Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

Mutations in the gene encoding for the K ؉ channel ␣-subunit KCNQ1 have been associated with long QT syndrome and deafness. Besides heart and inner ear epithelial cells, KCNQ1 is expressed in a variety of epithelial cells including renal proximal tubule and gastrointestinal tract epithelial cells. At these sites, cellular K ؉ ions exit through KCNQ1 channel complexes, which may serve to recycle K ؉ or to maintain cell membrane potential and thus the driving force for electrogenic transepithelial transport, e.g., Na ؉ ͞glucose cotransport. Employing pharmacologic inhibition and gene knockout, the present study demonstrates the importance of KCNQ1 K ؉ channel complexes for the maintenance of the driving force for proximal tubular and intestinal Na ؉ absorption, gastric acid secretion, and cAMP-induced jejunal Cl ؊ secretion. In the kidney, KCNQ1 appears dispensable under basal conditions because of limited substrate delivery for electrogenic Na ؉ reabsorption to KCNQ1-expressing mid to late proximal tubule. During conditions of increased substrate load, however, luminal KCNQ1 serves to repolarize the proximal tubule and stabilize the driving force for Na ؉ reabsorption. In mice lacking functional KCNQ1, impaired intestinal absorption is associated with reduced serum vitamin B12 concentrations, mild macrocytic anemia, and fecal loss of Na ؉ and K ؉ , the latter affecting K ؉ homeostasis.

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Extracellular ATP and adenosine have immunoregulatory roles during inflammation. Elevated extracellular ATP is known to exacerbate GVHD, and the pharmacologic activation of the adenosine A 2A receptor is protective. However, the role of endogenous adenosine is unknown. We used gene-targeted mice and a pharmacologic inhibitor to test the role of adenosine generated by CD73/ecto-5-nucleotidase in GVHD. In allogeneic transplants, both donor and recipient CD73 were protective, with recipient CD73 playing the dominant role. CD73 deficiency led to enhanced T-cell expansion and IFN-␥ and IL-6 production, and the migratory capacity of Cd73 ؊/؊ T cells in vitro was increased. However, the number of regulatory T cells and expression of costimulatory molecules on antigen-presenting cells were unchanged. A 2A receptor deficiency led to increased numbers of allogeneic T cells, suggesting that signaling through the A 2A receptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GVHD. IntroductionPatients with hematologic malignancies that are refractory to conventional chemotherapy have a chance of cure by allogeneic hematopoietic stem cell transplantation (allo-HCT). 1 However, the success of this treatment is limited by GVHD. 2 We have recently shown that extracellular ATP, which is released from dying or stressed cells and serves as an endogenous danger signal to evoke systemic inflammatory responses, enhances GVHD by activation of the purinergic receptor P2X 7 R. 3,4 The abundance of extracellular ATP is regulated by ecto-nucleotidases, such as CD39, which dephosphorylates ATP to ADP and AMP. Extracellular AMP is dephosphorylated to adenosine via the action of CD73, a glycosyl phosphatidylinositol-anchored glycoprotein with ecto-5Ј-nucleotidase enzyme activity. 5-7 CD73 is expressed on many cell types, including subsets of T lymphocytes, endothelial cells, and epithelial cells. 7,8 It is also present as a secreted form lacking a glycosyl phosphatidylinositol anchor. 9 CD73-generated adenosine can activate any of 4 G-protein-coupled 7-transmembrane-spanning adenosine receptors (ARs; A 1 , A 2A , A 2B and A 3 ) and can act as either a pro-or anti-inflammatory mediator depending on the physiologic setting and the type of AR engaged. [10][11][12] In most circumstances, A 1 and A 3 receptor triggering is proinflammatory, whereas activation of A 2A and A 2B receptors is antiinflammatory or tolerogenic. 13,14 The importance of CD73 in producing adenosine for AR signaling has been revealed through studies with CD73-deficient mice. For example, CD73-generated adenosine reduces inflammation and fibrosis in lungs of bleomycin-treated mice 15 and is tolerogenic for cardiac and airway allografts. 16,17 CD73-dependent A 2B AR signaling protects mice during renal ischemia, 18 inhibits systemic vascular leakage during hypoxia, 19,20 and is also required for cardioprotection as a result of ischemic preconditioning. 21 Extracellular adenosine inhibits platelet activation and leukocyte...