GENERATION OF LIPID-FREE RADICALS BY ADHERENT LEUKOCYTES FROM TRANSPLANTED RAT-LIVER

The production of free radicals in blood correlates with primary nonfu nction of transplanted livers, but the source of the free radicals is unknown. The purpose of this study was to determine if adherent leukoc ytes in the transplanted liver are responsible for the radicals detect ed in blood. First, a new method to harvest adherent leukocytes from t he liver without enzymatic digestion was developed and characterized b y transplanting livers from ethanol-treated rats, which increases prim ary nonfunction, and from saline-treated controls. Free radicals were then detected in isolated leukocytes using the spin-trapping technique and electron spin resonance (ESR) spin spectroscopy. Livers were perf used with a balanced salt solution (200 ml), followed by a Ca2+-free s olution containing EGTA and heparin (400 mi). Perfusion with Ca2+-free buffer removed greater than 90 % of all adherent leukocytes from sali ne-treated livers and nearly 80 % of all leukocytes from fatty livers without removing Kupffer cells. Transplanted fatty livers from rats gi ven ethanol contained significantly more adherent leukocytes (5.0 x 10 (7) cells/liver) than grafts from control donors (3.2 x 10(7) cells/li ver) and almost double the number of adherent neutrophils and monocyte s. Moreover, adherent white blood cells from transplanted livers produ ced the same three free radical species that have been detected previo usly in blood; however, cells from ethanol-treated livers produced abo ut five times more radical adducts. These data show that adherent whit e blood cells produce free radicals that are important in the mechanis m of primary graft nonfunction.