Endothelial IQGAP1 regulates efficient lymphocyte transendothelial migration

Abstract

Leukocyte movement from the blood to the tissues is a fundamental process in acute and chronic inflammatory diseases. While the role of endothelial cells (EC) to recruit leukocytes to sites of inflammation is well known, the mechanisms that control remodeling of EC shape and adhesive contacts during leukocyte transendothelial migration (TEM) are not completely understood. We studied the role of IQGAP1, an adaptor protein that binds to filamentous-actin and microtubules (MT) at interendothelial junctions, during lymphocyte TEM. EC IQGAP1 knockdown decreases MT tethered to the adherens junction, and decreases lymphocyte TEM to $70% (po0.05) versus control. Similarly, loss of adherens junction-associated MT induced by brief nocodazole (ND) treatment decreases lymphocyte TEM to $65% of control (po0.01). Confocal microscopy imaging indicates that EC IQGAP1 knockdown and MT depolymerization both result in lymphocyte accumulation above the vascular endothelial cadherin (VE-cadherin) junctions and reduces the fraction of adherent lymphocytes that complete diapedesis across interendothelial cell junctions. However, we observe no change in VE-cadherin gap formation underlying adherent lymphocytes among control, IQGAP1 knockdown, or MT depolymerised EC monolayers. These data indicate that IQGAP1 contributes to MT stability at endothelial junctions. Further, IQGAP1 is involved in junction remodeling required for efficient lymphocyte diapedesis, independent of VE-cadherin gap formation.Key words: Adherens junctions . Cytoskeleton . IQGAP1 Supporting Information available online IntroductionLeukocyte extravasation is fundamental to the development of many immune responses including solid-organ allograft rejection. In this process, leukocytes leave the bloodstream and migrate into tissues through the endothelial cells (EC) that line the walls of vessels, i.e. leukocytes undergo transendothelial migration (TEM). Whereas the specific adhesion molecules, chemoattractants, and possibly signaling pathways involved in TEM are unique among different subgroups of leukocytes and vascular beds, the interaction between leukocytes and EC during TEM can be generalized into a multicascade event, described in recent reviews [1][2][3]. EC and leukocyte adhesion molecules mediate tethering and rolling of leukocytes on EC followed by chemokinemediated leukocyte activation, then firm adhesion to the EC. Finally, adherent leukocytes crawl on the surface of endothelium, undergo diapedesis, and enter tissues by mechanisms that are not fully understood.Leukocyte transmigration may occur by either a transcellular, through EC, or paracellular route, between adjacent EC [4][5][6]. 204The latter is associated with structural changes in the interendothelial adhesion structures and EC cytoskeleton [5,7,8]. Cross-linking of adhesion molecules such as CD54 or CD106 is shown to mediate signals that lead to EC actin cytoskeleton remodeling [9][10][11][12]. These signaling cascades promote structural changes in interendothlelial junctions, which might be requ...

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