[en] We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy observations. To assert the specific role of reactive oxygen species, endothelial cells were treated by the hypoxanthine/xanthine oxidase (X/XOx) system (production of superoxide anion and hydrogen peroxide), and by hypochlorite (product of the activity of myeloperoxidase). A strong cytotoxicity was found with stimulated neutrophils; microscopic observations indicated a loss of 50% of the endothelial cells and morphological alterations in the remaining cells. The supernatant of stimulated neutrophils was cytotoxic, in correlation with the number of neutrophils used to obtain the supernatant, and with the supernatant concentration of myeloperoxidase. The cytotoxicity of the X/XOx system was weak, but was increased by myeloperoxidase. Hypochlorite was highly toxic. We concluded that the supernatant of stimulated neutrophils was sufficient to obtain cytotoxic effects on the endothelium, in the absence of a direct contact between endothelium and neutrophils, and that this cytotoxicity was mainly linked to the activity of myeloperoxidase. From these in vitro results, it can be extrapolated that in pathologies characterised by an important activation of neutrophils, damage can spread to cells and tissues away from the inflammation focus.
Research center :
CORD - Centre de l'Oxygène, Recherche et Développement - ULiège
Disciplines :
Veterinary medicine & animal health
Author, co-author :
Benbarek, H.
Grulke, Sigrid ; Université de Liège - ULiège > Département clinique des animaux de compagnie et des équidés > Département clinique des animaux de compagnie et des équidés
Deby-Dupont, G.
Deby, Christiane ; Centre Hospitalier Universitaire de Liège - CHU > Administration des patients - Admission des hospitalisés
Mathy-Hartert, M.
Caudron, I.
Dessy-Doize, C.
Lamy, Maurice ; Université de Liège - ULiège > Département des sciences cliniques > Anesthésie et réanimation
Serteyn, Didier ; Université de Liège - ULiège > Département clinique des animaux de compagnie et des équidés > Anesthésiologie gén. et pathologie chirurg. des grds animaux
Language :
English
Title :
Cytotoxicity of Stimulated Equine Neutrophils on Equine Endothelial Cells in Culture
Publication date :
July 2000
Journal title :
Equine Veterinary Journal
ISSN :
0425-1644
eISSN :
2042-3306
Publisher :
Equine Veterinary Journal Ltd, Newmarket, United Kingdom
Beilke, M.A. (1989) Vascular endothelium in immunology and infectious disease. Rev. Infect. Dis. 11, 273-283.
Carreras, M.C., Pargament, G.A., Catz, S.D., Podoroso, J.J. and Boveris, A. (1994) Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils. FEBS Lett. 341, 65-68.
Deby, C. and Goutier, R. (1990) New perspectives on the biochemistry of superoxide anion and the effiency of superoxide dismutases. Biochem. Pharmacol. 39, 399-405.
Deby-Dupont, G., Caudron, I., Mathy-Hartert, M., Benbarek, H., Grülke, S., Deby, C., Lamy, M. and Serteyn, D. (1998) Equine neutrophil myeloperoxidase in plasma: design of a radioimmunoassay and first results in septic pathologies. Vet. Immun. Immunopathol. 66, 257-271.
Eiserich, J.P., Hristova, M., Cross, C.B., Jones, D.A., Freeman, B.A. Halliwell, B. and Van der Vliet, A. (1998) Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 301, 393-397.
Freeman, B.A. and Crapo, J.D. (1982) Biology of disease: free radicals and tissue injury. Lab. Invest. 47, 412-426.
Grülke, S., Benbarek, H., Caudron, I., Deby-Dupont, G., Mathy-Hartert, M., Farnir, F., Deby, C., Lamy, M. and Serteyn, D. (1999) Plasma myeloperoxidase level and polymorphonuclear leucocyte activation in horses suffering from large intestinal obstruction requiring surgery: preliminary results. Can. J. vet. Res. 63, 142-147.
Hurst, K., Albrieh, J.M., Green, T.R., Rosen, H. and Klebanoff, S.J. (1984) Myeloperoxidase-dependent fluorescein clorination by stimulated neutrophils. J. Biol. Chem. 259, 4812-4821.
Jaffe, E.A., Nachman, R.I., Becker, C.G. and Monick, C.R. (1975) Culture of human endothelial cells derived from human umbilical veins. J clin. Invest. 52, 2747-2753.
Janoff, A. (1975) At least three human neutrophil lysosomal proteases are capable of degrading joint connective tissues. Ann. N. Y. Acad. Sci. 256, 402-408.
Karlsson, K.A. and Marklund, S.L. (1988) Extracellular superoxide dismutase in the vascular system of mammals. Biochem. J. 255, 223-228.
Kvietys, P.R., Inauen, W., Bacon, B.R. and Grisham, M.B. (1989) Xanthine-oxidase-induced injury to endothelium: role of intracellular iron and hydroxyl radical. Am. J. Physiol. 257, H1640-H1646.
MacEachern, K.E., Smith, G.L. and Nolan, A.M. (1997) Methods for the isolation, culture and characterization of equine pulmonary artery endothelial cells. Res. vet. Sci. 62, 147-152.
Mantovani, A., Bussolino, F. and Dejan, H. (1992) Cytokine regulation of endothelial cell function. FASEB J. 6, 2591-2599.
Mathy-Hartert, M., Bourgeois, F., Grülke, S., Deby-Dupont, G., Caudron, I., Deby, C., Lamy, M. and Serteyn, D. (1998) Purification of myeloperoxidase from equine polymorphonuclear leucocytes. Can. J. vet. Res. 62, 127-132.
Mathy-Hartert, M., Deby-Dupont, G., Deby, C., Jadoul, L., Vandenberghe, A. and Lamy, M. (1995) Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime. Mediat. Inflamm. 4, 437-443.
Moore, R.M., Bertone, A.L., Bailey, M.Q., Muir, W.W. and Beard, W.L. (1994) Neutrophil accumulation in the large colon of horses during low-flow ischemia and reperfusion. .Am. J. vet. Res. 55, 1454-1463.
Nakagima, K., Powers, J.C., Ashe, B.M. and Zimmerman, M. (1979) Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. J. Biol. Chem. 254, 4027-4032.
Nourshargh, S. and Williams, T.J. (1990) Neutrophils and neutrophil-endothelial cell interactions in vivo. In: The Endothelium: An Introduction to Current Research, Ed: J.B. Warren, Wiley-Liss, New York, pp 171-186.
Osborn, L. (1990) Leukocyte adhesion to endothelium in inflammation. Cell 62, 3-6.
Pober, J.S. and Cotran, R.S. (1990) The role of endothelium in inflammation. Transplantation 50, 537-544.
Pycock, J.K., Allen, W.E. and Morris, T.H. (1987) Rapid, single-step isolation of equine neutrophils on a discontinuous Percoll density gradient. Res. vet. Sci. 42, 411-412.
Ramos, C.L., Pou, S.P., Britigan, B.H., Cohen, M.S. and Rosen, G.M. (1992) Spin trapping evidence for myeloperoxidase dependent hydroxyl radical formation by human neutrophils and monocytes. J. Biol. Chem. 267, 8307-8312.
Rinaldo, J.E. and Gorry, M. (1990) Protection by deferoxamine from endothelial injury: a possible link with inhibition of intracellular xanthine oxidase. Am. J. respir. Cell. mol. Biol. 3, 525-533.
Rossi, F., Romeo, D. and Patriarca, P.J. (1972) Mechanism of phagocytosis-associated oxidative metabolism in polymorphonuclear leucocytes and macrophages. Reticuloendoth. Soc. 12, 127-149.
Sampson, J.B., Ye. Y., Rosen, H. and Beckmann, J.S. (1998) Myeloperoxidase and horseradish peroxidase catalyze tyrosin nitration in proteins from nitrite and hydrogen peroxide. Arch. Bioehem. Biophys. 356, 207-213.
Shi, X., Lenhart, A. and Mao, Y. (1994) ESR spin trapping investigation on peroxynitrite decomposition: no evidence for hydroxyl radical production. Biochem. Biophys. Res. Commun. 203, 1515-1521.
Shireman, P.K. and Pearce, W.H. (1996) Endothelial cell function: biologic and physiologic functions in health and disease. Am. J. Roentgenoi. 166, 7-13.
Tonnesen, M.G., Smedly, L.A. and Henson, P.M. (1984) Neutrophil-endothelial cell interactions. Modulation of neutrophil adhesiveness induced by complement fragments C5a and C5a des arg and formyl-methionyl-leucyl-phenylalanine in vitro. J. clin. Invest. 74, 1581-1592.
Turek, J.J., Templeton, C.B., Bottoms, G.D. and Fessier, J.F. (1985) Flunixin meglumine attenuation of endotoxin-induced damage to the cardiopulmonary vascular endothelium of the pony. Am. J. vet. Res. 46, 591-596.
Vane, J.R., Anggard, E.E. and Botting, R.M. (1990) Regulatory functions of the vascular endothelium. New Engl. J. Med. 323, 27-36.
Vercellotti, G.M., Moldow, C.F. Wickham, N.W.R. and Jacob, H.S. (1990) Endothelial cell platelet-activating factor primes neutrophil responses: amplification of endothelial activation by neutrophil products. J. lipid Med. 2, S23-S30.
Weiss, S.J. (1989) Tissue destruction by neutrophils. New Engl. J. Med. 320, 365-376.
Williams, T.J. and Hellewell, P.G. (1992) Endothelial cell biology. Adhesion molecules involved in the microvascular inflammatory response. Am. Rev. Respir. Dis. 146, S45-S50.
Zimmerman, G.A., Prescott, S.M. and McIntyre, T.M. (1992) Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol. Today 13, 93-99.
