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Updated: Aug 9, 2026
Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes
Published on: June 13, 2014
Cellular activation through the ligation of intercellular adhesion molecule-1
A Clayton1, R A Evans, E Pettit
1Institute of Nephrology, University of Wales College of Medicine, Cardiff Royal Infirmary, Cardiff CF2 1SZ, Wales, UK.
Insights
Leukocyte binding to cells upregulates intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. This process involves calcium influx and has implications for inflammatory disease progression.
Area of Science:
- Immunology
- Cell Biology
- Inflammation Research
Background:
- Intercellular adhesion molecule-1 (ICAM-1) is crucial for leukocyte infiltration at inflammatory sites.
- ICAM-1 mediates leukocyte adherence and migration across the endothelium.
- The role of interstitial cell ICAM-1 in controlling leukocyte infiltration requires further elucidation.
Purpose of the Study:
- To investigate the de novo synthesis of ICAM-1 mRNA and protein in response to leukocyte binding.
- To determine the role of ICAM-1-dependent activation in cultured cells.
- To explore the impact of ICAM-1 cross-linking on adhesion molecule expression and intracellular calcium levels.
Main Methods:
- Culturing human renal cortex and lung fibroblasts and endothelial cells.
- Inducing de novo synthesis of ICAM-1 via leukocyte binding and ICAM-1 cross-linking with antibodies.
- Measuring ICAM-1 mRNA and protein expression levels over time.
- Assessing VCAM-1 expression following ICAM-1 cross-linking.
- Monitoring cytosolic free calcium changes using calcium imaging and BAPTA-AM.
Main Results:
- Leukocyte binding and ICAM-1 cross-linking induced de novo synthesis of ICAM-1 mRNA and protein.
- ICAM-1 mRNA levels increased two- to threefold, peaking at 1-3 hours post-cross-linking.
- Peak ICAM-1 protein expression occurred between 18 and 48 hours.
- ICAM-1 cross-linking also led to increased VCAM-1 expression.
- Both leukocyte binding and ICAM-1 cross-linking triggered a rise in cytosolic free calcium, which was essential for adhesion molecule upregulation.
Conclusions:
- Direct cell surface interaction of ICAM-1 with inflammatory cell ligands stimulates endothelial cells and fibroblasts.
- Upregulated synthesis and expression of cellular adhesion molecules is a key phenotypic change.
- These findings suggest significant implications for controlling leukocyte infiltration and the progression of inflammatory diseases.
Abstract:
In addition to its role in mediating leukocyte adherence to and migration across the endothelium, intercellular adhesion molecule (ICAM)-1 on the surface of interstitial cells has been implicated as a principal adhesion molecule controlling leukocyte infiltration at inflammatory sites. The present study demonstrates that leukocyte binding to fibroblasts isolated from both the human renal cortex and lung and to endothelial cells induced the de novo synthesis of ICAM-1 mRNA and protein through the ICAM-1-dependent activation of the cultured cells. This was mimicked by specifically cross-linking the ICAM-1 receptor with anti-ICAM-1 antibodies. Following cross-linking there was a two- to threefold increase in the steady state levels of ICAM-1 mRNA isolated from the cells. The expression of this mRNA peaked at 1-3 hours and was dose-dependent on the concentration of secondary cross-linking antibody. Peak protein expression was between 18 and 48 hours after cross-linking. Additional data demonstrated a similar increase in the expression of VCAM-1 following ICAM-1 cross-linking. In contrast, there was no reponse as a result of incubation with an isotype-matched control antibody. Both the binding of leukocytes and the cross-linking of ICAM-1 triggered a rise in cytosolic free calcium as the result, in part, of a calcium influx from the extracellular medium. Using BAPTA-AM to chelate intracellular calcium ions indicated that this increase in cytosolic free calcium was directly involved in the initiation of adhesion molecule upregulation. The present study demonstrates that both endothelial cells and fibroblasts can be stimulated by the direct cell surface interaction of ICAM-1 with its ligands on inflammatory cells. One phenotypic change resulting from this interaction is the upregulated synthesis and expression of more cellular adhesion molecules. This may have profound implications for the control and persistence of a leukocyte infiltrate and the progression of inflammatory disease.
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