No abstract

The objective of this study was to identify the cellular source of the vascular oxidant stress in hepatic ischemia-reperfusion injury in male Fischer rats. Nonparenchymal cells (Kupffer cells, endothelial cells) and neutrophils were isolated from postischemic liver lobes by collagenase-pronase digestion followed by centrifugal elutriation. The spontaneous and stimulated generation of superoxide by these cells were subsequently quantified in vitro. Large Kupffer cells from the postischemic lobes spontaneously generated 300% more superoxide than similar cells from control animals. No difference in spontaneous superoxide formation was found when the small Kupffer cells were compared. No other cells isolated from the postischemic lobes or control liver including neutrophils released any detectable superoxide spontaneously. In contrast, small Kupffer cells and neutrophils from the postischemic liver generated significantly more superoxide after stimulation with phorbol ester or opsonized zymosan than the controls. The considerably higher response with zymosan stimulation compared to phorbol ester indicates a particular priming for a receptor-mediated signal transduction pathway during reperfusion. These studies demonstrate that Kupffer cells are the principal source of the oxidant stress during the initial reperfusion phase after hepatic ischemia. The priming of neutrophils during this time may be an important factor for the later neutrophil-induced injury phase.

The hypothesis that complement factors may be involved in the postischemic activation of Kupffer cells (KC) and polymorphonuclear neutrophils (PMN) was investigated in a model of hepatic ischemia (45 min) and reperfusion in male Fischer rats in vivo. Depletion of serum complement before ischemia resulted in a significant attenuation of the KC-induced oxidant stress (enhanced oxidation of plasma glutathione) and also prevented the accumulation of PMNs in the liver during the initial reperfusion period of 1 h. Complement activation through injection of cobra venom factor (CVF; 75 micrograms CVF/kg) also induced enhanced oxidation of plasma glutathione and accumulation of PMNs in the liver. Isolation of KC and PMNs from the liver 1 h after CVF treatment demonstrated a similar priming effect for stimulation with phorbol myristate acetate and opsonized zymosan as was observed in the postischemic liver. Complement-depleted animals and animals pretreated with the soluble human complement receptor type 1 (BRL 55730; 22.5 mg/kg) accumulated significantly less PMNs in the postischemic livers during longer reperfusion periods (24 h) and sustained significantly less injury. It is concluded that complement is involved in the induction of a KC-induced oxidant stress, the priming of KC and PMNs for enhanced reactive oxygen generation, and the continuous accumulation of PMNs in the liver during reperfusion.

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 (O2-) formation by large Kupffer cells (basal value 0.65 +/- 0.16 nmol/h/10(6) cells) was increased (up to 550%) during the entire reperfusion period. No enhanced O2- generation by the small Kupffer cell fraction was observed at any time. Control PMNs generated only small amounts of O2- spontaneously (0.25 +/- 0.05 nmol O2-/h/10(6) cells), but hepatic PMNs generated significantly more superoxide: 1.90 +/- 0.58 nmol O2-/h/10(6) cells at 5 h and similarly at 24 h of reperfusion. All cell types were significantly primed for enhanced O2- formation during reperfusion; the priming effect was consistently 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.