Inflammation Research

, Volume 60, Issue 7, pp 633–642 | Cite as

Inhibition of phosphodiesterase 9A reduces cytokine-stimulated in vitro adhesion of neutrophils from sickle cell anemia individuals

  • Lediana Iagalo Miguel
  • Camila B. Almeida
  • Fabiola Traina
  • Andreia A. Canalli
  • Venina M. Dominical
  • Sara T. O. Saad
  • Fernando F. Costa
  • Nicola ConranEmail author
Original Research Paper



Leukocyte adhesion to vessel walls may initiate vaso-occlusion in sickle cell anemia (SCA); however, the extent to which inflammation participates in this mechanism is not understood. This in vitro study investigated whether inflammatory molecules, commonly augmented in SCA, can affect neutrophil adhesive properties and whether cyclic guanosine monophosphate (cGMP)-elevating agents can inhibit such adhesion.

Subjects and methods

Effects of Interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines, BAY 73-6691 [phosphodiesterase (PDE)-9A-inhibitor], and BAY 41-2271 (guanylate-cylase stimulator) on the adhesive properties of neutrophils from healthy control (CON) and steady-state SCA individuals were determined using static-adhesion assays.


SCA neutrophils demonstrated increased adhesive properties, compared to CON neutrophils; IL-8, TNF-α and GM-CSF increased CON neutrophil adhesion and further increased SCA neutrophil adhesion to fibronectin (FN). The PDE9A inhibitor, BAY-73-6691, significantly reduced basal CON neutrophil and SCA neutrophil adhesion; this was accompanied by decreased SCA neutrophil surface expressions of the L-selectin and CD11b adhesion molecules. BAY-73-6691 also significantly reduced cytokine-stimulated CON neutrophil and SCA neutrophil adhesion to FN; however, this was not accompanied by alterations in adhesion-molecule presentation.


The chronic inflammatory nature of SCA may contribute to leukocyte adhesive functions in SCA. Furthermore, elevation of leukocyte cGMP may be an interesting approach for inhibition of leukocyte adhesion to the vessel wall, even in the presence of inflammatory stimuli.


cGMP Cytokine Inflammation Leukocyte Sickle cell anemia Vaso-occlusion 



The authors thank Fernanda Perreira Cunha for assistance with flow cytometry procedures. This study was funded by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Brazil. The Hematology and Hemotherapy Center, UNICAMP, forms part of the National Institute of Blood, Brazil (INCT de Sangue, CNPq/MCT/FAPESP).


  1. 1.
    Castro O, Brambilla DJ, Thorington B, Reindorf CA, Scott RB, Gillette P, et al. The acute chest syndrome in sickle cell disease: incidence and risk factors. The cooperative study of sickle cell disease. Blood. 1994;84:643–9.PubMedGoogle Scholar
  2. 2.
    Kinney TR, Sleeper LA, Wang WC, Zimmerman RA, Pegelow CH, Ohene-Frempong K, et al. Silent cerebral infarcts in sickle cell anemia: a risk factor analysis. The cooperative study of sickle cell disease. Pediatrics. 1999;103:640–5.PubMedCrossRefGoogle Scholar
  3. 3.
    Miller ST, Sleeper LA, Pegelow CH, Enos LE, Wang WC, Weiner SJ, et al. Prediction of adverse outcomes in children with sickle cell disease. N Eng J Med. 2000;342:83–9.CrossRefGoogle Scholar
  4. 4.
    Lanaro C, Franco-Penteado CF, Albuqueque DM, Saad ST, Conran N, Costa FF. Altered levels of cytokines and inflammatory mediators in plasma and leukocytes of sickle cell anemia patients and effects of hydroxyurea therapy. J Leukoc Biol. 2009;85:235–42.PubMedCrossRefGoogle Scholar
  5. 5.
    Chiang EY, Frenette PS. Sickle cell vaso-occlusion. Hematol Oncol Clin N Am. 2005;19:771–84.CrossRefGoogle Scholar
  6. 6.
    Conran N, Costa FF. Hemoglobin disorders and endothelial cell interactions. Clin Biochem. 2009;42:1824–38.PubMedCrossRefGoogle Scholar
  7. 7.
    Turhan A, Jenab P, Bruhns P, Ravetch JV, Coller BS, Frenette PS. Intravenous immune globulin prevents venular vaso-occlusion in sickle cell mice by inhibiting leukocyte adhesion and the interactions between sickle erythrocytes and adherent leukocytes. Blood. 2004;103:2397–400.PubMedCrossRefGoogle Scholar
  8. 8.
    Turhan A, Weiss LA, Mohandas N, Coller BS, Frenette PS. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm. Proc Natl Acad Sci USA. 2002;99:3047–51.PubMedCrossRefGoogle Scholar
  9. 9.
    Assis A, Conran N, Canalli AA, Lorand-Metze I, Saad ST, Costa FF. Effect of cytokines and chemokines on sickle neutrophil adhesion to fibronectin. Acta Haematol. 2005;113:130–6.PubMedCrossRefGoogle Scholar
  10. 10.
    Canalli AA, Franco-Penteado CF, Traina F, Saad ST, Costa FF, Conran N. Role for cAMP-protein kinase A signalling in augmented neutrophil adhesion and chemotaxis in sickle cell disease. Eur J Haematol. 2007;79:330–7.PubMedCrossRefGoogle Scholar
  11. 11.
    Fadlon E, Vordermeier S, Pearson TC, Mire-Sluis AR, Dumonde DC, Phillips J, et al. Blood polymorphonuclear leukocytes from the majority of sickle cell patients in the crisis phase of the disease show enhanced adhesion to vascular endothelium and increased expression of CD64. Blood. 1998;91:266–74.PubMedGoogle Scholar
  12. 12.
    Wun T, Cordoba M, Rangaswami A, Cheung AW, Paglieroni T. Activated monocytes and platelet–monocyte aggregates in patients with sickle cell disease. Clin Lab Haematol. 2002;24:81–8.PubMedGoogle Scholar
  13. 13.
    Kato GJ, Hebbel RP, Steinberg MH, Gladwin MT. Vasculopathy in sickle cell disease: biology, pathophysiology, genetics, translational medicine, and new research directions. Am J Hematol. 2009;84:618–25.PubMedCrossRefGoogle Scholar
  14. 14.
    Conran N, Ferreira HH, Lorand-Metze I, Thomazzi SM, Antunes E, de Nucci G. Nitric oxide regulates human eosinophil adhesion mechanisms in vitro by changing integrin expression and activity on the eosinophil cell surface. Br J Pharmacol. 2001;134:632–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Conran N, Gambero A, Ferreira HH, Antunes E, de Nucci G. Nitric oxide has a role in regulating VLA-4-integrin expression on the human neutrophil cell surface. Biochem Pharmacol. 2003;66:43–50.PubMedCrossRefGoogle Scholar
  16. 16.
    Canalli AA, Franco-Penteado CF, Saad STO, Conran N, Costa FF. Increased adhesive properties of neutrophils in sickle cell disease may be reversed by pharmacological nitric oxide donation. Haematologica. 2008;93:605–9.PubMedCrossRefGoogle Scholar
  17. 17.
    Mack AK, McGowan Ii VR, Tremonti CK, Ackah D, Barnett C, Machado RF, et al. Sodium nitrite promotes regional blood flow in patients with sickle cell disease: a phase I/II study. Br J Haematol. 2008;142:971–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Schmidt HH, Schmidt PM, Stasch JP. NO- and haem-independent soluble guanylate cyclase activators. Handb Exp Pharmacol. 2009;191:309–39.PubMedCrossRefGoogle Scholar
  19. 19.
    Bender AT, Beavo JA. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev. 2006;58:488–520.PubMedCrossRefGoogle Scholar
  20. 20.
    Francis SH, Busch JL, Corbin JD, Sibley D. cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol Rev. 2010;62:525–63.PubMedCrossRefGoogle Scholar
  21. 21.
    Almeida CB, Traina F, Lanaro C, Canalli AA, Saad ST, Costa FF, et al. High expression of the cGMP-specific phosphodiesterase, PDE9A, in sickle cell disease (SCD) and the effects of its inhibition in erythroid cells and SCD neutrophils. Br J Haematol. 2008;142:836–44.PubMedCrossRefGoogle Scholar
  22. 22.
    Stasch JP, Becker EM, Alonso-Alija C, Apeler H, Dembowsky K, Feurer A, et al. NO-independent regulatory site on soluble guanylate cyclase. Nature. 2001;410:212–5.PubMedCrossRefGoogle Scholar
  23. 23.
    Wunder F, Tersteegen A, Rebmann A, Erb C, Fahrig T, Hendrix M. Characterization of the first potent and selective PDE9 inhibitor using a cGMP reporter cell line. Mol Pharmacol. 2005;68:1775–81.PubMedGoogle Scholar
  24. 24.
    Montes RA, Eckman JR, Hsu LL, Wick TM. Sickle erythrocyte adherence to endothelium at low shear: role of shear stress in propagation of vaso-occlusion. Am J Hematol. 2002;70:216–27.PubMedCrossRefGoogle Scholar
  25. 25.
    Rodgers GP, Schechter AN, Noguchi CT, Klein HG, Nienhuis AW, Bonner RF. Periodic microcirculatory flow in patients with sickle-cell disease. N Eng J Med. 1984;311:1534–8.CrossRefGoogle Scholar
  26. 26.
    English D, Andersen BR. Single-step separation of red blood cells. Granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll–Hypaque. J Immunol Methods. 1974;5:249–52.PubMedCrossRefGoogle Scholar
  27. 27.
    Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol. 1982;78:206–9.PubMedCrossRefGoogle Scholar
  28. 28.
    Benkerrou M, Delarche C, Brahimi L, Fay M, Vilmer E, Elion J, et al. Hydroxyurea corrects the dysregulated l-selectin expression and increased H(2)O(2) production of polymorphonuclear neutrophils from patients with sickle cell anemia. Blood. 2002;99:2297–303.PubMedCrossRefGoogle Scholar
  29. 29.
    Finnegan EM, Barabino GA, Liu XD, Chang HY, Jonczyk A, Kaul DK. Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion. Am J Physiol Heart Circ Physiol. 2007;293:H1038–45.PubMedCrossRefGoogle Scholar
  30. 30.
    Finnegan EM, Turhan A, Golan DE, Barabino GA. Adherent leukocytes capture sickle erythrocytes in an in vitro flow model of vaso-occlusion. Am J Hematol. 2007;82:266–75.PubMedCrossRefGoogle Scholar
  31. 31.
    Zennadi R, Chien A, Xu K, Batchvarova M, Telen MJ. Sickle red cells induce adhesion of lymphocytes and monocytes to endothelium. Blood. 2008;112:3474–83.PubMedCrossRefGoogle Scholar
  32. 32.
    Conran N, Saad ST, Costa FF, Ikuta T. Leukocyte numbers correlate with plasma levels of granulocyte–macrophage colony-stimulating factor in sickle cell disease. Ann Hematol. 2007;86:255–61.PubMedCrossRefGoogle Scholar
  33. 33.
    Croizat H. Circulating cytokines in sickle cell patients during steady state. Br J Haematol. 1994;87:592–7.PubMedCrossRefGoogle Scholar
  34. 34.
    Canalli AA, Proenca-Ferreira R, Franco-Penteado CF, Traina F, Sakamoto TM, Saad ST, et al. Participation of the Mac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle cell disease neutrophils to endothelial layers, and reversal of adhesion by simvastatin. Haematologica. 2011 [Epub ahead of print].Google Scholar
  35. 35.
    Kobayashi Y. The role of chemokines in neutrophil biology. Front Biosci. 2008;13:2400–7.PubMedCrossRefGoogle Scholar
  36. 36.
    Hamilton JA. Colony-stimulating factors in inflammation and autoimmunity. Nat Rev Immunol. 2008;8:533–44.PubMedCrossRefGoogle Scholar
  37. 37.
    Lum AF, Wun T, Staunton D, Simon SI. Inflammatory potential of neutrophils detected in sickle cell disease. Am J Hematol. 2004;76:126–33.PubMedCrossRefGoogle Scholar
  38. 38.
    Okpala I. Leukocyte adhesion and the pathophysiology of sickle cell disease. Curr Opin Hematol. 2006;13:40–4.PubMedCrossRefGoogle Scholar
  39. 39.
    von Andrian UH, Chambers JD, McEvoy LM, Bargatze RF, Arfors KE, Butcher EC. Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo. Proc Natl Acad Sci USA. 1991;88:7538–42.CrossRefGoogle Scholar
  40. 40.
    Smalley DM, Ley K. l-selectin: mechanisms and physiological significance of ectodomain cleavage. J Cell Mol Med. 2005;9:255–66.PubMedCrossRefGoogle Scholar
  41. 41.
    Menezes GB, Lee WY, Zhou H, Waterhouse CC, Cara DC, Kubes P. Selective down-regulation of neutrophil Mac-1 in endotoxemic hepatic microcirculation via IL-10. J Immunol. 2009;183:7557–68.PubMedCrossRefGoogle Scholar
  42. 42.
    Luo BH, Carman CV, Springer TA. Structural basis of integrin regulation and signaling. Annu Rev Immunol. 2007;25:619–47.PubMedCrossRefGoogle Scholar
  43. 43.
    Fagerholm SC, Varis M, Stefanidakis M, Hilden TJ, Gahmberg CG. alpha-chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation. Blood. 2006;108:3379–86.PubMedCrossRefGoogle Scholar
  44. 44.
    Hogg N, Leitinger B. Shape and shift changes related to the function of leukocyte integrins LFA-1 and Mac-1. J Leukoc Biol. 2001;69:893–8.PubMedGoogle Scholar
  45. 45.
    Bischoff E, Stasch JP. Effects of the sGC stimulator BAY 41-2272 are not mediated by phosphodiesterase 5 inhibition. Circulation. 2004;110:e320–1. author reply e320-321.PubMedCrossRefGoogle Scholar
  46. 46.
    Mullershausen F, Russwurm M, Friebe A, Koesling D. Inhibition of phosphodiesterase type 5 by the activator of nitric oxide-sensitive guanylyl cyclase BAY 41-2272. Circulation. 2004;109:1711–3.PubMedCrossRefGoogle Scholar
  47. 47.
    Machado RF, Martyr S, Kato GJ, Barst RJ, Anthi A, Robinson MR, et al. Sildenafil therapy in patients with sickle cell disease and pulmonary hypertension. Br J Haematol. 2005;130:445–53.PubMedCrossRefGoogle Scholar
  48. 48.
    Huang LJ, Yoon MH, Choi JI, Kim WM, Lee HG, Kim YO. Effect of sildenafil on neuropathic pain and hemodynamics in rats. Yonsei Med J. 2010;51:82–7.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel AG 2011

Authors and Affiliations

  • Lediana Iagalo Miguel
    • 1
  • Camila B. Almeida
    • 1
  • Fabiola Traina
    • 1
  • Andreia A. Canalli
    • 1
  • Venina M. Dominical
    • 1
  • Sara T. O. Saad
    • 1
  • Fernando F. Costa
    • 1
  • Nicola Conran
    • 1
    • 2
    Email author
  1. 1.Hematology and Hemotherapy Center - Instituto Nacional de Ciência e Tecnologia do Sangue (INCTS)University of Campinas - UNICAMPCampinasBrazil
  2. 2.Hemocentro, Rua Carlos Chagas, 480Cidade UniversitáriaCampinasBrazil

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