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THU0023 A novel role for ELR+ CXC chemokine signaling in cartilage homeostasis
  1. J. Sherwood1,
  2. G. Nalesso2,
  3. J. Bertrand1,
  4. P. Achan2,
  5. L. Brandolini3,
  6. T. Pap1,
  7. C. Pitzalis2,
  8. F. Dell’Accio2
  1. 1Institute of Experimental Musculoskeletal Medicine, University Hospital Münster, Münster, Germany
  2. 2Experimental Medicine & Rheumatology, William Harvey Research Institute, Queen Mary, University of London, London, United Kingdom
  3. 3Dompé S.P.A., L’Aquila, Italy

Abstract

Background ELR+ CXC chemokine production is studied in arthritis and is thought to contribute to inflammation leading to articular cartilage breakdown and arthritis pathology. However, healthy articular chondrocytes express their own chemokine receptors and ligands. The function of CXC chemokine receptors in these cells is puzzling as chondrocytes are encased in a dense extracellular matrix and are not known to migrate in vivo.

Objectives This study aims to investigate the function of the CXCR1/2 signaling pathway in articular cartilage.

Methods CXCR1/2 expression in adult human articular chondrocytes was confirmed by semi-quantitative RT polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. Combined and individual functionality of CXCR1 and CXCR2 was tested using an in vitro calcium mobilisation assay. Validated blocking antibodies and siRNA were used to inhibit CXCR1/2 signaling at receptor level. Pertussis toxin, PI3K inhibitors and intracellular calcium chelators were used to block signaling at intracellular levels. The highly sulphated proteoglycan content of chondrocyte micromasses was analysed using Alcian blue staining and spectrophotometric quantification. Chondrocyte gene expression was assessed using real time RT-PCR. CXCL6 and CXCL8 were detected in heparitinase digested, chondroitinase ABC digested and undigested paraffin sections of human articular cartilage from healthy and osteoarthritic donors by immunohistochemistry. Finally, 8 week old CXCR2-/- mutant BALB/C mouse knee joint paraffin sections were analysed using Safranin Orange staining, Chambers scoring and ImageJ histomorphometry.

Results CXCR1/2 expression was confirmed in normal human articular cartilage. Individual blockade of either CXCR1 or CXCR2 did not inhibit downstream calcium mobilisation, indicating that CXCR1 and CXCR2 have more functional redundancy than that observed in neutrophils. CXCR1/2 signaling disruption at receptor level or by downstream blockade in chondrocytes resulted in reduced extracellular matrix sulphated glycosaminoglycan content and reduced expression of the chondrocyte differentiation markers COL2A1, Aggrecan, and SOX9. CXCL6 and CXCL8 were found in cartilage extracellular matrix in healthy tissue in distinct localisation patterns, which were disrupted in osteoarthritic tissue and following heparitinase digestion. In vivo analysis of 15 knockout and 15 wild type knees revealed that CXCR2-/- mice have significantly thinner epiphyseal growth plates and medial tibial plateaus, with a reduced sulphated proteoglycan content found in medial condyle articular cartilage.

Conclusions Our findings indicate that CXCR1/2 signaling is in fact required for maintenance of phenotypic stability in articular chondrocytes. Interactions with heparan sulphate proteoglycans and distribution patterns of ligands within the ECM, and their disruption during pathology, indicate the presence of a homeostatic mechanism whereby CXC chemokines are retained within the articular cartilage matrix via interactions with heparan sulphate proteoglycans, where they maintain chondrocyte phenotypic stability via an autocrine/paracrine signaling mechanism. In vivo analysis suggests that CXCR1/2 signaling may be specifically required during periods of high chondrocyte turnover, including within the growth plate.

Disclosure of Interest None Declared

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