Skip to main content
SpringerLink
Log in

Hühnergranulozyten: Ein Modell für die antimikrobielle Funktion peroxidasefreier menschlicher Granulozyten

  • Published:
Blut Aims and scope Submit manuscript

Zusammenfassung

Granulozyten von Hühnern besitzen natürlicherweise keine Myeloperoxidase. Sie gleichen darin den bei derAlius-Grignaschi-Anomalie gefundenen menschlichen Granulozyten. Sie können daher phagozytierte Mikroorganismen nicht mit Hilfe von Myeloperoxidase und einem oxidierbaren Kofaktor zerstören, wie es für normale menschliche Granulozyten postuliert wird. Um Einblicke in alternative antimikrobielle Mechanismen von Granulozyten zu erhalten, untersuchten wir die antimikrobiellen Potenzen intakter Hühnergranulozyten in vitro, isolierten ihre Granula, bestimmten den Enzymgehalt dieser Granula und lokalisierten einen antimikrobiellen Mechanismus in diesen Granula. Es zeigte sich:

  1. 1.

    Intakte Hühnergranulozyten sind wie menschliche Granulozyten in vitro in der Lage, vier verschiedene Stämme von Mikroorganismen zu phagozytieren und abzutöten.

  2. 2.

    Ein antimikrobieller Mechanismus von Hühnergranulozyten konnte in den großen Granula lokalisiert werden.

  3. 3.

    Dieser antimikrobielle Mechanismus besteht wahrscheinlich aus 5 basischen Proteinen. Eines dieser basischen Proteine ist Lysozym.

Die anderen konnten bisher nicht als Enzyme definiert werden. Die erhobenen Befunde zeigen, daß neben dem obengenannten antimikrobiellen Mechanismus von Granulozyten alternative Mechanismen bestehen. Bei diesen Alternativmechanismen spielen basische Proteine eine zentrale Rolle. In wieweit diese Befunde zum Verständnis der widersprüchlichen klinischen Beobachtungen an Patienten mit granulozytären Anomalien beitragen können, wird diskutiert.

Summary

Human subjects exhibiting the anomaly described byAlius andGrignaschi lack myeloperoxidase in their neutrophil polymorphonuclear leukocytes (PMN). Chicken heterophile granulocytes naturally lack myeloperoxidase as well. Therefore they must be incapable for killing ingested microorganisms by aid of H2O2, an oxidizable cofactor and myeloperoxidase, a mechanism proposed for human PMN.

To get closer insight into alternative bactericidal mechanisms we chose chicken PMN as a model. We checked the antimicrobial potencies of these cells in vitro, isolated their different classes of granules, determined their enzyme content and localized an antimicrobial mechanism within these granules.

The following results could be established:

  1. 1.

    Vital chicken PMN are not inferior to human PMN in respect to their antimicrobial potencies against four strains of microorganisms.

  2. 2.

    An antibacterial mechanism was located in the large granules of chicken heterophile PMN.

  3. 3.

    Evidently this antibacterial mechanism consists of five or more basic proteins. One of these is lysozyme. Up to now the other proteins could not be shown to be enzymes.

These results prove that there exists an alternative antimicrobial mechanism within PMN. Basic proteins appear to be of central importance for this mechanism. Our results might shed some light upon the controversial observations concerning the antimicrobial potencies of patients exhibiting the anomaly ofAlius andGrignaschi.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Literatur

  1. Andersch, M. A. and A. J. Szczypinski: Use of p-nitrophenyl phosphate as the substrate in determination of serum acid phosphatase. Amer. J. Clin. Path.17, 571 (1947).

    Google Scholar 

  2. Baggiolini, M., J. G. Hirsch and C. de Duve: Resolution of granules from rabbit heterophil leukocytes into distinct populations by zonal sedimentation. J. Cell Biol.40, 529 (1969).

    Google Scholar 

  3. Baehner, R. L., D. C. Nathan and M. L. Karnovsky: Correction of metabolic deficiencies in the leukocytes of patients with chronic granulomatous disease. J. Clin. Invest.49, 865 (1970).

    Google Scholar 

  4. Barka, T.: Studies of acid phosphatase. I. Electrophoretic separation of acid phosphatases of rat liver on polyacrylamide gels. J. Histochem. Cytochem.9, 542 (1961).

    Google Scholar 

  5. Bessey, O. A., O. H. Lowry and M. J. Brock: A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem.164, 321 (1946).

    Google Scholar 

  6. Cohn, Z. A. and J. G. Hirsch: The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leukocytes. J. Exp. Med.112, 983 (1960).

    Google Scholar 

  7. Brune, K., M. S. Leffell and J. K. Spitznagel: Microbicidal activity of peroxidaseless chicken heterophile leukocytes. Infect. Immun.5, 283 (1972).

    Google Scholar 

  8. Dhingra, L. D., W. B. Parrish and W. G. Venzke: Electron Microscopy of Granular Leukozytes of Chicken. Am. J. Vet. Res.30, 637 (1969).

    Google Scholar 

  9. Dhingra, L. D., W. B. Parrish and W. G. Venzke: Electron Microscopy of Nongranular Leukocytes and Thrombocytes of Chickens. Am. J. Vet. Res.30, 1837 (1969).

    Google Scholar 

  10. Davis, B. J.: Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sciences121, 404 (1964).

    Google Scholar 

  11. Davis, S. D., J. Schaller and R. J. Wedgwood: Job's Syndrome. Lancet1, 1013 (1966).

    Google Scholar 

  12. de Duve, C.: The lysosome in retrospect. In: Lysosomes in biology and pathology.1, 3 (1969). North-Holland, Amsterdam and London.

    Google Scholar 

  13. Ehrlich, P.: Über die specifischen Granulationen des Blutes. In: The collected papers of Paul Ehrlich,1, 117 (1879). (Hrsg. F. Himmelweit) Pergamon Press, London-New York.

    Google Scholar 

  14. Fishman, W. H.: Beta-glucuronidase. In: Methods of enzymatic analysis. pp. 869 (ed. H. U. Bergmeyer) Acad. Press, New York-London 1963.

    Google Scholar 

  15. Grignaschi, V. J., A. M. Sperperato, M. J. Etcheverry und A. J. L. Macario: Un nuevo cuadro citoquimico: negatividad espontanea de las reacciones de peroxidasas, oxidasas y lipido en la progenie neutrofila y en los monocitos de dos hermanos. Rev. Asoc. Med. Argent.77, 218 (1963).

    Google Scholar 

  16. Good, R. A., P. G. Quie, D. B. Winhorst, A. R. Page, G. E. Rodey, J. White, J. J. Wolfson and B. H. Holmes: Fatal (chronic) granulomatous disease of childhood: a hereditary defect of leukocyte function. Seminars Hematol.5, 215 (1968).

    Google Scholar 

  17. Hayashi, M., Y. Nakajima and W. H. Fishman: The cytologic demonstration of beta-glucuronidase employing naphthol As-BI glucuronide and hexazonium pararosanilin; A preliminary report. J. Histochem. Cytochem.12, 293 (1964).

    Google Scholar 

  18. Hermansky, F., V. Lodrova und V. Pössnerova: Zytochemische Untersuchungen an Leukozyten bei einigen Laboratoriums- und Haustieren. Folia Haemat.93, 249 (1970).

    Google Scholar 

  19. Hirsch, J.: Cinemicrographic observations on granule lysis in polymorphonuclear leukocytes during phagocytosis. J. Exp. Med.116, 827 (1962).

    Google Scholar 

  20. Holmes, B., A. R. Page and R. A. Good: Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J. Clin. Invest.46, 1422 (1967).

    Google Scholar 

  21. Jain, N. C.: Alkaline phosphatase activity in leukocytes of some animal species. Acta Haemat.39, 51 (1968).

    Google Scholar 

  22. Klebanoff, S. J.: Myeloperoxidase: Contribution to the microbicidal activity of intact leukocytes. Science169, 1095 (1970).

    Google Scholar 

  23. Klebanoff, S. J.: Iodination of bacteria: a bactericidal mechanism. J. Exp. Med.126, 1063 (1967).

    Google Scholar 

  24. Lehrer, R. I., J. Hanifin and M. J. Cline: Defective bactericidal activity in myeloperoxidase-deficient human neutrophils. Nature223, 78 (1969).

    Google Scholar 

  25. Lehrer, R. I. and M. J. Cline: Leukocyte myeloperoxidase deficiency and disseminated candidiasis: the role of myeloperoxidase in resistance to Candida infection. J. Clin. Invest.48, 1478 (1969).

    Google Scholar 

  26. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall: Protein mesurement with the folin phenol reagent. J. Biol. Chem.193, 265 (1951).

    Google Scholar 

  27. Lucas, A. M. and J. Jamroz: In: Atlas of avian haematology (ed. A. M. Lucas and D. Jamroz) Agriculture Monograph. Washington 1961.

  28. Lück, H.: Peroxidase. In: Methods of enzymatic analysis, pp. 895 (ed. H. U. Bergmeyer) Acad. Press Inc., New York 1963.

    Google Scholar 

  29. Lutzner, M. A.: Enzymatic specialization of organelles of blood cells, mast cells and platelets. Fed. Proc. Fed. Amer. Exp. Biol.23, 441 (1964).

    Google Scholar 

  30. McCall, C. E., L. R. De Chatelet, M. R. Cooper and P. Ashburn: The effects of ascorbic acid on bactericidal mechanisms of neutrophils. J. Infect. Dis.124, 194 (1971).

    Google Scholar 

  31. McRipley, R. J. and A. J. Sbarra: Role of the phagocyte in hostparasite interactions. XII. Hydrogen peroxide-myeloperoxidase bactericidal system in the phagocyte. J. Bacteriol.94, 1425 (1967).

    Google Scholar 

  32. Michell, R. H., M. J. Karnovsky and M. L. Karnovsky: The distribution of some granule-associated enzymes in guinea pig polymorphonuclear leukocytes. Biochem. J.116, 207 (1970).

    Google Scholar 

  33. Muschel, L. H. and H. P. Treffers: Quantitative studies on the bactericidal actions of serum and complement. J. Immunol.76, 1 (1956).

    Google Scholar 

  34. Olson, T.: Isolation of human leukocyte granules using colloidal silica-polysaccharide density gradients. Exptl. Cell Res.54, 325 (1969).

    Google Scholar 

  35. Orstein, L.: Enzyme analysis. Canalco Instruction Sheet. 1963.

  36. Press, E. M., R. R. Porter and T. Cebra: The isolation and properties of a proteolytic enzyme, cathepsin D, from bovine spleen. Biochem. J.74, 501 (1960).

    Google Scholar 

  37. Quie, P. G., J. G. White, B. Holmes and R. A. Good: In vitro bactericidal capacity of human polymorphonuclear leukocytes: diminished activity in chronic granulomatous disease of childhood. J. Clin. Invest.46, 668 (1967).

    Google Scholar 

  38. Saba, H. J., H. R. Roberts and J. C. Herion: The Anticoagulant Activity of Lysosomal Cationic Proteins from Polymorphonuclear Leukocytes. J. Clin. Invest.46, 580 (1967).

    Google Scholar 

  39. Schaefer, H. E., C. Kanfer und R. Fischer: Vergleichende fermentcytochemische Untersuchungen an Blut- und Knochenmarkzellen bei Laboratoriumstieren. Virchow. Arch. Abtl. B. Zellpath.4, 310 (1970).

    Google Scholar 

  40. Shugar, D.: The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim. Biophys. Acta8, 302 (1952).

    Google Scholar 

  41. Undritz, E.: Das Pelger-Huetsche Blutbild beim Tier und seine Bedeutung für die Entwicklungsgeschichte des Blutes. Schweiz. med. Wschr.69, 1177 (1939).

    Google Scholar 

  42. Undritz, E.: Haematologische Tafeln, p. 28 Sandoz, Basel. 1952.

    Google Scholar 

  43. Undritz, E.: Die Alius-Grignaschi-Anomalie: der erblichkonstitutionelle Peroxydasedefekt der Neutrophilen und Monozyten. Blut14, 129 (1968).

    Google Scholar 

  44. Vercauteren, R. E.: Contribution to the problem of the enzyme equipment of the specific granules of leukocytes. Enzymologia27, 369 (1964).

    Google Scholar 

  45. Welsh, I. R. H. and J. K. Spitznagel: Distribution of lysosomal enzymes, cationic proteins and bacterial substances in subcellular fractions of human polymorphonuclear leukocytes. Infect. Immun.4, 97 (1971).

    Google Scholar 

  46. Zeya, H. I. and J. K. Spitznagel: Arginie-rich proteins of polymorphonuclear leukocyte lysosomes. J. Exp. Med.127, 927 (1968).

    Google Scholar 

  47. Zeya, H. I. and J. K. Spitznagel: Cationic protein-bearing granules of polymorphonuclear leukocytes: Separation from enzyme-rich granules. Science163, 1069 (1969).

    Google Scholar 

  48. Zeya, H. I. and J. K. Spitznagel: Characterization of cationic protein-bearing granules of polymorphonuclear leukocytes. Lab. Invest.24, 229 (1971).

    Google Scholar 

  49. Zeya, H. I. and J. K. Spitznagel: Isolation of polymorphonuclear leukocyte granules from rabbit bone marrow. Lab. Invest.24, 237 (1971).

    Google Scholar 

Download references

Author information

Author notes

  1. Kay Brune

    Present address: Abteilung Pharmakologie, Biozentrum der Universität Basel, Klingelbergstr. 70, CH-4056, Basel

Authors and Affiliations

    Authors
    1. Kay Brune
      View author publications

      You can also search for this author in PubMed Google Scholar

    Additional information

    Diese Arbeit wurde mit dem Pappenheim-Preis 1972 der Deutschen Gesellschaft für Hämatologie ausgezeichnet.

    Rights and permissions

    Reprints and permissions

    About this article

    Cite this article

    Brune, K. Hühnergranulozyten: Ein Modell für die antimikrobielle Funktion peroxidasefreier menschlicher Granulozyten. Blut 26, 110–126 (1973). https://doi.org/10.1007/BF01635760

    Download citation

    • Received:

    • Issue Date:

    • DOI: https://doi.org/10.1007/BF01635760

    Search

    Navigation