TIMP-1 (Tissue Inhibitor of Metalloproteinases-1) is an inhibitor of matrix metallo proteinases (MMPs) that degrade extracellular matrix. Mice deficient for this protein were generated by targeted mutagenesis (Soloway et al. Submitted). In characterizing the phenotype of this mutant, it was observed that TIMP-1 deficient mice clear established Pseudomonas aeruginosa corneal infections far more efficiently than wild-type litter mates. Two days after infection, most mutant and wild-type mice have similar numbers of viable bacteria in infected corneas. But by five days after infection, the TIMP-1-deficient corneas contain between 2 and 7 orders of magnitude fewer viable bacteria than corneas from wild-type mice. One goal of this proposal is to identify the cellular and molecular basis of this mutant phenotype. There are three mechanisms under consideration. First, the ability of bacteria to grow in the eye may be reduced in mutant mice. This influence may be at the level of bacterial attachment, or rate of bacterial division in the cornea. Second, extracellular matrix structures that must be crossed by immune cells mobilized to combat the infection may be easier to cross in mutant mice, allowing more rapid entry of those cells into sites of infection, and hence, more rapid clearance of the bacteria. Third, some cellular and biochemical activities of immune cells mobilized to combat infections (primarily neutrophils and macrophages) may be enhanced in mice lacking TIMP-1. Both cell types produce matrix metalloproteinases, and their net activities are almost certainly increased in cells derived from mutant mice. Since (1) these activities have been implicated in the function of neutrophils and macrophages, (2) MMP inhibitors have been shown to have antiinflammatory properties, and (3) preliminary results suggest that growth of bacteria during the first few days is no different in mutant than wild-type eyes, the latter, immune response-dependent mechanism is considered the most likely basis of the mutant phenotype. Nonetheless, experiments to evaluate each possible mechanism are proposed. An additional goal of this proposal is to examine the responses of timp-1 mice to inflammation-inducing stimuli. Also, mice deficient for TIMP-2 will be developed, and their responses to P. aeruginosa-induced keratitis and inflammation will be assessed. And finally, the influence of the inflammation-associated cytokine, IL-6, on host response to bacterial keratitis, inflammation, and on the timp-1 phenotype, will be studied using mice deficient for IL-6, and both TIMP-1 and IL-6, using many of the same assays applied to the TIMP-1-deficient mice.
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