Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0508301 on October 23, 2008

Published online before print October 23, 2008
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(Journal of Leukocyte Biology. 2009;85:98-107.)
© 2009 by Society for Leukocyte Biology

Direct observations of the kinetics of migrating T cells suggest active retention by endothelial cells with continual bidirectional migration

Helen M. McGettrick*, Kirsty Hunter*, Paul A. Moss{dagger}, Christopher D. Buckley{ddagger}, G. Ed Rainger* and Gerard B. Nash*,1

* Divisions of Medical Sciences,
{dagger} Cancer Studies, and
{ddagger} Immunity and Infection, The Medical School, The University of Birmingham, Birmingham, United Kingdom

1 Correspondence: Department of Physiology, The Medical School, The University of Birmingham, Birmingham B15 2TT, UK. E-mail: g.nash{at}bham.ac.uk

ABSTRACT

The kinetics and regulatory mechanisms of T cell migration through the endothelium have not been fully defined. In experimental, filter-based assays in vitro, transmigration of lymphocytes takes hours, compared with minutes, in vivo. We cultured endothelial cell (EC) monolayers on filters, solid substrates, or collagen gels and treated them with TNF-{alpha}, IFN-{gamma}, or both prior to analysis of lymphocyte migration in the presence or absence of flow. PBL, CD4+ cells, or CD8+ cells took many hours to migrate through EC-filter constructs for all cytokine treatments. However, direct microscopic observations of EC filters, which had been mounted in a flow chamber, showed that PBL crossed the endothelial monolayer in minutes and were highly motile in the subendothelial space. Migration through EC was also observed on clear plastic, with or without flow. After a brief settling without flow, PBL and isolated CD3+ or CD4+ cells crossed EC in minutes, but the numbers of migrated cells varied little with time. Close observation revealed that lymphocytes migrated back and forth continuously across endothelium. Under flow, migration kinetics and the proportions migrating back and forth were altered little. On collagen gels, PBL again crossed EC in minutes and migrated back and forth but showed little penetration of the gel over hours. In contrast, neutrophils migrated efficiently through EC and into gels. These observations suggest a novel model for lymphoid migration in which EC support migration but retain lymphocytes (as opposed to neutrophils), and additional signal(s) are required for onward migration.

Key Words: lymphocytes • adhesion • flow • cytokines