BME Seminar: Ronen Alon, Ph.D.

Force regulated integrin activation in lymphocyte adhesion, motility and transendothelial migration

Lymphocyte arrest on specific target vascular sites is mediated by two major integrin receptors, VLA-4 and LFA-1 which exist in inactive states and need to undergo in situ reversible activation at the lymphocyte-endothelium interface. We propose that the conformational switches of these integrins involve both cytoplasmic (inside out) and extracellular (outside in) rearrangements that are mechanically regulated by external fluid forces. Without these forces, lymphocyte integrins fail to develop firm adhesion to their respective endothelial ligands, VCAM-1 and ICAM-1, even in the presence of stimulatory chemokine signals. Optimal force loading also governs spontaneous VLA-4 activation by ligand, a process necessary for effector immune cells to bypasss chemokine signals during arrest on highly inflamed vascular beds. External and internal force application on lymphocyte integrins also play key roles in coordinating their transendothelial migration (TEM). Specialized chemokine stimulated machineries activate the key lymphocyte integrin LFA-1 to coordinate post arrest motility (crawling) of lymphocytes from their original arrest sites to sites of TEM. Dissecting how endothelial chemokines trigger lymphocyte integrins to support shear-resistant crawling on and across endothelial barriers we found a critical role for high affinity (HA) LFA-1 integrin subsets in this process. Endothelial presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath the crawling lymphocytes. Shear forces dramatically enhanced filopodia density underneath crawling lymphocytes and a fraction of these adhesive filopodia invaded the endothelial cells prior to and during TEM. Transmigrating lymphocytes extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. We propose that shear forces exerted on LFA-1 stabilize LFA-1-ICAM-1 bonds at high affinity, trigger ICAM-1 clustering, facilitate endothelial invaginations, and thereby allow crawling lymphocytes to probe the endothelial barrier for chemotactic cues within the endothelial junctions and the basolateral compartment. Once in the tissue, motile lymphocytes silence their LFA-1 until encountering an antigenic stoppage signal. Our studies predict that LFA-1 and VLA-4 rapidly adopt distinct conformational states, depending on the chemokine cues and the availability of integrin ligands. In addition, we propose a new level of integrin conformational regulation, the state of integrin anchorage, which determines the extent by which a given integrin can undergo affinity modulation by force in response to ligand engagement.