ELR+ CXC chemokine signalling in cartilage homeostasis

Abstract

Purpose: The production of ELR+ CXC chemokines is widely studied in arthritis and is postulated to contribute to the inflammatory phenomena that lead to cartilage breakdown and arthritis pathology. Healthy articular chondrocytes however, also express their own chemokine receptors and ligands. The function of CXC chemokine receptors in these cells is puzzling because chondrocytes are encased in a dense extracellular matrix and are not known to migrate in vivo. This study aims to identify the function of this signaling mechanism in articular cartilage. Methods: Adult human articular chondrocytes (AHAC) were expanded in monolayer culture under standard conditions. Receptor expression was confirmed using semi quantitative RT polymerase chain reaction (RT-PCR), Westernblotandimmunohistochemistry.CXCR1/2combinedandindividual functionalitywastestedusinganinvitrocalciummobilisationassay.CXCR1/ 2 signaling was blocked at specific receptor level using validated blocking antibodies and siRNA, orat the downstream level using Pertussis toxin, PI3K inhibitors and intracellular calcium chelators. Chondrocyte phenotypic gene expression was assessed using real time RT-PCR. The content of highly sulphated proteoglycans in chondrocyte micromasses was analysed using Alcian blue staining and spectrophotometric quantification normalised for total protein content. CXCL6 and CXCL8 were detected in heparitinase digested, chondroitinase ABC digested and un-digested paraffin sections from healthy and osteoarthitis full thickness human articular cartilage using immunohistochemistry. 8 week old CXCR2-/- mutant mouse knee joint paraffin sections were analysed using Safranin Orange staining, followed by Chambers scoring and ImageJ histomorphometry. Results: Receptors were expressed in normal human articular cartilage. Blockade of either CXCR1 or CXCR2 individually did not inhibit downstream calcium mobilisation, indicating that CXCR1 and CXCR2 have a higher level of functional redundancy than that observed in neutrophils. Disruption of CXCR1/2 signaling 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 BALB/C controls revealed that CXCR2-/- mutant mice have significantly thinner epiphyseal growth plates and medial tibial plateaus. Conclusions: Our findings indicate that CXCR1/2 signaling is required for the maintenance of phenotypic stability in articular chondrocytes. Interactions with heparan sulphate proteoglycans and distribution patterns of ligands within the ECM, together with their disruption during pathology, indicate the presence of a homeostatic mechanism whereby CXCL8 is retained within the articular cartilage matrix via its interaction with heparan sulphate proteoglycans,contributingtochondrocytephenotypicstability.Invivoanalysissuggests