An assay for the analysis of lymphocyte migration across cerebral endothelium in vitro

We describe a recently developed assay for the analysis of leukocyte migration across cerebral endothelium in vitro. The endothelium is grown as monolayers on Goretex or Cyclopore membranes coated with extracellular matrix proteins and supported on inserts. This system permits the recovery and phenotyping of cells which migrate down through the endothelium. Using labelled lymphocytes we were able to differentiate four populations of cells, with differing degrees of mobility in the migration assay. We have compared the results from this system with those from conventional adhesion assays. Binding of cells to the endothelium is rapid, but is confined to a particular subpopulation of the applied lymphocytes. We have followed cell migration over 24 h in the system using normal and cytokine-activated endothelium and have found that whereas adhesion depends both on the state of lymphocyte activation and on the condition of the endothelium, the level of migration of stimulated lymphocytes is largely independent of endothelial activation. Moreover, whereas CD8+ cells bind well to the endothelium, it is the CD4+ cells which migrate most effectively. Comparison of brain and epididymal fat endothelium showed similar migration levels over 2 h, but migration was greater across epididymal fat endothelium at 24 h.