Clocking Leukocytes Reveal Dynamics of Integrin Braking

Abstract

In this issue, Willenbrock et al. (1)address how leukocytes achieve decel-eration during rolling and arrest onendothelium in the vasculature neartissue sites of inflammatory insult.While cell capture from the bloodstream and rolling is largely the func-tional domain of selectin adhesionreceptors that are constitutively ex-pressed on leukocytes and rapidlydeployed on endothelium to bindglycosylatedligands,theyarenotsuffi-cient to decelerate leukocytes. This isthe function of integrins, which aretransmembrane heterodimers consist-ing of a large head on two legsthat extend during cell activation tomediate ligand binding (2). Most ofthe heterodimer is expressed extracel-lularly, but both subunits traverse theplasma membrane and terminate inshort cytoplasmic domains wherethey dynamically assemble with cyto-skeletal and signaling complexes.Thus, ligand binding via integrinsfacilitates cell arrest in the stiff shearforce of blood flow and serves tobridge the extracellular milieu to theintracellular cytoskeleton—a criticalstep for initiating transendothelialmigration (3). Two decades haveelapsed since natural mutations werediscovered in the genes coding forintegrin and selectin receptors thatcause human leukocyte adhesion defi-ciency disease, LAD-I and LAD-II,respectively (4). Intense study of therelationship between structure andfunction has revealed that integrinseffectively serve as gatekeepers thatcontrol access of leukocytes by under-going an allosteric shape shift andlaterally redistribute on the membraneto modulate the affinity and valenceinbindingtoIg-superfamilycounterre-ceptors including ICAM-1 upregulatedon inflamed endothelium. Bondforma-tion on the outside has direct influenceon integrin linkage on the inside to thecortical cytoskeleton that in part deter-mines bond lifetime and adhesivestrength.The most recent immunodeficiencyassociated with impaired leukocyterecruitment is the LAD-III phenotype,characterized as a loss of functionmutation in Kindlin3, the predominantisoform expressed in leukocytes (5).This adaptor molecule is necessaryfor triggering the allosteric shift in in-tegrins and a rapid increase in densityof focal clusters on the plasmamembrane necessary for binding toICAM-1 in sufficient numbers todecelerate the cell. A recent flurry ofstudies have focused on the role ofKindlin3inintegrinfunction,revealingthat its participation in postcaptureadhesive events involves associationand the b-subunit cytoplasmic domainof LFA-1 and the cortical cytoskeletonvia talin (6). Evidence of the associa-tion of Kindlin3 with the cortical cyto-skeleton only provides a static readoutof how the brakes are deployed duringleukocyte touchdown and taxiing tosites of transmigration. To evaluatethe complex and overlapping mole-cular binding events and discriminatetheir respective contributions to decel-eration and arrest, it is necessary tocarefully track the position andvelocity of cells as a function of time.An automated solution, denoted‘‘Leukotrack’’, was developed byWillenbrock et al. to identify leuko-cytes captured by video microscopywhile rejecting streaks and smalldebristhatotherwisecanbiastheaccu-rate analysis of cell trajectory undershear flow. This program facilitatesanalysis of the diverse kinematics ex-hibited by leukocytes during exposureto hydrodynamic shear while interact-ing on a substrate. By storing theposition of each cell using a nearest-neighbor image analysis algorithm,Leukotrack effectively discriminatesrolling from arrested and migratingcell behaviors and facilitates a statis-tical comparison that otherwise wouldbe laborious, mind-numbing work.To address the specific defects inleukocyte rolling and deceleration toarrestinLAD-IIIpatients,Willenbrocket al. (1) employed Epstein Barr virus-transformed B lymphoblastoid cellsderived from peripheral blood mono-nuclear cells of Turkish LAD-IIIpatients. The adhesive behavior oftransformed B cells was comparedbetween two patients and parentEpstein Barr virus-transformed cells.B-cell rolling on E-selectin induced adistinct activation signal to shiftLFA-1toahighaffinitystatethatbindstightlytoitscognateendothelial ligandICAM-1 under shear flow. This tech-nique made possible direct imagingof LFA-1-mediated adhesion of B cellsand the Leukotrack program providedacquisition of kinetic data fromhundreds of cell trajectories, therebyfacilitating discrimination of the roleof Kindlin3 in LFA-1-mediated activa-tion and its participation in the multi-step process of recruitment.A key question addressed by theanalysis was how dynamic signalingvia E-selectin ligation allostericallytriggers the extension of LFA-1 andupshift from low to an intermediateand high affinity conformation througha mechanosensory mechanism pro-moted by shear-induced bond tension.Recent data from my laboratory andothers indicate that coupling ofLFA-1 to the actin cytoskeleton isrequired for high affinity and mul-tivalent binding to heterodimericICAM-1 (3,7). Remaining unresolvedis how Kindlin3 functions in leukocytedeceleration in the context of associa-tion with the cytoplasmic domain of