Biotinylated CXCL12α was synthesized by the Merrifield solid-phase method on a fully automated peptide synthesizer using Fmoc (N
-(9-fluorenyl)methoxycarbonyl) chemistry as described previously [17
]. Selective biotinylation at the carboxy-terminal position was achieved by incorporating a lysine residue (Lys68) bearing a 4,4-dimethyl-2,6-dioxocyclohex-1-ethylidene (Dde) protective group on the side chain. Coupling of biotin was performed on the peptide resin after Dde deprotection. Non-biotinylated native CXCL12α or 2/6 CXCL12α, in which the basic residues Lys24 and Lys27 were replaced by Ser (CXCL12α-K2427S), were synthesized similarly.
Synovial EC cultures were obtained from enzymatic cell suspensions of three RA and four osteoarthritis (OA) synovial tissues obtained at the time of joint replacement surgery. All patients gave informed consent, and the study was approved by the ethics committee of the Hospital 12 de Octubre. ECs were purified by two rounds of immunomagnetic enrichment with anti-CD105 coupled to magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany). ECs were cultured in medium199 (Life Technologies, Paisley, Renfrewshire, Scotland) with 10% FCS and the endothelial identity of cultured cells was confirmed by flow cytometry with UEA-rhodamine and anti-P1H12 antibodies (Chemicon, Temecula, CA, USA). Human umbilical vein ECs (HUVECs) were prepared from umbilical cord by digestion with collagenase and were propagated in medium199 with 20% FCS. Cultures displaying more than 90% cells positive for both EC markers were used between the third and seventh passages.
Cultured ECs were exposed to 300 to 1,000 nM CXCL12α peptides for 90 minutes in PBS buffer at 4°C and washed extensively in PBS. Surface presentation of exogenous CXCL12α was analysed by flow cytometry with K15C mAb and fluorescein isothiocyanate (FITC)-labelled secondary antibody or, in the case of biotinylated CXCL12α, with avidin-FITC (Pharmingen, San Diego, CA, USA). Expression of HSPGs in cultured ECs was studied with 10E4 anti-heparan sulfate-FITC mAb (Calbiochem, San Diego, CA, USA). Expression of CXCR4 in cultured ECs was studied with 12G5 mAb (Pharmingen) in 0.5% Tween 20 permeabilized or non-permeabilized ECs.
Where indicated, 500 μg/ml sodium heparin (Rovi S.A., Madrid, Spain) or 50 μg/ml T134 (a CXCR4 inhibitor) was added to the CXCL12α incubation medium. T134 specifically prevents the binding of CXCL12 to CXCR4 receptor at picomolar concentrations [20
]. Pretreatment of EC cultures with 60 mM sodium chlorate was performed for 24 hours before CXCL12α binding. Removal of surface HSPGs in cultured cells was performed by treatment with a cocktail of 100 mU/ml heparitinases I, II and III each, or 100 mU/ml chondroitin sulfate ABC lyase as a control for 90 minutes at 37°C (Sigma Aldrich Química S.A., Madrid, Spain). Where indicated, EC cultures were stimulated with either 25 ng/ml TNF-α or 10 ng/ml LT-α1/β2 (R&D Systems, Inc., Abingdon, Oxon, UK) for 16 hours before CXCL12α binding.
CXCL12 expression by ECs was studied by RT-PCR on cDNA synthesized from 1 μg of total RNA with the use of the CXCL12 oligonucleotides 5' -TCTGAGAGCTCGCTTGAGTG-3' (upstream) and 5' -GTGGATCGCATCTATGCATG-3' (downstream) and the β-actin oligonucleotides 5' -CTACCTCATGAAGATCCTCAC-3' (upstream) and 5' -GTCCACGTCACACTTCATGATG-3' (downstream). As a positive control for CXCL12 expression we used cDNA from RA synovial fibroblasts.
For immunolabelling of synovial tissues from patients with RA and control patients with OA, tissues were snap-frozen in optimal cutting temperature (OCT) compound and stored at -80°C. Double labelling for HSPGs and CXCL12, or MECA-79 and CXCL12, was performed as described previously [16
Flow cytometry data were expressed as mean fluorescence intensity (mean ± SD), normalized by the mean fluorescence intensity of the negative control. Statistical analysis was performed with Student's t test.