Critical role of hypoxia and A2A adenosine receptors in liver tissue-protecting physiological anti-inflammatory pathway

Abstract

Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene deleted mice to low oxygen containing inspired gas mixture allowed the investigation of the mechanism that controls inflammatory liver damage and protects the liver using a mouse model of T cell-mediated viral and autoimmune hepatitis. We tested the hypothesis that the inflammatory tissue damage-associated hypoxia and extracellular adenosine --> A2AR signaling plays an important role in the physiological anti-inflammatory mechanism that limits liver damage during fulminant hepatitis. After induction of T cell-mediated hepatitis, mice were kept in modular chambers either under normoxic (21% oxygen) or hypoxic (10% oxygen) conditions for 8 h. It was shown that the whole body exposure to hypoxic atmosphere caused tissue hypoxia in healthy animals as evidenced by a decrease in the arterial blood oxygen tension and increase of the plasma adenosine concentration (P < 0.05). This "hypoxic" treatment resulted in significantly reduced hepatocellular damage and attenuated levels of serum cytokines in mice with acute liver inflammation. The anti-inflammatory effects of hypoxia were not observed in the absence of A2AR in studies of A2AR gene-deficient mice or when A2AR have been pharmacologically antagonized with synthetic antagonist. The presented data demonstrate that total body hypoxia-triggered pathway provides protection in acute hepatitis and that hypoxia (upstream) and A2AR (downstream) function in the same immunosuppressive and liver tissue-protecting pathway.

Figure 1

Effects of Con A injection on serum liver enzyme activities in the absence and presence of A2 receptor antagonist ZM241385. Serum activities (I.U.) of alanine aminotransferase (ALT, Figure 1A), aspartate aminotransferase (AST, Figure 1B), and lactate dehydrogenase (LDH, Figure 1C) were determined 8 h after Con A (11.5 mg/kg i.v.) in the presence and absence of ZM241385 (10 mg/kg s.c. × 2). Sham treated animals had low and physiological liver enzyme activities (I.U.): Con A Sham: ALT 100 ± 10, AST 110 ± 10, LDH 150 ± 60; ZM241385 Sham: ALT 110 ± 15, AST 100 ± 10, LDH 160 ± 50 (Means ± SEM, n = 4 per group, paired t-test; + P < 0.05, * P < 0.01).

Figure 2

Hypoxia (10% O₂) reduces serum liver enzyme activities after Con A injection. As compared with the release of liver enzymes after Con A challenge in normo-oxygenated atmosphere (filled dark bars), hypoxia for 8 h after Con A injection (bright gray bars) results in lower liver enzyme activities of ALT, AST, and LDH. Sham 10%: ALT 110 ± 10, AST 110 ± 10, LDH 160 ± 84 (Means ± SEM, n = 9–11 per group, paired t-test; + P < 0.05; **P < 0.001).

Figure 3

Effects of pharmacological antagonism (ZM241385) or genetic “knockout” of A2AR gene on the hepatoprotective effects mediated by hypoxia. Mice of A2AR “wild type” (A2AR^+/+) either without (left black bar) or with ZM241385 (bright gray bar) and homozygotic A2AR “knock-out” mice (A2AR^−/−) (right dark gray bar) were subjected to Con A (11.5 mg/kg i.v.) injection and placed into hypoxic chambers (10% oxygen) for 8 h thereafter. Means ± SEM, n = 11–13 per group; paired t-test versus WT without ZM241385; + P < 0.05, *P < 0.01.

Figure 4

Effects of antagonism at the A2AR site on the mortality after Con A induced hepatitis under hypoxia. To verify the clinical effects of the antagonism of the hypoxia- > adenosine- > hepatoprotection pathway, mice were injected by Con A (11.5 mg/kg) and placed into 10% oxygen atmosphere for 8 h maximally. A2AR^+/+ wild type mice treated with ZM241385 revealed a significantly higher mortality with death mostly between 6–8 h after Con A injection. Differences between survival rates were tested by Chi-square test, P < 0.05.