The data shown in this report strongly support the newly emerging model that it is the tissue inflammation accompanying local tissue hypoxia that may represent the initial event in recruiting the tissue-protecting immunosuppressive mechanism.
It is shown that tissue hypoxia is associated with the accumulation of extracellular adenosine (28
), which then triggers A2AR and A2BR leading to the accumulation of immunosuppressive intracellular cAMP in activated immune cells (29
). Cyclic AMP, in turn, inhibits signaling pathways required for synthesis and secretion of pro-inflammatory and cytotoxic mediators by immune cells, terminates immune cells’ effector functions, and thereby protects remaining healthy tissues from continuous immune damage (reviewed in 9
This conclusion has implications for the regulation of immune response in general even though reported experiments have been performed in a model of acute liver inflammation. The pathogenesis of viral and autoimmune hepatitis is explained by activities of T-lymphocytes, tissue macrophages, and granulocytes which enhance tissue damage and impair the local microcirculation and blood supply by releasing inflammatory mediators, for example, IFN-γ and TNF-α. These processes mimicked in an acute model of Con A-induced liver injury are mediated by a T cell-initiated cascade of pro-inflammatory processes, which have been validated as reproducing the liver damage resembling autoimmune and viral hepatitis in detailed studies using different genetically altered mice, recombinant decoy cytokine receptors, and monoclonal antibodies (17
). These considerations led to the choice of this model and to testing the potentially tissue-protective role of hypoxia and endogenous adenosine in a standardized model of Con A-induced T cell activation and T cell-mediated tissue inflammation.
When animals were subjected to hypoxia of 10% instead of 21% oxygen for 8 h, Con A-induced hepatocellular damage was attenuated significantly as evidenced by the lower enzyme activities of the cytosolic enzyme ALT which has high organ specificity to the liver and also by lower levels of AST derived from the cell cytosol and mitochondria (31
). Similarly, the decrease was also seen in the activity of LDH in the blood. This result was expected as LDH levels were shown to reflect the degree of liver cell damage (32
). In addition, serum lactate concentrations were also reduced to the baseline levels of the sham-treated group when Con A-challenged mice were subjected to hypoxia. The measurements of lactate concentration are considered to be one of the most reliable biochemical parameters to determine the severity and outcome of acute liver failure and reflect the severity of liver injury (33
). Hyper-lactatemia develops under clinical conditions in approximately 80% of patients suffering from fulminant liver failure, for example, due to hepatitis-induced enhancement of hepatic and extrahepatic anaerobic glycolytic activity with higher lactate generation together with a reduced hepatocyte capacity and function to clear lactate, altogether resulting in a net hepatic lactate production (34
). In control, the hepatocellular integrity in the absence of Con A (sham) was not affected by hypoxia.
In addition to the suppression of liver damage, we found that serum cytokine levels were decreased significantly in mice subjected to hypoxia (). This result suggests that hypoxia can impair the initiation of inflammatory responses which eventually will lead to extensive liver damage. The decrease of cytokine levels such as IFN-γ, TNF-α, and IL-4 may, at least in part, explain the attenuation of liver damage by hypoxia, because these cytokines are shown to be essential in the induction of liver damage by Con A. Indeed, the importance of IFN-γ and TNF-α has been shown by the inhibition of Con A-induced liver damage after neutralization by anti-cytokine antibodies and in gene-deficient mice (17
). Furthermore, early IL-4 production from NKT cells is demonstrated to be essential in the induction of liver injury (23
). Therefore, the impairment of early IL-4 production by hypoxia may suggest inhibition of NKT cells activation by hypoxia-A2AR pathway as an additional inhibitory mechanism of Con A-induced liver injury. In correspondence, inhibition of NKT cells by an A2AR agonist is reported recently (36
As a major prerequisite to estimate the role of hypoxia and endogenous adenosine under inflammation, we confirmed first that exposure of the animals to 10% oxygen, with or without Con A treatment, resulted in decreased arterial blood pO2 values as compared with exposure to 21% oxygen. Hypoxic exposure (1.5 h) was paralleled by a significant increase of plasma concentrations of the purine nucleotide adenosine and its metabolites inosine and hypoxanthine in vehicle- and Con A-treated mice to the same degree, indicating that this increase was due to hypoxia treatment.
Hypoxia is associated with i) decrease in intracellular ATP; ii) increase in intracellular AMP; iii) inhibition of adenosine kinase; iv) accumulation of intracellular adenosine; and v) subsequent transport or diffusion of intracellular adenosine and accumulation of adenosine in extracellular space. The accumulation of extracellular adenosine from intracellular sources may be triggered by local tissue hypoxia that follows the excessive collateral immune damage to endothelial cells and microcirculation with ensuing interruption of normal blood and oxygen supply (9
). A recently described generation of extracellular adenosine by hypoxia-triggered ATPase/ADPase CD39 and 5′-ectonucleotidase CD73 represents another important pathway (37
). The CD39/ectonucleoside triphosphate diphosphohydrolase-type-1 (ENTPD1) is now recognized to be the key cell surface ectonucleotidase (39
The conditions of reduced oxygen delivery result in increased extracellular tissue concentrations of adenosine after hypoxic challenge in heart muscle (41
) and hippocampus (42
). We confirmed the elevation of adenosine concentration in the blood from healthy mice subjected to 10% oxygen for 1.5 h as compared with animals treated with 21% oxygen (). Observations of increased adenosine in the blood also were correlated with the proportional increase of its metabolite inosine. Because the increase of extracellular adenosine levels was observed in mice subjected to 10% oxygen in the absence of Con A (no liver damage according to liver enzyme assays) as well as after Con A injection, we conclude that the increase of adenosine is resulted from hypoxia even in the absence of tissue damage.
Because whole body hypoxia rendered mice resistant to the induction of liver injury, it had to be tested whether hypoxia-driven hepatoprotection is mediated by the hypoxia → extracellular adenosine → A2AR pathway or by yet unknown hypoxia → molecule “X” pathway. We hypothesized that if the anti-inflammatory mechanism triggered by hypoxia is dependent on the action of extracellular adenosine, then the antagonism of the A2AR or genetic deletion of A2AR in mice should inactivate the hypoxia-driven tissue protection. This, in turn, was expected to potentiate inflammatory liver damage even under hypoxic conditions (9
) because the hypoxia-induced accumulation of extracellular adenosine will not be transmitted further by A2AR and, therefore, liver tissues will be damaged continuously by inflammatory effectors (25
Here we show that the hepatoprotective effects of hypoxia during acute inflammation are abolished by pharmacological antagonism in mice treated with the selective antagonist ZM241385 as evidenced by significantly (P
< 0.01) elevated liver enzyme activities, lactate, and IFN-γ concentration. In agreement with these measurements, we also observed significantly higher mortality under hypoxia when the A2AR-mediated protection was antagonized (mortality: 10% oxygen Con A = 5.5% versus Con A + ZM = 33%, P
< 0.05). The lower survival rate emphasizes the importance of tissue hypoxia in control of liver tissue inflammation via adenosine and A2AR. Importantly, the genetic evidence for the crucial role of the A2AR in hypoxia-triggered hepatoprotection was provided by demonstrations of the aggravation of liver damage and inflammation even during hypoxia in mice with genetically deleted A2AR (). This indicates that the hypoxia → A2AR pathway is non-redundant and is critical in limiting liver inflammation as it is in lung inflammation (43
). Moreover, in contrast to our previous report testing hypoxia to control lung inflammation as initiated by bacterial toxin LPS (43
) which activates immune cells of innate immunity, here we provide evidence that Con A-induced T cell-dependent inflammatory liver damage is also under the control of hypoxia → A2AR pathway.
In conclusion, here we demonstrated that both hypoxia and A2AR are important in the downregulation of liver inflammation and that hypoxia and A2AR belong to the same non-redundant anti-inflammatory pathway. This could be an important and constitutive mechanism to control hepatic inflammation, because the liver is hypoxic even under normo-oxygenated conditions.