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

The studies outlined above leave some uncertainties about the rate at which migration
occurs, first through the endothelial monolayer and then away from it, and the requirement for
flow or for pre-activation of T-cells or endothelial cells for efficient transendothelial migration.
Hours are required for T-cells to cross EC/filter constructs or enter collagen gels below EC, as
opposed to minutes to cross EC alone, suggesting that assays are strongly influenced by
migration through the sub-endothelial matrix and/or filter itself. In vivo, it is likely that naive as
well as memory T-cells are recruited to peripheral tissue [17], and peripheral blood lymphocytes
(PBL) are evidently recruited without prior activation. While direct studies of the kinetics of
lymphocyte recruitment across inflamed endothelium are lacking, studies in the rat have shown
that T cells are recruited across the vessel wall in the peripheral lymph nodes and Peyers Patch
within 30-40mins of their infusion [18, 19]. To investigate these problems, we studied kinetics of
lymphocyte migration through endothelial cells cultured on filters, plastic wells or collagen gels,
and treated with different cytokines, and compared results in static and flow-based assays.
Previously we used similar approaches to analyse kinetics of neutrophil migration through
endothelial monolayers and filters [20-22]. Here we found that lymphocytes could cross
endothelial monolayers in minutes, with little evidence of a requirement for flow. The migrated
cells were highly motile, and the prolonged periods required to transit filter systems could not be
attributed to transendothelial migration itself. However, we made the novel observations that
some lymphocytes underwent multiple transits back and forth across endothelial monolayers, and
that few motile transmigrated lymphocytes entered collagen gels (e.g., compared to neutrophils).
We suggest that EC tend to retain migrating lymphocytes and that a separate signal is required to
overcome this and allow lymphocytes to move into the stroma.

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