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(Journal of Leukocyte Biology. 2001;69:860-866.)
© 2001 by Society for Leukocyte Biology

Novel chemokine functions in lymphocyte migration through vascular endothelium under shear flow

Guy Cinamon, Valentin Grabovsky, Eitan Winter, Suzanna Franitza, Sara Feigelson, Revital Shamri, Oren Dwir and Ronen Alon

Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Correspondence: Ronen Alon, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. Email: ronalon{at}wicc.weizmann.ac.il


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ABSTRACT
 
The recruitment of circulating leukocytes at vascular sites in target tissue has been linked to activation of Gi-protein signaling in leukocytes by endothelial chemokines. The mechanisms by which apical and subendothelial chemokines regulate leukocyte adhesion to and migration across endothelial barriers have been elusive. We recently found that endothelial chemokines not only stimulate integrin-mediated arrest on vascular endothelial ligands but also trigger earlier very late antigen (VLA)-4 integrin-mediated capture (tethering) of lymphocytes to vascular cell adhesion molecule 1 (VCAM-1)-bearing surfaces by extremely rapid modulation of integrin clustering at adhesive contact zones. This rapid modulation of integrin avidity requires chemokine immobilization in juxtaposition with the VLA-4 ligand VCAM-1. We also observed that endothelial-bound chemokines promote massive lymphocyte transendothelial migration (TEM). It is interesting that chemokine-promoted lymphocyte TEM requires continuous exposure of lymphocytes but not of the endothelial barrier to fluid shear. It is noteworthy that lymphocyte stimulation by soluble chemokines did not promote lymphocyte TEM. Our results suggest new roles for apical endothelial chemokines both in triggering lymphocyte capture to the endothelial surface and in driving post-arrest events that promote lymphocyte transmigration across endothelial barriers under shear flow.

Key Words: inflammation • trafficking • integrins • G proteins


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INTRODUCTION
 
Leukocyte recruitment at inflamed or lymphoid tissues is mediated by sequential adhesive interactions between multiple vascular receptors and their endothelial counter-ligands [1 2 3 ]. This adhesive cascade allows the circulating immune cell to arrest on the vessel wall at the site of emigration to the target tissue. Specific stimulatory signals are presented to the recruited leukocyte and up-regulate in situ its integrin avidity to the ligand, enabling the cell to generate shear-resistant adhesion to the vessel wall. Chemoattractive cytokines, primarily chemokines, and their Gi-protein-coupled receptors (GPCRs) on leukocytes, are critically involved in triggering leukocyte-integrin avidity at endothelial adhesive zones to vascular-endothelial-integrin ligands. This has been demonstrated over the past decade in numerous in vivo and in vitro studies [4 5 6 7 8 9 10 11 ]. These studies have conclusively shown that integrin-mediated arrest of hematopoietic cells on their target endothelium is effectively suppressed by blocking the Gi-protein pathway of the circulating cells. Subsequent to arrest on endothelium, leukocytes undergo transendothelial migration, a nonproteolytic process in which adherent blood-borne cells cross the endothelial lining of the vessel wall, usually through intercellular borders at or near their original site of recruitment on the vascular endothelium [12 13 14 ]. Endothelium-associated chemokines might participate in this process, by continuously signaling to the arrested leukocytes to trigger promigratory actin-remodeling events [15 ]. The cytoskeletal reorganization should allow the arrested leukocyte to move from the initial recruitment site, undergo morphological changes, initiate its TEM across the endothelial layer, and complete diapedesis—all while maintaining resistance to shear-induced detachment from the vessel wall.


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IMMOBILIZED ENDOTHELIAL CHEMOKINES INDUCE INTEGRIN CLUSTERING AND AVIDITY DURING SUBSECOND CONTACTS THROUGH Gi-PROTEIN ACTIVATION ON TETHERED LEUKOCYTES
 
Several potent lymphocyte chemokines, functionally displayed on cytokine-activated human umbilical-vein endothelial cells (HUVECs), can instantaneously augment VLA-4 integrin-dependent adhesion of lymphocytes and CD34+ hematopoietic progenitor cells captured from the blood flow, resulting in firm arrest of these cells [11 16 ]. This ability of lymphocytes and hematopoietic progenitor cells to develop shear-resistant adhesion to vascular cell adhesion molecule 1 (VCAM-1)-bearing surfaces has been recently linked by us to rapid potentiation of VLA-4 avidity at transient leukocyte-substrate contact zones containing immobilized chemokines [11 ]. Chemokine potentiation of VLA-4 avidity is facilitated by VLA-4 clustering triggered by a Gi-protein-signaling event that occurs within as little as <0.1 s, the fastest inside-out integrin-signaling event reported to date. It is interesting that saturating levels of soluble chemokines failed to trigger VLA-4 avidity to VCAM-1 under shear flow, suggesting that localized rather than global signals must be transmitted from occupied GPCRs to trigger VLA-4 clustering at adhesive zones (Fig. 1 ). Thus, chemokines need to be presented in juxtaposition to the endothelial ligand within the adhesive contact zone to rapidly transduce VLA-4 clustering within the zone [11 ]. It is therefore likely that endothelium-displayed chemokines, unlike soluble chemokines, trigger VLA-4 adhesion at dynamic endothelial sites by rapidly increasing the effective mobility and clustering properties of the integrin within local VCAM-1-containing contact sites (Fig. 1) . VLA-4 clustering induced within subseconds of contact with a ligand results in enhanced lymphocyte tethering to and rolling on VCAM-1-bearing surfaces under physiological shear flow [11 ]. This is the first demonstration that endothelial chemokines may function at an earlier stage than was previously realized in augmenting reversible leukocyte interactions with vascular endothelium before cell arrest on the vessel wall.



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  		Figure 1. Model for subsecond stimulation of VLA-4 clustering at adhesive contacts by chemokine binding to integrin-associated GPCRs. Fast changes in integrin clustering at short-lived adhesive contact zones must occur to convert nonfunctional binding events into functional tethers. An initial VLA-4–VCAM-1 bond that is too weak to support a functional tether (1) is followed by a local chemokine signal transmitted through the chemokine GPCR (2). The G-protein signal triggered by the GPCR is transmitted directly to the cytoplasmic tail of a neighboring VLA-4 molecule or to an effector target near this second VLA-4 (3). As a result, the mobility of the neighboring VLA-4 is increased such that it can cluster at the site of initial bond formation (4). A preformed GPCR–VLA-4 complex is probably stabilized within a raft domain on the tip of a leukocyte microvillus._art>

This fast coupling may depend on segregation of the GPCR and its target integrin within preformed supramolecular structures, possibly localized on leukocyte-surface microvilli (Fig. 1) —preferential sites of leukocyte-endothelial contacts under shear flow [17 , 18 ]. VLA-4 as well as other lymphocyte integrins, like lymphocyte-function-associated antigen 1 (LFA-1) and {alpha}4ß7, may be segregated at these or other surface locations, within specific lipid raft microdomains. Such domains are enriched with heterotrimeric G proteins, integrin-associated tetraspannins, and Src kinases, all putative modulators of integrin adhesiveness at dynamic contacts [19 20 21 22 23 ].

Because VLA-4 and LFA-1 are topographically segregated on the leukocyte surface, it is likely that each integrin is preferentially associated with a different subset of GPCRs capable of inducing its clustering. Consistent with this notion, chemokines can trigger reversible subsecond VLA-4 tethers to VCAM-1, which support lymphocyte capture and subsequent rolling interactions on VCAM-1-bearing surfaces, whereas chemokine-stimulated LFA-1 adhesion to intercellular-adhesion-molecule 1 (ICAM-1) results in irreversible adhesion and firm arrest [7 ]. Chemokine-triggered arrest is linked to augmentation of both LFA-1 clustering and binding affinity to ICAM-1 [10 ]. An interesting possibility is that the ability of VLA-4 to arrest lymphocytes on VCAM-1 might also be caused by high-affinity states preexistent on subsets of circulating lymphocytes [24 ]. However, VLA-4 affinity is internally controlled by the lymphocyte activation state [25 ] and is not modulated by chemokine [11 ]. Thus, to promote reversible leukocyte rolling, chemokine stimulation of VLA-4 should promote integrin adhesiveness without inducing high affinity to ligands. Chemokine stimulation of VLA-4-mediated rolling is consistent with the previously reported functional hierarchy of these integrins, indicating that VLA-4 adhesion precedes LFA-1 adhesion strengthening in various multistep, adhesive cascades of leukocytes on endothelial surfaces [26 ]. VLA-4 and LFA-1 are also differentially modulated by soluble chemokine signals. Whereas LFA-1 affinity and clustering can be triggered by soluble chemokines with similar efficiency to immobilized chemokines [10 ], VLA-4 fails to respond to such chemokines under physiological shear flow [11 ].

In another interesting finding, we demonstrated that subsecond signaling by chemokines does not involve Ca2+ mobilization from intracellular stores, phosphatidylinositol 3 (PI3)-kinase activity, or PTK activation [11 ]. Consistent with our results, chemokine-stimulation of LFA-1 affinity was also PI3-kinase independent and did not implicate a rise in intracellular free Ca2+, although the kinase was instrumental in LFA-1 mobility within the membrane and its polar patching on lymphocytes arrested on ICAM-1-bearing surfaces [10 ]. These relatively slow processes (>10 s) appeared therefore to contribute to LFA-1-mediated adhesion strengthening at a post-tethering level [10 ]. Thus, none of the classical effector systems triggered by chemokine signaling through GPCRs were necessary to promote rapid endothelial adhesion of lymphocytes mediated by either VLA-4 or LFA-1. Although Gi-protein activation of VLA-4 and LFA-1 adhesiveness has been linked to rapid stimulation of the guanosine triphosphatase RhoA [27 ], the role of this or other Rho-like guanosine triphosphatases in subsecond chemokine-induced VLA-4 clustering or in rapidly triggered LFA-1 affinity has not been demonstrated. It is possible that these rapid modulations of integrin activity are controlled by GPCR signaling to a subset of Rho, constitutively associated with preformed membranal-integrin–GPCR complexes. We raise this hypothesis because Rho is a cytoplasmic protein [27 ], and therefore its recruitment to the membrane would be too slow to account for its putative role in triggering integrin avidity at subsecond adhesive zones.


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ANALYSIS OF LEUKOCYTE TEM UNDER SHEAR FLOW BY AN ALTERNATIVE EXPERIMENTAL SYSTEM TO TRANSWELL CHEMOTAXIS CHAMBERS
 
In addition to stimulating integrin avidity to endothelial ligands, chemokines control leukocyte motility and microenvironmental localization within extravascular target tissues [28-31]. Chemotactic migration triggered by chemokines may predominate in extravascular tissues. Indeed, gradients of soluble chemokines introduced at basal compartments of endothelial cells (ECs) can direct leukocyte migration across endothelial barriers as well as extracellular matrices in vitro [32 33 34 ]. However, at present there is no in vivo evidence that putative chemotactic gradients across vascular endothelium play any physiological role in directing leukocyte transendothelial migration under vascular shear flow [32 , 35 ]. Transendothelial chemotaxis has been traditionally studied in transwell Boyden chamber assays in the absence of shear flow. The lack of a physiological mechanical context and the prolonged time scales of transwell assays provide a poor model for physiological leukocyte transendothelial migration (TEM). Also unclear is whether leukocytes adherent to vascular endothelium under shear flow can sense and respond to sublumenal tissue chemokines within the short time frame of their contact with the endothelium, translating these putative signals to efficient diapedesis towards the chemokine source. The notion that apical chemokines are critically involved in regulating leukocyte adhesion to vascular endothelial ligands allowed us to raise the intriguing possibility that apical chemokines could in fact signal promigratory stimuli to adherent leukocytes even in the absence of chemokine gradients, after leukocyte arrest on the endothelial-cell surface.

Leukocytes generally move from their original site of arrest on the endothelial surface to their site of diapedesis [36 ]. Even if previously arrested at sites of diapedesis, the adherent leukocyte must reorient its cell body and leading edge towards and within the endothelial-migration zone. Because shear flow exerts continuous disruptive forces on arrested leukocytes, these adhesive processes must be carefully balanced to maintain migrating leukocyte attachment to the vessel wall. Yet, at the same time, vascular adherence must not exceed a level that would restrain leukocyte spreading and locomotion to the site of diapedesis. Multiple proadhesive and motility signals must therefore be transmitted in a highly coordinated manner to the migrating leukocyte along its vascular migratory path. The molecular basis of leukocyte spreading, locomotion, and positioning at these endothelial sites of diapedesis under continuous disruptive shear flow has been obscure. As a multistep process, leukocyte TEM has been difficult to dissect in vivo or in vitro under physiological shear-flow settings. The existence of vascular endothelial models that lack endogenous chemokines for T lymphocytes and can be reconstituted with specific chemokines [16 ] enabled us to use these models to dissect the molecular basis of lymphocyte migration across vascular endothelium under physiological shear flow. We used cytokine-activated endothelial monolayers reconstituted with chemokines to dissect the steps by which apical chemokine presentation promotes lymphocyte migration on and across inflamed endothelium. The experimental set up illustrated in Figure 2A is based on individual cell tracking of freshly isolated peripheral blood lymphocytes (PBLs) accumulated on a monolayer of cytokine-activated primary ECs [tumor necrosis factor (TNF){alpha}-activated HUVECs] on which a chemokine of interest has been overlaid. Both morphological changes and migratory properties of the leukocytes adhering to the endothelial monolayers are monitored at a single-cell level by phase-contrast videomicroscopy [16 ]. Analysis of leukocyte motion and shape is conducted over a time scale of minutes, corresponding to in vivo diapedesis processes of leukocytes extravasating at postcapillary venules of lymphoid organs or inflamed peripheral tissues [37 ].



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  		Figure 2. Analysis of leukocyte TEM under physiological shear flow in a parallel-plate flow chamber assay. The parallel-plate flow chamber assay allowed us to resolve the separate steps in the migratory cascade of leukocytes accumulating on an endothelial monolayer under shear flow in the absence or presence of exogenous chemokine overlaid on the apical surface of the monolayer. The indicated steps (i through vi) were monitored by high-power videomicroscopy of all cells from their initial accumulation in the field of view at low physiological flow (i) until final diapedesis (vi): (i) rolling and arrest; (ii) detachment from original site; (iii) spreading; (iv) firm stationary adhesion; (v) locomotion over the ECs; (vi) transmigration through the ECs. The time course of various parts of the assay, i.e., accumulation phase and migration phase, are indicated. Below (B) are frames taken from time-lapse digitized video recordings of lymphocytes adhered on TNF-{alpha}-activated HUVEC monolayers overlaid with SDF-1 (100 ng/mL) and subjected to continuous shear stress of 5 dyn/cm2 for the indicated period. Lymphocytes are numbered in a counterclockwise manner, and numbers designate their central positions at t = 0'. The paths traveled by each lymphocyte from original point of arrest (t = 0') to final position underneath or over the EC monolayer (t = 6') are depicted with dashed lines and arrowheads. At t = 6', lymphocytes 4–6 had completed TEM, whereas lymphocytes 1–3 moved variable distances over the endothelial surface.


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APICAL CHEMOKINES PROMOTE LYMPHOCYTE LOCOMOTION TO SITES OF TRANSMIGRATION AND TRIGGER ROBUST TRANSENDOTHELIAL MIGRATION UNDER SHEAR FLOW
 
Stromal cell-derived factor (SDF)-1 overlaid on cytokine-activated HUVECs not only triggers firm integrin-dependent adhesion of PBLs but, within 1 min after recruitment, stimulates massive spreading of almost all arrested lymphocytes (Fig. 2) . The spread lymphocytes then begin to slowly migrate on the endothelial surface (Fig. 2B) . Neither cell spreading nor locomotion are directed with the direction of flow (Fig. 2B , lymphocytes 4–6), and almost all recruited lymphocytes move some distance from their original sites of arrest before initiating TEM (Fig. 2B) . Within 2–3 min of locomotion on the endothelial surface, lymphocytes are observed to migrate underneath the endothelial monolayer without returning to the apical endothelial surface (Fig. 2 and Fig. 3 ). Under optimal conditions of chemokine stimulation, about 60% of originally accumulated PBLs can transmigrate through the ECs during the first 20-min period (Fig. 3) . Similar observations are obtained with PBLs transmigrating through HUVECs overlaid with another potent T-cell chemokine, EBI-1 molecular ligand chemokine [38 ]. The average time required for a lymphocyte positioned at its site of emigration to complete the transendothelial passage is 1.5 ± 0.5 min. Although transmigrating lymphocytes migrate variable distances on ECs before initiating TEM, there is no correlation between the distance traveled by moving lymphocytes and their ability to undergo TEM. Chemokine signaling through GPCRs on the adherent lymphocytes is essential for both initial PBL arrest on the substrate and subsequent locomotion and transmigration, since pertussis toxin pretreatment of lymphocytes completely eliminates chemokine-triggered adhesion, locomotion, and transmigration across the endothelial barrier (Fig. 3 , third treatment). It is noteworthy that lymphocyte exposure to soluble chemokines at doses that activate GPCR signaling is insufficient to promote TEM [38 ]. Furthermore, triggering of TEM by apical SDF-1 requires a threshold density higher than that required for triggering VLA-4-dependent arrest of lymphocytes on the endothelial surface [38 ]. Thus, the ability of endothelial chemokines to promote lymphocyte TEM appears to depend on proper solid-state presentation to the migrating lymphocyte at the apical endothelial surface. This restricted requirement of chemokine presentation for both induction of integrin clustering and lymphocyte TEM suggests that locally presented chemokine signals may increase the effective mobility of integrins, as well as other vascular receptors, on migrating lymphocytes, which can facilitate their movement over and through endothelial sites.



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  		Figure 3. Apical endothelial chemokines promote lymphocyte TEM under physiological flow conditions. PBLs accumulated for 40 s at 0.75 dyn/cm2 on TNF-{alpha}-stimulated (24 h, 2 ng/mL) HUVECs alone or previously overlaid with SDF-1 (100 ng, 5 min) were subjected to physiological shear stress (5 dyn/cm2) for 20 min. Cells which remained bound to the ECs during the entire assay (cells per field) were divided into three categories: arrested, "locomoting" (without transmigration), and transmigrating (TEM; see Fig. 2 ). For inhibition of Gi-protein signaling, lymphocytes were incubated with pertussis toxin. The fractions of PBLs of the total number of accumulated lymphocytes that remained bound to the ECs during the entire assay are indicated on top of each set of bars. The percentages of TEM lymphocytes of initially accumulated lymphocytes are expressed on top of the filled bars. Results shown are the means ± SE of multiple independent experiments, i.e., using different donors and EC preparations. * and ** stand for P < 0.02 and 0.0001, respectively, compared with PBL TEM across HUVECs without adsorbed SDF-1.

These results are the first demonstration that prototypic lymphocyte chemokines displayed to adherent lymphocytes on the apical endothelial surface can trigger robust transendothelial migration of lymphocytes to extravascular spaces. We are intrigued that this mode of migration occurs despite the minute levels of spontaneous T-cell-stimulatory signals within this endothelial model (Fig. 3 , first treatment). These results thus indicate an essential role for apical chemokines in driving lymphocyte transmigration across endothelial barriers. Indeed, in this experimental setting, lymphocytes cross the ECs away from a lumenal endothelial surface, which displays high-density chemokines, towards a subendothelial compartment devoid of the chemokine, i.e., in a direction opposite to the chemokine gradient. Taxis away from high-concentration chemokine depot (chemofugetaxis) has been described in shear-free systems [39 ]. However, in our model of lymphocyte TEM, T cells failed to migrate from the apical (chemokine-rich) endothelial compartment to the sublumenal (chemokine-poor) compartment in the absence of shear flow (Fig. 4 ). Furthermore, leukocyte migration driven by chemofugetaxis is sensitive to inhibition of PI3-K signaling, whereas lymphocyte TEM under shear flow is not [38 ]. It thus appears that the chemokine-triggered lymphocyte TEM observed by us is not a repellent chemokine-dependent migratory mechanism such as fugetaxis.



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  		Figure 4. Shear stress applied on adherent lymphocytes promotes TEM triggered by apical chemokine. Lymphocytes accumulated for 40 s at 0.75 dyn/cm2 on SDF-1 overlaid TNF{alpha}-activated HUVECs were subjected for 15 min to shear stress of 5 dyn/cm2 or were left in shear-free conditions. The fractions of transmigrating lymphocytes of the originally accumulated lymphocyte population were determined at the indicated time points. Results are the means ± SE of five independent experiments. *, **, ***: P < 0.05, 0.005, and 0.001 respectively, compared with PBL TEM across HUVECs in the absence of shear flow.


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CONTINUOUSLY APPLIED WALL SHEAR FORCES PROMOTE CHEMOKINE-TRIGGERED LYMPHOCYTE TEM
 
Our recent results suggest that shear flow applied on lymphocytes migrating through chemokine-bound activated ECs is not merely permissive for their TEM but is in fact required for TEM initiation and progression. When T cells are left on chemokine-bound ECs in the absence of applied shear stress, minute levels of TEM are observed (Fig. 4) . This shear dependence of lymphocyte TEM is irrespective of the activation or differentiation properties of HUVEC preparation [38 ]. It is interesting that application of shear flow on the chemokine-bound ECs shortly before lymphocyte accumulation followed by stoppage of flow does not promote lymphocyte TEM. Furthermore, lymphocytes in the process of transmigration through SDF-1-bound HUVECs under continuous shear flow rapidly stop migrating when shear flow is halted [38 ]. Thus, to optimally promote lymphocyte TEM, shear stress must be continuously applied on the adherent lymphocyte at its apical endothelial contact with the adsorbed chemokine. It thus appears that external forces provided by applied shear stresses may trigger mechanoresponsive elements on migratory lymphocytes, which would combine with the localized biochemical signals transduced to the migrating cells by apical or junctional endothelial chemokines. Subsequent chemokine signals encountered by the extravasating lymphocytes are likely to facilitate its migration through the extracellular matrix (ECM) and navigation to its final target within the tissue (Fig. 5 ).



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  		Figure 5. Leukocyte transmigration across endothelial and stromal barriers requires distinct distribution patterns of chemokines. Under shear flow conditions, leukocyte adhesion to the vascular endothelial barrier is critically dependent on stimulation of leukocyte integrins by apical endothelial chemokine(s) (open circles). These chemokines alone or in conjunction with subendothelial chemokines (gray circles) also promote rapid TEM under continuous-shear-flow conditions. In the absence of shear flow, e.g., like in transstromal migration, leukocytes may cross the stromal barrier only in response to a basal chemokine (alone or when two distinct chemokines are sequentially displayed to the migrating leukocyte on the apical and basal surfaces of the barrier). Stimulation of shear-resistant leukocyte adhesion by apical chemokines is not required in transstromal migration, in contrast to its requirement in transendothelial migration. Although leukocyte TEM is shown to take place through intercellular borders, particular subsets of leukocytes may cross the endothelial lining by traversing the venular endothelium through a transcellular route [50 ]. Relative hypothesized rates of lymphocyte transmigration under these different experimental conditions are summarized in the table below. EC, endothelial cells; ECM, extracellular matrix; ST, stromal cells.


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POSTULATED MECHANISMS UNDERLYING NAVIGATION OF EXTRAVASATING LYMPHOCYTES TO TARGET TISSUES: REGULATION BY SEQUENTIAL CHEMOKINE SIGNALS COUPLED TO MECHANICAL SIGNALS OF SHEAR FLOW
 
Our findings of the key role played by apical endothelial chemokines in lymphocyte TEM may be extended to other leukocyte and EC systems. Similar functions of immobilized chemoattractants in promoting leukocyte TEM under physiological shear flow can be carried out with platelet-activating factor and interleukin (IL)-8 (e.g., neutrophil TEM [40–42]) as well as by IP-10 (e.g., TEM of IL-2-activated lymphoblasts) [43 , 44 ]. Our results lead us to predict that the threshold levels of chemokine signals sufficient to trigger integrin-dependent arrest on endothelial surfaces under flow are lower than those required to promote TEM of the same cells through identical endothelial surfaces [38 ]. Presentation of the chemokine on distinct regions of the EC lining (e.g., apical, junctional) may provide such functional specialization among serially triggered GPCRs on the migrating leukocytes. Reminiscent of these findings, peripheral-blood monocytes have been recently reported to use distinct chemokine interactions to trigger integrin-mediated arrest, subsequent spreading, or transendothelial migration across inflamed endothelium under shear flow [15 ]. Hierarchical involvement of particular chemokine-GPCRs has been suggested to prevail in distinct steps of the migratory cascade of lymphocyte subsets on and across endothelial barriers. In this respect, recent studies on extravascular-migration model systems predict that leukocytes can integrate multiple chemotactic signals and can navigate in a stepwise manner through chemoattractant arrays [28 , 45 ]. The presence of several apical chemokines or the combination of several apical and basal chemokines may be similarly necessary to promote successful emigration through both simultaneous and sequential engagement of respective GPCRs coexpressed on individual migrating cells (Fig. 5) . The in vitro experimental approach developed by us, combined with controlled monoclonal-antibody-blocking studies performed on different endothelial systems, should enable testing of how the order of chemokine presentation to defined subsets of migrating leukocytes controls the extent and rate of their TEM across various types of vascular endothelia.

Chemokines are cationic proteins, which bind to heparan sulfate and related glycosaminoglycan moieties of endothelial or matrix proteoglycans [46 , 47 ]. The idea that juxtacrine signaling by immobilized chemoattractants may regulate adhesion and migration was first demonstrated for neutrophils interacting with inflamed endothelium [40 ]. Haptotaxis was suggested as an alternative migratory mechanism to chemotaxis by which cells move to a region of highest adhesiveness [48 ]. Our results strongly suggest that immobilized chemokines presented on adhesive endothelial surfaces act as haptotactic and juxtacrine regulatory elements in lymphocytes and possibly other leukocytes migrating on and through endothelial surfaces. An attractive possibility is that these haptotactic functions of chemokines are not restricted to endothelial surfaces and might contribute to leukocyte motility through the ECM barriers underlying the vessel wall. Although haptotactic migration has not been demonstrated in vivo, recent observations suggest that particular chemokines can drive directional lymphocyte migration across ECMs, even when uniformly presented in immobilized states on these matrices [49 ]. Thus, sublumenal chemokines deposited at the site of leukocyte extravasation (Fig. 5) may provide haptotactic and chemotactic signals that up-regulate both leukocyte adhesion and motility through the underlying basal lamina. Subsequently, gradients of locally secreted chemokines may navigate the migrating leukocyte through the ECM to its final destination in a chemotactic manner [45 ]. In conclusion, our studies suggest that chemokines, commonly suggested to act as mere chemoattractants [31 ] might transmit both proadhesive and promigratory signals to recruited lymphocytes in a nonchemotactic manner (i.e., independently of a gradient and not implicating the classical Gi-protein-triggered effector systems) [11 , 38 ]. Taken together, these new notions and the novel mechanoregulatory role we find for wall shear forces in promoting chemokine-triggered lymphocyte migration suggest that chemokine regulation of leukocyte trafficking is much more versatile than was previously realized.


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ACKNOWLEDGEMENTS
 
R. Alon is the incumbent of the Tauro Career Development Chair in Biomedical Research. Parts of this work were supported by the Israel Science Foundation, the Minnerva Foundation, Germany, and the Abisch Frenkel Foundation. We thank Dr. S. Shwarzbaum for editorial assistance.

Received December 30, 2000; revised April 4, 2001; accepted April 5, 2001.


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