Basic and translational research Ann Rheum Dis: first published as 10.1136/annrheumdis-2014-205546 on 18 August 2014. Downloaded from EXTENDED REPORT A homeostatic function of CXCR2 signalling in articular cartilage Joanna Sherwood,1,2 Jessica Bertrand,2 Giovanna Nalesso,1 Blandine Poulet,3 Andrew Pitsillides,4 Laura Brandolini,5 Alexandra Karystinou,6 Cosimo De Bari,6 Frank P Luyten,7 Costantino Pitzalis,1 Thomas Pap,2 Francesco Dell’Accio1 Handling editor Tore K Kvien ABSTRACT Metalloproteinase and aggrecanase-mediated ▸ Additional material is Objective ELR+ CXC chemokines are heparin-binding extracellular matrix (ECM) degradation, and chon- published online only. To view cytokines signalling through the CXCR1 and CXCR2 drocyte apoptosis all contribute to cartilage break- please visit the journal online receptors. ELR+ CXC chemokines have been associated down and are initially compensated by chondrocyte (http://dx.doi.org/10.1136/ with inflammatory arthritis due to their capacity to proliferation and upregulation of SOX9, which dir- annrheumdis-2014-205546). attract inflammatory cells. Here, we describe an ectly regulates the synthesis of major ECM compo- fi – For numbered af liations see unsuspected physiological function of these molecules in nents including aggrecan and type II collagen.2 4 end of article. articular cartilage homeostasis. When such compensatory mechanisms are impaired Correspondence to Methods Chemokine receptors and ligands were or insufficient, cartilage breakdown progresses and Professor Francesco Dell’Accio, detected by immunohistochemistry, western blotting and ultimately leads to joint failure. Supporting the Centre for Experimental RT-PCR. Osteoarthritis was induced in wild-type and homeostatic response of cartilage can slow down or − − Medicine and Rheumatology, CXCR2 / mice by destabilisation of the medial even revert cartilage degeneration in animal William Harvey Research 56 Institute, Barts and the London meniscus (DMM). CXCR1/2 signalling was inhibited models. School of Medicine and in vitro using blocking antibodies or siRNA. Chondrocyte Enclosed within the cartilage matrix, chondro- Dentistry, Queen Mary phenotype was analysed using Alcian blue staining, cytes are not known to migrate in physiological University of London, UK; RT-PCR and western blotting. AKT phosphorylation and conditions. In spite of this, however, chondrocytes [email protected] SOX9 expression were upregulated using constitutively express several chemokine receptors, including TP and FDA shared co-senior active AKT or SOX9 plasmids. Apoptosis was detected by CXCR1 and CXCR2 and their cognate ligands that authorship. terminal deoxynucleotidyl transferase dUTP nick end have been extensively studied in the context of labelling (TUNEL) assay. arthritis.78 Received 11 March 2014 Results CXCL6 was expressed in healthy cartilage and ELR+ CXC chemokines are chemotactic cytokines Revised 12 June 2014 Accepted 20 July 2014 was retained through binding to heparan sulfate characterised by their glutamic acid-leucine-arginine −/− Published Online First proteoglycans. CXCR2 mice developed more severe (ELR+) motif. The chemokine receptor CXCR2 18 August 2014 osteoarthritis than wild types following DMM, with binds the human CXC chemokine ligands CXCL1, increased chondrocyte apoptosis. Disruption of CXCR1/2 CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and in human and CXCR2 signalling in mouse chondrocytes CXCL8 while CXCR1 binds only to CXCL6 http://ard.bmj.com/ led to a decrease in extracellular matrix production, and CXCL8.9 Mice lack Cxcl8 and express only one reduced expression of chondrocyte differentiation gene that shows homology to the human CXCL5 markers and increased chondrocyte apoptosis. CXCR2- and CXCL6 (hereafter referred to as mouse dependent chondrocyte homeostasis was mediated by CXCL6). Although a putative murine homologue of AKT signalling since forced expression of constitutively human CXCR1 has been identified,10 mouse active AKT rescued the expression of phenotypic markers CXCR2 is considered the main ELR+ CXC chemo- and the apoptosis induced by CXCR2 blockade. kine receptor because its function cannot be compen- on September 29, 2021 by guest. Protected copyright. Conclusions Our study demonstrates an important sated in neutrophil chemotaxis and wound – physiological role for CXCR1/2 signalling in maintaining healing.11 13 CXCR1 and CXCR2 activate intracellu- cartilage homeostasis and suggests that the loss of ELR+ lar calcium and lead to activation of the Pi3K/AKT CXC chemokines during cartilage breakdown in signalling pathway.14 15 osteoarthritis contributes to the characteristic loss of Although the biology and expression in vivo of chondrocyte phenotypic stability. CXCR1 and CXCR2 in intact normal human Open Access articular cartilage has not been reported, the Scan to access more free content expression of these receptors and various chemo- kines have been characterised in isolated arthritic INTRODUCTION chondrocytes.78ELR+ CXC chemokines have Osteoarthritis (OA) is a leading cause of chronic been studied and targeted in inflammatory arthritis disability, characterised by the breakdown of the because of their capacity to attract inflammatory articular cartilage, for which we have no cure. cells. CXCL8 was shown to stimulate, in vitro, the Whereas in inflammatory arthritides inflammation production of inflammatory mediators, metallopro- is the main driver of tissue destruction, in OA, teinases and the induction of hypertrophy and fi 16 To cite: Sherwood J, mechanical factors are the main drivers of cartilage calci cation. Bertrand J, Nalesso G, et al. breakdown while a low degree of synovitis detected Here, we report a novel, unsuspected homeo- Ann Rheum Dis only in a subset of patients may have an ancillary static role for CXCR1/2 signalling in the articular – 2015;74:2207 2215. role.1 cartilage where ELR+ CXC chemokines are Sherwood J, et al. Ann Rheum Dis 2015;74:2207–2215. doi:10.1136/annrheumdis-2014-205546 2207 Basic and translational research Ann Rheum Dis: first published as 10.1136/annrheumdis-2014-205546 on 18 August 2014. Downloaded from retained in the ECM through binding to GAGs and mouse AKT (Cell Signaling) 1:500 dilution or rabbit anti-mouse cell-autonomously support chondrocyte viability and differenti- CXCL6 (Biorbyt) 1:200 dilution in blocking solution at 4°C ation through AKT-dependent SOX9 expression. We suggest overnight. For more detailed protocols, see online supplemen- that disruption of CXCR1/2 signalling is an important event in tary methods. osteoarthritis, resulting in the loss of chondrocyte phenotypic stability and promoting OA-like changes. Immunofluorescence analysis of mouse and human cartilage Human cartilage explant and decalcified mouse knee joint sec- MATERIALS AND METHODS tions were deparaffinised and the subsequent steps including Mice blocking and pepsin antigen retrieval were performed as previ- 000.651 BALB/cJ and C.129S2(B6)-Cxcr2tm1Mwm/J CXCR2 ously described.22 Human sections and monolayer cells were (−/−) mice (BALB/cJ background) were obtained from The analysed using (anti-CXCR1, -CXCR2 or CXCL6 (R&D)) Jackson Laboratory and maintained in pathogen-free conditions. primary antibodies followed by Cy2 conjugated goat anti-mouse The Animal Use Committee for the University of Münster IgG secondary antibodies ( Jackson ImmunoResearch). Mouse (Landesamt für Natur, Umwelt und Verbraucherschutz, approval knee sections were incubated with rabbit anti-mouse -GCP2 number 84-02.04.2012.A189) approved all mouse procedures. (Biorbyt), -CXCR2 (R&D), -Col X (Abcam) or anti-Col II (Chemicon), followed by chicken anti-rabbit Alexa Fluor 488 or Destabilisation of the medial meniscus chicken anti-rabbit Alexa Fluor 594 secondary antibodies (Life − − Ten-week-old BALB/C or CXCR2 / male mice received desta- Technologies). Mouse isotype control IgG (Dako) or rabbit IgG bilisation of the medial meniscus (DMM) and sham surgery to (Abcam) were used as control primary antibodies. the contralateral limb as described.17 Apoptosis was detected by terminal deoxynucleotidyl trans- After 8 weeks, mice were killed and the joints were processed ferase dUTP nick end labelling (TUNEL) assay (Roche) accord- as previously described.18 A minimum of five sections per joint ing to the manufacturer’s instruction. were stained using toluidine blue for histological analysis and Images were acquired at 22°C by either Leica DM5500 Q Osteoarthritis Research Society International (OARSI) scoring Confocal microscope using 40× magnification/0.75 numerical for osteoarthritis severity by two independent investigators. aperture, or Olympus BX61 microscope with a fixed exposure using either 10×/0.4 or 20×/0.7 objective lenses using Cell-P soft- Cartilage harvest, chondrocyte isolation and culture ware. Images were enhanced using Adobe Photoshop for better Full-thickness human articular cartilage was obtained from the rendering without altering relationship of target to control images. femoral condyles of patients undergoing knee joint replacement surgery (ethics approval from the East London & The City Cartilage explant digestion Ethics Committee 3). Normal articular cartilage was obtained Hip caps from 4-week-old BALB/C wild-type mice were decellu- from postmortem and trauma surgery donors. Chondrocytes larised by repeated freeze thawing. Hip caps were incubated were isolated and cultured as previously described.19 Mouse overnight at 37°C either in phosphate buffered saline (PBS) or costal chondrocytes were obtained from the ribcages