SUPEROXIDE GENERATION BY NEUTROPHILS AND KUPFFER CELLS DURING INVIVO REPERFUSION AFTER HEPATIC ISCHEMIA IN RATS

Kupffer cells and polymorphonuclear leukocytes (PMNs) contribute to the severe reperfusion injury of the liver after ischemia at different time points. The objective of this study was to identify the cellular source(s) of reactive oxygen formation during the PMN-induced injury phase. Kupffer cells and PMNs were isolated from the liver after 45 min of ischemia and 5 h or 24 h of reperfusion using collagenase-pronase digestion and a centrifugal elutriation method. Spontaneous superoxide anion (02-) formation by large Kupffer cells (basal value 0.65 +/- 0.16 nmol/h/106 Cells) was increased (up to 550%) during the entire reperfusion period. No enhanced 02- generation by the small Kupffer cell fraction was observed at any time. Control PMNs generated only small amounts of 02- spontaneously (0.25 +/- 0.05 nmol 02-/h/10(6) cells), but hepatic PMNs generated significantly more superoxide: 1.90 +/- 0.58 nmol 02-/h/10(6) cells at 5 h and similarly at 24 h of reperfusion. All cell types were significantly primed for enhanced 02 formation during reperfusion; the priming effect was consistantly higher for stimulation with opsonized zymosan (receptor-mediated signal transduction pathway) compared to phorbol myristate acetate (protein kinase C activation). Our data support the hypothesis that PMNs and large Kupffer cells are predominantly responsible for the postischemic oxidant stress during the later reperfusion injury phase after hepatic ischemia in vivo.