Biomarkers of Neutrophil‐Mediated Liver Injury in Patients with Alcoholic Liver Disease

Abstract

Alcoholic liver disease (ALD) is a multi‐stage liver disorder associated with chronic ethanol abuse. The most serious syndrome associated with ALD is alcoholic hepatitis (AH), a type of acute‐on‐chronic liver failure that commonly occurs on a backdrop of alcoholic cirrhosis (AC). Neutrophil (PMN) accumulation in the liver is noted during AH, and is hypothesized to be a major mediator of the injury. This study was initiated to better define mechanisms of PMN mediated liver injury in human patients, using both mechanistic plasma biomarkers, and liver biopsies from AC and AH patients. We hypothesized PMN mediated ROS production exacerbated liver injury in patients with AH. Healthy volunteers, patients with clinically diagnosed AH, or abstinent AC patients were admitted to the IRB approved study, and whole blood was acquired for analysis of PMN activity and an array of mechanistic plasma biomarkers previously shown to provide insight into clinical liver injury, including cytokeratin‐18 levels, caspase‐3 activity, glutamate dehydrogenase (GDH) activity, and cytokine levels. In addition, hepatic cell death and PMN activation were measured in liver biopsies from patients with AH or AC via immunohistochemistry. Flow cytometry was used to ascertain the activation status of circulating PMNs. CD11b, a verified marker of neutrophil activation, was significantly elevated in neutrophils from AH patients, and was elevated in AC patients, but not significantly so. This was consistent with a substantial elevation in plasma IL‐8 levels, a known neutrophil chemoattractant, in the AH population. Baseline values of ROS production and phagocytic capacity were elevated in AC and AH patients, indicating low‐level basal stimulation of neutrophils. Surprisingly though, total hepatic neutrophils levels were similar in liver biopsies of AC and AH patients. Moreover, both groups stained positive for hypochlorous acid modified proteins, a gold standard histological marker for neutrophil activation, suggesting active neutrophils in both populations. Interestingly, plasma M30 levels, an apoptotic fragment of cytokeratin‐18 released during epithelial cell death, and plasma M65 levels, a necrotic fragment of cytokeratin‐18, were substantially elevated above control values, despite the fact that traditional markers of liver injury such as plasma ALT and plasma GDH were not elevated above controls, nor was there any detectable plasma caspase‐3 activity. Of note, AH patients had significantly higher plasma M65 values than AC patients, indicating increased total cell death. There was a small increase in TUNEL positive cells in AH patients (2–4 fold versus control), but this was not consistent with the dramatic elevation in M30/M65 levels (20–100 fold versus control). M30 staining of biopsies indicated only minor levels of M30 arose from the liver. In conclusion, neutrophil mediated hepatocyte cell death is likely a limited player in acute AH, although this does not preclude the possibility that neutrophil mediated ROS production contributes to the fibrotic or cirrhotic phenotype in these patients, as neutrophils are constitutively activated in the populations.Support or Funding InformationThis work was supported by a clinical pilot grant from Kansas University Medical Center. Additional support came from the “Training Program in Environmental Toxicology” T32 ES007079‐26A2 (to B.L.W.) from the National Institute of Environmental Health Sciences