C L Cubitt1, R N Lausch, J E Oakes. 1. University of South Alabama, College of Medicine, Department of Microbiology and Immunology, Mobile 36688, USA.
Abstract
PURPOSE: GRO alpha is an inducible neutrophil chemotactic factor synthesized in inflamed corneal tissues. In this study, the regulation of GRO alpha synthesis after exposure of human corneal cells to proinflammatory cytokines was investigated. METHODS: Pure cultures of human corneal epithelial cells and keratocytes were exposed to increasing concentrations of either interleukin-1 alpha (IL-1 alpha) or tumor-necrosis factor-alpha (TNF-alpha). At selected times postexposure, the amounts of GRO alpha produced by the cultures were quantitated by enzyme-linked immunosorbent assay. The RNA was harvested from stimulated cultures to monitor GRO alpha mRNA and pre-mRNA levels by the reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Both IL-1 alpha and TNF-alpha induced significant levels of GRO alpha synthesis in keratocytes. However, IL-1 alpha-stimulated cells produced > 10 times more GRO alpha protein and 12 times more GRO alpha mRNA than did TNF-alpha-stimulated keratocytes. In contrast to the log differences in mRNA levels and protein synthesis, there was a less than twofold difference between IL-1 alpha and TNF-alpha in their capacity to induce GRO alpha specific pre-mRNA synthesis. When actinomycin D was added to stimulated keratocytes to inhibit transcription, the half-life of GRO alpha mRNA in TNF-alpha-treated cells was < 1 hour, whereas the half-life of the GRO alpha mRNA synthesized in IL-1 alpha-stimulated cells was > or = 9 hours. In contrast to keratocytes, exposure of corneal epithelial cells to IL-1 alpha and TNF-alpha did not induce significant increases in steady-state levels of GRO alpha pre-mRNA, mRNA, or protein. CONCLUSIONS: The results suggest that keratocytes are the major producers of GRO alpha in the human cornea. The capacity of IL-1 alpha to stimulate the synthesis of significantly higher quantities of GRO alpha in keratocytes compared to TNF-alpha is caused by the fact that GRO alpha-specific transcripts are more than nine times more stable in cells exposed to IL-1 alpha than in cells exposed to TNF-alpha.
PURPOSE:GRO alpha is an inducible neutrophil chemotactic factor synthesized in inflamed corneal tissues. In this study, the regulation of GRO alpha synthesis after exposure of human corneal cells to proinflammatory cytokines was investigated. METHODS: Pure cultures of human corneal epithelial cells and keratocytes were exposed to increasing concentrations of either interleukin-1 alpha (IL-1 alpha) or tumor-necrosis factor-alpha (TNF-alpha). At selected times postexposure, the amounts of GRO alpha produced by the cultures were quantitated by enzyme-linked immunosorbent assay. The RNA was harvested from stimulated cultures to monitor GRO alpha mRNA and pre-mRNA levels by the reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Both IL-1 alpha and TNF-alpha induced significant levels of GRO alpha synthesis in keratocytes. However, IL-1 alpha-stimulated cells produced > 10 times more GRO alpha protein and 12 times more GRO alpha mRNA than did TNF-alpha-stimulated keratocytes. In contrast to the log differences in mRNA levels and protein synthesis, there was a less than twofold difference between IL-1 alpha and TNF-alpha in their capacity to induce GRO alpha specific pre-mRNA synthesis. When actinomycin D was added to stimulated keratocytes to inhibit transcription, the half-life of GRO alpha mRNA in TNF-alpha-treated cells was < 1 hour, whereas the half-life of the GRO alpha mRNA synthesized in IL-1 alpha-stimulated cells was > or = 9 hours. In contrast to keratocytes, exposure of corneal epithelial cells to IL-1 alpha and TNF-alpha did not induce significant increases in steady-state levels of GRO alpha pre-mRNA, mRNA, or protein. CONCLUSIONS: The results suggest that keratocytes are the major producers of GRO alpha in the human cornea. The capacity of IL-1 alpha to stimulate the synthesis of significantly higher quantities of GRO alpha in keratocytes compared to TNF-alpha is caused by the fact that GRO alpha-specific transcripts are more than nine times more stable in cells exposed to IL-1 alpha than in cells exposed to TNF-alpha.
Authors: Vira Bitko; Nicolle E Garmon; Tin Cao; Benjamin Estrada; John E Oakes; Robert N Lausch; Sailen Barik Journal: BMC Microbiol Date: 2004-07-15 Impact factor: 3.605
Authors: Matthew R Pennington; Amrita Saha; David F Painter; Christina Gavazzi; Ashrafali M Ismail; Xiaohong Zhou; James Chodosh; Jaya Rajaiya Journal: Microorganisms Date: 2019-09-13