underneath the monolayers at similar rates to the lymphocytes observed here, but a larger
proportion can be directly observed to migrate through filters in minutes in a flow assay [21]. In
static trans-filter assays, neutrophils can be collected as early as 15-30 minutes after addition to
the upper surface. Here, we found that neutrophils that had migrated through EC quickly moved
into collagen gels, and became uniformly dispersed with time. This suggests random diffusion
into the gel. Lymphocytes did not 'diffuse' in this way, and indeed it seems that most were
actually retained in or near the endothelial cell layer. The inefficiency of lymphocyte migration
away from the sub-endothelial space was linked with the tendency of many migrated cells to
move back through the monolayer to the luminal surface in minutes. We have not observed such
behaviour with neutrophils. Taken together, these observations suggest that lymphocytes
continually interacted with EC and that a critical signal was lacking in the in vitro models, which
was required to drive migration of lymphocytes away from endothelium into tissue. Here, the
addition of CXCL10 to the collagen under EC moderately enhanced lymphocyte migration into
the gel, and reduced the tendency to move back and forth across the endothelial monolayer. In
general, stromal chemoattractants may increase efficiency of migration into tissue for all
leukocytes, but it seems that neutrophils, as opposed to lymphocytes, do not need such a signal to
free themselves from endothelium. Given the heterogeneity in the chemokine receptors
expressed by different lymphocytes, more than one subendothelial chemokine might be required
to induce efficient migration of the entire population. Conversely, stromal production of a
restricted chemokine subset could provide a further 'subendothelial' level of control within the
recruitment cascade.
Reverse migration has been described for monocytes, over hours after migration though
HUVEC cultured on amniotic tissue [30] or over tens of minutes under flow after migration