AN ASSAY FOR THE ANALYSIS OF LYMPHOCYTE MIGRATION ACROSS CEREBRAL ENDOTHELIUM IN-VITRO

We describe a recently developed assay for the analysis of leukocyte m igration across cerebral endothelium in vitro. The endothelium is grow n as monolayers on Goretex or Cyclopore membranes coated with extracel lular matrix proteins and supported on inserts. This system permits th e recovery and phenotyping of eels which migrate down through the endo thelium. Using labelled lymphocytes we were able to differentiate four populations of cells, with differing degrees of mobility in the migra tion assay. We have compared the results from this system with those f rom 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 24h in the system u sing normal and cytokine-activated endothelium and have found that whe reas adhesion depends both on the state of lymphocyte activation and o n the condition of the endothelium, the lever of migration of stimulat ed lymphocytes is largely independent of endothelial activation. Moreo ver, whereas CD8(+) cells bind well to the endothelium, it is the CD4( +) cells which migrate most effectively. Comparison of brain and epidi dymal fat endothelium showed similar migration levels over 2h, but mig ration was greater across epididymal fat endothelium at 24h.