Expression of functional ICAM-1 on cultured human keratocytes induced by tumor necrosis factor-alpha.
OBJECTIVE
Leukocytes such as neutrophils contribute to the pathogenesis of corneal ulcer. The effect of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha on the expression of intercellular adhesion molecule (ICAM)-1 by cultured human keratocytes was investigated because the interaction of leukocytes with ICAM-1 expressed on the surface of structural cells mediates leukocyte infiltration into tissue at sites of inflammation.


METHODS
Cultured human keratocytes were incubated with various concentrations of TNF-alpha. The surface expression of ICAM-1 was evaluated by whole-cell enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry. The abundance of ICAM-1 mRNA in cell lysate was determined by quantitative reverse transcription and polymerase chain reaction analysis. Adhesion of neutrophils to corneal fibroblasts was assayed by measuring the fluorescence of Calcein-AM-labeled neutrophils.


RESULTS
Incubation of keratocytes with TNF-alpha induced increased expression of ICAM-1 on the surface of keratocytes in a dose- and time-dependent manner. The abundance of ICAM-1 mRNA in keratocytes was increased by the incubation of cells with TNF-alpha. Exposure of keratocytes to TNF-alpha increased the adherence of human neutrophils to these cells.


CONCLUSIONS
Stimulation of keratocytes with TNF-alpha resulted in an increase in the abundance of ICAM-1 mRNA, the cell surface expression of ICAM-1 protein, and enhanced adhesion of neutrophils to these cells. The expression of ICAM-1 on human keratocytes may thus contribute to leukocyte infiltration into the corneal stroma of individuals with inflammatory ocular diseases.
Department of Biomolecular Recognition and Ophthalmology, Ube City, Yamaguchi, Japan.
Naoki
N
Kumagai
Ken
K
Fukuda
Youichiro
Y
Fujitsu
Teruo
T
Nishida
D000293:Adolescent; D000328:Adult; D000368:Aged; D002448:Cell Adhesion; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003319:Corneal Stroma / Q000166:cytology*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009504:Neutrophils / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1