Activated Neutrophils Induce Epithelial Cell Apoptosis Through Oxidant-Dependent Tyrosine Dephosphorylation of Caspase-8

Activated neutrophils can injure host cells through direct effects of oxidants on membrane phospholipids, but an ability to induce apoptotic cell death has not previously been reported. We show that neutrophils activated in vivo in patients who have sustained multiple trauma or in vitro by exposure to bacterial lipopolysaccharide promote epithelial cell apoptosis through SHP-1-mediated dephosphorylation of epithelial cell caspase-8. Epithelial cell apoptosis induced by circulating neutrophils from patients who had sustained serious injury depended on the generation of neutrophil-derived reactive oxygen intermediates and was blocked by inhibition of NADPH oxidase or restoration of intracellular glutathione. Caspase-8 was constitutively tyrosine phosphorylated in a panel of resting epithelial cells, but underwent SHP-1-dependent dephosphorylation in response to hydrogen peroxide, activated neutrophils, or inhibition of Src kinases. Cells transfected with a mutant caspase-8 in which tyrosine residues at Tyr397 or Tyr465 are replaced by nonphosphorylatable phenylalanine underwent accelerated apoptosis, whereas either mutation of these residues to phosphomimetic glutamic acid or transfection with the Src kinases Lyn or c-Src inhibited hydrogen peroxide-induced apoptosis. Exposure to either hydrogen peroxide or lipopolysaccharide-stimulated neutrophils increased phosphorylation and activity of the phosphatase SHP-1, increased activity of caspases 8 and 3, and accelerated epithelial cell apoptosis. These observations reveal a novel mechanism for neutrophil-mediated tissue injury through oxidant-dependent, SHP-1-mediated dephosphorylation of caspase-8 resulting in enhanced epithelial cell apoptosis.