Endogenous interleukin-4 regulates glutathione synthesis following acetaminophen-induced liver injury in mice

Abstract

In a recent study, we reported that interleukin (IL)-4 had a protective role against acetaminophen (APAP)-induced liver injury (AILI), although the mechanism of protection was unclear. Here, we carried out more detailed investigations and have shown that one way IL-4 may control the severity of AILI is by regulating glutathione (GSH) synthesis. In the present studies, the protective role of IL-4 in AILI was established definitively by showing that C57BL/6J mice made deficient in IL-4 genetically (IL-4(-/-)) or by depletion with an antibody, were more susceptible to AILI than mice not depleted of IL-4. The increased susceptibility of IL-4(-/-) mice was not due to elevated levels of hepatic APAP-protein adducts but was associated with a prolonged reduction in hepatic GSH that was attributed to decreased gene expression of γ-glutamylcysteine ligase (γ-GCL). Moreover, administration of recombinant IL-4 to IL-4(-/-) mice postacetaminophen treatment diminished the severity of liver injury and increased γ-GCL and GSH levels. We also report that the prolonged reduction of GSH in APAP-treated IL-4(-/-) mice appeared to contribute toward increased liver injury by causing a sustained activation of c-Jun-N-terminal kinase (JNK) since levels of phosphorylated JNK remained significantly higher in the IL-4(-/-) mice up to 24 h after APAP treatment. Overall, these results show for the first time that IL-4 has a role in regulating the synthesis of GSH in the liver under conditions of cellular stress. This mechanism appears to be responsible at least in part for the protective role of IL-4 against AILI in mice and may have a similar role not only in AILI in humans but also in pathologies of the liver caused by other drugs and etiologies.

Figure 1

Interleukin-4 deficient mice were more susceptible than wild type mice to acetaminophen-induced death and liver injury. (A) Percent survival rates for WT and IL-4^−/− mice 24 h after APAP (200 mg/kg or 300 mg/kg) treatment. Data represent cumulative percent deaths following APAP treatment from two independent experiments (10 to 12 mice per group). *P<0.05 versus APAP-treated WT mice at the same dose. (B) Serum ALT activities in WT and IL-4^−/− mice before and after APAP (200 mg/kg) treatment. Results represent the mean ± SEM of 10 to 12 mice per group from two combined studies. *P<0.05 versus APAP-treated WT mice at the same time point. (C and D) WT mice were pre-treated with anti-IL-4 neutralizing antibody (anti-IL-4) or control antibody (IgG) 24 h prior to APAP (300 mg/kg) treatment. (C) serum IL-4 levels and (D) serum ALT activities were measured in WT mice treated with anti-IL-4+APAP or IgG+APAP. Results represent the mean ± SEM of 10 to 12 mice per group from two combined studies. *P<0.05 versus IgG+APAP-treated WT mice at the same time point.

Figure 2

Livers of interleukin-4 deficient mice showed more histopathological injury than wild type mice treated with acetaminophen. Representative photomicrographs of H&E stained liver sections from WT and IL-4^−/− mice before and after treatment with APAP (200 mg/kg). Liver sections of WT (A) and (B) IL-4^−/− mice before APAP treatment both exhibited normal liver architecture with no histopathological changes. (C) Liver section from a WT mouse at 8 h after APAP treatment showed moderate centrilobular coagulative necrosis with hemorrhaging at the lesion periphery. (D) Liver section from an IL-4^−/− mouse at 8 h after APAP treatment demonstrated marked to severe centrilobular coagulative necrosis with lesions expanding into the midzonal area, causing bridging between intact central veins along with moderate hemorrhaging in the lesion periphery, minimal ballooning hydropathy and moderate sinusoidal congestion. (E) Liver section from a WT mouse at 24 h after APAP treatment showed moderate to marked centrilobular coagulative necrosis with definite ballooning hydropathy at the lesion periphery and minimal sinusoidal congestion. (F) Liver section from an IL-4^−/− mouse at 24 h after APAP treatment was histopathologically similar to that of an 8 h APAP-treated IL-4^−/− mouse (D). CV = central vein. Original magnification, 40×.

Figure 3

Hepatic levels of protein adducts and glutathione were lower in interleukin-4 deficient mice than wild type mice after acetaminophen treatment. (A) Representative immunoblots showing APAP-protein adducts in livers (70 µg homogenate/lane) from WT and IL-4^−/− mice at 1 and 2 h after APAP (200 mg/kg) treatment. Immunoblots shows 2 mice per group, which are representative of a total of 5 mice used per group in this experiment. β-actin was used as a protein loading control. (B) Reduced GSH levels in APAP-treated WT and IL-4^−/− mice. Results for GSH analysis represent the mean ± SEM of 10 to 12 mice per group from two combined studies. † P<0.05 versus the earlier time point within the same treatment group. *P<0.05 versus APAP-treated WT mice at the same time point analyzed by ANOVA, while # P<0.05 versus WT mice at the same time point analyzed by Student’s t-test.

Figure 4

Hepatic levels of γ-glutamylcysteine ligase protein were lower in interleukin-4 deficient mice than wild type mice after acetaminophen treatment. (A) Representative immunoblot showing γ-GCL levels in the liver (100 µg homogenate/lane) of 2 mice per time point before and after APAP (200 mg/kg) treatment of WT and IL-4^−/− mice. β-actin was used as a protein loading control. (B) Densitometric analysis of γ-GCL protein levels from (A) normalized to β-actin levels. Results represent the mean ± SEM of 4 mice per group from two independent experiments. † P<0.05 versus the earlier time point within the same group. *P<0.05 versus WT mice at the same time point.

Figure 5

Hepatic levels of γ-glutamylcysteine ligase were lower in interleukin-4 deficient mice than wild type mice at 8 h after treatment with equivalent hepatotoxic doses of acetaminophen. APAP was administered to WT or IL-4^−/− mice at doses of 300 or 200 mg/kg, respectively. (A) ALT activities; results are representative of two independent experiments and represent the mean ± SEM of 5 mice per group. (B) Representative immunoblot showing hepatic levels (100 µg of homogenate/lane) of γ-GCL. Equal protein loading was confirmed by blotting for β-Actin.

Figure 6

Hepatic levels of γ-glutamylcysteine ligase mRNA and nuclear DNA binding activity of transcription factors were lower in livers of interleukin-4 deficient mice than wild type mice after acetaminophen treatment. WT and IL-4^−/− mice were treated with APAP (200 mg/kg). (A) qRT-PCR determination of γ-GCL mRNA levels. Enzyme-linked immunosorbant assay determinations of nuclear DNA binding activities of (B) Nrf-2 and (C) AP-1. Results represent the mean ± SEM of 7 to 9 mice per group. † P<0.05 versus the earlier time point within the same group. *P<0.05 versus APAP-treated WT mice at the same time point. Results represent the mean ± SEM of 6 mice per group. † P<0.05 versus the earlier time point within the same group. *P<0.05 versus APAP-treated WT mice at the same time point.

Figure 7

Recombinant IL-4 reduced susceptibility of interleukin-4 deficient mice to liver injury at 8 hours after acetaminophen treatment. IL-4^−/− mice were administered APAP (200 mg/kg) or saline vehicle, 3 h later mice were administered 100 pg of rIL-4 or vehicle (PBS, pH 7.4 containing 1% bovine serum albumin (PBS+BSA). (A) ALT activity; results represent the mean ± SEM of 6 mice per group. *P<0.05 versus APAP-treated mice. The APAP group was treated with APAP and PBS+BSA vehicle; the control group was treated with saline and PBS+BSA vehicles; the APAP+ rIL-4 group was treated with APAP and rIL-4; the rIL-4 group was treated with saline and rIL-4. Representative photomicrographs of H&E stained liver sections from mice treated with (B) APAP or (C) APAP + rIL-4 where n=6 for each group. The APAP + rIL-4 treatment group showed less centrilobular coagulative necrosis than the APAP treatment group. CV= central vein. Original magnification, 20×.

Figure 8

Recombinant IL-4 increased γ-glutamylcysteine ligase and GSH synthesis in interleukin-4 genetically deficient mice. IL-4^−/− mice were administered APAP (200 mg/kg) or saline vehicle, 3 h later, mice were administered 100 pg of rIL-4 or vehicle, PBS, pH 7.4 containing 1% bovine serum albumin (PBS+BSA). Eight hours after APAP treatment, hepatic (A) GSH levels and (B) γ-GCL mRNA levels were determined as was (C) densitometric analysis of γ-GCL protein levels analyzed by immunoblotting and normalized to β-actin levels. Results represent the mean ± SEM of 6 mice per group. *P<0.05 versus APAP-treated mice. The APAP group was treated with APAP and PBS+BSA vehicle; the control group was treated with saline and PBS+BSA vehicles; the APAP+ rIL-4 group was treated with APAP and rIL-4; the rIL-4 group was treated with saline and rIL-4.

Figure 9

Enhanced hepatic levels of phosphorylated-JNK2/1 in livers from IL-4^−/− mice relative to levels seen in WT mice after APAP-treatment. Liver homogenates were isolated from WT and IL-4^−/− mice before and after APAP treatment (200 mg/kg) and analyzed (100 µg/lane) by immunoblotting. Representative immunoblots of phosphorylated (P)-p54 JNK2 and P-p46 JNK1 protein levels. (A) P-p54 JNK2 and P-p46 JNK1 protein levels in livers from WT and IL-4^−/− mice before and after APAP. β-actin was used as a protein loading control. Scanned intensity for (B) P-p54 JNK2 and (C) P-p46 JNK1 normalized to β-actin levels. Results are presented as the mean ± SEM of 6 mice per group. Results represent the mean ± SEM of 6 mice per group. † P<0.05 versus the earlier time point within the same group. *P<0.05 versus APAP-treated WT mice at the same time point.