title: Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase.
abstract: The neutrophil has been implicated as an important mediator of vascular injury, especially after endotoxemia. This study examines neutrophil-mediated injury to human microvascular endothelial cells in vitro. We found that neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a, or lipopolysaccharide (LPS) (1-1,000 ng/ml) alone produced minimal endothelial injury over a 4-h assay. In contrast, neutrophils incubated with endothelial cells in the presence of low concentrations of LPS (1-10 ng/ml) could then be stimulated by FMLP or C5a to produce marked endothelial injury. Injury was maximal at concentrations of 100 ng/ml LPS and 10(-7) M FMLP. Pretreatment of neutrophils with LPS resulted in a similar degree of injury, suggesting that LPS effects were largely on the neutrophil. Endothelial cell injury produced by LPS-exposed, FMLP-stimulated neutrophils had a time course similar to that induced by the addition of purified human neutrophil elastase, and different from that induced by hydrogen peroxide (H2O2). Further, neutrophil-mediated injury was not inhibited by scavengers of a variety of oxygen radical species, and occurred with neutrophils from a patient with chronic granulomatous disease, which produced no H2O2. In contrast, the specific serine elastase inhibitor methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethyl ketone inhibited 63% of the neutrophil-mediated injury and 64% of the neutrophil elastase-induced injury. However, neutrophil-mediated injury was not inhibited significantly by 50% serum, 50% plasma, or purified alpha 1 proteinase inhibitor. These results suggest that, in this system, chemotactic factor-stimulated human neutrophil injury of microvascular endothelial cells is enhanced by small amounts of LPS and may be mediated in large part by the action of neutrophil elastase.
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D001769:Blood; D001798:Blood Proteins / Q000494:pharmacology; D003182:Complement C5 / Q000494:pharmacology; D015936:Complement C5a; D004727:Endothelium / Q000473:pathology*; D004731:Endotoxins / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D008833:Microcirculation; D008854:Microscopy, Electron; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000378:metabolism*; D013997:Time Factors; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D000515:alpha 1-Antitrypsin
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D001798:Blood Proteins; D003182:Complement C5; D004731:Endotoxins; D008070:Lipopolysaccharides; D000515:alpha 1-Antitrypsin; D014107:Tosyllysine Chloromethyl Ketone; D009240:N-Formylmethionine Leucyl-Phenylalanine; C026704:methoxysuccinyl-alanyl-alanyl-prolyl-valine chloromethyl ketone; D015936:Complement C5a; D006861:Hydrogen Peroxide; D010196:Pancreatic Elastase