implications for joint remodeling in AS

Introduction In 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. Methods We 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. Results The 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. Conclusions We 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

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

BACKGROUND: The 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. METHODS: DTHA 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. RESULTS: Bone 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. CONCLUSIONS: Anti-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

Histopathological characterization of the inflammation induced joint remodeling in patients with ankylosing spondylitis

Die Ankylosierende Spondylitis (AS) ist eine chronisch-entzündliche rheumatische Erkrankung ungeklärter Ätiologie, die zur Gruppe der Spondyloarthritiden gehört. Die AS ist primär durch eine Entzündung des Achsenskeletts und im weiteren Krankheitsverlauf durch eine Knochenneubildung, die zur Gelenkankylose und Syndesmophytenbildung führt, gekennzeichnet [1]. Die Zusammenhänge zwischen Entzündung und Knochenneubildung als auch die Mechanismen, die dabei zur Knochenneubildung und Gelenkankylose führen, sind wenig verstanden. Ziel dieser Arbeit war es daher, mittels immunhistologischer und histomorphometrischer Untersuchungen von Facettengelenken von AS-Patienten im Vergleich zu Autopsiekontrollen und OA-Patienten, die Sequenz des entzündungs-vermittelten Gelenkumbaus zu analysieren und beteiligte Mechanismen und Mediatoren zu identifizieren. Daneben sollte in Arthritis- und Spondylitis-Tiermodellen das Auftreten einer Knochenneubildung untersucht werden, mit dem Ziel, Modelle zu identifizieren, die für die Testung therapeutischer Ansätze zur Hemmung der Knochenneubildung geeignet sind. Als erstes wichtiges Ergebnis der systematischen Analyse der Facettengelenke von AS-Patienten konnten histomorphologische Stadien des Gelenkumbaus bei AS definiert werden. AS-Stadium 1: physiologische Gelenkmorphologie, AS-Stadium 2: Facettengelenke mit partieller oder kompletter Knorpelfusion, AS-Stadium 3: Facettengelenke mit knöcherner Fusion mit Erhalt vereinzelter Knorpelinseln, AS-Stadium 4: Facettengelenke mit totalem Verlust knorpeliger Gelenkbestandteile. Zweitens zeigte die stadienabhängige histomorphometrische Analyse der Facettengelenke den progredienten Verlust des hyalinen Gelenkknorpels (Abnahme der Knorpeldicke) und der subchondralen Endplatte (Abnahme der Dicke der Endplatte) bei AS. Daneben fand sich, koinzident mit der Knorpelfusion, das Auftreten subchondraler Knochenmarkveränderungen, das heißt eine Transformation des subchondralen Knochenmarkes in ein fibröses Pannusgewebe. Die immunhistologische Charakterisierung des Knorpelphänotyps deckte eine ausgeprägte Knorpeldegeneration in den Facettengelenken von AS-Patienten auf. Diese war charakterisiert durch eine erhöhte Apoptoserate der Chondrozyten und eine Abnahme des Proteoglykangehaltes des hyalinen Gelenkknorpels bei gleichzeitig reduzierter IL-10-, β-Catenin-, Wif-1-, Sclerostin-, Sox9-, BMP-2- und BMP-7-Expression der Chondrozyten. Anhalt für einen MMP-13-vermittelten aktiven Degradationsprozess ergab sich nicht, sodass die gezeigten Veränderungen als passive Knorpeldegeneration bewertet werden. Drittens wurde durch den direkten Nachweis Osteoklasten-vermittelter destruktiver, als auch Osteoblasten-vermittelter osteoproliferativer Eigenschaften des fibrösen Pannusgewebes die zentrale Rolle dieses Gewebes für den Umbauprozess der Facettengelenke bei AS pathophysiologisch untermauert. Durch den Nachweis Osteoblasten-vermittelter Knorpelossifikation konnte die Bedeutung der membranösen Ossifikation gezeigt werden, während sich kein Anhalt für die Beteiligung der enchondralen Ossifikation ergab (fehlende gesteigerte Expression der Hypertrophiemarker Runx2, MMP13 und COL10). Viertens zeigte die Analyse entzündlicher Veränderungen (Zellkomposition, Zellaggregate, entzündliche Mediatoren) im subchondralen Knochenmark der Facettengelenke eine stadienabhängige Anreicherung CD3+ T-Zellen sowie eine erhöhte Expression von TNFα, IL-23 und PGE2 bei AS. Diese immunologischen Veränderungen scheinen somit Bestandteil der Entzündungsreaktion zu sein und könnten Auslöser der Transformation des Knochenmarkes zu fibrösem Pannusgewebe bei AS sein. Mit diesen Ergebnissen verbessert diese Arbeit entscheidend das mechanistische Verständnis des entzündungs-induzierten Gelenkumbaus bei AS. Aufgrund der Analysen wurde das fibröse Pannusgewebe, inklusive der Osteoblasten und Osteoklasten, als neues Target für die Hemmung des Gelenkumbaus bei AS identifiziert. Als ein mögliches Modell zur Testung anti-osteoproliferativer Therapieansätze wurde das DTH-A-Mausmodell identifiziert. Dieses ist durch das Auftreten von entzündungsinduzierter membranöser und enchondraler Ossifikation gekennzeichnet, sodass eine Prüfung potenzieller neuer Wirkstoffe (wie zum Beispiel EP2- und EP4-Rezeptorantagonisten) auf beide Formen der Knochenneubildung möglich ist.Ankylosing spondylitis (AS) is a chronically inflammatory rheumatic disease with undetermined etiology, which belongs to the group of spondyloarthritides. AS is primarily characterized by the inflammation of the axial skeleton and in the course of the disease by new bone formation, which is leading to ankylosis and formation of syndesmophytes [1]. The correlation between inflammation and new bone formation as well as the mechanisms that lead to new bone formation and joint ankylosis are still poorly understood. Therefore, this study was constructed to discover an inflammatory mediated sequence of joint remodeling and the identification of involved mechanisms and mediators. In order to reach these goals facet joints of AS patients were immunohistochemically and histomorphometrical examined and compared with autopsy controls and patients with osteoarthritis. Furthermore, the occurrence of new bone formation was also investigated in animal models of arthritis and spondylitis to identify models, which are suitable for testing therapeutic strategies to inhibit this osteoproliferation. The first important conclusion of the systematic analysis of facet joints from patients with AS was the definition of histomorphological stages of the joint remodeling in AS. AS-stage 1: physiological joint morphology, AS-stage 2: facet joints with partially or completely fused cartilage, AS-stage 3: facet joints with bony fusion and a maintenance of isolated cartilage islands, AS-stage 4: facet joints with total loss of cartilaginous parts of the joint. Second, the stage-dependent histomorphometric analysis of the facet joints revealed the progressive loss of hyaline articular cartilage (decrease in cartilage thickness) and the subchondral bone plate (reduction of the thickness of the bone plate) in AS. In addition, subchondral bone marrow changes, i.e. the transformation of the subchondral bone marrow into a fibrous granulation tissue, occurred coincidentally with the fusion of the articular cartilage. The immunohistological characterization of the phenotype revealed a degeneration of the cartilage of facet joints from patients with AS. This phenomenon is characterized by an increase in apoptosis of the chondrocytes as well as a decrease in the proteoglycan content of the hyaline articular cartilage along with a reduced expression of IL-10, β-catenin, wif-1, sclerostin, Sox9, BMP-2 und BMP-7 by chondrocytes. Because of the lack of evidence for an MMP-13-mediated active process of degradation, the shown changes are assessed to be a kind of passive degeneration of the cartilage. Third, the importance of fibrous granulation tissue for the process of joint remodeling in facet joints from patients with AS could be proven for the first time through the direct detection of both, osteoclast-mediated destructive as well as osteoblast-mediated osteoproliferative properties of this tissue. Hence the proof of the osteoblast-mediated cartilage ossification the importance of the membranous ossification was verified, whereas no evidence for the involvement of the process of endochondral bone formation could be detected (lack of increased expression of the hypertrophy markers Runx2, MMP13 und COL10). Fourth, the analysis of inflammatory changes displayed (cell composition, cell aggregates, inflammatory mediators) an stage-dependent enrichment of CD3+ T-cells as well as an increased expression of TNFα, IL-23 und PGE2 within the subchondral bone marrow in facet joints from patients with AS. These immunological changes appear to be part of the inflammatory reaction and could be the trigger for the transformation of the subchondral bone marrow into the fibrous granulation tissue in AS. As a consequence of the gained results, this study significantly improves the mechanistic understanding of the pathological process of joint remodeling in AS. Due to the analysis the fibrous granulation tissue, including the osteoblasts and osteoclasts, could be identified as the new target for the inhibition of the process of joint remodeling in AS. The DTH-A mouse model was identified as a possible model for testing anti-osteoproliferative therapeutic approaches. This model is characterized by the occurrence of inflammatory induced endochondral and membranous ossification and therefore shows that the impact of potentially new substances (such as EP2 and EP4 receptor antagonists) can be examined on both forms of new bone formation