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. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.
Hummel
K M
KM
Center for Experimental Rheumatology, University Hospital, Zürich, Switzerland.
Neidhart
M
M
Vilim
V
V
Hauser
N
N
Aicher
W K
WK
Gay
R E
RE
Gay
S
S
Häuselmann
H J
HJ
D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D064236:Cartilage Oligomeric Matrix Protein; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism / Q000473:pathology; D017931:DNA Primers / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization; D064235:Matrilin Proteins; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D000074402:Synovectomy; D013582:Synovial Fluid / Q000378:metabolism*; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology
D064236:Cartilage Oligomeric Matrix Protein; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D064235:Matrilin Proteins; D012333:RNA, Messenger; C577553:TSP5 protein, human