Analysis of cartilage oligomeric matrix protein (COMP) in synovial fibroblasts and synovial fluids

We investigated the expression of cartilage oligomeric matrix protein(COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. Insitu hybridization (ISH) was conducted on synovial specimens from five RApatients applying specific probes for COMP or fibroblast collagen type I.ISH was combined with immunohistochemistry, applying antibodies to themacrophage marker CD68. Ribonuclease protection assay (RPA) and rapidamplification of 3'-cDNA ends (3'-RACE) were performed on total RNA fromnormal and RA synovial fibroblast cultures. Protein extracts fromfibroblasts and culture supernatants were compared with synovial fluids andprotein extracts from isolated chondrocytes by Western blot utilizingpolyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA wasdetected in fibroblasts of RA synovium by ISH, and in normal and RAsynovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homologyof chondrocyte and synovial fibroblast COMP along the coding sequence. COMPprotein was detected in synovial fibroblasts and culture supernatants byimmunoblot. Using polyclonal antibodies, the major portion of COMP fromfibroblasts and culture supernatants was present as low-molecular-weight(LMW) bands, corresponding to those found in synovial fluids. These LMWCOMP bands, however, were not detected in any of the cells or tissuestested using 18-G3 mAb. In protein extracts from chondrocytes and in COMPpurified from cartilage, these LMW bands could not be detected. Inconclusion, the data suggest that certain forms of COMP detected insynovial fluid are secreted from synovial fibroblasts and could bedistinguished by specific mAbs from COMP secreted by chondrocytes.