DOI: 10.1126/science.1092385, 1532 (2004);303 Science

, et al.Volker BrinkmannNeutrophil Extracellular Traps Kill Bacteria

This copy is for your personal, non-commercial use only.

clicking here.colleagues, clients, or customers by , you can order high-quality copies for yourIf you wish to distribute this article to others

here.following the guidelines can be obtained byPermission to republish or repurpose articles or portions of articles

): August 21, 2011 www.sciencemag.org (this infomation is current as ofThe following resources related to this article are available online at

http://www.sciencemag.org/content/303/5663/1532.full.htmlversion of this article at:

including high-resolution figures, can be found in the onlineUpdated information and services,

http://www.sciencemag.org/content/suppl/2004/03/04/303.5663.1532.DC1.html can be found at: Supporting Online Material

http://www.sciencemag.org/content/303/5663/1532.full.html#relatedfound at:

can berelated to this article A list of selected additional articles on the Science Web sites

343 article(s) on the ISI Web of Sciencecited by This article has been

http://www.sciencemag.org/content/303/5663/1532.full.html#related-urls100 articles hosted by HighWire Press; see:cited by This article has been

http://www.sciencemag.org/cgi/collection/immunologyImmunology

subject collections:This article appears in the following

registered trademark of AAAS. is aScience2004 by the American Association for the Advancement of Science; all rights reserved. The title

CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience

on A

ugus

t 21,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

Neutrophil Extracellular TrapsKill Bacteria

Volker Brinkmann,1 Ulrike Reichard,1,2 Christian Goosmann,1,2

Beatrix Fauler,1 Yvonne Uhlemann,2 David S. Weiss,2

Yvette Weinrauch,3 Arturo Zychlinsky2*

Neutrophils engulf and kill bacteria when their antimicrobial granules fuse withthe phagosome. Here, we describe that, upon activation, neutrophils releasegranule proteins and chromatin that together form extracellular bers that bindGram-positive and -negative bacteria. These neutrophil extracellular traps(NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivoin experimental dysentery and spontaneous human appendicitis, two examplesof acute inammation. NETs appear to be a form of innate response that bindsmicroorganisms, prevents them from spreading, and ensures a high local con-centration of antimicrobial agents to degrade virulence factors and kill bacteria.

In response to inflammatory stimuli, neutro-phils migrate from the circulating blood toinfected tissues, where they efficiently bind,engulf, and inactivate bacteria. Phagocytosedbacteria are killed rapidly by proteolytic en-zymes, antimicrobial proteins, and reactiveoxygen species (1, 2). Neutrophils also de-granulate, releasing antimicrobial factors intothe extracellular medium (3). Here, we show thatneutrophils generate extracellular fibers, or neu-trophil extracellular traps (NETs), which are struc-tures composed of granule and nuclear constitu-ents that disarm and kill bacteria extracellularly.

NETs were made by activated neutrophils.Although nave cells were round with somemembrane folds (Fig. 1, A and C), neutrophilsstimulated with interleukin-8 (IL-8), phorbolmyristate acetate (PMA), or lipopolysaccharide(LPS) became flat and formed membrane pro-trusions (Fig. 1B) as previously described (4).Surprisingly, we found that activated neutro-phils but not nave cells made prominent extra-cellular structures (arrows, Fig. 1, B and D).These fibers, or NETs, were very fragile, andspecimens had to be washed and fixed carefullyto preserve them. High-resolution scanningelectron microscopy (SEM) showed that theNETs contained smooth stretches with a diam-eter of 15 to 17 nm (Fig. 1E, arrowheads) andglobular domains of around 25 nm (Fig. 1E,arrows) that aggregated into larger threads withdiameters of up to 50 nm. Analysis of crosssections of the NETs by transmission electronmicroscopy (TEM) revealed they were not sur-rounded by membranes (Fig. 1F).

The composition of NETs was analyzed byimmunofluorescence. NETs contained proteins

from azurophilic (primary) granules (5, 6) suchas neutrophil elastase (Fig. 2A), cathepsin G,and myeloperoxidase (table S1). Proteins fromspecific (secondary) granules and tertiary gran-ules, such as lactoferrin and gelatinase, respec-tively, were also present (table S1). In contrast,CD63, a granule membrane protein, the cyto-plasmic markers annexin I (7), actin, tubulin,and various other cytoplasmic proteins wereexcluded from NETs (table S1).

DNA is a major structural component ofNETs, because several DNA intercalating dyesstained NETs strongly (Fig. 2B) and a brieftreatment with deoxyribonuclease (DNase) re-sulted in the disintegration of NETs (movie S1).Conversely, protease treatment left the DNA ofthe NETs intact (8). The NETs reacted withantibodies against histones H1, H2A, H2B, H3,and H4 (table S1) and against the H2A-H2B-DNA complex (9, 10) (Fig. 2C).

Double immunostaining of ultrathin cryo-sections for TEM (Fig. 2D) confirmed the pres-ence of neutrophil elastase (small gold particles,arrowheads) and H2A-H2B-DNA complexes(large gold particles, arrows) in NETs. Histoneand neutrophil elastase staining was found onglobular NET domains. Furthermore, immuno-staining of SEM samples (Fig. 2E) corroboratedthe localization of neutrophil elastase to theglobular domains of NETs. These data demon-strate that the structures visualized by differentmicroscopy approaches (immunofluorescence,TEM, and SEM) are identical. NET formationwas quantified in a fluorometer with the use ofa DNA dye that is excluded from cells. Neutro-phils release NETs as early as 10 min afteractivation, and the release depends on the doseof the activator (fig. S1).

Several lines of evidence indicate that neu-trophils make NETs actively: (i) Stimuli thatinduce NETs do not promote the release of thecytoplasmic marker lactate dehydrogenase(LDH), and activated cells exclude vital dyesfor at least two hours after stimulation (8). (ii)Stimuli such as IL-8 and LPS, which prolong

the life of neutrophils (11), can induce NETsefficiently. (iii) Incubation with DNA interca-lating dyes before neutrophil activation pre-vents NET formation but has no effect on theinduction of apoptosis by staurosporine or tu-mor necrosis factor (8). (iv) NETs are formedas early as 10 min after activation, a time coursefaster than apoptosis (fig. S1). (v) Time-lapsevideo microscopy (movie S2) shows that motilecells make NETs. Taken together, these datastrongly indicate that NETs are not the result ofleakage during cellular disintegration. We cannotexclude, however, the possibility that NET forma-tion is an early event in the neutrophil program forcell death. Neutrophils are terminally differentiat-ed cells that are programmed to die a few hoursafter they enter into circulation. Furthermore, iso-lated neutrophils are a heterogeneous populationwith respect to age, and a small portion of thisaged subpopulation is expected to die. Neutro-phils can undergo caspase-dependent (12) and-independent apoptosis in vitro (13), but the pro-cess that leads to neutrophil death in vivo is notknown. It is conceivable that NET formation is anearly event in cell death.

NETs associate with both Gram-positive(Staphylococcus aureus, shown in Fig. 3A) andGram-negative pathogens (Salmonella typhi-murium and Shigella flexneri, shown in Fig. 3,B and C, respectively). We have previouslyshown that neutrophil elastase degrades viru-lence factors of Gram-negative bacteria (14).Our finding that bacteria are trapped in NETsdecorated with neutrophil elastase prompted usto test whether bacterial virulence factors weretargeted extracellularly. Immunofluorescencestaining of IpaB, a virulence factor of S. flex-neri, was weaker in bacteria trapped in NETscompared to free Shigella (Fig. 3D, top left),although the bacteria and the NETs were clear-ly visible when DNA was stained. In contrast,when neutrophil protease activity was blockedby the secretory leukocyte proteinase inhibitor(SLPI), bacteria trapped in NETs containedhigh amounts of IpaB (Fig. 3D, bottom left).Interestingly, virulence factors from Gram-positive bacteria were also susceptible to neu-trophil proteases. Lower amounts of the S.aureus virulence factor toxin were found inNET-associated bacteria compared to that of freebacteria or when neutrophil proteases wereblocked with SLPI (fig. S2). These results suggestthat NETs can disarm a wide range of pathogens.

We corroborated that extracellular proteasesdegrade bacterial virulence factors by inhibitingneutrophil phagocytosis. This was accom-plished by incubating activated neutrophils withcytochalasin D. In the presence of cytochalasinD, an inhibitor of actin polymerization, NETspersisted and phagocytosis was blocked. Weinfected these neutrophils that have NETs butcannot phagocytose with S. flexneri. Extracel-lular neutrophil elastase, like purified elastase(14), degraded the virulence factors IcsA andIpaB but not the control OmpA, an outer mem-

1Microscopy Core Facility and 2Department of CellularMicrobiology, Max Planck Institute for Infection Biology,Schumannstrasse 21/22, 10117 Berlin, Germany. 3Depart-ment of Microbiology, New York University School ofMedicine, 540 First Avenue, New York, NY 10016, USA.

*To whom correspondence should be addressed. E-mail: zychlinsky@mpiib-berlin.mpg.de

R E P O R T S

5 MARCH 2004 VOL 303 SCIENCE www.sciencemag.org1532

on A

ugus

t 21,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

brane protein (Fig. 3E). This confirms that neu-trophil elastase presented in NETs actively tar-gets bacterial virulence factors.

Activated neutrophils incubated with cy-tochalasin D after formation of the NETs cankill about 30% of a S. flexneri or S. aureusinoculum (Fig. 3F, without DNase). We pro-pose the hypothesis that the NET structure isnecessary for this extracellular bactericidalactivity. Indeed, when NETs were dismantledwith DNase (movie S1), the killing of bacte-ria was negligible (Fig. 3F). In these experi-ments, the cultures were not washed aftertreatment with protease-free DNase, leavingthe total protein concentration unchanged.Hence, these data strongly suggest that thefibrous structure of NETs is necessary for thesequestration and killing of bacteria by deliv-ering a high local concentration of antimicro-bial molecules to the bound microbes.

In an alternative approach to demonstratethe antibacterial activity of NETs, we showedthat a monoclonal antibody against the H2A-H2B-DNA complex abrogated S. flexneri andS. aureus killing in infections of neutrophilspretreated with cytochalasin D after NET for-mation (Fig. 3G). An isotype control antibodyhad no effect on killing. The factors responsiblefor bacterial killing are likely to include granuleproteins like bactericidal permeability increas-ing protein (BPI) (table S1) and histones. Theantimicrobial activity of histones (15), evolu-tionarily conserved proteins that bind DNA toform the nucleosome complex, and peptidesderived from histones, is well established (16,17) Indeed, purified H2A killed S. flexneri, S.typhimurium, and S. aureus cultures with con-centrations as low as 2 g/ml (140 nM) in 30min (fig. S3). The concentration of H2A re-quired to kill bacteria is low compared withother antimicrobial proteins (18).

To determine whether NETs are present invivo, we analyzed samples from experimentalshigellosis in rabbits and spontaneous appendi-citis in humans. Staining of histological sec-tions clearly showed extracellular fibrous ma-terial that contains NET components: histones(Fig. 4, A and F), DNA (Fig. 4, C and G), andneutrophil elastase (Fig. 4E). In vivo, NETs trapbacteria as shown by the localization of Shigel-la (Fig. 4B) to the NETs. These results indicatethat NETs are abundant at inflammatory sites.

Neutrophils make NETs through an activemechanism that remains to be understood.NETs disarm pathogens with proteases suchas neutrophil elastase. NETs also kill bacteriaefficiently, and at least one of the NET com-ponents, histones, exerts antimicrobial activ-ity at surprisingly low concentrations. Thesedata correlate with previous findings showingthat neutrophil degranulation releases antimi-crobial factors extracellularly (3) and the ob-servation that inflammatory exudates rich inneutrophils, like pus, contain DNA, whichwas not known to play an active role in

antimicrobial defense. Also, these data are inaccord with recent findings proposing thatoxygen-independent mechanisms play an im-portant role in the control of infections (19).The data presented here indicate that granuleproteins and chromatin together form an ex-tracellular structure that amplifies the effec-tiveness of its antimicrobial substances byensuring a high local concentration. NETsdegrade virulence factors and/or kill bacteriaeven before the microorganisms are engulfedby neutrophils. In addition to their antimicro-bial properties, NETs may serve as a physicalbarrier that prevents further spread of bacte-ria. Moreover, sequestering the granule pro-teins into NETs may keep potentially nox-

ious proteins like proteases from diffusingaway and inducing damage in tissue adja-cent to the site of inflammation (20). NETsmight also have a deleterious effect on thehost, because the exposure of extracellularhistone complexes could play a role duringthe development of autoimmune diseaseslike lupus erythematosus.

References and Notes1. P. Elsbach, J. Weiss, in Inammation: Basic Principles and

Clinical Correlates, J. I. Gallin, I. M. Goldstein, R. Snyderman,Eds. (Raven Press, New York, ed. 2, 1992), pp. 603636.

2. S. J. Klebanoff, in Inammation: Basic Principles andClinical Correlates, J. I. Gallin, R. Snyderman, Eds.(Lippincott Williams & Wilkens, Philadelphia, PA, ed.3, 1999), pp. 721768.

3. P. E. Henson et al., in Inammation: Basic Principles

Fig. 1. Electron microscopical analysis of resting and activated neutrophils. (A) Resting neutrophilsare round and devoid of bers. (B) Upon stimulation with 25 nM PMA for 30 min, the cells atten,make many membrane protrusions, and form bers (NETs), arrows in (B) and (D). (C) TEM analysisof nave neutrophils in suspension. (D) Ultrathin section of neutrophils stimulated in suspensionwith 10 ng of IL-8 for 45 min. Bars in (A) to (D) indicate 10 m. The multilobular nuclei anddifferent granules are clearly visible in both gures. The activated cells in (D) have manypseudopods and show NETs (arrow). (E) High-resolution SEM analysis of NETs that consist ofsmooth bers (diameters of 15 to 17 nm, arrowheads) and globular domains (diameter around 25nm, arrow). Globular complexes can be aggregated to thick bundles or bers. (F) Ultrathin sectionsof NETs show that they are not membrane-bound. Neutrophils were stimulated as in (D). Bars in(E) and (F), 500 m.

R E P O R T S

www.sciencemag.org SCIENCE VOL 303 5 MARCH 2004 1533

on A

ugus

t 21,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

Fig. 2. Immunostaining ofNETs. Neutrophils were acti-vated with 10 ng of IL-8 for 30min and stained for neutrophilelastase (A), DNA (B), and thecomplex formed by H2A-H2B-DNA (C). Extracellular brousmaterial is stained brightly. Asexpected, we found granularstaining for neutrophil elastase(A) and nuclear staining for hi-stones and DNA [(B) and (C)].Samples were analyzed withthe use of a Leica TCS-SP(Beusheim, Germany) confocalmicroscope. The images areprojections of a z stack (origi-nal dimensions: x and y, 85.5m; z 6.3 m). Bar, 10 m.(D) Immunodetection of his-tones (large gold particles, ar-rows) and neutrophil elastase(small gold particles, arrow-heads) in ultrathin cryosec-tions of neutrophils stimulatedwith IL-8 (10 ng, 1 hour). Bar,200 nm. (E) Immuno-SEM,pseudocolored, of neutrophilstreated as in (A) to (C). Over-lay of images from secondaryelectron detector (red, topog-raphy) and backscattered elec-tron detector (green, elementsensitive, most back-scatteredelectrons from the site of gold binding). Bright yellow dots (arrows) show localization of 12-nm gold particles detecting neutrophil elastase. Bar, 200 nm.

Fig. 3. Gram-positive and Gram-negativebacteria associate with neutrophil bers. SEMof S. aureus (A), S. typhimurium (B), and S.exneri (C) trapped by NETs. Neutrophilswere treated with 100 ng of IL-8 for 40 minbefore infection. Bar, 500 nm. (D) Immuno-uorescence of neutrophils infected with S.exneri stained for the virulence factor IpaBand DNA. IpaB is degraded by neutrophilelastase and is only detectable on the bacte-ria (arrows) when neutrophil elastase isblocked with SLPI. DNA staining shows NETsand bacteria (arrows). (E) Western blotshowing that the virulence factors IcsA andIpaB but not OmpA were degraded by cy-tochalasin Dtreated neutrophils incubatedwith S. exneri. Lane 1, bacteria alone. Lane2, bacteria incubated with cytochalasin Dtreated neutrophils. (F)Extracellular bactericidal activity was greatly reduced in both S.exneri and S. aureus infections after incubation with DNase, whichdissociates NETs. (G) Extracellular bacterial killing by neutrophils was

reduced by addition of antibodies against histones. Neutrophils weretreated with cytochalasin D to prevent phagocytosis and infectedwith S. exneri or S. aureus. In the presence of antibody against H2A,bacterial killing was abrogated.

R E P O R T S

5 MARCH 2004 VOL 303 SCIENCE www.sciencemag.org1534

on A

ugus

t 21,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from

and Clinical Correlates, J. I. Gallin, R Snyderman, Eds.(Raven Press, New York, ed. 2, 1992), pp. 511539.

4. L. C. Junqueira, J. Carneiro, R. O. Kelley, Basic Histol-ogy (Appelton & Lange, Norwalk, CT, ed. 8, 1995).

5. D. F. Bainton, J. Immunol. Methods 232, 153 (1999).6. N. Borregaard, J. B. Cowland, Blood 89, 3503 (1997).7. C. Movitz, C. Dahlgren, Cell Biol. Int. 25, 963 (2001).8. V. Brinkmann et al., data not shown.9. M. J. Losman, T. M. Fasy, K. E. Novick, M. Monestier,

J. Immunol. 148, 1561 (1992).10. P. Salgame et al., Nucleic Acids Res. 25, 680 (1997).11. P. L. Sabroe et al., J. Immunol. 170, 5268 (2003).12. B. Fadeel et al., Blood 92, 4808 (1998).13. N. A. Maianski, D. Roos, T. W. Kuijpers, Blood 101,

1987 (2002).14. Y. Weinrauch et al., Nature 417, 91 (2002).15. J. G. Hirsch, J. Exp. Med. 108, 925 (1958).16. S. M. Zhdan-Pushkina, N. V. Dronova, Mikrobiologiia

45, 60 (1976).17. H. S. Kim, C. B. Park, M. S. Kim, S. C. Kim, Biochem.

Biophys. Res. Commun. 229, 381 (1996).18. P. Elsbach, J. Weiss, O. Levy, Trends Microbiol. 2, 324

(1994).19. E. P. Reeves et al., Nature 416, 291 (2002).20. V. Balloy et al.,Am. J. Respir. Cell Mol. Biol. 28, 746 (2003).21. The gift of M. Monestier, Temple University, the monoclo-

nal antibody against H2A-H2B-DNA, is gratefully acknowl-edged. The authors thank the help of M. Ingersoll, B.Raupach, C. Scharff, C. Heinz, and members of the Depart-ment of Cellular Microbiology, Max Planck Institute forInfection Biology. Supported in part by NIH grantAI037720.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/303/5663/1532/DC1Materials and MethodsFigs. S1 to S3Table S1Movies S1 and S2

9 October 2003; accepted 24 December 2003

Emerging Vectors in the Culexpipiens Complex

Dina M. Fonseca,1,2* Nusha Keyghobadi,1 Colin A. Malcolm,3

Ceylan Mehmet,3 Francis Schaffner,4 Motoyoshi Mogi,5

Robert C. Fleischer,1 Richard C. Wilkerson2

In the Old World, some mosquitoes in the Culex pipiens complex are excellentenzootic vectors of West Nile virus, circulating the virus among birds, whereasothers bite mainly humans and other mammals. Here we show that, in northernEurope, such forms differing in behavior and physiology have unique micro-satellite ngerprints with no evidence of gene ow between them, as would beexpected from distinct species. In the United States, however, hybrids betweenthese forms are ubiquitous. Such hybrids between human-biters and bird-bitersmay be the bridge vectors contributing to the unprecedented severity and rangeof the West Nile virus epidemic in North America.

Species in the Culex pipiens complex areconsidered to be the primary vectors ofWest Nile virus (WNV) in North Americabecause they are often the most commonmosquitoes in urban areas (1), because dis-ease outbreaks occur during their peakabundance period (2), because they arecompetent laboratory vectors of WNV (3),and because field populations in the UnitedStates have repeatedly been found infectedwith the virus (4, 5). In addition, they cantransmit the virus transovarially (6 ), so

overwintering mosquitoes can serve as asource of WNV to initiate an infectioncycle in the spring (7 ). Blood-meal analysishas revealed that Cx. pipiens in the UnitedStates bite both humans (anthropophagy)and birds, suggesting they may serve asbridge vectors of the disease from birds tohumans (2). Human WNV epidemics re-quire bridge vectors, because humans andother mammals do not usually generatehigh enough viremia to infect biting mos-quitoes (8). Although Cx. pipiens has been

Fig. 4. Analysis of tissue sections from experimental shigellosis inrabbits (A to D) and spontaneous human appendicitis (E to H). (A)Immunouorescence staining of histones reveals nuclear and extra-cellular localization that largely overlaps with staining for DNA (C).(B) Staining with an antibody against Shigella-specic LPS. (D) Theoverlay indicates that numerous Shigellae are closely associated to

brous material staining for histones and DNA. (E) Staining forneutrophil elastase in an area of neutrophil exudate in human spon-taneous appendicitis reveals brous extracellular material that alsostains for histone (F) and DNA (G). (H) Overlay of the images. Theimages are projections of confocal z stacks generated from sections of5 to 6 m thickness. Bar, 50 m.

R E P O R T S

www.sciencemag.org SCIENCE VOL 303 5 MARCH 2004 1535

on A

ugus

t 21,

201

1w

ww

.sci

ence

mag

.org

Dow

nloa

ded

from