Neutrophil depletion protects against murine acetaminophen hepatotoxicity: Another perspective
Authors: Hartmut Jaeschke, Jie Liu
Journal: Hepatology (2007)

In a recent issue of HEPATOLOGY, Liu et al. reported that a neutropenia-inducing monoclonal antibody protected against acetaminophen (APAP)-induced liver injury.1 This study,1 together with an independent paper published by Japanese authors at the same time,2 strongly suggests that a neutrophilic inflammatory component is critical for the overall liver injury and survival in these animals. Although the study is well designed and the conclusions appear to be justified by the presented data, some doubts remain. First, as discussed previously, there is a substantial body of experimental evidence published, which argues against a critical role of neutrophils in this particular model of drug-induced hepatotoxicity.3, 4 Second, when we treated animals with the same anti-Gr-1 antibody several hours after APAP administration but before neutrophil accumulation in the liver, we did not find any protection.5 These contradictory findings raised the important question: Why is the same antibody effective as a pretreatment for 24 hours1, 2 but not as a posttreatment after the metabolic activation of APAP?5 The most obvious explanation, i.e., inhibition of metabolic activation, was convincingly ruled out by Liu et al.1 The authors also demonstrated that the responsiveness of Kupffer cells to endotoxin was not significantly altered 24 hours after treatment of mice with the neutropenia antibody.1 However, another possible explanation remains unexplored. It is well known that after treatment with a neutropenia-inducing antibody, most antibody-tagged neutrophils end up in hepatic sinusoids and are eventually removed by Kupffer cells.6 This leads to at least a temporary activation of these tissue macrophages.6 Therefore, we hypothesized that during this early phagocytosis of tagged neutrophils, inflammatory mediators may have been generated, which triggered the induction of protective genes.3 To address this issue, we treated C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) intraperitoneally with a dose of 250 μg and 100 μg per mouse with the anti-Gr-1 antibody (RB6-8C5; BD Biosciences, San Diego) at 24 hours and 4 hours before killing the animals. Controls received either identical doses of an isotype-matched rat IgG2b or saline (200 μl per mouse). The protocol was identical to the one described by Liu et al.1 Although there was no evidence of liver cell damage based on plasma ALT values, using real-time PCR as described,5 we found a moderate increase (200%-300% of baseline) of several cytokines and chemokines in the liver (data not shown) but also of heme oxygenase-1 (HO-1) (173 ± 24% of baseline; P < 0.05). However, most striking was the 14-fold and 35-fold increase of metallothionein 1 (MT-1) and MT-2 mRNA, respectively, in the anti-Gr-1–treated mice (Fig. 1A). Using a cadmium saturation assay,7 we confirmed that the increased mRNA levels translated into 18-fold higher MT protein levels in these livers (Fig. 1B). Metallothionein (MT) mRNA (A) and protein (B) expression in livers of C57BL/6 mice after treatment with the anti-Gr-1 antibody (doses: 250 μg and 100 μg per mouse) at 24 h and 4 h before sacrificing the animals. Controls received either identical doses of an isotype-matched rat IgG2b or saline (200 μl per mouse). The mRNA levels of MT-1 and MT-2 were determined by real-time RT-PCR and normalized to RNA levels of β-actin. The MT-to-actin ratio of saline treated animals was set as 100%. Hepatic MT protein levels were determined with the cadmium saturation assay and expressed as μg MT/g liver weight. All data represent means ± SE of n = 3 animals per group. *P < 0.05 (compared to the saline or IgG group) Because a previous report from one of us7 demonstrated a substantial aggravation of liver injury within 24 hours after APAP in MT-1/MT-2 knockout mice and a protection with prior induction of MT, our data raise the possibility that MT induction could have been involved in the mechanism of protection by the anti-Gr-1 antibody treatment. MT can act as powerful scavenger of reactive oxygen or nitrogen species,7 which are critical mediators of APAP-induced cell death.8 This conclusion does not preclude the additional involvement of HO-1 or other yet unidentified protective genes. Thus, our new data suggest that besides neutropenia, the induction of MT and other stress-response genes may have been contributing factors in the effect of anti-Gr-1 pretreatment for 24 hours. More detailed mechanistic studies are required to evaluate the contribution of neutrophils and of each individual gene in the pathophysiology. Our data also suggest that even beyond the recently published APAP studies1, 2 other conclusions based solely on anti-Gr-1 treatment, especially when neutropenia was induced 24 hours before the experiment, may not be as solid as previously assumed.9 Hartmut Jaeschke*, Jie Liu , * Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS, Laboratory of Comparative Carcinogenesis, NCI at NIEHS, Research Triangle Park, NC.