Neutrophil-dependent hepatic platelet accumulation and liver injury revealed by acetaminophen dose-response studies

Abstract

Background: Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure (ALF). Neutrophil activation has been associated with poor outcomes in patients with ALF and is proposed to amplify coagulation in this context. However, the precise role of neutrophils in APAP-induced liver injury is not known.

Methods: We used a dual antibody-mediated neutrophil depletion strategy to determine the role of neutrophils in mice challenged with different doses of APAP (300 or 600 mg/kg) that produce hepatotoxicity and ALF-like pathology.

Results: Flow cytometry confirmed depletion of neutrophils in whole blood prior to APAP challenge. Mice given isotype control and challenged with 300 mg/kg APAP developed marked hepatocellular necrosis and showed an increase in biomarkers of coagulation cascade activation. Neutrophil depletion (anti-Ly6G) did not affect either liver injury or coagulation activation in mice challenged with 300 mg/kg APAP. Mice given isotype control and challenged with 600 mg/kg APAP developed hepatic necrosis alongside marked hemorrhage and congestion indicative of vascular injury. Interestingly, hepatic neutrophil and platelet accumulation were increased in mice given 600 mg/kg APAP compared with those given the lower APAP dose. Neutrophil depletion significantly reduced the severity of liver necrosis in mice challenged with 600 mg/kg APAP, without significantly impacting biomarkers of coagulation activity. Notably, neutrophil depletion significantly reduced hepatic platelet accumulation in mice challenged with 600 mg/kg APAP.

Conclusion: The results indicate a role of neutrophils in APAP-induced liver injury that is dependent on the APAP dose and suggest involvement of neutrophil-platelet interactions in promoting hepatic injury in experimental APAP-induced ALF.

Keywords: blood coagulation; chemical and drug induced liver injury; mice; neutrophils.

Figure 1

Confirmation of neutrophil depletion using flow cytometry and impact of neutrophil depletion on liver injury induced by 300 mg/kg acetaminophen (APAP) challenge. Male C57Bl/6J mice were treated with 50 μg of anti-Ly6G or isotype control antibody and then 2 hours later with 100 μg of anti-rat IgGk. (A) Anticoagulated whole blood was collected 22 hours after anti-Ly6G antibody administration to confirm neutrophil (SSC^high CD11b⁺ F4/80⁻ CD115⁻ Ly6c^mid) depletion by flow cytometry. Neutrophils, determined as a percentage of singlet, live, CD45⁺ cells, are shown (A). Mice were challenged with 300 mg/kg APAP 24 hours after administration of anti-Ly6G antibody. Twenty-four hours after APAP challenge, (B) plasma alanine-transaminase (ALT) activity and (C) hepatic necrosis were determined (see Methods). (D) Representative photomicrographs depicting hepatocellular necrosis in hematoxylin and eosin–stained liver sections. N = 9 to 12 mice per group. Data points for individual mice are shown, and the results are expressed as mean ± SEM. ∗∗∗∗P < .001.

Figure 2

Impact of neutrophil depletion on coagulation in mice challenged with 300 mg/kg acetaminophen (APAP). Male C57Bl/6J mice were treated with 50 μg of anti-Ly6G or isotype control antibody and then 2 hours later with 100 μg of anti-rat IgGk. Mice were challenged with 300 mg/kg APAP 24 hours after administration of anti-Ly6G antibody. Twenty-four hours after APAP challenge, (A) plasma thrombin-antithrombin (TAT) complexes, (B) plasma fibrinogen, (C) hepatic insoluble fibrinogen (Fib)β chain, and (D) high-molecular-weight (HMW) crosslinked insoluble Fibα (>250 kD) chain and (E) Fibγ chain (>100 kD) levels were determined (see Methods). (F) Hepatic CD41 levels were measured using Western blot (chemiluminescence, film), and total protein (Revert 700, Licor) was quantified and expressed as a ratio. N = 8 to 12 mice per group. Data points for individual mice are shown, and the results are expressed as mean ± SEM.

Figure 3

Impact of neutrophil depletion on liver injury in mice challenged with different doses of acetaminophen (APAP). Male C57Bl/6J mice were treated with 50 μg of anti-Ly6G or isotype control antibody and then 2 hours later with 100 μg of anti-rat IgGk. Mice were challenged with 300 or 600 mg/kg APAP 24 hours after administration of anti-Ly6G antibody. Liver and plasma samples were collected 24 hours after APAP challenge. (A) Representative photomicrographs showing liver sections stained for neutrophils (Ly6G) and (B) quantification of Ly6G area, expressed as a percentage of positive area to total area. (C) Plasma alanine aminotransferase (ALT) and (E) area of hepatic necrosis were determined as described (see Methods). (D) Representative photomicrographs depicting hepatocellular necrosis in hematoxylin and eosin–stained liver sections. N = 5 mice per group. Data points for individual mice are shown, and the results are expressed as mean ± SEM.

Figure 4

Impact of neutrophil depletion on coagulation in mice challenged with different doses of acetaminophen (APAP). Male C57Bl/6J mice were treated with 50 μg of anti-Ly6G or isotype control antibody and then 2 hours later with 100 μg of anti-rat IgGk. Mice were challenged with 300 or 600 mg/kg APAP 24 hours after administration of anti-Ly6G antibody. Twenty-four hours after APAP challenge, (A) plasma thrombin-antithrombin (TAT) complexes, (B) plasma fibrinogen, (C) hepatic insoluble fibrinogen (Fib)β chain, and (D) high-molecular-weight (HMW) crosslinked insoluble Fibα chain (>250 kD) and (E) Fibγ chain (>100 kD) levels were determined (see Methods). N = 5 mice per group. Data points for individual mice are shown, and the results are expressed as mean ± SEM. ∗P < .05.

Figure 5

Role of neutrophils in hepatic platelet accumulation after challenge of mice with different doses of acetaminophen (APAP). Male C57Bl/6J mice were challenged with 300 or 600 mg/kg APAP or vehicle (saline), and hepatic CD41 levels were measured by Western blotting 24 hours after APAP challenge. (A) CD41 Western blot (chemiluminescence) and (B) total protein (no-stain total protein) were visualized and quantified using iBright software. N = 5 mice per group. (C and D) Male wild-type mice were treated with 50 μg of anti-Ly6G or isotype control antibody and then 2 hours later with 100 μg of anti-rat IgGk. Mice were challenged with 300 or 600 mg/kg APAP 24 hours after administration of anti-Ly6G antibody. (C) Representative photomicrographs showing immunohistochemical staining for CD41 (brown) that was quantified (D) using QuPath (see Methods). N = 4 to 5 mice per group. Data points for individual mice are shown, and the results are expressed as mean ± SEM.