Complement activation in acetaminophen-induced liver injury in mice

Abstract

Overdose with acetaminophen (APAP) results in acute liver failure in humans and experimental animals. Complement comprises more than 30 proteins that can participate in tissue injury and/or repair, but the role of complement activation in APAP-induced hepatotoxicity has not been evaluated. Treatment of male, C57BL6J mice with APAP (200-400 mg/kg) resulted in liver injury as evidenced by increased activity of alanine aminotransferase (ALT) in plasma and hepatocellular necrosis. Plasma concentration of the complement component C3 was significantly reduced 6 h after treatment with APAP, indicating complement activation, and C3b (detected by immunostaining) accumulated in the centrilobular areas of liver lobules. Pretreatment with cobra venom factor (CVF; 15 U/mouse) to deplete complement components abolished APAP-mediated C3b accumulation, and this was accompanied by reductions in plasma ALT activity, hepatocellular necrosis, hepatic neutrophil accumulation, and expression of inflammatory genes (interleukin-6, interleukin-10, and plasminogen activation inhibitor-1) at 24 h after APAP treatment. Loss of hepatocellular GSH was similar in APAP-treated mice pretreated with either saline or CVF, suggesting that CVF pretreatment did not affect APAP bioactivation. Mice with a genetic deficiency in C3 had reduced ALT activity 6 and 12 h after APAP administration compared with wild-type animals. These results reveal a key role for complement activation in hepatic inflammation and progression of injury during the pathogenesis of APAP-induced hepatotoxicity.

Fig. 1.

C3 concentration in plasma from mice treated with APAP. Mice were treated with saline vehicle (0 APAP) or APAP (200, 300, or 400 mg/kg) after treatment with saline or CVF as described under Materials and Methods. C3 concentration in plasma was determined 6 h (left) or 24 h (right) later. Data are expressed as mean ± S.E.M. *, significantly different from respective value in the absence of APAP. #, significantly different from respective value in the absence of CVF. n = 3–5 per group, p < 0.05.

Fig. 2.

Hepatic C3b deposition. Mice were treated with saline vehicle (0 APAP) or APAP (300 mg/kg) after treatment with saline or CVF as described under Materials and Methods. Twenty four hours later livers were harvested and frozen. Frozen liver sections were immunostained with C3b antibody as described under Materials and Methods. Arrows represent C3b in the centrilobular area. Images were acquired at 100× magnification using an Olympus IX-70 fluorescent microscope.

Fig. 3.

GSH concentration in livers from APAP-treated mice. GSH was determined 1 h after APAP (300 mg/kg) or saline administration in mice pretreated with CVF or saline vehicle as described under Materials and Methods. Data represent mean ± S.E.M. (n = 4 animals per group). *, significantly different from respective value in the absence of APAP (p < 0.05).

Fig. 4.

Liver injury after APAP treatment. Mice were treated with saline vehicle (0 APAP) or APAP (200, 300, or 400 mg/kg) after treatment with saline or CVF as described under Materials and Methods. A, plasma ALT activity at 6 h (left) or 24 h (right) after APAP. B, representative images from hematoxylin and eosin-stained liver sections from livers harvested at 24 h. a to d, treatment with APAP [0 (vehicle) (a), 200 (b), 300 (c), or 400 (d) mg/kg)] after treatment with saline. e to h, treatment with APAP [0 (vehicle) (e), 200 (f), 300 (g), or 400 (h) mg/kg)] after treatment with CVF. C, ALT activity in plasma at 6, 12, or 24 h after treatment with CVF or saline and APAP (300 mg/kg) or saline vehicle as described under Materials and Methods. Values for saline/saline are obscured by values for CVF/saline. Data are expressed as mean ± S.E.M. *, significantly different from respective value in the absence of APAP. #, significantly different from respective value in the absence of CVF. n = 3–5 per group, p < 0.05.

Fig. 5.

Liver injury in C3(−/−) mice treated with APAP. A, plasma ALT activity at 6 or 12 h after APAP (400 mg/kg) administration. Data are expressed as mean ± S.E.M. *, significantly different from wild type at the indicated time (p < 0.05). n = 3–5 per group. B, representative liver sections stained with hematoxylin and eosin. Wild-type (a) or C3(−/−) (b) mice were treated with APAP (400 mg/kg), and livers were harvested 12 h after APAP administration.

Fig. 6.

Hepatic neutrophil (PMN) accumulation after APAP administration. A, representative liver sections from mice treated with saline (a) or CVF (b) then given APAP (300 mg/kg); livers were harvested 24 h after treatment with APAP. Liver sections were immunohistochemically stained for infiltrating PMNs (red chromagen; arrows) and counterstained with hematoxylin as described under Materials and Methods. B, morphometric determination of PMN accumulation in sections of liver 6 or 24 h after APAP treatment. Data represent mean ± S.E.M. of PMNs counted in 10 microscopic fields at 100× magnification. *, significantly different from respective value in the absence of APAP. #, significantly different from respective value in the absence of CVF.

Fig. 7.

Cytokine mRNA expression or protein concentration after APAP administration. Mice were pretreated with CVF or saline then given APAP (300 mg/kg) or saline vehicle. A, mRNA expression of IL-6 and IL-10 was determined in livers harvested 24 h after APAP administration. Expression levels were normalized to that of the GAPDH housekeeping gene and represented as percentage of saline control. B, TNF-α concentration in plasma was determined 6 or 12 h after administration of APAP. C, PAI-1 concentration in plasma was determined 6 or 24 h after administration of APAP. Data represent mean ± S.E.M. *, significantly different from respective value in the absence of APAP. #, significantly different from respective value in the absence of CVF. n = 3–5 per group; p < 0.05.

Fig. 8.

Hepatocellular viability after APAP treatment. Mice were pretreated with CVF or saline then given APAP (300 mg/kg) and killed 24 or 48 h after APAP administration. A, plasma ALT activity measurement. B, hepatic mRNA expression 24 h after APAP administration. Expression levels were normalized to that of the GAPDH housekeeping gene and represented as percentage of Sal/Sal (not shown) control. C, mice pretreated with CVF or saline and then treated with APAP (300 mg/kg) or saline were given BrdU (50 mg/kg) 2 h before euthanasia at 24 or 48 h after APAP administration. Liver sections were immunohistochemically stained for nuclear incorporation of BrdU (brown chromagen) and counterstained with hematoxylin as described under Materials and Methods. a and b, mice treated with Sal/APAP with their livers harvested at 24 h (a) and 48 h (b). c and d, mice treated with CVF/APAP with their livers harvested at 24 h (c) and 48 h (d). D, morphometric determination of BrdU-positive cells in section of livers 24 or 48 h after APAP treatment. Data represent mean ± S.E.M. of BrdU-positive cells counted in 10 microscopic fields at 100× magnification. In A, B, and D, data represent mean ± S.E.M. *, significantly different from respective value in the absence of APAP. #, significantly different from respective value in the absence of CVF. n = 3–5 per group; p < 0.05.