DOI: 10.1002/ame2.70155
Ligand-complex-based quantification of β2-integrin-mediated affinity and avidity of murine T cells
M.Therre1
A.A.Kuhnle2
H.M.Arndt3
N.Frey2
M.H.Konstandin2
N.V.Bogert2
1.Department of Cardiology,University Hospital Heidelberg,Ruprecht-Karls-University Heidelberg,Heidelberg,Germany;German Centre for Cardiovascular Research(DZHK),Partner Site Heidelberg/Mannheim,Berlin,Germany;Department of Cardiology,Angiology and Intensive Care Medicine,Saarland University Hospital,Homburg,Germany2.Department of Cardiology,University Hospital Heidelberg,Ruprecht-Karls-University Heidelberg,Heidelberg,Germany;German Centre for Cardiovascular Research(DZHK),Partner Site Heidelberg/Mannheim,Berlin,Germany3.Department of Gynecology and Obstetrics,University Hospital Heidelberg,Heidelberg,Germany
摘要：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 inflam-mation 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 mu-rine whole blood and consecutive stimulation with PMA and Mg2+/EGTA.Underlying signaling pathways in β2-integrin-mediated adhesiveness upon chemokine stimula-tion with CCL-19 were identified by incubation with reducing substances,and a Ca2+chelator and ROS and Ca2+measurements were carried out.
Results:Incubation of murine whole blood with PMA leads to 30-fold and Mg2+/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 Ca2+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 Ca2+dependent and significantly elevated after myocardial infarction.Our refined and robust assay may be of particular use in phenotyping in-volved mechanisms in T cell activation in atherosclerotic cardiovascular disease.
机标关键词：quantificationintegrinligandcellsaffinityaviditybasedcomplex
论文发表日期：2026-02-28
在线出版日期：2026-03-31（本平台首次上网日期，不代表文献的发表时间）
页数：10( 319-328 )
英文信息