An increase in endothelial intracellular calcium and F-actin precedes the extravasation of interleukin-2-activated lymphocytes.
OBJECTIVE
Interleukin-2 (IL-2) induces protein leakage from the microcirculation 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, permeability to albumin, and extravasation of lymphocytes.


METHODS
IL-2 activated lymphocytes (IL-2 LYMPH) or unstimulated lymphocytes (LYMPH) were co-incubated with human microvascular endothelial cells (HMVEC). Endothelial albumin permeability, lymphocyte extravasation intracellular 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 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 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 peripheral 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.
Center for Applied Microcirculatory Research, University of Louisville, School of Medicine 40292, USA.
W D
WD
Ehringer
M J
MJ
Edwards
K A
KA
Wintergerst
A
A
Cox
F N
FN
Miller
D000199:Actins / Q000378:metabolism*; D000328:Adult; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004730:Endothelium, Vascular / Q000378:metabolism*; D016257:Fura-2 / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008833:Microcirculation; D012709:Serum Albumin / Q000378:metabolism
D000199:Actins; D001798:Blood Proteins; D007376:Interleukin-2; D012709:Serum Albumin; D002118:Calcium; D016257:Fura-2