The basis of the immunomodulatory activity of malaria pigment (hemozoin)

  • Clare K. Carney
  • Alexandra C. Schrimpe
  • Kristin Halfpenny
  • Reese S. Harry
  • Crystal M. Miller
  • Malgorzata Broncel
  • Sarah L. Sewell
  • Jacob E. Schaff
  • Ravinder Deol
  • Melissa D. Carter
  • David W. WrightEmail author
Original Paper


The most common and deadly form of the malaria parasite, Plasmodium falciparum, is responsible for 1.5–2.7 million deaths and 300–500 million acute illnesses annually [Bremen in J. Trop. Med. Hyg. 64:1–11 (2001); World Health Organization (2002)]. Hemozoin, the biomineral formed to detoxify the free heme produced during parasitic hemoglobin catabolism, has long been suspected of contributing to the pathological immunodeficiencies that occur during malarial infection. While there is a growing consensus in the literature that native hemozoin maintains immunosuppressive activity, there is considerable controversy over the reactivity of the synthetic form, β-hematin (BH). Given the emerging importance of hemozoin in modulating a host immune response to malarial infection, a careful examination of the effects of the constitutive components of the malaria pigment on macrophage response has been made in order to clarify the understanding of this process. Herein, we present evidence that BH alone is unable to inhibit stimulation of NADPH oxidase and inducible nitric oxide synthase, the key enzymes involved in oxidative burst, and is sensitive to the microbicidal agents of these enzymes both in vitro and in vivo. Further, by systematically examining each of the malaria pigment’s components, we were able to dissect their impact on the immune reactivity of a macrophage model cell line. Reactions between BH and red blood cell (RBC) ghosts effectively reconstituted the observed immunomodulatory reactivity of native hemozoin. Together, these results suggest that the interaction between hemozoin and the RBC lipids results in the generation of toxic products and that these products are responsible for disrupting macrophage function in vivo.


Hemozoin β-Hematin Malaria Lipid peroxidation Immunomodulation 





Dichlorofluorescein diacetate


Diethylamine/nitric oxide sodium complex


Dimethyl sulfoxide


Ferric protoporphyrin IX


Fourier transform IR


15-S-Hydroxyeicosatetraenioc acid


N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid




Inducible nitric oxide synthase






Phosphate-buffered saline


Piperazine-1,4-bis(2-ethanesulfonic acid)




Red blood cell


Reactive nitrogen species


Reactive oxygen species



D.W.W. thanks NIH for financial support through NIH (NIAID) grant 1R03AI060827. Confocal images using a Zeiss LSM 510 Meta inverted confocal microscope were performed in part through the use of the VUMC Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, HD15052, DK59637, and EY08126). The Wright group would also like to thank the laboratory of Virginia Shepherd for access to cell lines and experimental equipment, expert advice and helpful discussions during this project.

Supplementary material


  1. 1.
    Bremen J (2001) Am J Trop Med Hyg 64:1–11Google Scholar
  2. 2.
    World Health Organization (2002)Google Scholar
  3. 3.
    Sherman IW (1998) ASM Press, WashingtonGoogle Scholar
  4. 4.
    Greenwood B, Mutabingwa T (2002) Nature 415:670–672PubMedCrossRefGoogle Scholar
  5. 5.
    Goldberg DE (1993) Semin Cell Biol 4:355–358PubMedCrossRefGoogle Scholar
  6. 6.
    Goldberg DE, Slater AFG, Cerami A, Henderson GB (1990) Proc Natl Acad Sci USA 87:2931–2935PubMedCrossRefGoogle Scholar
  7. 7.
    Francis SE, Sullivan DJ Jr, Goldberg DE (1997) Annu Rev Microbiol 51:97–123PubMedCrossRefGoogle Scholar
  8. 8.
    Green MD, Xiao L, Lal AA (1996) Mol Biochem Parasitol 83:183–188PubMedCrossRefGoogle Scholar
  9. 9.
    Oliveira MR, Timm BL, Machado EA, Kildare M, Attias M, Silva JR, Dansa-Petretski M, de Oliveira MA, De Souza W, Pinhal NM, Sousa JJF, Vugman NV, Oliveira PL (2002) FEBS Lett 512:139–144PubMedCrossRefGoogle Scholar
  10. 10.
    Atamna H, Ginsberg H (1993) Mol Biochem Parasitol 61:231–241PubMedCrossRefGoogle Scholar
  11. 11.
    Orjih AU, Banyal HS, Chevli R, Fitch CD (1981) Science 214:667–669PubMedCrossRefGoogle Scholar
  12. 12.
    Carney CK, Harry RS, Sewell S, Wright DW (2006) In: Naka K (ed) Biomineralization. Springer, Berlin Heidelberg New YorkGoogle Scholar
  13. 13.
    Pagola S, Stephens PW, Bohle DS, Kosar AD, Madsen SK (2000) Nature 404:307–310PubMedCrossRefGoogle Scholar
  14. 14.
    Hurst JK, Lymar SV (1999) Acc Chem Res 32:520–528CrossRefGoogle Scholar
  15. 15.
    Schwarzer E, Bellomo G, Giribaldi G, Ulliers D, Arese P (2001) Parasitology 123:125–131PubMedCrossRefGoogle Scholar
  16. 16.
    Schwarzer E, Turrini F, Ulliers D, Giribaldi G, Ginsberg H, Arese P (1992) J Exp Med 176:1033–1041PubMedCrossRefGoogle Scholar
  17. 17.
    Schwarzer E, Arese P (1996) Biochim Biophys Acta 1316:169–175PubMedGoogle Scholar
  18. 18.
    Reiner NE (1994) Immunol Today 15:374–381PubMedCrossRefGoogle Scholar
  19. 19.
    Fiori PL, Rappelli P, Mirkarimi SN, Ginsburg H, Capuccinelli P, Turrini F (1993) Parasite Immunol 15:647–655PubMedCrossRefGoogle Scholar
  20. 20.
    Schwarzer E, Alessio M, Ulliers D, Arese P (1998) Infect Immun 66:1601–1606PubMedGoogle Scholar
  21. 21.
    Schwarzer E, Muller O, Arese P, Siems WG, Grune T (1996) FEBS Lett 388:119–122PubMedCrossRefGoogle Scholar
  22. 22.
    Schwarzer E, Ludwig P, Valente E, Arese P (1999) Parassitologoa 41:199–202Google Scholar
  23. 23.
    Schwarzer E, Kuhn H, Valente E, Arese P (2003) Blood 101:722–728PubMedCrossRefGoogle Scholar
  24. 24.
    Miller CM, Carney CK, Schrimpe AC, Wright DW (2005) Inorg Chem 44:2134–2136PubMedCrossRefGoogle Scholar
  25. 25.
    Pichyangkul S, Saengkrai P, Webster H (1994) Am J Trop Med Hyg 51:430–435PubMedGoogle Scholar
  26. 26.
    Prada J, Malinowsky J, Muller S, Bienzle U, Kremsner P (1995) Eur Cytokine Netw 6:109–112PubMedGoogle Scholar
  27. 27.
    Sherry BA, Alava G, Tracey KJ, Martiney J, Cerami A, Slater AFG (1995) J Inflamm 45:85–96PubMedGoogle Scholar
  28. 28.
    Taramelli D, Basilico N, Pagani E, Grande R, Monti D, Ghionew M, Olliaro P (1995) Exp Parasitol 81:501–511PubMedCrossRefGoogle Scholar
  29. 29.
    Taramelli D, Basilico N, De Palma AM, Sarasella M, Ferrante P (1998) Trans R Soc Trop Med Hyg 92:57–62PubMedCrossRefGoogle Scholar
  30. 30.
    Taramelli D, Recalcati S, Basilico N, Olliaro P, Cairo G (2000) Lab Invest 80:1781–1788PubMedCrossRefGoogle Scholar
  31. 31.
    Biswas S, Karmarkar MG, Sharma YD (2001) Microbiol Lett 194:175–179CrossRefGoogle Scholar
  32. 32.
    Jaramillo M, Gowda DC, Radzioch D, Olivier M (2003) J Immunol 171:4243–4253PubMedGoogle Scholar
  33. 33.
    Bohle DS, Helms JB (1993) Biochem Biophys Res Commun 193:504–508PubMedCrossRefGoogle Scholar
  34. 34.
    Slater AFG, Swiggard WJ, Orton BR, Flitter WD, Goldberg DE, Cerami A, Henderson GB (1991) Proc Natl Acad Sci USA 88:325–329PubMedCrossRefGoogle Scholar
  35. 35.
    Blauer G, Akkawi M (1997) J Inorg Biochem 66:145–152PubMedCrossRefGoogle Scholar
  36. 36.
    Basilico N, Pagani E, Monti D, Olliaro P, Taramelli D (1998) J Antimicrob Chemother 42:55PubMedCrossRefGoogle Scholar
  37. 37.
    Noland GS, Briones N, Sullivan DJ Jr (2003) Mol Biochem Parasitol 130:91PubMedCrossRefGoogle Scholar
  38. 38.
    Nagano T (1999) Lumin 14:283–290CrossRefGoogle Scholar
  39. 39.
    Huckaba CE, Keys FG (1948) J Am Chem Soc 70:1640–1644CrossRefPubMedGoogle Scholar
  40. 40.
    Ditz H, Rudolf M (1930) Z Anal Chem 79:333–345CrossRefGoogle Scholar
  41. 41.
    Derham BK, Ellory JC, Bron AJ, Harding JJ (2003) Eur J Biochem 270:2605–2611PubMedCrossRefGoogle Scholar
  42. 42.
    Steck TL, Kant JA (1974) Methods Enzymol 31Google Scholar
  43. 43.
    Babior BM (1999) Blood 93:1464–1476PubMedGoogle Scholar
  44. 44.
    Vignais PV (2002) Cell Mol Life Sci 59:1428–1459PubMedCrossRefGoogle Scholar
  45. 45.
    DeLeo FR, Quinn MT (1996) J Leukoc Biol 60:677–691PubMedGoogle Scholar
  46. 46.
    Jaramillo M, Godbout M, Olivier M (2005) J Immunol 174:475–484PubMedGoogle Scholar
  47. 47.
    Keller CC, Kremsner PG, Hittner JB, Misukonis MA, Weinberg JB, Perkins DJ (2004) Infect Immun 72:4868–4873PubMedCrossRefGoogle Scholar
  48. 48.
    Prada J, Malinowsky J, Muller S, Bienzle U, Kremsner PG (1996) Am J Trop Med Hyg 54:620–624PubMedGoogle Scholar
  49. 49.
    Tenhunen R, Marver RS (1969) J Biol Chem 244:6388–6394PubMedGoogle Scholar
  50. 50.
    Chen MM, Shi L, Sullivan DJ Jr (2001) Mol Biochem Parasitol 113:1–8PubMedCrossRefGoogle Scholar
  51. 51.
    Schaefer WH, Harris TM, Guengerich FP (1985) Biochemistry 24:3254–3263PubMedCrossRefGoogle Scholar
  52. 52.
    Groves JT, Haushalter RC, Nakamura M, Nemo TE, Evans BJ (1981) J Am Chem Soc 103:2884–2886CrossRefGoogle Scholar
  53. 53.
    Nagababu E, Rifkind JM (2004) Antioxid Redox Signal 6:967–978PubMedGoogle Scholar
  54. 54.
    Nakamoto K (1997) In: Nakamoto K (ed) Infrared and Raman spectra of inorganic and coordination compounds part B: applications in coordination, organometallic, and bioinorganic chemistry. Wiley, New York, pp 319–377Google Scholar
  55. 55.
    Decatur SM, Franzen S, Depillis GD, Dyer RB, Woodruff WH, Boxer SG (1996) Biochemistry 35:4939–4944PubMedCrossRefGoogle Scholar
  56. 56.
    Bohle DS, Kosar AD, Stephens PW (2003) Can J Chem 81:1285–1291CrossRefGoogle Scholar
  57. 57.
    Albrich JM, McCarthy CA, Hurst JK (1981) Proc Natl Acad Sci USA 78:210–214PubMedCrossRefGoogle Scholar
  58. 58.
    Arese P, Schwarzer E (1997) Ann Trop Med Parasitol 91:501–516PubMedCrossRefGoogle Scholar
  59. 59.
    Schwarzer E, De Matteis F, Giribaldi G, Ulliers D, Valente E, Arese P (1999) Mol Biochem Parasitol 100:61–72PubMedCrossRefGoogle Scholar
  60. 60.
    Schwarzer E, Muller O, Arese P, Siems WG, Grune T (1996) FEBS Lett 388:119–122PubMedCrossRefGoogle Scholar
  61. 61.
    Neely MD, Amarnath V, Weitlauf C, Montine TJ (2002) Chem Res Toxicol 15:40–47PubMedCrossRefGoogle Scholar
  62. 62.
    Dianzani C, Parrini M, Ferrara C, Fantozzi R (1996) Cell Biochem Funct 14:193–200PubMedCrossRefGoogle Scholar
  63. 63.
    Poli G, Schaur RJ (2000) Life 50:315–321PubMedGoogle Scholar
  64. 64.
    Schwarzer E, Turrini F, Giribaldi G, Cappadoro M, Arese P (1993) Biochim Biophys Acta 1181:51–54PubMedGoogle Scholar
  65. 65.
    Goldie P, Roth EF Jr, Oppenheim J, Vanderberg JP (1990) Am J Trop Med Hyg 43:584–596PubMedGoogle Scholar
  66. 66.
    Coban C, Ishii KJ, Hemmi H, Sato S, Uematsu S, Yamamoto M, Takeuchi O, Itagaki S, Kumar N, Horri T, Akira S (2005) J Exp Med 201:19–25PubMedCrossRefGoogle Scholar
  67. 67.
    Millington OR, Di Lorenzo C, Phillips RS, Garside P, Brewer JM (2006) J Biol 5:5PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2006

Authors and Affiliations

  • Clare K. Carney
    • 1
  • Alexandra C. Schrimpe
    • 1
  • Kristin Halfpenny
    • 1
  • Reese S. Harry
    • 1
  • Crystal M. Miller
    • 1
  • Malgorzata Broncel
    • 1
  • Sarah L. Sewell
    • 1
  • Jacob E. Schaff
    • 1
  • Ravinder Deol
    • 1
  • Melissa D. Carter
    • 1
  • David W. Wright
    • 1
    Email author
  1. 1.Department of ChemistryVanderbilt UniversityNashvilleUSA

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