Role of A(2A) adenosine receptors in regulation of opsonized E. coli-induced macrophage function

Abstract

Adenosine is a biologically active molecule that is formed at sites of metabolic stress associated with trauma and inflammation, and its systemic level reaches high concentrations in sepsis. We have recently shown that inactivation of A(2A) adenosine receptors decreases bacterial burden as well as IL-10, IL-6, and MIP-2 production in mice that were made septic by cecal ligation and puncture (CLP). Macrophages are important in both elimination of pathogens and cytokine production in sepsis. Therefore, in the present study, we questioned whether macrophages are responsible for the decreased bacterial load and cytokine production in A(2A) receptor-inactivated septic mice. We showed that A(2A) KO and WT peritoneal macrophages obtained from septic animals were equally effective in phagocytosing opsonized E. coli. IL-10 production induced by opsonized E. coli was decreased in macrophages obtained from septic A(2A) KO mice as compared to WT counterparts. In contrast, the release of IL-6 and MIP-2 induced by opsonized E. coli was higher in septic A(2A) KO macrophages than WT macrophages. These results suggest that peritoneal macrophages are not responsible for the decreased bacterial load and diminished MIP-2 and IL-6 production that are observed in septic A(2A) KO mice. In contrast, peritoneal macrophages may contribute to the suppressive effect of A(2A) receptor inactivation on IL-10 production during sepsis.

Keywords: Adenosine; Cytokines; Inflammation; Macrophages; Phagocytosis; Sepsis.

Fig. 1

Phagocytic activity of A_2A WT and KO CLP-elicited peritoneal macrophages. Peritoneal macrophages were harvested from A_2A WT and KO mice after 16 h of polymicrobial sepsis induced by cecal ligation and puncture. Cells were seeded at 10⁶/ml density in 96-well plates. Peritoneal macrophages were then stimulated with IgG-coated, FITC-labeled E. coli for 2.5, 5 or 16 h, and phagocytosis was quantitated by measuring fluorescence. Results (mean ± SEM, n = 5–8) shown are one representative experiment from three separate studies

Fig. 2

IL-10 release is decreased by peritoneal macrophages obtained from A_2A KO vs. WT mice. Peritoneal macrophages were obtained from A_2A WT and KO mice 16 h after cecal ligation and puncture. Cells were plated at a density of 10⁶/ml in 96-well plates. Cells were stimulated with IgG-coated FITC-labeled E. coli for the indicated time periods, and IL-10 release was measured using ELISA. Results (mean ± SEM) depicted are from n = 30–48 wells from six separate experiments. *P < 0.05, ***P < 0.005 vs. A_2A WT group

Fig. 3

Effect of genetic inactivation of the A_2A receptor on IL-6 production by CLP-elicited peritoneal macrophages. Peritoneal macrophages were obtained from A_2A WT and KO mice 16 h after cecal ligation and puncture. Cells were plated at a density of 10⁶/ml in 96-well plates. Cells were stimulated with IgG-coated FITC-labeled E. coli for the indicated time periods, and IL-6 release was measured using ELISA. Results (mean ± SEM) depicted are from n = 30–48 wells from six separate experiments. ***P < 0.005 vs. A_2A WT group

Fig. 4

Effect of genetic inactivation of the A_2A receptor on MIP-2 production by CLP-elicited peritoneal macrophages. Peritoneal macrophages were obtained from A_2A WT and KO mice 16 h after CLP. Cells were plated at a density of 10⁶/ml in 96-well plates. Cells were stimulated with IgG-coated FITC-labeled E. coli for the indicated time periods, and MIP-2 release was measured using ELISA. Results (mean ± SEM) depicted are from n = 30–48 wells from six separate experiments. ***P < 0.05 vs. A_2A WT group