AN INCREASE IN ENDOTHELIAL INTRACELLULAR CALCIUM AND F-ACTIN PRECEDESTHE EXTRAVASATION OF INTERLEUKIN-2-ACTIVATED LYMPHOCYTES

Objective: Interleukin-2 (IL-2) induces protein leakage from the micro circulation and activates lymphocytes; yet it is unclear how it alters endothelial barrier function. Here, we report of a new continuous mon itoring system that allows for the continuous measurement and correlat ion of endothelial calcium, permeability to albumin, and extravasation of lymphocytes. Methods: IL-2 activated lymphocytes (IL-2 LYMPH) or u nstimulated lymphocytes (LYMPH) mere co-incubated with human microvasc ular endothelial cells (HMVEC). Endothelial albumin permeability, lymp hocyte extravasation; intracellular calcium mobilization, and f-actin distribution were examined using a new continuous monitoring system. R esults: The clearance rate of fluorescein isothiocyanate-labeled-human serum albumin (FITC-PISA) in the presence of IL-2 LYMPH peaked at 20 minutes, whereas the clearance rate of LYMPH peaked at 40 minutes. App roximately 40 minutes after the peak in the clearance rate to albumin, extravasation of carboxyfluorescein-labeled lymphocytes was detected, peak clearance rates for the extravasation of IL-2 LYMPH occurred at approximately 40 minutes after the addition of the lymphocytes to the HMVEC, whereas the peak clearance rate for the LYMPH occurred at 60 mi nutes after their addition. Both FITC-HSA and lymphocyte extravasation were measured concurrent to endothelial intracellular calcium mobiliz ation by FURA-2. There was an increase in calcium activation after the addition of IL-2 stimulated lymphocytes (71 +/- 5.1 nmol/L to 185 +/- 18.9 nmol/L) compared viith unstimulated lymphocytes (71 +/- 5.1 nmol /L to 110 +/- 12.2 nmol/L). The addition of IL-2 had little or no effe ct on endothelial actin, whereas the unstimulated lymphocytes and, to a greater extent, IL-2 LYMPH increased the presence of transversing st ress fibers and decreased peripheral actin. Conclusions: The findings reported here suggest that the permeability and extravasation events t hat occur upon addition of lymphocytes proceeds by a calcium- and acti n-dependent mechanism and that incubation of lymphocytes with IL-2 enh ances normal lymphocyte mechanisms of extravasation.