Research Of Kupffer Cell Function In Acetaminophen-induced Hepatotoxicity

liver is the largest organ in the body. Previously, the immune function of theliver has been neglected by scholars, in recent years, more and more research revealsthe important role of the liver in the immune system. Liver immune system consists ofinnate immunity, adaptive immunity and other components in circulation, and theinnate immunity is the most important one. Kupffer cell (KC) is an importantcomponent of innate immunity of liver. KC’ biological functions are very extensive,and they play an important role in hepatic immune defense. KC has a strongphagocytic, which can remove endotoxin and exogenous substances from thecirculation, also be used to remove the body cells, cell debris, old virus, bacteria,apoptosis cells and some tumor cells. Initiating inflammation after damage andinfection. KC are professional antigen presenting cells, which can activate helper Tcells involved in triggering the adaptive immune response. In addition, KC is also themost secretory activity cells, can secrete several active mediators to modulatehomeostasis, host resistance, inflammatory reaction. Notably, KC also tightly relatedwith a lot of liver disease occurrence and development.Acetaminophen (APAP), also konw as paracetamol, is one of the widely usedanalgesic and antipyretic drug. In American and European countries, about45%acuteliver failure patients caused by APAP overdose each year. In China, APAP is widelyused, except direct application, it also as an important component in other drugs,about80%of the approved clodrex containing APAP. Due to the lack of knowledge of medical and pharmaceutical cognitive, overdose or cross use ingredients containingAPAP drugs are still frequent. Leading to liver and kidney serious damage and evendeath. Previously, the mechanism of liver injury induced by APAP mainly focus onthe biochemical and metabolic events occurring in early stage. However, more andmore evidence that the innate immune response plays an important role in promotingand aggravation of liver injury induced by APAP.Significant effect of KC has been demonstrated in a variety of drug-induced liverdamage model including APAP. In inflammation, cell signals and damage associatedmolecular patterns released by damaged liver cells and dead cell can activating KC.Activated KC can release proinflammatory cytokines, chemokines, ROS and RNS tofurther promote the hepatic injury and inflammation. On the other hand, KC can alsorelease cytokines IL-6and anti-inflammatory cytokine IL-10, which havehepatoprotective effect. Therefore, the role of KC in hepatotoxicity induced by APAPin is very complex. In recent year, for evaluating the role of KC in APAP inducedinjury, scholars use gadolinium chloride and/or lipo-clodronate (CLO) to depletedKC in mice/rats’ livers and observe liver injury after APAP challenge. However, theexperimental conclusion is not unified, KC in APAP induced liver injury and in therepair of the injured are still controversial. The purpose of this study is to explore KCfunction in APAP-induced liver injury.In this experiment, we have to set up KC-defected-APAP-induced-liver-injuryanimal model and then we i.p. injected500mg/kg of APAP dissolved in PBS toinduce liver injury. KC were depleted by i.v. injection of150ul of liposomalclodronate (clodrosome, CLO，5mg/ml)24h prior to APAP challenge; liposome wasused as vehicle control. SerumALT and histology were used for liver damage analysis.Hepatic non-parenchymal cells and leukocytes were isolated and assessed for cellsubtypes by flowcytometry (FACS). Immunohistochemistry(IHC) of liver sections for F4/80were performed to identify KC and confirm CLO effects.Fluorescent-Quantitation Polymerase Chain Reaction Assay for Cytokines（TNF-α,IFN-γ, IL-6, IL-1-α, IL-1-β） and chemokines（CCL2，CXCL1）. IHC for PCNA andwestern blot for Cyclin D1were used for detecting proliferating hepatocytes. To roleout the effect of CLO on metabolism of APAP, hepatic GSH concentrations wasmeasured using the method of Tietze, CYP2E1detected by western blot.The results display KC were effectively depleted at24h after CLO treatment,which was confirmed by hepatic F4/80IHC staining and FACS data. CLO treatmenthad no effect on the numbers of hepatic NK, NKT, T and B cells. Compared withAPAP-treated control mice, KC depletion by CLO pretreatment conferred protectionagainst APAP-induced early liver injury at6h evidenced by significantly reducedlevels of serum ALT (1598±257U/L in Controls vs.808±164U/L in CLO, p<0.05)and centrilobular necrosis area (35.5±7.0%vs.14.5±7.7%, p<0.05). The findingshowed decreased hepatic infiltration of inflammatory monocytes (CD11b+，Ly6Chi),recrument marophages (CD11b+, F4/80+) and neutrophils (CD11b+，Ly6Ghi),and reduced hepatic mRNA expressions for chemokines (CCL2, CXCL1) andcytokines (IL-6, TNF-a, IFN-γ) in CLO-treated mice at6h after APAP injected.However, at24h after APAP, there were no significant differences of liver injury interms of serum ALT and liver histology between these two groups. Interestingly, thenumbers of hepatic inflammatory monocytes, macrophages and neutrophils inCLO-treated mice reached to the similar levels of APAP-treated control mice at24hafter APAP. APAP-induced liver injury was resolved by72h in both groups and nodifferences of serum ALT and liver histology were observed at48h and72h despitethe continued absence of KC in the liver of CLO-treated mice. Furthermore, thePCNA positive hepatocytes in the liver displayed no differences at24h,48h and72hafter APAP overdose. Hepatic CYP2E1proteins and GSH depletions after APAP exhibited no differences between control and CLO-treated mice.These findings suggest that KC contribute to the early development ofAPAP-induced liver injury by producing inflammatory chemokines/cytokines torecruit inflammatory monocytes, macrophages and neutrophils, but their role in thelate phase of liver injury and regeneration could be replaced by newly recruitedinflammatory monocytes/macrophages. In a word, our results indicate that KCpromoting injury in the early phase of APAP overdose, which provides a newresearch thought of early inflammatory damage control in APAP-induced livertoxicity. In addition, point out the close links between innate immunity and liverdrug-induced liver injury, and liver as an immune organ worthy of our continuedexploration.