Synovial tissues have been isolated from eight RA individuals undergoing total knee replacement surgery. Isolation of synovial fibroblasts Synovial fibroblasts have been 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 aspect have been purchased from R D Systems. Partheno lide, curcurmin and cyclosporin A had been obtained from Sigma Chemical Co. LY294002, SB203580, SP600125, PD98059, and AG490 were obtained from Calbiochem. Anti human IL 1b, TNF a, IL six, 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 were 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 were obtained from individuals, fixed in 4% paraf ormaldehyde resolution overnight at four C, dehydrated with alcohol, washed, embedded in paraffin, and sectioned into slices 7 um thick. The sections were inhibitor Microtubule Inhibitors depleted of endogenous peroxidase activity by adding methanolic H2O2 and had been blocked with standard serum for 30 min utes. Following overnight incubation at four C with polyclonal anti human RANKL and anti MIF antibodies, the samples had been incubated using the acceptable secondary antibo dies biotinylated anti rabbit IgG or biotinylated anti goat IgG for 20 minutes and then incubated with streptavi din peroxidase for one hour followed by incubation with 3,3 diaminobenzidine for 5 minutes.
The sec tions have been counterstained with hematoxylin. Samples have been photographed straight from the source with an Olympus photomicroscope. Synovial fibroblasts had been grown in 150 mm dishes in DMEM full medium, plated at a density of 1 104 cells cm2 onto glass coverslips, and stimulated with rhMIF. Cells had been fixed in 4% paraformaldehyde for immuno histochemical evaluation using anti RANKL antibody 72 hours after the addition of rhMIF.Expression of RANKL mRNA measured by true time reverse transcription polymerase chain reaction amplification RA synovial fibroblasts had been stimulated with rhMIF. For signal pathway evaluation of RANKL, synovial fibroblasts had been incubated in the pre sence or absence of LY294002, SB203580, SP600125, PD98059, AG490, cyclosporin A, parthenolide, or curcumin for one particular hour ahead of the addition of rhMIF. Right after incubation for 72 hours, mRNA was extracted applying RNAzol B as outlined by the producers instruc tions. RT PCR of 2 ug of total mRNA was carried out at 42 C applying the SuperScript reverse transcription sys tem.