94%) of OA joints showed replacement of bone marrow by fat marrow of various extend (up 100% of the bone marrow area/slide) while 0/16 (0%) of control joints, 12/29 (41.4%) of AS joints and 14/17 (82%) of OA joints showed transformation into fibrous tissue. Among AS joints, replacement of bone marrow by fibrous tissue was found more often in joints with cartilaginous fusion (5/6) compared to joints with bony fusion (4/15, p<0.05) while there was no difference for the incidence of fat marrow (p>0.05). At sites of subchondral bone and cartilage erosion we always found fibrous tissue. Runx2+ cells were detectable at the borders of fibrous tissue in AS and OA joints. The lack of collagen type X and MMP13 expression but osteocalcin and collagen type I expression of these cells proved the osteoblastic nature of these cells. Formation of new bone was found at contact areas between fibrous tissue and eroded articular cartilage – a phenomenon occurring more often in AS joints than in OA joint, i.e. in 92% of AS joints and 27% of OA joints (p<0.003) with fibrous tissue-cartilage contacts. Conclusions In AS, osteoblasts lining the fibrous tissue facilitate bone formation at sites of bone and cartilage erosions which promotes joint ankylosis. Fat marrow is rather found within the adjacent subchondral bone marrow. References Maksymowych WP, Wichuk S, Chiowchanwisawakit P, Lambert RG, Pedersen SJ: Fat metaplasia and backfill are key intermediaries in the development of sacroiliac joint ankylosis in patients with ankylosing spondylitis. Arthritis Rheumatol 2014, 66(11):2958-2967. Disclosure of Interest None declared

SAT0559 High Expression of Cyclooxygenase-2, Prostaglandin E2 and Prostaglandin E2 Receptor Ep4 in Zygapophyseal Joints of Patients with Ankylosing Spondylitis

Background Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment for patients with ankylosing spondylitis (AS). They are effective in reducing signs and symptoms, and increasing evidence suggests that their sustained use may also retard radiographic progression [1, 2]. The equal efficacy of cyclooxygenase (COX)-2 selective inhibitors in AS suggests that COX-2-dependent PGE2 synthesis is involved in the pathogenesis of AS. Objectives We studied here the expression of COX-2 and PGE2 and of the PGE2 receptors EP2 and EP4 in zygapophyseal joints of AS patients. Methods COX-2, PGE2, EP2 and EP4 were determined by immunohistochemistry in zygapophyseal joints from 19 AS, 11 osteoarthritis patients (OA) and 12 autopsy controls (CO). COX-2+, PGE+, EP+ and EP+ cells were detected at four different sites: within hyaline cartilage, subchondral bone plate, fibrous tissue and subchondral bone marrow. Results Within the subchondral bone marrow, numbers of COX-2+ and PGE+ cells were higher in joints of AS patients compared to controls (p=0.0394, p=0.0138). The number of cells expressing EP2 and EP4 were also but non-significantly elevated in the bone marrow of AS patients (p=0.0630; p=0.0786). In contrast, we found in preliminary analysis very low expression of COX-2 within the cartilage, the subchondral bone and the subchondral fibrous tissue. Within the subchondral bone plate, numbers of PGE+ and EP+ cells were higher in AS (and OA) patients compared to controls (p<0.05 for all), while no significant differences were seen within the cartilage comparing AS patients and controls. Comparably very high numbers of PGE+ and EP+ cells were detected within the fibrous tissue of AS and OA patients (p=0.1902, p=0.3726). Table 1 COX-2 PGE2 EP2 EP4 Bone marrow cells (No. +cells per HPF; mean ± SEM) AS 358.03±196.21 AS 874.28±330.80 AS 736.97±154.80 AS 1071.45±236.91 CO 157.73±67.32 CO 421.25±332.52 CO 542.74±137.45 CO 824.84±199.52 AS/CO* AS/CO* AS/CO ns AS/CO ns Osteocytes (%; mean ± SEM) under investigation AS 41.80±16.42 AS 13.36±11.64 AS 52.44±20.21 OA 44.82±15.47 OA 28.63±13.90 OA 65.38±23.11 CO 19.94±16.05 CO 7.13±10.36 CO 18.28±12.01 AS/CO* AS/CO ns AS/CO* AS/OA ns AS/OA* AS/OA ns Chondrocytes (%; mean ± SEM) under investigation AS 61.92±22.90 AS 50.77±24.26 AS 89.29±6.12 OA 77.32±12.04 OA 77.11±14.65 OA 81.02±10.56 CO 48.13±19.87 CO 37.99±19.94 CO 84.50±7.98 AS/CO ns AS/CO ns AS/CO ns AS/OA ns AS/OA* AS/OA ns Fibroblasts (%; mean ± SEM) under investigation AS 84.82±9.00 AS 56.17±21.14 AS 88.21±6.44 OA 87.88±13.38 OA 80.90±10.18 OA 91.02±5.24 AS/OA ns AS/OA* AS/OA ns * p<0.05; ns = not significant. Conclusions According to these data, COX-2 dependent PGE2 synthesis is primarily seen in the subchondral bone marrow and elevated in AS patients, and might be the target site for drugs inhibiting COX-2. References Poddubnyy D, Rudwaleit M, Haibel H, Listing J, Marker-Hermann E, Zeidler H, et al. Effect of non-steroidal anti-inflammatory drugs on radiographic spinal progression in patients with axial spondyloarthritis: results from the German Spondyloarthritis Inception Cohort. Annals of the rheumatic diseases. 2012 Oct; 71(10):1616-1622. Wanders A, Heijde D, Landewe R, Behier JM, Calin A, Olivieri I, et al. Nonsteroidal antiinflammatory drugs reduce radiographic progression in patients with ankylosing spondylitis: a randomized clinical trial. Arthritis and rheumatism. 2005 Jun; 52(6):1756-1765. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3576