The Tangled Webs That Neutrophils Weave

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Science  05 Mar 2004:
Vol. 303, Issue 5663, pp. 1477-1478
DOI: 10.1126/science.1095484

Neutrophils [HN1] are the body's first line of defense against invading microorganisms. The high incidence of bacterial infections in neutropenic [HN2] patients, and the associated high mortality rate among these patients, underscore the importance of neutrophils for microbial clearance (1). Most studies of microbial killing by neutrophils investigate the ability of these immune cells to engulf pathogens by phagocytosis [HN3], and the complex armamentarium they deploy to eliminate engulfed pathogens within intracellular phagocytic vacuoles. Such antimicrobial tools include toxic oxygen radicals, cationic proteins that disrupt the pathogen membrane, and an assortment of lytic enzymes that contribute to the death and degradation of invading microorganisms. These cationic peptides and lytic enzymes are packaged in specialized membrane-bound granules that fuse with the phagocytic vacuoles, delivering their lytic contents to the vacuole interior (2). However, neutrophils can kill extracellular pathogens in the absence of phagocytosis. The killing of extracellular bacteria by neutrophils may seem advantageous, providing a more rapid and disseminated means of controlling infection. On the other hand, extracellular killing may be riskier, because neutrophils can potentially damage surrounding host tissues. Brinkmann and colleagues (3) [HN4] describe, on page 1532 of this issue, a tool that neutrophils produce to kill extracellular bacteria in a way that minimizes damage to host cells.

Brinkmann et al. observed that when neutrophils are stimulated with cytokines [HN5] or bacterial endotoxin [HN6], these cells generate a web of extracellular fibers composed of DNA, histones [HN7], and granule proteins such as elastase [HN8] (see the figure). These fibers, nicknamed neutrophil extracellular traps (NETs), bind to both Gram-positive and Gram-negative bacteria and display bactericidal activity. The authors also demonstrate that the elastase component of NETs effectively degrades bacterial virulence factors. Dispersal of NETs with the enzyme deoxyribonuclease (DNase) [HN9], which degrades extracellular DNA, eliminates their antimicrobial properties. The investigators postulate that NETs immobilize a high concentration of extracellular antimicrobial peptides and, by trapping and killing invading bacteria, provide rapid control of infection.

Making NETs.

Neutrophils generate extracellular fibers called NETs that kill bacterial pathogens without the need for phagocytosis. A resting neutrophil becomes activated (1), leading to the release of DNA, histones, and granule proteins that assemble into NETs (3). The cellular constituents of neutrophils are released either through necrotic cell death (2a) or by exocytosis of granule contents coupled with active extrusion of DNA via an unknown mechanism (2b).


Although neutrophils are known to secrete granule proteins, the finding that NETs contain DNA and histones raises an obvious question: How are NETs formed? In particular, what are the mechanisms by which neutrophils might release some of their nuclear contents? Neutrophils are terminally differentiated cells and therefore do not divide; they normally die within hours through a tightly regulated and noninflammatory process of programmed cell death called apoptosis [HN10]. This process, however, seems not to be involved in the generation of NETs. Brinkmann et al. note that NET formation occurs much more quickly than apoptosis, and that stimuli known to delay apoptosis in fact induce NET formation. Explosive neutrophil death by necrosis [HN11] would therefore seem a more likely alternative. However, the authors do not favor this mechanism, as they failed to detect abundant cytoplasmic proteins, such as actin and tubulin, in the NETs. Instead they speculate that neutrophils may have developed an active process of extrusion of nuclear material while conserving cellular integrity (see the figure). This daring hypothesis needs to be validated by direct observation of the extrusion event and by detection of intact neutrophil remnants selectively depleted of their nuclear contents.

However they are formed, the discovery of NETs may help to explain some puzzling observations about what neutrophils do. Elastase, cathepsin G [HN12], and other proteases secreted by leukocytes have been postulated to be important not only for microbial killing, but also for cell migration and tissue remodeling. But uncontrolled activity by these degradative enzymes poses the risk of diffuse tissue damage. Endogenous soluble inhibitors may constrain the range of action of elastase and the cathepsins (4). Meanwhile, NETs may contribute to focusing the activity of proteases by precluding their diffusion. Although not addressed in the paper, it is also possible that NETs sequester cytokines in the local environment of the neutrophil, preventing untoward dissemination of the inflammatory response and thus minimizing damage to surrounding host tissues (5).

The proposed antimicrobial role of NETs seems to be at odds with the use of DNase in mucolytic therapy for treating patients with cystic fibrosis [HN13]. The hallmark of this common inherited lung disease is tenacious and purulent sputum, whose components include a complex of DNA and elastase (6), reminiscent of NETs. The abundance of proteases immobilized by extracellular DNA in the sputum would appear to be ideally suited to limiting the chronic respiratory infections suffered by cystic fibrosis patients. Hence, degradation of DNA in the sputum by inhaled DNase (which destroys NETs) would be expected to aggravate the disease, not only by impairing these bactericidal tools, but also by releasing elastase and other mediators that could exacerbate the inflammatory condition (7). Paradoxically, therapy with inhaled DNase reduces the symptoms of cystic fibrosis and improves pulmonary function (8). It is possible that excessive NET formation may be counterproductive, preventing proper mechanical clearance of the airways, which is driven by ciliary motion. The cilia themselves may get tangled in the NETs.

The discovery of NETs raises a number of other questions. Do all activated neutrophils make NETs, and if not, what determines which ones do? At what stage of the activation cascade are the NETs “woven”? These questions are important because the notion of activated neutrophils laying down a dense web of extracellular fibers is difficult to reconcile with their need to migrate by chemotaxis [HN14] toward sites of infection. Indeed, NETs could conceivably act as a barrier to the recruitment of more white blood cells and thereby impede the clearance of chronic infections, as is the case with the formation of abscesses. Along the same lines, it is not apparent how NETs might be dismantled when the infection has been cleared.

The observations of Brinkmann et al. suggest the crucial nature of extracellular killing of bacteria by neutrophils. Whether this antimicrobial action is the result of an active, targeted process by live neutrophils or an altruistic post mortem contribution to the well-being of the organism remains to be defined. Complex though these NETs may be, they should yield to further unraveling during future studies.

HyperNotes Related Resources on the World Wide Web

General Hypernotes

Dictionaries and Glossaries

The On-line Medical Dictionary is provided by CancerWeb.

The Dictionary of Cell and Molecular Biology is made available by J. Dow, Division of Molecular Genetics, University of Glasgow.

D. Glick's Glossary of Biochemistry and Molecular Biology is made available by Portland Press.

An immunology glossary is provided by the Microbiology & Immunology Web page of the Department of Microbiology and Immunology, University of Leicester, UK.

Web Collections, References, and Resource Lists

The Open Directory Project offers a collection of links to immunology resources.

The WWW Virtual Library of Cell Biology is provided by the Fenteany Research Group, Department of Chemistry, University of Illinois at Chicago.

OMNI, a guide to Internet resources in health and medicine provided by the University of Nottingham, UK, includes annotated links to immunology resources.

The Karolinska Institutet Library, Stockholm, provides links to Web resources related to immunology as well as other biomedical resources.

The ExPASy (Expert Protein Analysis System) Molecular Biology Server provides the Swiss-Prot protein knowledgebase, the ENZYME nomenclature database, and other resources.

Online Texts and Lecture Notes

The companion Web site for the third edition of Biochemistry by C. Mathews, K. van Holde, and K. Ahern provides introductions to concepts, molecules, and enzymes.

J. Kimball maintains Kimball's Biology Pages, an online biology textbook and glossary.

The Immunology Book Case is a resource for students provided by the Pathology, Immunology and Microbiology Unit, Dalhousie University Medical School.

K. Todar, Department of Bacteriology, University of Wisconsin, provides lecture notes for a bacteriology course on host-parasite interactions.

J. Decker, Department of Veterinary Science and Microbiology, University of Arizona, provides tutorials for an immunology course. A presentation on innate immunity is included.

Microbiology and Immunology On-line is a Web textbook provided by the Department of Microbiology and Immunology, University of South Carolina School of Medicine. A chapter on non-specific immunity is included.

General Reports and Articles

The Bookshelf provided by the National Center for Biotechnology Information (NCBI) makes available the fifth edition of Immunobiology by C. Janeway, P. Travers, M. Walport, and M. Shlomchik.

The 1 November 1998 issue of Blood had a review article by M. Hampton, A. Kettle, and C. Winterbourn titled “Inside the neutrophil phagosome: Oxidants, myeloperoxidase, and bacterial killing.”

The eMedicine Web site makes available a review article on neutrophils by N. D. Nader and H. Arora.

The 26 April 2002 issue of Science had an Enhanced Perspective by D. Roos and C. C. Winterbourn titled “Lethal weapons.”

Numbered Hypernotes

1. Neutrophils. Neutrophil is defined in the pathology tutorial offered by the Department of Pathology and Laboratory Medicine, University of Kansas Medical Center (KUMC). An introduction to neutrophils is provided by the University of Leicester's Microbiology & Immunology Web page. The Department of Pathology, Medical School, University of Birmingham, offers an introduction to the role of the neutrophil polymorph. C. Davis, Department of Biology, Western Kentucky University, provides lecture notes on the cells of the immune system for an immunology course. A section on neutrophils is included in a presentation on inflammation provided by the Histology Web site of the Southern Illinois University School of Medicine. Immunobiology has a section on neutrophils. The Periodontics Information Center of the University of California, Los Angeles, makes available K. Miyasaki's lecture notes on neutrophils. Inflammation and Fever, an online text by V. Stvrtinová, J. Jakubovsky, and I. Hulín (Faculty of Medicine, Comenius University, Bratislava, Slovak Republic) has a section on neutrophils.

2. Neutropenia is defined in the On-line Medical Dictionary. The Merck Manual of Diagnosis and Therapy has a section on neutropenia; the Home Edition also includes information about neutropenia. Online Mendelian Inheritance in Man (OMIM) has an entry for severe congenital neutropenia.

3. Phagocytosis.Phagocytosis is defined in the KUMC pathology tutorial. Cells Alive! offers a video of phagocytosis. An introduction to phagocytosis is provided for a course on human anatomy and physiology at the University of Washington. An introduction to phagocytosis is provided by the University of Leicester's Microbiology & Immunology Web page. L. Henderson, Department of Biochemistry, University of Bristol, UK, offers a section on phagocytosis in lecture notes on phagocytes and immunology.

4. Volker Brinkmann, Ulrike Reichard, Christian Goosmann, Beatrix Fauler are at the Microscopy Core Facility, Max-Planck Institute for Infection Biology, Berlin. Ulrike Reichard, Christian Goosmann, Yvonne Uhlemann, David S. Weiss, and Arturo Zychlinsky are in the Department for Cellular Microbiology, Max-Planck Institute for Infection Biology. Yvette Weinrauch is in the Department of Microbiology, New York University School of Medicine.

5. Cytokines. The Immunology Book Case provides an introduction to cytokines. J. Decker offers a tutorial on cytokines for an immunology course. Lecture notes on cytokines for a general pathology course are provided by the Department of Pathology and Laboratory Medicine, New Jersey Medical School. H. Ibelgaufts' Cytokines Online Pathfinder Encyclopaedia (COPE) is a hypertext reference covering all aspects of cytokine research.

6. Bacterial endotoxins. A presentation on bacterial endotoxins is provided by S. W. Joseph, Department of Cell Biology and Molecular Genetics, University of Maryland, for a course on pathogenic microbiology. Doc Kaiser's Microbiology Home Page offers lecture notes on bacterial endotoxins. K. Todar provides lecture notes on bacterial endotoxins for a bacteriology course on host-parasite interactions.

7. Histones.Histones are defined in the Dictionary of Cell and Molecular Biology. The Columbia Encyclopedia includes an entry on histones. An entry on histones is included in the Biochemistry companion Web site. The Molecular Biology Web Book includes an introduction to histones. Molecular Biology of the Cell (available on the NCBI Bookshelf) includes a section on histones.

8. Elastase.Elastase is defined in the Dictionary of Cell and Molecular Biology. Entries for leukocyte elastase are included in IUBMB Enzyme Nomenclature and in the ExPASy ENZYME and Swiss-Prot databases. OMIM has an entry for elastase.

9. Deoxyribonuclease (DNase).Deoxyribonuclease is defined in the Dictionary of Cell and Molecular Biology. ExPASy's PROCITE defines deoxyribonuclease. IUBMB Enzyme Nomenclature has an entry for deoxyribonuclease. ExPASy's ENZYME has an entry for deoxyribonuclease.

10. Apoptosis.Cells Alive! provides a video of neutrophils undergoing apoptosis. An introduction to apoptosis is provided by the Cell Death Society. An entry on apoptosis is included in the Biochemistry companion Web site. Kimball's Biology Pages include a presentation on apoptosis. The NIH Apoptosis Interest Group offers a presentation on apoptosis. Information about apoptosis, with a comparison of necrosis and apoptosis, is offered by the Neuromuscular Disease Center at Washington University. COPE has an article on apoptosis.

11. Necrosis is defined by the Cell Death Society. For a course on cancer, P. Hall, Department of Pathology, Queen's University Belfast, defines and contrasts necrosis and apoptosis and offers a chart summarizing the differences

12. Cathepsins are defined on the Biochemistry Companion Web site. ExPASy's PROCITE has an entry about the serine proteases from the trypsin family. Kimball's Biology Pages offer an introduction to serine proteases. An entry for cathepsin G is included in Swiss-Prot. OMIM has an entry for cathepsin G.

13. DNase in cystic fibrosis therapy. The March 2001 issue of Thorax had a review article by P. Robinson titled “Cystic fibrosis” (6). The Merck Manual of Diagnosis and Therapy has a section on cystic fibrosis. MedlinePlus provides a collection of links to Internet resources about cystic fibrosis. MedlinePlus Drug Information includes an entry for DNase (Dornase Alfa) in treating cystic fibrosis. The Cystic Fibrosis Medicine Web site offers a presentation about recombinant DNase. The Yale New Haven Health System Web site provides information about cystic fibrosis and DNase for cystic fibrosis. The UK Health Technology Assessment Programme makes available a 2002 monograph by R. Suri et al. titled “A comparative study of hypertonic saline, daily and alternate-day rhDNase in children with cystic fibrosis.”

14. Chemotaxis. Cells Alive! offers a video of chemotaxis. COPE has an article on chemotaxis. L. Henderson offers an introduction to chemotaxis.

15. Warren L. Lee is in the Division of Respirology, Department of Medicine, University of Toronto, and in the Programme in Cell Biology, Hospital for Sick Children, Toronto.

16. Sergio Grinstein is in the Programme in Cell Biology, Hospital for Sick Children, Toronto.


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