Generation of lipid free radicals by adherent leukocytes from transplanted rat liver

The production of free radicals in blood correlates with primary nonfunction of transplanted livers, but the source of the free radicals is unknown. The purpose of this study was to determine if adherent leukocytes in the transplanted liver are responsible for the radicals detected in blood. First, a new method to harvest adherent leukocytes from the liver without enzymatic digestion was developed and characterized by transplanting livers from ethanol-treated rats, which increases primary 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 perfused with a balanced salt solution (200 ml), followed by a Ca(2+)-free solution containing EGTA and heparin (400 ml). Perfusion with Ca(2+)-free buffer removed greater than 90% of all adherent leukocytes from saline-treated livers and nearly 80% of all leukocytes from fatty livers without removing Kupffer cells. Transplanted fatty livers from rats given ethanol contained significantly more adherent leukocytes (5.0 x 10(7) cells/liver) than grafts from control donors (3.2 x 10(7) cells/liver) and almost double the number of adherent neutrophils and monocytes. Moreover, adherent white blood cells from transplanted livers produced the same three free radical species that have been detected previously in blood; however, cells from ethanol-treated livers produced about five times more radical adducts. These data show that adherent white blood cells produce free radicals that are important in the mechanism of primary graft nonfunction.