Chlorotyrosine protein adducts are reliable biomarkers of neutrophil-induced cytotoxicity in vivo.

INTRODUCTION: A limitation for investigating the pathophysiological role of neutrophils in vivo is the lack of a reliable biomarker for neutrophil cytotoxicity in the liver. Therefore, we investigated if immunohistochemical detection of chlorotyrosine protein adducts can be used as a specific footprint for generation of neutrophil-derived hypochlorous acid in vivo. METHODS: C3Heb/FeJ mice were treated with 100 micrograms/kg endotoxin (ET) alone or in combination with 700 mg/kg galactosamine (Gal/ET). Some animals received additionally two doses of 10 mg/kg of the pancaspase inhibitor Z-VAD-fmk. An antibody against chlorotyrosine was used for the immunohistochemical analysis. RESULTS: At 6 h after Gal/ET, hepatocellular apoptosis was evident without increase in plasma ALT activities. Neutrophils accumulated in sinusoids but there was no evidence for chlorotyrosine staining. At 7 h after Gal/ET, about 54% of the sequestered neutrophils had extravasated, there was extensive necrosis and increased plasma ALT activities. Extensive immunostaining for chlorotyrosine, mainly colocalized with neutrophils, could be observed. Treatment with Z-VAD-fmk eliminated apoptosis, necrosis and the increase in plasma ALT values. Neutrophil extravasation was prevented but the overall number of neutrophils in the liver was unchanged. Chlorotyrosine staining was absent in these samples. After ET alone (7 h), sinusoidal neutrophil accumulation was similar to Gal/ET treatment but there was no apoptosis, neutrophil extravasation, ALT release or chlorotyrosine staining. CONCLUSIONS: Chlorotyrosine staining in liver samples correlated well with evidence of neutrophil-induced liver injury in the endotoxemia model. These results indicate that assessment of chlorotyrosine protein adduct formation by immunohistochemistry could be a useful marker of neutrophil-induced liver cell injury in vivo.