Zhang-Xu Liu

Neutrophil depletion protects against murine acetaminophen hepatotoxicity

We previously reported that liver natural killer (NK) and NKT cells play a critical role in mouse model of acetaminophen (APAP)‐induced liver injury by producing interferon gamma (IFN‐γ) and modulating chemokine production and subsequent recruitment of neutrophils into the liver. In this report, we examined the role of neutrophils in the progression of APAP hepatotoxicity. C57BL/6 mice were given an intraperitoneal toxic dose of APAP (500 mg/kg), which caused severe acute liver injury characterized by significant elevation of serum ALT, centrilobular hepatic necrosis, and increased hepatic inflammatory cell accumulation. Flow cytometric analysis of isolated hepatic leukocytes demonstrated that the major fraction of increased hepatic leukocytes at 6 and 24 hours after APAP was neutrophils (Mac‐1+Gr‐1+). Depletion of neutrophils by in vivo treatment with anti‐Gr‐1 antibody (RB6‐8C5) significantly protected mice against APAP‐induced liver injury, as evidenced by markedly reduced serum ALT levels, centrilobular hepatic necrosis, and improved mouse survival. The protection was associated with decreased FasL‐expressing cells, cytotoxicity against hepatocytes, and respiratory burst in hepatic leukocytes. In intracellular adhesion molecule (ICAM)‐1–deficient mice, APAP caused markedly reduced liver injury when compared with wild‐type mice. The marked protection in ICAM‐1–deficient mice was associated with decreased accumulation of neutrophils in the liver. Hepatic GSH depletion and APAP‐adducts showed no differences among the antibody‐treated, ICAM‐1–deficient, and normal mice. In conclusion, accumulated neutrophils in the liver contribute to the progression and severity of APAP‐induced liver injury. (HEPATOLOGY 2006;43:1220–1230.)

Immune-mediated drug-induced liver disease

Drug-induced immune-mediated hepatic injury is an adverse immune response against the liver that results in a disease with hepatitic, cholestatic, or mixed clinical features. Drugs such as halothane, tienilic acid, dihydralazine, and anticonvulsants trigger a hepatitic reaction, and drugs such as chlorpromazine, erythromycins, amoxicillin-calvulanic acid, sulfonamides and sulindac trigger a cholestatic or mixed reaction. Unstable metabolites derived from the metabolism of the drug may bind to cellular proteins or macromolecules, leading to a direct toxic effect on hepatocytes. Protein adducts formed in the metabolism of the drug may be recognized by the immune system as neoantigens. Immunocyte activation may then generate autoantibodies and cell-mediated immune responses, which in turn damage the hepatocytes. Cytochromes 450 are the major oxidative catalysts in drug metabolism, and they can form a neoantigen by covalently binding with the drug metabolite that they produce. Autoantibodies that develop are selectively directed against the particular cytochrome isoenzyme that metabolized the parent drug. The hapten hypothesis proposes that the drug metabolite can act as a hapten and can modify the self of the individual by covalently binding to proteins. The danger hypothesis proposes that the immune system only responds to a foreign antigen if the antigen is associated with a danger signal, such as cell stress or cell death. Most clinically overt adverse hepatic events associated with drugs are unpredictable, and they have intermediate (1 to 8 weeks) or long latency (up to 12 months) periods characteristic of hypersensitivity reactions. Immune-mediated drug-induced liver disease nearly always disappears or becomes quiescent when the drug is removed. Methyldopa, minocycline, and nitrofurantoin can produce a chronic hepatitis resembling AIH if the drug is continued.

NK Cells Cause Liver Injury and Facilitate the Induction of T Cell-Mediated Immunity to a Viral Liver Infection

NK cells are a relatively rare cell population in peripheral lymphoid organs but are abundant in the liver, raising questions as to their function in immune responses to infections of this organ. To investigate this, cell-mediated immunity to viral liver infection induced by a type 5, replication-defective, adenovirus was examined. It is shown that NK cells in the absence of T cells cause hepatocyte apoptosis in virus-infected livers associated with an increase in liver enzymes in the serum. Concomitantly, NK cells induce production of IFN-γ, inhibitable by their elimination before infection. NK cells are shown to be necessary for optimal priming of virus-specific T cells, assessed by delayed-type hypersensitivity response and CTL activity, consistent with their ability to secrete IFN-γ. The conclusion is drawn that NK cells mediate two important functions in the liver: they induce cell death in the infected organ and concomitantly stimulate the induction of T cell-mediated immunity by release of IFN-γ.

Role of innate immunity in acetaminophen-induced hepatotoxicity

Acetaminophen (APAP) hepatotoxicity is currently the single most important cause of acute liver failure in the US, and is associated with a significant number of deaths. The toxic response to APAP is triggered by a highly reactive metabolite N-acetyl-p-benzoquinone-imine. Following the hepatocellular initiation events, such as glutathione depletion and covalent binding, intracellular stress simultaneously activates signal transduction and transcription factor pathways that are protective or toxic (directly or through sensitisation). Subsequently, pro- and anti-inflammatory cascades of the innate immune system are simultaneously activated, the balance of which plays a major role in determining the progression and severity of APAP-induced hepatotoxicity. The threshold and susceptibility to APAP hepatotoxicity is determined by the interplay of injury promoting and inhibiting events downstream of the initial production of toxic metabolite. The environmental and genetic control of these intracellular and intercellular responses to toxic metabolites may be of critical importance in determining susceptibility to APAP hepatotoxicity and presumably idiosyncratic drug hepatotoxicity.

Toll-like receptor 4 mediates alcohol-induced steatohepatitis through bone marrow-derived and endogenous liver cells in mice

BACKGROUND Excessive alcohol intake causes an increase in intestinal permeability that induces translocation of gut-derived lipopolysaccharide (LPS) to the portal vein. Increased LPS in the portal vein stimulates Kupffer cells through Toll-like receptor (TLR) 4 in the liver. Activated TLR4 signaling in Kupffer cells induces various inflammatory mediators including TNF-α, IL-1β, and reactive oxygen species, resulting in liver injury. Hepatic stellate cells (HSCs) also express TLR4. This study investigates whether TLR4 on bone marrow (BM)-derived cells, including Kupffer cells, or non-BM-derived endogenous liver cells, including HSCs, contributes to the progression of alcohol-induced steatohepatitis and fibrogenesis in mice. METHODS TLR4 BM chimera (wild-type [WT] mice with TLR4(-/-) BM or TLR4(-/-) mice with WT BM) were generated by the combination of liposomal clodronate injection with whole body irradiation and BM transplantation, followed by treatment with intragastric alcohol feeding. RESULTS WT mice transplanted with WT BM exhibited liver injury, steatosis, inflammation, and a fibrogenic response. Conversely, TLR4(-/-) mice with TLR4(-/-) BM displayed less steatosis, liver injury, and inflammation. Notably, steatosis, macrophage infiltration, and alanine aminotransferase levels in both TLR4-chimeric mice showed intermediate levels between WT mice transplanted with WT BM and TLR4(-/-) mice transplanted with TLR4(-/-) BM. Hepatic mRNA expression of fibrogenic markers (collagen α1(I), TIMP1, TGF-β1) and inflammatory cytokines (IL-1β, IL-6) were markedly increased in WT mice with WT BM, but there was less of an increase in both TLR4-chimeric mice and in TLR4(-/-) mice transplanted with TLR4(-/-) BM. CONCLUSIONS TLR4 signaling in both BM-derived and non-BM-derived liver cells is required for liver steatosis, inflammation, and a fibrogenic response after chronic alcohol treatment.

Fas-Mediated Apoptosis Causes Elimination of Virus-Specific Cytotoxic T Cells in the Virus-Infected Liver

Immunity to allogeneic MHC Ags is weak in rodent livers, raising questions as to the mechanisms that might control responses in this organ. Infection with an adenovirus vector reveals that T cell-mediated immunity to nonself-Ags in the liver is self-limiting. Virus-induced liver injury decreases and coincides with disappearance of virus-specific CTL, concomitant to an increase of apoptotic T cells early after infection. But whereas death in CD4 cells is independent of Fas, perforin, and TNF-α, that of CD8 cells requires Fas and not perforin or TNF-α pathways. Fas ligand is expressed on liver-infiltrating cells, pointing to death by fratricide that causes almost complete disappearance of virus-specific CTL 4 wk after infection. CTL elimination is virus dose dependent, and high doses induced high alanine aminotransferase values, elevated expression of Fas ligand on CD8 cells, and increased CD8 cell migration into the infected liver.

IP-10 and Mig facilitate accumulation of T cells in the virus-infected liver.

Viral infection of the liver causes accumulation of T cells in the infected organ, raising the question as to the signals that mediate this response. Employing an adenovirus induced hepatitis model in mice, we show that IP-10 and Mig are essential for T cell recruitment and that induction of the two chemokines occurs concomitant to production of IFNgamma. It is shown that while IFNgamma induces IP-10 and Mig in hepatocytes, for optimal chemokine induction, a co-stimulatory signal mediated by cross-linking of Fas on hepatocytes is required. Moreover, cross-linking of Fas by injection of anti-Fas antibody into mice triggers induction of IP-10 and Mig in the liver. The cells providing the two signals are shown to express NK1.1 and AsGM1; elimination of these cells leads to inhibition of IFNgamma and chemokine transcript induction. The conclusion is drawn that both NK cells and T cells provide the two signals for induction of IP-10 and Mig in the liver.

A Cell Surface ADP-ribosyltransferase Modulates T Cell Receptor Association and Signaling

ART-1, a cell surface ADP-ribosyltransferase, is imbedded in the membrane by a glycosylphosphatidylinositol anchor. Function of this enzyme in mouse T lymphocytes is to transfer ADP-ribose groups from NAD to arginine residues, exposed on the extracellular domain of cell surface molecules. As a consequence, T cell responses are modulated. To explore the precise action of the enzyme, the T cell lymphoma EL-4 was transfected with the ART-1 gene, and its effects were examined. It is shown that ART-1 ADP-ribosylates distinct cell surface molecules, causing inhibition of T cell receptor signaling, concomitant to suppression of p56 lck kinase activation. These effects are explained by failure of T cell receptors and co-receptors to associate into a contiguous and functional receptor cluster.

Mechanisms of Adaptation and Progression in Idiosyncratic Drug Induced Liver Injury, Clinical Implications

In the past decade our understanding of idiosyncratic drug induced liver injury (IDILI) and the contribution of genetic susceptibility and the adaptive immune system to the pathogenesis of this disease process has grown tremendously. One of the characteristics of IDILI is that it occurs rarely and only in a subset of individuals with a presumed susceptibility to the drug. Despite a clear association between single nucleotide polymorphisms in human leukocyte antigen (HLA) genes and certain drugs that cause IDILI, not all individuals with susceptible HLA genotypes develop clinically significant liver injury when exposed to drugs. The adaptation hypothesis has been put forth as an explanation for why only a small percentage of susceptible individuals develop overt IDILI and severe injury, while the majority with susceptible genotypes develop only mild abnormalities that resolve spontaneously upon continuation of the drug. This spontaneous resolution is referred to as clinical adaptation. Failure to adapt or defective adaptation leads to clinically significant liver injury. In this review we explore the immuno‐tolerant microenvironment of the liver and the mechanisms of clinical adaptation in IDILI with a focus on the role of immune‐tolerance and cellular adaptive responses.

Extracellular Nicotinamide Adenine Dinucleotide Induces T Cell Apoptosis In Vivo and In Vitro

Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3+, CD4+, and CD8+ cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69+ T cells.