Rabbit polyclonal antibody sera were raised against the peptides SVVYGL (anti-OPN-L), SLAYGLR (anti-OPN-R), and VDTYDRGDSVVYGLR (anti-OPN) conjugated to KLH by the standard protocol of Covance (Denver, PA). Antibodies cross-reactive to OPN-L in anti-OPN-R were removed by adsorption on sepharose-linked SLAYGL. The human OPN Quantikine ELISA Kit, MAB14331 (anti-human OPN monoclonal antibody), AF1433 (goat anti-human OPN antibody), and purified recombinant eukaryotic human and mouse OPN were purchased from R&D Systems (Minneapolis, MN). 10A16 anti-OPN antibody was from IBL-America (Minneapolis, MN). Peroxidase- or FITC-conjugated goat anti-rabbit antibody, FITC-conjugated donkey anti-mouse antibody, and Cy3-conjugated goat anti-mouse IgG antibody, were from Jackson Immunological (West Grove, PA). The AminoLink Plus Immobilization Kit was from Pierce (Rockford, IL). Human thrombin and pCPB were from Haematologic Technologies (Essex Junction, VA). The ACTICHROME CPB activity kit and anti-thrombomodulin and anti-pCPB monoclonal antibodies were from American Diagnostica (Stamford, CT). Potato carboxypeptidase inhibitor (CPI) and all other reagents and chemicals, unless stated, were from Sigma (Atlanta, GA). The OPN peptides were synthesized by the peptide synthesis facility at Beckman Center, Stanford University School of Medicine. High protein-binding 96-well ELISA plates were from Granier Bio-One (Monroe, NC).
Preparation of OPN-R and OPN-L from human milk-derived OPN and recombinant OPN
Full length human milk (23
) or recombinant human OPN (OPN-FL, 10 μg each), quantitated by extinction at 280 nm, were digested with 100 nM thrombin for OPN-R or thrombin then 100 nM CPB for OPN-L in 20 mM HEPES pH 7.6, 1 mM CaCl2
, 150 mM NaCl, 0.1% PEG8000
. Thrombin was quenched by PPACK (5 μM); no further treatment of CPB was required due to its thermal instability. OPN-R and OPN-L were used without further purification.
Development of specific ELISAs for human OPN-R and OPN-L
A commercial antibody (MAB14331, 500 ng/well) was used for the capture of OPN-FL, OPN-R and OPN-L. OPN-FL was measured using the R&D Systems Quantikine OPN ELISA kit. Captured OPN-R and OPN-L were detected by anti-OPN-R or anti-OPN-L in conjunction with peroxidase-conjugated goat-anti-rabbit antibody and TMB substrate (Alpha Diagnostic, San Antonio, TX). Recombinant OPN-FL, OPN-R and OPN-L (0.023 – 1.5 nM; 0.625 ng/mL – 50 ng/mL) were used to construct the calibration curves.
Epitope mapping of anti-OPN-R and anti-OPN-L antibodies
The sandwich ELISA system using milk-derived OPN-R or OPN-L at 10 ng/mL was used for epitope mapping. Molar excesses of peptides to antibody (50 fold) were pre-incubated for 2 h then added to the wells. Peptides SAAYGLR, SLGYGLR, SLAAGLR, SLAYALR, SLAYGAR, SLAYGLA, SLAYGLR-NH2 and RLGYALS were incubated with anti- OPN-R antibody and SAVYGL, SVAYGL, SVVAGL, SVVYAL, SVVYGA, SVVYGL-NH2 and RLGYALS with anti-OPN-L antibody (substituted residue in bold font). The decrease in binding of the antibody to the immobilized OPN-R or OPN-L by the cognate unsubstituted peptide was defined as 100% inhibition, and results from the substituted peptides were normalized to this.
Detection of OPN-FL and its cleaved forms in RA, OA, and PSA synovial fluid
Synovial fluid specimens were obtained from 26 RA, 18 osteoarthritis (OA), and 10 psoriatic arthritis (PsA) patients following approved human subjects protocols at Stanford University Medical Center and Brigham and Women’s Hospital, and stored frozen at −80°C until analysis. Samples were thawed on ice, and clarified by centrifugation at 400x g for 10 min at 4°C. The various forms of OPN were measured using the specific ELISAs. Wilcoxon Rank test was performed, and P values < 0.05 were considered significant.
Multiplex cytokine analysis of synovial fluid
A 12-cytokine Beadlyte kit (Millipore, Billerica, MA) and the Luminex xMAP 100IS platform (Austin, TX) were used. To block non-specific cross-linking by rheumatoid factor, synovial fluid samples were pre-incubated with 3 μg/ml HeteroBlock (Omega Biologicals Inc, Bozeman, MT). The Wilcoxon rank test was used to compare the median cytokine levels in RA vs. OA. The correlation between cytokines and OPN was performed using Spearman correlation analysis, and all reported values have a Spearman’s rho P value < 0.01.
Immunofluorescence labeling of fibroblast-like synoviocytes
Fibroblast-like synoviocytes (FLS) obtained from human synovial fluid samples were cultured in DME with 10% FBS. Cells at passages 6–8 were used in immunofluorescence studies using standard procedures.
RT-PCR for pCPB detection in FLS
Total RNA (~1 μg) prepared from FLS was converted to cDNA using an oligo dT primer and superscript II (Invitrogen). The specific primers used for amplifying a 454bp pCPB fragment were CGTTTCAGAGTGGCCAAGTT (forward) and GGCATTTTTGGCTGTTTGTT (reverse). Annealing temperature used in the PCR reaction was 55°C and 35 cycles applied.
Activation of pCPB by thrombin in the presence of cultured FLS
The functional activity of thrombomodulin on the surface of FLS was determined by adding pCPB (40 nM) and thrombin (10 nM) in 100 μL PBS and incubating at room temperature (RT) for 30 min. The reactions were stopped by PPACK (10 μM). CPB activity was assessed using a chromogenic assay (Actichrome CPB kit). CPI (10 μg/mL) was added to inhibit CPB activity in some assays.
Direct ELISA of pCPB, OPN-R and OPN-L
Synoviocytes were cultured in a 96-well plate, washed, and agonists added at 37°C for 30 min. Aliquots of supernatants were transferred to a new 96-well plate and coated at RT for 2 h. Non-specific binding sites were blocked by incubation with BSA (2%) for 1 h, followed by anti-pCPB, anti-OPN-R or anti-OPN-L antibodies for 1 h and then developed as described in the OPN ELISAs.
Immunohistochemical detection of OPN-FL and OPN-R in RA synovium
Synovial tissue samples were obtained with informed consent from RA patients during total knee replacement surgery under human subjects protocols approved at Stanford University Medical Center. The tissue specimens were snap-frozen then embedded. For immunofluorescence analyses, cryosections were stained with anti-OPN-R or preimmune rabbit IgG. All cryosections were co-stained with monoclonal anti-OPN antibody (10A16). FITC-conjugated goat anti-rabbit IgG antibody was used to detect anti-OPN-R staining, and Cy3-conjugated goat anti-mouse IgG antibody to detect 10A16 staining. Some cryosections were pre-incubated with thrombin (100 nM) for 30 min before fixation to generate OPN-R in situ. Peptide quenching was performed by pre-incubation of anti-OPN-R with the peptide SLAYGLR in a 100:1 peptide:antibody molar ratio for 1.5 h. H&E staining was performed according to standard procedures.
Neutrophil apoptosis assay
Neutrophils were isolated from buffy coats (Stanford Blood Center) using density gradient ficoll separation, washed with RPMI 1640 with 10% FBS, and seeded into a 24-well plate at 106 cells/mL. Cells were incubated with various concentrations of recombinant WT and RGD-mutated human OPN-FL, OPN-R, OPN-L for 22 h at 37°C in the presence of 5% CO2. Cells were then washed and labeled with annexin-FITC and PI (BD Apoptosis Assay kit II), and analyzed by FACS.
Adhesion Assay of FLS to OPN-FL, OPN-R and OPN-L
Recombinant WT and RAA-substituted OPN-FL, OPN-R and OPN-L (100nM) as well as RGDS, GRGES, SVVYGLR, and SVVYGL peptides (0.1–1000 μM) were coated onto 96-well micro black fluorescence plates (Thermo Scientific, Waltham, MA). The recombinant proteins were prepared as previously described (20
). Wells were washed and blocked using 2% BSA for 1 h. FLS were labeled with CFDA cell tracer dye (Invitrogen, Carlsbad, CA) and 5000 cells/well were incubated for 1 h at 37°C. The wells were washed, and the plate read at excitation wavelength of 488 nm and emission of 538 nm using Fluoroskan Ascent (Thermo Scientific, Waltham, MA); data were recorded as relative fluorescence unit (RFU).