International Journal of Hematology

, Volume 96, Issue 5, pp 600–610

Role of CD61+ cells in thrombocytopenia of dengue patients

  • Sansanee Noisakran
  • Nattawat Onlamoon
  • Kovit Pattanapanyasat
  • Hui-Mien Hsiao
  • Pucharee Songprakhon
  • Nasikarn Angkasekwinai
  • Kulkanya Chokephaibulkit
  • Francois Villinger
  • Aftab A. Ansari
  • Guey Chuen Perng
Original Article

Abstract

Although hematological disorders with salient features of thrombocytopenia have been well documented in dengue patients, the role of CD61-expressing platelets and the megakaryocytic cell lineage in the pathogenesis of dengue virus (DENV) infection remains largely unexplored. A prospective observational study was performed using blood samples and PBMCs from dengue-confirmed patients, as well as from rhesus monkeys (RM) experimentally infected with DENV. Immunohistochemical staining and FACS techniques were applied to evaluate the frequencies of CD61+ cells that contained DENV antigen. Highly enriched population of CD61+ cells was also isolated from acute DENV-infected RM and assayed for DENV RNA by quantitative RT-PCR. Results revealed that DENV antigen was found in small vesicles of varying size, and more frequently in anucleated cells associated with platelets in dengue patients. The DENV antigen-containing cells were CD61+ and appeared to share characteristics of megakaryocytes. Kinetic profiles of CD61+ cells from DENV-infected RM revealed a transient increase in CD61+CD62P+ cells early after DENV infection. DENV RNA in a highly enriched population of CD61+ cells from the infected RM was observed during acute stage. Our results indicate that virus containing CD61+ cells may be directly linked to the platelet dysfunction and low platelet count characteristics of dengue patients.

Keywords

Dengue Viremia Thrombocytopenia Fever Megakaryocytes DF DHF 

Supplementary material

12185_2012_1175_MOESM1_ESM.pptx (50 kb)
Supplementary Figure 1. A schematic diagram depicts an isolation method for megakaryocyte-like cells in circulation. Giant cells or megakaryocytic-like cells from whole blood of dengue patients was performed by applying a method developed by Wilde et al {Wilde, 1997 #2295}. Whole blood (0.3 ml) was passed down by gravity through an assembled syringe filter holder containing a nucleopore polycarbonate membrane of 5 µm pore diameter. Following two washes with saline, the membrane was removed and left to dry thoroughly before being processed for immunohistochemical staining (IHC). (PPTX 49 kb)
12185_2012_1175_MOESM2_ESM.pptx (212 kb)
Supplementary Figure 2. A gating strategy of flow cytometry-based cell sorting for CD41+CD61+ cells . PBMCs from dengue virus-infected rhesus monkeys on days 3 and 5 post infection were stained with a panel of fluorochromes-conjugated monoclonal antibodies with specificity for CD3, CD20, CD14, CD16, CD41 and CD61 and processed for a FACSAria II cell sorter. CD3-CD20-CD14-CD16- cells were selected from the total stained cell population and those with CD41+CD61+, CD41-CD61+ and CD41-CD61- subsets were collected in each sample for further investigation. Pre-sorted and post-sorted cell populations were analyzed for the purity of cell sorting. (PPTX 211 kb)
12185_2012_1175_MOESM3_ESM.pptx (382 kb)
Supplementary Figure 3. Detection of dengue viral antigen in CD61+ cells. Blood smears prepared from dengue patients were subjected to double immunofluorescence staining for dengue viral antigen and CD61 as described in the Methods section. The stained cells were mounted with DAPI and observed using a Zeiss fluorescence microscope equipped with an Axis 5 digital camera. Dengue viral antigen was observed in granular cells with CD61 marker. Results show bright field, fluorescent field and merged images from blood smears of 2 randomly selected dengue patients. Dengue viral antigen (red); CD61 (green); nucleus (blue); co-localization of dengue viral antigen with CD61 (yellow). (PPTX 382 kb)
12185_2012_1175_MOESM4_ESM.pptx (772 kb)
Supplementary Figure 4. The presence of giant platelets and DENV antigen-positive platelets in dengue patients. Whole blood from dengue patients was smeared onto slides and subjected to Wright’s stain. The stained cells were observed using a light microscope with 40× (A) and 100× (B) magnification of objective lenses. Results revealed the presence of giant platelets with budding vesicles (red arrows) in the samples from dengue patients. To detect dengue viral antigens in the platelets, double immunohistochemical staining was performed using platelets isolated from whole blood of dengue patients. A small proportion of the platelets were found to contain dengue viral antigen as indicated by red arrows (C and D). Representative images from two dengue patients are shown. (PPTX 772 kb)
12185_2012_1175_MOESM5_ESM.pptx (802 kb)
Supplementary Figure 5. Dengue viral antigen was observed in anucleated cells and irregular shaped-nucleus containing cells with loose cytoplasm. Immunohistochemical staining for dengue viral antigen was performed on PBMC smears from dengue patients as described in the Materials and Methods. Mouse anti-dengue E antibody (A and C) and isotype-matched control antibody (B and D) were used in the primary staining step. Counterstaining with hematoxylin was applied to all the stained samples. Representative images taken from different areas on the slides are shown (dengue viral antigen, brown). (PPTX 802 kb)
12185_2012_1175_MOESM6_ESM.pptx (367 kb)
Supplementary Figure 6. CD41+ cells with budding platelets and proplatelet formation contained dengue viral antigen. PBMC smears prepared from dengue patients were processed for double immunohistochemical staining for dengue viral antigen (red) and CD41(dark blue), a marker for platelets and megakaryocytes, as described in the Materials and Methods. Mouse anti-dengue E antibody (A and B) and isotype-matched control antibody (C) were utilized in the primary staining step. The stained cells were mounted with Hoechst and observed using a fluorescence microscope. Results show bright field, fluorescent field and merged image of the stained cells captured from different areas on the slides. (PPTX 367 kb)
12185_2012_1175_MOESM7_ESM.pptx (565 kb)
Supplementary Figure 7. Dengue viral antigen was observed in CD41+ cells with low cytoplasm to nucleus ratio in association with platelets. Double immunohistochemical staining was performed to determine dengue viral antigen (red) and CD41(dark blue), a marker for platelets and megakaryocytes as described in the Materials and Methods using PBMC smears from dengue patients. A and B show two representative images captured from different areas on the stained slides. (PPTX 565 kb)
12185_2012_1175_MOESM8_ESM.pptx (432 kb)
Supplementary Figure 8. A gating strategy of multicolor flow cytometric analysis for CD61+CD62P+ cells in leukocyte subpopulation. Whole blood from dengue virus-infected rhesus monkeys were stained with a panel of fluorochromes-conjugated monoclonal antibodies with specificity for CD45, CD3, CD20, CD14, CD61 and CD62P. The stained cells were analyzed by flow cytometry for the frequency of CD61+CD62+ cells in the following leukocyte subpopulations: granulocytes (CD45+CD14-CD3-CD20-), monocytes (CD45+CD14+CD3-CD20-), T lymphocytes (CD45+CD14-CD3+CD20-), B lymphocytes (CD45+CD14-CD3-CD20+) and non-T, non-B lymphocytes (CD45+CD14-CD3-CD20-). (PPTX 431 kb)
12185_2012_1175_MOESM9_ESM.docx (12 kb)
Supplementary material 9 (DOCX 12 kb)

References

  1. 1.
    Morens DM, Fauci AS. Dengue and hemorrhagic fever: a potential threat to public health in the United States. JAMA. 2008;299:214–6.PubMedCrossRefGoogle Scholar
  2. 2.
    CDC. Locally acquired Dengue—Key West, Florida, 2009–2010. MMWR 2010;59:577–581.Google Scholar
  3. 3.
    Gregory CJ, Santiago LM, Arguello DF, et al. Clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic area—Puerto Rico, 2007–2008. Am J Trop Med Hyg. 2010;82:922–9.PubMedCrossRefGoogle Scholar
  4. 4.
    WHO. Dengue: guidelines for diagnosis, treatment, prevention and control. 2009.Google Scholar
  5. 5.
    WHO. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. World Health Organization. Geneva; 2008.Google Scholar
  6. 6.
    Tsai J-J, Liu L-T, Chang K, et al. The importance of hematopoietic progenitor cells in dengue. Ther Adv Hematol. 2011;3(1) 59–71.Google Scholar
  7. 7.
    Srichaikul T, Nimmannitya S. Haematology in dengue and dengue haemorrhagic fever. Baillieres Best Pract Res Clin Haematol. 2000;13:261–76.PubMedCrossRefGoogle Scholar
  8. 8.
    Nelson ER, Bierman HR, Chulajata R. Hematologic findings in the 1960 hemorrhagic fever epidemic (Dengue) in Thailand. Am J Trop Med Hyg. 1964;13:642–9.PubMedGoogle Scholar
  9. 9.
    La Russa VF, Innis BL. Mechanisms of dengue virus-induced bone marrow suppression. Baillieres Clin Haematol. 1995;8:249–70.PubMedCrossRefGoogle Scholar
  10. 10.
    Na-Nakorn S, Suingdumrong A, Pootrakul S, Bhamarapravati N. Bone-marrow studies in Thai haemorrhagic fever. Bull World Health Organ. 1966;35:54–5.PubMedGoogle Scholar
  11. 11.
    Theofilopoulos AN, Wilson CB, Dixon FJ. The Raji cell radioimmune assay for detecting immune complexes in human sera. J Clin Invest. 1976;57:169–82.PubMedCrossRefGoogle Scholar
  12. 12.
    Saito M, Oishi K, Inoue S, et al. Association of increased platelet-associated immunoglobulins with thrombocytopenia and the severity of disease in secondary dengue virus infections. Clin Exp Immunol. 2004;138:299–303.PubMedCrossRefGoogle Scholar
  13. 13.
    Oishi K, Saito M, Mapua CA, et al. Dengue illness: clinical features and pathogenesis. J Infect Chemother. 2007;13:125–33.PubMedCrossRefGoogle Scholar
  14. 14.
    Boonpucknavig S, Vuttiviroj O, Bunnag C, et al. Demonstration of dengue antibody complexes on the surface of platelets from patients with dengue hemorrhagic fever. Am J Trop Med Hyg. 1979;28:881–4.PubMedGoogle Scholar
  15. 15.
    Noisakran S, Chokephaibulkit K, Songprakhon P, et al. A re-evaluation of the mechanisms leading to dengue hemorrhagic fever. Ann N Y Acad Sci. 2009;1171(Suppl 1):E24–35.PubMedCrossRefGoogle Scholar
  16. 16.
    Noisakran S, Gibbons RV, Songprakhon P, et al. Detection of dengue virus in platelets isolated from dengue patients. Southeast Asian J Trop Med Public Health. 2009;40:253–62.PubMedGoogle Scholar
  17. 17.
    Noisakran S, Onlamoon N, Hsiao HM, et al. Infection of bone marrow cells by dengue virus in vivo. Exp Hematol. 2012;40(250–259):e254.Google Scholar
  18. 18.
    Wang S, He R, Patarapotikul J, et al. Antibody-enhanced binding of dengue-2 virus to human platelets. Virology. 1995;213:254–7.PubMedCrossRefGoogle Scholar
  19. 19.
    Onlamoon N, Noisakran S, Hsiao HM, et al. Dengue virus-induced hemorrhage in a nonhuman primate model. Blood. 2010;115:1823–34.PubMedCrossRefGoogle Scholar
  20. 20.
    Institute of Laboratory Animal Research CoLS, National Research Council. Guide for the Care and Use of Laboratory Animals. Washington, DC: The National Academic Press; 1996.Google Scholar
  21. 21.
    Wilde NT, Burgess R, Keenan DJ, et al. The effect of cardiopulmonary bypass on circulating megakaryocytes. Br J Haematol. 1997;98:322–7.PubMedCrossRefGoogle Scholar
  22. 22.
    Tsai JJ, Jen YH, Chang JS, et al. Frequency alterations in key innate immune cell components in the peripheral blood of dengue patients detected by FACS analysis. J Innate Immun. 2012;3:59–71.Google Scholar
  23. 23.
    WHO. Summaries of Papers Presented at the WHO Inter-Regional Seminar on Mosquito-borne Haemorrhagic Fevers in the South-East Asia and Western Pacific Regions. Bulletin 1966;35.Google Scholar
  24. 24.
    Bierman HR, Nelson ER. Hematodepressive virus diseases of Thailand. Ann Intern Med. 1965;62:867–84.PubMedGoogle Scholar
  25. 25.
    Nelson ER, Tuchinda S, Bierman HR, Chulajata R. Haematology of Thai haemorrhagic fever (dengue). Bull World Health Organ. 1966;35:43–4.PubMedGoogle Scholar
  26. 26.
    Nardi M, Tomlinson S, Greco MA, et al. Complement-independent, peroxide-induced antibody lysis of platelets in HIV-1-related immune thrombocytopenia. Cell. 2001;106:551–61.PubMedCrossRefGoogle Scholar
  27. 27.
    Honda S, Saito M, Dimaano EM, et al. Increased phagocytosis of platelets from patients with secondary Dengue virus infection by human macrophages. Am J Trop Med Hyg. 2009;80:841–5.PubMedGoogle Scholar
  28. 28.
    Avirutnan P, Mehlhop E, Diamond MS. Complement and its role in protection and pathogenesis of flavivirus infections. Vaccine. 2008;26(Suppl 8):I100–7.PubMedCrossRefGoogle Scholar
  29. 29.
    Boonnak K, Slike BM, Burgess TH, et al. Role of dendritic cells in antibody-dependent enhancement of dengue virus infection. J Virol. 2008;82:3939–51.PubMedCrossRefGoogle Scholar
  30. 30.
    Alonzo MT, Lacuesta TL, Dimaano EM, et al. Platelet apoptosis and apoptotic platelet clearance by macrophages in secondary dengue virus infections. J Infect Dis. 2012;205(8):1321–9.Google Scholar
  31. 31.
    Lipschultz CA, Yee A, Mohan S, et al. Temperature differentially affects encounter and docking thermodynamics of antibody–antigen association. J Mol Recognit. 2002;15:44–52.PubMedCrossRefGoogle Scholar
  32. 32.
    Sobel AT, Bokisch VA, Muller-Eberhard HJ. C1q deviation test for the detection of immune complexes, aggregates of IgG, and bacterial products in human serum. J Exp Med. 1975;142:139–50.PubMedCrossRefGoogle Scholar
  33. 33.
    Ruangjirachuporn W, Boonpucknavig S, Nimmanitya S. Circulating immune complexes in serum from patients with dengue haemorrhagic fever. Clin Exp Immunol. 1979;36:46–53.PubMedGoogle Scholar
  34. 34.
    Vaughn DW, Green S, Kalayanarooj S, et al. Dengue in the early febrile phase: viremia and antibody responses. J Infect Dis. 1997;176:322–30.PubMedCrossRefGoogle Scholar
  35. 35.
    Oishi K, Inoue S, Cinco MT, et al. Correlation between increased platelet-associated IgG and thrombocytopenia in secondary dengue virus infections. J Med Virol. 2003;71:259–64.PubMedCrossRefGoogle Scholar
  36. 36.
    Lin CF, Wan SW, Cheng HJ, et al. Autoimmune pathogenesis in dengue virus infection. Viral Immunol. 2006;19:127–32.PubMedCrossRefGoogle Scholar
  37. 37.
    Mitrakul C, Poshyachinda M, Futrakul P, et al. Hemostatic and platelet kinetic studies in dengue hemorrhagic fever. Am J Trop Med Hyg. 1977;26:975–84.PubMedGoogle Scholar
  38. 38.
    Phanichyakarn P, Pongpanich B, Israngkura PB, et al. Studies on dengue hemorrhagic fever. III. Serum complement (C3) and platelet studies. J Med Assoc Thai. 1977;60:301–6.PubMedGoogle Scholar
  39. 39.
    Emlen W, Carl V, Burdick G. Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes. Clin Exp Immunol. 1992;89:8–17.PubMedCrossRefGoogle Scholar
  40. 40.
    Tambyah PA, Koay ES, Poon ML, et al. Dengue hemorrhagic fever transmitted by blood transfusion. N Engl J Med. 2008;359:1526–7.PubMedCrossRefGoogle Scholar
  41. 41.
    Scott RM, Nisalak A, Cheam-U-Dom U, Seridhoranakul S, Nimmannitya, S. A preliminary report on the isolation of viruses from the platelets and leukocytes of dengue patients. J Infect Dis. 1980:141(1):1–6.Google Scholar
  42. 42.
    Carballal G, Rodriguez M, Frigerio MJ, et al. Junin virus infection of guinea pigs: electron microscopic studies of peripheral blood and bone marrow. J Infect Dis. 1977;135:367–73.PubMedCrossRefGoogle Scholar
  43. 43.
    Shang X, Cancelas JA, Li L, et al. R-Ras and Rac GTPase cross-talk regulates hematopoietic progenitor cell migration, homing, and mobilization. J Biol Chem. 2011;286:24068–78.PubMedCrossRefGoogle Scholar
  44. 44.
    Lapidot T, Petit I. Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol. 2002;30:973–81.PubMedCrossRefGoogle Scholar
  45. 45.
    Bhamarapravati N, Boonyapaknavik V, Nimsomburana P. Pathology of Thai haemorrhagic fever: an autopsy study. Bull World Health Organ. 1966;35:47–8.PubMedGoogle Scholar
  46. 46.
    Piyaratn P. Pathology of Thailand epidemic hemorrhagic fever. Am J Trop Med Hyg. 1961;10:767–72.PubMedGoogle Scholar
  47. 47.
    Semple JW, Freedman J. Platelets and innate immunity. Cell Mol Life Sci. 2010;67:499–511.PubMedCrossRefGoogle Scholar
  48. 48.
    Clemetson KJ. Platelets and pathogens. Cell Mol Life Sci. 2010;67:495–8.PubMedCrossRefGoogle Scholar
  49. 49.
    Flaujac C, Boukour S, Cramer-Borde E. Platelets and viruses: an ambivalent relationship. Cell Mol Life Sci;67:545–556.Google Scholar
  50. 50.
    Satchell CS, Cotter AG, O’Connor EF, et al. Platelet function and HIV: a case–control study. AIDS. 2010;24:649–57.PubMedCrossRefGoogle Scholar
  51. 51.
    Zucker-Franklin D. The effect of viral infections on platelets and megakaryocytes. Semin Hematol. 1994;31:329–37.PubMedGoogle Scholar
  52. 52.
    White JG, Clawson CC. Effects of small latex particle uptake on the surface connected canalicular system of blood platelets: a freeze-fracture and cytochemical study. Diagn Histopathol Publ Assoc Pathol Soc Great Britain Irel. 1982;5:3–10.Google Scholar
  53. 53.
    Youssefian T, Drouin A, Masse JM, et al. Host defense role of platelets: engulfment of HIV and Staphylococcus aureus occurs in a specific subcellular compartment and is enhanced by platelet activation. Blood. 2002;99:4021–9.PubMedCrossRefGoogle Scholar
  54. 54.
    Michelson AD. Platelets. San Diego, CA: Academic Press, Elsevier Inc.; 2007.Google Scholar
  55. 55.
    Marchette NJ, Halstead SB, Falkler WA Jr, et al. Studies on the pathogenesis of dengue infection in monkeys. 3. Sequential distribution of virus in primary and heterologous infections. J Infect Dis. 1973;128:23–30.PubMedCrossRefGoogle Scholar
  56. 56.
    Hombach J, Cardosa MJ, Sabchareon A, et al. Scientific consultation on immunological correlates of protection induced by dengue vaccines report from a meeting held at the World Health Organization 17–18 November 2005. Vaccine. 2007;25:4130–9.PubMedCrossRefGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2012

Authors and Affiliations

  • Sansanee Noisakran
    • 1
    • 5
  • Nattawat Onlamoon
    • 6
  • Kovit Pattanapanyasat
    • 6
  • Hui-Mien Hsiao
    • 1
  • Pucharee Songprakhon
    • 6
    • 7
  • Nasikarn Angkasekwinai
    • 8
  • Kulkanya Chokephaibulkit
    • 9
  • Francois Villinger
    • 2
    • 3
  • Aftab A. Ansari
    • 4
  • Guey Chuen Perng
    • 1
    • 10
  1. 1.Department of Pathology and Laboratory Medicine, Emory Vaccine CenterEmory University School of MedicineAtlantaUSA
  2. 2.Department of Pathology and Laboratory Medicine, Emory Vaccine CenterEmory University School of MedicineAtlantaUSA
  3. 3.Division of Pathology, Yerkes National Primate Research CenterAtlantaUSA
  4. 4.Department of Pathology and Laboratory Medicine, Emory Vaccine CenterEmory University School of MedicineAtlantaUSA
  5. 5.Medical Biotechnology Research UnitNational Center for Genetic Engineering and Biotechnology, National Science and Technology Development AgencyPathumthaniThailand
  6. 6.Center of Excellence for Flow CytometryMahidol UniversityBangkokThailand
  7. 7.Office for Research and DevelopmentMahidol UniversityBangkokThailand
  8. 8.Department of Medicine, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand
  9. 9.Department of Pediatrics, Faculty of Medicine Siriraj HospitalMahidol UniversityBangkokThailand
  10. 10.Department of Microbiology and ImmunologyCollege of Medicine, NCKUTainan CityTaiwan

Personalised recommendations