Circulating adenosine increases during human experimental endotoxemia but blockade of its receptor does not influence the immune response and subsequent organ injury

Sepsis, the systemic inflammatory response syndrome that occurs during infection, is associated with considerable morbidity and mortality in non-cardiac intensive care units [1]. During sepsis, the initial inflammatory response can be overwhelming, leading to significant collateral damage to normal tissues.

During systemic inflammation, the extracellular concentration of the endogenous nucleoside adenosine increases rapidly [2, 3], with concentrations increasing up to 10-fold in septic shock patients [2]. Animal studies have shown that subsequent stimulation of adenosine receptors, mainly the adenosine A_2A receptor, on various immune cells potently reduces the inflammatory response [4, 5]. In humans, however, evidence that adenosine can limit the inflammatory response or prevent tissue injury is limited [6].

Interestingly, a genetic loss-of-function variant of the enzyme adenosine monophosphate deaminase (AMPD1) was recently shown to improve prognosis in patients with coronary artery disease [7], most likely because of augmented adenosine formation during ischemia in these patients [8]. It is unknown whether subjects with this polymorphism have an altered immune response or whether these individuals are protected from inflammation-induced organ injury.

In the present study, we addressed three major questions, illustrated in Figure 1. First, does systemic inflammation induced by experimental human endotoxemia increase the circulating adenosine concentration in vivo? Second, does this enhanced increase in circulating adenosine modulate the innate immune response? Third, does this increase reduce end-organ damage? We addressed these questions in healthy volunteers after systemic administration of lipopolysaccharide (LPS) with or without concomitant administration of the adenosine receptor antagonist caffeine. In addition, we separately studied healthy volunteers with the 34C > T variant of the AMPD1 gene to test the third hypothesis (that is, that the inflammation-induced increase in circulating adenosine is augmented and organ damage is attenuated in these subjects).

In the present study, we show for the first time that acute systemic inflammation induced by human experimental endotoxemia results in an increase in circulating endogenous adenosine in humans in vivo. Apparently, the systemic inflammatory response during experimental endotoxemia is sufficient to stress the body to a level that induces adenosine release. These results are in accordance with those of previous findings demonstrating increased plasma adenosine concentrations in humans with septic shock [2, 3, 20]. We found no evidence that circulating adenosine exerted immune modulatory effects or tissue-protective effects during inflammation. Pretreatment with the adenosine receptor antagonist caffeine did not potentiate the inflammatory response or the inflammation-induced subclinical organ damage, suggesting that this increased adenosine concentration does not act as a negative feedback signal to temper inflammation and organ damage in this model.

Previous in vitro and animal studies have provided robust evidence that endogenous adenosine plays a pivotal role in the limitation of excessive tissue injury in situations of inflammation, mainly by activation of adenosine A_2A receptor [5]. In humans in vivo, however, data on the effect of inflammation on the endogenous adenosine concentration are limited to only one small study in which the plasma adenosine concentration was significantly higher in patients with septic shock compared with control patients [2].

In this study, we studied the effect of inflammation on circulating adenosine in a well-validated model of systemic inflammation [21] and used a previously described method to measure the plasma adenosine concentration [15]. Our results show that, during endotoxemia, the endogenous adenosine concentration increases in time, with a maximum concentration reached 2 hours after LPS administration. Recently, measuring circulating adenosine in 10 septic shock patients who were admitted to the intensive care unit, we found a median (IQR) adenosine concentration of 30.9 [24.1 to 39.8] ng/mL (BPR, NPR, PvdB, JGvdH, PS, and PP, unpublished observations). The adenosine concentration was lower in the LPS-treated volunteers, probably indicating that the less severe and shorter duration of the inflammatory response during experimental endotoxemia induces a smaller insult compared with septic shock. In addition, in septic shock patients, not only the inflammatory response but also tissue hypoperfusion may play a role in the formation of adenosine. We subsequently aimed to demonstrate that this increased circulating adenosine could act as a negative feedback molecule, which attenuates the inflammatory response and ameliorates end-organ dysfunction. To this end, subjects were pretreated with the nonselective adenosine receptor antagonist caffeine [22] in a dose previously shown to completely block the cardiovascular effects of adenosine [13]. Subjects were asked to refrain from caffeine ingestion for the 48-hour period prior to the experiment in order to reveal any effects of adenosine receptor stimulation [23]. At the moment of LPS administration, the plasma caffeine concentration averaged 6.0 mg/L, which is a concentration previously shown to effectively antagonize adenosine receptor stimulation [14, 24]. In more detail, we recently showed that an intravenous dose of caffeine of 4 mg/kg, similar to the dose of the present study, completely blunted ischemic preconditioning, which is mediated by adenosine receptor stimulation [13]. In addition, our group has demonstrated, in the past, that caffeine in a plasma concentration of 5 mg/L significantly antagonizes the hemodynamic effects of adenosine administration [14].

Previous studies in animal models have shown that caffeine is able to potentiate the production of pro-inflammatory cytokines both in vitro [25, 26] and in vivo [27] and that caffeine exacerbates tissue injury during inflammation [5, 24]. In contrast to these results, in our human endotoxemia model, caffeine did not augment the immune response nor did it increase (subclinical) organ damage. There are several potential explanations for this finding. First, endogenous adenosine may not have an important anti-inflammatory potential in humans in vivo. However, this is not likely, given the consistent findings in animal studies and isolated cell studies and given the observation that administration of exogenous adenosine can limit the IL-6 response during human experimental endotoxemia [6]. Second, the limited increase in adenosine in our model might not be sufficient to induce significant anti-inflammatory effects. Recently, Soop and colleagues [28] demonstrated that the administration of 40 μg/kg per minute adenosine attenuated the release of the soluble RAGE (receptor for advanced glycation end products) but was unable to decrease the pro-inflammatory response. Unfortunately, no endogenous adenosine concentrations were measured in that study, although it was speculated that blood adenosine levels were at the submicromolar range. Finally, it needs to be realized that caffeine only blocks the adenosine A₁, A_2A, and A_2B receptors in the dose we used. Therefore, stimulation of the adenosine A₃ receptor, which also exerts anti-inflammatory potential, may have counteracted the pro-inflammatory effects of caffeine [29‐31]. Specific adenosine subtype receptor antagonists are being developed but are not currently available for human use.

We studied the effect, in a separate group of healthy volunteers, of the common 34C > T variant of the AMPD1 gene on the adenosine concentration and subclinical end-organ damage during endotoxemia. In Caucasians, approximately 20% of subjects are heterozygous for this variant allele, encoding a premature stopcodon, which results in a dysfunctional enzyme [32]. AMPD catalyzes the intracellular conversion of AMP into IMP (inosine monophosphate). Subjects heterozygous for this variant allele appear to have a 50% reduction in enzyme activity [33]. Interestingly, heterozygosity was recently associated with an improved cardiovascular prognosis in patients with coronary artery disease, probably because of an increased conversion of AMP into adenosine with subsequent increased adenosine concentrations and subsequent organ protection during ischemia [8]. Considering the beneficial cardiovascular effects of adenosine receptor stimulation in subjects with AMPD deficiency [7, 34], we hypothesized that endotoxemia-induced adenosine formation and subsequent adenosine receptor stimulation would also be potentiated. Although the LPS-induced increase in adenosine concentrations tended to be most strongly potentiated in the AMPD1 heterozygous group (with a mean increase of 71% versus 59% and 53% in the placebo and caffeine groups, respectively), this difference between groups did not reach statistical significance. Moreover, we did not observe an attenuation of organ damage in subjects heterozygous for the AMPD1 variation. A different route of adenosine formation during inflammation as compared with situations of ischemia could be an explanation. During ischemia/hypoxia, an increased intracellular degradation of ATP significantly contributes to the increase in extracellular adenosine. In this situation of increased intracellular AMP availability, a reduction of AMPD activity could have an important effect on adenosine formation. In contrast, during inflammation, the main source of adenosine formation following endotoxemia is the extracellular hydrolysis of ATP instead of an intracellular increase in AMP. Previous studies have suggested that inflammation directly leads to active release of adenine nucleosides, such as ATP, as well as passive release due to endothelial cell damage [35]. ATP is then quickly converted into adenosine. During sepsis, tissue hypoxia will most likely also play an important role in the accumulation of adenosine [36, 37]; however, this is unlikely during the relatively mild model of experimental endotoxemia. This could explain why the AMPD1 polymorphism did not influence the inflammation-induced increase in extracellular adenosine concentration. The lack of a significantly more pronounced increase in circulating adenosine in AMPD1 subjects may also be explained by the fact that adenosine is produced locally in the tissue and the endothelium acts as an active metabolic barrier for adenosine. Thus, circulating adenosine concentrations may not correctly reflect the inflammation-induced adenosine increase in the interstitial compartment. Pharmacological interventions, such as dipyridamole, an adenosine re-uptake inhibitor that increases the local adenosine concentration [38], or pentoxifylline, of which the immunomodulatory effects depend on sufficient levels of adenosine [20], may represent new therapeutic interventions to modulate the immune response.

Human experimental endotoxemia results in systemic inflammation and increases the circulating endogenous adenosine concentration. Pharmacological blockade of the adenosine receptors, however, does not augment the innate immune response or its resultant (subclinical) organ injury. In addition, organ damage is not reduced in subjects with the AMPD1 polymorphism, despite the tendency to a more pronounced LPS-induced increase in endogenous adenosine in these subjects. Given these observations, we conclude that, during human endotoxemia, endogenous adenosine does not act as a negative feedback molecule to limit the inflammatory response and subsequent tissue injury.