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. 1985 Oct;76(4):1427–1435. doi: 10.1172/JCI112120

Defensins. Natural peptide antibiotics of human neutrophils.

T Ganz, M E Selsted, D Szklarek, S S Harwig, K Daher, D F Bainton, R I Lehrer
PMCID: PMC424093  PMID: 2997278

Abstract

We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens.

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Selected References

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  1. Ambruso D. R., Johnston R. B., Jr Lactoferrin enhances hydroxyl radical production by human neutrophils, neutrophil particulate fractions, and an enzymatic generating system. J Clin Invest. 1981 Feb;67(2):352–360. doi: 10.1172/JCI110042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnold R. R., Cole M. F., McGhee J. R. A bactericidal effect for human lactoferrin. Science. 1977 Jul 15;197(4300):263–265. doi: 10.1126/science.327545. [DOI] [PubMed] [Google Scholar]
  3. Bainton D. F., Ullyot J. L., Farquhar M. G. The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J Exp Med. 1971 Oct 1;134(4):907–934. doi: 10.1084/jem.134.4.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bjornson A. B., Altemeier W. A., Bjornson H. S. Comparison of the in vitro bactericidal activity of human serum and leukocytes against bacteroides fragilis and Fusobacterium mortiferum in aerobic and anaerobic environments. Infect Immun. 1976 Sep;14(3):843–847. doi: 10.1128/iai.14.3.843-847.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins M. S., Pappagianis D. Inhibition by lysozyme of growth of the spherule phase of Coccidioides immitis in vitro. Infect Immun. 1974 Sep;10(3):616–623. doi: 10.1128/iai.10.3.616-623.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cramer E., Pryzwansky K. B., Villeval J. L., Testa U., Breton-Gorius J. Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold. Blood. 1985 Feb;65(2):423–432. [PubMed] [Google Scholar]
  7. Drazin R. E., Lehrer R. I. Fungicidal properties of a chymotrypsin-like cationic protein from human neutrophils: adsorption to Candida parapsilosis. Infect Immun. 1977 Aug;17(2):382–388. doi: 10.1128/iai.17.2.382-388.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elsbach P., Weiss J. A reevaluation of the roles of the O2-dependent and O2-independent microbicidal systems of phagocytes. Rev Infect Dis. 1983 Sep-Oct;5(5):843–853. doi: 10.1093/clinids/5.5.843. [DOI] [PubMed] [Google Scholar]
  9. Elsbach P., Weiss J., Franson R. C., Beckerdite-Quagliata S., Schneider A., Harris L. Separation and purification of a potent bactericidal/permeability-increasing protein and a closely associated phospholipase A2 from rabbit polymorphonuclear leukocytes. Observations on their relationship. J Biol Chem. 1979 Nov 10;254(21):11000–11009. [PubMed] [Google Scholar]
  10. Gadebusch H. H., Johnson A. G. Natural host resistance to infection with Cryptococcus neoformans. IV. The effect of some cationic proteins on the experimental disease. J Infect Dis. 1966 Dec;116(5):551–565. doi: 10.1093/infdis/116.5.551. [DOI] [PubMed] [Google Scholar]
  11. Griffiths G., Brands R., Burke B., Louvard D., Warren G. Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport. J Cell Biol. 1982 Dec;95(3):781–792. doi: 10.1083/jcb.95.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. HIRSCH J. G. Antimicrobial factors in tissues and phagocytic cells. Bacteriol Rev. 1960 Mar;24(1):133–140. doi: 10.1128/br.24.1.133-140.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. HIRSCH J. G. Phagocytin: a bactericidal substance from polymorphonuclear leucocytes. J Exp Med. 1956 May 1;103(5):589–611. doi: 10.1084/jem.103.5.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heyderman E., Neville A. M. A shorter immunoperoxidase technique for the demonstration of carcinoembryonic antigen and other cell products. J Clin Pathol. 1977 Feb;30(2):138–140. doi: 10.1136/jcp.30.2.138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ingham H. R., Sisson P. R., Middleton R. L., Narang H. K., Codd A. A., Selkon J. B. Phagocytosis and killing of bacteria in aerobic and anaerobic conditions. J Med Microbiol. 1981 Nov;14(4):391–399. doi: 10.1099/00222615-14-4-391. [DOI] [PubMed] [Google Scholar]
  16. Lehrer R. I., Daher K., Ganz T., Selsted M. E. Direct inactivation of viruses by MCP-1 and MCP-2, natural peptide antibiotics from rabbit leukocytes. J Virol. 1985 May;54(2):467–472. doi: 10.1128/jvi.54.2.467-472.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lehrer R. I. Functional aspects of a second mechanism of candidacidal activity by human neutrophils. J Clin Invest. 1972 Oct;51(10):2566–2572. doi: 10.1172/JCI107073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lehrer R. I., Ladra K. M., Hake R. B. Nonoxidative fungicidal mechanisms of mammalian granulocytes: demonstration of components with candidacidal activity in human, rabbit, and guinea pig leukocytes. Infect Immun. 1975 Jun;11(6):1226–1234. doi: 10.1128/iai.11.6.1226-1234.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mandell G. L. Bactericidal activity of aerobic and anaerobic polymorphonuclear neutrophils. Infect Immun. 1974 Feb;9(2):337–341. doi: 10.1128/iai.9.2.337-341.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Modrzakowski M. C., Cooney M. H., Martin L. E., Spitznagel J. K. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes. Infect Immun. 1979 Mar;23(3):587–591. doi: 10.1128/iai.23.3.587-591.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Modrzakowski M. C., Spitznagel J. K. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes: antagonism of granule cationic proteins by lipopolysaccharide. Infect Immun. 1979 Aug;25(2):597–602. doi: 10.1128/iai.25.2.597-602.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Odeberg H., Olsson I. Antibacterial activity of cationic proteins from human granulocytes. J Clin Invest. 1975 Nov;56(5):1118–1124. doi: 10.1172/JCI108186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Odeberg H., Olsson I. Mechanisms for the microbicidal activity of cationic proteins of human granulocytes. Infect Immun. 1976 Dec;14(6):1269–1275. doi: 10.1128/iai.14.6.1269-1275.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okamura N., Spitznagel J. K. Outer membrane mutants of Salmonella typhimurium LT2 have lipopolysaccharide-dependent resistance to the bactericidal activity of anaerobic human neutrophils. Infect Immun. 1982 Jun;36(3):1086–1095. doi: 10.1128/iai.36.3.1086-1095.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Olsson I., Venge P. Cationic proteins of human granulocytes. II. Separation of the cationic proteins of the granules of leukemic myeloid cells. Blood. 1974 Aug;44(2):235–246. [PubMed] [Google Scholar]
  26. Reichlin M. Use of glutaraldehyde as a coupling agent for proteins and peptides. Methods Enzymol. 1980;70(A):159–165. doi: 10.1016/s0076-6879(80)70047-2. [DOI] [PubMed] [Google Scholar]
  27. Rest R. F., Cooney M. H., Spitznagel J. K. Bactericidal activity of specific and azurophil granules from human neutrophils: studies with outer-membrane mutants of Salmonella typhimurium LT-2. Infect Immun. 1978 Jan;19(1):131–137. doi: 10.1128/iai.19.1.131-137.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rest R. F., Cooney M. H., Spitznagel J. K. Susceptibility of lipopolysaccharide mutants to the bactericidal action of human neutrophil lysosomal fractions. Infect Immun. 1977 Apr;16(1):145–151. doi: 10.1128/iai.16.1.145-151.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rest R. F., Fischer S. H., Ingham Z. Z., Jones J. F. Interactions of Neisseria gonorrhoeae with human neutrophils: effects of serum and gonococcal opacity on phagocyte killing and chemiluminescence. Infect Immun. 1982 May;36(2):737–744. doi: 10.1128/iai.36.2.737-744.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Root R. K., Cohen M. S. The microbicidal mechanisms of human neutrophils and eosinophils. Rev Infect Dis. 1981 May-Jun;3(3):565–598. doi: 10.1093/clinids/3.3.565. [DOI] [PubMed] [Google Scholar]
  31. Selsted M. E., Brown D. M., DeLange R. J., Harwig S. S., Lehrer R. I. Primary structures of six antimicrobial peptides of rabbit peritoneal neutrophils. J Biol Chem. 1985 Apr 25;260(8):4579–4584. [PubMed] [Google Scholar]
  32. Selsted M. E., Harwig S. S., Ganz T., Schilling J. W., Lehrer R. I. Primary structures of three human neutrophil defensins. J Clin Invest. 1985 Oct;76(4):1436–1439. doi: 10.1172/JCI112121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Selsted M. E., Szklarek D., Ganz T., Lehrer R. I. Activity of rabbit leukocyte peptides against Candida albicans. Infect Immun. 1985 Jul;49(1):202–206. doi: 10.1128/iai.49.1.202-206.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Selsted M. E., Szklarek D., Lehrer R. I. Purification and antibacterial activity of antimicrobial peptides of rabbit granulocytes. Infect Immun. 1984 Jul;45(1):150–154. doi: 10.1128/iai.45.1.150-154.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shafer W. M., Casey S. G., Spitznagel J. K. Lipid A and resistance of Salmonella typhimurium to antimicrobial granule proteins of human neutrophil granulocytes. Infect Immun. 1984 Mar;43(3):834–838. doi: 10.1128/iai.43.3.834-838.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spitznagel J. K. Nonoxidative antimicrobial reactions of leukocytes. Contemp Top Immunobiol. 1984;14:283–343. doi: 10.1007/978-1-4757-4862-8_10. [DOI] [PubMed] [Google Scholar]
  37. Spitznagel J. K., Okamura N. Oxygen independent microbicidal mechanisms of human polymorphonuclear leukocytes. Adv Exp Med Biol. 1983;162:5–17. doi: 10.1007/978-1-4684-4481-0_2. [DOI] [PubMed] [Google Scholar]
  38. Stenberg P. E., Shuman M. A., Levine S. P., Bainton D. F. Redistribution of alpha-granules and their contents in thrombin-stimulated platelets. J Cell Biol. 1984 Feb;98(2):748–760. doi: 10.1083/jcb.98.2.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  40. Vel W. A., Namavar F., Verweij A. M., Pubben A. N., MacLaren D. M. Killing capacity of human polymorphonuclear leukocytes in aerobic and anaerobic conditions. J Med Microbiol. 1984 Oct;18(2):173–180. doi: 10.1099/00222615-18-2-173. [DOI] [PubMed] [Google Scholar]
  41. Weiss J., Elsbach P., Olsson I., Odeberg H. Purification and characterization of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes. J Biol Chem. 1978 Apr 25;253(8):2664–2672. [PubMed] [Google Scholar]
  42. Westbrook E. M., Lehrer R. I., Selsted M. E. Characterization of two crystal forms of neutrophil cationic protein NP2, a naturally occurring broad-spectrum antimicrobial agent from leukocytes. J Mol Biol. 1984 Sep 25;178(3):783–785. doi: 10.1016/0022-2836(84)90252-3. [DOI] [PubMed] [Google Scholar]
  43. Winterbourn C. C. Lactoferrin-catalysed hydroxyl radical production. Additional requirement for a chelating agent. Biochem J. 1983 Jan 15;210(1):15–19. doi: 10.1042/bj2100015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zeya H. I., Spitznagel J. K. Arginine-rich proteins of polymorphonuclear leukocyte lysosomes. Antimicrobial specificity and biochemical heterogeneity. J Exp Med. 1968 May 1;127(5):927–941. doi: 10.1084/jem.127.5.927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zeya H. I., Spitznagel J. K. Cationic proteins of polymorphonuclear leukocyte lysosomes. II. Composition, properties, and mechanism of antibacterial action. J Bacteriol. 1966 Feb;91(2):755–762. doi: 10.1128/jb.91.2.755-762.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zeya H. I., Spitznagel J. K. Isolation of polymorphonuclear leukocyte granules from rabbit bone marrow. Lab Invest. 1971 Mar;24(3):237–245. [PubMed] [Google Scholar]

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