ELR plus CXC chemokine signaling is required for phenotypic stability of human articular chondrocytes

Sherwood J, Nalesso G, Bertrand J, Pap T, Achan P, Pitzalis C, Dell'Accio F

Abstract

IntroductionThe production of ELR+ CXC chemokines iswidely studied in arthritis and is postulated to contribute tothe inflammatory phenomena that lead to cartilage break-down. Healthy articular chondrocytes however, also expresstheir own chemokine receptors and ligands. The function ofCXC chemokine receptors in these cells is puzzling becausechondrocytes are encased in a dense extracellular matrix andare not known to migrate in vitro. This study aims to identifythe function of this signalling mechanism in articular carti-lage.Materials and MethodsAdult human articular chondrocytes(AHAC) were expanded in monolayer culture under standardconditions. Receptor expression was confirmed using semiquantitative RT polymerase chain reaction (RT-PCR), Wes-tern blot and immunohistochemistry. CXCR1/2 combinedand individual functionality was tested using an in vitrocalcium mobilisation assay. CXCR1/2 signalling was blockedat specific receptor level using validated blocking antibodiesand siRNA, or at the downstream G-protein level usingPertussis toxin. Chondrocyte phenotypic gene expression wasassessed using real time RT-PCR. The content of highlysulphated proteoglycans in chondrocyte micromasses wasanalysed using Alcian blue staining followed by guanidineHCl extraction and spectrophotometric quantification.CXCL6 and CXCL8 were detected in heparitinase digested,chondroitinase ABC digested and un-digested paraffin sec-tions from healthy and osteoarthritis full thickness humanarticular cartilage using immunohistochemistry.ResultsReceptors were expressed in normal human articularcartilage. Disruption of CXCR1/2 signaling at receptor levelor by downstream blockade in chondrocytes resulted inreduced extracellular matrix sulphated glycosaminoglycancontent, and reduced expression of the chondrocyte differen-tiation markers COL2A1, Aggrecan, and SOX9. CXCL6 andCXCL8 were found in cartilage extracellular matrix inhealthy tissue in distinct localisation patterns, which weredisrupted in osteoarthritic tissue. CXCL8 was lost fromterritorial matrix following heparitinase digestion, but notfollowing digestion in chondroitinase ABC.DiscussionOur findings indicate that CXCR1/2 signalling isrequired for the maintenance of phenotypic stability inarticular chondrocytes. Interactions with heparan sulphateproteoglycans and distribution patterns of ligands within theECM, together with their disruption during pathology,indicate the presence of a homeostatic mechanism whereby CXCL8 is retained within the articular cartilage matrix via itsinteraction with heparan sulphate proteoglycans, contributingto chondrocyte phenotypic stability.