Detrimental effects of adenosine signaling in sickle cell disease

Article metrics


Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A2B receptor (A2BR)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A2BR has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Increased adenosine levels contribute to sickling and hemolysis in SCD transgenic mice.
Figure 2: In vivo effects of PEG-ADA treatment on multiple organ damage and renal dysfunction in SCD transgenic mice.
Figure 3: PEG-ADA treatment attenuates hypoxia-reoxygenation–induced acute sickle crisis in SCD transgenic mice.
Figure 4: Excess adenosine acts through A2BRs to induce 2,3-DPG and subsequent sickling in SCD transgenic mice.
Figure 5: Adenosine levels are elevated in individuals with SCD and A2BR-mediated elevation of 2,3-DPG concentrations is required for hypoxia-induced human erythrocyte sickling.


  1. 1

    Ingram, V.M. A specific chemical difference between the globins of normal human and sickle-cell anaemia haemoglobin. Nature 178, 792–794 (1956).

  2. 2

    Madigan, C. & Malik, P. Pathophysiology and therapy for haemoglobinopathies. Part I: sickle cell disease. Expert Rev. Mol. Med. 8, 1–23 (2006).

  3. 3

    Urbinati, F., Madigan, C. & Malik, P. Pathophysiology and therapy for haemoglobinopathies. Part II: thalassaemias. Expert Rev. Mol. Med. 8, 1–26 (2006).

  4. 4

    Christoph, G.W., Hofrichter, J. & Eaton, W.A. Understanding the shape of sickled red cells. Biophys. J. 88, 1371–1376 (2005).

  5. 5

    de Montalembert, M. Management of sickle cell disease. BMJ 337, a1397 (2008).

  6. 6

    de Montalembert, M. Advances in sickle cell disease. Bull. Acad Natl. Med. 192, 1375–1381, discussion 1381 (2008).

  7. 7

    Chui, D.H. & Dover, G.J. Sickle cell disease: no longer a single gene disorder. Curr. Opin. Pediatr. 13, 22–27 (2001).

  8. 8

    Bunn, H.F. et al. Molecular and cellular pathogenesis of hemoglobin SC disease. Proc. Natl. Acad. Sci. USA 79, 7527–7531 (1982).

  9. 9

    Ferrone, F.A. Polymerization and sickle cell disease: a molecular view. Microcirculation 11, 115–128 (2004).

  10. 10

    Hebbel, R.P. Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology. Blood 77, 214–237 (1991).

  11. 11

    Hebbel, R.P., Osarogiagbon, R. & Kaul, D. The endothelial biology of sickle cell disease: inflammation and a chronic vasculopathy. Microcirculation 11, 129–151 (2004).

  12. 12

    Pászty, C. Transgenic and gene knock-out mouse models of sickle cell anemia and the thalassemias. Curr. Opin. Hematol. 4, 88–93 (1997).

  13. 13

    Pászty, C. et al. Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease. Science 278, 876–878 (1997).

  14. 14

    Fredholm, B.B. Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ. 14, 1315–1323 (2007).

  15. 15

    Hershfield, M.S. PEG-ADA replacement therapy for adenosine deaminase deficiency: an update after 8.5 years. Clin. Immunol. Immunopathol. 76, S228–S232 (1995).

  16. 16

    Chan, B. et al. Long-term efficacy of enzyme replacement therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). Clin. Immunol. 117, 133–143 (2005).

  17. 17

    Blackburn, M.R. et al. The use of enzyme therapy to regulate the metabolic and phenotypic consequences of adenosine deaminase deficiency in mice. Differential impact on pulmonary and immunologic abnormalities. J. Biol. Chem. 275, 32114–32121 (2000).

  18. 18

    Scheinman, J.I. Sickle cell disease and the kidney. Nat. Clin. Pract. Nephrol. 5, 78–88 (2009).

  19. 19

    Carbonaro, D.A. et al. Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion. Blood 111, 5745–5754 (2008).

  20. 20

    Carbonaro, D.A. et al. In vivo transduction by intravenous injection of a lentiviral vector expressing human ADA into neonatal ADA gene knockout mice: a novel form of enzyme replacement therapy for ADA deficiency. Mol. Ther. 13, 1110–1120 (2006).

  21. 21

    Wallace, K.L. et al. NKT cells mediate pulmonary inflammation and dysfunction in murine sickle cell disease through production of IFN-gamma and CXCR3 chemokines. Blood 114, 667–676 (2009).

  22. 22

    Narita, H., Yanagawa, S., Sasaki, R. & Chiba, H. Synthesis of 2,3-bisphosphoglycerate synthase in erythroid cells. J. Biol. Chem. 256, 7059–7063 (1981).

  23. 23

    Sasaki, R. & Chiba, H. Functions and metabolism of 2,3-bisphosphoglycerate in erythroid cells. Tanpakushitsu Kakusan Koso 28, 957–973 (1983).

  24. 24

    Sasaki, R. & Chiba, H. Role and induction of 2,3-bisphosphoglycerate synthase. Mol. Cell. Biochem. 53–54, 247–256 (1983).

  25. 25

    Chiba, H. & Sasaki, R. Functions of 2,3-bisphosphoglycerate and its metabolism. Curr. Top. Cell. Regul. 14, 75–116 (1978).

  26. 26

    Poillon, W.N., Kim, B.C., Labotka, R.J., Hicks, C.U. & Kark, J.A. Antisickling effects of 2,3-diphosphoglycerate depletion. Blood 85, 3289–3296 (1995).

  27. 27

    Poillon, W.N. & Kim, B.C. 2,3-Diphosphoglycerate and intracellular pH as interdependent determinants of the physiologic solubility of deoxyhemoglobin S. Blood 76, 1028–1036 (1990).

  28. 28

    Poillon, W.N., Kim, B.C., Welty, E.V. & Walder, J.A. The effect of 2,3-diphosphoglycerate on the solubility of deoxyhemoglobin S. Arch. Biochem. Biophys. 249, 301–305 (1986).

  29. 29

    Fredholm, B.B., AP, I.J., Jacobson, K.A., Klotz, K.N. & Linden, J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol. Rev. 53, 527–552 (2001).

  30. 30

    Stiles, G.L. Adenosine receptor subtypes: new insights from cloning and functional studies. in Purinergic Approaches in Experimental Therapeutics (eds. Jacobson, K.A. & Jarvis, M.F.) 29–37 (Wiley-Liss, New York, 1997).

  31. 31

    Palmer, T.M. & Stiles, G.L. Identification of an A2a adenosine receptor domain specifically responsible for mediating short-term desensitization. Biochemistry 36, 832–838 (1997).

  32. 32

    Eltzschig, H.K. et al. Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism. Blood 104, 3986–3992 (2004).

  33. 33

    Eltzschig, H.K. et al. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors. J. Exp. Med. 198, 783–796 (2003).

  34. 34

    Sebastiani, P. et al. A network model to predict the risk of death in sickle cell disease. Blood 110, 2727–2735 (2007).

  35. 35

    Fung, E.B. et al. Morbidity and mortality in chronically transfused subjects with thalassemia and sickle cell disease: A report from the multi-center study of iron overload. Am. J. Hematol. 82, 255–265 (2007).

  36. 36

    Lanzkron, S., Haywood, C. Jr., Segal, J.B. & Dover, G.J. Hospitalization rates and costs of care of patients with sickle-cell anemia in the state of Maryland in the era of hydroxyurea. Am. J. Hematol. 81, 927–932 (2006).

  37. 37

    Ballas, S.K. Current issues in sickle cell pain and its management. Hematology Am. Soc. Hematol. Educ. Program 2007, 97–105 (2007).

  38. 38

    Mi, T. et al. Excess adenosine in murine penile erectile tissues contributes to priapism through A2B adenosine receptor signaling. J. Clin. Invest. 118, 1491–1501 (2008).

  39. 39

    Wallace, K.L. & Linden, J. Adenosine A2A receptors induced on iNKT and NK cells reduce pulmonary inflammation and injury in mice with sickle cell disease. Blood 116, 5010–5020 (2010).

  40. 40

    Sreekumar, A. et al. Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 457, 910–914 (2009).

  41. 41

    Knudsen, T.B. et al. Effects of (R)-deoxycoformycin (pentostatin) on intrauterine nucleoside catabolism and embryo viability in the pregnant mouse. Teratology 45, 91–103 (1992).

  42. 42

    Perumbeti, A. et al. A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction. Blood 114, 1174–1185 (2009).

  43. 43

    Ericson, A. & de Verdier, C.H. A modified method for the determination of 2,3-diphosphoglycerate in erythrocytes. Scand. J. Clin. Lab. Invest. 29, 84–90 (1972).

  44. 44

    Zhou, C.C. et al. Angiotensin receptor agonistic autoantibodies induce pre-eclampsia in pregnant mice. Nat. Med. 14, 855–862 (2008).

Download references


Supported by US National Institute of Health grants DK077748 (to Y.X.), DK083559 (to Y.X.), HL070952 (to M.R.B.) and HL092188 (to H.K.E.) and by China National Science Foundation Scholarship Council 2008637068 (to J.W.). We thank T. Krahn (Bayer HealthCare AG) for the adenosine receptor A2BR agonist BAY 60-6583. Adenosine receptor–deficient mice were obtained from the following sources: A1R-deficient mice (J. Schnermann, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health); A2AR-deficient mice (J.-F. Chen, Boston University School of Medicine); A2BR-deficient mice (M.R. Blackburn, University of Texas–Houston Medical School); and A3R-deficient mice (M. Jacobson, Merck Research Laboratories).

Author information

Y.Z. carried out the measurement of adenosine and 2,3-DPG in humans and mice, analysis of sickling, hemolysis and lifespan of mouse RBCs, histological analysis of multiple tissues and image quantification, ELISA analysis of inflammatory cytokines in the lung homogenates and immunostaining of lung tissues with neutrophil markers, human erythrocyte culture and analysis of sickling under hypoxic conditions, and immunostaining of A2BRs on human and mouse RBCs, and contributed to generation of figures. Y.D. conducted PEG-ADA purification, treatment of mice with PEG-ADA or PSB1115 and proteinuria measurement, and isolation of multiple organs and blood from mice. J.W. was involved in the purification of PEG-ADA and treatment of mice with PEG-ADA or PSB1115, and performed heme content measurement and histological analysis of kidneys. W.Z. was involved in the purification of PEG-ADA; treated mice with PEG-ADA or PSB1115 and contributed to immunostaining of lung tissues with neutrophil markers. A.G. treated normal human erythrocyte cultures with A2BR or A2AR agonists. D.C.A. and M.V.M. conducted metabolomic screens in blood of wild-type and SCD transgenic mice. L.C.-D. provided expertise in confocal analysis of A2BR expression on RBCs. D.E.L. provided expertise in flow cytometry to measure the lifespan of RBCs. W.Z. assisted with urine osmolality analysis. H.S., L.T. and G.L. provided expertise in hemolytic disorders and kidney dysfunction. H.K.E. assisted A.G. with experiments on the effects of A2AR and A2BR agonists on 2,3-DPG induction in normal RBCs. R.E.K. provided expertise in adenosine signaling and helped edit the manuscript; M.R.B. provided mice deficient in each of the four types of adenosine receptors; H.S.J. provided expertise in hemolytic disorders, procured human subjects' approval and maintained the database of de-identified human subject information. Y.X. was the principal investigator, oversaw the design of experiments and interpretation of results, wrote and organized the manuscript, including the text and figures, and edited the manuscript.

Correspondence to Yang Xia.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Tables 1 and 2 and Supplementary Figures 1–5 (PDF 941 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading