through 10μm-thick porous filters or into collagen gels. Interestingly, a significant proportion of
PBL could be seen to migrate back and forth across the endothelial monolayer, sometimes
repeatedly. In consequence, the proportion under the endothelium did not vary much over about
15 minutes. The above phenomena could be observed for CD3+ T-cells (which would be
expected to make up the great majority of PBL) and the CD4+ T-cell subset, as well as PHA-
activated cells. In non-visual, static, filter-based assays, little transmigration was detected within
hours, and 24 hours were needed to obtain a proportion of lymphocytes under the filter
comparable to that seen in minutes during direct observations of transendothelial migration. After
24 hours, penetration of gels by lymphocytes was inefficient (e.g., compared to neutrophils).
Given the speed at which lymphocytes were seen to migrate under EC, it seems that EC tended to
retain lymphocytes in their vicinity, and that a signal to migrate through stroma and/or across the
filters was lacking. Studies in which exogenous chemokine was added to collagen gels, and
penetration of the matrix was increased, supported this concept.
Efficient migration of T-cells through EC treated with TNF and IFN (reaching ~40% of
those adherent) has been described previously under flow conditions similar to those used here
[5]. In studies using TNF alone, Luscinskas et al., observed that migration occurred in minutes
but did not quantify the proportion of cells migrating [3]. Cinamon et al., found that only ~5% of
adherent T-cells migrated through TNF-treated EC under flow, but this proportion increased
greatly when SDF was added to the endothelial surface [6]. Most striking was their observation
that if flow was stopped, there was negligible transmigration. In each of these flow-based
studies, isolated T-cells were pre-incubated for prolonged periods, presumably to induce
activation and greater migration, although this was only explicitly stated by Piali et al. [5].