Antileukoproteinase protects against hepatic inflammation, but not apoptosis in the response of D-galactosamine-sensitized mice to lipopolysaccharide

Abstract

Background and purpose: There is major evidence for the strong bi-directional interrelation of parenchymal cell apoptosis and leukocyte accumulation and inflammation in acute liver injury. Therefore, the aim of this in vivo study was to investigate the anti-apoptotic and anti-inflammatory potential of antileukoproteinase (ALP) in a murine model of acute liver failure.

Experimental approach: C57BL/6J mice were given galactosamine (D-GalN) and E. coli lipopolysaccharide (LPS) followed by administration of saline or ALP. Besides survival rate, hepatic tissue damage and inflammatory response were analyzed by intravital fluorescence microscopy 6 hours after treatment. In addition, immunohistochemical analysis of NFkappaB-p65 and hepatocellular apoptosis, plasma levels of AST/ALT, TNF-alpha and IL-10 were determined.

Key results: Administration of D-GalN/LPS provoked hepatic damage, including marked leukocyte recruitment and microvascular perfusion failure, as well as hepatocellular apoptosis and enzyme release. NFkappaB-p65 became increasingly detectable in hepatocellular nuclei, accompanied by a rise of TNF-alpha and IL-10 plasma levels. ALP markedly reduced intrahepatic leukocyte accumulation, nuclear translocation of NFkappaB and plasma levels of TNF-alpha and IL-10. Moreover, liver enzyme levels indicated the absence of necrotic parenchymal cell death. In contrast, ALP failed to block both apoptosis and caspase-3 levels and the mortality rate of ALP-treated animals was comparable to that of saline-treated mice.

Conclusions and implications: ALP could effectively prevent D-GalN/LPS-associated intrahepatic inflammatory responses by inhibition of NFkappaB activity, but not apoptosis-driven mortality. Thus, a protease-inactivating approach such as application of ALP seems to be inadequate in damaged liver where apoptosis represents the predominant mode of cell death.

Figure 1

Representative intravital fluorescence microscopic images (upper panels, original magnification × 424) of liver tissue after staining of leukocytes with rhodamine6G (a, b) and after contrast enhancement with sodium fluorescein (c) as well as quantitative analysis of sinusoidal leukocyte stasis (a), venular leukocyte adherence (b) and sinusoidal perfusion (c). For induction of acute liver injury, animals were injected with D-GalN (720 mg kg⁻¹ i.p.) and LPS (10 μg kg⁻¹ i.p.) and received either saline (G-L) or ALP (15 mg kg⁻¹; G-L/ALP). Control animals received isotonic saline only (C). Values are given as means±s.e.m; ANOVA and Dunn's test; ^*P<0.05 vs C; ^#P<0.05 vs G-L.

Figure 2

Representative Western blot analysis (a) and densitometry analysis (b) of cleaved caspase-3 protein expression. Signals were corrected with that of β-actin serving as internal control. For induction of acute liver injury, animals were injected with D-GalN (720 mg kg⁻¹ i.p.) and LPS (10 μg kg⁻¹ i.p.) and received either saline (G-L) or ALP (15 mg kg⁻¹, G-L/ALP). Control animals received isotonic saline only (C). Values are given as means±s.e.m.; ANOVA and Dunn's test; ^*P<0.05 vs C.

Figure 3

Quantitative analysis of TUNEL histochemistry, shown as number of TUNEL-positive cells per mm² differentiating between hepatocellular (a) and non-parenchymal cell apoptosis (b) as well as representative images of a saline- (c), G-L- (d) and G-L/ALP-treated animal (e). Bars represent 50 μm. For induction of acute liver injury, animals were injected with D-GalN (720 mg kg⁻¹ i.p.) and LPS (10 μg kg⁻¹ i.p.) and received either saline (G-L) or ALP (15 mg kg⁻¹; G-L/ALP). Control animals received isotonic saline only (C). Values are given as means±s.e.m.; ANOVA and Dunn's test; ^*P<0.05 vs C.

Figure 4

Plasma activities of ALT (a) and AST (b) at 4 and 6 h after induction of acute liver injury. Animals were injected with D-GalN (720 mg kg⁻¹ i.p.) and LPS (10 μg kg⁻¹ i.p.) and received either saline (G-L) or ALP (15 mg kg⁻¹; G-L/ALP). Control animals received isotonic saline only (C). Values are given as means±s.e.m.; ANOVA and Dunn's test; ^*P<0.05 vs C. ^#P<0.05 vs G-L at the respective time points. For analysis of survival rate (c), mortality was assessed every hour during the first 24 h and every 12 h over a period of 5 days.

Figure 5

Quantitative analysis of NFκB-p65 immunohistochemistry, shown as number of NFκB-p65-positive hepatocytes per mm² (a), and representative images of a saline- (b), G-L- (c) and G-L/ALP-treated animal (d). Bars represent 50 μm. For induction of acute liver injury, animals were injected with D-GalN (720 mg kg⁻¹ i.p.) and LPS (10 μg kg⁻¹ i.p.) and received either saline (G-L) or ALP (15 mg kg⁻¹; G-L/ALP). Control animals received isotonic saline only (C). Values are given as means±s.e.m.; ANOVA and Dunn's test; ^*P<0.05 vs C, ^#P<0.05 vs G-L.

Figure 6

Plasma concentrations of TNF-α (a), IL-10 (b) and IL-5 (c). For induction of acute liver injury, animals were injected with D-GalN (720 mg kg⁻¹ i.p.) and LPS (10 μg kg⁻¹ i.p.) and received either saline (G-L) or ALP (15 mg kg⁻¹; G-L/ALP). Control animals received isotonic saline only (C). Values are given as means±s.e.m.; ANOVA and Dunn's test; ^*P<0.05 vs C, ^#P<0.05 vs G-L.