Defensins and Host Defense

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Science  15 Oct 1999:
Vol. 286, Issue 5439, pp. 420-421
DOI: 10.1126/science.286.5439.420

The production of antimicrobial peptides and proteins HN1 is an important means of host defense in eukaryotes (1). The larger antimicrobial proteins are often lytic enzymes, nutrient-binding proteins or proteins containing sites that target specific microbial macromolecules. The smaller antimicrobial peptides (defined here as peptides containing fewer than 100 amino acids) act, at least in part, by disrupting the structure or function of microbial cell membranes. In the last 20 years, hundreds of antimicrobial peptides have been found in plants and in the cells and body fluids of multicellular animals, from mollusks to humans. Some antimicrobial peptides are produced constitutively; others are induced in response to infection or inflammation. Studies of the regulation of antimicrobial peptide synthesis in Drosophila have been particularly fruitful, providing new directions for the analysis of mammalian host defense HN2 (2). Although antimicrobial peptides display a variety of shapes and amino acid compositions, many of those found in vertebrates are defensins HN3, 3- to 6-kD β-sheet peptides that contain three disulfides and are encoded by related genes (3). Structurally and functionally similar defensin-like peptides also abound in insects, other invertebrates, and plants. Now, two reports in this issue (4, 5) and a third published 2 weeks ago (6) provide new insights into the biology of vertebrate defensins.

Like most antimicrobial peptides, defensins are cationic (polar) molecules with spatially separated hydrophobic and charged regions. This arrangement allows them to insert themselves into phospholipid HN4 membranes so that their hydrophobic regions are buried within the oily membrane interior and their cationic regions interact with anionic phospholipid head groups and water. In the membrane, some defensins assemble into multimeric pores. Defensins and other antimicrobial peptides preferentially disrupt bacterial membranes that are rich in negatively charged phospholipids. Conversely, the lower anionic phospholipid content of the cell membranes of higher animals may provide relative protection from collateral damage.

Depending on the specific pattern of their cysteine spacing and disulfide connections, vertebrate defensins fall into two structural classes, α and β HN5. The molecular shapes of the two classes are similar, and their genes reside in the same gene cluster, indicating a common evolutionary origin. In vitro, defensins (at micromolar concentrations) have a broad spectrum of antimicrobial activity against bacteria, fungi, and even some enveloped viruses. In mammals and birds, defensins are among the most abundant polypeptides secreted by phagocytic white cells involved in host defense against bacteria and fungi (7). During phagocytosis HN6, ingested microbes are exposed to very high concentrations of defensins. The function of defensin-rich Paneth cells (8)—specialized secretory cells in the small intestine—is less certain. In addition to defensins, Paneth cells also contain lysozyme and secretory phospholipase A2, antimicrobial enzymes that clearly mark them as host defense cells (see figure below). The location of Paneth cells in intestinal crypts (narrow pits that harbor stem cells for the continual regeneration of the intestinal surface) suggests that Paneth cell secretions protect stem cells from pathogenic microbes HN7. In many other epithelia, defensins are produced constitutively or induced in response to infection or inflammation.

Defense of the realm.

A model of defensin activity in intestinal crypts. Paneth cells release defensins (red) and other antimicrobial substances into the crypt in response to microbial penetration. The critically important intestinal stem cells (green) are exposed to the highest concentrations of defensins and may be protected against infection. Defensins may act as signaling molecules in the gut lumen where their concentration is much lower.

The expression of defensins in tissues varies markedly among animal species. Rats but not mice have defensins in their white cells, but both species produce Paneth cell defensins in the gut and epithelial β-defensins in other epithelia. The peculiar defensin distribution in mice and the many closely related defensin genes have complicated attempts to study their function by engineering defensin-deficient mice.

Wilson et al. HN8 (6) report an alternative approach to ablating defensin genes: they instead disrupted the gene for matrilysin, which is required for activation of Paneth cell defensins. These peptides are initially synthesized as inactive prodefensins with an NH2-terminal anionic propiece that is cleaved off by matrilysin (a tissue metalloproteinase) HN9 to generate the much smaller, mature defensin peptide (9). Matrilysin is expressed in Paneth cell granules together with perhaps more than 20 different α-defensins (cryptdins). Disruption of the matrilysin gene prevents the normal posttranslational proteolytic activation of intestinal α-prodefensins. The investigators found that, relative to normal mice, matrilysin-deficient mice were less able to kill exogenous Escherichia coli bacteria in the gut and were more susceptible to death after infection with orally administered Salmonella typhimurium (6). The observed host defense defects are certainly consistent with the proposed antimicrobial role of defensins in the small intestine. However, it remains to be seen whether the micromolar concentrations of defensins required for in vitro antimicrobial activity are generated when the Paneth cells release the contents of their granules into the gut. Possibly additional polypeptides that depend on activation by matrilysin contribute to host defense, and sublethal concentrations of defensins may act synergistically with other substances in the intestinal environment. Moreover, by acting as signaling molecules, some defensins could increase resistance to microbial infection by activating other host defenses.

In their report on page 525, Yang et al. HN10 (4) investigated the chemoattractant activity of two recently identified human β-defensins, HBD-1 and HBD-2 HN11 (10-12). HBD-1 is constitutively expressed in kidney tubules and to a lesser extent in the pancreas and other epithelial sites; HBD-2 is induced in skin and other epithelia during inflammation (see figure below). At submicromolar concentrations, these defensins attracted both immature dendritic cells and memory T cells HN12, which initiate a primary and recall immune response, respectively. The effect was evidently mediated by the CCR6 chemokine receptor HN13 because β-defensins effectively competed with the receptor's ligand, MIP-3α. If the same mechanism functions in vivo, the release of these two defensins from injured epithelial cells would recruit dendritic cells and memory T cells to infected tissues, thereby promoting the development of adaptive (antibody and T cell-mediated) immunity HN14. Like the β-defensins, human neutrophil α-defensins HN15 also attract T cells. In addition, some defensins also block the adrenocorticotrophin HN16 receptor and could inhibit the production of the immunosuppressive adrenal steroid hormones during acute infection (HN13). The ability of some defensins to act as signaling molecules could explain their biological effects when their concentrations are too low to be directly microbicidal.

Inflamed defenders.

A model of defensin activity in an infected epithelium. Epithelial cells synthesize antimicrobial defensins (red) both constitutively and in response to infectious and inflammatory stimuli. Other defensins are introduced by the influx of phagocytic cells that use them to kill ingested microbes. Released defensins attract dendritic cells and memory T cells, setting the stage for the adaptive phase of the immune response.

Comparison of defensin genes across vertebrate species indicates that they are rapidly evolving. As variations in defensin sequences and patterns of tissue expression bring out distinct aspects of defensin biology in each species, studies of defensins in diverse animals have often provided crucial insights. The article by Tang et al. HN17 on page 498 (5) reports on studies of white cell defensins in the rhesus monkey. Several of the defensins isolated from rhesus white cells resembled their human counterparts, but one was unique and initially refractory to conventional analysis. The puzzle was solved brilliantly when the investigators determined that they were dealing with a cyclic peptide generated by head-to-tail peptide splicing of the products of two similarly truncated α-defensin genes! Although otherwise similar to α-defensins with their six cysteines, these novel defensin genes contained a “premature” stop codon in the segment after the third cysteine. This resulted in generation of two abbreviated defensin molecules that each donated 9 amino acids to the final, 18-amino acid cyclic product: a θ-defensin stabilized by three parallel disulfide bonds. This remarkable feat of posttranslational processing could be reconstituted by transfecting HL-60 human leukemia cells with the two defensin cDNAs. This elegant model should provide the requisite tools that will allow the processing and splicing mechanism to be worked out. The new θ-defensin molecule resembles porcine white cell protegrins HN18, hairpin-shaped peptides with 16- to 18-amino acid residues and two disulfide bonds (14, 15). Like protegrins, θ-defensins retain full activity at salt concentrations present in blood, a feature that makes them interesting candidates for development as antibiotics.

Despite advances in prevention, diagnosis, and treatment, infectious diseases continue to challenge us. We are facing the limitations of vaccine-based immunization strategies and the increasing resistance of microbes to existing antibiotics. The renaissance of research into innate host defense mechanisms HN19 that do not depend on specific recognition of individual antigens offers the promise that some of the many substances that mediate the innate resistance of plants and animals to infections may prove useful as templates for new antibiotics or immunostimulants.

HyperNotes Related Resources on the World Wide Web

General Hypernotes

Cell & Molecular Biology Online, maintained by P. Gannon, is a well-organized collection of annotated links to Internet resources.

The On-line Medical Dictionary is made available by CancerWEB.

Biology Links, maintained by the Department of Molecular and Cellular Biology, Harvard University, includes a section on Immunology Web resources.

The Department of Veterinary Pathobiology, Texas A&M College of Veterinary Medicine, provides a glossary of immunology by I. Tizard.

The Virtual Explorer from the Wilson Group at the University of California, San Diego, offers an illustrated introduction to the cells and the molecules and proteins of the immune system.

The Immunology Book Case is a reference on immunological topics offered by the Dalhousie University Medical School.

Understanding the Immune System is an educational presentation made available by the National Cancer Institute's Patients, Public, and Mass Media Web site.

The American Institute of Immunology offers an introduction to immunology with definitions provided.

C. Hewitt, Department of Microbiology and Immunology, University of Leicester, UK, offers an illustrated introduction to immunology in lecture notes for a course on infection and immunity. A glossary of immunology is provided by the Department's Infection & Immunity Web page.

P. Millikin, Department of Pathology, University of Illinois College of Medicine at Peoria, provides a review of immune defenses in lecture notes for a tutorial on 21st century infections.

P. Bugl, Department of Mathematics, University of Hartford, CT, presents an overview of the immune system for a course on epidemics and AIDS; a link to additional course materials on immunity by R. Aloisi is provided.

The Department of Microbiology and Immunology, University of Western Ontario, provides lecture notes for a course on the biology of infection and immunity. Four lectures by S. Galsworthy on host resistance to infection are included.

Immunology Overview by A. Goldman and B. Prabhakar, which is a chapter in S. Baron's Medical Microbiology Web textbook, is a detailed illustrated introduction to the immune system.

J. Pérez, Natural Toxins Research Initiative at Texas A&M University, Kingsville, makes available a series of immunology lectures.

The contents of the February 1998 issue of Current Opinion in Immunology, which offered a collection of articles on innate immunity and antimicrobial proteins, are available on the Web (in Adobe Acrobat format).

Numbered Hypernotes

1. A Gordon Research Conference on Antimicrobial Peptides was held in April 1999 in Italy. The R.E.W. Hancock Laboratory, Department of Microbiology and Immunology, University of British Columbia, presents information about their research on cationic antimicrobial peptides; a compilation of research methods used by the laboratory is provided.

2. The Interactive Fly includes a section on Drosophila host defense. CNRS (Centre National de la Recherche Scientifique) issued research information summaries in June 1999 (longer French version) and January 1997 (longer French version) about the insect immunology research of J. Hoffmann and colleagues at the Institut de Biologie Moléculaire et Cellulaire, Institut Fédératif de Recherche du CNRS, Strasbourg, France. U. Theopold, Department of Applied and Molecular Ecology, University of Adelaide Waite Campus, Australia, offers a presentation on insect immunity.

3. The American Institute of Immunology offers a definition of defensins. A definition of defensins is provided in a list of keywords for the section on inflammation of a pathology course offered by the Department of Pathology and Laboratory Medicine, University of Kansas School of Medicine. The Defensins Homepage is a class project by K. Fung for a biochemistry course taught by T. Richmond, Claremont Colleges, CA. A presentation by B. Dominy on defensin with 3D illustrations is provided in a collection of lab notes for immunobiology courses taught by J. Quintans, Department of Pathology, Division of Biological Sciences, University of Chicago. The SCOP (Structural Classification of Proteins) database has an entry for defensin. The PROSITE database of protein families and domains, available on the ExPASy Molecular Biology Server has entries for mammalian defensins signatures and arthropod defensins signatures. H. Ibelgaufts' COPE (Cytokines Online Pathfinder Encyclopaedia) has an article on defensins.

4. Kimball's Biology Pages provides a definition of hydrophobic and a presentation on phospholipids.

5. Cysteine is defined in the On-line Medical Dictionary. The Molecular Biology Notebook, maintained by the Bioinformatics Department, Institute of Arable Crops Research, UK, provides an introduction to protein structure and diversity. A Web presentation on protein structure by J. Yao was a class project for a biochemistry course offered by the Department of Biochemistry, University of Nebraska.

6. The Infection & Immunity Web page offered by the Department of Microbiology and Immunology, University of Leicester, offers a presentation on phagocytosis. The Macrophage Home Page from the Gordon laboratory, School of Pathology, University of Oxford, provides an introduction to phagocytosis. K. Todar, Department of Bacteriology, University of Wisconsin, offers lecture notes on inflammation and phagocytosis for a bacteriology course on host-parasite interactions.

7. Paneth cell is defined and a photomicrograph provided in the glossary of a histology tutorial offered by the University of Florida College of Medicine. An introduction to Paneth cells is presented by T. Caceci for a veterinary histology course at the Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA. “Villi, crypts and the life cycle of small intestinal enterocytes” is the title of a section of a hypertextbook on the pathophysiology of the digestive system by R. Bowen, Department of Physiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University. Lysozyme and phospholipase are defined in the On-line Medical Dictionary. R. Hurlbert, Washington State University, briefly discusses lysozymes in lecture notes on nonspecific immunity for a microbiology course. The On-line Medical Dictionary defines stem cells.

8. C. Wilson and W. Parks are in the Division of Allergy and Pulmonary Medicine, Department of Pediatrics, Washington University, St. Louis. L. Matrisian is in the Department of Cell Biology, Vanderbilt University School of Medicine, Nashville. A. Ouellette is in the Department of Pathology, School of Medicine, University of California, Irvine.

9. Jackson Laboratory's Mouse Genome Informatics database has an entry for MM7 (matrilysin). COPE has an entry for matrilysin and an article on matrix metalloproteinases. The Weizman Institute's GeneCards has an entry for MM7 (matrilysin) in humans with links to information in other databases.

10. D. Yang, J. Wang, and J. Oppenheim are at the Laboratory of Molecular Immunoregulation, Frederick Cancer Research and Development Center of the National Cancer Institute.

11. OMIM has entries for beta-defensin-1 and beta-defensin-2.

12. The American Institute of Immunology provides a definition of memory T cells. The PharmInfoNet glossary defines dendritic cell and memory cell. The On-line Medical Dictionary defines dendritic cells and memory T cells. The JAMA HIV/AIDS Information Center provides a background briefing on dendritic cells by D. Blakeslee. The 16 October 1998 issue of Science had an Enhanced Perspective about dendritic cells by K. Shortman and E. Maraskovsky; the 19 February 1999 issue had a Perspective by K. Bottomly titled “T cells and dendritic cells get intimate.”

13. OMIM has an entry for CCR6. COPE provides an article on chemokines and includes an entry for the CCR-6 chemokine receptor. An article by A. Luster titled “Chemokines reveal important roles in lymphocyte and dendritic cell trafficking” is presented by Research Diagnostics Inc.

14. S. Arkins, Department of Biological Sciences, Illinois State University, provides lecture notes on adaptive immunity for an immunology course.

15. The American Institute of Immunology provides a definition of neutrophils. The On-line Medical Dictionary defines neutrophils. The Histology Lessons tutorial from the Department of Cell Biology, Vanderbilt University Medical Center, provides a description and illustration of neutrophils. The Immunology Book Case presents an introduction to neutrophils. OMIM has entries for alpha-defensin-1, apha-defensin-4, alpha-defensin-5, and alpha-defensin-6.

16. Adrenocorticotrophic hormone is defined in the On-line Medical Dictionary.

17. Y.-Q. Tang, J. Yuan, G. Ösapay, K. Ösapay, D. Tran, A. Ouellette, and M. Selsted are in the Department of Pathology, School of Medicine, University of California, Irvine. C. Miller is at the California Regional Primate Research Center and Center for Comparative Medicine, School of Veterinary Medicine, University of California, Davis.

18. W. Wimley, Department of Biochemistry, Tulane University School of Medicine, provides an illustration of the structure of porcine protegrin.

19. The Introduction to Immunology Tutorial from the Biology Project of the University of Arizona defines the difference between innate and adaptive immunity. C. Hewitt provides an illustrated presentation on innate and adaptive immunity for a course on infection and immunity. S. Arkins provides lecture notes on innate immunity for an immunology course. The International Study Group on New Antimicrobial Strategies consists of biomedical scientists who do research on host defense mechanisms; an overview of research priorities for developing new antimicrobial strategies is provided that includes a section on defensins.

20. T. Ganz is at the Will Rogers Pulmonary Research Laboratory, Department of Medicine, UCLA School of Medicine.


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