Endothelial cell calpain activity facilitates lymphocyte diapedesis

Lymphocyte infiltration of tissue is a cardinal feature of solid-organ allograft rejection. Vascular endothelial cells (EC) participate in lymphocyte recruitment through the display of adhesion molecules and chemokines to promote leukocyte extravasation. Moreover, EC reorganize the Cytoskeleton and cytoskeleton-associated structures during leukocyte diapedesis. We examined the role of EC (Ca+2)i and the calcium-sensitive protease, calpain, during lymphocyte diapedesis through a human EC monolayer under physiologic shear stress in vitro. We observed that lymphocyte transendothelial migration (TEM) was inhibited by chelating EC cytosolic calcium, or depleting EC endoplasmic reticulum calcium stores by inhibition of the endoplasmic reticulum Ca ATPase. Further, inhibition of EC phospholiase C also decreased lymphocyte TEM. We determined that EC constitutively exhibit calpain activity, using fluorescence generation from a calpain substrate to report calpain activity in individual live cells. Moreover, EC adjacent to a transmigrating lymphocyte showed increased calpain activity. Further, lymphocyte TEM was inhibited by agents that block calpain activity. Inhibition of lymphocyte TEM occurs at the lumenal EC surface and correlates with impaired development of intercellular adhesion molecule 1 (ICAM-1)-rich docking structures by the EC. We conclude EC calcium and calpain activity facilitates lymphocyte TEM, and participates in the assembly of the docking structure.