An Increase in Endothelial Intracellular Calcium and F-Actin Precedes the Extravasation of Interleukin-2– Activated Lymphocytes WILLIAM D. EHRINGER,* MICHAEL J. EDWARDS,* ,† KUPPER A. WINTERGERST, † ABIGAIL COX † AND FREDERICK N. MILLER* ,‡ *Center for Applied Microcirculatory Research, and Departments of † Surgery and ‡ Physiology, University of Louisville, School of Medicine, Louisville, KY, USA ABSTRACT Objective: Interleukin-2 (IL-2) induces protein leakage from the microcircula- tion and activates lymphocytes; yet it is unclear how it alters endothelial barrier function. Here, we report of a new continuous monitoring system that allows for the continuous measurement and correlation of endothelial calcium, permeabil- ity to albumin, and extravasation of lymphocytes. Methods: IL-2 activated lymphocytes (IL-2 LYMPH) or unstimulated lympho- cytes (LYMPH) were co-incubated with human microvascular endothelial cells (HMVEC). Endothelial albumin permeability, lymphocyte extravasation, intra- cellular calcium mobilization, and f-actin distribution were examined using a new continuous monitoring system. Results: The clearance rate of fluorescein isothiocyanate-labeled–human serum albumin (FITC-HSA) in the presence of IL-2 LYMPH peaked at 20 minutes, whereas the clearance rate of LYMPH peaked at 40 minutes. Approximately 40 minutes after the peak in the clearance rate to albumin, extravasation of car- boxyfluorescein-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 minutes after their addition. Both FITC-HSA and lymphocyte extravasation were measured concurrent to endothelial intracellular calcium mobilization 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 with unstimulated lymphocytes (71 ± 5.1 nmol/L to 110 ± 12.2 nmol/L). The addition of IL-2 had little or no effect on endothelial actin, whereas the unstimulated lymphocytes and, to a greater extent, IL-2 LYMPH increased the presence of transversing stress fibers and decreased pe- ripheral actin. Conclusions: The findings reported here suggest that the permeability and extravasation events that occur upon addition of lymphocytes proceeds by a calcium- and actin-dependent mechanism and that incubation of lymphocytes with IL-2 enhances normal lymphocyte mechanisms of extravasation. KEY WORDS: endothelial, permeability, calcium, f-actin, IL-2, lymphocytes INTRODUCTION Interleukin-2 (IL-2) is a 15-kD glycoprotein that induces protein leakage from the microcirculation (5). The effects of IL-2 on vascular endothelium is well documented (1,10,22), yet conflicting hypoth- eses still remain concerning the mechanism(s) in- volved. It has been proposed that high levels of IL-2 increase circulating levels of tumor necrosis factor- Supported in part by The American Heart Association (Kentucky affiliate), The Louisville Institute for Heart and Lung Research, Jewish Hospital, National Institute of Health, and Alliant Health System. For reprints of this article, contact William D. Ehringer, PhD, Center for Applied Microcirculatory Research, Health Science Center, Building A, Room 1110, University of Louisville, School of Medicine, 500 S. Preston Street, Louisville, KY 40292, USA. Received 2 June 1997; accepted 4 October 1997 Microcirculation (1998) 5, 71–80 © 1998 Stockton Press All rights reserved 1073-9688/98 $12.00 http://www.stockton-press.co.uk