Short CommunicationAnalysis of A2a receptor-deficient mice reveals no significant compensatory increases in the expression of A2b, A1, and A3 adenosine receptors in lymphoid organs
Introduction
Inflammation is a defensive response of organisms against various pathogens; such processes are crucial for survival. However, the destruction of pathogens may also involve collateral tissue damage due to actions of proinflammatory molecules [1], [2]. Extended or excessive inflammation may lead to septic shock or autoimmune diseases and is implicated in the pathogenesis of cardiovascular diseases and some cancers [3], [4], [5], [6], [7]. Powerful anti-inflammatory mechanisms regulate inflammation by controlling the balance between pro- and anti-inflammatory molecules [8], [9]. It was shown recently that extracellular Ado-mediated signaling is an important anti-inflammatory mechanism involving anti-inflammatory cytokines, prostaglandins [10], [11], lipoxins [8], and glucocorticoids [12].
Intensive studies of the potential role of extracellular Ado in inflammation were facilitated by identification and cDNA cloning of four types of Ado receptors. A1 and A3 Ado receptors are Gi protein coupled, while A2a and A2b are Gs protein-coupled receptors that can activate adenylate cyclase and cause accumulation of intracellular cAMP [13]. Accumulation of Ado during ischemia and inflammation can protect normal tissues from injury due to immunosuppressive signaling through Ado receptors [14], [15], [16], [17], [18]. Similar protection from excessive inflammatory tissue injury by A2a receptor signaling was demonstrated in vivo using A2a receptor-deficient (Adora2a−/−) mice [19]. It was shown that sub-threshold doses of inflammatory stimulus, which caused minimal damage in normal mice, induced prolonged high levels of proinflammatory cytokines that led to extensive tissue damage and death of Adora2a−/− mice or normal mice treated with antagonists of the A2a receptor [19]. The role of A2a receptors seemed to be non-redundant. It is now believed that excessive inflammatory tissue damage leads to accumulation of extracellular Ado, activation of Gs-coupled A2a receptors on the surface of immune cells, and increases in immunosuppressive cAMP. This, in turn, leads to inhibition of proinflammatory cytokine secretion as well as to attenuation of inflammation. Both the A2a and A2b receptors are capable of elevating cAMP levels and thereby modulating the immune response [20]; it was therefore somewhat surprising that, in studies of acute liver inflammation, sepsis, and infected wound models, the A2b receptor was incapable of compensating for A2a receptor absence in protection from tissue damage [19]. It is not clear whether lack of compensation is due to the insufficient presence of A2b receptors in effector immune cells or to their inability to up-regulate expression sufficiently to compensate for the absence of A2a receptors. We also investigated the levels of expression of A1 and A3 receptors in Adora2a−/− mice because these receptors were also shown to have anti-inflammatory capability [20].
We designed experiments to clarify whether there are changes in expression of mRNA by cAMP-elevating A2b receptors and of Gi-coupled A1 and A3 receptors in immune cells of Adora2a−/− mice. In addition, we compared Ado-induced cAMP accumulation in immune cells from wild-type and Adora2a−/− mice. We conclude that the non-redundancy of A2a receptors in protection from excessive inflammatory tissue damage could be at least partially due to the inability of immune cells of Adora2a−/− mice to significantly up-regulate other immunosuppressive Ado receptors to compensate for their lack of A2a receptors.
Section snippets
Mice
Two-month-old male C57BL/6 Adora2a−/− mice [21] and age-matched wild-type mice were maintained in pathogen-free NIH animal facilities. Three animals were used in each experiment. Mice were treated and killed according to Federal rules, regulations, and policies pertaining to the care and use of animals in medical research.
Cell subset purification
Macrophage T cell and B cell subsets from the spleen were purified using immunomagnetic separation on the AutoMACS (Miltenyi Biotec). Single cell splenocyte preparations were
Results and discussion
Real-time RT–PCR was used to evaluate the transcriptional levels of Ado receptors in cells from different lymphoid and non-lymphoid organs. Because the interpretation of studies of mRNA expression depends on standardization of samples and assays, the choice of the housekeeping gene is very important, and dictates the careful comparison of commonly used internal standards [23], [24]. This was done by performing real-time RT–PCR with individual samples of GAPDH mRNA, β-actin mRNA, L32 mRNA, and
Acknowledgements
We are grateful to N. Shulman for help in preparing the manuscript.
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