Michael P. Bevilacqua

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.

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.

Endothelial-Dependent Mechanisms of Leukocyte Adhesion

Mononuclear phagocytes interact with vascular endothelium in a variety of pathological and physiological settings. Reversible adhesive interactions with blood-vessel walls lead to mar-gination of a substantial proportion of the circulating monocyte pool (1). Constitutive emigration into uninflamed tissues occurs through mechanisms that remain obscure. Increasing evidence suggests that local activation of vascular endothelium plays an important role in initial adhesion and subsequent emigration of monocytes at inflammatory sites, and that similar mechanisms promote monocyte accumulation in atherosclerotic plaques (2).

Identification and Characterization of Endothelial-Leukocyte Adhesion Molecule 1

Increasing evidence suggests that the vascular endothelium actively contributes to a variety of proinflammatory processes, including leukocyte extravasation. In particular, recent in vitro studies have supported the hypothesis that inflammatory/immune mediators can act directly on vascular endothelial cells to increase the adhesion of blood leukocytes (1–14). In addition, in vivo studies have suggested that local activation of the vascular endothelium may contribute to accumulation of leukocytes at sites of inflammation in various pathophysiological contexts (15–18). Our laboratory has used standardized in vitro monolayer adhesion assays to study factors that alter endothelial-leukocyte adhesion and to investigate the mechanisms of these interactions. Initially, human monocyte derived IL-1 was found to act on cultured human endothelial cells (HEC) in a time- and protein-synthesis dependent fashion to increase the adhesion of isolated human blood polymorphonuclear leukocytes (PMN), monocytes and the related cell lines HL-60 and U937 (1). Subsequently, other cytokines including recombinant IL-1-α, IL-1-β, tumor necrosis factor (TNF) and lymphotoxin, as well as bacterial endotoxin (lipopo-lysaccharide, LPS) (2,3) were shown to induce endothelial-dependent mechanisms of leukocyte adhesion.