Key points are not available for this paper at this time.
A role of membrane microparticles (MP) released by vascular cells in endothelial cell (EC) activation was investigated. Flow cytofluorimetric analysis of blood samples from normal volunteers revealed the presence of an heterogeneous MP population, which increased by ∼2-fold after inflammatory stimulation with the chemotactic peptide, N -formyl-Met-Leu-Phe (2,799 ± 360 versus 5241 ± 640, p < 0.001). Blood-derived MP stimulated release of EC cytokines interleukin (IL)-6 (377 ± 68 pg/ml) and MCP-1 (1, 282 ± 79) and up-regulatedde novo expression of tissue factor on the EC surface. This was associated with generation of a factor Xa-dependent procoagulant response (2.28 ± 0.56 nm factor Xa/min/104 cells), in a reaction inhibited by a monoclonal antibody to tissue factor. Fluorescent labeling with antibodies to platelet GPIbα or leukocyte lactoferrin demonstrated that circulating MP originated from both platelets and leukocytes. However, depletion of platelet MP with an antibody to GPIbα did not reduce EC IL-6 release, and, similarly, MP from thrombin-stimulated platelets did not induce IL-6 release from endothelium. EC stimulation with leukocyte MP did not result in activation of the transcription factor NF-κB and was not associated with tyrosine phosphorylation of extracellular signal-regulated protein kinase, ERK1. In contrast, leukocyte MP stimulated a sustained, time-dependent increased tyrosine phosphorylation of ∼46-kDa c-Jun NH2-terminal kinase (JNK1) in EC. These findings demonstrate that circulating leukocyte MP are up-regulated by inflammatory stimulation in vivo and activate a stress signaling pathway in EC, leading to increased procoagulant and proinflammatory activity. This may provide an alternative mechanism of EC activation, potentially contributing to dysregulation of endothelial functions during vascular injury. A role of membrane microparticles (MP) released by vascular cells in endothelial cell (EC) activation was investigated. Flow cytofluorimetric analysis of blood samples from normal volunteers revealed the presence of an heterogeneous MP population, which increased by ∼2-fold after inflammatory stimulation with the chemotactic peptide, N -formyl-Met-Leu-Phe (2,799 ± 360 versus 5241 ± 640, p < 0.001). Blood-derived MP stimulated release of EC cytokines interleukin (IL)-6 (377 ± 68 pg/ml) and MCP-1 (1, 282 ± 79) and up-regulatedde novo expression of tissue factor on the EC surface. This was associated with generation of a factor Xa-dependent procoagulant response (2.28 ± 0.56 nm factor Xa/min/104 cells), in a reaction inhibited by a monoclonal antibody to tissue factor. Fluorescent labeling with antibodies to platelet GPIbα or leukocyte lactoferrin demonstrated that circulating MP originated from both platelets and leukocytes. However, depletion of platelet MP with an antibody to GPIbα did not reduce EC IL-6 release, and, similarly, MP from thrombin-stimulated platelets did not induce IL-6 release from endothelium. EC stimulation with leukocyte MP did not result in activation of the transcription factor NF-κB and was not associated with tyrosine phosphorylation of extracellular signal-regulated protein kinase, ERK1. In contrast, leukocyte MP stimulated a sustained, time-dependent increased tyrosine phosphorylation of ∼46-kDa c-Jun NH2-terminal kinase (JNK1) in EC. These findings demonstrate that circulating leukocyte MP are up-regulated by inflammatory stimulation in vivo and activate a stress signaling pathway in EC, leading to increased procoagulant and proinflammatory activity. This may provide an alternative mechanism of EC activation, potentially contributing to dysregulation of endothelial functions during vascular injury. endothelial cell(s) microparticle(s) platelet-rich plasma tissue factor interleukin tumor necrosis factor polymorphonuclear leukocyte(s) phosphate-buffered saline formyl-methionyl-leucyl-phenylalanine fluorescein isothiocyanate enzyme-linked immunosorbent assay monoclonal antibody Vascular endothelial cells (EC)1 respond to environmental and cellular stimuli with profound changes of adhesive, procoagulant, and inflammatory phenotypes (1Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar, 2Pober J.S. Cotran R.S. Physiol. Rev. 1990; 70: 427-451Crossref PubMed Scopus (1134) Google Scholar, 3Cines D.B. Pollak E.S. Buck C.A. Loscalzo J. Zimmerman G.A. McEver R.P. Pober J.S. Wick T.M. Konkle B.A. Schwartz B.S. Barnathan E.S. McCrae K.R. Hug B.A. Schmidt A.M. Stern D.M. Blood. 1998; 91: 3527-3561PubMed Google Scholar). This process of EC activation results in release of inflammatory and chemotactic cytokines IL-6, IL-8, and MCP-1 (2Pober J.S. Cotran R.S. Physiol. Rev. 1990; 70: 427-451Crossref PubMed Scopus (1134) Google Scholar, 4Rot A. Immunol. Today. 1992; 13: 291-294Abstract Full Text PDF PubMed Scopus (413) Google Scholar, 5Schall T.J. Cytokine. 1991; 3: 165-183Crossref PubMed Scopus (639) Google Scholar, 6Tanaka Y. Adams D.H. Hubscher S. Hirano H. Siebenlist U. Shaw S. Nature. 1993; 361: 79-82Crossref PubMed Scopus (846) Google Scholar), expression of procoagulant tissue factor (TF) (3Cines D.B. Pollak E.S. Buck C.A. Loscalzo J. Zimmerman G.A. McEver R.P. Pober J.S. Wick T.M. Konkle B.A. Schwartz B.S. Barnathan E.S. McCrae K.R. Hug B.A. Schmidt A.M. Stern D.M. Blood. 1998; 91: 3527-3561PubMed Google Scholar, 7Napoleone E. Di Santo A. Lorenzet R. Blood. 1997; 89: 541-549Crossref PubMed Google Scholar), and enhanced leukocyte recruitment via expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 (1Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar). These responses may originate from signal transduction by released cytokines,i.e. TNFα (8Modur V. Zimmerman G.A. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1996; 271: 13094-13102Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 9Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (1002) Google Scholar), shear stress during vascular remodeling (10Tardy Y. Resnick N. Nagel T. Gimbrone M.A. Dewey C.F. Arterioscler. Throm. Vasc. Biol. 1997; 17: 3102-3106Crossref PubMed Scopus (223) Google Scholar), or cross-talk with different vascular cells, including leukocytes and platelets (11Evangelista V. Manarini S. Sideri R. Rotondo S. Martelli N. Piccoli A. Totani L. Piccardoni P. Vestweber D. de Gaetano G. Cerletti C. Blood. 1999; 93: 876-885Crossref PubMed Google Scholar). Although of critical importance to preserve immune inflammatory responses and leukocyte trafficking (1Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar, 12Butcher E.C. Cell. 1991; 67: 1033-1036Abstract Full Text PDF PubMed Scopus (2522) Google Scholar), dysregulated EC activation may contribute to vascular injury and exacerbate the onset and progression of atherosclerosis in vivo (13Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9990) Google Scholar). Considerable interest has recently focused on alternative mechanisms of EC activation by vascular cells. Recent work has suggested that released membrane microparticles (MP) from platelets (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar) or leukocytes (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar) may provide such an alternative pathway of EC activation. In these studies, platelet or leukocyte MP stimulated increased expression of various adhesion molecules on EC, up-regulation of inflammatory and chemotactic cytokines, and increased monocyte adhesiveness (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar, 16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). For platelet MP, this pathway was recapitulated by arachidonic acid, consistent with the presence of bioactive lipids in platelet MP, and their ability to influence gene expression in target cells (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar). Although the existence of platelet MP in vivo and their potential contribution to procoagulant and/or anticoagulant responses have long been established (17Tans G. Rosing J. Thomassen M.C. Heeb M.J. Zwaal R.F.A. Griffin J.H. Blood. 1991; 77: 2641-2648Crossref PubMed Google Scholar, 18George J. Thoi L.L. McManus L.M. Reimann T.A. Blood. 1982; 60: 834-840Crossref PubMed Google Scholar, 19Shattil S.J. Cunningham M. Hoxie J.A. Blood. 1987; 70: 307-315Crossref PubMed Google Scholar, 20Abrams C.S. Ellison N. Budzynski A.Z. Shattil S.J. Blood. 1990; 75: 128-138Crossref PubMed Google Scholar), little is known about leukocyte MP or their potential ability to stimulate ECin vivo . In this study, we sought to investigate a potential role of leukocyte MP in EC responses and to identify signaling requirements involved in gene expression. We found that although both platelet and leukocyte MP are present in the normal circulation in vivo , only the leukocyte fraction initiates signal transduction and stimulates inflammatory and procoagulant responses in the endothelium. Polymorphonuclear leukocytes (PMN) were isolated from heparin sodium-anticoagulated blood drawn after informed consent from normal healthy volunteers by differential centrifugation on Ficoll/Hypaque gradient and dextran sedimentation as described (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). Human umbilical vein EC were prepared by collagenase treatment, maintained in medium 199 (BioWhittaker, Walkersville, MD) supplemented with 20% heat-inactivated fetal bovine serum (BioWhittaker), l-glutamine (2 mm), and 1% endothelial cell growth supplement, pH 7.4, and used between passages 2 and 4. Heparin sodium-anticoagulated blood was drawn from normal healthy volunteers after informed consent. Aliquots (1.5 ml) of undiluted blood or samples diluted 1:5 in PBS, pH 7.4, were incubated with the fluorescent labeling dye, quinacrine mustard (mepacrine, 0.1 mm; Sigma), in the presence or in the absence of fMLP (1 μm) for 2 h at 37 °C. Blood samples were centrifuged at 1500 × g for 20 min at 22 °C, and the cell-free supernatant was collected and analyzed by flow cytometry. In some experiments, cell-free supernatants prepared as described above were passed through 100-kDa cut-off nanospins (Millipore, Bedford, MA) according to the manufacturer's specifications. Aliquots of 0.5 ml were analyzed on a Becton-Dickinson (Mountain View, CA) flow cytometer as described (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). To identify the MP population, samples were gated according to their fluorescence (mepacrine) labeling. With the light scatter and fluorescence channels set at logarithmic gain, samples were analyzed for 6-s intervals for forward light scatter, right angle light scatter, and mepacrine fluorescence. light scatter of MP demonstrated light scatter population, in with (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). results were blood samples were with or In some experiments, of diluted blood were incubated with of M. antibody or for min at 22 in the was according to the manufacturer's were stimulated or not with nm fMLP and for h at 22 and centrifuged at 1500 × g for 20 min to the various cellular and the supernatants were collected and at × g for MP were with PBS, pH 7.4, in 0.5 ml of PBS, and analyzed by flow cytometry. were analyzed for a of for and fluorescein light as described In experiments, supernatants collected after with the various antibodies as described above were for of endothelial IL-6 release by MP were by flow a fluorescent dye, This and a fluorescence. (2 × were with μm) according to the manufacturer's specifications. cells were in 199 medium and with or CA) for 2 h at 37 °C. were stimulated with fMLP or for a 2 h at 37 in the presence of supernatants were isolated and analyzed by flow as described was analyzed for a of A was to for fluorescence. Blood was collected from healthy volunteers a and with was prepared by centrifugation at × g for min at 22 °C. MP were isolated after platelet by and with (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar). a at 37 °C, platelet were at 1500 × g for 20 and the supernatants were diluted 1:5 in PBS, pH 7.4, and analyzed by flow as described of fMLP (1 or (1 cell-free supernatants from blood or samples were to EC to in a at 37 °C, EC supernatants were centrifuged at × g for and analyzed for released IL-6 and by as described In experiments, of blood were incubated in the absence or presence of 20 or for min at 22 °C. fMLP cellular of the samples were by centrifugation at 1500 × g for 20 and of the supernatants were to EC for of release, as described In experiments, cell-free supernatants from fMLP (1 × were with or for min at 22 to EC for and of IL-6 release, as described In experiments, cell-free supernatants from or blood samples were collected and through 100-kDa cut-off membrane of to EC and of released IL-6 and For cytofluorimetric analysis of in EC, cell-free supernatants from or fMLP (1 were to EC in a for h at 37 °C. or EC were and with serum for min on by and with 20 of T. S. CA) or EC were incubated with a of and analyzed by flow cytometry. In experiments, EC were stimulated with TNFα and analyzed for as described In experiments, EC in at were incubated with medium or with or cell-free supernatant from × for h at 37 °C. was by and with × or transcription reaction of EC or of 0.5 of or 2 × in the presence of and was incubated for min at °C. the of the samples were for min at °C, on with of for 20 min at 37 °C, and by reaction with and from the of of were in a with for at °C, for at °C, and for min at °C. was used at a of reaction were analyzed on 1% by In of experiments, EC in a were and incubated in the presence or absence of cell-free supernatant from × or MP prepared as described above for h at 37 °C. EC were incubated with of medium nm factor nm factor and 2 for 20 min at 37 °C. In some experiments, EC were with or for min at 22 of and of factor was by of to were to a and factor was by of the at a of were at nm a of factor the various was by with a with of factor EC in were stimulated with × supernatants for intervals at 37 °C. EC were and with 1% and EC were centrifuged at × g for with protein and with antibodies (1 to extracellular signal-regulated kinase c-Jun NH2-terminal kinase or CA) for 2 h at °C. immune were on a to and with antibody or the various were by of and by enhanced For EC were or incubated with × supernatant or TNFα for min at 37 °C. were prepared as described C. A. R. J. Immunol. 1991; Google Scholar). were for protein and were incubated with of for 20 min at 22 and for min with in a of used in these for the was was with kinase were on a in pH acid, 20 were to and were by of isolated with stimuli fMLP or release of an heterogeneous membrane MP population, as by flow and in with (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). with A. J. C. D. S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) did not reduce MP release from or Flow cytofluorimetric analysis of blood supernatants revealed the presence of a with forward scatter consistent with MP A , of blood samples with fMLP in an ∼2-fold in the MP 2 A , right in with (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). In experiments, fMLP stimulation increased the of fluorescent MP mepacrine labeling of blood samples 2 A , and right with these in vivo cytofluorimetric analysis of cell-free supernatants collected from normal volunteers revealed the presence of an MP in blood samples ± of blood samples with fMLP in a ∼2-fold in the of MP by flow 5241 ± p 2 of EC with cell-free supernatants from blood samples did not result in release of cytokines, IL-6, and MCP-1 A In contrast, cell-free supernatants from blood supernatants a increased release of cytokines MCP-1 ± pg/ml) and IL-6 (377 ± 68 A A in release was the of cell-free supernatants through 100-kDa to with EC the release of IL-6 and MCP-1 by EC A in with (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). with these of cell-free supernatants in depletion of the blood MP population, by forward and scatter and by flow In experiments, of MP with a to TNFα did not reduce EC IL-6 release In contrast, the antibody inhibited by EC IL-6 release by TNFα stimulation TNFα A of EC to supernatants from in consistent increased expression of as by flow cytofluorimetric with A results were MP EC stimulation with MP in de novo expression of as by of a by reaction of EC A , In experiments, EC stimulation with TNFα in in expression and A in with N. J.H. U. S. A. 1990; PubMed Scopus Google Scholar). In experiments, EC stimulation with or MP in the generation of ± 0.56 and ± nm of factor Xa/min/104 EC, This response was in the absence of factor or by of ± nm of factor Xa/min/104 In contrast, EC with did not reduce EC procoagulant In experiments, EC stimulation with TNFα in the of ± nm factor Xa/min/104 EC in with N. J.H. U. S. A. 1990; PubMed Scopus Google Scholar). To identify the potential cell(s) of of released MP in vivo , flow cytofluorimetric were with antibodies to platelet GPIbα or leukocyte and the in MP (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar) as and D. C. In these experiments, MP with antibodies to GPIbα and as with antibody fMLP stimulation increased by the of the MP with the lactoferrin in the of were with or samples of blood samples with 20 in a in the of MP released after fMLP stimulation A In contrast, in MP release were in the presence of or the antibody to lactoferrin A In experiments, of cell-free supernatants with antibodies to lactoferrin or GPIbα did not reduce EC IL-6 release, as with samples or with A potential differential ability of platelet or leukocyte MP to induce EC IL-6 release was investigated. stimulation of in MP release, as by flow in with However, thrombin-stimulated platelet MP or supernatant did not stimulate EC IL-6 release and not In contrast, TNFα stimulation of EC in the generation of ± IL-6 after a at 37 EC stimulation with TNFα in activation of as with EC as by assay A In contrast, leukocyte MP did not induce NF-κB activation A in tyrosine phosphorylation of were in EC with leukocyte MP, as with or In contrast, and of EC with antibody revealed a time-dependent and increased tyrosine phosphorylation of a ∼46-kDa in EC, as with of the ∼46-kDa was investigated. of EC with an antibody to by with antibody revealed a in tyrosine phosphorylation of in not in EC In contrast, EC serum did not result in tyrosine phosphorylation of In experiments, of demonstrated a of the various In this study, we have that MP in the normal and are up-regulated by inflammatory not MP stimulate release of cytokines and expression of tissue factor in EC, in a pathway associated with phosphorylation of ∼46-kDa that vascular cells release MP in response to environmental stimuli is established and has been for platelets T. 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Mesri et al. (Sun,) studied this question.
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