Ligand‐complex–based quantification of β2‐integrin–mediated affinity and avidity of murine T cells

Abstract Background Integrins facilitate binding to the extracellular matrix and other cells. Their subunit β2 is exclusively expressed by leukocytes, binds to the intercellular cell adhesion molecule 1 (ICAM‐1), and is pivotal for their recruitment to sites of inflammation such as the atherosclerotic plaque. Methods To investigate β2‐integrin–mediated adhesiveness, a well‐established assay for human whole blood was adapted for the analysis of murine T cell subsets. Changes in avidity and affinity were assessed by incubation of murine complexes ICAM‐1 in murine whole blood and consecutive stimulation with PMA and Mg 2+ /EGTA. Underlying signaling pathways in β2‐integrin–mediated adhesiveness upon chemokine stimulation with CCL‐19 were identified by incubation with reducing substances, and a Ca 2+ chelator and ROS and Ca 2+ measurements were carried out. Results Incubation of murine whole blood with PMA leads to 30‐fold and Mg 2+/ EGTA to 65‐fold increase in β2‐integrin–mediated adhesiveness of T cells. Specificity of the assay was proven by preincubation of a blocking antibody, leading to a 60% reduction in adhesion capacity. ROS species and Ca 2+ are crucial for chemokine‐mediated β2‐integrin activation. In vivo relevance was proven by induction of T cell adhesiveness in whole blood of mice upon myocardial infarction. Conclusions Our assay allows specific quantification of β2‐integrin–mediated affinity and avidity of T cells in whole blood samples. In congruence to human adhesion, these mechanisms are ROS and Ca 2+ dependent and significantly elevated after myocardial infarction. Our refined and robust assay may be of particular use in phenotyping involved mechanisms in T cell activation in atherosclerotic cardiovascular disease.