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Expression of functional ICAM-1 on cultured human keratocytes induced by tumor necrosis factor-alpha
Naoki Kumagai1, Ken Fukuda, Youichiro Fujitsu
1Department of Biomolecular Recognition and Ophthalmology, Ube City, Yamaguchi, Japan.
Insights
Tumor necrosis factor-alpha (TNF-alpha) increases intercellular adhesion molecule-1 (ICAM-1) expression in corneal cells. This promotes neutrophil adhesion, contributing to corneal ulcer pathogenesis in inflammatory eye diseases.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Leukocytes, particularly neutrophils, play a role in corneal ulcer development.
- Intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte infiltration into inflamed tissues.
- Understanding the regulation of ICAM-1 expression in corneal cells is crucial for inflammatory eye disease research.
Purpose of the Study:
- To investigate the effect of tumor necrosis factor-alpha (TNF-alpha) on intercellular adhesion molecule-1 (ICAM-1) expression in cultured human keratocytes.
- To determine if TNF-alpha influences the adhesion of neutrophils to corneal cells.
Main Methods:
- Cultured human keratocytes were treated with varying concentrations of TNF-alpha.
- ICAM-1 surface expression was measured using enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry.
- ICAM-1 mRNA levels were quantified via reverse transcription and polymerase chain reaction.
- Neutrophil adhesion to keratocytes was assessed using fluorescence-labeled neutrophils.
Main Results:
- TNF-alpha significantly increased ICAM-1 surface expression on keratocytes in a dose- and time-dependent manner.
- The abundance of ICAM-1 mRNA in keratocytes was elevated following TNF-alpha stimulation.
- Exposure to TNF-alpha enhanced the adherence of human neutrophils to keratocytes.
Conclusions:
- TNF-alpha stimulation upregulates ICAM-1 mRNA and protein expression in human keratocytes.
- Increased ICAM-1 expression on keratocytes promotes neutrophil adhesion.
- These findings suggest ICAM-1 on human keratocytes contributes to leukocyte infiltration in inflammatory ocular conditions.
Purpose:
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.
