P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively, of CD4+ T lymphocytes on tumor necrosis factor alpha-activated vascular endothelium under flow.

This report examines the adhesive interactions of human CD4+ T lymphocytes with tumor necrosis factor alpha-activated human endothelial cell monolayers in an in vitro model that mimics microcirculatory flow conditions. Resting CD4+ T cell interactions with activated endothelium consisted of initial attachment followed by rolling, stable arrest, and then spreading and transendothelial migration. P-selectin, but not E-, or L-selectin, mediated most of this initial contact and rolling, whereas beta 1-integrins (alpha 4 beta 1), interacting with endothelial-expressed vascular cell adhesion molecule 1, participated in rolling and mediated stable arrest. In contrast, beta 2-integrins were primarily involved in spreading and transmigration. These findings highlight an important role for P-selectin and suggest discrete functions for beta 1- and beta 2-integrins during lymphocyte recruitment to sites of immune-mediated inflammation.

Monocyte attachment to activated human vascular endothelium in vitro is mediated by leukocyte adhesion molecule-1 (L-selectin) under nonstatic conditions.

The receptors that mediate monocyte adhesion to cytokine-stimulated endothelial monolayers were assessed using a nonstatic (rotating) cell-attachment assay. In this system, leukocyte adhesion molecule-1 (LAM-1) (L-selectin) mediated a major portion (87 +/- 15% at 37 degrees C) of monocyte attachment to activated endothelium. mAb blocking of endothelial leukocyte adhesion molecule-1 (41% inhibition), CD18 (36%), and vascular cell adhesion molecule-1 (25%) function had lesser effects on attachment. These results suggest that LAM-1 may serve an important role in monocyte attachment to endothelium at sites of inflammation.

Acquisition of selectin binding and peripheral homing properties by CD4(+) and CD8(+) T cells.

Different T cell subsets exhibit distinct capacities to migrate into peripheral sites of inflammation, and this may in part reflect differential expression of homing receptors and chemokine receptors. Using an adoptive transfer approach, we examined the ability of functionally distinct subsets of T cells to home to a peripheral inflammatory site. The data directly demonstrate the inability of naive T cells and the ability of effector cells to home to inflamed peritoneum. Furthermore, interleukin (IL)-12 directs the differentiation of either CD4(+) or CD8(+) T cells into effector populations that expresses functional E- and P-selectin ligand and that are preferentially recruited into the inflamed peritoneum compared with T cells differentiated in the presence of IL-4. Recruitment can be blocked by anti-E- and -P-selectin antibodies. The presence of antigen in the peritoneum promotes local proliferation of recruited T cells, and significantly amplifies the Th1 polarization of the lymphocytic infiltrate. Preferential recruitment of Th1 cells into the peritoneum is also seen when cytokine response gene 2 (CRG-2)/interferon gamma-inducible protein 10 (IP-10) is used as the sole inflammatory stimulus. We have also found that P-selectin binds only to antigen-specific T cells in draining lymph nodes after immunization, implying that both antigen- and cytokine-mediated signals are required for expression of functional selectin-ligand.

Endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the vascular endothelial-cadherin complex at endothelial cell-to-cell junctions.

Although several adhesion molecules expressed on leukocytes (beta1 and beta2 integrins, platelet endothelial cell adhesion molecule 1 [PECAM-1], and CD47) and on endothelium (intercellular adhesion molecule 1, PECAM-1) have been implicated in leukocyte transendothelial migration, less is known about the role of endothelial lateral junctions during this process. We have shown previously (Read, M.A., A.S. Neish, F.W. Luscinskas, V.J. Palambella, T. Maniatis, and T. Collins. 1995. Immunity. 2:493-506) that inhibitors of the proteasome reduce lymphocyte and neutrophil adhesion and transmigration across TNF-alpha-activated human umbilical vein endothelial cell (EC) monolayers in an in vitro flow model. The current study examined EC lateral junction proteins, principally the vascular endothelial (VE)-cadherin complex and the effects of proteasome inhibitors (MG132 and lactacystin) on lateral junctions during leukocyte adhesion, to gain a better understanding of the role of EC junctions in leukocyte transmigration. Both biochemical and indirect immunofluorescence analyses of the adherens junction zone of EC monolayers revealed that neutrophil adhesion, not transmigration, induced disruption of the VE-cadherin complex and loss of its lateral junction localization. In contrast, PECAM-1, which is located at lateral junctions and is implicated in neutrophil transmigration, was not altered. These findings identify new and interrelated endothelial-dependent mechanisms for leukocyte transmigration that involve alterations in lateral junction structure and a proteasome-dependent event(s).

The cutaneous lymphocyte antigen is an essential component of the L-selectin ligand induced on human vascular endothelial cells.

L-selectin mediates leukocyte rolling on vascular endothelium during inflammation. Although vascular endothelium can be activated with inflammatory cytokines to express functional L-selectin ligands, these ligands have not been well characterized. In this study, fucosyltransferase VII cDNA (Fuc-TVII) transfection of the EA.hy926 human vascular endothelial cell line (926-FtVII) induced functional L-selectin ligand expression and expression of sialyl Lewisx (sLex), as defined by HECA-452 (cutaneous lymphocyte antigen; CLA) and CSLEX-1 mAbs. Cytokine activation of human umbilical vein endothelial cells (HUVEC) also induced functional L-selectin ligand expression, with increased CLA expression and Fuc-TVII transcription. The majority of L-selectin-dependent lymphocyte attachment to activated HUVEC and 926-FtVII cells was blocked specifically by treating the endothelial cells with the HECA-452 mAb, but not the CSLEX-1 mAb. CLA-bearing ligands on vascular endothelium also required sulfation and appropriate molecular scaffolds for functional activity, but were distinct from the L-selectin ligands previously identified by the MECA-79 mAb. These findings demonstrate that the HECA-452- defined antigen, CLA, is an essential carbohydrate component of vascular L-selectin ligands.

Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules.

Cytokines such as interleukin 1 (IL-1) promote adhesiveness in human umbilical vein endothelial cells for leukocytes including basophils, eosinophils, and neutrophils, and induce expression of adherence molecules including ICAM-1 (intercellular adhesion molecule-1), ELAM-1 (endothelial-leukocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1). In the present study, blocking monoclonal antibodies (mAb) recognizing ICAM-1, ELAM-1, and VCAM-1 have been used to compare their roles in IL-1-induced adhesion of human basophils, eosinophils, and neutrophils. IL-1 treatment of endothelial cell monolayers for 4 hours induced a four- to eight-fold increase in adhesion for each cell type. Treatment of endothelial cells with either anti-ICAM-1 or anti-ELAM-1 mAb inhibited IL-1-induced adherence of each cell type. In contrast, treatment with anti-VCAM-1 mAb inhibited basophil and eosinophil (but not neutrophil) adhesion, and was especially effective in blocking eosinophil adhesion. The effects of these mAb were at least additive. Indirect immunofluorescence and flow cytometry demonstrated expression of VLA-4 alpha (very late activation antigen-4 alpha, a counter-receptor for VCAM-1) on eosinophils and basophils but not on neutrophils. These data document distinct roles for ICAM-1, ELAM-1, and VCAM-1 during basophil, eosinophil, and neutrophil adhesion in vitro, and suggest a novel mechanism for the recruitment of eosinophils and basophils to sites of inflammation in vivo.

Monocytes induce reversible focal changes in vascular endothelial cadherin complex during transendothelial migration under flow.

The vascular endothelial cell cadherin complex (VE-cadherin, alpha-, beta-, and gamma-catenin, and p120/p100) localizes to adherens junctions surrounding vascular endothelial cells and may play a critical role in the transendothelial migration of circulating blood leukocytes. Previously, we have reported that neutrophil adhesion to human umbilical vein endothelial cell (HUVEC) monolayers, under static conditions, results in a dramatic loss of the VE-cadherin complex. Subsequent studies by us and others (Moll, T., E. Dejana, and D. Vestweber. 1998. J. Cell Biol. 140:403-407) suggested that this phenomenon might reflect degradation by neutrophil proteases released during specimen preparation. We postulated that some form of disruption of the VE-cadherin complex might, nonetheless, be a physiological process during leukocyte transmigration. In the present study, the findings demonstrate a specific, localized effect of migrating leukocytes on the VE-cadherin complex in cytokine-activated HUVEC monolayers. Monocytes and in vitro differentiated U937 cells induce focal loss in the staining of VE-cadherin, alpha-catenin, beta-catenin, and plakoglobin during transendothelial migration under physiological flow conditions. These events are inhibited by antibodies that prevent transendothelial migration and are reversed following transmigration. Together, these data suggest that an endothelial-dependent step of transient and focal disruption of the VE-cadherin complex occurs during leukocyte transmigration.

Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, beta 1-integrins, and beta 2-integrins.

Leukocyte interactions with vascular endothelium at sites of inflammation can be dynamically regulated by activation-dependent adhesion molecules. Current models, primarily based on studies with polymorphonuclear leukocytes, suggest the involvement of multiple members of the selectin, integrin, and immunoglobulin gene families, sequentially, in the process of initial attachment (rolling), stable adhesion (arrest), spreading and ultimate diapedesis. In the current study, IL-4-activated human umbilical vein endothelium, which selectively expresses VCAM-1 and an L-selectin ligand but not E-selectin, and appropriate function blocking monoclonal antibodies, were used to study monocyte-endothelial interactions in an in vitro model that mimics microcirculatory flow conditions. In this system, L-selectin mediates monocyte rolling and also facilitates alpha 4 beta 1-integrin-dependent arrest, whereas beta 2-integrins are required for spreading of firmly attached monocytes on the endothelial cell surface but not their arrest. These findings provide the first in vitro evidence for human monocyte rolling on cytokine-activated endothelium, and suggest a sequential requirement for both beta 1- and beta 2-integrin-dependent adhesive mechanisms in monocyte-endothelial interactions.

Isolated P-selectin glycoprotein ligand-1 dynamic adhesion to P- and E-selectin.

Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x-containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1-mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.

Cultured human endothelial cells stimulated with cytokines or endotoxin produce an inhibitor of leukocyte adhesion.

Activation of cultured human endothelial cells (HEC) by inflammatory stimuli, such as interleukin 1 (IL-1), tumor necrosis factor (TNF), and bacterial endotoxin (lipopolysaccharide, LPS), increases their surface adhesiveness for blood leukocytes and related cell lines. We now report that activated HEC also generate a soluble leukocyte adhesion inhibitor (LAI), which accumulates in conditioned media from IL-1-, TNF-, or LPS-treated, but not sham-treated, HEC cultures. LAI significantly inhibits the adhesion of PMN and monocytes to activated, but not unactivated, HEC. In contrast, LAI has no effect on the adhesion of lymphocytes, the promyelocytic cell line HL-60 or the monocyte-like cell line U937 to HEC monolayers. LAI appears to act directly on the leukocyte, but does not inhibit either agonist-induced responses in PMN (membrane depolarization, changes in cytosolic calcium concentration, superoxide production) or PMN attachment to serum-coated plastic surfaces. Endothelial generation of LAI is blocked by actinomycin D but not by aspirin or indomethacin. Preliminary biochemical characterization indicates that LAI is a soluble, protein-containing molecule that is heat- and acid-stable. Fractionation by HPLC gel filtration yields a single peak of LAI activity (14,000 less than Mr greater than 24,000). Thus, in addition to proadhesive cell surface changes, the endothelium may also actively contribute to the regulation of endothelial-leukocyte interactions at sites of inflammation in vivo through the production of soluble adhesion inhibitors such as LAI.

Memory B lymphocytes from secondary lymphoid organs interact with E-selectin through a novel glycoprotein ligand.

Recirculation of B lymphocytes through the secondary lymphoid organs is key for recognition and response to foreign antigen. B lymphocytes within secondary lymphoid organs comprise a heterogeneous population of cells at distinct differentiation stages. To ascribe a particular adhesive behavior to discrete B-cell subsets within secondary lymphoid organs, we investigated their functional interaction with endothelial selectins under flow. We describe herein the characterization of a subset of human tonsillar B cells that interact with E-selectin but not P-selectin. E-selectin-interacting B cells had a phenotype of non-germinal center (CD10(-), CD38(-), CD44(+)), memory (IgD-) cells. Furthermore, FucT-VII was expressed selectively in CD44(+) E-selectin-adherent B lymphocytes. B-cell rolling on E-selectin required sialic acid but was independent of previously described selectin ligands. A novel glycoprotein ligand of 240 kDa carrying N-linked glycans was isolated from B-cell membranes by an E-selectin immunoadhesin. Binding of this protein was strictly Ca2+ dependent, was inhibited by a cell adhesion-blocking mAb against E-selectin, and required the presence of sialic acid but not N-linked carbohydrates. Our results enable us to assign to resident memory B lymphocytes a novel adhesion function, the rolling on E-selectin, that provides insights on the adhesion pathways involved in homing of memory B cells to tertiary sites.

Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes.

PURPOSE: A critical early event in the pathogenesis of diabetic retinopathy is leukocyte adhesion to the diabetic retinal vasculature. The process is mediated, in part, by intercellular adhesion molecule-1 (ICAM-1) and results in blood-retinal barrier breakdown and capillary nonperfusion. This study evaluated the expression and function of the corresponding ICAM-1-binding leukocyte beta2-integrins in experimental diabetes. METHODS: Diabetes was induced in Long Evans rats with streptozotocin. The expression of the surface integrin subunits CD11a, CD11b, and CD18 on rat neutrophils isolated from peripheral blood was quantitated with flow cytometry. In vitro neutrophil adhesion was studied using quantitative endothelial cell-neutrophil adhesion assays. The adhesive role of the integrin subunits CD11a, CD11b, and CD18 was tested using specific neutralizing monoclonal antibodies. CD18 bioactivity was blocked in vivo with anti-CD18 F(ab')2 fragments, and the effect on retinal leukocyte adhesion was quantitated with acridine orange leukocyte fluorography. RESULTS: Neutrophil CD11a, CD11b, and CD18 surface integrin levels were 62% (n = 5, P = 0.006), 54% (n = 5, P = 0.045), and 38% (n = 5, P = 0.009) greater in diabetic versus nondiabetic animals, respectively. Seventy-five percent more neutrophils from diabetic versus nondiabetic animals adhered to rat endothelial cell monolayers (n = 6, P = 0.02). Pretreatment of leukocytes with either anti-CD11b or anti-CD18 antibodies lowered the proportion of adherent diabetic neutrophils by 41% (n = 6, P = 0.01 for each treatment), whereas anti-CD11a antibodies had no significant effect (n = 6, P = 0.5). In vivo, systemic administration of anti-CD18 F(ab')2 fragments decreased diabetic retinal leukostasis by 62% (n = 5, P = 0.001). CONCLUSIONS: Neutrophils from diabetic animals exhibit higher levels of surface integrin expression and integrin-mediated adhesion. In vivo, CD18 blockade significantly decreases leukostasis in the diabetic retinal microvasculature. Integrin adhesion molecules may serve as therapeutic targets for the treatment and/or prevention of early diabetic retinopathy.

Ca2+ mobilization with leukotriene A4 and epoxytetraenes in human neutrophils.

The biosynthesis of leukotrienes and lipoxins involves epoxide-containing intermediates which may be subject to several routes of transcellular metabolism. We have examined the capacity of leukotriene A4 (LTA4) and 15S-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid [5(6)-epoxytetraene] to stimulate the mobilization of free cytosolic calcium [( Ca2+]i) in human blood neutrophils. To gain insight into structure-activity relationships, a putative intermediate in lipoxin biosynthesis, 5S-hydroxy-14,15-oxido-6,10,12-trans-8-cis-eicosatetraenoic acid [14(15)-epoxytetraene], was prepared by total synthesis. When added to fura-2 loaded neutrophils, each of these compounds provoked a rapid and transient increase in [Ca2+]i (maximum by 8 sec) which returned to baseline within 60-90 sec. Ca2+ mobilization with LTA4 was dose dependent and, at 1 microM, the efficacies of LTA4 and LTB4 were quantitatively similar. The 5(6)-epoxytetraene and 14(15)-epoxytetraene were less potent than LTA4. Prior exposure of the cells to ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) (60 sec, 3 mM) did not diminish either the amplitude or the extent of [Ca2+]i elicited by LTA4. Methyl esters of LTA4 and 5(6)-epoxytetraene were less potent than their corresponding free acids, whereas the free acid of 14(15)-epoxytetraene and its methyl ester were quantitatively similar. Results from alcohol trapping studies showed that these epoxides were intact during the initial phase of Ca2+i mobilization (t0-10 sec) stimulated by LTA4, 5(6)-epoxytetraene, and 14(15)-epoxytetraene. In addition, the individual mixtures of products formed upon aqueous hydrolysis of each of the epoxides did not stimulate changes in [Ca2+]i. In each case, the products formed were identified by physical methods including reverse phase high pressure liquid chromatography, ultraviolet spectroscopy and gas liquid chromatography-mass spectrometry. These results indicate that, when added to human neutrophils, LTA4, 5(6)-epoxytetraene and 14(15)-epoxytetraene each stimulate a rapid mobilization of [Ca2+]i. Moreover, they suggest that intermediates in the biosynthesis of leukotrienes and lipoxins possess intrinsic activities that may serve to amplify cellular responses within their cell of origin or act on adjacent cells during their transcellular metabolism.

C-C and C-X-C chemokines trigger firm adhesion of monocytes to vascular endothelium under flow conditions.

In summary, our findings indicate that specific chemokines that are elaborated by endothelial cells after cytokine or endotoxin activation can play an essential role in monocyte recruitment beyond their chemoattractant activities. We show that this action is to translate initial monocyte tethering into firm adhesion via rapid leukocyte integrin activation. The in vitro model presented here provides a sensitive system for investigating the modulating ability of chemokines and reveals an important biological effect that is not predicted by results in simpler in vitro assays, such as measurement of calcium transients or chemotaxis. The surprising finding that the C-X-C chemokine IL-8 can trigger monocyte firm adhesion to vascular endothelium suggests a potential role for this chemokine in monocyte recruitment and underscores the biological complexity of the chemokine family.

Current in vitro models of leukocyte migration : methods and interpretation.

A large component of airway inflammatory disease is the recruitment of activated leukocytes (primarily eosinophils and T lymphocytes) from the lung vasculature into the bronchial walls resulting in lung edema. Ultimately, many of the infiltrating leukocytes progress across the airway epithelium into respiratory bronchioles, compromising lung capacity (1,2). In the case of an infection, such as pneumonia, leukocytes (primarily neutrophils and monocyte/macrophages) are recruited to alveolar air spaces reducing the capacity for gaseous exchange. In both cases, resident leukocytes then release further factors that promote additional leukocyte recruitment. During an inflammatory response in the peripheral microvasculature leukocyte recruitment takes place predominantly in the postcapillary venules via the multistep adhesion cascade (reviewed in 3,4,5). In the lung, however, leukocyte extravasation takes place via capillaries. This may be due to the specialized architecture of the lung vasculature (e.g., large numbers of branch points), or because of the differing expression of surface adhesion molecules that are required for leukocyte recruitment (1,6). In addition, local concentrations of cytokines, chemokines or other chemoattractant factors will play a role in the site and degree of leukocyte infiltration (7,8) through acute local activation of endothelial cells.

Integrins as dynamic regulators of vascular function.

The vascular endothelium lines the entire cardiovascular system and serves as a nonthrombogenic and selectively permeable boundary between the blood-stream and extravascular space. Endothelial cells are polar cells that are continuously subjected to fluid-generated forces on their luminal surface whereas their abluminal surface resides on basement membranes/extracellular matrix. The integrin family of cell-surface heterodimeric glycoproteins is located along both of these surfaces and participates in maintaining the normal endothelium and in the dynamic changes associated with the pathophysiology of the endothelium. Endothelial cell beta 1 and beta 3 integrins function together with other families of adhesion molecules during vasculogenesis, angiogenesis, inflammation, and wound healing. Leukocyte beta 1 and beta 2 integrins, in conjunction with members of the Ig and selectin gene families expressed on endothelium, mediate leukocyte recruitment to sites of inflammation. The structural and functional properties of integrins make them uniquely suited to mediate these essential and complex processes in the vasculature.

Endothelial-dependent mechanisms in chronic inflammatory leukocyte recruitment.

Peripheral blood leukocytes interact with the vascular endothelium in a wide range of physiologic and pathophysiologic situations. A current working concept is that activation of vascular endothelium is an important event during the inflammatory response, conferring spatial and temporal localization and leukocyte-type selectivity to the recruitment process. This chapter highlights recent advances in our understanding of the endothelial-dependent molecular mechanisms that mediate recruitment of mononuclear leukocytes (lymphocytes and monocytes) and discusses these advances in the context of chronic inflammatory diseases and their potential therapeutic interventions.

Monoclonal antibody blockade of L-selectin inhibits mononuclear leukocyte recruitment to inflammatory sites in vivo.

L-selectin interacting with inducible endothelial counterreceptors mediates in part the initial adhesive interactions, termed rolling, between circulating blood leukocytes and vascular endothelium. While blockade of L-selectin function in in vivo models of inflammation reduces both neutrophil and lymphocyte influx at early times, little is known concerning the role of L-selectin in leukocyte recruitment at later times (> 24 hours). Using an in vivo murine model of experimentally induced inflammation of the peritoneum, the role of L-selectin in recruitment of mononuclear leukocytes to chronic sites of inflammation (48 hours) was investigated. Saturating levels of function blocking anti-L-selectin monoclonal antibody (MEL-14) or control rat IgG were maintained for 48 hours using surgically implanted mini-osmotic pumps; this treatment did not alter the circulating leukocyte cell count or differential. In animals receiving MEL-14 monoclonal antibody (MAb), macrophage and lymphocyte accumulation in response to thioglycollate was reduced by 60% (P < or = 0.0002) and > 90% (P < 0.001), respectively, at 48 hours as compared with animals implanted with pumps containing saline. Similarly, MEL-14 MAb dramatically inhibited granulocyte influx by 80% (P < 0.03) at 6 hours; recruitment at 24 and 48 hours was reduced by 50%. In contrast, the effects of purified rat IgG was not significantly different from saline. Our results suggest L-selectin, interacting with its inducible endothelial counterreceptor(s), plays an important role in circulating mononuclear leukocyte extravasation at sites of inflammation.