Linda F. Thompson

Crucial Role for Ecto-5′-Nucleotidase (CD73) in Vascular Leakage during Hypoxia

Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5′-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73 −/− mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O2), Cd73 −/− mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73 −/− mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5′-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo.

Cardioprotection by Ecto-5'-Nucleotidase (CD73) and A2B Adenosine Receptors

Background— Ecto-5′-nucleotidase (CD73)–dependent adenosine generation has been implicated in tissue protection during acute injury. Once generated, adenosine can activate cell-surface adenosine receptors (A1AR, A2AAR, A2BAR, A3AR). In the present study, we define the contribution of adenosine to cardioprotection by ischemic preconditioning. Methods and Results— On the basis of observations of CD73 induction by ischemic preconditioning, we found that inhibition or targeted gene deletion of cd73 abolished infarct size-limiting effects. Moreover, 5′-nucleotidase treatment reconstituted cd73−/− mice and attenuated infarct sizes in wild-type mice. Transcriptional profiling of adenosine receptors suggested a contribution of A2BAR because it was selectively induced by ischemic preconditioning. Specifically, in situ ischemic preconditioning conferred cardioprotection in A1AR−/−, A2AAR−/−, or A3AR−/− mice but not in A2BAR−/− mice or in wild-type mice after inhibition of the A2BAR. Moreover, A2BAR agonist treatment significantly reduced infarct sizes after ischemia. Conclusions— Taken together, pharmacological and genetic evidence demonstrate the importance of CD73-dependent adenosine generation and signaling through A2BAR for cardioprotection by ischemic preconditioning and suggests 5′-nucleotidase or A2BAR agonists as therapy for myocardial ischemia.

Physiological roles for ecto-5'-nucleotidase (CD73).

Nucleotides and nucleosides influence nearly every aspect of physiology and pathophysiology. Extracellular nucleotides are metabolized through regulated phosphohydrolysis by a series of ecto-nucleotidases. The formation of extracellular adenosine from adenosine 5’-monophosphate is accomplished primarily through ecto-5’-nucleotidase (CD73), a glycosyl phosphatidylinositol-linked membrane protein found on the surface of a variety of cell types. Recent in vivo studies implicating CD73 in a number of tissue protective mechanisms have provided new insight into its regulation and function and have generated considerable interest. Here, we review contributions of CD73 to cell and tissue stress responses, with a particular emphasis on physiologic responses to regulated CD73 expression and function, as well as new findings utilizing Cd73-deficient animals.

Ecto‐enzyme and signaling functions of lymphocyte CD 7 3

Summary: CD73 or ecto‐5′‐nucleotidase (S‐NT) is a widely expressed ecto‐enzyme which catalyzes the dephosphorylation of AMP and other nucleoside monophosphates. CD 73 participates in purine salvage through this enzymatic activity, supplying cells with precursors for energy metabolism and nucleic acid biosynthesis. As an enzyme that produces adenosine, CD 73 can also regulate adenosine receptor engagement in many tissues. However, CD73 also has functions independent of its enzyme activity like many glycosyl phosphatidylinositol (GPI)‐anchored molecules, it transmits potent activation signals in T cells when ligated by antibodies. Less compelling evidence suggests that CD 73 may function as a cell adhesion molecule. In the human immune system, CD73 is expressed on subsets of T and B cells, on germinal center follicular dendritic cells, and on thymic medullary reticular fibroblasts and epithelial cells. Many challenging areas remain to be explored before the role of CD73 in the immune system will be fully understood. These include an evaluation of the role of adenosine receptors in lymphoid development, the identification of physiological CD73 ligands, a functional assessment of the GPI anchor, and an analysis of the intricate cell‐type‐specific and developmental regulation of CD 73 expression.

Identification of very early lymphoid precursors in bone marrow and their regulation by estrogen

Estrogen is a negative regulator of lymphopoiesis and provides an experimental tool for probing relationships between lymphocyte precursors and stem cells. We found that expression of lymphocyte-associated genes and immunoglobulin (Ig) gene rearrangement occurred before CD45R acquisition. Lymphoid-restricted progenitors that were Lin−IL-7Rα+c-kitloTdT+ (lineage marker−, interleukin receptor 7α+, c-kitlo and terminal deoxynucleotidyl transferase+) were selectively depleted in estrogen-treated mice; within a less differentiated Lin−c-kithi fraction, functional precursors of B and T, but not myeloid, cells were also selectively depleted. TdT and an Ig heavy chain transgene were detected within a hormone-regulated Lin−c-kithiSca-1+CD27+Flk-2+IL-7Rα− subset of this multipotential progenitor population. Identification of these extremely early lymphoid precursors should facilitate investigation of the molecular mechanisms that control lineage-fate decisions in hematopoiesis.

CD73 is required for efficient entry of lymphocytes into the central nervous system during experimental autoimmune encephalomyelitis

CD73 is a cell surface enzyme of the purine catabolic pathway that catalyzes the breakdown of AMP to adenosine. Because of the strong immunosuppressive and antiinflammatory properties of adenosine, we predicted that cd73−/− mice would develop severe experimental autoimmune encephalomyelitis (EAE), an animal model for the central nervous system (CNS) inflammatory disease, multiple sclerosis. Surprisingly, cd73−/− mice were resistant to EAE. However, CD4 T cells from cd73−/− mice secreted more proinflammatory cytokines than wild-type (WT) mice and were able to induce EAE when transferred into naïve cd73+/+ T cell-deficient recipients. Therefore, the protection from EAE observed in cd73−/− mice was not caused by a deficiency in T cell responsiveness. Immunohistochemistry showed that cd73−/− mice had fewer infiltrating lymphocytes in their CNS compared with WT mice. Importantly, susceptibility to EAE could be induced in cd73−/− mice after the transfer of WT CD73+CD4+ T cells, suggesting that CD73 must be expressed either on T cells or in the CNS for disease induction. In the search for the source of CD73 in the CNS that might facilitate lymphocyte migration, immunohistochemistry revealed a lack of CD73 expression on brain endothelial cells and high expression in the choroid plexus epithelium which regulates lymphocyte immunosurveillance between the blood and cerebrospinal fluid. Because blockade of adenosine receptor signaling with the A2a adenosine receptor-specific antagonist SCH58261 protected WT mice from EAE induction, we conclude that CD73 expression and adenosine receptor signaling are required for the efficient entry of lymphocytes into the CNS during EAE development.

Much Ado about Adenosine: Adenosine Synthesis and Function in Regulatory T Cell Biology

Recent studies have reported that adenosine is a significant mediator of regulatory T cell (Treg) function. Indeed, activation of the adenosine receptor subtypes expressed by a broad range of immune and inflammatory cells attenuates inflammation in several disease models. This anti-inflammatory response is associated with an increase in intracellular cAMP that inhibits cytokine responses of many immune/inflammatory cells, including T cells and APCs. Thus, adenosine produced by Tregs can provide a paracrine feedback that shapes the host response following an immunologic provocation. This review discusses the evidence that adenosine is an integral part of Treg biology and presents some of the mechanisms that may account for its contribution to the resolution of inflammation and the regulation of the immune/inflammatory cell phenotype.

CD73 has distinct roles in nonhematopoietic and hematopoietic cells to promote tumor growth in mice

CD73 is overexpressed in many types of human and mouse cancers and is implicated in the control of tumor progression. However, the specific contribution from tumor or host CD73 expression to tumor growth remains unknown to date. Here, we show that host CD73 promotes tumor growth in a T cell-dependent manner and that the optimal antitumor effect of CD73 blockade requires inhibiting both tumor and host CD73. Notably, enzymatic activity of CD73 on nonhematopoietic cells limited tumor-infiltrating T cells by controlling antitumor T cell homing to tumors in multiple mouse tumor models. In contrast, CD73 on hematopoietic cells (including CD4⁺CD25⁺ Tregs) inhibited systemic antitumor T cell expansion and effector functions. Thus, CD73 on hematopoietic and nonhematopoietic cells has distinct adenosinergic effects in regulating systemic and local antitumor T cell responses. Importantly, pharmacological blockade of CD73 using its selective inhibitor or an anti-CD73 mAb inhibited tumor growth and completely restored efficacy of adoptive T cell therapy in mice. These findings suggest that both tumor and host CD73 cooperatively protect tumors from incoming antitumor T cells and show the potential of targeting CD73 as a cancer immunotherapy strategy.

Ecto-5′-Nucleotidase (CD73)-Mediated Adenosine Production Is Tissue Protective in a Model of Bleomycin-Induced Lung Injury

Adenosine signaling has diverse actions on inflammation and tissue injury. Levels of adenosine are rapidly elevated in response to tissue injury; however, the mechanisms responsible for adenosine production in response to injury are not well understood. In this study, we found that adenosine levels are elevated in the lungs of mice injured by the drug bleomycin. In addition, increased activity of ecto-5′-nucleotidase (CD73) was found in the lungs in conjunction with adenosine elevations. To determine the contribution of CD73 to the generation of adenosine in the lung, CD73−/− mice were subjected to bleomycin challenges. Results demonstrated that CD73−/− mice challenged with bleomycin no longer accumulated adenosine in their lungs, suggesting that the primary means of adenosine production following bleomycin injury resulted from the release and subsequent dephosphorylation of adenine nucleotides. CD73−/− mice challenged with bleomycin exhibited enhanced pulmonary inflammation and fibrosis as well as exaggerated expression of proinflammatory and profibrotic mediators in the lung. Intranasal instillations of exogenous nucleotidase restored the ability of lungs of CD73−/− mice to accumulate adenosine following bleomycin challenge. Furthermore, these treatments were associated with a decrease in pulmonary inflammation and fibrosis. CD73+/+ animals challenged with bleomycin and supplemented with exogenous nucleotidase also exhibited reduced inflammation. Together, these findings suggest that CD73-dependent adenosine production contributes to anti-inflammatory pathways in bleomycin-induced lung injury.

P-selectin (CD62) binds to subpopulations of human memory T lymphocytes and natural killer cells

P-selectin (CD62) is a Ca(2+)-dependent lectin expressed on activated platelets and endothelium. Although P-selectin is known to function as a receptor for myeloid cells, previous studies indicated that P-selectin also bound to a subset of lymphocytes. Using a multi-color immunofluorescence assay we found that purified P-selectin bound to 12.2 +/- 4.1% of peripheral blood lymphocytes and that P-selectin could mediate adhesion of activated platelets to lymphocytes. A subpopulation of CD4+, CD8+, and CD16+ lymphocytes bound P-selectin. There was a marked preference for P-selectin binding to memory cells (CD45RO+) in both the CD4+ and CD8+ populations. Binding to all cell types was Ca(2+)-dependent and blocked by pretreatment of the cells with sialidase. These data suggest that P-selectin may play a role in the recruitment of specific lymphocyte populations to sites of inflammation.