Receptors for cold-insoluble globulin (plasma fibronectin) on human monocytes.

This investigation focused on the role played by cold-insoluble globulin (CIg, plasma fibronectin) in monocyte function. Surface-bound CIg mediated a concentration-dependent of human blood monocytes to gelatin-coated surfaces. CIg also mediated the binding of gelatin-coated particles such as latex beads or tanned erythrocytes to surface-bound human monocytes. However, CIg did not mediate particle ingestion. Subfractionated CIg that was highly enriched in monomeric forms (zone II CIg, mol wt 190,000-235,000) was less effective than were fractions enriched in dimeric forms (zone I CIg, mol wt 450,000) in promoting monocyte attachment. Binding of CIg to a gelatin or plastic surface occurred in the absence of divalent cations, but monocyte attachment to CIg-coated surfaces required divalent cations, Mg++ being much more effective than Ca++. Cation-dependent cell attachment was reversible in that bound cells could be released by treatment with EDTA. Serum-mediated binding of monocytes to gelatin-coated plastic dishes was a result of its content of CIg because the binding activity was abolished by removal of CIg from serum, and could be restored by readdition of purified CIg. Treatment of monocytes with trypsin abolished subsequent cell attachment to CIg-gelatin surfaces or particles. Expression of certain other known monocyte membrane receptors (Fc and C3b) was markedly enhanced as a result of CIg-monocyte interaction. These several observations indicate that monocytes bear membrane receptors (termed receptor cold-insoluble globulin) for surface-bound CIg.

Inducible cell adhesion molecule 110 (INCAM-110) is an endothelial receptor for lymphocytes. A CD11/CD18-independent adhesion mechanism.

Inducible cell adhesion molecule 110 (INCAM-110) is a 110-kD glycoprotein expressed on cytokine-activated human vascular endothelial cells. mAb blocking studies indicate that INCAM-110 and intercellular adhesion molecule 1 (ICAM-1) independently support the adhesion of lymphocytes to activated human umbilical vein endothelial cell monolayers. Anti-CD11a/CD18 antibodies with anti-INCAM-110 mAb E1/6 produce greater inhibition of lymphocyte adhesion than either reagent alone, suggesting that INCAM-110 and LFA-1 are not an obligate receptor-ligand pair. Blood monocytes, but not polymorphonuclear leukocytes, also appear to bind endothelial INCAM-110. Endothelial expression of INCAM-110 is upregulated at sites of inflammation, suggesting a role in the recruitment of mononuclear leukocytes.

Induction and detection of a human endothelial activation antigen in vivo.

We used a murine mAb, H4/18, raised by immunization with IL-1-treated human umbilical vein endothelial cell cultures, to localize an endothelial activation antigen in induced human delayed hypersensitivity reactions (DHR) and in pathological tissues. We used streptococcus varidase to elicit DHR in human skin and we examined sequential skin biopsies with the immunoperoxidase technique. There was no staining for H4/18 binding antigen in normal endothelium of skin and other tissues; strong positive staining, localized to vascular endothelium, was seen at 16 and 23 h but disappeared by 6 d, when the DHR had faded. H4/18 binding antigen, also confined to endothelium, was detected in lymph nodes, skin, and other tissues exhibiting immune/inflammatory reactions. The studies indicate that H4/18 is a useful marker for activated endothelium in vivo and they support the relevance of in vitro studies on inducible endothelial cell functions.

Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells.

Human monocyte-derived interleukin 1 (IL-1) was found to be a potent inducer of procoagulant activity in cultured human vascular endothelium. IL-1-induced human umbilical vein endothelial cell procoagulant activity (HEC-PCA) was transiently expressed, manifest in intact cell monolayers, and required protein synthesis. Data obtained with coagulation factor-deficient plasma and a goat anti-human apoprotein III antiserum suggested that most, if not all, of IL-1-induced endothelial cell procoagulant activity is tissue factor-like. IL-1 induction of HEC-PCA may be important in the pathogenesis of intravascular coagulation in a variety of immunological and inflammatory conditions.

An inducible endothelial cell surface glycoprotein mediates melanoma adhesion.

Hematogenous metastasis requires the arrest and extravasation of blood-borne tumor cells, possibly involving direct adhesive interactions with vascular endothelium. Cytokine activation of cultured human endothelium increases adhesion of melanoma and carcinoma cell lines. An inducible 110-kD endothelial cell surface glycoprotein, designated INCAM-110, appears to mediate adhesion of melanoma cells. In addition, an inducible endothelial receptor for neutrophils, ELAM-1, supports the adhesion of a human colon carcinoma cell line. Thus, activation of vascular endothelium in vivo that results in increased expression of INCAM-110 and ELAM-1 may promote tumor cell adhesion and affect the incidence and distribution of metastases.

Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins.

Focal adhesion of leukocytes to the blood vessel lining is a key step in inflammation and certain vascular disease processes. Endothelial leukocyte adhesion molecule-1 (ELAM-1), a cell surface glycoprotein expressed by cytokine-activated endothelium, mediates the adhesion of blood neutrophils. A full-length complementary DNA (cDNA) for ELAM-1 has now been isolated by transient expression in COS cells. Cells transfected with the ELAM-1 clone express a surface structure recognized by two ELAM-1 specific monoclonal antibodies (H4/18 and H18/7) and support the adhesion of isolated human neutrophils and the promyelocytic cell line HL-60. Expression of ELAM-1 transcripts in cultured human endothelial cells is induced by cytokines, reaching a maximum at 2 to 4 hours and decaying by 24 hours; cell surface expression of ELAM-1 protein parallels that of the mRNA. The primary sequence of ELAM-1 predicts an amino-terminal lectin-like domain, an EGF domain, and six tandem repetitive motifs (about 60 amino acids each) related to those found in complement regulatory proteins. A similar domain structure is also found in the MEL-14 lymphocyte cell surface homing receptor, and in granule-membrane protein 140, a membrane glycoprotein of platelet and endothelial secretory granules that can be rapidly mobilized (less than 5 minutes) to the cell surface by thrombin and other stimuli. Thus, ELAM-1 may be a member of a nascent gene family of cell surface molecules involved in the regulation of inflammatory and immunological events at the interface of vessel wall and blood.

Adhesion of human B cells to germinal centers in vitro involves VLA-4 and INCAM-110.

Human B lymphocytes localize and differentiate within the microenvironment of lymphoid germinal centers. A frozen section binding assay was developed for the identification of those molecules involved in the adhesive interactions between B cells and lymphoid follicles. Activated human B cells and B cell lines were found to selectively adhere to germinal centers. The VLA-4 molecule on the lymphocyte and the adhesion molecule INCAM-110, expressed on follicular dendritic cells, supported this interaction. This cellular interaction model can be used for the study of how B cells differentiate.

Regulation of the fibrinolytic system of cultured human vascular endothelium by interleukin 1.

We examined the effects of human interleukin 1 (IL-1) on the production of fibrinolytic components by cultured human vascular endothelium. Conditioned media collected from IL-1-treated (5 U/ml, 24 h) monolayers exhibited decreased tissue-type plasminogen activator (tPA) activity and increased plasminogen activator inhibitor (PAI) activity, as assessed by fibrin and reverse fibrin-autography. Quantitative immunological assays revealed a 35% decrease in tPA antigen and a 360% increase in active PAI antigen, after incubation for 24 h with 0.6 U/ml IL-1. Maximal effects (approximately 50% decrease in tPA antigen; 400-800% increase in active PAI antigen) were observed with 2.5-5 U/ml IL-1. Changes in tPA and PAI reached a maximum at approximately 24 h and persisted for greater than 48 h. IL-1 induction of endothelial procoagulant activity was more rapid and transient, peaking by 6 h and subsiding by 24 h. Natural monocyte-derived IL-1 and two species of recombinant IL-1 had comparable effects. Heat and polymyxin-B treatments differentiated IL-1 actions from those of endotoxin, which promoted similar endothelial alterations. IL-1 effects on endothelial procoagulant and fibrinolytic activities may contribute to the generation and maintenance of fibrin in pathophysiological settings in vivo.

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.

Higher-affinity oligosaccharide ligands for E-selectin.

A series of synthetic oligosaccharides based on sialyl Lewis x (sLex; Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc) and sialyl Lewis a (sLea; Neu5Ac alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc) was used to study the binding interactions of selectins. E-selectin-immunoglobulin fusion protein (E-selectin-Ig) bound to immobilized bovine serum albumin (BSA)-neoglycoproteins containing sLex or sLea in a Ca(2+)-dependent manner. Solution-phase sLex tetrasaccharide blocked this interaction by 50% at a concentration of 750 +/- 20 microM (IC50). sLea was more effective (IC50 = 220 +/- 20 microM), while nonsialylated, nonfucosylated derivatives showed little or no activity at concentrations up to 1 mM. Attachment of an 8-methoxycarbonyloctyl aglycone in a beta linkage to the anomeric carbon of the GlcNAc of sLex or sLea increased their blocking activity nearly twofold. Finally, replacement of the 2-N-acetyl substituent of the GlcNAc by an azido or amino group resulted in substantial increases in activity, with the most potent inhibitor being amino substituted sLea, which was 36-fold more active (IC50 = 21 +/- 3 microM) than the reducing tetrasaccharide sLex. In contrast to results obtained with E-selectin-Ig, P-selectin-Ig binding to immobilized BSA-sLea was blocked modestly by most oligosaccharides at 1 mM, with no substantial differences among them. IC50 values of soluble oligosaccharides determined in competitive binding studies accurately predicted blocking of leukocyte adhesion to recombinant E-selectin-Ig and to cytokine-activated endothelium.

Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines.

Increased leukocyte adhesion to the endothelial lining of blood vessels is an essential event in inflammation and the pathogenesis of certain vascular diseases. We have studied the effect of interleukin 1 (IL-1), an inflammatory/immune mediator, on endothelial-leukocyte adhesion using quantitative in vitro assays. Selective pretreatment of cultured human umbilical vein endothelial monolayers with IL-1 (5 U/ml, 4 h) resulted in an 18.3 +/- 2.6-fold increase in human peripheral blood polymorphonuclear leukocyte (PMN) adhesion (mean +/- SEM, n = 16) and a 2.6 +/- 0.3-fold increase in monocyte adhesion (n = 7) over basal levels. IL-1-treated endothelial monolayers also supported increased adhesion of the promyelocytic cell line HL-60 and the monocytelike cell line U937 (33.0 +/- 6.0-fold, n = 6 and 4.9 +/- 0.5-fold, n = 15, respectively). In contrast, selective IL-1 pretreatment of leukocytes, or the addition of IL-1 during the adhesion assay, did not alter endothelial-leukocyte adhesion. Conditioned medium from IL-1-treated endothelial cultures also did not promote leukocyte adhesion to untreated monolayers. IL-1 induction of endothelial adhesivity was concentration dependent (maximum, 10 U/ml), time dependent (peak, 4-6 h), and reversible, was blocked by cycloheximide (10 micrograms/ml) or actinomycin D (5 micrograms/ml) but not by acetylsalicylic acid (100 microM), and occurred without detectable endothelial cell damage. IL-1 treatment of SV40-transformed human endothelial cells and dermal fibroblasts did not increase their adhesivity for leukocytes. These data suggest that IL-1 can act selectively on human vascular endothelium to increase its adhesivity for circulating blood leukocytes, and thus to localize leukocyte-vessel wall interactions at sites of inflammation in vivo.

Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin-type glycoproteins.

E-, P-, and L-selectin support the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate ligands, which often contain sialylated, fucosylated lactosamines such as sialyl Lewis x [sLex; Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-]. E-selectin expressed by activated endothelium has been shown to support the adhesion of sLex-bearing colon cancer cells. In the present study, we examine the interactions of multiple colon cancer cell lines with all three selectins. Three colon cancer cell lines (LS 180, T84, and COLO 205) bound to recombinant purified E-, P-, and L-selectin. The colon cancer line COLO 320 bound to P- and L-selectin but not E-selectin; conversely, HT-29 cells bound E-selectin but not P- and L-selectin. Caco-2 showed little or no interaction with any of the three selectins. Treatment of the cells with O-sialoglycoprotease from Pasteurella haemolytica, an enzyme that selectively cleaves mucin-type O-linked glycoproteins, reduced binding to purified P- and L-selectin in all cases. In addition, recombinant soluble P- and L-selectin bound to affinity-purified mucins from all adherent tumor cell lines. Of the four tumor cell lines that interacted with E-selectin, O-glycoprotease treatment substantially diminished adhesion of LS 180 and T84, had little effect on COLO 205, and failed to inhibit the binding of HT-29. As predicted by these data, E-selectin showed substantial binding only to mucins purified from LS 180 and T84. These findings suggest that L- and P-selectin interact primarily with mucin-type ligands on colon cancers, whereas E-selectin can recognize both mucin and nonmucin ligands. Binding of the colon cancer lines to purified selectins correlates with their adhesion to activated endothelial cells (E-selectin-dependent), platelets (P-selectin-dependent), and neutrophils (L-selectin-dependent). These differential tumor cell-selectin interactions may influence metastatic spread and may also contribute to the observed variability in host response to tumor progression.

Endothelial-leukocyte adhesion molecules in inflammation and metastasis.

Vascular endothelium is an active tissue. In response to various mediators, it expresses specific cell surface glycoproteins that support the binding and extravasation of blood leukocytes. These events are essential for host defense against pathogens and for repair of tissue damage. Unfortunately, it appears that a number of blood borne tumor cells may be able to use the same adhesive structures to facilitate their metastasis. The development of agents designed to block the adhesion of leukocytes (or tumor cells) to the vessel wall may prove valuable in the treatment of a variety of human disease processes, including adult respiratory distress syndrome (ARDS), ischemic reperfusion injury, autoimmunity, and metastatic spread of cancer.

Endothelial-leukocyte adhesion molecules.

One decade ago, vascular endothelium was commonly considered a "non-stick" lining of blood vessels that functioned only to prevent blood coagulation and to separate the vascular space from tissues. By comparison to many other cell types, endothelial cells were thought to be less active, less complex, and less interesting. Since that time, research concerning the endothelium has expanded dramatically and produced a new image of the vascular lining as an active participant in a wide variety of pathophysiological processes, including inflammation and immunity. Nowhere has the excitement been more intense than in the study of the molecular mechanisms of leukocyte adhesion to endothelium. Recent efforts resulted in the identification, characterization, and cloning of multiple endothelial cell-surface glycoproteins that support adhesion through an interaction with specific ligands (or counter-receptors) on leukocytes. The selectins, two of which are found on endothelium and one on leukocytes, support adhesion through the recognition of carbohydrates. Endothelial members of the immunoglobulin superfamily including ICAM-1 and VCAM-1/INCAM-110 bind to leukocyte cell-surface integrins. In various combinations, these and other molecules support leukocyte adhesion to the vessel wall and extravasation, key steps in our response to infection and tissue injury.

Inducible endothelial functions in inflammation and coagulation.

The studies discussed have established that inflammatory or immune cytokines, such as IL-1, TNF, and LT, as well as bacterial endotoxin, can act directly on vascular endothelial cells to modulate two important functional properties. The first of these, the inducible expression of E-LAMs, provides a mechanism for the local regulation of leukocyte-vessel wall interactions. This endothelial-dependent mechanism may be relevant to a broad spectrum of pathologic processes, including inflammation, delayed hypersensitivity reactions, and atherogenesis. The second, modulation of endothelial tissue factor PCA and fibrinolytic components, has important implications for the local balance of prothrombotic and antithrombotic influences at the blood-vessel wall interface. Thus, under the influence of inflammatory stimuli, vascular endothelial cells may actively contribute to the development and maintenance of intravascular or perivascular fibrin. Although the endothelial effector mechanisms of these functional alterations are distinct, their induction by similar stimuli points to important interrelationships of leukocyte-vessel wall adhesion and thrombosis. Further understanding of the regulation of endothelial expression of E-LAMs and coagulant properties should contribute to our understanding of vascular disease.

Tumor cell-endothelial interactions. Increased adhesion of human melanoma cells to activated vascular endothelium.

The authors examined the adhesion of seven human melanoma cell lines to cultured human umbilical vein endothelial cells (HEC) that were activated by cytokines or bacterial endotoxin. The adhesion of Hs 294T and MEL-24 cells was markedly increased (approximately 2 to 12-fold) after pretreatment of HEC monolayers for 6 hours with tumor necrosis factor, interleukin-1, or endotoxin. Smaller increases were noted with the cell lines RPMI 7951, HT 144, Malme-3M, MEL-2, and no significant increase was observed with MEL-5. Cytokine and endotoxin effects on melanoma-HEC adhesion were concentration- and time-dependent, with onset by 2 hours, peak at 6-8 hours and maintenance through 48 hours. Cytokine induction of increased HEC adhesiveness for melanoma cells was blocked by actinomycin-D or cycloheximide, suggesting the requirement for RNA and protein synthesis. Interaction of melanoma cells with subendothelial matrix did not appear to play a primary role because: 1) phase contrast and electron microscopy revealed direct contact between tumor cells and endothelial cells in standardized monolayer adhesion assays; 2) increased adhesion (rosette formation) of tumor cells to activated HEC was also observed after nonenzymatic resuspension of HEC, and 3) the matrix peptide GRGDSP partially blocked (approximately 45%) Hs 294T cell adhesion to subendothelial matrix, but had little or no effect on adhesion to activated HEC monolayers. Taken together, these data suggest that inducible HEC surface changes may mediate the adhesion of certain melanoma cells, thereby exerting an active influence over the metastatic process.

Cytokine-induced beta-galactoside alpha-2,6-sialyltransferase in human endothelial cells mediates alpha 2,6-sialylation of adhesion molecules and CD22 ligands.

Sialic acids decorating blood and cell surface proteins can play important roles in various biological processes. The inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1, as well as bacterial lipopolysaccharide, can activate vascular endothelium, increasing expression of several surface glycoproteins. Here we show that treatment of cultured human endothelial cells (HEC) with TNF-alpha, interleukin-1, or lipopolysaccharide causes increased expression of the enzyme beta-galactoside alpha-2,6-sialytransferase (alpha 2-6STN). TNF-alpha was most effective, inducing a 3.5-fold enhancement of cell-associated sialytransferase activity by 72 h. In addition, activated HEC secreted a large portion of the induced sialyltransferase activity into the medium. Analysis of labeled HEC showed both a relative and an absolute increase of alpha 2,6-linked sialic acid on N-linked oligosaccharides after TNF-alpha stimulation. This coincided with increased expression of endothelial glycoproteins bearing N-linked glycans with alpha 2,6-linked sialic acid detected by the lectin Sambucus nigra agglutinin. The cytokine-inducible endothelial cell adhesion molecules E-selectin, ICAM-1, and VCAM-1 are among these glycoprotein substrates for alpha 2-6STN. These changes also correlated with a substantial increase in binding sites for CD22 beta, a mammalian lectin known to recognize oligosaccharides carrying multiple copies of alpha 2,6-linked sialic acid. Northern analysis revealed increased levels of mRNA encoding alpha 2-6STN. Thus, activation of endothelial cells during inflammatory and immunological processes may induce alpha 2-6STN, which can participate in sialylation of other activation-dependent molecules.