|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 1 March 2006, Vol. 107, No. 5, pp. 2101-2111. Prepublished online as a Blood First Edition Paper on November 8, 2005; DOI 10.1182/blood-2005-06-2303.
PHAGOCYTES Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endotheliumFrom the Department of Biomedical Engineering, Genome and Biomedical Sciences Facility, University of California at Davis, Davis, CA; and ICOS, Bothell, WA.
Polymorphonuclear leukocyte (PMN) recruitment to vascular endothelium during acute inflammation involves cooperation between selectins, G-proteins, and  2-integrins. LFA-1 (CD11a/CD18) affinity correlates with specific adhesion functions because a shift from low to intermediate affinity supports rolling on ICAM-1, whereas high affinity is associated with shear-resistant leukocyte arrest. We imaged PMN adhesion on cytokine-inflamed endothelium in a parallel-plate flow chamber to define the dynamics of 2-integrin function during recruitment and transmigration. After arrest on inflamed endothelium, high-affinity LFA-1 aligned along the uropod-pseudopod major axis, which was essential for efficient neutrophil polarization and subsequent transmigration. An allosteric small molecule inhibitor targeted to the I-domain stabilized LFA-1 in an intermediate-affinity conformation, which supported neutrophil rolling but inhibited cell polarization and abrogated transmigration. We conclude that a shift in LFA-1 from intermediate to high affinity during the transition from rolling to arrest provides the contact-mediated signaling and guidance necessary for PMN transmigration on inflamed endothelium.
Neutrophils are recruited at vascular sites of acute inflammation by the sequential binding of selectins, CXC chemokines, and 2-integrins that function cooperatively to elicit rolling, arrest, and transmigration. From observations of neutrophil recruitment in the murine microcirculation and on endothelial monolayers grown in tissue culture, a number of rules of engagement have emerged. First, Mac-1 ( M2) and LFA-1 (L2) are necessary and sufficient for neutrophil arrest and transmigration, with each subunit providing distinct adhesive contributions throughout the process from rolling to transmigration.1-3 Second, polymorphonuclear leukocyte (PMN) rolling on a monolayer of cells coexpressing E-selectin and intercellular adhesion molecule-1 (ICAM-1) is sufficient to induce selectin ligand clustering (PSGL-1 and L-selectin) and to signal a shift in LFA-1 and Mac-1 from low to high affinity to bind ICAM-1. This process is synergistic with chemokine signaling on rolling PMN to amplify the efficiency of arrest.4,5 LFA-1 appears to function early in this process in that it participates in tethering to ICAM-1 as it shifts from low to intermediate and high affinity.6,7 How changes in conformation of the heterodimer result in changes in affinity to interact with ICAM-1 and mediate rolling, arrest, and outside-in signaling is only partially defined.8
Structural studies of LFA-1 reveal that extension and activation of ICAM-1 binding involves an inserted or I-domain on the
Coinciding with a shift to high affinity, the stimulation of leukocytes elicits redistribution of LFA-1 into submicron clusters necessary for efficient arrest and transmigration at the region of contact with ICAM-1.14,18,25-27 Current data suggest that these high-density clusters of LFA-1 provide outside-in signaling of subsequent functions, including recruitment of high affinity LFA-1, F-actin polymerization, microtubule rearrangements, Rho GTPase activation, and up-regulation of In this study we used phase and fluorescence microscopy to observe in real time the dynamics of neutrophil recruitment in shear flow on inflamed human venular endothelium. These studies define how rolling velocity and its variance change as a function of LFA-1 affinity regulated through the I-domain. We discovered that a shift in LFA-1 from intermediate to high affinity plays a regulatory role in the steps leading from neutrophil arrest to cell polarization and diapedesis.
Isolation of human neutrophils Whole blood was obtained from healthy adults by venipuncture into sterile syringes with heparin (10 U/mL blood; Elkins-Sinn, Cherry Hill, NJ) according to protocol number 200311635-6 of the University of California at Davis institutional review board. Informed consent was provided according to the Declaration of Helsinki. Neutrophils were isolated in an unactivated state (Figure S1, available on the Blood website; see the Supplemental Figure link at the top of the online article) using a one-step Ficoll-Hypaque density gradient (Robbins Scientific, Sunnyvale, CA). Neutrophils were maintained at room temperature in a calcium-free HEPES buffer and were assessed for viability by trypan blue exclusion (greater than 98% viable). Cell culture and reagents Human umbilical vein endothelial cells (HUVECs; Cascade Biologics, Portland, OR) were maintained in Medium 200 (Cascade Biologics, Portland, OR). Transfected L cells coexpressing human E-selectin and ICAM-1 (L-E/I), as described previously, were maintained in a modified RPMI medium (Invitrogen, Carlsbad, CA).5 Agonists, inhibitors, and antibodies
Recombinant human ICAM-1/IgG (ICOS, Bothell, WA) is a chimer with two full-length ICAM-1 spliced onto the Fab domains and was used at 10 µg/mL.18 Recombinant LFA-1 (rLFA-1) heterodimer is composed of a leucine zipper motif inserted between the Flow cytometric detection of PMN activation and adhesion
To examine specific adhesion by the
Analysis of neutrophil rolling, arrest, and transmigration kinetics
Neutrophil adhesion to IL-1
Quantitation of neutrophil polarization To differentiate the roles of E-selectin and IL-8 in eliciting neutrophil shape change, a novel polydimethylsiloxane (PDMS) microflow channel device was constructed from a photo-etched silicon master with dimensions of 200 µm x 100 µm x 2 mm (3 µL volume). Neutrophils were incubated with IC487475 (1 µM), BIRT (1 µM), or anti-LFA-1 for 10 minutes at 23°C, then perfused into the microflow channels over L-E/I using a syringe pump (Harvard Apparatus) at 1 dyne/cm2 for 3 minutes. The perfusion buffer was then adjusted to 1 nM IL-8, as indicated. Polarization of adherent neutrophils on HUVECs was assayed using the parallel-plate flow chamber, as described for rolling, arrest and transmigration kinetics. To determine the rate and extent of neutrophil polarization on HUVECs or L-E/I, individual neutrophil membrane perimeters were defined using an automated polygon region of interest tool at 1-second intervals throughout the video sequence. The aspect ratio of the cell was then quantitated using Image Pro Plus version 4.5 (Media Cybernetics) software by the following equation: aspect ratio = major radius/minor radius. A neutrophil was considered polarized if the aspect ratio exceeded 1.4. The automated polygon region of interest tool was also used to track the centroid of vehicle- or IC487475-treated neutrophils at 5-second intervals at the start of migration. Centroid movement was tracked for 30 seconds and analyzed for total migration distance, net migration distance, and average velocity. Total migration distance was determined by summing migration step distances every 5 seconds throughout the video sequence. Net migration distance was quantitated as the direct distance between the start of migration and the end of migration. Real-time immunofluorescence microscopy The topography of LFA-1, Mac-1, and high-affinity CD18 was imaged in real time during neutrophil rolling and arrest in shear flow. Neutrophils were preincubated with nonblocking fluorescent mAbs and allosteric inhibitors, as indicated, at 37°C for 10 minutes and then perfused into the parallel-plate flow chamber at 2 dyne/cm2. Inhibitors were present in the perfusion buffer throughout the period of acquisition. Immunofluorescence microscopy coupled with phase-contrast microscopy images were acquired at 1 frame/s using a Nikon TE2000-S inverted microscope with a 60 x Plan-Apo oil immersion objective (NA = 1.4), an automated bright-field source shutter (Vincent Associates, Rochester, NY), and a optical excitation filter wheel (Sutter Instrument, Novato, CA) with filters appropriate for Alexa-488, Alexa-546, and PE labels. Images were captured with a digital CCD camera (ORCA, Hamamatsu Photonics KK, Hamamatsu, Japan) and Simple PCI acquisition software (Compix, Imaging Systems, Cranberry, PA). A 2-D blind deconvolution algorithm was applied to all fluorescent images using Simple PCI software (Compix). Fluorescent pixel intensity of CD18 at leading cell projection and the uropod were quantitated using Image Pro Plus version 4.5 (Media Cybernetics) software. Pixel bit maps were averaged to produce a mean fluorescence intensity at either the cell front or uropod and then were normalized by the total fluorescence intensity over the entire cell surface. Fluorescence intensity of 327C at contact on arrest was determined by quantitating the pixel intensity within defined regions around fluorescent clusters, with a maximum value set at 255. Statistical analysis Data analysis was performed using GraphPad Prism version 4.0 software (GraphPad Software, San Diego, CA.). All data are reported as mean ± SD or mean ± SE, as indicated. Nonparametric group data were analyzed by analysis of variance (ANOVA) and secondary analysis for significance with Tukey posttests. Gaussian-distributed mean values were analyzed by Student t test. Group comparisons were deemed significant for 2-tailed P values below .05.
IC487475 inhibits LFA-1 adhesion to ICAM-1 by stabilizing an extended conformation To evaluate the specificity and dose dependence of allosteric inhibition with IC487475, human neutrophils were sheared with fluorescence-labeled dimeric ICAM-1/IgG- or ICAM-1-coated latex beads, and binding was assayed by flow cytometry (Figure 1). Activation of CD18 was initiated allosterically by binding of mAb 240Q, as previously described.18 In the absence of 240Q activation, ICAM-1/IgG was not detected on PMNs, nor did treatment with IC487475 or anti-Mac-1 induce binding (Figure 1A). In contrast, ICAM-1/IgG binding increased uniformly on populations of activated PMNs, which were inhibited approximately 50% in the presence of anti-Mac-1 or IC487475 alone and were blocked to baseline in combination (Figure 1A-B). PMNs stimulated with IL-8 and sheared in suspension captured ICAM-1-derivatized latex beads through LFA-1 (Figure 1C). Treatment with anti-LFA-1 reduced bead capture to unstimulated control, whereas anti-Mac-1 blocked only residual ICAM-1 adhesion.18 Targeting the IDAS with IC487475 blocked ICAM-1 bead capture with an IC50 of approximately 50 nM and reduced PMN adhesion to baseline at 100 nM (Figure 1C). IC487475 was specific for LFA-1 because Mac-1-dependent capture of albumin-coated latex beads (ACLBs) was not affected by IC487475 treatment (data not shown).18
We next examined the effect of the allosteric inhibitors IC487475 and BIRT on shifting the conformation of
LFA-1 conformation regulates the microkinetics of PMN rolling We next examined the influence of steric and allosteric inhibition of LFA-1 on the microkinetics of PMN rolling on IL-1-stimulated HUVECs in a parallel-plate flow chamber. Untreated PMNs rolled in the direction of fluid flow at an average velocity of approximately 3 µm/s and exhibited subsecond deviations in velocity averaging approximately 4 µm/s (Figure 3). Allosteric stabilization of LFA-1 at intermediate affinity in the presence of IC487475 increased rolling velocity by approximately 1-fold and velocity deviation by approximately 50%. Low-affinity LFA-1 in the presence of BIRT increased mean velocity and its deviation approximately 2-fold above control and 50% above IC487475 treatment. Steric inhibition of LFA-1 by blocking of the I-domain with mAb TS1/22 elicited rolling kinetics equivalent to BIRT treatment, with frequent subsecond excursions at velocity greater than approximately 30 µm/s. Despite these alterations in the rolling kinetics, the efficiency of capture as determined by the total number of PMNs interacting with the cell monolayer was comparable between untreated and antibody or allosteric inhibition. In fact, the increase in rolling velocity and velocity deviation in the presence of TS1/22 and BIRT was equivalent to PMN rolling on an L-cell monolayer transfected to express only E-selectin.5 Thus, LFA-1 conformation and availability are key parameters in the regulation of PMN rolling velocity and stability. PMN arrest and transmigration are regulated by LFA-1 conformation
The efficiency of PMN recruitment, as determined by the conversion from rolling to arrest in postcapillary venules, was recapitulated on cytokine-stimulated HUVECs in that approximately 80% of rolling PMNs transitioned to arrest within seconds of capture in the parallel-plate flow chamber (Figure 4A-B). To determine the role of LFA-1 conformation and affinity in neutrophil recruitment, we preincubated PMNs with mAbs targeting
LFA-1 and CXCR cooperate to signal polarization of arrested PMNs in shear flow Previous reports suggest that leukocyte rolling on inflamed HUVECs facilitates G-protein-coupled chemotactic receptor (GPCR) signaling and activation of CD18 binding to ICAM-1.45 Binding of high-affinity LFA-1 to ICAM-1 appears to provide an important co-signal that activates T-cell migration.29 We examined the role of LFA-1 affinity and binding to ICAM-1 in providing a co-signal leading to cytoskeletal activation and contact guidance during PMN transendothelial migration. Within seconds of arrest on cytokine-stimulated HUVECs, virtually all PMNs destined to transmigrate exhibited significant shape changes (Figure 5A). By 2 minutes, virtually all PMNs adopted a polarized morphology as they projected and retracted multiple pseudopods during migration to a site of TEM. Blocking LFA-1 with TS1/22 did not alter the rate or extent of shape change in PMNs that achieved arrest, despite the efficacy of TS1/22 in diminishing arrest and TEM by approximately 60% (Figure 5A). In contrast, allosteric inhibition of LFA-1 with IC487475 or BIRT significantly retarded the onset of shape change and ultimately inhibited polarization in 50% of arrested PMNs. We next examined the kinetics of shape change after PMN recruitment on L-E/I (Figure 5B-C). Studies were performed in a custom microfluidic flow channel engineered to introduce PMNs onto the monolayer, followed by injection of IL-8 at a defined position in the flow field. This model system provided a means of observing PMN rolling to arrest on L-E/I after activation through GPCR ligation. As with the interaction on inflamed HUVECs, most rolling PMNs achieved arrest on L-E/I in the microchannels.5 A significant difference was that PMNs remained spherical after arrest, indicating that neither tethering on E-selectin nor adhesion through high-affinity CD18 binding to ICAM-1 was sufficient to activate PMN shape change (Figure 5B-C). Perfusion of IL-8 into the microchannel elicited rapid and uniform shape changes such that within 1 minute virtually the entire PMN population achieved polarization. As observed on HUVECs, allosteric inhibition significantly decreased the rate and extent of IL-8-induced polarization (Figure 5B-C). It is important to note that PMNs activated with chemokine in suspension retained the capacity to form pseudopods in the presence or absence of IDAS inhibitors (data not shown). We conclude that a conformational shift in LFA-1 to high affinity cooperates with CXCR signaling in the activation of PMN polarization after rolling and arrest on E-selectin and ICAM-1. High-affinity LFA-1 coordinates PMN directional guidance during migration Allosteric inhibition of LFA-1 was found to attenuate PMN polarization and effectively abrogate subsequent TEM, suggesting that LFA-1 may provide a regulatory function in contact guidance to the site of diapedesis. To further examine how LFA-1 affinity regulates this process, PMN migration on inflamed HUVECs was tracked for untreated PMN and PMN with LFA-1 stabilized with IC487475 at intermediate affinity. In a 30-second interval, PMNs traveled a mean total distance of 9.1 µm, with a point-to-point net migration distance of 7.0 µm (Figure 6; Table 1). A representative sequence illustrates that PMNs migrated to the site of TEM with high persistence (Figure 6). In contrast, allosteric inhibition with IC487475, or BIRT (data not shown), diminished the total migration distance to 7.2 µm and the net migration distance to 4.9 µm (Figure 6; Table 1). PMNs treated with anti-LFA-1 mAb TS1/22 migrated to the site of TEM with trajectories similar to those of untreated control, whereas allosteric inhibition resulted in less persistent, more random migration, as indicated by reduced net movement. Migration velocity over the time course was equivalent between untreated PMNs and in the presence of LFA-1 inhibitors (Table 1). These data clearly demonstrate that a high-affinity conformation of LFA-1 is necessary for persistent migration and subsequent diapedesis.
LFA-1 redistributes to high-affinity sites of CD18 during PMN arrest and polarization
A final set of experiments was initiated to image in real time the dynamics of Membrane distribution of high-affinity CD18 was imaged with a fluoroconjugate of mAb 327C (Figure 7A). High-affinity CD18 typically formed clusters at sites of contact that aligned along the lamellipod-uropod major axis of polarized PMNs. This topography corresponded to that of LFA-1 at the base of forming lamellipodia, and less mobile focal clusters of LFA-1 and Mac-1 stabilized at the uropod. Active CD18 was equally distributed at the front and rear of the PMN during the process of migration, suggesting its role in orientation and guidance during diapedesis (Figure 7B). Treatment with allosteric inhibitors reduced 327C fluorescence intensity at arrest by approximately 3-fold compared with control but did not abrogate expression of 327C within the adhesive contact of arrested PMNs (Figure 7A). Despite this accumulation of active CD18, bipolar clusters of 327C did not form in the presence of BIRT or IC487475. As depicted in the bottom sequences of Figure 7A, neither active CD18 nor LFA-1 (data not shown) was observed to redistribute during lamellipod extension. These data reveal a pattern of LFA-1 membrane redistribution at the site of adhesive contact that is associated with a shift to high affinity and subsequent signaling and contact guidance of PMNs to the site of transendothelial migration.
In the present study, we demonstrate that the affinity of LFA-1 is a key determinant in the efficiency of PMN arrest and transmigration on inflamed endothelium. We show that (1) a shift in LFA-1 from low to intermediate to high affinity cooperates with selectins in reducing the velocity of PMN rolling in shear flow, (2) a shift in LFA-1 to high affinity during slow rolling is necessary for efficient arrest, and (3) high-affinity LFA-1 redistributes along the major axes of polarized PMNs and is necessary in contact guidance and subsequent TEM. A key observation was that antibody blocking of LFA-1 binding did not alter PMN shape change, nor did it significantly diminish migration, which was partially supported by Mac-1. However, allosterically stabilizing the I-domain with IC487475 in an intermediate-affinity, or locking LFA-1 in a low-affinity, state with BIRT377 abrogated TEM by perturbing PMN polarization. These data are the first to demonstrate that dynamic shifts in LFA-1 affinity for ICAM-1 can serve as a gatekeeper by providing a critical outside-in signal to direct contact guidance during the transition from arrest to transmigration in shear flow.
Experimental approaches using site-directed mutagenesis and allosteric small molecule targeting have begun to reveal how shifts in the conformation of LFA-1 mediate profound changes in equilibrium binding of ICAM-1. Soluble monomeric ICAM-1 binding to low-affinity, wild-type LFA-1 or to an I-domain mutant locked in a closed conformation dissociates at rates of 5.5 seconds-1 and 2.8 seconds-1, respectively.46 Stabilizing an intermediate-affinity conformation of LFA-1 by displacement of the
We describe here the function of IC487475, a class of P-arylthio cinnamides that targets the IDAS and stabilizes the I-domain with intermediate affinity, corresponding to an ICAM-1 dissociation rate of approximately 0.14 second-1.37 This is the first report of an allosteric antagonist that downshifts I-domain affinity independent of global shifts in the - heterodimer conformation. Closing of the I-domain was confirmed by blocking the binding of mAb R3.1, which reports on I-domain occupancy, in the presence of BIRT377 or IC487475. IC487475 did not alter heterodimer extension, as determined by binding of KIM127 to activated CD18. In contrast, BIRT induced KIM127 displacement from activated CD18. The activity of IC487475 is similar to that of XVA143, which also stabilizes an intermediate affinity of LFA-1 by reportedly altering - intersubunit signaling.12,47 XVA143 enhanced lymphocyte rolling on purified ICAM-1 and high endothelial venules and virtually abrogated lymphocyte arrest by stabilizing LFA-1 in the extended conformation.22 Other P-arylthio cinnamides or diarylsulfide analogs are also capable of stabilizing the I-domain into low-affinity conformation, as determined by nuclear magnetic resonance (NMR) and X-ray crystallography (O.E.S., unpublished data, June 2005). Our data demonstrate for the first time that the affinity of wild-type LFA-1 can be downshifted allosterically by a small molecule binding to the IDAS independently of global conformational changes in the - heterodimer. Neutrophils treated with IC487475 or BIRT were recruited to HUVECs with equal efficiency, yet rolling velocity supported by each was dramatically different. LFA-1 locked in a low-affinity conformation by BIRT or blocked from binding ICAM-1 by TS1/22 exhibited unsteady rolling with frequent velocity spikes. Published data show that LFA-1, or isolated I-domain, can support cell rolling on ICAM-1 at shear stress in excess of 10 dyne/cm2; however, in all cases, this required an initial interaction period of static or very low shear (less than 0.3 dyne/cm2).15-17 In this regard, mice deficient in E- and P-selectin lack the capacity for leukocyte capture and rolling.50 Thus, selectins are necessary for capture at venular shear rates, yet optimal rolling requires cooperativity with LFA-1 because leukocyte rolling on monolayers coexpressing E-selectin and ICAM-1, or in vivo within the microcirculation of ICAM-1(-/-) mice, is markedly increased relative to ICAM-1 expressed alone.5,51 Our data suggest that intermediate-affinity LFA-1 stabilized by IC487475 can participate in neutrophil capture and rolling; however, further up-regulation to high affinity by other activation mechanisms, including selectins, chemokines, and inflammatory lipids, is critical for the transition to arrest.5,18,52
Ligation of neutrophil chemotactic receptors is thought to be a primary mechanism directing polarization and contact-mediated guidance to sites of transendothelial migration.53,54 Integrins also function as transmembrane signaling receptors that can activate kinases, cytoskeletal reorganization, and cell motility.55 In particular, LFA-1 binding to ICAM-1 on lymphocytes in Mg2+-containing buffer induces conformational extension of extracellular domains, unclasping of integrin cytoplasmic domains, F-actin polymerization, microtubule reorganization, and activation of signaling molecules such as PKC- A recent report from Shamri et al6 supports a model in which chemokine and ICAM-1 must be juxtaposed on the substrate of a flow channel to activate LFA-1 and to arrest lymphocytes in shear. In this model, only chemokine immobilized on a substrate, but not in solution, effectively stabilized activated LFA-1 on T cells, as reported by binding to activation reporters such as 327C and KIM127. Shamri et al6 conclude that chemokine signals only transiently convert low-affinity LFA-1 to an extended intermediate-affinity conformation in which it is poised to bind ICAM-1. Interaction of extended I-domain with ICAM-1 must then occur within a fraction of a second to mediate shear-resistant stable adhesion. This model of T-cell adhesion highlights several potential differences between LFA-1 function on lymphocytes and that on PMNs. Stimulation of PMNs in suspension with chemokine, or rolling on E-selectin, is sufficient to activate high-affinity LFA-1 and stable adhesion to ICAM-1 in shear flow. Our data support a model in which LFA-1 in intermediate, but not low, affinity promotes deceleration during PMN rolling. A conversion to high affinity and clustering of LFA-1 within the site of contact is essential for efficient transition to arrest with endothelial ICAM-1, and this can be | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
