lymphocyte arrest requires instantaneous induction of an extended lfa-1 conformation mediated by...

Lymphocyte arrest requires instantaneous inductionof an extended LFA-1 conformation mediated byendothelium-bound chemokines

Revital Shamri1, Valentin Grabovsky1, Jean-Marc Gauguet2, Sara Feigelson1, Eugenia Manevich1,Waldemar Kolanus3, Martyn K Robinson4, Donald E Staunton5, Ulrich H von Andrian2 & Ronen Alon1

It is widely believed that rolling lymphocytes require successive chemokine-induced signaling for lymphocyte function–

associated antigen 1 (LFA-1) to achieve a threshold avidity that will mediate lymphocyte arrest. Using an in vivo model

of lymphocyte arrest, we show here that LFA-1-mediated arrest of lymphocytes rolling on high endothelial venules bearing

LFA-1 ligands and chemokines was abrupt. In vitro flow chamber models showed that endothelium-presented but not soluble

chemokines triggered instantaneous extension of bent LFA-1 in the absence of LFA-1 ligand engagement. To support lymphocyte

adhesion, this extended LFA-1 conformation required immediate activation by its ligand, intercellular adhesion molecule 1.

These data show that chemokine-triggered lymphocyte adhesiveness involves a previously unrecognized extension step that

primes LFA-1 for ligand binding and firm adhesion.

The arrest of rolling leukocytes on target endothelium is a keyregulatory step for successful emigration. In lymphocyte homing tolymph nodes and/or peripheral tissues, lymphocyte function–associated antigen 1 (LFA-1 (aLb2 subunits)) is the dominant integrininvolved in firm arrest1. LFA-1 is a heterodimer that transitionsbetween inactive bent conformations and variable extended confor-mations with intermediate and high affinity to ligand2. LFA-1 prevailsin inactive states on most circulating lymphocytes3,4 and must beactivated by endothelium-displayed chemokines through specific Gprotein–coupled receptors (GPCRs) on the leukocyte surface1,5. Atpresent, the paradigm of integrin activation on rolling leukocytesproposes that the leukocyte integrates successive GPCR signals as itmoves along the vessel wall to reach a threshold activation of integrinavidity. However, in vivo evidence for such stepwise activation hasbeen lacking. A difficulty in this postulated activation mode is that itassumes random modulation of integrin affinity and clustering overthe entire surface of the rolling leukocyte5. As many blood-borne cellsexpress L-selectin and LFA-1 ligands on their surface, random LFA-1activation on adherent leukocytes could result in pathological inter-cellular adhesion molecule 1 (ICAM-1)–mediated attachment offlowing leukocytes to these leukocytes. Another difficulty with thismodel is that chemokine activation of LFA-1 would start 100 mm awayfrom the final site of arrest, the typical distance passed by lymphocytes

rolling until arrested. Such activation would therefore involve undesir-able premature integrin activation signals remote from the actualtarget site of emigration.

We sought to address these fundamental issues using the wellestablished model of lymphocyte arrest on high endothelial venulesbearing LFA-1 ligands and chemokines such as CCL21 (also calledSLC) and CXCL12 (also called SDF-1)1,6. Microdynamic analysis ofrolling lymphocytes in this in vivo model indicated lymphocytesabruptly arrested via LFA-1-mediated interactions without any notice-able gradual deceleration. In vitro flow chamber models using distinctendothelial monolayers, isolated ICAM-1 and LFA-1 conformation‘reporter’ monoclonal antibodies (mAbs) further indicated thatchemokine triggering of LFA-1-mediated arrest had a t1/2 of 0.5 sand involved the instantaneous extension of bent LFA-1. This exten-sion primed the integrin to a subsequent ligand-induced activation ofthe LFA-1 inserted domain (I-domain), the key ligand-bindingmodule in the headpiece of LFA-1. Notably, soluble chemokines failedto prime this critical integrin extension and therefore poorly stimu-lated adhesiveness under shear flow. These and other mechanistic datarefine the three-step model of lymphocyte recruitment, suggesting thatchemokine stimulation of b2-integrins occurs locally and bidirection-ally rather than through random stepwise accumulation of high-affinity LFA-1 during the rolling phase.

Published online 17 April 2005; doi:10.1038/ni1194

1Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. 2CBR Institute for Biomedical Research and the Department of Pathology,Harvard Medical School, Boston Massachusetts 02115, USA. 3Department of Cellular Biochemistry, Institute of Molecular Physiology and Developmental Biology,University of Bonn, Bonn D-53115, Germany. 4Celltech Group, Slough SL1 4EN, UK. 5ICOS, Bothell, Washington 98021, USA. Correspondence should be addressedto R.A. ([email protected]).

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RESULTS

Triggering of instantaneous LFA-1 adhesiveness

We initially analyzed the arrest of rolling lymphocytes in highendothelial venules of peripheral lymph nodes, a classical model oflymphocyte-endothelial interactions, in vivo using intravital micro-scopy. A substantial fraction of lymphocytes captured and rolling onL-selectin ligands arrested on these vessels exclusively via LFA-1 and ina Gi protein-dependent way (Fig. 1 and data not shown). Microkineticanalysis of multiple rolling paths preceding final arrest showed abruptrather than gradual deceleration of rolling lymphocytes arrested onthese vessels (Fig. 1 and Supplementary Fig. 1 online). Rolling wasinterrupted by short LFA-1-independent pauses of less than 0.3 s(Fig. 1 and Supplementary Fig. 1 online), but the irregularity ininstantaneous rolling velocities of individual lymphocytes did notdecrease with rolling time (Supplementary Fig. 1 online). Thesefindings suggested that LFA-1 activation during rolling occurs locallyand abruptly at the final arrest site.

Activation of leukocyte integrins by circulating chemokines may beunfavorable, as it would trigger integrin-mediated arrest remote fromchemokine secretion sites. In vitro results have indicated that solublechemokines trigger robust b2-integrin–mediated lymphocyte arrest onendothelial ligands under shear flow7. Nevertheless, rolling or recentlytethered lymphocytes efficiently arrested via their LFA-1 (data notshown) on endothelium-like monolayers expressing physiologicalL-selectin ligands and ICAMs only when they encountered theimmobilized prototypic chemokines CXCL12, CCL21 and CXCL9(also known as MIG; Fig. 2a) but not when they were briefly pre-exposed to the soluble chemokine counterparts. Similarly, LFA-1-mediated arrest of lymphocytes captured via E-selectin on tumornecrosis factor–stimulated human umbilical vein endothelial cells wastriggered only by surface-bound CXCL12, not by its soluble counter-part (Supplementary Fig. 2 online). Soluble chemokines failed, evenat saturating concentrations and with short exposure periods, totrigger LFA-1-dependent arrest, even though they did not interferewith selectin-dependent functions (Fig. 2a, Supplementary Fig. 2online and data not shown). As a considerable fraction of selectin-captured lymphocytes arrested via LFA-1 binding immediately afterencountering immobilized chemokine (Fig. 2a and SupplementaryFig. 2 online), we considered that lymphocyte LFA-1 may undergoimmediate activation even without a prior rolling step. Notably, allprototypic lymphocyte chemokines capable of arresting lymphocytescaptured through selectins (Fig. 2a and Supplementary Fig. 2 online)triggered robust LFA-1-mediated capture and arrest on purifiedICAM-1, the prototypic LFA-1 ligand expressed in high abundanceon lymph node high endothelial venules5,8 (Fig. 2b). The ability ofLFA-1 to arrest flowing lymphocytes was highest at a subphysiologicalshear stress yielding lymphocyte transport velocity lower than150 mm/s over the substrate containing both chemokine and ICAM-1,within the upper velocity range of lymphocyte rolling in vivo.Consistent with results using ICAM-1-bearing endothelial mono-layers, all three soluble chemokines poorly triggered LFA-1-dependentlymphocyte capture or arrest on intact ICAM-1 under shear flow,despite their ability to stimulate LFA-1 avidity to bead-bound ICAM-1under shear-free conditions (Fig. 2c and data not shown). Thefraction of transient LFA-1 tethers in total capturing events triggeredby a given chemokine increased with either reduced ICAM-1 densities(Fig. 2b, right, and data not shown) or reduced mean expression ofLFA-1 (Fig. 2b,d). Both transient and firm tethers triggered by allthree chemokines tested were eliminated by LFA-1 blockade using theTS1/22 mAb specific for blocking the I-domain, and none of theimmobilized chemokines supported any adhesive interactions on their

own (data not shown). These dynamic results collectively suggest thatall three prototypic chemokines can trigger LFA-1 activation in vitroduring contact with ICAM-1 lasting less than a second withoutstepwise integrin priming by chemokine signals encountered duringthe rolling phase.

Soluble chemokines trigger high-affinity LFA-1 conformations

Two ‘neoepitopes’, recognized by mAbs 327C and 327A, in the keyregulatory I-like domain of the b2-subunit are associated with high-affinity LFA-1 conformations (Supplementary Fig. 3 online), both onleukocytes and on cell-free LFA-1 (refs. 9,10). The proadhesivechemokines CXCL12, CCL21 and CXCL9 potently triggered theseconformations (Fig. 3a–c and data not shown). At concentrations of10–100 nM, CXCL12 and CCL21 activated the neoepitopes to anextent comparable to maximal LFA-1 activation induced by the potentprotein tyrosine kinase C (PKC) agonist phorbol-12-myristate-13-acetate (PMA) or by conformational integrin activation in the presenceof Mg2+ and EGTA (Fig. 3a,b and data not shown). The Mg2+-inducedI-like epitope detected by mAb m24 ‘reports’ integrin extension ratherthan a high-affinity state4. This epitope was efficiently triggered byPMA but only poorly by the three proadhesive chemokines (Fig. 3dand data not shown). In contrast, the KIM127 mAb, another reporterof integrin extension directed to the b-subunit integrin-EGF2domain11 (Supplementary Fig. 3 online), was weakly but substantiallyinduced by both chemokine and PMA signals (Fig. 3e). The a-subunit‘leg’ extension reporter mAb NKI-L16 (ref. 12) was highly expressedon peripheral blood lymphocytes (PBLs) and could not be furtherinduced by either soluble chemokine signals or PMA (Fig. 3f).

Immobilized chemokines induce extended integrin conformations

To address induction of the conformational epitopes by functionallyproadhesive immobilized chemokines, we developed an assay inwhich immobilized mAb reporters were coated on a flow chamber–assembled substrate to monitor in situ induction by chemokine signalsof their integrin neoepitope targets on flowing lymphocytes (Fig. 4a).We monitored in situ neoepitope induction under shear stress‘permissive’ for chemokine-triggered LFA-1 adhesiveness to ICAM-1(Fig. 2b). Consistent with the flow cytometry data (Fig. 3a,c), solubleCXCL12 triggered both the 327C and 327A neoepitopes to interact

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Figure 1 Rolling lymphocytes do not undergo gradual deceleration before

firm arrest. (a) Data represent the average of instantaneous normalized

rolling velocities (Vroll) of 11 representative lymphocytes in order IV

peripheral lymph node vessels1. Lymphocyte rolling was monitored for 7 s

or less before arrest (time 0). Instantaneous normalized rolling velocities

of individual lymphocytes are in Supplementary Figure 1 online. Inset,

average of instantaneous normalized rolling velocities of 10 LFA-1-blocked

lymphocytes in identical venules. Non-normalized velocities were not altered

by LFA-1 blockade (data not shown). All data are expressed as mean 7 s.d.(b) Arrested fractions among all rolling lymphocytes pretreated with control

mAb (Control) or mAb blocking LFA-1 (Anti-LFA-1).

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with their respective immobilized mAb reporters during both contactsof less than a second and in prolonged stationary contacts (Fig. 4b–dand data not shown). Notably, immobilized CXCL12, in contrast,failed to trigger either of these neoepitopes in situ (Fig. 4b,d) or evenafter prolonged stationary contacts (Fig. 4c and data not shown). Weobtained similar findings with CCL21 and CXCL9 (data not shown).Instead, all immobilized chemokines that triggered LFA-1 adhesive-ness to ICAM-1 under shear flow robustly triggered the LFA-1extension–associated epitope KIM127 (Fig. 4e). ImmobilizedCXCL12 signals also triggered both the a- and b-subunit-specificextension epitopes NKI-L16 and m24, respectively, although to amuch lower extent than with the KIM127 epitope (Fig. 4e–g; four- to

sixfold for the first two epitopes; 20-fold for KIM127; n ¼ 6,P o 0.02). The KIM127 mAb failed to stimulate LFA-1 adhesivenessto ICAM-1 (not shown), supporting the idea that it serves as areporter rather than an inducer of LFA-1 extension. Notably, solublechemokines poorly triggered all a- or b-extension-associated epitopes(Fig. 4e–g). Thus, the ability of chemokines to rapidly induceextended LFA-1 states during contacts lasting less than a secondtightly correlated with their ability to trigger functionally adhesiveLFA-1 in these contacts. Induction of high-affinity b2-subunit I-likedomain neoepitopes by soluble chemokines, in contrast, generatedLFA-1 species incapable of promoting functional adhesive tethers onICAM-1-bearing surfaces specifically under shear flow conditions.

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Figure 2 Immobilized but not soluble chemokines stimulate LFA-1 adhesiveness to ICAM-1 under shear flow. (a) Frequency of PBLs rolling (Rolling) andarrested immediately (Immediate arrest) or after short rolling phase (Rolling then arrest) on ICAM-1-expressing monolayers also expressing sulfated L-selectin

ligands (ECV-304). Imm. Chemokine, CXCL12, CCL21 and CXCL9 were each preimmobilized on the monolayer. Pretreatment, PBLs were prestimulated

with 10 nM of each soluble chemokine, were washed and were immediately perfused over the ECV monolayer at a shear stress of 1.75 dyn/cm2. Data are

expressed as the mean 7 range of two fields of view and are representative of four experiments each. (b) Frequency and strength of PBL tethers to ICAM-1–

Fc, coated at a density of 190 sites/mm2 (left) or 60 sites/mm2 (right), with heat-inactivated (–) or functional CXCL12, CCL21 or CXCL9 at a shear stress of

0.5 dyn/cm2. Pretreatment, PBLs were also pretreated with soluble chemokine (10 nM) as described in a. Inset, PBLs were pretreated with pertussis toxin

(PTX) to block all Gi protein signaling. Data are expressed as the mean 7 range of two fields of view and are representative of five experiments each.

(c) PBLs were stimulated with 10 nM chemokine and were immediately mixed with ICAM-1–Fc–coated beads for 1 min. Bottom row (+): PBLs were

preincubated for 10 min at 25 1C in binding medium with a b2-subunit-blocking mAb (TS1/18 mAb; 10 mg/ml) before chemokine stimulation. Results are

an average of three experiments. P o 0.005, bead binding to intact versus chemokine-treated PBLs. (d) Flow cytometry of LFA-1 in PBLs double-stained

for the integrin and anti-CXCR3, anti-CXCR4 or anti-CCR7. Inset, percentage of LFA-1hi cells above 50 mean fluorescence intensity, as indicated by the

horizontal bracketed bar in d, in the population of CXCR3-, CXCR4- or CCR7-expressing cells. Results are the average 7 range of two experiments.


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Figure 3 Soluble chemokines activate b2-subunit I-like domain neoepitopes associated with high affinity. (a) Flow cytometry of PBLs stained with the high-

affinity reporter mAb 327C in the absence (Basal) or presence of CXCL12 (CXCL12; gray shading) or PMA (PMA). Mg2+ + EGTA, staining after artificial

activation by Mg2+ and EGTA. (b) Flow cytometry of PBLs stained with mAb 327C in the absence or presence of CXCL9 or CCL21. (c,d) Flow cytometry

of PBLs treated with various agonists (keys) and stained with the high-affinity reporter 327A mAb (c) or the extension reporter mAb m24 (d). (e,f) Flow

cytometry of PBLs treated with various agonists (keys) and stained with the LFA-1 extension reporter mAbs KIM127 (e) and NKI-L16 (f). Data are

representative of three experiments each.


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LFA-1 triggering does not involve cytoskeletal release

To gain further insights into GPCR mechanisms involved in LFA-1activation by immobilized chemokines, we next focused on CXCR4,the most broadly expressed and functionally used GPCR on freshlyisolated T lymphocytes (Fig. 2d and data not shown). Diacylglycerol-dependent PKCs have been proposed to release LFA-1 from cytoske-letal mobility constraints13 and have been linked to the activation ofboth high-affinity integrin and extension neoepitopes (Fig. 3a,c–e).Nevertheless, chemokine-triggered LFA-1 adhesiveness under shearflow could not be inhibited by the potent diacylglycerol-PKC inhibitorbisindolylmaleimide I (Fig. 5a), suggesting that diacylglycerol-depen-dent PKCs are not involved in rapid CXCR4-mediated LFA-1 activa-tion. In contrast, PMA-triggered adhesiveness and induction ofextension or high-affinity neoepitopes were fully blocked by thisinhibitor (data not shown). CXCL12 triggering of LFA-1 adhesivenessto both high- and low-density ICAM-1 under shear flow as well asat stationary contacts also did not indicate activation of phosphati-dylinositol 3 kinase (Fig. 5a,b), which has been suggested to promoteLFA-1 mobility and ‘macroclustering’5,10. Chemokine activationof LFA-1 adhesiveness both under continuous shear flow and in

stationary contacts (Fig. 5a,b) and chemokine-induction of theextended KIM127 conformation (Fig. 5c) were all highly susceptibleto moderate disruption of the actin cytoskeleton by the actin poly-merization blockers cytochalasin D or latrunculin A (data not shown),which augments T cell adhesion to isolated ICAM-1 at stationarycontacts14,15. Basal LFA-1-mediated T cell tethering to ICAM-1 wasalso substantially impaired by these inhibitors, indicating intrinsicdependence of LFA-1–ICAM-1 tethers on intact cytoskeletal integrinassociations (Fig. 5a). Notably, cytochalasin D tested at concentrationsranging from 1 nM to 10 mM failed to augment LFA-1 adhesiveness toICAM-1 for contacts lasting less than a second (data not shown).Consistent with the pharmacological findings with the substrate ofICAM-1 plus CXCL12, chemokine activation of the extended LFA-1conformation did not require diacylglycerol-dependent PKC or phos-phatidylinositol 3 kinase activation (Fig. 5c). These results collectivelysuggest that activation of extended LFA-1 conformations lasting lessthan a second by immobilized chemokine signals and subsequentLFA-1 rearrangement by surface-bound ICAM-1 require integrinassociations with the actin cytoskeleton rather than the release ofcytoskeletal integrin constraints.

Figure 4 Immobilized chemokines stimulate

the induction of extended (intermediate-affinity)

rather than high-affinity LFA-1 conformations.

(a) Cellular interactions with the reporter mAb at

contacts lasting less than a second by soluble

(left) or immobilized (right) chemokine signals.

(b) Frequency and strength of tethers irradiated

by PBLs interacting with the b2-subunit reportermAb 327C coimmobilized with heat-inactivated

(–) or functional (+) CXCL12 measured at a shear

stress rate 0.5 dyn/cm2. Pretreatment, PBLs were

pretreated with soluble CXCL12 (10 nM) as

described in Figure 2b. Data are expressed as

the mean 7 range of two fields of view and

are one representative of five experiments.

(c) Prolonged PBL adhesion to the 327C reporter

coimmobilized with heat-inactivated (–) or

functional (+) CXCL12. Data represent the

fraction of originally settled cells (1 min) that

resisted detachment by a shear stress of 0.5 dyn/cm2 for 5 s. Pretreatment (CXCL12), PBLs pretreated with 10 nM soluble CXCL12. (d) Frequency and

strength of tethers mediated by PBLs interacting with mAb 327A coimmobilized with heat-inactivated (–) or functional (+) CXCL12 as described in b.

(e) Frequency and strength of tethers mediated by PBLs interacting with the KIM127 mAb reporter coimmobilized with identical amounts of heat-inactivated

CXCL12 (–) or active CXCL12, CCL21 or CXCL9. (f,g) Frequency and strength of tethers mediated by PBLs interacting with the aL-subunit extension reporter

mAb NKI-L16 (f) or the b2-subunit extension reporter mAb m24 (g), each coimmobilized with CXCL12, as described in b. Data in c–g are expressed as the

mean 7 range of two fields of view and are representative of four each.

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Figure 5 Cytoskeletal anchorage of LFA-1 is

required for integrin activation by immobilized

chemokines. (a) Frequency and strength of

tethers mediated by PBLs interacting at a

shear stress of 0.5 dyn/cm2 with ICAM-1–Fc

(density, 190 sites/mm2) coimmobilized with

heat-inactivated CXCL12 (–), a nonsignaling

CXCL12 mutant (P2G) or functional CXCL12 (+).

Pretreatment, PBLs pretreated with cytochalasin

D (Cyto) wortmanin (Wort) or bisindolylmaleimide

I (Bis). Data are expressed as the mean 7 range

of two fields of view and are one representative of

three experiments. (b) Adhesion of intact or inhibitor-treated PBLs to ICAM-1 at stationary contacts. PBLs were pretreated with inhibitors and were perfused

on ICAM-1–Fc coated at low density (20 sites/mm2) on protein A coimmobilized with heat-inactivated (–) or functional (+) CXCL12 as described in a.Data represent the fraction of cells originally settled for 1 min that resisted detachment with a shear stress of 0.5 dyn/cm2 applied for 5 s. Data are one

representative of two experiments. (c) Frequency and strength of tethers mediated by PBLs interacting with the KIM127 mAb to b2-subunit coimmobilized

with heat-inactivated (–) or functional (+) CXCL12 under a shear stress of 0.5 dyn/cm2. Pretreatment, PBLs pretreated with inhibitors as described in a.

Data are expressed as the mean 7 range of two fields of view and are one representative of three experiments.

BisWortBisBis WortWort CytoCytoCytoCytoCyto PretreatmentPretreatmentPretreatment

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Soluble chemokines do not prime integrins

Although LFA-1 activation was not induced during L-selectin-mediated rolling (Figs. 1,2), lymphocytes approaching target endothe-lial sites may be activated by locally secreted chemokines at nearby‘upstream’ sites. We therefore tested if prior LFA-1 activation bysoluble chemokines augmented subsequent integrin stimulation byimmobilized chemokine signals. Unexpectedly, soluble CCL21, whichmaximally induced high-affinity LFA-1 neoepitopes (Fig. 3b), did notenhance the extent to which LFA-1 underwent in situ activation byimmobilized CXCL12 signals (Fig. 6a). CXCR4 signaling was also notperturbed by the prior CCL21 signal (Fig. 6a, inset). Thus, inductionof high-affinity LFA-1 conformation by soluble CCL21 did not primethe integrin to undergo more-efficient LFA-1 activation of adhesive-ness to ICAM-1 by a subsequent CXCL12 signal via CXCR4. Similarly,artificial activation of high LFA-1 affinity by Mg2+ and EGTA did notaugment LFA-1 responsiveness to in situ–immobilized CXCL12 signals(Fig. 6b). Notably, high-affinity induction by Mg2+ and EGTA wasinsufficient to enhance LFA-1 adhesiveness to ICAM-1 present atdensities below 200 sites/mm2 (Fig. 6b), whereas ICAM-1 coated atdensities as low as 30 sites/mm2 allowed robust stimulation of LFA-1by immobilized chemokines (Fig. 2b and data not shown). Asartificial activation of the LFA-1 headpiece by Mg2+ only marginallytriggered LFA-1 extension in PBLs (Fig. 3e and data not shown),Mg2+-activated LFA-1 was probably not extended and was notaccessible to support firmer adhesive tethers below this threshold ofICAM-1 density. Collectively, these results suggest that prior integrinactivation by soluble chemokines or Mg2+ does not prime the integrinto undergo in situ activation by subsequent immobilized chemokinesignals, possibly because of the poor ability of these agonists tostimulate proper integrin extension (Fig. 4e–g).

Rolling lymphocytes can be exposed to multiple costimulatorysignals triggering diacylglycerol-dependent PKCs shortly before enter-ing sites of arrest and emigration16. We next assessed whether shortstimulation of diacylglycerol-dependent PKCs by PMA augmentedLFA-1 responsiveness to a subsequent chemokine signal. PMA notonly failed to augment subsequent LFA-1 activation by chemokine butalso, in contrast to soluble chemokines or Mg2+, reduced LFA-1activation by immobilized chemokine signals at both high- and low-density ICAM-1 (Fig. 6c), possibly because of desensitization of GPCRresponsiveness by PKCs17. Collectively, these results suggest thatneither GPCR nor PKC stimulatory signals encountered by LFA-1upstream of its target site of arrest can increase LFA-1 responsivenessto local chemokine signals encountered at the final target site.

Arrest requires allosteric LFA-1 activation by ICAM-1

LFA-1-mediated arrest on ICAM-1 under shear flow involves activationof the LFA-1 a-subunit I-domain4. Soluble ICAM-1 rearranges thisdomain and transforms a low-affinity closed I-domain conformationto a high-affinity open conformation through shape-shifting rearran-gements18. As immobilized chemokine signals activated extendedrather than high-affinity LFA-1 conformations (Fig. 4b–g), we nexttested whether autonomous activation of this domain by surface-bound ICAM-1 occurred during contacts lasting less than a second.An inhibitor to the I-domain allosteric site (IDAS)19 ‘froze’ theI-domain in the closed inactive state at nanomolar concentrations(D.E.S., unpublished data). At a concentration of 2.4 nM, the inhibitorblocked 50% of all types of LFA-1-mediated tethers to high-densityICAM-1, whereas tethering frequency and strength were not affected bya control compound or the carrier (Fig. 7a and data not shown). Atdecreasing ICAM-1 densities, the effect of the inhibitor on LFA-1 tetherformation was minimal (Fig. 7b), but the ‘lifetime’ of tethers decreased

by up to 50% (Fig. 7b, inset). Thus, LFA-1 with a fully activatedI-domain can dissociate from surface-bound ICAM-1 with lifetime of0.5 s (Fig. 7b, inset). As the lifetime of tethers mediated by allostericallyblocked LFA-1 was only 0.2–0.3 s (Fig. 7b, inset), I-domain activationby surface-bound ICAM-1 must occur in this time frame. Notably,allosteric blockade of LFA-1 activation also resulted in a nearlycomplete inhibition of chemokine-triggered LFA-1-mediated tethersto ICAM-1 (Fig. 7c). The lifetime of chemokine-triggered LFA-1tethers (0.5 s) closely matched the lifetime of tethers mediated byLFA-1 whose I-domain underwent ICAM-1-induced activation(Fig. 7b, inset). Notably, the allosteric inhibitor had no effect on thepriming of LFA-1 to an extended state by immobilized chemokinesignals, even at a concentration of 10 nM (Fig. 7d and data not shown).This result separates chemokine-triggered LFA-1 extension from LFA-1I-domain activation, further suggesting that the latter activation ismediated by ICAM-1-induced I-domain rearrangements. Correspond-ingly, I-domain allosteric inhibition by the IDAS inhibitor alsosuppressed the ability of rolling lymphocytes to arrest on endothelialICAM-1 in response to immobilized CXCL12 (Fig. 7e). Thus, to fullyactivate LFA-1 and arrest lymphocytes on ICAM-1, allosteric activationof LFA-1 must follow, in less than a second, LFA-1 extension triggeredby immobilized chemokine signals.

Partial talin suppression impairs chemokine stimulation of LFA-1

Talin is a key adaptor linking integrins to the actin cytoskeleton. Thetalin head domain has been proposed to unclasp LFA-1 subunit tailassociations essential for maintaining LFA-1 in low-affinity conforma-tions20,21. Talin cleavage by calpain was predicted to drive this ‘inside-out’ affinity regulation, but in agreement with published results5, wecould find no evidence for involvement of this cytoplasmic proteolytic

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Figure 6 PBL priming by a soluble chemokine does not enhance LFA-1activation by a second stimulus from a distinct immobilized chemokine.

(a) PBLs were prestimulated with soluble CCL21 (10 nM), were washed

and were perfused over ICAM-1 (density, 270 sites/ mm2) coimmobilized

with heat-inactivated (–) or functional (+) CXCL12 as described in

Figure 2b. Data are expressed as the mean 7 range of two fields of view

and are one representative of three experiments. Inset, CCL21 does not

desensitize PBL response to CXCL12 in a Transwell chemotaxis assay. PBLs

were pretreated (Pre treat.) with 10 nM CCL21 or CXCL12 and were allowed

to migrate toward 10 nM CXCL12. Migration fraction is expressed as the

mean 7 range of two experiments. (b) PBLs were perfused in binding

medium (1 mM Ca2+ plus Mg2+; Ca + Mg) or with medium containing

2 mM Mg2+ plus EGTA (Mg + EGTA) over ICAM-1 (density, 190 sites/mm2)

coimmobilized with heat-inactivated (–) or functional (+) CXCL12 as

described in a. Inset, Mg2+ plus EGTA augments tethering frequency

and strength on higher-density ICAM-1 (270 sites/mm2). (c) PBLs were

prestimulated with PMA and were immediately perfused over ICAM-1

coimmobilized at various site densities (bottom line) with heat-inactivated

(–) or functional (+) CXCL12. The frequency and strength of PBL tetherswere measured in a–c under a shear stress of 0.5 dyn/cm2. Data in a–c are

expressed as the mean 7 range of two fields of view and are representative

of five experiments each.


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event in chemokine-triggering of LFA-1 affinity under shear flow(Fig. 8a). As we found an intact actin cytoskeleton to be essentialin LFA-1-mediated adhesions both under shear flow and stasis(Figs. 5a,b), we next sought to determine if intact talin was requiredin these processes. Suppression of talin was achieved by small inter-fering RNA (siRNA)-mediated silencing of talin1, the main talinisoform expressed in T cells22. The maximal extent of talin suppressionat which cell viability was maintained was 60% (Fig. 8b). Althoughboth LFA-1 expression and basal expression of high-affinity LFA-1subsets were fully retained, this partial talin suppression nearlycompletely inhibited LFA-1-mediated adhesion to ICAM-1 at stasis(Fig. 8c–e). Consistent with involvement in ‘outside-in’ LFA-1 activa-tion, LFA-1 tethering to low-density ICAM-1 under shear flow did notrequire intact talin, whereas at increasing ICAM-1 densities, thecontribution of talin increased (Fig. 8f). Notably, chemokine-primedLFA-1 could still tether to ICAM-1 even in talin-suppressedT cells, although it was reduced (Fig. 8g). However, at ICAM-1densities that were twofold higher, this partial talin suppression didnot interfere with CXCL12 stimulation of LFA-1-mediated tethering(not shown). Thus, partial talin suppression abolishes ICAM-1-mediated outside-in activation of LFA-1 at stasis but only partiallyreduces chemokine-stimulation of LFA-1 adhesiveness under shearflow and does so independently of talin cleavage.

DISCUSSION

The multistep paradigm of lymphocyte homing to lymphoid andperipheral sites predicts that rolling leukocytes integrate successivechemokine signals along their rolling path until their integrins acquire

high avidity for endothelial ligands23,24. Our in vivo and in vitro resultssuggest that LFA-1 activation under shear flow in lymphocytesinvolves abrupt, spatially confined integrin activation that does notdepend on a prior chemokine encounter during selectin-mediatedrolling. Thus, stepwise activation of LFA-1 by chemokine signalsaccumulated by the rolling lymphocyte does not seem necessary forfirm LFA-1-dependent arrest on vascular endothelium. Studyinglymphocyte responses to prototypic endothelial chemokines thatsignal through the three main integrin stimulatory GPCRs on circu-lating T cells, CXCR4, CCR7 and CXCR3, we have found that GPCRsignals to integrins must instantaneously couple with local integrinoccupancy to productively rearrange integrin headpieces and rapidlystimulate adhesiveness under shear flow. The most notable result wasthat rather than inducing the fully active high-affinity conformations,endothelial chemokine signals transiently converted inactive LFA-1into an extended conformation in the absence of full I-domain and I-like domain activation detected by the 327C and 327A reporter mAbs.Thus, when I-domain activation is blocked by an allosteric I-domain-specific inhibitor, conformational switch to the extended state stilloccurs. The extended state, detected by mAbs KIM127 and m24,determines LFA-1 binding to surface-bound ICAM-1 under shearflow4. The unfolded LFA-1 is predicted to extend 25 nm over the cellsurface, compared with the inactive folded state, which extends only5 nm over the surface and is practically inaccessible to ligandimmobilized on a ‘counter surface’25. We found that the chemo-kine-triggered extended LFA-1 was necessary for full LFA-1 activationby surface-bound ICAM-1, suggesting that it is sterically poised toundergo I-domain activation lasting less than a second after ICAM-1

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Figure 7 Allosteric activation of the aL-subunit I-domain is critical for LFA-1 tether formation and

stabilization by ICAM-1 and chemokine signals. (a) Dose-dependent inhibition of LFA-1 mediated

tethering to high-density ICAM-1 (1,900 sites/mm2) by the I-domain IDAS inhibitor A308926.

PBLs were perfused over ICAM-1 in the presence of the IDAS inhibitor (I) or its control compoundA270412 (C; concentrations, below graph). The frequency and strength of PBL tethers were

determined at a shear stress of 0.5 dyn/cm2. (b) Left, effect of the IDAS inhibitor on LFA-1

tethering. Tethers of PBLs treated with inhibitor or the control compound (2.4 nM each) to ICAM-1

were determined at a shear stress of 0.5 dyn/cm2. Right, effect of the IDAS inhibitor on the

dissociation kinetics of LFA-1-mediated tethers to ICAM-1 at a density of 760 sites/mm2 (top) or

380 sites/mm2 (bottom). Diagonal lines, first-order dissociation fit; slope ¼ –Koff. Top right, ICAM-1,

760 sites/mm2: Koff for control tethers, 2.1/s (linear regression (r2) ¼ 0.958); Koff for inhibitor-

treated tethers, 3.25/s (r2 ¼ 0.973). Bottom right, ICAM-1, 380 sites/mm2: Koff for control tethers,

2.21/s (r2 ¼ 0.962); Koff for inhibitor-treated tethers, 4.61/s (r2 ¼ 0.943). (c) Effect of the IDAS

inhibitor on chemokine-stimulated LFA-1 tethers. Left, PBL tethers to low-density ICAM-1 (114

sites/mm2) coimmobilized with heat-inactivated (–) or functional (+) CXCL12 at a shear stress of

0.5 dyn/cm2. Right, effect of the inhibitor on first-order dissociation kinetics (Koff) of transient

CXCL12-triggered LFA-1 tethers to the ICAM-1–plus–CXCL12 cosubstrate tested at left. Koff for

control tethers, 2.17/s (r2 ¼ 0.958); Koff for inhibitor-treated tethers, 2.68/s (r2 ¼ 0.958).

(d) Effect of the IDAS inhibitor on CXCL12-stimulated PBL tethers to the KIM127 reporter mAb

coimmobilized with heat-inactivated (–) or functional (+) CXCL12, determined at a shear stress

of 0.5 dyn/cm2. (e) Rolling and arrest of PBLs treated with carrier (Carr.), control compound

(C) or the IDAS inhibitor (I) on an ECV monolayer, left intact (–) or ‘preoverlaid’ with CXCL12 (+).Experiments were done as described in Figure 2a. Data (a–e) are expressed as the mean 7 range

of two fields and are representative of three experiments each.


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encounter. The ability of immobilized chemokine signals to induce anincrease of up to 20-fold in the number of extended LFA-1 conforma-tional states therefore increases the effective site density of surface-bound ICAM-1 encountered by passing T cells by the same amount.Indeed LFA-1 adhesion to low-density ICAM-1 stimulated by coim-mobilized chemokine is similar to LFA-1 adhesion to ICAM-1 coatedalone at densities 30-fold higher. In conclusion, our results suggestthat chemokine activation of LFA-1 in rolling lymphocytes is a‘biconditional’ process that involves temporally and physically coupledinside-out and outside-in rearrangements of individual LFA-1 mole-cules. A global signal induced by soluble chemokines fails to inducethis key state even though it induces high-affinity LFA-1 subsetscapable of binding soluble ICAM-1 (ref. 5). Artificial induction ofthis extended state on the entire pool of surface LFA-1 by an a/b I-likeintegrin antagonist can slow down L-selectin-mediated rolling4. Thus,the lack of LFA-1 contribution to rolling we noted further suggests thatwhen encountering a premature signal from immobilized chemokineduring rolling on L-selectin ligand coexpressed with chemokine, LFA-1extension occurs only transiently and abortively. Thus, signals fromsurface-bound endothelial chemokines, if triggered in immediateproximity to ICAM-1 occupancy events, couple to yield full bidirec-tional LFA-1 activation at confined leukocyte-endothelial contacts.

Although endothelium-bound chemokines have been postulated tobe the physiological agonists encountered by vessel-captured leuko-cytes, we have demonstrated here that even in vitro, soluble chemo-kines do not transmit adequate adhesive signals to integrins operatingunder shear stress. Although soluble chemokines can trigger LFA-1-mediated lymphocyte arrest on ICAM-1-bearing substrates5,7,

we found that soluble chemokines perfused with cells on suchsubstrates were instantaneously adsorbed onto these substrates andthus functioned in partially immobilized forms. In contrast, wepreexposed lymphocytes to soluble chemokines and verified highaffinity induction of their LFA-1 as well as induced binding ofICAM-1-bearing beads, yet those cells only poorly arrested onICAM-1 under shear flow. Thus, we conclude that even in vitro,immobilized chemokines are far more efficient than their solublecounterparts in triggering LFA-1 adhesiveness to ICAM-1 under shearstress conditions. Our results also predict that LFA-1 on circulatingresting lymphocytes preexposed to a functional chemokine remotefrom their target site is not primed to respond more efficiently to asubsequent signal from a second immobilized proadhesive chemokineat the target site. Thus, although induction of high affinity by solublechemokines has been suggested as a paradigm for rapid inside-out signaling from GPCRs to integrins20, our results suggest thatsignals from immobilized chemokines trigger an extended LFA-1,which must immediately rearrange with immobilized ICAM-1 toachieve full and productive LFA-1 activation4. Surface-bound chemo-kines, but not their soluble counterparts, along with juxtaposedendothelial ICAM-1 may stabilize this extended LFA-1 in a pro-per cytoskeletally anchored state, suggested by our findings to beinstrumental to adhesive tethers generated under disruptive shearstress. This specialized and synchronized bidirectional activationmechanism may restrict random activation of high-affinity LFA-1on the entire surface of the rolling leukocyte and thereby eliminatenonspecific adherence of ICAM-bearing leukocytes to rolling orarrested lymphocytes.

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Figure 8 Talin suppression but not cleavage impairs outside-in ICAM-1 signaling and SDF-1-stimulated LFA-1

adhesion to ICAM-1. (a) Effect of calpain inhibition of chemokine-stimulated LFA-1 tethers to ICAM-1 under

shear flow. PBLs were pretreated with the calpain inhibitor calpeptin or with carrier (–) and were perfused over

ICAM-1 (density, 60 sites/mm2) coimmobilized with CXCL12 as described in Figure 2. The frequency and

strength of PBL tethers were measured at a shear stress of 0.5 dyn/cm2. Data are expressed as the mean 7range of two fields and are one representative of three experiments. (b) PBLs were transfected with talin1-

specific or control luciferase siRNA and total lysates of each group were immunoblotted with mAb to talin.

(c) CXCR4, aL-subunit or b2-subunit expression on PBLs transfected with control siRNA (top) or talin siRNA

(bottom), probed with mAbs 12G5, NKI-L15 and TS1/18, respectively. (d) Shear-resistant adhesion established

in 1-minute contacts on high-density ICAM-1 (2,280 sites/ mm2) by PBLs transfected with control siRNA or talin

siRNA. b2-subunit blocking mAb, PBLs pretreated with the b2-subunit function–blocking mAb TS1/18. (e) Flow

cytometry of PBLs transfected with control siRNA (top) or talin siRNA (bottom) and stained with the b2-subunit

high-affinity reporter mAb 327C. (f) Tethering frequency and strength of PBLs transfected with control siRNA (C)or talin siRNA (T) interacting with ICAM-1 at a shear stress of 0.5 dyn/cm2. (g) Effect of talin siRNA treatment

on chemokine-stimulated PBL tethers to ICAM-1 (190 sites/mm2) coimmobilized with heat-inactivated (–) or

functional (+) CXCL12 determined at a shear stress of 0.5 dyn/cm2. *, P ¼ 0.19; **, P o 0.003. Data in b–g

are expressed as the mean 7 range of two fields and are representative of four experiments each.


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Many studies have suggested that LFA-1 mobility and ‘macroclus-tering’ can be promoted by release of the integrin from cytoskeletalconstraints13,14. Although this mode of LFA-1 activation is probablyinvolved in the reorganization of immune and some endothelialsynapses25, our results suggest that rapid LFA-1-mediated lymphocytearrest does not involve GPCR-transduced machineries linked to therelease of integrins from such constraints, such as diacylglycerol-dependent PKCs or phosphatidylinositol 3 kinase5. Instead, disruptionof the actin cytoskeleton is highly suppressive to LFA-1 adhesivenessinduced by immobilized chemokine signals. Actin cytoskeleton dis-ruption may also impair intrinsic GPCR signaling26,27. Althoughdisruption of the actin cytoskeleton with cytochalasins may alsoreduce microvilli size and number28, LFA-1 is mostly excluded fromthese substructures29. Thus, LFA-1 availability is expected to increasein cytochalasin- or latunculin A–treated lymphocytes. As the oppositeoccurred, we suggest that cytochalasin interferes with LFA-1 adhesive-ness by blocking essential cytoskeletal associations. In this context,talin is a key integrin adaptor which constitutively links LFA-1 to theactin cytoskeleton30 and modulates integrin subunit separation20,headpiece activation and high-affinity binding to ligand via its headdomain21 generated by calpain-mediated cleavage31. We have con-firmed published results ruling out involvement of calpain-mediatedtalin cleavage in rapid LFA-1 affinity modulation by chemokines5. Wehave also shown that partial suppression of talin expression incultured PBLs impaired ICAM-1-induced LFA-1 activation byICAM-1 at stasis to a higher extent than it suppressed chemokineactivation of LFA-1 lasting less than a second under shear flow. Theseresults suggest that other tail-binding proteins such as RAPL32,33

compensate for partial loss of talin during rapid activation of LFA-1by chemokine signals. LFA-1 conformational changes by chemokinesignals also indicate involvement of the cytoskeleton-remodelingGTPases RhoA34 and Rap-1 (ref. 35). Indeed, a defect in Rap-1activation by CXCL12 in a rare inherited adhesion deficiency syn-drome, LAD-III, results in a loss of LFA-1 and VLA-4 activation bymultiple chemokines under shear flow36,37.

Membrane LFA-1 and ICAM-1 can form homodimers, and dimericICAM-1–LFA-1 complexes have been isolated18. Intermediate- andlow-affinity LFA-1–ICAM-1 bonds dissociate in the high micromolarrange18,38, and dimerization may thus be key for rapid ligand rebindingand stabilization of nascent integrin bonds15. The purified ICAM-1analyzed in our initial studies is oligomeric and most of our subsequentstudies therefore used a dimeric ICAM-1 fused to an immunoglobulinFc stalk. As ligand-induced activation of the LFA-1 headpiece iscoupled to separation of the subunit tails and transmembranedomains20, the ligand-induced activation step we propose may furtherreinforce homo-oligomerization of a- and b-subunits via their trans-membrane helix associations39. Thus, after binding, microclustering15

rather than chemokine-triggered integrin mobility is likely to furtherincrease LFA-1 avidity to endothelial ICAM-1 and possibly to otherweaker endothelial ligands. The type and distribution of the GPCReffectors, their integrin cytoskeletal targets and their membranal plat-forms in distinct lymphocyte subsets will determine if and how specificchemokine signals will combine with juxtaposed integrin ligandoutside-in signals to arrest these subsets on target endothelial sites.

METHODSAntibodies and reagents. The 327C and 327A mouse mAbs specific for human

b2-subunit I-like domain and the TS1/18 and TS1/22 mAbs blocking LFA-1

function (a gift from T. Springer, Harvard University, Cambridge, Massachu-

setts) have been described9,10,40. The m24 mAb to human b2-subunit41 was a

gift from N. Hogg (Cancer Research Institute, London, UK). The NKI-L15 and

NKI-L16 mAbs to human aL-subunit42 were a gift from C.G. Figdor (Uni-

versity Medical Center St Radboud, Nijmegen, The Netherlands). All mAbs

were used as purified immunoglobulin G (IgG). Human CCL21 was purchased

from R&D Systems. Human CXCL9 was purchased from Pepro Tech. Human

CXCL12 and its nonsignaling mutant P2G43 were a gift from F. Baleux (Pasteur

Institute, Paris, France). Human ICAM-1–Fc, a recombinant ICAM-1–IgG1

chimera containing five-immunoglobulin ICAM-1 fused to the Fc region of

IgG1, was obtained from R&D Systems. Purified spleen ICAM-1 was a gift

from l. Klickstein (Brigham and Women’s Hospital, Boston, Massachusetts).

Phycoerythrin-conjugated mouse antibody to human CXCR3 (anti–human

CXCR3), anti–human CXCR4 and anti–human CCR7 were purchased from

R&D Systems. Phycoerythrin-conjugated goat anti-mouse was purchased from

Jackson Laboratories. The mAbs to talin and actin were purchased from Sigma.

PMA, protein A and Ca2+- and Mg2+-free Hank’s balanced salt solution and

latrunculin A were obtained from Sigma. Cytochalasin D, calpeptin, worth-

manin, bisindolylmaleimide I, human serum albumin and pertussis toxin were

all purchased from Calbiochem.

Cells. Human PBLs were isolated from citrate-anticoagulated whole blood

from healthy donors as described43 and consisted of more than 90% CD3+

T lymphocytes. Cells were maintained in binding medium (Hank’s balanced

salt solution containing 2 mg/ml of BSA, 10 mM HEPES, pH 7.4, and Ca2+ and

Mg2+ at 1 mM each) and were used within 1 h. Primary cultures of human

umbilical vein endothelial cells were established as described44 and passages 3–4

were grown in monolayers and then were used for adhesion experiments as

described43. Human umbilical vein endothelial cells were left intact or were

stimulated with tumor necrosis factor (2 ng/ml, 200 units/ml; R&D Systems)

for 24 h before experiments. The human umbilical vein endothelial cell–derived

line ECV-304 (LS12), stably transfected with FucTVII and N-acetylglucosamine

6-O-sulfotransferase and constitutively expressing functional sulfated L-selectin

ligands and low amounts of ICAM-1 (refs. 45,26) was a gift from R. Kannagi

(Aichi Cancer Center, Nagoya, Japan). These cells were found to capture

and functionally present all the main chemokines displayed by high endo-

thelial venules, including CCL21, CXCL12, CXCL9 and CCL19. Cells were

cultured in F12 medium supplemented with 10% FCS, 2 mM L-glutamine

and antibiotics (Biological Industries) and were used without stimulation for

adhesion experiments.

Transfection of peripheral blood T lymphocytes with siRNA. Silencing of

talin expression in primary T lymphocytes was achieved by siRNA. A talin1-

specific 21-nucleotide siRNA sequence was synthesized by Dharmacon. The

sense siRNA sequence was 5¢-AAUCGUGAGGGUACUGAAACU-3¢, corres-

ponding to positions 6,043–6,063 relative to the talin1 mRNA start codon.

Control transfections used a fluorescein-labeled 21-nucleotide duplex directed

to the luciferase gene (Dharmacon). T cells were transfected by electroporation

using the Nucleofection system (Amaxa), according to the manufacturer’s

protocols. Transfection efficiency was evaluated by measurement of the

incorporation of fluorescein-labeled siRNA into cells by flow cytometry.

Transfected cells were maintained in culture medium (RPM1 1640 medium

with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate and antibiotics)

supplemented with interleukin 2 (25 units/ml; Sigma). Talin expression,

monitored by immunoblot, was maximally suppressed 96 h after transfection,

the time point chosen for all assays. Statistical comparisons were made with an

unpaired, two-tailed Student’s t-test.

Flow cytometry. For integrin and GPCR staining, cells were incubated for

30 min at 4 1C with primary antibody (10 mg/ml), were washed and were

incubated with secondary antibody, and then were analyzed immediately with a

FACScan flow cytometer (Becton Dickinson). For detection of LFA-1 activation

epitopes, PBLs (intact, chemokine or PMA-stimulated) were suspended for

10 min at 37 1C in binding medium in the presence of the various reporter

mAbs at 10 mg/ml. For artificial integrin activation, PBLs were washed in 5 mM

EDTA and were suspended in binding medium containing 2 mM each of Mg2+

and EGTA. Washed cells were resuspended with phycoerythrin-conjugated

antibodies to mouse for an additional 30 min at 4 1C.

Bead-immobilized ICAM-1 binding assays. Protein A magnetic M-280

Dynabeads (Dynal Biotech) were left intact or were coated with 50 mg/ml of


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ICAM-1–Fc, according to the manufacturer’s instructions. PBLs were preincu-

bated for 10 min at 25 1C in binding medium with or without blocking mAbs

(10 mg/ml) and then were left unstimulated or were stimulated for 30 s with

10 nM of chemokines, then were washed and were immediately mixed for

1 min with ICAM beads at a bead/cell ratio of 8:1. Cells were diluted threefold

and bead binding was determined by cellular side scatter, analyzed with a

FACScan flow cytometer. Binding specificity was confirmed by complete

absence of binding of protein A bead to cells.

Intravital microscopy. Intravital microscopy of inguinal lymph nodes from

young adult (6–12 weeks of age) female wild-type (C57BL/6–129J mixed

background) mice was done as described46. Mice were housed and bred in a

specific pathogen–free animal facility. All experiments were done in accordance

with National Institutes of Health guidelines and approved by the Committees

on Animals of both Harvard Medical School and the CBR Institute for

Biomedical Research. After induction of surgical anesthesia by intraperitoneal

injection of 10 ml/kg of saline containing xylazine (1 mg/ml) and ketamine

(5 mg/ml), an incision was made to expose a left abdominal skin flap, which

was stretched over a glass slide immobilized on a Plexiglas microscope stage.

Calcein-labeled lymphocytes injected into a right femoral artery catheter were

visualized in the peripheral lymph node microvasculature by stroboscopic

epifluroescence illumination with an intravital microscope (IV-500; Micron

Instruments) and images were recorded on a Hi8 VCR (Sony)46. For calculation

of instantaneous velocities, lymphocyte displacements were measured in

0.3-second intervals using software for random line-length measurements47.

Instantaneous velocities were measured in order IV venules as described48.

Calculation of instantaneous normalized rolling velocities (Vroll) was done by

division of a lymphocyte’s instantaneous velocity by its mean Vroll over the

entire rolling period.

For inhibition of LFA-1, lymphocytes were incubated for 10 min with

50 mg/ml of M17/4 mAb to LFA-1 (anti-mouse aL-subunit; Pharmingen).

Before lymphocytes were injected, recipient mice received 50 mg anti-LFA-1

through the femoral artery catheter.

In vitro shear flow assays. The preparation of all chemokine-containing

adhesive substrates and endothelial monolayers in a parallel plate flow chamber

(260-mm gap) were done as described26,27,43. ICAM-1–Fc–coated substrates

were prepared by plating of polystyrene dishes with 1 mg/ml of protein A

followed by coimmobilization of either heat-inactivated or intact chemokines

and human serum albumin, each at a concentration of 2 mg/ml. The protein A–

chemokine substrates were overlaid overnight at 4 1C with various concentra-

tions of ICAM-1–Ig (in PBS and 20 mg/ml BSA). ICAM-1 site densities were

assessed using 125I-labeled ICAM-1–Fc, as described49. Human umbilical vein

endothelial cells or ECV-304 cells were plated at confluence on tissue culture

dishes, and chemokines (1 mg/ml) were overlaid on the monolayers for 5 min

and plates were washed extensively with binding medium (Hank’s balanced salt

solution containing 2 mg/ml of BSA, 10 mM HEPES, pH 7.4, and Ca2+ and

Mg2+ at 1 mM each). Less than 5% of the overlaid chemokine remained stably

adsorbed on the monolayers, as determined using radiolabeled chemokines.

PBLs were prestimulated for 30 s with soluble chemokines (10 nM) in binding

medium at 25 1C, were washed and were immediately perfused into the

chamber. The total time elapsing from chemokine exposure to the introduction

into the chamber did not exceed 3 min, a period found to retain high integrin

affinity, as detected by the 327C and 327A reporters (Fig. 4b–d). Lymphocyte

cytoskeletons were disrupted by pretreatment of cells for 20 min at 37 1C with

10 mM cytochalasin D or 1 mM latrunculin A. The inhibitors were maintained

at 1/10 of the concentrations throughout the shear flow assay. For calpain- and

diacylglycerol-dependent PKC inhibition, PBLs were pretreated for 30 min

at 37 1C with 100 mg/ml of calpeptin or 5 mM bisindolylmaleimide I,

respectively. For phosphatidylinositol 3 kinase inhibition, cells were pretreated

for 30 min with 100 nM wortmanin. For blockade of Gi protein signaling,

PBLs were cultured for 15 h at 37 1C with 100 ng/ml of pertussis toxin in

culture medium. The LFA-1 I-domain was inhibited by pretreatment of cells

for 5 min with the allosteric IDAS-specific inhibitor A308926 (ref. 9) or

its control compound A270412 or carrier alone (dimethyl sulfoxide).

Heterologous chemokine desensitization between CXCR4 and CCR7 was ruled

out by testing of chemotactic migration of intact or CCL21-treated

PBLs toward 10 nM CXCL12 across a 5-mm Transwell filter (Corning) at

37 1C, as described44.

All flow experiments were done at 37 1C. The entire period of cell perfusion

was recorded as described43. All cellular interactions with the adhesive

substrates were determined by computerized tracking of individual cell motion

in at least two fields of view (each 0.17 mm2 in area). Transient tethers were

defined as cells attached briefly (less than 2 s) to the substrate; arrest (firm)

tethers were defined as tethered cells immediately stopping for at least 3 s

(ref. 43). Frequencies of adhesive categories in differently pretreated cells were

determined as a percentage of cells flowing along 0.9-mm paths in the focal

plane of the substrates. For assessment of the generation of integrin adhesive-

ness at stationary contacts, cells were allowed to settled on to the substrate for

1 min. For assessment of in situ conformational changes of lymphocyte-surface

LFA-1 at contacts lasting less than a second, PBLs were perfused over surface-

bound mAb to LFA-1 (coated at a concentration of 0.2 mg/ml with 2 mg/ml of

inactivated or active chemokine), as described43. Data are expressed as the

mean 7 range of two fields of view.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank S. Schwarzbaum for editorial help. Supported by the German-IsraeliFoundation for Scientific Research and Development, the Israel ScienceFoundation and MAIN, the EU6 Program for Migration and Inflammation.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 29 December 2004; accepted 18 March 2005

Published online at http://www.nature.com/natureimmunology/

1. Warnock, R.A., Askari, S., Butcher, E.C. & von Andrian, U.H. Molecular mechanisms oflymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187, 205–216 (1998).

2. Shimaoka, M., Takagi, J. & Springer, T.A. Conformational regulation of integrinstructure and function. Annu. Rev. Biophys. Biomol. Struct. 31, 485–516 (2002).

3. Carman, C.V. & Springer, T.A. Integrin avidity regulation: are changes in affinity andconformation underemphasized? Curr. Opin. Cell Biol. 15, 547–556 (2003).

4. Salas, A. et al. Rolling adhesion through an extended conformation of integrin aLb2 andrelation to aI and bI-like domain interaction. Immunity 20, 393–406 (2004).

5. Constantin, G. et al. Chemokines trigger immediate b2 integrin affinity and mobilitychanges: differential regulation and roles in lymphocyte arrest under flow. Immunity 13,759–769 (2000).

6. Stein, J.V. et al. The CC chemokine thymus-derived chemotactic agent 4 (TCA-4,secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) triggers lymphocytefunction-associated antigen 1-mediated arrest of rolling T lymphocytes in peripherallymph node high endothelial venules. J. Exp. Med. 191, 61–76 (2000).

7. Campbell, J.J., Hedrick, J., Zlotnik, A., Siani, M.A. & Thompson, D.A. Chemokines andthe arrest of lymphocytes rolling under flow conditions. Science 279, 381–384 (1998).

8. Sasaki, K., Okouchi, Y., Rothkotter, H.J. & Pabst, R. Ultrastructural localization of theintercellular adhesion molecule (ICAM-1) on the cell surface of high endothelialvenules in lymph nodes. Anat. Rec. 244, 105–111 (1996).

9. Beals, C.R., Edwards, A.C., Gottschalk, R.J., Kuijpers, T.W. & Staunton, D.E. CD18activation epitopes induced by leukocyte activation. J. Immunol. 167, 6113–6122(2001).

10. Lum, A.F., Green, C.E., Lee, G.R., Staunton, D.E. & Simon, S.I. Dynamic regulation ofLFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) inshear flow. J. Biol. Chem. 277, 20660–20670 (2002).

11. Beglova, N., Blacklow, S.C., Takagi, J. & Springer, T.A. Cysteine-rich module structurereveals a fulcrum for integrin rearrangement upon activation. Nat. Struct. Biol. 9,282–287 (2002).

12. Xie, C. et al. The integrin a-subunit leg extends at a Ca2+-dependent epitope in the thigh/genu interface upon activation. Proc. Natl. Acad. Sci. USA 101, 15422–15427 (2004).

13. Kucik, D.F., Dustin, M.L., Miller, J.M. & Brown, E.J. Adhesion-activating phorbol esterincreases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes. J. Clin.Invest. 97, 2139–2144 (1996).

14. Lub, M., van Kooyk, Y., van Vliet, S.J. & Figdor, C.G. Dual role of the actin cytoskeletonin regulating cell adhesion mediated by the integrin lymphocyte function-associatedmolecule-1. Mol. Biol. Cell 8, 341–351 (1997).

15. Kim, M., Carman, C.V., Yang, W., Salas, A. & Springer, T.A. The primacy of affinity overclustering in regulation of adhesiveness of the integrin aLb2. J. Cell Biol. 167, 1241–1253 (2004).

16. Alon, R. & Feigelson, S. From rolling to arrest on blood vessels: leukocyte tap dancingon endothelial integrin ligands and chemokines at sub-second contacts. Semin.Immunol. 14, 93–104 (2002).

17. Zhang, N., Hodge, D., Rogers, T.J. & Oppenheim, J.J. Ca2+-independent protein kinaseCs mediate heterologous desensitization of leukocyte chemokine receptors by opioidreceptors. J. Biol. Chem. 278, 12729–12736 (2003).


A R T I C L E S©

2005

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

eim

mun

olog

y

18. Shimaoka, M. et al. Structures of the aL I domain and its complex with ICAM-1 reveal ashape-shifting pathway for integrin regulation. Cell 112, 99–111 (2003).

19. Huth, J.R. et al. NMR and mutagenesis evidence for an I domain allosteric site thatregulates lymphocyte function-associated antigen 1 ligand binding. Proc. Natl. Acad.Sci. USA 97, 5231–5236 (2000).

20. Kim, M., Carman, C.V. & Springer, T.A. Bidirectional transmembrane signaling bycytoplasmic domain separation in integrins. Science 301, 1720–1725 (2003).

21. Tadokoro, S. et al. Talin binding to integrin b tails: a final common step in integrinactivation. Science 302, 103–106 (2003).

22. Monkley, S.J., Pritchard, C.A. & Critchley, D.R. Analysis of the mammalian talin2 geneTLN2. Biochem. Biophys. Res. Commun. 286, 880–885 (2001).

23. Jung, U., Norman, K.E., Scharffetter-Kochanek, K., Beaudet, A.L. & Ley, K. Transittime of leukocytes rolling through venules controls cytokine-induced inflammatory cellrecruitment in vivo. J. Clin. Invest. 102, 1526–1533 (1998).

24. Ley, K., Allietta, M., Bullard, D.C. & Morgan, S. The importance of E-selectin for firmleukocyte adhesion in vivo. Circ. Res. 83, 287–294 (1998).

25. Dustin, M.L., Bivona, T.G. & Philips, M.R. Membranes as messengers in Tcell adhesionsignaling. Nat. Immunol. 5, 363–372 (2004).

26. Grabovsky, V., Dwir, O. & Alon, R. Endothelial chemokines destabilize L-selectin-mediated lymphocyte rolling without inducing selectin shedding. J. Biol. Chem. 277,20640–20650 (2002).

27. Shamri, R. et al. Chemokine-stimulation of lymphocyte a4 integrin avidity but not ofLFA-1 avidity to endothelial ligands under shear flow requires cholesterol membranerafts. J. Biol. Chem. 277, 40027–40035 (2002).

28. Finger, E.B., Bruehl, R.E., Bainton, D.F. & Springer, T.A. A differential role for cellshape in neutrophil tethering and rolling on endothelial selectins under flow.J. Immunol. 157, 5085–5096 (1996).

29. Abitorabi, M.A., Pachynski, R.K., Ferrando, R.E., Tidswell, M. & Erle, D.J. Presentationof integrins on leukocyte microvilli: a role for the extracellular domain in determiningmembrane localization. J. Cell Biol. 139, 563–571 (1997).

30. Sampath, R., Gallagher, P.J. & Pavalko, F.M. Cytoskeletal interactions with theleukocyte integrin b2 cytoplasmic tail. Activation-dependent regulation of associationswith talin and a-actinin. J. Biol. Chem. 273, 33588–33594 (1998).

31. Yan, B., Calderwood, D.A., Yaspan, B. & Ginsberg, M.H. Calpain cleavage promotestalin binding to the b3 integrin cytoplasmic domain. J. Biol. Chem. 276, 28164–28170 (2001).

32. Pfaff, M., Liu, S., Erle, D.J. & Ginsberg, M.H. Integrin b cytoplasmic domainsdifferentially bind to cytoskeletal proteins. J. Biol. Chem. 273, 6104–6109 (1998).

33. Katagiri, K., Maeda, A., Shimonaka, M. & Kinashi, T. RAPL, a Rap1-binding moleculethat mediates Rap1-induced adhesion through spatial regulation of LFA-1.Nat. Immunol. 4, 741–748 (2003).

34. Giagulli, C. et al. RhoA and z PKC control distinct modalities of LFA-1 activation bychemokines critical role of LFA-1 affinity triggering in lymphocyte in vivo homing.Immunity 20, 25–35 (2004).

35. Shimonaka, M. et al. Rap1 translates chemokine signals to integrin activation, cellpolarization, and motility across vascular endothelium under flow. J. Cell Biol. 161,417–427 (2003).

36. Alon, R. et al. A novel genetic leukocyte adhesion deficiency in subsecond tri-ggering of integrin avidity by endothelial chemokines results in impairedleukocyte arrest on vascular endothelium under shear flow. Blood 101, 4437–4445(2003).

37. Kinashi, T. et al. LAD-III, a leukocyte adhesion deficiency syndrome associated withdefective Rap1 activation and impaired stabilization of integrin bonds. Blood 103,1033–1036 (2004).

38. Lupher, M.L., Jr et al. Cellular activation of leukocyte function-associated antigen-1and its affinity are regulated at the I domain allosteric site. J. Immunol. 167, 1431–1439 (2001).

39. Li, R. et al. Activation of integrin aIIb3 by modulation of transmembrane helixassociations. Science 300, 795–798 (2003).

40. Huang, C. & Springer, T.A. A binding interface on the I domain of lymphocyte function-associated antigen-1 (LFA-1) required for specific interaction with intercellular adhe-sion molecule 1 (ICAM-1). J. Biol. Chem. 270, 19008–19116 (1995).

41. Cabanas, C. & Hogg, N. Ligand intercellular adhesion molecule 1 has a necessary rolein activation of integrin lymphocyte function-associated molecule 1. Proc. Natl. Acad.Sci. USA 90, 5838–5842 (1993).

42. van Kooyk, Y. et al. Activation of LFA-1 through a Ca2+-dependent epitope stimulateslymphocyte adhesion. J. Cell Biol. 112, 345–354 (1991).

43. Grabovsky, V. et al. Subsecond induction of a4 integrin clustering by immobilizedchemokines enhances leukocyte capture and rolling under flow prior to firm adhesion toendothelium. J. Exp. Med. 192, 495–505 (2000).

44. Cinamon, G., Shinder, V. & Alon, R. Shear forces promote lymphocyte migrationacross vascular endothelium bearing apical chemokines. Nat. Immunol. 2, 515–522(2001).

45. Maly, P. et al. The Fuc-TVII a1,3 fucosyltransferase controls leukocyte traffickingthrough an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell 86, 643–653 (1996).

46. Gauguet, J.M., Rosen, S.D., Marth, J.D. & Von Andrian, U.H. Core 2 branching b1,6-N-acetylglucosaminyltransferase and high endothelial cell N-acetylglucosamine-6-sulfotransferase exert differential control over B and T lymphocyte homing to peripherallymph nodes. Blood 104, 4104–4112 (2004).

47. Pries, A.R. A versatile video image analysis system for microcirculatory research. Int. J.Microcirc. Clin. Exp. 7, 327–345 (1988).

48. M’Rini, C. et al. A novel endothelial L-selectin ligand activity in lymph nodemedulla that is regulated by a1,3-fucosyltransferase-IV. J. Exp. Med. 198, 1301–1312(2003).

49. Sigal, A. et al. The LFA-1 integrin supports rolling adhesions on ICAM-1 underphysiological shear flow in a permissive cellular environment. J. Immunol. 165,442–452 (2000).


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lymphocyte arrest requires instantaneous induction of an extended lfa-1 conformation mediated by...

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