Endothelial leukocyte adhesion molecule 1: direct expression cloning and functional interactions

Area of Science:

• Molecular Biology
• Immunology
• Cell Biology

Background:

• Cytokine treatment induces adhesion molecules on endothelial cells, crucial for leukocyte recruitment.
• Endothelial leukocyte adhesion molecule 1 (ELAM-1) is an inducible adhesion molecule involved in inflammatory responses.
• Understanding ELAM-1's function requires its isolation and characterization.

Purpose of the Study:

• To isolate the cDNA for endothelial leukocyte adhesion molecule 1 (ELAM-1).
• To characterize the binding properties and ligand interactions of ELAM-1.
• To develop a novel cloning strategy for adhesion molecules.

Main Methods:

• Subtracted cDNA library construction from cytokine-treated human umbilical vein endothelial cells (HUVECs).
• Transient expression in COS-7 cells and selection of ELAM-1-expressing clones via adhesion to HL-60 cells.
• Functional assays including Ca2+ dependence, temperature independence, carbohydrate inhibition, and antibody blocking (anti-CD18).

Main Results:

• Successful isolation of ELAM-1 cDNA using a non-antibody, non-ligand-dependent cell adhesion selection method.
• ELAM-1-expressing COS cells demonstrated Ca2+-dependent, temperature-independent binding to HL-60 cells, neutrophils, and HT-29 cells.
• Binding was not inhibited by simple carbohydrates or anti-CD18 antibodies, suggesting a novel ligand interaction.

Conclusions:

• A novel and efficient method for cloning adhesion molecules based on cell-cell interactions was established.
• ELAM-1 mediates adhesion through a mechanism distinct from classical lectin-carbohydrate interactions and CD11/CD18-mediated adhesion.
• The findings provide insights into the molecular basis of endothelial cell adhesion during inflammation.