Hydrogen peroxide pretreatment of perfused canine vessels induces ICAM-1 and CD18-dependent neutrophil adherence.
BACKGROUND
Cytotoxic products of neutrophils (polymorphonuclear leukocytes, PMNs) contribute to ischemia-reperfusion injury of several tissues. Hydrogen peroxide (H2O2), one of the cytotoxic products of PMNs, also promotes the adherence of PMNs to cultured vascular endothelial cells in vitro. The present study was undertaken to determine if H2O2 also augmented adhesion of PMNs to intact vessels perfused ex vivo and to determine if H2O2-induced PMN adherence to intact canine carotid arteries and external jugular veins or to cultured canine venous endothelium is mediated by specific adherence ligands on the neutrophil and/or the endothelium.


RESULTS
Vessels were perfused for 20 minutes with oxygenated Krebs-Henseleit bicarbonate buffer with and without H2O2, washed with buffer alone, and then exposed to 111In-labeled isolated PMNs (10(7) cells/vessel) under static conditions for up to 20 minutes before being washed again. Residual radioactivity retained by the washed vessel was counted as an index of PMN retention. The adherence of unlabeled PMNs to cultured endothelial cells was determined by a visual assay method after pretreatment of the endothelium with H2O2 for brief periods followed by washing. Perfusion of vessels with H2O2 produced a transient, concentration-dependent increase in PMN adhesion to both canine carotid arteries and external jugular veins that was two to four times that of control values at 1 mmol/l and declined at higher H2O2 concentrations. Peak retention of PMNs by canine carotid arteries occurred 10 minutes after exposure to 1 mmol/l H2O2 and then rapidly declined to control values; this effect was replicated by a second 20-minute exposure of canine carotid arteries to 1 mmol/l H2O2 60 minutes after the first exposure. Scanning and transmission electron microscopy revealed not only adherence of PMNs to but migration through the vascular endothelium of the carotid artery after H2O2 perfusion. The endothelium was intact in H2O2-treated arteries not exposed to PMNs. H2O2-induced PMN retention was completely inhibited by addition of catalase or the hydroxyl radical scavenger dimethylthiourea to the perfusate by incubation of the PMN with a monoclonal antibody (Mab) against CD18 (R15.7) or by perfusion of the H2O2-treated vessel with CL18/6, a Mab against canine ICAM-1 (intercellular adhesion molecule-1). Similar effects of Mabs on PMN adhesion to H2O2-pretreated cultured endothelium were noted. The retention of PMNs by vessels mechanically denuded of endothelial cells was markedly increased. H2O2 pretreatment of these vessels did not further augment PMN adherence, and no inhibitory effect of R15.7 was noted. Incubation of carotid arteries and PMNs with a specific platelet-activating factor antagonist, WEB2086, completely inhibited the H2O2-induced increased PMN retention by these vessels.


CONCLUSIONS
These results indicate that H2O2 in the absence of evidence for permanent endothelial cell injury, can induce a transient, reversible, platelet-activating factor-dependent adherence of PMNs to vessels by mechanisms that depend on an intact endothelium and involve CD18 on the PMN and ICAM-1 on the endothelium.
Center for Experimental Therapeutics, Baylor College of Medicine, Houston, TX 77030.
A C
AC
Gasic
G
G
McGuire
S
S
Krater
A I
AI
Farhood
M A
MA
Goldstein
C W
CW
Smith
M L
ML
Entman
A A
AA
Taylor
D000818:Animals; D015703:Antigens, CD / Q000502:physiology*; D018821:CD18 Antigens; D002339:Carotid Arteries; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000502:physiology; D004285:Dogs; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D016023:Integrins; D018799:Intercellular Adhesion Molecule-1; D007601:Jugular Veins; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D009504:Neutrophils / Q000502:physiology*; D010972:Platelet Activating Factor / Q000502:physiology
D015703:Antigens, CD; D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D016023:Integrins; D010972:Platelet Activating Factor; D018799:Intercellular Adhesion Molecule-1; D006861:Hydrogen Peroxide