Equine TIMP-1 and TIMP-2: identification, activity and cellular sources.

Matrix metalloproteinases (MMPs) are the main enzymes involved in connective tissue turnover. Regulation of MMPs is achieved by controlling production, activation of the pro-enzymes 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 ability of the fluid to inhibit equine MMP-9 activity using a gelatin degradation ELISA. The cellular source of the TIMPs was determined using culture supernatants of resident articular cells (chondrocytes and synovial fibroblasts) and invading inflammatory cells (polymorph neutrophils [PMN] and peripheral blood monocytes [PBM]). The TIMPs were characterised further using reverse zymography, affinity chromatography and N-terminal amino acid sequencing. Synovial fluid was recovered from horses with articular sepsis and aseptic joint disease (AJD) and compared 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 from synovial fibroblasts, chondrocytes and PBMs contained TIMP activity, although supernatants from PMN cell culture did not. Reverse zymography of synovial fluid recovered from normal and AJD horses showed two protein bands, 22 and 28 kDa in size, exhibiting inhibitory activity against MMP-9. Reverse zymography of culture supernatants of synovial fibroblasts and chondrocytes gave similar results whereas the culture supernatants from PMNs and PBMs showed the presence of only the 28 kDa protein. The N-terminal amino acid sequence was obtained for the 22 kDa protein and revealed a 66% homology with human TIMP-2. The identification of TIMPs in equine synovial fluids and cell culture supernatants suggest that they may have a fundamental role in the homeostasis of the normal joint and in the excess proteolysis which occurs in articular disease in the horse.