Evaluation of components of the extracellular purinergic signaling system in human sepsis

Abstract

Abstract Objective Extracellular purines such as ATP, UTP and UDP and the ATP degradation product adenosine are biologically active signaling molecules, which accumulate at sites of metabolic stress in sepsis. They have potent immunomodulatory effects by binding to and activating P1 or adenosine and P2 receptors on the surface of leukocytes. Here we assessed the levels of extracellular purines, their receptors, metabolic enzymes and cellular transporters in leukocytes of septic patients. Methods Peripheral blood mononuclear cells (PBMCs), neutrophils and plasma were isolated from blood obtained from septic patients and healthy control subjects. RNA was isolated from cells and mRNA levels for purinergic receptors, enzymes, and transporters were measured. ATP, UTP, UDP and adenosine levels were evaluated in plasma. Results ATP levels were lower in septic patients than in healthy individuals and levels of the other purines were comparable between the two groups. Levels of P1 and P2 receptors did not differ between the two patient groups. mRNA levels of ectonucleoside triphosphate diphosphohydrolase (NTPDase)1 or CD39 increased while those of NTPDase2, -3 and -8 decreased in PBMCs of septic patients when compared to healthy controls. CD73 mRNA was lower in PBMCs of septic than healthy individuals. Equilibrative nucleoside transporter (ENT)1 mRNA concentrations were higher and ENT2, 3, and 4 mRNA concentrations were lower in PBMCs of septic subjects when compared to healthy subjects. Concentrative nucleoside transporter (CNT)1 mRNA levels were higher in PBMCs of septic vs. healthy subjects whereas the mRNA levels of CNT2, 3, and 4 did not differ. We failed to detect differences in mRNA levels of purinergic receptors, enzymes and transporters in neutrophils of septic vs. healthy subjects. Conclusion Since CD39 degrades ATP to AMP, the lower ATP levels in septic individuals may be the result of increased CD39 expression. This increased degradation of ATP did not lead to increased adenosine levels, which may be explained by the decreased expression of CD73, which converts AMP to adenosine. Altogether, our results demonstrate differential regulation of components of the purinergic system in PBMCs during human sepsis.