Synovial tissues had been isolated from eight RA individuals undergoing total knee replacement surgery. Isolation of synovial fibroblasts Synovial fibroblasts were isolated by enzymatic digestion of synovial tissues obtained from RA sufferers undergoing total joint replacement surgery, as described previously. Reagents Recombinant human MIF, rhRANKL and rh mono cyte colony stimulating factor were bought from R D Systems. Partheno lide, curcurmin and cyclosporin A were obtained from Sigma Chemical Co. LY294002, SB203580, SP600125, PD98059, and AG490 had been obtained from Calbiochem. Anti human IL 1b, TNF a, IL 6, RANKL and MIF had been bought from R D Systems. Determination of concentrations of soluble RANKL and MIF by sandwich ELISA Concentrations of soluble RANKL and MIF in sera and synovial fluids had been measured by sandwich ELISA as described previously.
Immunohistochemistry of RA synovium and synovial fibroblasts Immunohistochemical staining for RANKL and MIF was performed on sections of synovium. Briefly, synovium samples had been obtained from individuals, fixed in 4% paraf ormaldehyde option overnight at four C, dehydrated with alcohol, washed, embedded in paraffin, and sectioned into slices 7 um thick. The sections were selleckchem depleted of endogenous peroxidase activity by adding methanolic H2O2 and had been blocked with normal serum for 30 min utes. Just after overnight incubation at four C with polyclonal anti human RANKL and anti MIF antibodies, the samples were incubated using the proper secondary antibo dies biotinylated anti rabbit IgG or biotinylated anti goat IgG for 20 minutes and then incubated with streptavi din peroxidase for one particular hour followed by incubation with 3,three diaminobenzidine for five minutes.
The sec tions were counterstained with hematoxylin. Samples were photographed read full report with an Olympus photomicroscope. Synovial fibroblasts have been grown in 150 mm dishes in DMEM complete medium, plated at a density of 1 104 cells cm2 onto glass coverslips, and stimulated with rhMIF. Cells were fixed in 4% paraformaldehyde for immuno histochemical analysis using anti RANKL antibody 72 hours immediately after the addition of rhMIF.Expression of RANKL mRNA measured by true time reverse transcription polymerase chain reaction amplification RA synovial fibroblasts have been stimulated with rhMIF. For signal pathway evaluation of RANKL, synovial fibroblasts have been incubated within the pre sence or absence of LY294002, SB203580, SP600125, PD98059, AG490, cyclosporin A, parthenolide, or curcumin for a single hour just before the addition of rhMIF. Following incubation for 72 hours, mRNA was extracted employing RNAzol B in accordance with the suppliers instruc tions. RT PCR of two ug of total mRNA was carried out at 42 C applying the SuperScript reverse transcription sys tem.