Activation of endothelium by Borrelia burgdorferi in vitro enhances transmigration of specific subsets of T lymphocytes.

Lyme disease, caused by Borrelia burgdorferi, is characterized by the accumulation of lymphocytes and monocytes in the affected tissue. Endothelial cells line the blood vessel walls and control the trafficking of inflammatory leukocytes from the blood into the surrounding tissues. A model of the blood vessel wall, consisting of human umbilical vein endothelial cells (HUVEC) grown on amniotic connective tissue, was utilized to examine the effects of B. burgdorferi on the transendothelial migration of T lymphocytes. Maximal migration occurred when the HUVEC-amnion cultures were preincubated with B. burgdorferi for 24 h and T lymphocytes were added for an additional 4 h, yielding a two- to fourfold increase compared to migration across unstimulated cultures. The number of T lymphocytes that migrated was proportional to the number added. The anti-inflammatory cytokine interleukin 10 (IL-10), added during activation of the HUVEC, significantly diminished (by an average of 70% +/- 21%) the migration of T lymphocytes across endothelium stimulated for 8 or 24 h with B. burgdorferi, but not IL-1. Compared to the initially added population of T lymphocytes, the population that migrated across untreated endothelium or HUVEC activated with B. burgdorferi or IL-1 contained a significantly smaller percentage of CD45RA+RO- (naïve) cells and a greater proportion of CD45RA+RO+ cells. The migratory population was also enriched for CD8+ T lymphocytes when the endothelium was incubated with either control medium or B. burgdorferi, but not IL-1. B. burgdorferi thus activates endothelium in a manner that promotes the transmigration of T lymphocytes, and IL-10 inhibits this activation. These data further suggest that endothelium plays an active role in promoting the recruitment of specific subpopulations of T lymphocytes.

Extracts of smokeless tobacco induce pro-inflammatory changes in cultured human vascular endothelial cells.

Habitual use of smokeless tobacco leads to accumulation of inflammatory leukocytes at the site of placement, which may contribute to tissue damage. Recruitment of leukocytes is facilitated by the endothelial lining of blood vessels, which can be activated to express adhesion molecules and to produce chemoattractants. The ability of aqueous extracts of chewing tobacco, dry snuff, and moist snuff to stimulate such changes was investigated using cultured human umbilical vein endothelial cells (HUVEC). All three extracts caused HUVEC to express the adhesion molecule E-selectin and to produce the chemokines interleukin-8 and monocyte chemoattractant protein-1. Neutrophils migrated avidly across HUVEC monolayers that had been previously exposed to the extracts, whereas migration across unstimulated monolayers was negligible. The smokeless tobacco extracts contained relatively high concentrations of bacterial lipopolysaccharide (LPS). Although LPS appeared to be the major stimulatory component in extracts of chewing tobacco, it accounted for only part of the activity found in extracts of moist and dry snuffs. These observations suggest that smokeless tobacco may induce inflammatory changes in vivo by activating endothelium in a manner that promotes recruitment of leukocytes.

The relapsing fever spirochaete, Borrelia crocidurae, activates human endothelial cells and promotes the transendothelial migration of neutrophils.

The blood-borne, erythrocyte-aggregating Borrelia crocidurae, the causative agent of African relapsing fever, have been shown to induce severe cellular lesions in mice. In this paper, we present the first report of how the endothelium is stimulated during an African relapsing fever B. crocidurae infection. B. crocidurae co-incubated with cultured human umbilical vein endothelial cells (HUVECs) activated endothelium in such way that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose- and time-dependent fashion, as determined by a whole-cell enzyme-linked immunosorbent assay (ELISA). The upregulation was reduced by treatment that killed the bacteria, suggesting that viability is important for the stimulation of HUVECs by B. crocidurae. Furthermore, conditioned medium from HUVECs stimulated with B. crocidurae contained interleukin (IL)-8, which is a chemotactic agent for neutrophils. Activation of HUVECs by B. crocidurae resulted in migration of subsequently added neutrophils across the endothelial monolayers, and this migration was inhibited by antibodies to IL-8. The activation of endothelium by B. crocidurae may constitute a key pathophysiological mechanism in B. crocidurae-induced vascular damage.

Borrelia burgdorferi and interleukin-1 promote the transendothelial migration of monocytes in vitro by different mechanisms.

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% +/- 3%, 65% +/- 2%, or 25% +/- 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold +/- 2-fold increase in the migration of human CD4(+) T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4(+) T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.

Production of interleukin-8 (IL-8) by cultured endothelial cells in response to Borrelia burgdorferi occurs independently of secreted [corrected] IL-1 and tumor necrosis factor alpha and is required for subsequent transendothelial migration of neutrophils.

Previous studies have shown that Borrelia burgdorferi, the spirochetal agent of Lyme disease, promotes inflammation by stimulating endothelial cells to upregulate adhesion molecules for leukocytes and to produce a soluble agent that is chemotactic for neutrophils. We determined that interleukin-8 (IL-8) was the chemotactic agent for neutrophils present in conditioned media from cultured human umbilical vein endothelial cells stimulated with B. burgdorferi. As few as one spirochete per endothelial cell stimulated production of IL-8 within 8 h of coincubation. When 10 spirochetes per endothelial cell were added, IL-8 was detected after 4 h of coculture. Production of IL-8 continued in a linear fashion for at least 24 h. Neutralizing antibodies against IL-8 reduced migration of neutrophils across spirochete-stimulated endothelial monolayers by 93%. In contrast, pretreatment of neutrophils with antagonists of platelet-activating factor did not inhibit migration. Increases in production of IL-8 and expression of the adhesion molecule E-selectin by endothelial cells in response to B. burgdorferi were not inhibited by IL-1 receptor antagonist or a neutralizing monoclonal antibody directed against tumor necrosis factor alpha, used either alone or in combination. These results suggest that activation of endothelium by B. burgdorferi is not mediated through the autocrine action of secreted IL-1 or tumor necrosis factor alpha. Rather, it appears that B. burgdorferi must stimulate endothelium either by a direct signaling mechanism or by induction of a novel host-derived proinflammatory cytokine.

Outer surface lipoproteins of Borrelia burgdorferi activate vascular endothelium in vitro.

Previously, we reported that activation of vascular endothelium by the Lyme disease pathogen Borrelia burgdorferi results in enhanced expression of endothelial cell adhesion molecules and promotion of the transendothelial migration of neutrophils in vitro. To investigate the role of spirochetal lipoproteins in this process, we assessed the ability of a synthetic lipohexapeptide corresponding to the N terminus of B. burgdorferi outer surface protein A (OspA) to activate human umbilical vein endothelial cells (HUVEC). Using a whole-cell enzyme-linked immunosorbent assay, we demonstrated that OspA lipopeptide activated endothelium in a dose-dependent fashion, as measured by upregulation of E-selectin. Near-maximal stimulation was achieved with 100 micromolar lipopeptide. In addition, the lipopeptide increased expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Similar results were obtained with 25 nM native OspA or lipidated recombinant OspA or OspB. Incubation of HUVEC with nonlipidated OspA peptide, nonlipidated recombinant OspA or OspB, or tripalmitoyl-S-glyceryl-cysteine had little or no effect on expression of these adhesion molecules. A mutant strain of B. burgdorferi that lacked OspA and OspB upregulated expression of E-selectin to the same degree as its wild-type counterpart, indicating that other spirochetal components also possess the ability to activate endothelium. Conditioned medium from HUVEC incubated with OspA lipopeptide or lipidated recombinant OspA induced chemotaxis of neutrophils in Boyden chamber assays, whereas the OspA preparations alone were devoid of chemotactic activity. When HUVEC grown on connective tissue substrates were treated with OspA lipopeptide, subsequently added neutrophils migrated across the endothelial monolayers. These results implicate the outer surface lipoproteins of B. burgdorferi as potential effector molecules in the promotion of a host inflammatory response.

Mononuclear phagocytes egress from an in vitro model of the vascular wall by migrating across endothelium in the basal to apical direction: role of intercellular adhesion molecule 1 and the CD11/CD18 integrins.

Little is known about how mononuclear phagocytes (MP) are cleared from sites of inflammation as inflammatory lesions resolve. In this study, the possibility that MP could be cleared from tissues by migrating across endothelium in the basal to apical direction was investigated. In an in vitro model of a blood vessel wall consisting of human umbilical vein endothelial cells (HUVEC) grown on amniotic tissue, a majority of MP that initially transmigrated into the amnion later exited by migrating back across the endothelium in the basal to apical direction. MP that egressed from these cultures adhered to the apical surface of the endothelium or were found nonadherent in the medium above the endothelium. Egression of MP continued throughout the 4-d period examined, displaying higher than first order kinetics and a t(1/2) of approximately 24 h. These kinetics were decreased by increasing the volume of medium bathing the cultures, suggesting that a soluble factor(s) regulates the rate of egression. In contrast, the kinetics were accelerated by pretreating the endothelium with IL-1. The initial phase of this increased rate of egression was inhibited by antibodies to inter- cellular adhesion molecule 1 (ICAM-1) or CD18 by 100 and 71%, respectively. Immunostaining revealed that ICAM-1 was present on the apical and basal surfaces of umbilical vein endothelium in vitro and in situ. These data demonstrate that MP can traverse endothelium in the basal to apical direction, and lend insight into the mechanisms by which this process occurs.

Borrelia burgdorferi upregulates expression of adhesion molecules on endothelial cells and promotes transendothelial migration of neutrophils in vitro.

The accumulation of leukocytic infiltrates in perivascular tissues is a key step in the pathogenesis of Lyme disease, a chronic inflammatory disorder caused by Borrelia burgdorferi. During an inflammatory response, endothelial cell adhesion molecules mediate the attachment of circulating leukocytes to the blood vessel wall and their subsequent extravasation into perivascular tissues. Using cultured human umbilical vein endothelial cells (HUVEC) in a whole-cell enzyme-linked immunosorbent assay, we demonstrated that B. burgdorferi activated endothelium in a dose- and time-dependent fashion as measured by upregulation of the adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1). As few as one spirochete per endothelial cell stimulated increased expression of these molecules. Expression of E-selectin peaked after spirochetes and HUVEC were coincubated for 4 h and returned to near-basal levels by 24 h. In contrast, expression of VCAM-1 and ICAM-1 peaked at 12 h and remained elevated at 24 h. HUVEC monolayers cultured on acellular amniotic tissue were used to investigate the consequences of endothelial cell activation by spirochetes. After incubation of HUVEC-amnion cultures with B. burgdorferi, subsequently added neutrophils migrated across the endothelial monolayers. This process was mediated by E-selectin and by CD11/CD18 leukocytic integrins. The extent of migration depended on both the number of spirochetes used to stimulate the HUVEC and the length of the coincubation period. These results raise the possibility that B. burgdorferi induces a host inflammatory response and accompanying perivascular damage through activation of vascular endothelium.

A soluble gradient of endogenous monocyte chemoattractant protein-1 promotes the transendothelial migration of monocytes in vitro.

Chemokines secreted by endothelium may promote diapedesis of leukocytes by a gradient-dependent chemotactic mechanism or by stimulating random motility so that leukocytes transmigrate in a gradient-independent manner. Alternatively, chemokines may bind to endothelium and extracellular matrix to stimulate haptotactic migration. We first analyzed the role of the chemokine monocyte chemoattractant protein-1 (MCP-1) in the migration of human monocytes across untreated or IL-1-stimulated HUVEC monolayers cultured on human amnion. Then we further examined whether MCP-1-dependent transmigration occurred through a chemokinetic, chemotactic, or haptotactic mechanism. A neutralizing mAb against MCP-1 inhibited passage of monocytes across untreated or IL-1-stimulated HUVEC by 74 +/- 3% and 45 +/- 4%, respectively. Addition of MCP-1 itself to the apical compartment of unstimulated HUVEC/amnion cultures also reduced the transmigration of monocytes, in this instance by 73 +/- 9%. MCP-1 suppressed diapedesis only when present above the endothelium at a concentration equal to or greater than that endogenously deposited beneath the endothelium, and its inhibitory action could be overcome by addition of more concentrated MCP-1 below the HUVEC cultures. As much as 90% of the MCP-1 secreted into the underlying basement membrane and connective tissue could be washed out of HUVEC/amnion cultures; this procedure decreased transmigration by 69 +/- 4%. These data indicate that MCP-1 promotes transmigration of monocytes, but only when present in a gradient across endothelial monolayers. They further suggest that this gradient is predominantly soluble, rather than haptotactic.

Borrelia burgdorferi binds plasminogen, resulting in enhanced penetration of endothelial monolayers.

Several strains of Borrelia burgdorferi and Borrelia hermsii can bind human Lys-plasminogen specifically. Affinity blots using 125I-labeled plasminogen showed that numerous polypeptides of all the strains and species tested could bind via lysine residues to the plasminogen molecule since binding could be completely inhibited by the lysine analog epsilon-aminocaproic acid. Binding analysis using 125I-labeled plasminogen on live intact organisms showed that the organisms possess two binding sites for plasminogen: a high-affinity site with a Kd of 24 +/- 12 pM and 106 +/- 14 binding sites per spirochete and a low-affinity site with a Kd of 20 +/- 4 nM and 2,683 +/- 36 binding sites per spirochete. Indirect immunofluorescence and confocal microscopy showed a generalized but punctate pattern of plasminogen binding to the spirochete surface. Exogenously provided urokinase-type plasminogen activator converted B. burgdorferi surface-bound plasminogen to enzymatically active plasmin as demonstrated by the breakdown of the chromogenic plasmin substrate S2251. Plasmin-coated organisms showed an enhanced ability to penetrate endothelial cell monolayers grown on connective tissue substrates compared to untreated controls (P < 0.001). This functional assay demonstrated that enzymatically active plasmin on the surface of spirochetes can lead to greater invasion of tissues.

Chemokines and tissue injury.

Accumulation of leukocytes at sites of inflammation is essential for host defense, yet secretory products of the white cells may augment injury by damaging surrounding healthy tissues. Members of the chemokine family of chemotactic cytokines play a fundamental role in this process by attracting and stimulating specific subsets of leukocytes. In vitro studies suggest that chemokines participate in at least three phases of leukocyte recruitment. First, they foster tight adhesion of circulating leukocytes to the vascular endothelium by activating leukocytic integrins. Second, because of their chemoattractant properties, chemokines guide leukocytes through the endothelial junctions and underlying tissue to the inflammatory focus. Finally, chemokines activate effector functions of leukocytes, including production of reactive oxygen intermediates and exocytosis of degradative enzymes. Animal studies in which antibodies are used to neutralize the activity of individual members of the chemokine family confirm that these mediators contribute to the development of both acute and chronic inflammatory conditions. A number of mechanisms may operate in vivo to limit the proinflammatory properties of chemokines. Therapies that target chemokines directly or enhance the body's mechanisms for controlling their activity may prove to be reasonable approaches for treatment of inflammatory diseases.

The adhesion molecules used by monocytes for migration across endothelium include CD11a/CD18, CD11b/CD18, and VLA-4 on monocytes and ICAM-1, VCAM-1, and other ligands on endothelium.

CD11/CD18 and VLA-4 integrins mediate interactions of monocytes with HUVEC cultured on human amniotic tissue. In the present study, the roles of individual CD11/CD18 integrins and endothelial adhesion molecules were examined using blocking mAbs and peptides. After 20 min of incubation, monocyte adhesion to and migration across unstimulated endothelium was dependent primarily on CD11a/CD18. When incubation was extended to 2 h to allow for completion of migration, either CD11a/CD18 or CD11b/CD18 could be used. Similarly, either CD11a/CD18 or CD11b/CD18 could be used by monocytes to bind to and traverse IL-1 beta-stimulated endothelium. Although both CD11a/CD18 and CD11b/CD18 are known to bind to ICAM-1, results of Ab-mixing experiments suggest that alternative ligands on HUVEC for CD11/CD18 integrins also may be used during transendothelial migration of monocytes. Our previous studies indicate that VLA-4 on monocytes interacts primarily with VCAM-1 on unstimulated endothelium. In contrast, migration of monocytes across IL-1 beta-stimulated endothelium was less dependent on VCAM-1. mAbs directed against binding sites for VLA-4 in domain 1 and domain 4 of VCAM-1 did not, by themselves, inhibit interactions of monocytes with stimulated HUVEC. VLA-4-dependent migration across IL-1 beta-stimulated endothelium was markedly inhibited only when mAbs to VCAM-1 were added in combination with peptides of fibronectin. Therefore, VLA-4 can interact with either VCAM-1 or alternative ligands on IL-1 beta-stimulated HUVEC-amnion cultures to mediate transendothelial migration of monocytes.

Monocytes use either CD11/CD18 or VLA-4 to migrate across human endothelium in vitro.

Monocytes traverse the endothelial lining of blood vessels and migrate into both normal and inflamed tissues. An in vitro model of a vascular wall, consisting of HUVEC cultured on acellular human amniotic tissue, was employed to examine the roles of several adhesion molecules in diapedesis of monocytes. Approximately half of the monocytes added to this system traversed the endothelium in a time-dependent fashion, completing their migration within 2 h. Pretreatment of HUVEC with IL-1 beta for 4 h increased the rate of adhesion of monocytes, but did not alter the number that ultimately migrated. A mAb to CD18, ts1/18, greatly inhibited adhesion and migration of monocytes when the monocytes were incubated with unstimulated HUVEC monolayers for 20 min. Much less inhibition was observed when the incubation period was increased to 2 h or when HUVEC were pretreated with IL-1 beta. A mAb to VLA-4, HP1/2, had little or no inhibitory effect in all cases. The combination of ts1/18 and HP1/2 greatly inhibited (up to 98%) adhesion and migration of monocytes across both unstimulated and IL-1 beta-stimulated monolayers. Additional inhibition experiments indicated that VLA-4 interacted with unstimulated endothelium by binding to VCAM-1 and, to a much lesser extent, fibronectin. These results suggest that monocytes are capable of interacting with endothelium during diapedesis via either CD11/CD18- or VLA-4-dependent pathways.

Type IV collagenase/gelatinase (MMP-2) is not increased in plasma of patients with cancer.

We have developed a sensitive and specific sandwich-type enzyme-linked immunosorbent assay to detect M(r) 72,000 type IV collagenase [matrix metalloproteinase 2 (MMP-2)] in human plasma. As a result of the linkage between MMP-2 production by cancer cells and the metastatic phenotype, we undertook this study to compare plasma MMP-2 levels in healthy individuals, patients with various types of cancer, and hospitalized patients with chronic diseases other than cancer. The results demonstrate that MMP-2 levels are not increased in cancer patients regardless of the extent of disseminated malignancy. In an effort to explain this data, we compared MMP-2 secretion by human umbilical vein endothelial cells and lung cancer cells passaged as cell lines. Endothelial cells secreted higher levels of MMP-2 than did lung cancer cells propagated in vitro. We propose that blood vessel lining cells make a sizable contribution to plasma levels of MMP-2 and may thereby obfuscate the detection of increased levels of MMP-2 originating from extravascular sources such as solid tumors.

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Monoclonal antibodies to leukocyte integrins CD11a/CD18 and CD11b/CD18 or intercellular adhesion molecule-1 inhibit chemoattractant-stimulated neutrophil transendothelial migration in vitro.

Intercellular adhesion molecule-1 (ICAM-1) is present on the endothelium and binds to one or more members of the CD11/CD18 family of leukocyte surface integrins. To assess the role of these molecules in mediating chemotaxis of neutrophils across the endothelium, an in vitro model consisting of monolayers of human umbilical vein endothelial cells (HUVEC) grown on amniotic connective tissue was used. Neutrophils placed on the apical sides of these cultures migrated across the endothelium in response to chemoattractants added basally. Monoclonal antibodies (MoAbs) to CD11a, CD11b, and CD18 on the neutrophils inhibited this migration by 52% +/- 11%, 29% +/- 19%, and 90% +/- 7%, respectively. An MoAb to ICAM-1 inhibited transendothelial chemotaxis of the leukocytes by 55% +/- 16%. Inhibition was mediated by binding of the MoAb to ICAM-1 on the HUVEC, rather than by any direct effect of the antibody on the neutrophils. When used in combination, MoAbs to CD11a and to CD11b inhibited migration in a nearly additive fashion. A similar additive effect was observed when MoAbs to CD11b and to ICAM-1 were used together. In contrast, MoAbs to CD11a and to ICAM-1 produced no more inhibition when used in combination than when added singly. These results show that ICAM-1, CD11a/CD18, and CD11b/CD18 all participate in controlling migration of neutrophils across endothelial monolayers in response to chemotactic agents.

Macrovascular and microvascular endothelium during long-term hypoxia: alterations in cell growth, monolayer permeability, and cell surface coagulant properties.

In bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose-dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a approximately 3-fold increase in the total number of high-affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia-mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin-based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro- and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions.

The effect of hypoxia on capillary endothelial cell function: modulation of barrier and coagulant function.

As the cells forming the luminal vascular surface, endothelium is exposed to alterations in the vascular microenvironment, such as hypoxaemia. In this report we demonstrate that hypoxia, with pO2 as low as 12-14 mmHg, was not toxic to endothelium, but reversibly modulated central cellular functions essential for maintenance of homeostasis: permeability of monolayers to solutes increased in a dose-dependent manner, and cell surface coagulant properties were shifted to promote activation of coagulation. The anticoagulant cofactor thrombomodulin was suppressed and an apparently novel activator of factor X, distinct from the classical extrinsic and intrinsic systems, was induced. The hypoxia-induced factor X activator was cell surface-associated, had properties of a cysteine protease, had Mr corresponding to approximately 100 kDa, based on sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE), and isoelectric point (pI) approximately 5.0. These findings indicate that hypoxia dynamically modulates endothelial function providing insights into the contribution of microvascular endothelial dysfunction in the pathogenesis of vascular lesions.

Interaction between Borrelia burgdorferi and endothelium in vitro.

During the pathogenesis of Lyme disease, Borrelia burgdorferi spreads hematogenously from the site of a tick bite to several tissues throughout the body. The specific mechanism of spirochete emigration is presently unknown. Using cultured human umbilical vein endothelial cells, we found that Borrelia burgdorferi bound to the endothelial cells and to the subendothelial matrix. Low passage isolates adhered 22-30-fold greater than a strain maintained in culture continuously. Spirochete binding to subendothelial matrix was inhibited 48-63% by pretreatment of the matrix with anti-fibronectin antiserum. Spirochete migration across endothelial monolayers cultured on amniotic membrane was increased when the monolayers were damaged by chemical or physical means. Electron microscopic examination of spirochete-endothelial interactions demonstrated the presence of spirochetes in the intercellular junctions between endothelial cells as well as beneath the monolayers. Scanning electron microscopy identified a mechanism of transendothelial migration whereby spirochetes pass between cells into the amniotic membrane at areas where subendothelium is exposed.