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Abstract
Matrix metalloproteinase 9 (MMP-9) degrades type IV collagen, gelatin, type V collagen and type XI collagen. We measured proMMP-9 and proMMP-9-TIMP-1 complex in sera and joint fluids by sandwich ELISA, and immunohistochemically examined the expression of this enzyme in joint tissues from patients with rheumatoid arthritis (RA). ProMMP-9 was purified from the culture medium of HT 1080 cells by the three steps of chromatography. Purified proMMP-9 and activated MMP-9 by aminophenylmercuric acetate showed two bands of 92 and 67 kDa on gelatin zymography. We raised two monoclonal antibody clones, named 2G9 and 8G7, against proMMP-9. 2G9 and 8G7 reacted with proMMP-9 in western blotting and these clones reacted not only with proMMP-9, but also with proMMP-9-TIMP-1 complex in sandwich ELISA, respectively. The proMMP-9 concentration in 86 sera (749.4 ± 940.2 ng/ml) and 54 joint fluids (4539.9 ± 7681.5 ng/ml) from patients with RA was significantly higher than those of patients with osteoarthritis (15 sera: 139.0 ± 149.6 ng/ml; 16 joint fluids: 655.0 ± 1982.8 ng/ml) and control (37 sera: 266.7 ± 120.4 ng/ml; three joint fluids: 0 ng/ml). The immunohistochemistry with 2G9 monoclonal antibody showed that proMMP-9 were expressed in the neutrophils and the monocytes-macrophages which diffusely infiltrated in the sublining layer of rheumatoid synovium. In addition, the osteoclasts along subchondral bone were also intensively stained. The proMMP-9 concentration in joint fluids from 39 RA patients was positively correlated to the count of proMMP-9 positive cells in RA synovium (r = 0.607) and to the score of diffuse infiltrates of lymphocytes (r = 0.720). However, it did not show correlation to the stage and the class defined by Steinbrocker and to the other clinical laboratory data. Our results suggest that proMMP-9 actively participates in joint destruction of RA through the expression of neutrophils and monocytes-macrophages and is regulated by lymphocytes.