, Volume 18, Issue 11, pp 1319–1331 | Cite as

A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation

  • Christopher J. Coates
  • Tim Whalley
  • Michael Wyman
  • Jacqueline Nairn
Original Paper


Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.


Phosphatidylserine Apoptosis Phagocytosis Innate immunity Limulus polyphemus Damage-associated patterns 



This work was supported by the University of Stirling. With thanks to Alex Mühlhölzl, C.T.O., Marine Biotech Limited, for providing access to L. polyphemus. We are grateful to Prof. Seamus J. Martin and Conor M. Henry (Molecular Cell Biology Laboratory, Trinity College Dublin) for kindly providing the pProEx.Htb.annexin V plasmid.

Conflict of interest

The authors declare no financial or other potential conflicts of interest.


  1. 1.
    Brinkmann V, Reichard U, Goosmann C et al (2004) Neutrophil extracellular traps kill bacteria. Science 303:1532–1535PubMedCrossRefGoogle Scholar
  2. 2.
    Wang XW, Tan NS, Ho B, Ding JL et al (2006) Evidence for the ancient origin of the NF-kappaB/IkappaB cascade: its archaic role in pathogen infection and immunity. Proc Natl Acad Sci USA 103:4204–4209PubMedCrossRefGoogle Scholar
  3. 3.
    Kroemer G, Galluzzi L, Vandenabeele P et al (2009) Classification of cell death: recommendations of the nomenclature committee on cell death 2009. Cell Death Differ 16:3–11PubMedCrossRefGoogle Scholar
  4. 4.
    Brown GC, Neher JJ (2012) Eaten alive! Cell death by primary phagocytosis: ‘phagoptosis’. Trends Biochem Sci 37:325–332PubMedCrossRefGoogle Scholar
  5. 5.
    Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516PubMedCrossRefGoogle Scholar
  6. 6.
    Cooper DM, Mitchell-Foster K (2011) Death for survival: What do we know about innate immunity and cell death in insects? ISJ 8:162–172Google Scholar
  7. 7.
    Menze MA, Fortner G, Nag S, Hand SC et al (2010) Mechanisms of apoptosis in crustacea: What conditions induce versus suppress cell death? Apoptosis 15:293–312PubMedCrossRefGoogle Scholar
  8. 8.
    Kiss T (2010) Apoptosis and its functional significance in molluscs. Apoptosis 15:313–321PubMedCrossRefGoogle Scholar
  9. 9.
    DeLeo FR (2004) Modulation of phagocyte apoptosis by bacterial pathogens. Apoptosis 9:399–413PubMedCrossRefGoogle Scholar
  10. 10.
    Thi EP, Lambertz U, Reiner NE (2012) Sleeping with the enemy: how intracellular pathogens cope with macrophage lifestyle. PLoS Pathog 8:e1002551PubMedCrossRefGoogle Scholar
  11. 11.
    Frankenberg T, Kirschnek S, Hacker H, Hacker G (2008) Phagocytosis-induced apoptosis of macrophages is linked to uptake, killing and degradation of bacteria. Eur J Immunol 38:204–215PubMedCrossRefGoogle Scholar
  12. 12.
    Kirschnek S, Ying S, Fischer SF et al (2005) Phagocytosis-induced apoptosis is mediated by up-regulation and activation of the Bcl-2 homology domain 3-only protein Bim. J Immunol 174:671–679PubMedGoogle Scholar
  13. 13.
    Kennedy AD, DeLeo FR (2009) Neutrophil apoptosis and the resolution of infection. Immunol Res 43:25–61PubMedCrossRefGoogle Scholar
  14. 14.
    Decker H, Jaenicke E (2004) Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins. Devel Comp Immunol 28:673–687CrossRefGoogle Scholar
  15. 15.
    Jiang N, Tan NS, Ho B, Ding JL (2007) Respiratory protein generated reactive oxygen species as an antimicrobial strategy. Nature Immunol 8:1114–1122CrossRefGoogle Scholar
  16. 16.
    Zhang Y, Yan F, Hu Z, Zhao X et al (2009) Hemocyanin from shrimp Litopenaeus vannamei shows hemolytic activity. Fish Shellfish Immunol 27:33–335CrossRefGoogle Scholar
  17. 17.
    Coates CJ, Kelly SM, Nairn J (2011) Possible role of phosphatidylserine-hemocyanin interaction in the innate immune response of Limulus polyphemus. Dev Comp Immunol 35:155–163PubMedCrossRefGoogle Scholar
  18. 18.
    Zhao X, Guo L, Zhang Y, Liu Y et al (2012) SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei. FEBS Lett 586:403–410PubMedCrossRefGoogle Scholar
  19. 19.
    Coates CJ, Whalley T, Nairn J (2012) Phagocytic activity of Limulus polyphemus amebocytes in vitro. J Invert Pathol 111:205–210CrossRefGoogle Scholar
  20. 20.
    Altman SA, Randers L, Rao G (1993) Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinants. Biotech Prog 9:671–674CrossRefGoogle Scholar
  21. 21.
    Logue SE, Elgendy M, Martin SJ (2009) Expression, purification and use of recombinant annexin V for the detection of apoptotic cells. Nat Protoc 4:1383–1395PubMedCrossRefGoogle Scholar
  22. 22.
    Evans TC Jr, Nelsestuen GL (1994) Calcium and membrane-binding of monomeric and multimeric annexin II. Biochemistry 33:13231–13238PubMedCrossRefGoogle Scholar
  23. 23.
    Ding JL, Thangamani S, Kusuma N, Seow WK et al (2005) Spatial and temporal coordination of expression of immune genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda. Genes Immun 6:557–574PubMedCrossRefGoogle Scholar
  24. 24.
    Coates CJ, Bradford EL, Krome CA, Nairn J (2012) Effect of temperature on biochemical and cellular properties of Limulus polyphemus. Aquaculture 334–337:30–38CrossRefGoogle Scholar
  25. 25.
    Armstrong PB (1980) Adhesion and spreading of Limulus blood cells on artificial surfaces. J Cell Sci 44:243–262PubMedGoogle Scholar
  26. 26.
    Conrad ML, Pardy RL, Wainwright N, Child A, Armstrong PB (2006) Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga. Comp Biochem Physiol Part A 144:423–428CrossRefGoogle Scholar
  27. 27.
    Li H, Zhu H, Xu C-J, Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491–501PubMedCrossRefGoogle Scholar
  28. 28.
    Marchetti P, Castedo M, Susin SA, Zamzami N et al (1996) Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp Med 184:1155–1160PubMedCrossRefGoogle Scholar
  29. 29.
    Jagasia R, Grote P, Westermann B, Condradt B (2005) DRP-1-mediated mitochondrial fragmentation during EGL-1-induced cell death in C. elegans. Nature 433:754–760PubMedCrossRefGoogle Scholar
  30. 30.
    Henry CM, Hollville E, Martine SJ (2013) Measuring apoptosis by microscopy and flow cytometry. Methods 61:90–97PubMedCrossRefGoogle Scholar
  31. 31.
    Green DR, Oberst A, Dillon CP, Weinlich R, Salvesen GS (2011) RIPK-dependent necrosis and its regulation by caspases: a mystery in five acts. Mol Cell 44:9–16PubMedCrossRefGoogle Scholar
  32. 32.
    Baird S, Kelly SM, Price NC, Jaenicke E et al (2007) Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation. Biochim Biophys Acta 1774:1380–1394PubMedCrossRefGoogle Scholar
  33. 33.
    Watson RW, Redmond HP, Wang JH, Condron C, Bouchier-Hayes D (1996) Neutrophils undergo apoptosis following ingestion of Escherichia coli. J Immunol 156:3986–3992PubMedGoogle Scholar
  34. 34.
    Colamussi ML, White MR, Crouch E, Hartshorn KL (1999) Influenza A virus accelerates apoptosis and markedly potentiates apoptotic effects of bacteria. Blood 93:2395–2403PubMedGoogle Scholar
  35. 35.
    Rotstein D, Parodo J, Taneja R, Marshall JC (2000) Phagocytosis of Candida albicans induces apoptosis of human neutrophils. SHOCK 14:278–283PubMedCrossRefGoogle Scholar
  36. 36.
    Zhang B, Hirahashi J, Cullere X, Mayadas TN (2003) Elucidation of the molecular events leading to neutrophil apoptosis following phagocytosis. J Biol Chem 278:28443–28454PubMedCrossRefGoogle Scholar
  37. 37.
    Krzyzowska M, Schollenberger A, Skierski J, Niemialtowski M (2002) Apoptosis during ectromelia orthopoxvirus infection is DEVDase dependent: in vitro and in vivo studies. Microb Infect 4:599–611CrossRefGoogle Scholar
  38. 38.
    Phongdara A, Wanna W, Chotigeat W (2006) Molecular cloning and expression of caspase from white shrimp Penaeus merguiensis. Aquaculture 252:114–120CrossRefGoogle Scholar
  39. 39.
    Song Z, McCall K, Steller H (1997) DCP-1, a Drosophila cell death protease essential for development. Science 275:536–540PubMedCrossRefGoogle Scholar
  40. 40.
    Albee L, Shi B, Perlman H (2007) Aspartic protease and caspase 3/7 activation are central to macrophage apoptosis following infection with Escherichia coli. J Leuk Biol 81:229–237CrossRefGoogle Scholar
  41. 41.
    Gille Ch, Leiber A, Mundle I, Spring B et al (2009) Phagocytosis and postphagocytic reaction of cord blood and adult blood monocyte after infection with green fluorescent protein-labelled Escherichia coli and group B Streptococci. Cytom Part B 76B:271–284CrossRefGoogle Scholar
  42. 42.
    Dreschers S, Gille C, Haas M, Grosse-Ophoff J et al (2013) Infection-induced bystander apoptosis of monocytes is TNF-alpha-mediated. PLoS ONE 8:e53589PubMedCrossRefGoogle Scholar
  43. 43.
    Smith VJ (2010) Immunology of invertebrates: cellular. Encyclopedia of life sciences (ELS). Wiley, Chicester. doi: 10.1002/9780470015902.a0002344.pub2 Google Scholar
  44. 44.
    Franc NC (2002) Phagocytosis of apoptotic cells in mammals, Caenorhabditis elegans and Drosophila melanogaster: molecular mechanisms and physiological consequences. Front Biosci 7:1298–1313CrossRefGoogle Scholar
  45. 45.
    Hillman MR (2004) Strategies and mechanisms for host and pathogen survival in acute and persistent viral infections. Proc Nat Acad Sci, USA 101:14560–14566CrossRefGoogle Scholar
  46. 46.
    McLean JE, Ruck A, Shirazian S, Pooyaei-Mehr F, Zakeri ZF (2008) Viral manipulation of cell death. Cur Pharm Des 14:198–220CrossRefGoogle Scholar
  47. 47.
    Watthanasurorot A, Jiravanichpaisal P, Soderhall K, Soderhall I (2013) A calreticulin/gC1qR complex prevents cells from dying: a conserved mechanism from arthropods to humans. J Mol Cell Biol 5:120–131PubMedCrossRefGoogle Scholar
  48. 48.
    Hsu J-P, Huang C, Liao C-M, Hsuan S-L, Hung H–H, Chien M-S (2005) Engulfed pathogen-induced apoptosis in haemocytes of giant freshwater prawn, Macrobrachium rosenbergii. J Fish Dis 28:729–735PubMedCrossRefGoogle Scholar
  49. 49.
    Lavrov DV, Boore JL, Brown WM (2000) The complete mitochondrial DNA sequence of the horseshoe crab Limulus polyphemus. Mol Biol Evol 17:813–824PubMedCrossRefGoogle Scholar
  50. 50.
    Wu Y, Tibrewal N, Birge RB (2006) Phosphatidylserine recognition by phagocytes: a view to a kill. Trends Cell Biol 16:189–197PubMedCrossRefGoogle Scholar
  51. 51.
    Clark MR (2011) Flippin’ lipids. Nat Immunol 12:373–375PubMedCrossRefGoogle Scholar
  52. 52.
    Lee SH, Meng XW, Flatten KS, Loegering DA, Kaufmann SH (2013) Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm. Cell Death Differ 20:64–76PubMedCrossRefGoogle Scholar
  53. 53.
    Levin J, Bang FB (1968) Clottable protein in Limulus: its localization and kinetics of its coagulation by endotoxin. Thrombosis et Diathesis Haemorrhagica 19:186–197PubMedGoogle Scholar
  54. 54.
    Nagai T, Kawabata S (2000) A link between blood coagulation and prophenoloxidase activation in the arthropod host defence. J Biol Chem 275:29264–29267PubMedCrossRefGoogle Scholar
  55. 55.
    Nellaiappan K, Sugumaran M (1996) On the presence of prophenoloxidase in the hemolymph of the horseshoe crab, Limulus. Comp Biochem Phys Part B 113:163–168CrossRefGoogle Scholar
  56. 56.
    Nagai T, Osaki T, Kawabata S (2001) Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. J Biol Chem 276:27166–27170PubMedCrossRefGoogle Scholar
  57. 57.
    Meers P, Mealy T (1993) Calcium-dependent annexin V binding to phospholipids: stoichiometry, specificity, and the role of negative charge. Biochemistry 32:11711–11721PubMedCrossRefGoogle Scholar
  58. 58.
    Pigault C, Follenius-Wund A, Schmutz M, Freyssinet J-M, Brisson A (1994) Formation of two-dimensional arrays of annexin V on phosphatidylserine-containing liposomes. J Mol Biol 236:199–208PubMedCrossRefGoogle Scholar
  59. 59.
    Wright J, McCaskill-Clark W, Cain JA, Patterson A, Coates CJ, Nairn J (2012) Effects of known phenoloxidase inhibitors on hemocyanin-derived phenoloxidase from Limulus polyphemus. Comp Biochem Physiol Part B 163:303–308CrossRefGoogle Scholar
  60. 60.
    Martin CJ, Booty MG, Rosebrock TR (2012) Efferocytosis is an innate antibacterial mechanism. Cell Host Microbe 12:289–300PubMedCrossRefGoogle Scholar
  61. 61.
    Zwaal RFA, Comfurius P, Bevers EM (1998) Lipid-protein interactions in blood coagulation. Biochim Biophys Acta 1376:433–453PubMedCrossRefGoogle Scholar
  62. 62.
    Stace CL, Ktistakis NT (2006) Phosphatidic acid-and phosphatidylserine-binding proteins. Biochim Biophys Acta 1761:913–926PubMedCrossRefGoogle Scholar
  63. 63.
    Nakamura T, Tokunaga F, Morita T et al (1988) Intracellular serine-protease zymogen, factor C, from horseshoe crab hemocytes. Eur J Biochem 176:89–94PubMedCrossRefGoogle Scholar
  64. 64.
    Bidla A, Hauling T, Dushay MS, Theopold U (2009) Activation of insect phenoloxidase after injury: endogenous versus foreign elicitors. J Innat Immun 1:301–308CrossRefGoogle Scholar
  65. 65.
    Vance JE, Steenbergen R (2005) Metabolism and functions of phosphatidylserine. Prog Lipid Res 44:207–234PubMedCrossRefGoogle Scholar
  66. 66.
    Zaini NAM, Osman A, Hamid AA, Ebrahimpour A, Saari N (2013) Purification and characterisation of membrane-bound Polyphenoloxidase (mPPO) from snake fruit [Salacca zalacca (Gaertn.) Voss]. Food Chem 136:407–414PubMedCrossRefGoogle Scholar
  67. 67.
    Sokolova IM (2009) Apoptosis in molluscan immune defense. ISJ 6:49–58Google Scholar
  68. 68.
    Bidla A, Dushay MS, Theopold U (2007) Crystal rupture after injury in Drosophila requires JNK pathway, small GTPases and the TNF homolog Eiger. J Cell Sci 120:1209–1215PubMedCrossRefGoogle Scholar
  69. 69.
    Jiravanichpaisal P, Lee BL, Soderhall K (2006) Cell-mediated immunity in arthropods: Hemoatopoiesis, coagulation, melanisation and opsonisation. Immunbiol 211:213–236CrossRefGoogle Scholar
  70. 70.
    Kawabata S, Koshiba T, Shibata T (2009) The lipopolysaccharide-activated innate immune response network of the horseshoe crab. ISJ 6:59–77Google Scholar
  71. 71.
    MacPherson JC, Pavlovich JG, Jacobs RS (1998) Phospholipid composition of the granular amebocyte from the horseshoe crab, Limulus polyphemus. Lipids 33:931–940PubMedCrossRefGoogle Scholar
  72. 72.
    Armstrong P, Levin J (1979) In vitro phagocytosis by Limulus blood cells. J Invert Pathol 34:145–151CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Biological and Environmental Sciences, School of Natural SciencesUniversity of StirlingStirlingScotland, UK

Personalised recommendations