Neutrophil Extracellular Traps Kill Bacteria

Volker Brinkmann, et al.

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

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Neutrophil Extracellular Traps

Kill 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 with
the phagosome. Here, we describe that, upon activation, neutrophils release
granule proteins and chromatin that together form extracellular bers that bind
Gram-positive and -negative bacteria. These neutrophil extracellular traps
(NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo
in experimental dysentery and spontaneous human appendicitis, two examples
of acute inammation. NETs appear to be a form of innate response that binds
microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.
In response to inflammatory stimuli, neutrophils migrate from the circulating blood to
infected tissues, where they efficiently bind,
engulf, and inactivate bacteria. Phagocytosed
bacteria are killed rapidly by proteolytic enzymes, antimicrobial proteins, and reactive
oxygen species (1, 2). Neutrophils also degranulate, releasing antimicrobial factors into
the extracellular medium (3). Here, we show that
neutrophils generate extracellular fibers, or neutrophil extracellular traps (NETs), which are structures composed of granule and nuclear constituents that disarm and kill bacteria extracellularly.
NETs were made by activated neutrophils.
Although nave cells were round with some
membrane folds (Fig. 1, A and C), neutrophils
stimulated with interleukin-8 (IL-8), phorbol
myristate acetate (PMA), or lipopolysaccharide
(LPS) became flat and formed membrane protrusions (Fig. 1B) as previously described (4).
Surprisingly, we found that activated neutrophils but not nave cells made prominent extracellular structures (arrows, Fig. 1, B and D).
These fibers, or NETs, were very fragile, and
specimens had to be washed and fixed carefully
to preserve them. High-resolution scanning
electron microscopy (SEM) showed that the
NETs contained smooth stretches with a diameter of 15 to 17 nm (Fig. 1E, arrowheads) and
globular domains of around 25 nm (Fig. 1E,
arrows) that aggregated into larger threads with
diameters of up to 50 nm. Analysis of cross
sections of the NETs by transmission electron
microscopy (TEM) revealed they were not surrounded by membranes (Fig. 1F).
The composition of NETs was analyzed by
immunofluorescence. NETs contained proteins
Microscopy Core Facility and 2Department of Cellular
Microbiology, Max Planck Institute for Infection Biology,
Schumannstrasse 21/22, 10117 Berlin, Germany. 3Department of Microbiology, New York University School of
Medicine, 540 First Avenue, New York, NY 10016, USA.

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

1532

from azurophilic (primary) granules (5, 6) such

as neutrophil elastase (Fig. 2A), cathepsin G,
and myeloperoxidase (table S1). Proteins from
specific (secondary) granules and tertiary granules, such as lactoferrin and gelatinase, respectively, were also present (table S1). In contrast,
CD63, a granule membrane protein, the cytoplasmic markers annexin I (7), actin, tubulin,
and various other cytoplasmic proteins were
excluded from NETs (table S1).
DNA is a major structural component of
NETs, because several DNA intercalating dyes
stained NETs strongly (Fig. 2B) and a brief
treatment with deoxyribonuclease (DNase) resulted in the disintegration of NETs (movie S1).
Conversely, protease treatment left the DNA of
the NETs intact (8). The NETs reacted with
antibodies against histones H1, H2A, H2B, H3,
and H4 (table S1) and against the H2A-H2BDNA complex (9, 10) (Fig. 2C).
Double immunostaining of ultrathin cryosections for TEM (Fig. 2D) confirmed the presence of neutrophil elastase (small gold particles,
arrowheads) and H2A-H2B-DNA complexes
(large gold particles, arrows) in NETs. Histone
and neutrophil elastase staining was found on
globular NET domains. Furthermore, immunostaining of SEM samples (Fig. 2E) corroborated
the localization of neutrophil elastase to the
globular domains of NETs. These data demonstrate that the structures visualized by different
microscopy approaches (immunofluorescence,
TEM, and SEM) are identical. NET formation
was quantified in a fluorometer with the use of
a DNA dye that is excluded from cells. Neutrophils release NETs as early as 10 min after
activation, and the release depends on the dose
of the activator (fig. S1).
Several lines of evidence indicate that neutrophils make NETs actively: (i) Stimuli that
induce NETs do not promote the release of the
cytoplasmic marker lactate dehydrogenase
(LDH), and activated cells exclude vital dyes
for at least two hours after stimulation (8). (ii)
Stimuli such as IL-8 and LPS, which prolong

the life of neutrophils (11), can induce NETs

efficiently. (iii) Incubation with DNA intercalating dyes before neutrophil activation prevents NET formation but has no effect on the
induction of apoptosis by staurosporine or tumor necrosis factor (8). (iv) NETs are formed
as early as 10 min after activation, a time course
faster than apoptosis (fig. S1). (v) Time-lapse
video microscopy (movie S2) shows that motile
cells make NETs. Taken together, these data
strongly indicate that NETs are not the result of
leakage during cellular disintegration. We cannot
exclude, however, the possibility that NET formation is an early event in the neutrophil program for
cell death. Neutrophils are terminally differentiated cells that are programmed to die a few hours
after they enter into circulation. Furthermore, isolated neutrophils are a heterogeneous population
with respect to age, and a small portion of this
aged subpopulation is expected to die. Neutrophils can undergo caspase-dependent (12) and
-independent apoptosis in vitro (13), but the process that leads to neutrophil death in vivo is not
known. It is conceivable that NET formation is an
early event in cell death.
NETs associate with both Gram-positive
(Staphylococcus aureus, shown in Fig. 3A) and
Gram-negative pathogens (Salmonella typhimurium and Shigella flexneri, shown in Fig. 3,
B and C, respectively). We have previously
shown that neutrophil elastase degrades virulence factors of Gram-negative bacteria (14).
Our finding that bacteria are trapped in NETs
decorated with neutrophil elastase prompted us
to test whether bacterial virulence factors were
targeted extracellularly. Immunofluorescence
staining of IpaB, a virulence factor of S. flexneri, was weaker in bacteria trapped in NETs
compared to free Shigella (Fig. 3D, top left),
although the bacteria and the NETs were clearly visible when DNA was stained. In contrast,
when neutrophil protease activity was blocked
by the secretory leukocyte proteinase inhibitor
(SLPI), bacteria trapped in NETs contained
high amounts of IpaB (Fig. 3D, bottom left).
Interestingly, virulence factors from Grampositive bacteria were also susceptible to neutrophil proteases. Lower amounts of the S.
aureus virulence factor toxin were found in
NET-associated bacteria compared to that of free
bacteria or when neutrophil proteases were
blocked with SLPI (fig. S2). These results suggest
that NETs can disarm a wide range of pathogens.
We corroborated that extracellular proteases
degrade bacterial virulence factors by inhibiting
neutrophil phagocytosis. This was accomplished by incubating activated neutrophils with
cytochalasin D. In the presence of cytochalasin
D, an inhibitor of actin polymerization, NETs
persisted and phagocytosis was blocked. We
infected these neutrophils that have NETs but
cannot phagocytose with S. flexneri. Extracellular neutrophil elastase, like purified elastase
(14), degraded the virulence factors IcsA and
IpaB but not the control OmpA, an outer mem-

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antimicrobial defense. Also, these data are in
accord with recent findings proposing that
oxygen-independent mechanisms play an important role in the control of infections (19).
The data presented here indicate that granule
proteins and chromatin together form an extracellular structure that amplifies the effectiveness of its antimicrobial substances by
ensuring a high local concentration. NETs
degrade virulence factors and/or kill bacteria
even before the microorganisms are engulfed
by neutrophils. In addition to their antimicrobial properties, NETs may serve as a physical
barrier that prevents further spread of bacteria. Moreover, sequestering the granule proteins into NETs may keep potentially nox-

ious proteins like proteases from diffusing

away and inducing damage in tissue adjacent to the site of inflammation (20). NETs
might also have a deleterious effect on the
host, because the exposure of extracellular
histone complexes could play a role during
the development of autoimmune diseases
like lupus erythematosus.
References and Notes

1. 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 and
Clinical 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

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brane protein (Fig. 3E). This confirms that neutrophil elastase presented in NETs actively targets bacterial virulence factors.
Activated neutrophils incubated with cytochalasin D after formation of the NETs can
kill about 30% of a S. flexneri or S. aureus
inoculum (Fig. 3F, without DNase). We propose the hypothesis that the NET structure is
necessary for this extracellular bactericidal
activity. Indeed, when NETs were dismantled
with DNase (movie S1), the killing of bacteria was negligible (Fig. 3F). In these experiments, the cultures were not washed after
treatment with protease-free DNase, leaving
the total protein concentration unchanged.
Hence, these data strongly suggest that the
fibrous structure of NETs is necessary for the
sequestration and killing of bacteria by delivering a high local concentration of antimicrobial molecules to the bound microbes.
In an alternative approach to demonstrate
the antibacterial activity of NETs, we showed
that a monoclonal antibody against the H2AH2B-DNA complex abrogated S. flexneri and
S. aureus killing in infections of neutrophils
pretreated with cytochalasin D after NET formation (Fig. 3G). An isotype control antibody
had no effect on killing. The factors responsible
for bacterial killing are likely to include granule
proteins like bactericidal permeability increasing protein (BPI) (table S1) and histones. The
antimicrobial activity of histones (15), evolutionarily conserved proteins that bind DNA to
form the nucleosome complex, and peptides
derived from histones, is well established (16,
17) Indeed, purified H2A killed S. flexneri, S.
typhimurium, and S. aureus cultures with concentrations as low as 2 g/ml (140 nM) in 30
min (fig. S3). The concentration of H2A required to kill bacteria is low compared with
other antimicrobial proteins (18).
To determine whether NETs are present in
vivo, we analyzed samples from experimental
shigellosis in rabbits and spontaneous appendicitis in humans. Staining of histological sections clearly showed extracellular fibrous material that contains NET components: histones
(Fig. 4, A and F), DNA (Fig. 4, C and G), and
neutrophil elastase (Fig. 4E). In vivo, NETs trap
bacteria as shown by the localization of Shigella (Fig. 4B) to the NETs. These results indicate
that NETs are abundant at inflammatory sites.
Neutrophils make NETs through an active
mechanism that remains to be understood.
NETs disarm pathogens with proteases such
as neutrophil elastase. NETs also kill bacteria
efficiently, and at least one of the NET components, histones, exerts antimicrobial activity at surprisingly low concentrations. These
data correlate with previous findings showing
that neutrophil degranulation releases antimicrobial factors extracellularly (3) and the observation that inflammatory exudates rich in
neutrophils, like pus, contain DNA, which
was not known to play an active role in

Fig. 1. Electron microscopical analysis of resting and activated neutrophils. (A) Resting neutrophils
are 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 analysis
of nave neutrophils in suspension. (D) Ultrathin section of neutrophils stimulated in suspension
with 10 ng of IL-8 for 45 min. Bars in (A) to (D) indicate 10 m. The multilobular nuclei and
different granules are clearly visible in both gures. The activated cells in (D) have many
pseudopods and show NETs (arrow). (E) High-resolution SEM analysis of NETs that consist of
smooth bers (diameters of 15 to 17 nm, arrowheads) and globular domains (diameter around 25
nm, arrow). Globular complexes can be aggregated to thick bundles or bers. (F) Ultrathin sections
of NETs show that they are not membrane-bound. Neutrophils were stimulated as in (D). Bars in
(E) and (F), 500 m.

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Fig. 2. Immunostaining of
NETs. Neutrophils were activated with 10 ng of IL-8 for 30
min and stained for neutrophil
elastase (A), DNA (B), and the
complex formed by H2A-H2BDNA (C). Extracellular brous
material is stained brightly. As
expected, we found granular
staining for neutrophil elastase
(A) and nuclear staining for histones and DNA [(B) and (C)].
Samples were analyzed with
the use of a Leica TCS-SP
(Beusheim, Germany) confocal
microscope. The images are
projections of a z stack (original dimensions: x and y, 85.5
m; z 6.3 m). Bar, 10 m.
(D) Immunodetection of histones (large gold particles, arrows) and neutrophil elastase
(small gold particles, arrowheads) in ultrathin cryosections of neutrophils stimulated
with IL-8 (10 ng, 1 hour). Bar,
200 nm. (E) Immuno-SEM,
pseudocolored, of neutrophils
treated as in (A) to (C). Overlay of images from secondary
electron detector (red, topography) and backscattered electron detector (green, element
sensitive, most back-scattered
electrons 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-negative

bacteria associate with neutrophil bers. SEM
of S. aureus (A), S. typhimurium (B), and S.
exneri (C) trapped by NETs. Neutrophils
were treated with 100 ng of IL-8 for 40 min
before infection. Bar, 500 nm. (D) Immunouorescence of neutrophils infected with S.
exneri stained for the virulence factor IpaB
and DNA. IpaB is degraded by neutrophil
elastase and is only detectable on the bacteria (arrows) when neutrophil elastase is
blocked with SLPI. DNA staining shows NETs
and bacteria (arrows). (E) Western blot
showing that the virulence factors IcsA and
IpaB but not OmpA were degraded by cytochalasin Dtreated neutrophils incubated
with S. exneri. Lane 1, bacteria alone. Lane
2, 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, which
dissociates NETs. (G) Extracellular bacterial killing by neutrophils was

1534

reduced by addition of antibodies against histones. Neutrophils were

treated with cytochalasin D to prevent phagocytosis and infected
with S. exneri or S. aureus. In the presence of antibody against H2A,
bacterial killing was abrogated.

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Fig. 4. Analysis of tissue sections from experimental shigellosis in

rabbits (A to D) and spontaneous human appendicitis (E to H). (A)
Immunouorescence staining of histones reveals nuclear and extracellular localization that largely overlaps with staining for DNA (C).
(B) Staining with an antibody against Shigella-specic LPS. (D) The
overlay indicates that numerous Shigellae are closely associated to

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The gift of M. Monestier, Temple University, the monoclonal antibody against H2A-H2B-DNA, is gratefully acknowledged. The authors thank the help of M. Ingersoll, B.
Raupach, C. Scharff, C. Heinz, and members of the Department of Cellular Microbiology, Max Planck Institute for
Infection Biology. Supported in part by NIH grant
AI037720.

Supporting Online Material

www.sciencemag.org/cgi/content/full/303/5663/1532/DC1
Materials and Methods
Figs. S1 to S3
Table S1
Movies S1 and S2
9 October 2003; accepted 24 December 2003

brous material staining for histones and DNA. (E) Staining for
neutrophil elastase in an area of neutrophil exudate in human spontaneous appendicitis reveals brous extracellular material that also
stains for histone (F) and DNA (G). (H) Overlay of the images. The
images are projections of confocal z stacks generated from sections of
5 to 6 m thickness. Bar, 50 m.

Emerging Vectors in the Culex

pipiens 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 excellent
enzootic vectors of West Nile virus, circulating the virus among birds, whereas
others bite mainly humans and other mammals. Here we show that, in northern
Europe, such forms differing in behavior and physiology have unique microsatellite ngerprints with no evidence of gene ow between them, as would be
expected from distinct species. In the United States, however, hybrids between
these forms are ubiquitous. Such hybrids between human-biters and bird-biters
may be the bridge vectors contributing to the unprecedented severity and range
of the West Nile virus epidemic in North America.
Species in the Culex pipiens complex are
considered to be the primary vectors of
West Nile virus (WNV) in North America
because they are often the most common
mosquitoes in urban areas (1), because disease outbreaks occur during their peak
abundance period (2), because they are
competent laboratory vectors of WNV (3),
and because field populations in the United
States have repeatedly been found infected
with the virus (4, 5). In addition, they can
transmit the virus transovarially (6 ), so

overwintering mosquitoes can serve as a

source of WNV to initiate an infection
cycle in the spring (7 ). Blood-meal analysis
has revealed that Cx. pipiens in the United
States bite both humans (anthropophagy)
and birds, suggesting they may serve as
bridge vectors of the disease from birds to
humans (2). Human WNV epidemics require bridge vectors, because humans and
other mammals do not usually generate
high enough viremia to infect biting mosquitoes (8). Although Cx. pipiens has been

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