Neutrophil-endothelial cell interactions on endothelial monolayers grown on micropore filters.

Taylor, Robert F.; Price, Thomas H.; Schwartz, Stephen M.; Dale, David C.

doi:10.1172/jci110071

Cited by 76 publications

(20 citation statements)

References 19 publications

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“…For transmigration studies, we used a similar system as that described by Taylor et al 40 Human ECs (2ϫ10 5 /cm 2 ) were seeded onto polycarbonate membranes (5.0-m pore size) of transwell inserts closely fitted into 24-well plates (Nunc) and allowed to grow for a further 48 hours. Then, medium containing E-LDL, ox-LDL, or LDL at 50 g/mL was introduced beneath the filter supporting the EC monolayer for 4 hours.…”

Section: Transmigration Assaysmentioning

confidence: 99%

Enzymatically Modified, Nonoxidized LDL Induces Selective Adhesion and Transmigration of Monocytes and T-Lymphocytes Through Human Endothelial Cell Monolayers

Klouche

May

Hemmes

et al. 1999

ATVB

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Abstract-Circulating monocytes and T lymphocytes extravasate through the endothelium at sites of developing atheromatous lesions, where they tend to accumulate and mediate the progression of the disease. We have previously demonstrated the presence of an enzymatically degraded, nonoxidized form of LDL (E-LDL) in early human fatty streaks, which possesses major biological properties of an atherogenic lipoprotein. The effects of E-LDL on human endothelial cells have now been studied with respect to adhesion and transmigration of monocytes and T lymphocytes. E-LDL induced a rapid and dose-dependent selective adhesion of monocytes and T lymphocytes to endothelial cell monolayers within 30 minutes of incubation. Maximal increases in the number of adherent monocytes (8-fold) and of adherent T lymphocytes (4-fold) were observed after treatment with 50 g/mL E-LDL. E-LDL was more active than oxidized LDL (ox-LDL), whereas native LDL produced only minor adhesive effects. Both E-LDL and ox-LDL enhanced transmigration of monocytes and of T lymphocytes through endothelial monolayers. Again, E-LDL was more potent than ox-LDL, inducing transmigration to a similar extent as N-formyl-Met-Leu-Phe. In endothelial cells, E-LDL stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cells adhesion molecule-1 (PECAM-1), P-selectin, and E-selectin with distinct kinetics. Analyses with blocking antibodies indicated that ICAM-1 and P-selectin together mediated approximately 70% of cell adhesion, whereas blocking of PECAM-1 had no effect on adhesion but reduced transmigration to less than 50% of controls. E-LDL also upregulated expression of ICAM-1 in human aortic smooth muscle cells, and this correlated with increased adhesion of T lymphocytes. E-LDL is thus able to promote the selective adhesion of monocytes and T lymphocytes to the endothelium, stimulate transmigration of these cells, and foster their retention in the vessel wall by increasing their adherence to smooth muscle cells. These findings underline the potential significance of E-LDL in the pathogenesis of atherosclerosis.

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Section: Transmigration Assaysmentioning

confidence: 99%

Enzymatically Modified, Nonoxidized LDL Induces Selective Adhesion and Transmigration of Monocytes and T-Lymphocytes Through Human Endothelial Cell Monolayers

Klouche

May

Hemmes

et al. 1999

ATVB

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“…Polycarbonate filters (13 mm in diameter, 5-ftm pore size, Nucleopore Corp.) were gelatin-impregnated by the technique described by Postlethwaite (1976) and modified by Taylor et al (1981).…”

Section: B Preparation Of Filtersmentioning

confidence: 99%

Role of endothelial cell cytoskeleton in control of endothelial permeability.

Shasby¹,

Shasby²,

Sullivan³

et al. 1982

Circulation Research

295

150

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SUMMARY. Increased permeability of the pulmonary microvasculature is felt to cause acute noncardiogenic lung edema, and histological studies of edematous lungs show gaps between apparently healthy endothelial cells. To determine whether alterations in endothelial cell cytoskeletons would alter endothelial permeability, we exposed monolayers of pulmonary artery endothelial cells grown on micropore filters to cytochalasin B or D. Cytochalasin exposed monolayers demonstrated a 2-to 3-fold increase in endothelial permeability that was readily reversible by washing the monolayers free of cytochalasins. Parallel phase contrast and fluorescence microscopy demonstrated retraction of cell cytoplasm and disruption of bundles of microfilaments in cytochalasin exposed cells. These changes also were readily reversed after washing the cells free of cytochalasins. To test the relevance of these findings to an in situ microvasculature, we added cytochalasin B to the perfusate of isolated rabbit lungs and observed that cytochalasin B caused a high permeability lung edema. These studies suggest that endothelial cell cytoskeletons may be important determinants of endothelial permeability. (Circ Res 51: 657-661, 1982) ACUTE noncardiogenic lung edema is believed to result from increased permeability of the pulmonary microvasculature (Staub, 1978). Although many animal and ex vivo models of noncardiogenic lung edema have been studied, the mechanism of increased endothelial permeability remains unknown. Several studies of the systemic circulation have suggested that increased endothelial permeability induced by histamine is associated with endothelial cells pulling apart and gaps forming between cells (Majno and Palade, 1961;Majno et al., 1967). Hurley (1982) has provided evidence that similar gaps in the continuity of the endothelium may be important in high permeability lung edema.The permeability of epithelial surfaces has been studied more thoroughly than that of endothelial surfaces, and several studies have suggested a role of microfilaments of the cytoskeleton in regulating epithelial permeability (Meza et al., 1980;Bentzel et al., 1980;Duffey et al., 1981). We hypothesized that the cytoskeleton might also contribute to regulation of endothelial permeability. To test this, we added the microfilament disrupting agents, cytochalasin B and D, to monolayers of endothelial cells grown on micropore filters, and we observed that cytochalasins caused a readily reversible increase in endothelial permeability. The increased permeability was associated with disruption of the microfilament apparatus and formation of gaps between adjacent endothelial cells, whereas the recovery was associated initially with the localization of actin to the junction of adjacent cells and later reorganization into the normal pattern of microfilaments for endothelial cells. To support the relevance of these in vitro findings to an intact microvasculature, we added cytochalasin B to the perfusate of isolated lungs, and we found that cytochalasin B increased ...

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“…Chemotaxis assays were performed in modified blind well chambers as previously described (14). Monocyte migration was measured by using a modification of the radiolabeled chemotaxis assay of Gallin et aL (15).…”

Section: Methodsmentioning

confidence: 99%

Human arterial wall cells secrete factors that are chemotactic for monocytes.

Mazzone¹,

Jensen²,

Chait³

1983

Proc. Natl. Acad. Sci. U.S.A.

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Macrophages and arterial smooth muscle cells comprise the cellular components of the atherosclerotic plaque. The vessel wall accumulation of macrophages occurs by a process of increased circulating monocyte migration into the vessel wall.In these studies it is demonstrated that human macrophages and arterial smooth muscle cells in culture secrete potent chemotactic factors for freshly isolated human monocytes. In contrast, human fibroblast-conditioned medium has no chemotactic activity. The effect of macrophage-conditioned medium is a function of macrophage differentiation and can be potentiated by macrophage activation. These results suggest that secretory products of human macrophages and arterial smooth muscle cells may be important stimuli for increased monocyte migration into the vessel wall in vivo.Atherosclerotic plaques are characterized by vascular wall collections of lipid-laden cells referred to as "foam cells." There is now substantial evidence that these cells originate from arterial smooth muscle cells and from monocyte-derived macrophages (1-5). Intimal accumulation of smooth muscle cells occurs as a result of cell division (5, 6), which may be induced by locally active mitogens such as those released by platelets (7), macrophages (8), or endothelial cells (9). Intimal accumulation of macrophages probably results from the migration of blood-borne monocytes into the developing plaque (10), but little is known of the factors that might induce the migration of monocytes into the vascular wall. We now report that conditioned media from cultured human macrophages and arterial smooth muscle cells are chemotactic for freshly isolated human monocytes. These results indicate that cells that are present in the nascent plaque can secrete factors that, if present in vivo, could mobilize monocytes into the vascular wall, thereby promoting growth of the lesion. MATERIALS AND METHODSCell Culture. Human monocytes were prepared from human blood by counterflow centrifugation as described (11 microscopy (12). Cultured human skin fibroblasts were grown from skin biopsies obtained from the anterior thighs of normal volunteers as described (13). Arterial smooth muscle cells and fibroblasts were grown in modified Dulbecco-Vogt medium containing 10% pooled human serum. Cells from the third or greater subculture were allowed to reach near confluence in 35-mm dishes before collection of conditioned media.Prior to the collection of conditioned medium, arterial smooth muscle cells, macrophages, or fibroblasts were washed three times with RPMI 1640 medium and were then incubated in medium alone or in medium containing 0.2% bovine serum albumin. After a 24-hr incubation, media were collected, spun, and frozen at -200C until used in the chemotaxis assay. Unless otherwise specified, macrophage-conditioned medium was collected from day 5-6 cultures and arterial smooth muscle celland fibroblast-conditioned media were collected when cells reached near confluence.For experiments testing the effect of macrophage stimulation...

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Neutrophil-endothelial cell interactions on endothelial monolayers grown on micropore filters.

Abstract: A B S T R A C T We have developed

Cited by 76 publications

References 19 publications

Enzymatically Modified, Nonoxidized LDL Induces Selective Adhesion and Transmigration of Monocytes and T-Lymphocytes Through Human Endothelial Cell Monolayers

Enzymatically Modified, Nonoxidized LDL Induces Selective Adhesion and Transmigration of Monocytes and T-Lymphocytes Through Human Endothelial Cell Monolayers

Role of endothelial cell cytoskeleton in control of endothelial permeability.

Human arterial wall cells secrete factors that are chemotactic for monocytes.

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