The transfer of VT-1-FITC bound to PMNs to human GMVECs was studied by flow cytometry.

Panels A, C, and F: FACS analysis of nonstimulated GMVECs incubated for 4 hours with PMNs loaded with VT-FITC on ice. (A) Forward scatter and side scatter of nonstimulated GMVECs are shown. (C) No positive staining for CD16-PE was observed, indicating that all PMNs were removed by washing the monolayers. (F) Nonstimulated GMVECs did not bind any VT-FITC after incubation with VT-FITC–loaded PMNs. Panels B, D, E, G, and H: TNF-α–stimulated GMVECs retained 2.8%-3.6% of PMNs (present in the gated area P in panel B) after 4 hours of incubation and washing of the monolayers. (D, E) PMNs were distinguished from GMVEC using CD16-PE; panel D represents all cells, whereas panel E reflects the gated area. (G) TNF-α–treated GMVECs incubated with VT-FITC-loaded PMNs were able to bind 30%-50% of VT-FITC after the incubation period of 4 hours (all cells minus the gated area P). (H) PMNs showed no positive staining for VT-FITC, indicating that ligand passing of VT-FITC from PMNs to GMVECs had occurred.