Herpes stromal keratitis [HSK] is characterized by a histopathologically district pattern of infiltration by neutrophils, Langerhans cells, monocytes and lymphocytes. We have recently demonstrated that HSV-1 infection of human corneal keratocytes [HCK] but not human corneal epithelial cells [HCE] Is associated with an enhanced synthesis of IL-8. Since IL-8 is a chemotactic factor for neutrophils, these results suggest that HSV-1 infection of keratocytes induces the synthesis of a cytokine which can induce infiltration of these cells into the stroma. The first goal of this study is to determine if HSV-1 infection of cells found in the human corneal stroma enhances expression of the genes for GM- CSF and lL-6, two cytokines which in addition to lL-B could influence the histopathologically distinct pattern of inflammation seen in HSV keratitis. This goal will be accomplished by infecting pure cultures of HCK and HCE established in tissue culture by recent techniques developed in our laboratory. Reverse-transcriptase PCR and ELISA will then be used to test the hypothesis that HSV-1 infection enhances lL-6 and GM-CSF mRNA levels and protein synthesis in HCK but not in HCE. The second goal of the study is to identify mechanisms by which HSV-1 enhances cytokine gene expression in HCK. We will first determine if the rate of transcription of specific cytokine genes and/or the stability of their messages is increased following HSV-1 infection of HCK. Specific inhibitors of viral gene expression will be used to determine if HSV specified immediate early regulatory proteins synthesized after infection are responsible for enhancing select cytokine gene expression. Co- transfection experiments with vectors expressing specific HSV-1 regulatory genes and reporter plasmids driven by cytokine promoters will then be used to identify specific HSV-1 regulatory proteins which have the capacity to activate specific cytokine genes within human corneal cells. Identification of the cytokines induced by HSV-1 infection in the human cornea and the mechanisms of their induction by virus encoded proteins can lead to new targets for anti-inflammatory drugs designed to reduce the inflammation which leads to the vision loss seen in herpes stromal keratitis.
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