Annelid Humoral Immunity: Cell Lysis in Earthworms

  • Edwin L. Cooper
  • Ellen Kauschke
  • Andrea Cossarizza
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 484)


Earthworms and other organisms have deployed several strategies essential for maintaining and perpetuating species and one is the immune system that functions effectively against microbes. Effector activity against experimental antigens such as cancer cells is mediated by leukocytes and molecules that they synthesize and secrete (Cooper, et al., 1995; Cossarizza et al., 1995; 1996; Quaglino, et al., 1996; Cooper et al., 1992; 1999; Bilej et al., 1995; Kauschke et al., 1997). Current work emphasizes the capacity of coelomic fluid to effect lysis by means of a protein which has been referred to as Eiseniapore (Lange et al., 1997). Clearly the earthworm’s importance in understanding this aspect of invertebrate humoral immunity is significant as indicated by the recent increase in molecular studies relevant to lysis, (Lassegues et al., 1997; Milochau et al., 1997; Sekizawa, et al., 1997; Yamaji et al., 1998), its apparent regulation by serine proteases (Roch et al., 1997) and its possible relation to humoral agglutinins (H1, H2, H3), (Eue et al., 1997) and those antimicrobial peptides that are not hemolytic (Cho et al., 1998). These research groups propose the names fetidin, lysin, and now Eiseniapore, and perhaps perforin (Komiyama et al., 1997). From molecular analyses, these groups have cloned cDNAs which encode the cytolytic proteins and confirmed the activities of recombinant proteins. Certain common characteristics seem to be shared. As an example, their work reveals that the 40kDa fetidin has at least four isoforms with different isoelectric points and lysenin represents two isoforms of molecular masses of 41 and 42 kDa. This suggests that coelomic fluid of Eisenia fetida contains several sphingomyelin-binding cytolytic proteins with molecular masses around 40 kDa.


Natural Killer Cell Serine Protease Hemolytic Activity Serine Protease Inhibitor Large Granular Lymphocyte 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. Acha-Orbea, H., Scarpellino, L., Hertig, S., Dupuis, M., and Tschopp J. (1990) Inhibition of lymphocyte mediated cytotoxicity by perform antisense oligonucleotides. EMBO J., 9: 3815–3819.PubMedGoogle Scholar
  2. Bcschin A, Bilej M, Hanssens F, Raymakers J, Van Dyck E, Revets H, Brys L, Gomez J, De Baetselier P, and Timmermans M. (1998) Identification and cloning of a glucan-and lipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade. J Bio Chem 273:24948–24954.CrossRefGoogle Scholar
  3. Bilej, M., Brys, L., Beschin, A., Lucas, R., Vercauteren, E., Hanusova, R., and De Baetselier, P. (1995) Identification of a cytolytic protein in the coelomic fluid of Eisenia foetida earthworms. Immunol. Lett. 45:123–128.Google Scholar
  4. Brumback, R.A. (1981) The neuralmuscular junction. Part 1: physiology and the effects of drugs and toxins. Am. Fam. Physician 23, 188–192.PubMedGoogle Scholar
  5. Canicatti, C. (1990) Hemolysis: Pore-forming proteins in invertebrates. Experientia 46: 239–244.PubMedCrossRefGoogle Scholar
  6. Cho J. H., Park, C. B., Yoon, Y. G., and Kim, S. C. (1998) Lumbricin I, a novel proline-rich antimicrobial peptide from the earthworm: purification, eDNA cloning and molecular characterization. Biochem Biophys Acta. 1408:67–76.PubMedCrossRefGoogle Scholar
  7. Cooper, E. L., Cossarizza, A., Suzuki, M. M., Salvioli, S., Capri, M., Quaglino, D., and Franceschi, C. (1995) Autogeneic but not allogeneic earthworm effector coelomocytes kill the mammalian tumor target K562. Cell. Immunol. 166:113–122.PubMedCrossRefGoogle Scholar
  8. Cooper, E.L., and Roch, P. (1992). The capacities of earthworms to heal wounds and to destroy allografts are modified by polychlorinated biphenyls (PCB). J. Invert. Pathol. 60: 59–63.CrossRefGoogle Scholar
  9. Cooper, E.L., Cossarizza, A., Kauschke, E., and Franceschi, C. (1999) Cell adhesion and the immune system: a case study using earthworms. Micro. Res. Tech. 44, 237–253.Google Scholar
  10. Cooper, E.L., Rinkevich, B., Uhlenbruck, G., and Valembios, P. (1992) Invertebrate immunity: another viewpoint. Scand. J. Immunol. 35, 247–26.Google Scholar
  11. Cossarizza, A., Cooper, E. L., Quaglinio, D., Salvioli, S., Kalachnikova, G., and Franceschi, C., (1995) Mitochondrial mass and membrane potential in coelomocytes from the earthworm Eisenia foetida: Studies with fluorescent probes in single intact cells. Biochem. Biophys. Res. Comm. 214: 503–510.Google Scholar
  12. Cossarizza, A., Cooper, E. L., Suzuki, M. M., Salvioli, S., Capri, M., Gri, G., Quaglino, D., and Franceschi, C. (1996) Earthworm leukocytes that arc not phagocytic and cross-react with several human epitopes can kill human tumor cell lines. Exptl. Cell Res. 224: 174–182.PubMedCrossRefGoogle Scholar
  13. Dennert, G. and Podack, E.R. (1983) Cytolysis by H-2-specific T killer cells: Assembly of tubular complexes on target membranes. J. Exp. Med. 157: 1483–1495.PubMedCrossRefGoogle Scholar
  14. Dourmashkin, R.R., Deteix, P., Simone C.B., and Henkart, P. (1980) Electron microscopic demonstration of lesions in target cell membranes associated with antibody-dependent cellular cytotoxicity. Clin. Exp. Immunol. 42: 554–60.PubMedGoogle Scholar
  15. Eue, I., Kauschke, E., Mohrig, W., and Cooper, E. L. (1998) Isolation and characterization of earthworm hemolysins and agglutinins. Dev. Comp. Immunol., 22: 13–25.Google Scholar
  16. Ferrarini, M. and Grossi, C.E. (1985). Ultrastmcture and cytochemistry of the human large granular lymphocytes. In: Immunobiology of natural killer cells. Lotzová, E. and Herbermann, R. B. (eds) CRC Press, Boca Raton, FL. 1: 33–43.Google Scholar
  17. Garcia-Sanz, J.A., Plaetinck, G., Velotti, F., Masson, D., Tschopp, J., MacDonald, H.R., and Nabholz, M. (1987) Perforin is present only in normal activated Lyt2+ T-lymphocytes and not in L3T4+ cells, but serine protease granzyme A is made by both subsets. EMBO J. 6: 933–938.PubMedGoogle Scholar
  18. Gordon, D., Martin-Eauclaire, M.F., Cestele, S., Kopeyan, C., Cartier, E., Khalifa, R.B., Pelhate, M., and Rochat, H. (1996) Scorpin toxins affecting sodium current inactivation bind to distinct homologous receptor sites on rat brain and insect sodium channels. J. Biol. Chem. 271, 8034–8045.PubMedCrossRefGoogle Scholar
  19. Henkart, P.A. (1985) Mechanisms of lymphocyte-mediated cytotoxicity. Ann. Rev. Immun. 3: 31–58.CrossRefGoogle Scholar
  20. Jenne, D.E and Tschopp, J. (1988) Granzymes a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation. Immunol. Rev. 103: 53–71.Google Scholar
  21. Kägl, D., Ledermann, B., Burkl, K., Seller, P., Odermatt, B., Olsen, K. J., Podack, E. R., Zinkemagel, R. M., and Hengartner, H. (1994) Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perform -deficient mice. Nature. 369: 31–37.CrossRefGoogle Scholar
  22. Kaminski, H.J., Suarez, J.I., and Ruff, R.L. (1997) Neuromuscular junction physiology in myasthemia gravis: isoforms of the acetylcholine receptor in extraocular muscle and the contribution of sodium channels to the safety factor. Neurology 48, 8–17.CrossRefGoogle Scholar
  23. Kauschke, E., Pagliara, E, Stabili, L., and Cooper, E. L. (1997) Characterization of proteolytic activity in coelomic fluid of Lumbricus terrestris. Comp. Biochim. Physiol. 116B: 235–242.Google Scholar
  24. Kawasaki, A., Shinkai, Y., Yagita, H., and Okumura, E. (1992) Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo. Eur. J. Immun. 22: 1215–1219.CrossRefGoogle Scholar
  25. Komiyama, K., Yoshimura, M., Iwase, T., Sato, J, Okumura, K., Cooper, E. L., and Moro, I. (1997) Identification of perforin gene and its protein in the earthworm coelomocytes. Dev. Comp. Immunol. 21:115.Google Scholar
  26. Kupfer, A., Dennert, G., and Singer, S.J. (1985) The reorientation of the Golgi apparatus and the microtubuleorganizing center in the cytotoxic effector cell is a prerequisite in the lysis of bound target cells. J. Mol. Cell. Immunol. 2: 37–49.PubMedGoogle Scholar
  27. Lange, S., Kauschke, E., Mohrig, W., and Cooper, E. L. (1999) Biochemical characteristics of eiseniapore, a pore forming protein in the coelomic fluid of earth worms. Eu. J. Biol. Chem. 262: 1–11.CrossRefGoogle Scholar
  28. Lange, S., Nübler, Kauschke, E., Lutsch, G., Cooper, E. L., and Herrmann, A. (1997) Interactions of earthworm hemolysin with lipid membranes requires sphingolipids. J. Biol. Chem. 272: 20884–20892.Google Scholar
  29. Lassegues, M., Milochau, A., Doignon, F., Du Pasquier, L., and Valembois, P. (1997) Sequence and expression of an Eisenia-fetida-derived eDNA clone that encodes the 40-kDa fetidin antibacterial protein Eur. J. Biochem. 246: 756–762.PubMedCrossRefGoogle Scholar
  30. Leipner, C., Tuckova, L., Rejnek, J., and Langner, J. (1993) Serine proteases in coelomic fluid of annelids Eisenia fetida and Lumbricus terrestris. Comp. Biochim. Physiol. 105B: 637–641).Google Scholar
  31. Lichtenheld, M. G., Olsen, K. J., Ping, L., Lowrey, D.M., Hameed, A., Hengartner, H., and Podack, E.R. (1988) Structure and function of human perforin. Nature 335: 448–451.PubMedCrossRefGoogle Scholar
  32. Mazumder, P.K. and Dube, S. N. (1996) Marine toxins as molecular probes for biological interactions: a review. Indian J. Physiol. Allied Sci. 50, 34–47.Google Scholar
  33. Milochau, A., Lassegues, M., and Valembois, P. (1997) Purification, characterization and activities of two hemolytic and antibacterial proteins from coelomic fluid of the annelid Eisenia fetida andrei. Biochem. Biophys, Acta. 1337: 123–132.CrossRefGoogle Scholar
  34. Mueller, C., Gershenfeld, H.K., Lobe, C.G., Okada, C.V., Bleackley, R.C., and Weissman, I.L. (1988) A high proportion ofT lymphocytes that infiltrate H-2-incompatible heart allografts in vivo express genes encoding cytotoxic cell-specific serine proteases, but do not express the MEL-14-defined lymph node homing receptor. J. Exp. Med. 167: 1124–1136.PubMedCrossRefGoogle Scholar
  35. Mueller, C., Kagi, D., Aebischer, T., Odermatt, B., Held, W., Podack, E.R., Zinkemagel, R.M., and Hengartner, H. (1989) Detection of perforin and granzyme A mRNA in infiltrating cells during infection of mice with lymphocytic choreomeningitis virus. Eur.J. Immunol. 19: 1253–1259.CrossRefGoogle Scholar
  36. Nastuk, W.L. (1971) Mechanisms of neuromuscular blockade. Ann. N.Y. Acad. Sci. 183, 171–182PubMedCrossRefGoogle Scholar
  37. Pagliara, P., Canicatti, C., and Cooper, E.L. (1993) Structure and enzyme content of sea urchin cytolytic granules. Comp. Biochim. Physiol. 106B: 813–818.Google Scholar
  38. Pennington, M.W., Mahnir, V.M., Krafle, D.S., Zaydenberg, I., Byrens, M.E., Khaytin, I., Crowler, K., and Kern, W.R. (1996) Identification of three separate binding sites of SHK toxin, a potent inhibitor of voltage dependent potassium channels in human T-lymphocytes and rat brain. Biochem Biophys. Res. Commun. 219,696–701.Google Scholar
  39. Podack, E.R. (1985) The molecular mechanism of lymphocyte-mediated tumor cell lysis. Immunol. Today 6: 21–27.Google Scholar
  40. Podack, E.R., Hengartner, H., and Lichtenheld, M.G. (1991) A central role of perforin in cytolysis. Ann. Rev. Immunol. 9: 129–157.CrossRefGoogle Scholar
  41. Podack, E.R., Lowrey, D.M., Lichtenheld, M., Olsen, K.J., Aebischer, T., Binder, D., Rupp, F., and Hengartner, H. (1988) Structure, function and expression of murine and human perform 1 (P1). Immun. Rev. 103: 203–211.Google Scholar
  42. Polanowski, A. and Wilusz, T. (1996) Serine proteinase inhibitors from insect hemolymph. Acta Biochim Polinica 43: 445–454.Google Scholar
  43. Porchet-Henneré, E., Dugimont, T., and Fischer, A. (1992) Natural killer cells in a lower invertebrate, Nereis diversicolor. Eur. J. Cell Biol. 58: 99–107.PubMedGoogle Scholar
  44. Quaglino, D., Cooper, E. L., Salvioli, S., Capri, M., Suzuki, M. M., Pasquali-Ronchetti, I., Franceschi C., and Cossarizza, A. 1996 Earthworm coelomocytes in vitro: cellular features and “granuloma” formation during cytotoxic activity against the mammalian tumor cell target K562. Eu. J. Cell Biol. 70:278–288.Google Scholar
  45. Roch, P., Ville, P., and Cooper, E. L. (1998) Characterization of a 14 kDa plant-related serine protease inhibitor and regulation of cytotoxic activity in earthworm coelomic fluid. Dev. Comp. Immunol. Dev. Comp. Immunol. 22: 1–12.CrossRefGoogle Scholar
  46. Roch, P., Ville, P., and Cooper, E. L. (1998) Characterization of a 14 kDa plant-related serine protease inhibitor and regulation of cytotoxic activity in earthworm coelomic fluid. Dev. Comp. Immunol. Dev. Comp. Immunol. 22: 1–12.CrossRefGoogle Scholar
  47. Roch, Ph., Stabili, L., and Pagliara, P. (1991) Purification of three serine proteases from the coelomic cells of earthworms (Eisenia foetida). Comp. Biochim. Physiol. 98B:597–602.Google Scholar
  48. Rouvier, E., Luciani, M.F., and Golstein, P. (1993) Fas involvement in Ca2+-independent T cell-mediated cytotoxicity. J. Exp. Med. 177: 195–200.PubMedCrossRefGoogle Scholar
  49. Russell, J.H. (1983) Internal disintegration model of cytotoxic lymphocyte-induced target damage. Immunol. Rev. 72: 97–118.Google Scholar
  50. Sekizawa, Y., Kubo, T., Kobayashi, H., Nakajima, T., and Natori, S. (1997) Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of rat vascular smooth muscle. Gene 191: 97–102.PubMedCrossRefGoogle Scholar
  51. Shinkai, Y., Takio, K., and Okumura, K. (1988) Homology of perforin to the ninth component of complement (C9). Nature 334: 525–527.PubMedCrossRefGoogle Scholar
  52. Shiver, J.W., Su, L., and Henkart, P.A. (1992) Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysis and granzyme A. Cell 71: 315–321.PubMedCrossRefGoogle Scholar
  53. Tschopp, J. and Nabholz, M. (1990) Perform mediated target cell lysis by cytolytic T lymphocytes. Annu. Rev. Immunol. 8: 279–302.CrossRefGoogle Scholar
  54. Walsh, C.M., Matloubian, M., Liu, C., Ueda, R., Kurahara, C., Christensen, J., Huang, M.T.F., Young, J.D., Ahmed, R., and Clark, W.R. (1994) Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA 91: 10854–10858.PubMedCrossRefGoogle Scholar
  55. Yamaji A, Sekizawa Y, Emoto K, Sakuraba H, Inoue K, Kobayashi H, and Umeda M. (1998) Lysenin, a novel sphingomyclin-specific binding protein. J. Biol Chem 273: 5300–5306.PubMedCrossRefGoogle Scholar
  56. Young, J.D.E. (1989) Killing of target cells by lymphocytes: A mechanistic view. Physiol. Rev. 69: 250–314.Google Scholar
  57. Young, J.D.E., Hengartner, H., Podack, E.R., and Cohn, Z.A. (1986) Purification and Characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity. Cell 44: 849–859.PubMedCrossRefGoogle Scholar
  58. Young, L.H., Joag, S.V., Zheng, L.M., Lee, C.D., Lee, Y.S., and Young, J.D.E. (1990) Perform mediated myocardial damage in acute myocarditis. Lancet 336: 1019–1021.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2001

Authors and Affiliations

  • Edwin L. Cooper
    • 1
  • Ellen Kauschke
    • 2
  • Andrea Cossarizza
    • 3
  1. 1.Laboratory of Comparative Immunology, Department of Neurobiology, School of MedicineUniversity of California Los AngelesUSA
  2. 2.Zoological Institute and Museum University of GreifswaldGermany
  3. 3.Department of Biomedical Sciences, Section of General PathologyUniversity of Modena, Via CampiItaly

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