Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
CXCR1/2 Signalling Is Required but Is Not Sufficient To Maintain the Phenotypic Stability of Human Articular Chondrocytes.
Sherwood1, Joanna C., Achan2, Pramod, Nalesso2, Giovanna, Pitzalis2, Costantino, Dell'Accio2, Francesco
The production of ELR+ CXC chemokines is widely studied in arthritis and has been postulated to contribute to the inflammatory phenomena that eventually lead to cartilage breakdown. Articular chondrocytes however, also express their own chemokine receptors. 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 vitro. In this study we have hypothesized that ELR+ CXC chemokine signalling via the receptors CXCR1 and CXCR2 in chondrocytes is required for the phenotypic stability of human articular chondrocytes, but is not sufficient to maintain the stable phenotype of these chondrocytes during in vitro expansion.
Adult human articular chondrocytes (AHAC) were enzymatically released and expanded in monolayer culture under standard conditions for blockade experiments and in the presence of CXCL6 or CXCL8 for phenotype preservation experiments. The expression of CXCR1 and CXCR2 was confirmed using PCR, Western blot and immunocytochemistry, and functionality was tested using an in vitro calcium influx assay. CXCR1/2 signalling was blocked at a downstream level using pertussis toxin and at specific receptor level using blocking antibodies. The expression of molecular markers associated with the cartilage phenotype was assessed using real time PCR. The content of highly sulphated proteoglycans in chondrocyte micromasses was analysed using Alcian blue staining at pH0.2 followed by guanidine extraction and quantification.
Early passage articular chondrocytes expressed CXCL1, CXCL8 and CXCL6 as well as their receptors CXCR1 and CXCR2. Receptor expression was confirmed at protein level by Western blotting. Confocal microscopy confirmed the localization of both receptors in both in vitro cultured cells and in human cartilage explants at the cell membrane as well as within the cytoplasm, as expected for the well known internalization and recycling of these receptors and for the autocrine/paracrine production of their ligands. The blockade of CXCR1/2 signalling at both G protein and at receptor level in early passage AHAC resulted in a downregulation of some but not all molecular markers associated with the cartilage forming capacity of AHAC, including type II collagen, aggrecan and the cartilage specific transcription factor SOX9. Both methods resulted in the decrease of sulphated proteoglycan levels of cells cultured in micromass. However, the addition of CXCR1 and CXCR2 ligands during cell expansion did not allow for the maintenance of phenotypic molecular markers at gene expression level.
CXCR1 and CXCR2 are expressed and are functional in adult human articular chondrocytes.
CXCR1 and CXCR2 blockade results in the downregulation of some but not all molecular markers associated with the capacity of AHAC to form stable cartilage in vivo.
CXCR signalling is required for the phenotypic stability of articular chondrocytes in vitro and therefore may play a role in cartilage homeostasis in vivo.
CXCR1 and CXCR2 signalling are not sufficient to maintain the phenotypic stability of articular chondrocytes during in vitro expansion.
To cite this abstract, please use the following information:
Sherwood, Joanna C., Achan, Pramod, Nalesso, Giovanna, Pitzalis, Costantino, Dell'Accio, Francesco; CXCR1/2 Signalling Is Required but Is Not Sufficient To Maintain the Phenotypic Stability of Human Articular Chondrocytes. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1476