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P2X receptor stimulation amplifies complement-induced haemolysis

  • Signaling and Cell Physiology
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Abstract

Activation of the complement system evokes cell damage by insertion of membrane attack complexes, which constitute the basis of the pathogenesis of various haemolytic disorders. Recently, we found that haemolysis caused by other types of membrane pore-forming proteins such as α-haemolysin (HlyA) from Escherichia coli, α-toxin from Staphylococcus aureus and leukotoxin from Aggregatibacter actinomycetemcomitans inflict their cytotoxic effects through P2 receptor activation. Here we show that similar to haemolysis induced by HlyA, leukotoxin and α-toxin, complement-induced haemolysis is amplified through ATP release and subsequent P2 receptor activation. Similar results were found both in murine, sensitised ovine and human erythrocytes, with either human plasma or guinea pig serum as complement donors. Non-selective P2 antagonists (PPADS and suramin) concentration-dependently inhibited complement-induced haemolysis. More specific P2 receptor antagonists imply that P2X1 and P2X7 are the main receptors involved in this response. Moreover, complement activation produces a sustained increase in [Ca2+]i, which initially triggers significant erythrocyte shrinkage, most likely mediated by KCa3.1-dependent K+ efflux. These results indicate that complement, similar to HlyA and α-toxin, requires purinergic signalling for full haemolysis and that activation of erythrocyte volume regulation protracts the process. This finding points to several new pathways to interfere with haemolytic diseases and implies that P2 receptor antagonists potentially can be used to prevent intravascular haemolysis.

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References

  1. Davidson JS, Baumgarten IM, Harley EH (1986) Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid. Biochem Biophys Res Commun 134:29–36

    Article  PubMed  CAS  Google Scholar 

  2. De Franceschi L, Rivera A, Fleming MD, Honczarenko M, Peters LL, Gascard P, Mohandas N, Brugnara C (2005) Evidence for a protective role of the Gardos channel against hemolysis in murine spherocytosis. Blood 106:1454–1459

    Article  PubMed  Google Scholar 

  3. Dunkelberger JR, Song WC (2010) Complement and its role in innate and adaptive immune responses. Cell Res 20:34–50

    Article  PubMed  CAS  Google Scholar 

  4. Foller M, Bobbala D, Koka S, Boini KM, Mahmud H, Kasinathan RS, Shumilina E, Amann K, Beranek G, Sausbier U, Ruth P, Sausbier M, Lang F, Huber SM (2010) Functional significance of the intermediate conductance Ca2+-activated K+ channel for the short-term survival of injured erythrocytes. Pflugers Arch 460:1029–1044

    Article  PubMed  Google Scholar 

  5. Fong JS, Good RA (1972) Suramin—a potent reversible and competitive inhibitor of complement systems. Clin Exp Immunol 10:127–138

    PubMed  CAS  Google Scholar 

  6. Iglesias R, Spray DC, Scemes E (2009) Mefloquine blockade of pannexin1 currents: resolution of a conflict. Cell Commun Adhes 16:131–137

    Article  PubMed  CAS  Google Scholar 

  7. Kelk P, Abd H, Claesson R, Sandstrom G, Sjostedt A, Johansson A (2011) Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin. Cell Death Dis 2:e126

    Article  PubMed  CAS  Google Scholar 

  8. Kroes BH, Beukelman CJ, van den Berg AJ, Wolbink GJ, van Dijk H, Labadie RP (1997) Inhibition of human complement by beta-glycyrrhetinic acid. Immunology 90:115–120

    Article  PubMed  CAS  Google Scholar 

  9. Larsen CK, Skals M, Wang T, Cheema MU, Leipziger J, Praetorius HA (2011) Python erythrocytes are resistant to alpha-hemolysin from Escherichia coli. J Membr Biol 244:131–140

    Article  PubMed  CAS  Google Scholar 

  10. Locovei S, Bao L, Dahl G (2006) Pannexin 1 in erythrocytes: function without a gap. Proc Natl Acad Sci USA 103:7655–7659

    Article  PubMed  CAS  Google Scholar 

  11. Munksgaard PS, Vorup-Jensen T, Reinholdt J, Söderström CM, Poulsen K, Leipziger J, Praetorius HA, Skals M (2012) Leukotoxin from Aggregatibacter actinomycetemcomitans causes shrinkage and P2X receptor-dependent lysis of human erythrocytes. Cell Microbiol. doi:10.1111/cmi.12021

  12. Orth D, Wurzner R (2010) Complement in typical hemolytic uremic syndrome. Semin Thromb Hemost 36:620–624

    Article  PubMed  CAS  Google Scholar 

  13. Roumenina LT, Loirat C, Dragon-Durey MA, Halbwachs-Mecarelli L, Sautes-Fridman C, Fremeaux-Bacchi V (2011) Alternative complement pathway assessment in patients with atypical HUS. J Immunol Methods 365:8–26

    Article  PubMed  CAS  Google Scholar 

  14. Saez C, Thielens NM, Bjes ES, Esser AF (1999) Association of terminal complement proteins in solution and modulation by suramin. Biochemistry 38:6807–6816

    Article  PubMed  CAS  Google Scholar 

  15. Silverman W, Locovei S, Dahl G (2008) Probenecid, a gout remedy, inhibits pannexin 1 channels. Am J Physiol Cell Physiol 295:C761–C767

    Article  PubMed  CAS  Google Scholar 

  16. Skals MG, Jorgensen NR, Leipziger J, Praetorius HA (2009) α-Hemolysin from Escherichia coli uses endogenous amplification through P2X receptor activation to induce hemolysis. Proc Natl Acad Sci USA 106:4030–4035

    Article  PubMed  CAS  Google Scholar 

  17. Skals M, Jensen UB, Ousingsawat J, Kunzelmann K, Leipziger J, Praetorius HA (2010) Escherichia coli alpha-hemolysin triggers shrinkage of erythrocytes via K Ca 3.1 and TMEM16A channels with subsequent phosphatidylserine exposure. J Biol Chem 285:15557–15565

    Article  PubMed  CAS  Google Scholar 

  18. Skals M, Leipziger J, Praetorius HA (2011) Haemolysis induced by α-toxin from Staphylococcus aureus requires P2X receptor activation. Pflugers Arch 462:669–679

    Article  PubMed  CAS  Google Scholar 

  19. Sluyter R, Shemon AN, Barden JA, Wiley JS (2004) Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X 7 receptor. J Biol Chem 279:44749–44755

    Article  PubMed  CAS  Google Scholar 

  20. Tanneur V, Duranton C, Brand VB, Sandu CD, Akkaya C, Kasinathan RS, Gachet C, Sluyter R, Barden JA, Wiley JS, Lang F, Huber SM (2006) Purinoceptors are involved in the induction of an osmolyte permeability in malaria-infected and oxidized human erythrocytes. FASEB J 20:133–135

    PubMed  CAS  Google Scholar 

  21. Wang L, Olivecrona G, Gotberg M, Olsson ML, Winzell MS, Erlinge D (2005) ADP acting on P2Y 13 receptors is a negative feedback pathway for ATP release from human red blood cells. Circ Res 96:189–196

    Article  PubMed  CAS  Google Scholar 

  22. Wiedmer T, Hall SE, Ortel TL, Kane WH, Rosse WF, Sims PJ (1993) Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria. Blood 82:1192–1196

    PubMed  CAS  Google Scholar 

  23. Zalman LS, Brothers MA, Muller-Eberhard HJ (1985) A C9 related channel forming protein in the cytoplasmic granules of human large granular lymphocytes. Biosci Rep 5:1093–1100

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank for the skilled technical assistance from Helle Jakobsen and Edith Bjoern Moeller. This project is financially supported by The Danish Medical Research Council, Danish National Research Foundation and The Aarhus University Research Foundation.

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Authors and Affiliations

  1. Department of Biomedicine - Physiology, Aarhus University, Aarhus, Denmark

    J. L. Hejl, M. Skals, J. Leipziger & H. A. Praetorius

  2. Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Build. 1160, 8000, Aarhus, Denmark

    H. A. Praetorius

Authors
  1. J. L. Hejl
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  2. M. Skals
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  3. J. Leipziger
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  4. H. A. Praetorius
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Corresponding author

Correspondence to H. A. Praetorius.

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Supplementary figure 1

Preincubation of human erythrocytes with 240 μl ml−1 guinea pig serum and 80 μl ml−1 purified guinea pig complement for 0, 10 and 15 minutes. After preincubation the samples where washed and incubated with 300 μM suramin for the remaining of 60 minutes. The graph illustrates the degree of inhibition by suramin by this procedure. (PDF 34 kb)

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Hejl, J.L., Skals, M., Leipziger, J. et al. P2X receptor stimulation amplifies complement-induced haemolysis. Pflugers Arch - Eur J Physiol 465, 529–541 (2013). https://doi.org/10.1007/s00424-012-1174-z

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  • DOI: https://doi.org/10.1007/s00424-012-1174-z

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