Activation of endothelial extracellular signal-regulated kinase is essential for neutrophil transmigration: potential involvement of a soluble neutrophil factor in endothelial activation

During an inflammatory response induced by infection or injury, leukocytes traverse the endothelial barrier into the tissue space. Extravasation of leukocytes is a multistep process involving rolling, tethering, firm adhesion to the endothelium, and finally, transendothelial migration, the least characterized step in the process. The resting endothelium is normally impermeable to leukocytes; thus, during inflammation, intracellular signals that modulate endothelial permeability are activated to facilitate the paracellular passage of leukocytes. Using a static in vitro assay of neutrophil transmigration across human umbilical vein endothelium, a panel of inhibitors of intracellular signaling was screened for their ability to inhibit transmigration. PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK) 1/2 activation, inhibited both transmigration across TNF-alpha-activated endothelium and transmigration induced by the chemoattractant fMLP in a dose-dependent manner. PD98059 did not inhibit neutrophil chemotaxis in the absence of an endothelial barrier nor neutrophil adhesion to the endothelium, suggesting that its effect was on the endothelium, and furthermore, that endothelial ERK activation may be important for transmigration. We demonstrate in this study that endothelial ERK is indeed activated during neutrophil transmigration and that its activation is dependent on the addition of neutrophils to the endothelium. Further characterization showed that the trigger for endothelial ERK activation is a soluble protein of molecular mass approximately 30 kDa released from neutrophils after activation.     

Leukotriene B4-induced neutrophil-mediated endothelial leakage in vitro and in vivo.

The vascular leakage of macromolecules seen in several models after application of leukotriene B4 (LTB4) is mediated by neutrophil granulocytes. We describe here an in vitro assay for this event. Human umbilical vein endothelial cells were grown on polycarbonate filters separating luminal and abluminal compartments of fluid. Both clearance rate of fluorescein isothiocyanate albumin and neutrophil migration through the endothelial monolayer were increased when LTB4 (10-100 nM) was added to the abluminal compartment. However, if LTB4 was instead added to the luminal compartments together with the neutrophils, no migration or change in clearance could be detected. These findings were confirmed in vivo in the cheek pouches of anesthetized hamsters, where extravascular application of LTB4 induced intravascular adhesion of neutrophils, accompanied by neutrophil-dependent vascular leakage. On the other hand, intravascular deposition of LTB4 with micropipettes induced adhesion of leukocytes but no leakage. In conclusion, the presence of neutrophils adhering to endothelium does not necessarily imply the development of neutrophil-mediated vascular leakage. Instead, the leakage appears connected to the process of neutrophil chemotaxis.     

The effect of anti-intercellular adhesion molecule-1 on phorbol-ester-induced rabbit lung inflammation

The role of the CD18 complex of leukocyte glycoproteins in adhesion-dependent functions of human leukocytes in vitro has been well documented. A ligand, intercellular adhesion molecule-1 (ICAM-1), for at least one member of the CD18 complex has been identified. This molecule is inducible on many cell types including vascular endothelium and keratinocytes by inflammatory mediators such as IL-1, TNF, and IFN-gamma. ICAM-1 has been shown to mediate, in part, the in vitro adhesion of lymphocytes and neutrophils to endothelial cells expressing ICAM-1. In the present study we have shown that mAb's to the human CD18 complex and to human ICAM-1 cross react with rabbit cells and that both anti-CD18 and anti-CD11b but neither anti-CD11a nor anti-ICAM-1 mAb's inhibit neutrophil migration, an adhesion-dependent function, in vitro. Pretreatment of rabbits with anti-CD18 and anti-ICAM-1 but not anti-CD11a mAb inhibited by greater than 60% neutrophil migration into PMA-induced inflamed rabbit lungs. This effect of anti-ICAM-1 mAb on pulmonary neutrophil influx after PMA injection has important implications. Specifically, that ICAM-1 can function as a ligand for CD18 and can mediate, at least in part, the migration of neutrophils to inflammatory sites.     

E-selectin (endothelial-leukocyte adhesion molecule-1) is not required for the migration of neutrophils across IL-1-stimulated endothelium in vitro.

Treatment of vascular endothelial cells with inflammatory cytokines stimulates surface expression of E-selectin (previously known as endothelial-leukocyte adhesion molecule-1) and promotes the transendothelial migration of neutrophils. To assess participation of E-selectin in cytokine-mediated neutrophil migration, an in vitro model consisting of monolayers of human umbilical vein endothelial cells (HUVEC) grown on amniotic connective tissue was used. When HUVEC-amnion cultures were stimulated for 4 h with relatively low concentrations of IL-1 (0.1 to 0.15 U/ml), mAb BB11 or H18/7 to E-selectin partially inhibited migration of subsequently added neutrophils. However, when the cultures were stimulated with 15 U/ml of IL-1 for 4 or 24 h, little to no inhibition was observed. mAb to E-selectin also failed to inhibit migration of neutrophils across HUVEC-amnion cultures treated with low doses of IL-1 when the leukocytes were additionally stimulated by the chemoattractant leukotriene B4. In contrast, migration of neutrophils across IL-1-treated HUVEC was profoundly inhibited by mAb to CD11/CD18 leukocytic integrins under all conditions tested. Results of these studies suggest that participation of E-selectin is not essential for migration of neutrophils across cytokine-stimulated HUVEC in vitro; rather, E-selectin can be bypassed in favor of CD11/CD18-dependent mechanisms under appropriate circumstances.     

IFN-gamma alters the response of Borrelia burgdorferi-activated endothelium to favor chronic inflammation

Borrelia burgdorferi, the agent of Lyme disease, promotes proinflammatory changes in the endothelium that lead to the recruitment of leukocytes. The host immune response to infection results in increased levels of IFN-gamma in the serum and lesions of Lyme disease patients that correlate with greater severity of disease. Therefore, the effect of IFN-gamma on the gene expression profile of primary human endothelial cells exposed to B. burgdorferi was determined. B. burgdorferi and IFN-gamma synergistically augmented the expression of 34 genes, 7 of which encode chemokines. Six of these (CCL7, CCL8, CX3CL1, CXCL9, CXCL10, and CXCL11) attract T lymphocytes, and one (CXCL2) is specific for neutrophils. Synergistic production of the attractants for T cells was confirmed at the protein level. IL-1beta, TNF-alpha, and LPS also cooperated with IFN-gamma to induce synergistic production of CXCL10 by the endothelium, indicating that IFN-gamma potentiates inflammation in concert with a variety of mediators. An in vitro model of the blood vessel wall revealed that an increased number of human T lymphocytes traversed the endothelium exposed to B. burgdorferi and IFN-gamma, as compared with unstimulated endothelial monolayers. In contrast, addition of IFN-gamma diminished the migration of neutrophils across the B. burgdorferi-activated endothelium. IFN-gamma thus alters gene expression by endothelia exposed to B. burgdorferi in a manner that promotes recruitment of T cells and suppresses that of neutrophils. This modulation may facilitate the development of chronic inflammatory lesions in Lyme disease.     

Genetic evidence for functional redundancy of Platelet/Endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1-dependent and PECAM-1-independent functions

Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31), a member of the Ig superfamily, is expressed strongly at endothelial cell-cell junctions, on platelets, and on most leukocytes. CD31 has been postulated to play a role in vasculogenesis and angiogenesis, and has been implicated as a key mediator of the transendothelial migration of leukocytes. To further define the physiologic role of CD31, we used targeted gene disruption of the CD31 gene in embryonic stem cells to generate CD31-deficient mice. CD31-deficient mice (CD31KO) are viable and born at the expected Mendelian frequency, remain healthy, and exhibit no obvious vascular developmental defects. In response to inflammatory challenge, polymorphonuclear leukocytes of CD31KO mice are arrested between the vascular endothelium and the basement membrane of inflammatory site mesenteric microvessels, confirming a role for CD31 in the migration of neutrophils through the subendothelial extracellular matrix. Normal numbers of leukocytes are recovered from inflammatory sites in CD31KO mice, however, suggesting that the defect in leukocyte migration across basal lamina observed in the absence of CD31 may be compensated for by the use of other adhesion molecules, or possibly an increased rate of migration. Homing of T lymphocytes in vivo is normal, and CD31KO mice are able to mount a cutaneous hypersensitivity response normally. In addition, CD31-mediated homophilic adhesion does not appear to play a role in platelet aggregation in vitro. This study provides genetic evidence that CD31 is involved in transbasement membrane migration, but does not play an obligatory role in either vascular development or leukocyte migration.     

CD99 is essential for leukocyte diapedesis in vivo

Recruitment of leukocytes into inflamed tissue requires migration of leukocytes from the blood stream across the endothelial lining and the basement membrane of the local blood vessels. CD99 in humans is a 32-kDa highly O-glycosylated cell surface protein expressed on most leukocytes. The authors recently found CD99 to be expressed in leukocytes and at human endothelial cell contacts. Human CD99 is involved in homophilic interaction between the two cell types and participates in the transendothelial migration of monocytes and polymorphonuclear neutrophils (PMNs) in vitro. To test the role of CD99 in vivo, the authors cloned murine CD99 (muCD99), expressed it in vitro, and generated a blocking monoclonal antibody against it. We first showed that muCD99 is expressed on mouse leukocytes as well as enriched at the endothelial cell borders. Transfection of cells with muCD99 imparts on them the ability to aggregate in a CD99-dependent homophilic manner. Cells expressing muCD99 did not bind to cells expressing murine or human platelet endothelial call adhesion molecule (PECAM) or human CD99. In the thioglycollate peritonitis model of inflammation, anti-CD99 monoclonal antibody blocked the recruitment of neutrophils and monocytes by over 40% and 80%, respectively, at 18 h. Microscopy showed that this blocking occurred at the luminal surface of venules. The authors conclude that CD99 plays a major role in the emigration of leukocytes in vivo.     

The effect of short-term, high glucose concentration on endothelial cells and leukocytes in a 3D in vitro human vascular tissue model

Efforts to determine a link between diabetes and atherosclerosis have involved examining the effect of high glucose levels on the adhesion and migration of circulating leukocytes, mostly monocytes and T lymphocytes. Leukocyte differentiation and proliferation within the subendothelial space can also be investigated by the use of a 3D in vitro human vascular tissue model. This model was used to study the effect of short-term, high glucose concentration on certain cell behavior associated with the early stages of atherosclerosis. Samples were exposed to either a 30- or 5.6-mM glucose concentration for 9 h to represent either hyperglycemic or normoglycemic conditions, respectively. There was a significant increase in vascular cell adhesion molecule-1 expression on the endothelial cells exposed to a 30-mM compared to a 5.6-mM glucose concentration. There was no significant difference in either intercellular adhesion molecule-1 or E-selectin expression on the endothelial cells exposed to a 30-mM compared to a 5.6-mM glucose concentration. After the endothelium was exposed to 30 mM glucose concentration, there was a 70% increase in the number of monocytes (CD14⁺) migrating across the endothelium and a 28% increase in the number of these monocytes differentiating into macrophages, compared to cell migration and differentiation across the endothelium exposed to 5.6 mM glucose concentration. Also, for the endothelium exposed to 30 mM glucose concentration, there were nearly 2.5 times more T lymphocytes that migrated across the endothelium, along with significant cell proliferation, compared to cell migration across the endothelium exposed to 5.6 mM glucose concentration.     

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sj?gren's syndrome (SS).     

Quantitative assessment of the adherence of normal human blood mononuclear cells to vascular endothelial cell monolayers

We have developed a method to reliably quantitate the in vitro adherence of ⁵¹Cr-labeled blood mononuclear leukocytes to cultured monolayers of vascular endothelial cells from human umbilical veins. Normal mononuclear leukocytes adhered to endothelial cells more than to cover glass at all studied time periods over 4 hr with major differences seen at 2 hr (9.7 ± 1.2% vs 3.7 ± 1.1%; P < 0.01). Only a minority of cells adhering to endothelium were esterase positive. Similar patterns of binding were seen using varying concentrations of suspended mononuclear cells (1–4 × 10⁶/ml) simulating that occurring in vivo in different clinical states. This approach shows promise for in vitro approaches to lymphocyte-vascular endothelial interactions in human immune/inflammatory disorders.     

Molecular events during leukocyte diapedesis

The recruitment of leukocytes from the circulation into tissues requires leukocyte migration through the vascular endothelium. The mechanisms by which leukocytes attach and firmly adhere to the endothelial cell surface have been studied in detail. However, much less is known about the last step in this process, the diapedesis of leukocytes through the vascular endothelium. This minireview focuses on the interactions between leukocyte and endothelial cell adhesion molecules that are important during leukocyte extravasation. In the past few years a series of endothelial cell surface and adhesion molecules have been identified that are located at endothelial cell contacts and found to participate in leukocyte diapedesis. These junctional cell adhesion molecules are believed to have an active role in controlling the opening and closure of endothelial cell contacts to allow the passage of leukocytes between adjacent endothelial cells. Alternatively, leukocytes can cross the endothelium at nonjunctional locations, with leukocytes migrating through a single endothelial cell. Further work is clearly needed to understand, in greater detail, the molecular mechanisms that allow leukocytes to cross the endothelium via either the paracellular or the transcellular pathway.     

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.     

Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment

Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-α, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D₂ (PGD₂) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD₃. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD₂ receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD₂ signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.     

Migrational guidance of neutrophils is mechanotransduced via high-affinity LFA-1 and calcium flux

Acute inflammation triggers the innate immune response of neutrophils that efficiently traffic from the bloodstream to concentrate at high numbers at the site of tissue infection or wounding. A gatekeeper in this process is activation of β(2) integrins, which form bond clusters with ICAM-1 on the endothelial surface. These bond clusters serve dual functions of providing adhesive strength to anchor neutrophils under the shear forces of blood flow and directional guidance for cell polarization and subsequent transmigration on inflamed endothelium. We hypothesized that shear forces transmitted through high-affinity LFA-1 facilitates the cooperation with the calcium release-activated channel Orai1 in directing localized cytoskeletal activation and directed migration. By using vascular mimetic microfluidic channels, we observed neutrophil arrest on a substrate of either ICAM-1 or allosteric Abs that stabilize a high- or low-affinity conformation of LFA-1. Neutrophils captured via low-affinity LFA-1 did not exhibit intracellular calcium flux, F-actin polymerization, cell polarization, or directional migration under shear flow. In contrast, high-affinity LFA-1 provided orientation along a uropod-pseudopod axis that required calcium flux through Orai1. We demonstrate how the shear stress of blood flow can transduce distinct outside-in signals at focal sites of high-affinity LFA-1 that provide contact-mediated guidance for neutrophil emigration.     

The peripheral lymph node homing receptor, LECAM-1, is involved in CD18-independent adhesion of human neutrophils to endothelium.

The binding of polymorphonuclear granulocytes (PMN) to activated vascular endothelium is a crucial step in the recruitment of PMN to an inflammatory site. Studies employing cytokine-activated endothelium in culture have shown that PMN binding involves the CD18 family of leukocyte integrins, but also CD18-independent adhesion mechanism(s) on PMN that have not been defined. We unify here two previously disparate approaches to study cell adhesion events between endothelial cells and leukocytes. We show that antibodies to human LECAM-1, the peripheral lymph node homing receptor that is also expressed on PMN, partially inhibit the adhesion of human PMN not only to HEV in frozen sections of lymph node tissue, but also to cytokine-activated human umbilical vein endothelium in vitro. Inhibition with anti-LECAM-1 antibodies and anti-CD18 antibodies is additive. Furthermore, the anti-LECAM-1 antibodies inhibit the adhesion of CD18-deficient PMN to cytokine activated human endothelial cells. These findings indicate that LECAM-1 and CD18-mediated binding mechanisms are independent, and act coordinately or sequentially to mediate PMN attachment to cytokine activated endothelium.     

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.     

Cell-cell junctions of dermal microvascular endothelial cells contain tight and adherens junction proteins in spatial proximity

Endothelial cell-cell contacts control the vascular permeability, thereby regulating the flow of solutes, macromolecules, and leukocytes between blood vessels and interstitial space. Because of specific needs, the endothelial permeability differs significantly between the tight blood-brain barrier endothelium and the more permeable endothelial lining of the non-brain microvasculature. Most likely, such differences are due to a differing architecture of the respective interendothelial cell contacts. However, while the molecules and junctional complexes of macrovascular endothelial cells and the blood-brain barrier endothelium are fairly well characterized, much less is known about the organization of intercellular contacts of microvascular endothelium. Toward this end, we developed a combined cross-linking and immunoprecipitation protocol which enabled us to map nearest neighbor interactions of junctional proteins in the human dermal microvascular endothelial cell line HMEC-1. We show that proteins typically located in tight or adherens junctions of epithelial cells are in the proximity in HMEC-1 cells. This contrasts with the separation of the different types of junctions observed in polarized epithelial cells and "tight" endothelial layers of the blood-brain barrier and argues for a need of the specific junctional contacts in microvascular endothelium possibly required to support an efficient transendothelial migration of leukocytes.     

A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them

The basic route and mechanisms for leukocyte migration across the endothelium remain poorly defined. We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel "cuplike" transmigratory structure. This endothelial structure was comprised of highly intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-enriched vertical microvilli-like projections that surrounded transmigrating leukocytes and drove redistribution of their integrins into linear tracks oriented parallel to the direction of diapedesis. Disruption of projections was highly correlated with inhibition of transmigration. These findings suggest a novel mechanism, the "transmigratory cup", by which the endothelium provides directional guidance to leukocytes for extravasation.     

Endothelial Cell Adhesion Molecule Expression and Lymphocyte Adhesion to Endothelial Cells: Effect of Nitric Oxide

Interactions between circulating leukocytes and vascular endothelial cells are of fundamental importance in controlling normal recirculation and migration of cells into sites of inflammation. Nitric oxide (NO), which is synthesized by vascular endothelial cells, has been reported to decrease the binding of platelets, monocytes, macrophages, and neutrophils to endothelial cells. Using NO donors and inhibitors of the enzyme NO synthase, we found no evidence that physiologically relevant levels of NO alter adhesion of purified lymphocytes to an endothelial cell line derived from human umbilical vein endothelial cells (SGHEC-7). In addition, NO donors did not alter the cell surface expression of VCAM-1, ICAM-1, or E-selectin on SGHEC-7 cells.