EQUINE TIMP-1 AND TIMP-2 - IDENTIFICATION, ACTIVITY AND CELLULAR SOURCES

Matrix metalloproteinases (MMPs) are the main enzymes involved in conn ective tissue turnover. Regulation of MMPs is achieved by controlling production, activation of the proenzymes together with the presence of inhibitors, such as, tissue inhibitors of metalloproteinases (TIMPS), The presence of TIMPs in equine synovial fluid was assessed by the ab ility of the fluid to inhibit equine MMP-9 activity using a gelatin de gradation ELISA, The cellular source of the TIMPs was determined using culture supernatants of resident articular cells (chondrocytes and sy novial fibroblasts) and invading inflammatory cells (polymorph neutrop hils [PMN] and peripheral blood monocytes [PBM]), The TIMPs were chara cterised further using reverse zymography, affinity chromatography and N-terminal amino acid sequencing. Synovial fluid was recovered from h orses with articular sepsis and aseptic joint disease (AJD) and compar ed with that from normal horses (n = 4), TIMP activity was minimal in articular sepsis but significantly increased, albeit a small increase, in AJD when compared to normal (P<0.05). Cell culture supernatants fr om synovial fibroblasts, chondrocytes and PBMs contained TLMP activity , although supernatants from PMN cell culture did not, Reverse zymogra phy of synovial fluid recovered from normal and AJD horses showed two protein bands, 22 and 28 kDa in size, exhibiting inhibitory activity a gainst MMP-9, Reverse zymography of culture supernatants of synovial f ibroblasts and chondrocytes gave similar results whereas the culture s upernatants from PMNs and PBMs showed the presence of only the 28 kDa protein. The N-terminal amino acid sequence was obtained for the 22 kD a protein and revealed a 66% homology with human TIMP-2, The identific ation of TIMPs in equine synovial fluids and cell culture supernatants suggest that they may have a fundamental role in the homeostasis of t he normal joint and in the excess proteolysis which occurs in articula r disease in the horse.