https://doi.org/10.1007/s00392-025-02625-4
1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; 2Universitätsklinikum des Saarlandes Innere Medizin III - Kardiologie, Angiologie und internistische Intensivmedizin Homburg/Saar, Deutschland
Background
Integrin β2 is pivotal in T cell activation and adhesion, playing a crucial role in inflammatory processes as atherosclerosis. By binding to intercellular adhesion molecules (ICAMs) on endothelial cells, integrin β2 mediates firm adhesion and trans-endothelial migration of T cells, promoting their recruitment to the side of inflammation. A ligand-complex-based adhesion assay (LC-AA) to evaluate affinity and avidity changes of integrins has been already established for human leukocytes in whole blood samples. This method enables the identification of activated subpopulations of T cells in patients with acute coronary syndrome preceding myocardial infarction.
Methods
To investigate β2-integrin–mediated adhesiveness of T cells in murine whole blood, the well-established assay for human samples (Ligand complex-based adhesion assay, LC-AA) was adapted for the analysis of murine T cell subsets (mLC-AA). T cell subsets were identified by costaining for CD3, CD4, CD44, and CD62L and followed by evaluation in flow cytometry. Changes in adhesiveness of T cell subsets were assessed upon stimulation with CCL-19, PMA and Mg2+/EGTA. To prove β2-integrin specificity, function blocking antibodies were used.
Results
β2-integrin-mediated adhesiveness of T Cells is increased after incubating murine whole blood with CCL-19 (7-fold increase), PMA (30-fold increase) and Mg2+/EGTA (65-fold increase). Preincubation with CD18 specific IgG (clone GAME) leads to a 60% reduction of adhesion capacity, which confirmed the specificity of the assay. Interestingly, after CCL-19 stimulation only naive T cells show strong increment of β2-integrin mediated adhesiveness but not memory T cells. Furthermore, incubation with reducing substances NAC, APO and DPI as well as calcium chelator BAPTA attenuated the adhesion capacity, confirming the significance of ROS species and Calcium for murine β2 integrin activation. Lastly, myocardial infarction by LAD ligation in mice induced significant β2-integrin activation in T cell subsets compared to sham control mice reflecting T cell activation upon myocardial ischemia.
Conclusions
mLC-AA was successfully established to monitor β2 integrin activation of T cells in murine whole blood. The assay opens the possibility to quantify integrin adhesiveness in small sample size to further our understanding in leukocyte recruitment e.g. upon myocardial infarction or other inflammatory disease states.