Leukocyte Capture Research Articles

Leukocyte Adhesion: A Suitable Target for Anti-Inflammatory Drugs

Inflammatory responses in all tissue compartments require the emigration of leukocytes from the microvasculature through endothelial cells into the respective microenvironment. Adhesion to endothelial cells is the most crucial step in order to facilitate selective and effective capture of leukocytes. The sequence of adhesions events, e.g. rolling, tethering, and firm adhesion are tightly regulated by a variety of molecules expressed by endothelial cells and leukocytes either constitutively or after induction by mainly inflammatory mediators. In diseases with a prominent inflammatory response such as psoriasis, rheumatoid arthritis, or Crohn's disease, interference with leukocyte adhesion and/or emigration may be of substantial clinical effect. A number of therapeutic approaches by using monoclonal antibodies, designed molecules, and other modulators of adhesion molecule expression have been investigated in clinical trials. This review aims to give an overview about the current knowledge of targeting adhesion molecules as a therapeutic strategy to treat inflammatory diseases.

Basic Fibroblast Growth Factor (bFGF, FGF-2) Potentiates Leukocyte Recruitment to Inflammation by Enhancing Endothelial Adhesion Molecule Expression

Basic fibroblast growth factor (bFGF, FGF-2) is a potent angiogenic factor and endothelial cell mitogen. Although bFGF levels are increased in chronically inflamed tissue, its role in inflammation is unclear. We investigated the effect of bFGF on acute dermal inflammation and the recruitment of monocytes, T cells, and neutrophils. Leukocyte recruitment to inflamed sites was quantified with radiolabeled leukocytes. Intradermal injection of bFGF in rats did not induce leukocyte recruitment or inflammation. However, the recruitment of leukocytes to inflammation induced by tumor necrosis factor-alpha, interferon-gamma, C5a, or a delayed hypersensitivity reaction was enhanced by bFGF by 55 to 132% (P < 0.05). Either acute or prolonged bFGF treatment of dermal sites had this effect. The potentiating effect of bFGF on leukocyte recruitment was also seen in joints. There was no associated modulation of vascular permeability, blood flow, or angiogenesis in the sites by bFGF. However, the expression of the endothelial cell adhesion molecules (CAMs) for leukocytes, P-selectin, E-selectin, and ICAM-1, was significantly up-regulated in the inflamed tissue by bFGF, as quantified by radiolabeled anti-CAM antibody binding in vivo. Thus, although not directly proinflammatory, bFGF synergistically potentiates inflammatory mediator-induced leukocyte recruitment, at least in part, by enhancing CAM up-regulation on endothelium.

Nano-to-Microscale Mechanical Switches and Fuses Mediate Adhesive Contacts between Leukocytes and the Endothelium

As a vital part of the inflammatory response, the leukocyte adhesion cascade involves the capture of white blood cells at the vascular endothelium, a progressively slowing, “rolling” motion of cells along the vessel walls, firm cell adhesion at target sites, and eventually, the transmigration of activated cells into the inflamed tissue1-4 (for an instructive overview see also http://bme.virginia.edu/ley/). Studies aiming to reveal the biophysical mechanisms underlying these processes face the difficult challenge of covering a large range of relevant scales: nanoscale interactions between adhesive molecules, local deformations of subcellular structures under point loads, and whole-cell activation and motion. Yet multiscale approaches also offer particularly rewarding insights, for example, by providing an understanding of single-molecule interactions within their natural, larger-scale biological context and, conversely, by tracing macroscopically observed cellular phenomena directly to their molecular origins. The present work focuses on nano-to-microscale regulatory aspects that mediate the initial stages of the adhesion cascade. Early events, such as the capture of leukocytes and their initial rolling exploration at the endothelium, occur usually too fast to be regulated biologically, i.e., through the expression of proteins. We thus expected that most mechanisms governing initial leukocyte adhesion under a broad range of blood-flow conditions had to be “preprogrammed” into molecular-to-subcellular structures and that they had to be primarily mechanical. Accordingly, our experimental approach was to study in vitro the nano-to-micromechanical response of individual leukocytes that were loaded at a point with a rapidly increasing force. The adhesion bond between P-selectin (expressed in vivo on the endothelium and on activated platelets), and its ligand PSGL-1 (resident in the leukocyte membrane) is a key player in the initial adhesion stages.4,5 We exploited this bond to form point attachments between polymorphonuclear leukocytes (PMNs) and functionalized microspheres that had been coated with P-selectin. To distinguish the dynamic properties of an individual P-selectin:PSGL-1 bond from mechanisms governed by other cellular structures, we also tested this bond on its own using isolated PSGL-1 that had been immobilized on a second batch of microspheres. Both types of interaction, i.e., P-selectin:PSGL-1 as well as P-selectin:PMN, were characterized by dynamic force spectroscopy6,7 over exceptionally wide ranges of forceloading rates. Complementary test surfaces were first brought into soft, feedback-controlled contact to allow for the formation of bonds between immobilized, reactive molecules. Then, while moving the surfaces apart at preset velocities, we recorded the force experienced by attachments between them. Piconewton forces were reported by the biomembrane force probe,8,9 a prototypical biophysical tool that is uniquely suited to study the dynamics of biologically relevant, ultraweak interactions. This approach enabled us to inspect in detail two possible candidates for principal regulatory mechanisms in early leukocyte adhesion. One is a serial molecular attachment consisting of (i) the extracellular P-selectin:PSGL-1 bond and (ii) a putative, weak biochemical link that anchors the intracellular tail domain of the transmembrane PSGL-1 to the cortical cytostructure.10 The other is a hierarchy of rheological cell responses to point loads: from slow viscoelastic deformation of the whole cell, and a soft-elastic displacement of the cell cortex, to the extrusion of thin membrane tubes (“tethers”) that can easily growsat quasiconstant pulling forcessto several micrometers in length. The formation of tethers from fluid membranes under point loads is a ubiquitous phenomenon, as evidenced by a large body of work on lipid vesicles11-14 and on various types of biological cells.15-18 A number of pioneering studies also investigated the mechanical properties of PMN tethers19-21 and their importance in leukocyte rolling.22,23 Perhaps even more interesting than these regulatory mechanisms by themselves is the intriguing interplay between them. For example, the lifetime of weak attachments under stress depends critically on the history of force application. In the present case where the leukocyte itself links adhesive molecules to rigid surfaces, any subcellular soft structures will act to dampen the sudden impact of force and thus modulate the force history experienced by the actual adhesion bonds. Therefore, the lifetime, or strength, of adhesive attachments is directly affected by the rheology of cell deformation under point loads. On the other hand, the extrusion of tethers from PMNs appears to commence only * Corresponding author e-mail: vheinrich@ucdavis.edu. † Boston University. # Current address: Department of Biomedical Engineering, University of California, Davis, 451 East Health Sciences Drive, Davis, CA 95616. ‡ Department of Pathology, University of British Columbia. § Department of Physics and Astronomy, University of British Columbia. 1482 J. Chem. Inf. Model. 2005, 45, 1482-1490

Engagement of PSGL-1 upregulates CSF-1 transcription via a mechanism that may involve Syk

PSGL-1, the optimal selectin ligand demonstrated by in vivo studies to date, is an essential adhesive molecule mediating the rolling of leukocytes on the endothelial cells and the recruitment of leukocytes to the inflamed tissue. Recent studies demonstrated, in addition to its direct role in capture of leukocytes from the bloodstream, PSGL-1 also functions as a signal-transducing receptor and initiates a series of intracellular signal events during the activation of leukocytes. Our present work showed antibody engagement of PSGL-1 upregulated the transcriptional activity of CSF-1 promotor and increased the endogenous expression of CSF-1 mRNA in Jurkat cell. Overexpression of wild-typed non-receptor tyrosine kinase Syk, but not kinase dead mutant of Syk, promoted the upregulation of the transcriptional activity of CSF-1 promoter caused by antibody engagement of PSGL-1. Additionally, special inhibitor of Syk Piceatannol suppressed the increase of CSF-1 mRNA caused by the engagement of PSGL-1. The results suggest that signal transducted by PSGL-1 upregulate the transcriptional activity of CSF-1, and non-receptor tyrosine kinase Syk participates in this pathway.

Thematic Review Series: The Immune System and Atherogenesis. Molecular mechanisms regulating monocyte recruitment in atherosclerosis

Cardiovascular disease, a progressive disorder characterized by the accumulation of lipids in the artery wall, is a leading cause of death in Western societies. One of the initial events in atherogenesis involves the recruitment of inflammatory cells from the circulation into the developing lesion. Studies during the past decade have underscored the role of inflammatory mediators in disease initiation and progression. Critical progress has been made in our understanding of the complex mechanisms by which monocytes, macrophages, and T-cells accumulate in atherosclerotic plaques. Experimental research has identified several candidate adhesion proteins and chemokines that are critically involved in the recruitment process, and encouraging data provide a mechanistic framework for new therapeutic targets. This review provides an overview of our current understanding of the mechanisms that direct the recruitment of monocytes to, and their retention in, atherosclerotic lesions.

A new twist to the leukocyte adhesion cascade: intimate cooperation is key

Leukocyte migration from blood into lymphoid and non-lymphoid tissues requires a highly orchestrated series of adhesive and signaling events. A network of adhesion molecules with overlapping functions mediate the capture, rolling and firm adhesion of leukocytes to the vascular endothelium. A new study adds a novel twist to this paradigm by demonstrating that two adhesion molecules, CD44 and VLA-4, must be physically associated with each other on activated T cells to mediate efficient rolling and firm adhesion.

Cooperativity Between Selectins and 2-Integrins Define Neutrophil Capture and Stable Adhesion in Shear Flow

A cooperative, sequential process of molecular recognition governs leukocyte capture, rolling, and arrest on inflamed endothelium. Flowing neutrophils are captured via heterotypic adhesive interactions mediated by endothelial E-selectin, whereas homotypic interactions between neutrophils are mediated by L-selectin. To elucidate how each selectin facilitates the transition to CD18-mediated stable adhesion, E-selectin and L-selectin were expressed at defined site density in a murine pre-B-cell line. Direct observation of two-body collisions revealed that 30% of neutrophil interactions with E-selectin transfectants formed doublets at low shear rate G = 14 s(-1) whereas a threshold shear rate 14 s(-1) < or = G < or = 10 s(-1) was necessary for L-selectin adhesion. Adhesion via L-selectin resisted rupture at high shear stress, while E-selectin tethered doublets remained intact longer once formed. Moreover, higher expression of L-selectin (1100 sites/microm2) than that of E-selectin (220 sites/microm2) was required for comparable heterotypic adhesion efficiency. With a threefold rise in active CD18 upregulated on chemotactically stimulated neutrophils, homotypic adhesion efficiency increased 10-fold compared to less than 5-fold for heterotypic adhesion to selectin transfectants. Co-expression of E-selectin and ICAM-1 boosted adhesion efficiency threefold more than either receptor alone over the range of active CD18 expression. These data are the first to quantify adhesion efficiency mediated by selectin tethering and conformational activation of beta2-integrin in neutrophils in shear flow.

Mutagenesis of the Ezrin-Radixin-Moesin Binding Domain of L-selectin Tail Affects Shedding, Microvillar Positioning, and Leukocyte Tethering

L-selectin is a cell adhesion molecule that mediates the initial capture (tethering) and subsequent rolling of leukocytes along ligands expressed on endothelial cells. We have previously identified ezrin and moesin as novel binding partners of the 17-amino acid L-selectin tail, but the biological role of this interaction is not known. Here we identify two basic amino acid residues within the L-selectin tail that are required for binding to ezrin-radixinmoesin (ERM) proteins: arginine 357 and lysine 362. L-selectin mutants defective for ERM binding show reduced localization to microvilli and decreased phorbol 12-myristate 13-acetate-induced shedding of the L-selectin ectodomain. Cells expressing these L-selectin mutants have reduced tethering to the L-selectin ligand P-selectin glycoprotein ligand-1, but rolling velocity on P-selectin glycoprotein ligand-1 is not affected. These results suggest that ERM proteins are required for microvillar positioning of L-selectin and that this is important both for leukocyte tethering and L-selectin shedding.

017 Lower Expression of P‐Selectin may Modulate the Fetal Inflammatory Response:

Fetal dermal wound healing is characterized by a minimal inflammatory response and absence of scar. In order to determine the factors contributing to the minimal inflammatory response in the fetus, the role of P‐selectin in the interaction and transmigration of leukocytes through vascular endothelium was investigated. Methods: Primary endothelial monolayers were established from adult porcine central veins and from umbilical veins of mid‐gestation porcine fetuses. The ability of the endothelial cells to capture and enable transmigration of adult leukocytes was studied under flow conditions of 4 dynes/sec, with and without prior stimulation of the endothelial cells with TNF‐α or IL‐1β. In addition, P‐selectin mRNA expression was determined by quantitative real time PCR. All data were analyzed by ANOVA. Results: In response to IL‐1β 10 ng/ml stimulation, adult endothelial cells manifested a 10 fold increase in P‐selectin mRNA expression while fetal endothelial cells mounted only a 3.5 fold increase. 100 ng/ml was required to mount a 10 fold increase in fetal P‐selectin expression while no further increase in adult P‐selectin expression was noted at this dose. Leukocyte capture, demonstrated by rolling of neutrophils over the endothelial surface, was more efficient on adult monolayers compared to the fetus. This is inversely related to the rolling velocity which was significantly slower in the adult (5.3 ± 0.6 vs 12.4 ± 1.0 μm/mm2). Ultimately, the number of neutrophils transmigrating across adult endothelial monolayers under flow conditions was significantly higher compared to the fetal monolayer (199 ± 18 vs 72 ± 9 cells/mm2). Conclusion: Lower P‐selectin expression on fetal endothelial cells may account, in part, for the minimal inflammatory response noted following fetal dermal injury.

Avidity enhancement of L-selectin bonds by flow: shear-promoted rotation of leukocytes turn labile bonds into functional tethers.

L-selectin is a key lectin essential for leukocyte capture and rolling on vessel walls. Functional adhesion of L-selectin requires a minimal threshold of hydrodynamic shear. Using high temporal resolution videomicroscopy, we now report that L-selectin engages its ligands through exceptionally labile adhesive bonds (tethers) even below this shear threshold. These tethers share a lifetime of 4 ms on distinct physiological ligands, two orders of magnitude shorter than the lifetime of the P-selectin–PSGL-1 bond. Below threshold shear, tether duration is not shortened by elevated shear stresses. However, above the shear threshold, selectin tethers undergo 14-fold stabilization by shear-driven leukocyte transport. Notably, the cytoplasmic tail of L-selectin contributes to this stabilization only above the shear threshold. These properties are not shared by P-selectin– or VLA-4–mediated tethers. L-selectin tethers appear adapted to undergo rapid avidity enhancement by cellular transport, a specialized mechanism not used by any other known adhesion receptor.

Rolling adhesion kinematics of yeast engineered to express selectins.

Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewis-x ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast "walk" across the surface, alternately pivoting about bud and parent. Small-budded yeast "wobble" across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces.

Indirect capture augments leukocyte accumulation on P-selectin in flowing whole blood

Leukocytes are captured directly by E- and P-selectin on activated endothelium and by indirect means, which includes attached leukocytes capturing free-flowing leukocytes. However, controversy exists as to whether the latter mechanism occurs in the presence of red blood cells. We analyzed leukocyte capture mechanisms on P-selectin under circulatory hydrodynamics using whole blood. The selective disruption of leukocyte-leukocyte interactions with an L-selectin monoclonal antibody reduced leukocyte accumulation by >50% under various stringencies (substrate concentrations and shear stresses). In addition, a direct analysis of leukocyte capture events revealed that 69% were indirect. Our data indicate that in the presence of red blood cells, P-selectin-attached leukocytes, individually and as a monolayer, augment leukocyte accumulation by indirect capture. This mechanism may contribute to increasing the density of leukocytes on discrete areas of activated endothelial cells at sites of inflammation. These findings are significant since L-selectin accounts for the majority of the leukocyte rolling flux in small venules at diverse inflammatory settings. Yet, the primary mechanism by which L-selectin mediates leukocyte accumulation remains unresolved.

The State Diagram for Cell Adhesion Mediated by Two Receptors

Leukocyte recruitment from the bloodstream to surrounding tissues is an essential component of the immune response. Capture of blood-borne leukocytes onto vascular endothelium proceeds via a two-step mechanism, with each step mediated by a distinct receptor-ligand pair. Cells first transiently adhere, or “roll” (via interactions between selectins and sialyl-Lewis-x), and then firmly adhere to the vascular wall (via interactions between integrins and ICAM-1). We have reported that a computational method called adhesive dynamics (AD) accurately reproduces the fine-scale dynamics of selectin-mediated rolling. This paper extends the use of AD simulations to model the dynamics of cell adhesion when two classes of receptors are simultaneously active: one class (selectins or selectin ligands) with weakly adhesive properties, and the other (integrins) with strongly adhesive properties. AD simulations predict synergistic functions of the two receptors in mediating adhesion. At a fixed density of surface ICAM-1, increasing selectin densities lead to greater pause times and an increased tendency toward firm adhesion; thus, selectins mechanistically facilitate firm adhesion mediated by integrins. Conversely, at a fixed density of surface selectin, increasing ICAM-1 densities lead to greater pause times and an increased tendency to firm adhesion. We present this relationship in a two-receptor state diagram, a map that relates the densities and properties of adhesion molecules to various adhesive behaviors that they code, such as rolling or firm adhesion. We also present a state diagram for neutrophil activation, which relates β 2-integrin density and integrin-ICAM-1 kinetic on rate to neutrophil adhesive behavior. The predictions of two-receptor adhesive dynamics are validated by the ability of the model to reproduce in vivo neutrophil rolling velocities from the literature.

Rheological properties of the blood influencing selectin-mediated adhesion of flowing leukocytes

We studied how the rheological properties of blood influenced capture and rolling adhesion of leukocytes as well as their margination in the bloodstream. When citrated, fluorescently labeled blood was perfused through glass capillaries coated with P-selectin, leukocytes formed numerous rolling attachments. The number of attached leukocytes increased as the hematocrit was increased between 10% and 30% and was essentially constant from 30% to 50%. In EDTA-treated blood, adhesion was absent, and the flux of marginated cells varied little with increasing hematocrit. However, the velocity of marginated leukocytes increased monotonically, whereas the volumetric flow rate was constant, implying that the flow velocity profile became blunted and wall shear rate increased. Thus increasing hematocrit promoted attachment for a given total flow rate, without increasing margination, even though wall shear rate and blood viscosity increased. Blood was diluted to 20% hematocrit with plasma, 40-kDa dextran (to reduce red blood cell aggregation), or 500-kDa dextran (to enhance aggregation). Increasing aggregation correlated with increasing leukocyte adhesion and with more slow-flowing leukocytes near the wall. Thus flowing erythrocytes promote leukocyte adhesion, either by causing margination of leukocytes or by initiating and stabilizing attachments that follow.

Forming a Maternal Attachment

The process of embryo implantation during mammalian reproduction involves signaling between the embryo and the uterus. Genbacev et al. (see the Perspective by Fazleabas and Kim) report that selectin adhesion systems, which operate in leukocyte capture from bloodstream, function during embryo implantation, too. On the maternal side, selectin oligosaccharide ligands are expressed in the uterine epithelium, and on the fetal side, trophoblast cells express L-selectin receptors. In both mouse and human models, this receptor-ligand pair is shown to mediate successful uterine attachment. O. D. Genbacev, A. Prakobphol, R. A. Foulk, A. R. Krtolica, D. Ilic, M. S. Singer, Z.-Q. Yang, L. L. Kiessling, S. D. Rosen, S. J. Fisher, Trophoblast L-selectin-mediated adhesion at the maternal-fetal interface. Science 299 , 405-408 (2003). [Abstract] [Full Text] A. T. Fazleabas, J. J. Kim, What makes an embryo stick? Science 299 , 355-356 (2003). [Summary] [Full Text]

Trophoblast L-selectin-mediated adhesion at the maternal-fetal interface.

Trophoblast adhesion to the uterine wall is the requisite first step of implantation and, subsequently, placentation. At the maternal-fetal interface, we investigated the expression of selectin adhesion systems that enable leukocyte capture from the bloodstream. On the maternal side, human uterine epithelial cells up-regulated selectin oligosaccharide-based ligands during the window of receptivity. On the fetal side, human trophoblasts expressed L-selectin. This ligand-receptor system was functional, because beads coated with the selectin ligand 6-sulfo sLe(x) bound to trophoblasts, and trophoblasts bound to ligand-expressing uterine luminal epithelium in tissue sections. These results suggest that trophoblast L-selectin mediates interactions with the uterus and that this adhesion mechanism may be critical to establishing human pregnancy.

A Functional Role for Circulating Mouse L-Selectin in Regulating Leukocyte/Endothelial Cell Interactions In Vivo

L-selectin mediates the initial capture and subsequent rolling of leukocytes along inflamed vascular endothelium and mediates lymphocyte migration to peripheral lymphoid tissues. Leukocyte activation induces rapid endoproteolytic cleavage of L-selectin from the cell surface, generating soluble L-selectin (sL-selectin). Because human sL-selectin retains ligand-binding activity in vitro, mouse sL-selectin and its in vivo relevance were characterized. Comparable with humans, sL-selectin was present in adult C57BL/6 mouse sera at approximately 1.7 micro g/ml. Similar levels of sL-selectin were present in sera from multiple mouse strains, despite their pronounced differences in cell surface L-selectin expression levels. Adhesion molecule-deficient mice prone to spontaneous chronic inflammation and mice suffering from leukemia/lymphoma had 2.5- and 20-fold increased serum sL-selectin levels, respectively. By contrast, serum sL-selectin levels were reduced by 70% in Rag-deficient mice lacking mature lymphocytes. The majority of serum sL-selectin had a molecular mass of 65-75 kDa, consistent with its lymphocyte origin. Slow turnover may explain the relatively high levels of sL-selectin in vivo. The t(1/2) of sL-selectin, assessed by transferring sera from wild-type mice into L-selectin-deficient mice and monitoring serum sL-selectin levels by ELISA, was >20 h, and it remained detectable for longer than 1 wk. Short-term in vivo lymphocyte migration assays demonstrated that near physiologic levels ( approximately 0.9 micro g/ml) of sL-selectin decreased lymphocyte migration to peripheral lymph nodes by >30%, with dose-dependent inhibition occurring with increasing sL-selectin concentrations. These results suggest that sL-selectin influences lymphocyte migration in vivo and that the increased sL-selectin levels present in certain pathologic conditions may adversely affect leukocyte migration.

Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether.

L-selectin is a leukocyte lectin that mediates leukocyte capture and rolling in the vasculature. The cytoplasmic domain of L-selectin has been shown to regulate leukocyte rolling. In this study, the regulatory mechanisms by which this domain controls L-selectin adhesiveness were investigated. We report that an L-selectin mutant generated by truncation of the COOH-terminal 11 residues of L-selectin tail, which impairs association with the cytoskeletal protein α-actinin, could capture leukocytes to glycoprotein L-selectin ligands under physiological shear flow. However, the conversion of initial tethers into rolling was impaired by this partial tail truncation, and was completely abolished by a further four-residue truncation of the L-selectin tail. Physical anchorage of both cell-free tail-truncated mutants within a substrate fully rescued their adhesive deficiencies. Microkinetic analysis of full-length and truncated L-selectin–mediated rolling at millisecond temporal resolution suggests that the lifetime of unstressed L-selectin tethers is unaffected by cytoplasmic tail truncation. However, cytoskeletal anchorage of L-selectin stabilizes the selectin tether by reducing the sensitivity of its dissociation rate to increasing shear forces. Low force sensitivity (reactive compliance) of tether lifetime is crucial for selectins to mediate leukocyte rolling under physiological shear stresses. This is the first demonstration that reduced reactive compliance of L-selectin tethers is regulated by cytoskeletal anchorage, in addition to intrinsic mechanical properties of the selectin–carbohydrate bond.

Importance of primary capture and L-selectin-dependent secondary capture in leukocyte accumulation in inflammation and atherosclerosis in vivo.

In the multistep process of leukocyte extravasation, the mechanisms by which leukocytes establish the initial contact with the endothelium are unclear. In parallel, there is a controversy regarding the role for L-selectin in leukocyte recruitment. Here, using intravital microscopy in the mouse, we investigated leukocyte capture from the free flow directly to the endothelium (primary capture), and capture mediated through interactions with rolling leukocytes (secondary capture) in venules, in cytokine-stimulated arterial vessels, and on atherosclerotic lesions in the aorta. Capture was more prominent in arterial vessels compared with venules. In venules, the incidence of capture increased with increasing vessel diameter and wall shear rate. Secondary capture required a minimum rolling leukocyte flux and contributed by approximately 20-50% of total capture in all studied vessel types. In arteries, secondary capture induced formation of clusters and strings of rolling leukocytes. Function inhibition of L-selectin blocked secondary capture and thereby decreased the flux of rolling leukocytes in arterial vessels and in large (>45 microm in diameter), but not small (<45 microm), venules. These findings demonstrate the importance of leukocyte capture from the free flow in vivo. The different impact of blockage of secondary capture in venules of distinct diameter range, rolling flux, and wall shear rate provides explanations for the controversy regarding the role of L-selectin in various situations of leukocyte recruitment. What is more, secondary capture occurs on atherosclerotic lesions, a fact that provides the first evidence for roles of L-selectin in leukocyte accumulation in atherogenesis.

Severe impairment of leukocyte rolling in venules of core 2 glucosaminyltransferase–deficient mice

Leukocyte capture and rolling are mediated by selectins expressed on leukocytes (L-selectin) and the vascular endothelium (P- and E-selectin). To investigate the role of core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I) for synthesis of functional selectin ligands in vivo, leukocyte rolling flux and velocity were studied in venules of untreated and tumor necrosis factor-alpha (TNFalpha)-pretreated autoperfused cremaster muscles of C2GlcNAcT-I-deficient (core 2(-/-)) and littermate control mice. In untreated core 2(-/-) mice, leukocyte rolling was dramatically reduced with markedly increased rolling velocities (81 +/- 4 microm/s vs 44 +/- 3 microm/s). The reduced rolling in core 2(-/-) mice was due mainly to severely impaired binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1). Some rolling remained after blocking PSGL-1 in controls but not in core 2(-/-) mice. In TNFalpha-pretreated mice, rolling was markedly reduced in core 2(-/-) mice owing to impaired P-selectin- and E-selectin-mediated rolling. Rolling velocities in core 2(-/-) mice treated with an E-selectin-blocking monoclonal antibody (59 +/- 4 microm/s) were significantly higher than in controls (14 +/- 1 microm/s), which provides further evidence for the severe impairment in P-selectin-mediated rolling. In conclusion, P-selectin ligands including PSGL-1 are largely C2GlcNAcT-I dependent. In addition, E-selectin-mediated rolling in vivo is partially dependent on the targeted C2GlcNAcT-I. (Blood. 2001;97:3812-3819)