Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells

Abstract

The arrest of rolling leukocytes on various target vascular beds is mediated by specialized leukocyte integrins and their endothelial immunoglobulin superfamily (IgSF) ligands. These integrins are kept in largely inactive states and undergo in situ activation upon leukocyte-endothelial contact by both biochemical and mechanical signals from flow-derived shear forces. In vivo and in vitro studies suggest that leukocyte integrin activation involves conformational alterations through inside-out signaling followed by ligand-induced rearrangements accelerated by external forces. This activation process takes place within fractions of seconds by in situ signals transduced to the rolling leukocyte as it encounters specialized endothelial-displayed chemoattractants, collectively termed arrest chemokines. In neutrophils, selectin rolling engagements trigger intermediate affinity integrins to support reversible adhesions before chemokine-triggered arrest. Different leukocyte subsets appear to use different modalities of integrin activation during rolling and arrest at distinct endothelial sites.

Figure 1

Rapid GPCR mediated integrin activation by endothelial-immobilized chemokine; involvement of Rap-1 and Rho GTPases. A proposed intracellular signaling cascade from chemokine (blue ball) bound to GPCR to the LFA-1 integrin (blue heterodimer) within a single microvillus of a lymphocyte rolling on L-selectin ligands. The front microvillus is occupied by L-selectin ligand whereas integrin activation takes place during occupancy and stretching of the rear microvillus, which may be co-occupied via L-selectin (not drawn). Canonical bidirectional activation of LFA-1 from inactive bent state to a semi-active extended state (unbent ectodomain with the two headpiece I-domains in closed conformation with low affinity to ligand) and to high affinity extended state (unbent integrin with both of the I-domains stabilized in an open state, see the ribbon diagram of the various LFA-1 headpiece states in the lower panels) is shown. Upon initial encounter of the proper endothelial-bound chemokine, the leukocyte GPCR is activated within milliseconds and transmits signals converting a nearby inactive (folded) integrin to its extended conformation, likely through Gβγ-triggered phospholipase C (PLC) mediated hydrolysis of PIP2, activation of CalDAG-GEFI (CDGI), its target Rap-1 and the downstream effector talin (steps 1,2). GPCR-triggered GEFs of RhoA and possibly of Rac1 co-activate downstream PIP2-generating kinases (PIPKIα,β,γ). Local increase of PIP2 directly activates talin binding to and unclasping of the inactive integrin, rendering it extended (step 2) and facilitating its full outside in activation by ICAM-1 (step 3). Force exerted on the ligand-occupied integrin facilitates this outside-in activation by opening the I-domains on both the α and β subunits (lower panels) and both CDG-I activated Rap-1 and PIP2 activated talin may further stabilize the unclasped integrin. Integrin homodimers may interact with preexistent ligand dimers (not shown) and increase binding avidity. Similar events can take place in lymphocytes and other leukocytes rolling on P and E selectins (not shown). The degree of integrin extension, headpiece activation by ligand, force loading and headpiece rearrangements, and ligand induced dimerization is likely to differ among distinct integrins, GPCRs, cell types and species. The lower panels were modified from [17].

Figure 2. Accumulating vs, abrupt switches in integrin avidity states in rolling leukocytes

Upper panel: A lymphocytes rolls via PSGL-1 or other endothelial selectin ligands (not shown) but fails to undergo integrin activation by successive rolling engagements due to insufficient triggering of kinases, PLCs and secondary messengers. When lymphocytes encounter high density of arrest chemokines juxtaposed to integrin ligands, their integrins are locally and instantaneously activated by Gα_iβγ signals as delineated in figure 1. Lower panel: a rolling neutrophil and possibly other myeloid leukocytes can integrate weak integrin activation signals through engagements of E-selectin ligands including PSGL-1, which trigger phosphorylation and activation of spleen tyrosine kinase Syk [8,58]. Within seconds, LFA-1 and possibly other integrins are stabilized in an extended intermediate affinity state on the entire plasma membrane. These integrins can form reversible adhesive bonds with endothelial ICAM-1 sufficient to slow down selectin mediated rolling. Like the lymphocyte, when the rolling neutrophil encounters high density of arrest chemokines, its integrins can undergo robust bidirectional activation via G_i signals. Post arrest, ligand-driven integrin microclustering can immediately follow to further stabilize the integrin-mediated contact. Co-ligation of multiple selectin ligands and integrins trigger both Src and Syk kinases to further activate integrin avidity and adhesion strengthening [59].