title: Differential induction of GRO alpha gene expression in human corneal epithelial cells and keratocytes exposed to proinflammatory cytokines.
abstract: OBJECTIVE
  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.affiliation: University of South Alabama, College of Medicine, Department of Microbiology and Immunology, Mobile 36688, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Cubitt
authors.forename: R N
authors.initials: RN
authors.lastname: Lausch
authors.forename: J E
authors.initials: JE
authors.lastname: Oakes
mesh_terms: D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000235:genetics*; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016207:Cytokines / Q000494:pharmacology*; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation; D006131:Growth Inhibitors / Q000235:genetics*; D006133:Growth Substances / Q000235:genetics*; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology*; D036341:Intercellular Signaling Peptides and Proteins; D007375:Interleukin-1 / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D016207:Cytokines; D006131:Growth Inhibitors; D006133:Growth Substances; D018836:Inflammation Mediators; D036341:Intercellular Signaling Peptides and Proteins; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha