Mouse intercellular adhesion molecule 1 (ICAM-1) isoforms demonstrate different binding affinities to mouse macrophage-1 antigen (Mac-1) and preliminary evidence for alternatively-spliced variants of ICAM-1 in humans

Full-length intercellular adhesion molecule 1 (ICAM-1) is an immunoglobulin-like expressed in various tissues, including the endothelium. The binding of ICAM-1 on an endothelial cell to lymphocyte function-associated molecule 1 (LFA-1) and macrophage-1 antigen (Mac-1) on neutrophils is important for neutrophil recruitment. ICAM-1 has five extracellular immunoglobulin domains and most investigations have studied neutrophil recruitment with fulllength ICAM-1. Binding between LFA-1: ICAM-1 and Mac-1: ICAM-1 differ slightly in function and binding site; LFA-1 binds domain 1 of ICAM-1 while Mac-1 binds domain 3. Six alternatively-spliced ICAM-1 isoforms were previously discovered in ICAM-1-exon-deletion mice: full-length ICAM-1, ICAM-1(2-4), ICAM-1(2-6), ICAM-1(3-6), ICAM-1(4-6), and ICAM-1(2-5), named for the spliced exons. To better understand the role of ICAM-1 isoforms in Mac-1: ICAM-1-dependent neutrophil recruitment to the liver, we studied the relative binding of five ICAM-1 isoforms to Mac-1. These isoforms were transiently expressed in HEK293T cells using customized plasmids. A V-bottomed adhesion assay assessed the binding of these cells to Mac-1. The ICAM-1(2-4) isoform demonstrated the highest relative binding percentage to Mac-1 (relative binding percentage of 42.3% in the presence of Mg/EGTA), while ICAM-1(2-6) demonstrated the lowest (relative binding percentage of 9.9% in the presence of Mg/EGTA). We also began assessment of the existence of ICAM-1 isoforms in human blood, liver, and lung RNA samples using reverse transcription polymerase chain reaction (RT-PCR). Additional experiments are required to evaluate the binding of the six ICAM-1 isoforms to Mac-1 and LFA1 using the V-bottomed adhesion assay, and will confirm the presence of ICAM-1 isoforms in humans. Greenwald 5 Introduction ICAM-1 and neutrophil recruitment Intercellular adhesion molecule 1 (ICAM-1) is a cell surface adhesion molecule expressed on endothelial cells and other immune cells, and its major role is to facilitate adhesion between two different cells during immune responses. ICAM-1 is a ligand for lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1), both of which are adhesion molecules expressed on the surface of neutrophils. During neutrophil recruitment, neutrophils leave the blood stream to go to a site of infection. In this process, shown in Figure 1, neutrophils adhere to the endothelium via binding of ICAM-1 to LFA-1 or to Mac-1 prior to transmigration through the blood vessel. Neutrophil recruitment is a multistep process mediated by various molecules; this process is categorized into steps of rolling, adhesion, and transmigration, which facilitate migration of the neutrophils to a site of infection in order to fight a causative pathogen. When the body begins to defend against a potential threat, circulating neutrophils are activated by various proinflammatory mediators, including chemoattractants, which are protein messengers produced by the host during inflammation. Figure 1 depicts the process of neutrophil-to-endothelial cell adhesion and accompanies the following explanation. Neutrophils express LFA-1 and/or Mac-1 on the cell surface, and these adhesion molecules are activated by various chemoattractants and cytokines. After activation, the neutrophils begin rolling along the endothelium. Neutrophils adhere to the endothelial cell via high-affinity binding between activated LFA-1 and/or Mac-1 expressed on the surface of a neutrophil and ICAM-1 expressed on the surface of an endothelial cell. Then, neutrophils can transmigrate through the endothelium and move to the site of inflammation. ICAM-1 thus plays a major role in neutrophil recruitment.