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Regular Article
PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS
Eosinophil extracellular DNA trap cell death mediates lytic release of
free secretion-competent eosinophil granules in humans
Shigeharu Ueki,1,2 Rossana C. N. Melo,1,3 Ionita Ghiran,1 Lisa A. Spencer,1 Ann M. Dvorak,4 and Peter F. Weller1
1
Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 2Department of
Infection, Allergy, Clinical Immunology and Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan; 3Laboratory of Cellular Biology,
Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil; and 4Department of Pathology, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, MA
Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal
degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases
of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil
• This research is the first to
cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil
document that human
degranulation are poorly understood. We demonstrate that activated human eosinophils
eosinophils undergo
can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free
extracellular DNA trap cell
eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunodeath.
globulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol
• This research revealed
myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidasea process by which
dependent manner. Initially, nuclear lobular formation is lost and some granules are
eosinophils undergo cytolysis
released by budding off from the cell as plasma membrane–enveloped clusters.
to liberate intact cell-free and
Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms
secretion-competent granules. weblike extracellular DNA nets and releases free intact granules. EETosis-released
eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated
to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent
mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretioncompetent granules and the formation of extracellular DNA nets. (Blood. 2013;121(11):2074-2083)
Key Points
Introduction
Human eosinophils, leukocytes notably associated with allergic,
anthelmintic parasite, and other immune responses,1–3 contain an
abundant singular population of crystalloid-bearing granules. As
intracellular organelles, these granules are central to the functional
responses of eosinophils in that eosinophil granules house preformed
protein stores of (1) 4 major cationic proteins, including eosinophil
cationic protein (ECP), major basic protein (MBP), and eosinophil
peroxidase (EPO); (2) hydrolytic enzymes; and (3) over 4 dozen
cytokines, chemokines, and growth factors.4,5 Intact eosinophils
may secrete their granule proteins by classic exocytosis (principally on the surfaces of large, multicellular helminths) or more
commonly by piecemeal degranulation.4
Unlike granules from other granule-containing leukocytes, eosinophil granules have long been recognized to be present extracellularly
in tissues and sputum associated with diverse eosinophil-associated
diseases. Extracellular eosinophil granules have been detected by
immunostaining for their cationic proteins and/or by ultrastructural
studies that demonstrated intact granules still bound by their
granule-delimiting membranes. Free extracellular eosinophil granules have been documented in diverse diseases, including atopic
dermatitis and nasal allergy, and they have been correlated with
the severity of urticaria.4,6 Since the late 1800s, free eosinophil
granules have been noted in the sputum of asthmatics, and clusters of
free eosinophil granules (termed “Cfegs”) have been documented in
human asthma and experimental guinea pig models of asthma.7
The release of intact, membrane-bound granules occurs via an
enigmatic mode of eosinophil “degranulation” that arises from the
cytolysis of eosinophils. Prior in vivo studies revealed that (1) lytic
eosinophils, noted ultrastructurally by chromatolysis and loss of
plasma membrane integrity, were frequently observed in human
airway specimens rather than apoptotic cells,8,9 (2) the numbers of
free granules increased severalfold within an hour after allergeninduced airway provocation,7 and (3) released granules exhibited little
evidence of loss of their granule contents.10 Collectively, the many in
vivo findings of free extracellular eosinophil granules suggested that
a process exists for human eosinophils to undergo nonapoptotic but
cytolytic cell death that liberated intact extracellular granules.
Defining the means by which eosinophils may release extracellularly intact granules is more cogent with our recent findings, in
which isolated eosinophil granules remain secretion competent.
Cell-free eosinophil granules express ligand-binding cytokine,
chemokine, and eicosanoid receptors on the surface of their delimiting
Submitted May 25, 2012; accepted December 20, 2012. Prepublished online
as Blood First Edition paper, January 9, 2013; DOI 10.1182/blood-2012-05432088.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2013 by The American Society of Hematology
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BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
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BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
membranes.11,12 Cell-free human and mouse eosinophil granules
with receptor-mediated activation of intragranule signaling pathways
can directly secrete selected granule-derived proteins, including ECP,
EPO, ribonucleases, and cytokines, for example, the interleukins
IL-4 and IL-6.12-14 Thus, the local tissue release of cell-free,
secretion-competent eosinophil granules, secondary to eosinophil
lysis, might constitute a means by which postmortem eosinophils
could continue to provide immunoregulatory, pro-inflammatory,
and other immunopathogenic stimuli. Despite these observations,
the mechanisms of the eosinophil cytolytic release of their intact
granules are not well known.
Recently, an active form of cell death, namely, extracellular DNA
trap cell death (called ETosis15), has been recognized in neutrophils16 and mast cells.17 In these cells, ETosis develops with time,
often over 1 or more hours, and is morphologically distinct from
other classic cell death processes, including apoptosis and necrosis.18
In contrast to apoptosis, nuclear condensation and DNA fragmentation do not occur. Instead, nuclear chromatin decondenses in the
cytoplasm.19 Finally, rupture of the plasma membrane releases
nuclear DNA to form extracellular DNA nets, which for neutrophils
bind free antimicrobial molecules such as granule proteins and
histones.20 Reduced NADP (NADPH)-oxidase-mediated production of reactive oxygen species (ROS) has an essential role in the
activation of ETosis.15 To date, while it has been shown that IL-5–
or interferon-g2primed human eosinophils, in response to certain
agonists, can very rapidly and noncytolytically release mitochondrial, and not nuclear, DNA to form extracellular DNA nets,21
eosinophil ETosis has not been recognized.
Prior studies reported that the immobilized immunoglobulins
(IgG, IgA) and the calcium ionophore A23187 induced eosinophil
cell death morphologically, similar to lytic degranulation.22,23 In the
present study, we evaluated whether eosinophil cytolysis is mediated
by ETosis. We have now shown that activated human eosinophils
undergo NADPH-oxidase-dependent ETosis and that during ETosismediated cytolysis, eosinophil granules are liberated both when
contained within plasma membrane–bound clusters and as cell-free
granules. Cell-free eosinophil granules retained their cationic
proteins, and some were functionally competent to secrete in
response to the chemokine ligand CCL11 (also known as eotaxtin-1).
Material and methods
Sample collection and cell preparations
Eosinophils were purified from 320 mL of blood from mildly atopic and healthy
donors by negative selection, as described in Neves et al.14 In accordance with
the Declaration of Helsinki, informed consent was obtained under Institutional
Review Board–approved protocols. Briefly, after blood was collected into a
6% dextran saline solution (Pharmacosmos, Holbaek, Denmark) and red
blood cells (RBCs) were sedimented, the upper leukocyte-containing region
was collected and centrifuged over Ficoll-Paque (GE Healthcare, Pittsburgh,
PA). Eosinophils were isolated from granulocyte pellets by incubation with a
depletion antibody (Ab) cocktail (StemSep, Stemcell Technologies, Vancouver, BC, Canada) followed by passage over magnetized columns (Miltenyi
Biotec, Auburn, CA). The purity of isolated eosinophils was .97% of
nucleated cells with <3% contaminating RBCs, and eosinophil viability
was .99%, as determined by microscopic analyses. Separately, RBCs
(.99% purity) were purified from the dextran sedimented fraction, as
described in Aoshiba et al.24 Tissue biopsy samples were obtained during
clinical evaluations from the left frontal sinus and the skin from a patient with
allergic sinusitis and a patient with hypereosinophilic syndrome (negative
for FIP1-like 1/platelet-derived growth factor-a mutation), respectively.
EOSINOPHIL ETOSIS RELEASES GRANULES
2075
Detection of extracellular chromatin structure and cell death
Purified eosinophils were stimulated with A23187 (Sigma-Aldrich, St. Louis,
MO), PMA (phorbol 12-myristate 13-acetate, Sigma-Aldrich), IL-5 (R&D
Systems, Minneapolis, MN), GM-CSF (R&D Systems), and/or platelet
activating factor (PAF; Enzo Life Sciences, Farmingdale, NY) in poly-Llysine–coated chambered cover glasses (Nalge Nunc, Rochester, NY) or
96-well, flat-bottomed tissue culture plates in 0.1% bovine serum albumin
(BSA, wt/vol; Sigma-Aldrich) or as stated in fetal bovine serum (FBS;
Gibco, Grand Island, NY) containing phenol red-free HEPES-buffered
RPMI 1640 medium. Human IgG or IgA (Sigma-Aldrich), diluted with
phosphate-buffered saline (Gibco) in indicated concentrations, was coated
on culture plates for 3 hours at 37°C. For detection of extracellular
chromatin structure, eosinophils (2 3 105 cells per well) were seeded on
chamber slides and stimulated with the indicated conditions followed by
fixation with 3% paraformaldehyde (PFO, Electron MIcroscopy Sciences,
Hatfield, PA) without permeabilization. Eosinophils were incubated with
mouse Abs against histones (recognizing histones H1, H2A, H2B, and H4,
1:500, 45 minutes at room temperature (RT); United States Biological,
Swampscott, MA) followed by goat antimouse IgG (1:100, 30 minutes at
RT; Invitrogen, Carlsbad, CA) and propidium iodide (PI; Invitrogen). For
quantification of cell death, CYTOX green (Invitrogen) was added to the
medium, and CYTOX intensity was measured using the GloMax-Multi
detection system (Promega, Madison, WI). The increase of CYTOX intensity
was concurrent with cell death (confirmed using lactate dehydrogenase
release). In some experiments, diphenyleneiodonium chloride (DPI, 20
mM; Sigma-Aldrich) was added to the culture medium.
Scanning electron microscopy (SEM)
Eosinophils, added to round coverslips coated with poly-L-lysine in culture
plates, were stimulated with A23187 for 1 hour. Cells were fixed (1% PFO,
1.25% glutaraldehyde in 1 M sodium cacodylate buffer, pH 7.4) for 30
minutes at RT. Coverslip-adherent cells were postfixed with 1% osmium
tetroxide in water for 1 hour and dehydrated in an ascending ethanol series
from 50% (vol/vol) to absolute ethanol (10 minutes per step). Cells were
then critical point dried in carbon dioxide. Coverslips were mounted on
aluminum holders, sputtered with 5 nm gold, and analyzed in a scanning
electron microscope (Quanta 200 FEG; FEI, Eindhoven, The Netherlands).
Immunostaining for eosinophil granule MBP
Eosinophils, seeded in 8-well Lab-Tek II chamber slides (Nalge Nunc) or
immunoglobulin-coated Millicell EZ slides (Millipore), were stimulated for
indicated time points and then fixed with 3% PFO for 10 minutes. For MBP
staining, cells were permeabilized with 0.1% saponin for 10 minutes,
incubated with primary mouse antihuman MBP Ab (5 mg/mL, clone AHE2, 60 minutes at 4°C; BD Pharmingen), and followed by Alexa Fluor 488
conjugated Ab (goat antimouse IgG, 1:300, 30 minutes at RT; Invitrogen).
Control Abs and PI were used for each experiment.
Τransmission electron microscopy (TEM)
Blood eosinophils were added to chamber slides, stimulated with 2 mM of
A23187 for 1 hour, and immediately fixed in a mixture of freshly
prepared aldehydes (1% PFO, 1.25% glutaraldehyde) in 1 M sodium
cacodylate buffer, pH 7.4, for 30 minutes at RT. To obtain optimal
morphology, cells directly on the slide surface were postfixed in 1%
osmium tetroxide and processed as described in Melo et al.25,26 Biopsy
samples were fixed for 4 hours at RT using the same fixative and
processed for electron microscope as before.25,26 Sections were mounted
on uncoated 200-mesh copper grids (Ted Pella, Redding, CA) before staining
with lead citrate, and then viewed with a transmission electron microscope
(CM10; Philips, Eindhoven, The Netherlands) at 60 kV.
Time-lapse microscopy
Eosinophils in chambered cover glasses were stimulated with immobilized
IgG (1 mg/mL) for 40 minutes or 2 mM A23187 for 20 minutes in a carbon
dioxide incubator. Chambered cover glasses were sealed with 37°C mineral
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2076
BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
UEKI et al
oil before microscopic imaging, and morphological changes and granule
release were evaluated using an inverted microscope (Eclipse TE300;
Nikon, Tokyo, Japan) equipped with a digital camera in conjunction with
iVision 4.0 image analysis software (BioVision, Exton, PA). The stage was
preheated at 37°C, and phase-contrast images were recorded for 30 minutes
(every 3 seconds) with a 1003 objective. Images were sequenced into movies
(every 0.03 seconds per image).
Evaluation of released eosinophil subcellular structures
by using flow cytometry
Through the use of a modified fluorescent bead-count–based assay for
microparticles,27 subcellular structures in eosinophil culture medium were
counted and studied using FACScan (BD Biosciences, San Jose, CA). In
some experiments, subcellular structures from eosinophils loaded with
acridine orange (AO, 3 mM, 15 minutes; Sigma-Aldrich) and structures
stained with annexin V (Invitrogen) were examined. Data were analyzed by
Flowjo (version 9.03; Tree Star, Ashland, OR). Details are available in
supplemental “Materials” (supplemental Figure 1).
Staining of granule-rich subcellular structures
After eosinophils were stimulated with 2 mM A23187 for 1 hour, culture
medium was gently collected. Contaminating cells and DNA were removed
by centrifugation at 200 3 g for 10 minutes, followed by incubation for 10
minutes with DNase I (20 U/mL; New England Biolabs, Ipswich, MA).
Thereafter, supernatants were centrifuged at 2500 3 g for 10 minutes, and
pelleted subcellular structures were stained as previously described in
Neves et al.11 The gating strategy for flow cytometry was the same as for
supplemental Figure 1. CellMask, LysoTracker, and CellTracker (Invitrogen) were used for microscopic analyses as described in supplemental
“Materials” (supplemental Figure 7).
ECP measurements
ECP levels in supernatants and extraction buffer14 (Biosource, Camarillo,
CA)-disrupted subcellular structures were quantified by ECP enzymelinked immunosorbent assay kits (Medical and Biological Labs, Nagoya,
Japan). Samples were frozen at 220°C until measurement.
Stimulation of subcellular granule-associated structures
with CCL11
Eosinophils (5 to 7 3 106 cells) were stimulated with 2 mM A23187 for 1
hour in 0.1% BSA containing RPMI medium, followed by the addition of
20 U/mL DNase I for 30 minutes. Culture medium was collected, residual
intact eosinophils were removed by centrifugation at 200 3 g for 10
minutes, and granule-rich subcellular structures were pelleted by 2500 3 g
centrifugation for 10 minutes. Pelleted structures were washed and
resuspended in 0.1% BSA RPMI medium. The absence of live, intact
eosinophil contamination was confirmed by trypan blue staining. Released
granule-associated structures were incubated with the indicated concentrations of CCL11 (R&D Systems) or 2 mM A23187 for 1 hour at 37°C.
Activation of granule-associated subcellular structures derived
from AO-loaded eosinophils
After AO loading, eosinophils were stimulated for 1 hour with 2 mM A23187,
and ETosis-released granule-rich subcellular structures were isolated by
centrifugation. Cell-free AO-stained granules were stimulated as described
previously in Neves et al with some modifications.14 After recovered structures
were washed and resuspended in 0.1% BSA RPMI medium, they were spread
on poly-L-lysine–coated slides and coverslipped. Stimuli (800 ng/mL CCL11
or 4 mM A23187) or control medium were introduced in 2-ml drops at the
edge of the coverslips, enabling stimuli to diffuse under the coverslip. The
responses of AO-stained granules were monitored by fluorescence microscopy
using the fluorescein isothiocyanate band fluorescence filter. To prevent AO
quenching during time-lapse microscopy, 3 neutral-density filters were used
during data acquisition. Fluorescence images were recorded for 100 seconds
(100 frames total) and acquired using a BX62 microscope (Olympus).
Figure 1. Transmission electron micrographs of human tissue eosinophils
from (A) allergic and (B) hypereosinophilic patients. Biopsy samples from the left
frontal sinus and the skin were obtained from a patient with allergic sinusitis and
a patient with hypereosinophilic syndrome (negative for FIP1-like 1/platelet-derived
growth factor-a mutation), respectively. In (Ai), a biopsy of the frontal sinus shows
infiltrated eosinophils. Note the disrupting nuclei (Nu) and extracellular free secretory
granules. (Aii) is the boxed area of (Ai) seen at higher magnification. In (B), both free
granules (Gr) and disrupted nuclei (Nu) are also observed in eosinophils from a skin
biopsy. Arrowheads in (Aii) and (B) indicate releasing decondensed chromatin.
Samples were fixed in a mixture of glutaraldehyde and PFO and prepared for
conventional TEM as in “Materials and methods.” Scale bars represent 1.2mm (Ai),
700 nm (Aii), 600 nm (B).
Statistical analysis
Results were expressed as mean 6 SD. The differences of groups were
evaluated using 2-tailed Student t test, with the level of statistical significance
taken as P , .05.
Results
Human tissue eosinophils in vivo exhibit cytolysis
accompanied by nuclear disruption and extracellular
granule release
Disruptions of nuclear and plasma membranes (PMs) in cytolytic
eosinophils have been observed in varied allergic and parasitic
diseases.28,29 To ascertain the ultrastructural characteristics of
eosinophil cytolysis in vivo, tissue-infiltrated eosinophils were
evaluated using TEM (Figure 1). Eosinophils within biopsies from
the sinus and the skin (from patients with allergic sinusitis and
hypereosinophilic syndrome, respectively) showed similar characteristics of cytolytic degranulation with disrupted PMs and nuclear
envelope membranes. In contrast to the nuclear chromatin condensation observed in apoptotic eosinophils,30 both distinct nuclear
heterochromatin decondensation and release of nuclear contents
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BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
EOSINOPHIL ETOSIS RELEASES GRANULES
2077
Figure 2. EETosis releases weblike chromatin
structures from lytic cells. (A) Eosinophils were
stimulated with the indicated stimuli in 0.1% BSA
medium for 120 minutes or in A23187 for 60 minutes
and fixed. EETosis was detected using extracellular
histone staining without permeabilization, as described
in “Materials and methods” and supplemental Figure 3.
Green indicates histones, and red indicates DNA.
Images were obtained using a BX62 microscope (203,
equipped with a Qimaging Rolera EM-C2 cooled digital
camera (Surrey, BC, Canada) in conjunction with
SlideBook 5.0 image analysis software (Intelligent
Imaging Innovations, Denver, CO), or equipped with
a Qimaging Retiga EXi cooled digital camera in
conjunction with iVision image analysis software).
Experiments were repeated with eosinophils from 3
independent donors with similar results. (B) Eosinophils were stimulated with 2 mM A23187 for 60 minutes
and processed for SEM. Large DNA nets were
released from lytic cells and often were associated
with originating cells (outlined by arrows, left). Weblike
DNA nets consisted of 25- to 35-nm diameter fibers
aggregated into larger fibers (right). Scale bars
represent 20 mm (A), 10 mm (B, left panel), and
500 nm (B, right panel).
into the extracellular space (Figure 1, arrowheads) were observed.
Moreover, eosinophil granules released in vivo were intact and
exhibited little evidence of loss of their granule contents (Figure 1).
Eosinophil ETosis (EETosis) releases nuclear DNA in response
to various stimuli
In neutrophils, ETosis releases nuclear contents as filamentous
chromatin structures, and neither apoptosis nor necrosis induces
DNA nets.19 To test the hypothesis that immobilized IgG or IgA23
and A23187,22 previously shown to elicit eosinophil cytolysis,
elicit ETosis, eosinophils were stained with antibodies against
histones and fixed-cell permeable PI. Using nonpermeabilized
cells, we were able to distinguish intact cells and extracellular
histones (supplemental Figure 2). Immobilized IgG and IgA,
like A23187, elicited the release of histone-positive DNA from
disrupted cells (Figure 2A). In addition, PAF, in combination with
IL-5 or GM-CSF, induced EETosis (Figure 2A, supplemental
Figure 2A). PAF itself was a less potent and somewhat donordependent EETosis inducer, and IL-5 or GM-CSF alone did not
induce EETosis (data not shown). PMA, an ETosis inducer for
neutrophils,16 also induced EETosis.
To ascertain differences among apoptosis, necrosis, and EETosis,
we identified conditions that led to ;20% dead cells (supplemental
Figure 3A) mediated by necrosis, apoptosis, or EETosis by
inducing cell death by brief heating, anti-Fas Abs,31 or A23187,
respectively. Morphological changes were evaluated using phasecontrast microscopy and concomitant staining for annexin V and PI
(supplemental Figure 3B). Anti-Fas–activated cells showed typical
apoptotic morphology (cytoplasmic and nuclear condensation,
budding) with bright annexin V–positive membranes and abundant
released apoptotic bodies. Heated cells showed bleb formation,
characteristic of necrotic cells.32 EETosis exhibited completely
different appearances, releasing filamentous DNA and cell-free
granules, with only weak staining for annexin V relative to Fasactivated cells. In heated or EETosis cells, surface annexin V
single-positive cells were rarely observed, indicating that the
phosphatidylserine (PS) redistribution typical during apoptosis was
lacking. Of note, extracellular filamentous DNA was never observed
in heated or Fas-activated cells. As assessed by the release of
lactate dehydrogenase and histone staining, A23187-stimulated
eosinophils exhibited cytolytic EETosis in an A23187 concentrationdependent manner (supplemental Figure 3C).
To study the process of nuclear shape changes during EETosis,
we loaded live eosinophils with Hoechst 33342 dye, which stained
nuclear DNA; cells were fixed at different time points. As shown in
supplemental Figure 4, supplemental Video 1, and supplemental
Video 2, initially the bilobular form of the eosinophil nucleus started
to lose its shape, and thereafter the nuclear envelope disintegrated
and DNA filled the cytoplasm. Finally, the PM ruptured, allowing
the extrusion of the DNA net (confirmed by DNase treatment; data
not shown). This process of nuclear shape change and dissolution
was entirely consistent with neutrophil ETosis.19 A23187-induced
EETosis developed more rapidly when compared with immobilized
IgG and PMA (supplemental Figure 4). High-resolution SEM
showed that DNA nets were associated with lytic cells (Figure 2B,
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2078
UEKI et al
BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
Figure 3. EETosis developing within 120 minutes in an NADPH oxidase-dependent manner. Eosinophils (in 0.1% BSA and CYTOX containing RPMI medium) incubated
with (A) 1 mg/mL immobilized IgG and (B) 2 mM A23187 with the indicated stimuli concentrations induced temporal EETosis cytolysis (extracellular CYTOX intensity)
attributable to DPI-inhibitable NADPH oxidase activity. For (A), data are means 6 SD, from 3 different donors, and for (B), data are means 6 SD from 4 different donors.
left, arrows). DNA nets formed weblike structures that consisted mostly
of fibers with diameters of 25 to 35 nm in conjunction with larger fibers
(Figure 2B, right). These results indicate that activated eosinophil cell
death is EETosis, which is distinct from necrosis and apoptosis.
EETosis is NADPH oxidase-dependent cell death
Neutrophil ETosis is dependent on ROS and inhibited by the
NADPH oxidase inhibitor DPI.18 To monitor the kinetics of cell
death and the effect of DPI, eosinophils were stimulated with
immobilized IgG and A23187, and cell death was measured every
15 minutes (Figure 3). CYTOX intensity progressively increased
45 minutes after immobilized IgG stimulation in a concentrationdependent manner, and DPI completely abolished the effect of
IgG- and A23187-stimulated eosinophils to undergo DPI-inhibitable
cell death. Similar inhibition was observed with DPI when stimulated
with other EETosis-inducing stimuli we tested (data not shown). In
line with the time course of nuclear shape change (supplemental
Figure 4), A23187-induced EETosis developed ;30 minutes
earlier when compared with IgG-induced EETosis (Figure 3B). As
reported for ETosis in mast cells and neutrophils,17,19 both fetal
bovine serum and BSA had concentration-dependent protective
effects on A23187-induced EETosis (supplemental Figure 5A), likely
based on their capacities to scavenge ROS. To further evaluate
whether the generation of extracellular ROS was critical for
EETosis, eosinophils were stimulated in the presence of the
cell-impermeable ROS scavenging enzyme, catalase. Catalase
inhibited A23187-induced eosinophil cytolysis and death (supplemental Figure 5B). RBCs, also known to scavenge ROS,24
likewise inhibited A23187-induced eosinophil cytolysis (supplemental Figure 5C).
EETosis releases intact granules and DNA nets predominantly
free of granule proteins
Neutrophils, diverse granule-derived proteins, many with antimicrobial activities, have been recognized to be bound as free proteins to
extracellular DNA nets.16,33 Likewise, for the elicited release of
mitochondrial DNA from eosinophils, eosinophil granule proteins
have been found bound to extracellular DNA nets.21 To assess
whether specific eosinophil granule proteins were released free or
were otherwise bound to eosinophil DNA nets, EETosis cells were
immunostained for MBP (Figure 4A). Notably, many regions of
extracellular DNA nets lacked linear immunostaining for free
MBP, and immunostaining was localized to smaller punctate foci
or cell-associated domains, suggestive that eosinophil granule proteins
might still be within granules. Immunostaining for EPO showed
similar results (data not shown). Indeed, TEM analyses of EETosis
revealed the presence of clusters of intact eosinophil granules
(Figure 4B, arrowheads), and some were associated with DNA nets.
In residual intact eosinophils, granules exhibited various degrees of
electron density, likely indicating secretion of some of their contents.
Ultrastructurally, cell-free eosinophil granules released from cytolytic cells retained their typical morphologies with intact granuledelimiting membranes and intact crystalloid cores and granule
matrices (Figure 4B, insert).
EETosis releases eosinophil granules both bound within
plasma membranes and later as free granules following plasma
membrane lysis
To study the EETosis processes that liberate intact eosinophil
granules extracellularly, we monitored eosinophils with timelapse, phase-contrast microscopy (Figure 5A, supplemental
Video 1, and supplemental Video2). Eosinophils undergoing
ETosis showed common morphological behaviors, starting with
the arrest of their chemokinetic migration and with enhanced,
rapid intracytoplasmic movements of their granules. As shown
in supplemental Figure 4, the eosinophil’s bilobed nucleus
progressively condensed into a single round shape. PM protrusions
developed, and at times 1 to 3 granules were released together
with enveloping PMs by budding off from the PM protrusions. In
addition, larger collections of granules were released from
eosinophils into PM-bound clusters. Thus, the budding release of
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BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
Figure 4. Intact granules and limited granule protein localization at extracellular DNA nets released through EETosis-mediated cell lysis. (A) Eosinophil
EETosis was induced using 1 mg/mL immobilized IgG (120 minutes) or 2 mM A23187
(60 minutes) and fixed and permeabilized, then stained for MBP (green) and DNA (PI,
red). Most released DNA nets did not contain free granule MBP protein and instead
showed punctate or cell-associated staining. Images were obtained with a BX62
Olympus upright microscope, 203 UPlanApo objective with a numerical aperture of
0.70, coupled to a Hamamatsu Orca-AG fire-wire cooled digital camera (Hamamatsu
Photonics, Hamamatsu, Japan; images were acquired using iVision software). Data are
representative of .3 experiments from independent donors with similar results. (B)
Cells were processed for TEM directly on slide surfaces. A23187-induced (2 mM, 60
minutes) EETosis eosinophils exhibit disrupted plasma membranes with clusters of
released secretory granules (arrows) and DNA nets (Nets). Extracellular free granules
(B, insert) show their typical morphologies with full crystalloid cores and matrices and
intact granule-delimiting membranes. Some granules were entrapped in DNA nets.
Scale bars represent 20 mm (A), 1 mm (B), and 700 nm (B, insert).
small numbers of granules and the extrusion of larger clusters of
granules resulted in the production of a variety of sizes of PMbound, granule-packed structures. The release of granule structures typically precedes by two to five minutes nuclear membrane
disintegration and nuclear chromatolysis. With the subsequent lysis
of the eosinophils and the disruption of their PMs, the remaining
intracellular granules were liberated.
In other cells, activated cells can induce vesicle-shedding without
affecting cell viability.34 To verify that the release of intact granules
from eosinophils was associated with EETosis, we counted the
absolute number of released subcellular structures (<4 mm with
high granularity) in the culture medium by using a flow cytometer
(supplemental Figure 1). Notably, the release of extracellular free
granule structures with A23187 was completely inhibited by DPI
(Figure 5B). Enhanced release of granule structures was associated
with increasing EETosis, as evidenced by concentration dependency
(supplemental Figure 6A) and time course (supplemental Figure 6B).
Moreover, extracellular structures were not liberated at 4°C
(supplemental Figure 6C), indicating an active cellular process. To
confirm the stability of released cell-free granules, we loaded
eosinophil granules with AO, a lysosomal granule dye, followed
by stimulation with A23187. After 1 hour, abundant cell-free, AOretained granules were found in culture medium (supplemental
Figure 7A). Consistent with Figure 5A, granules were found as
EOSINOPHIL ETOSIS RELEASES GRANULES
2079
single or grouped structures as well as PM-enveloped clusters
containing different numbers of granules. Released subcellular
structures were further subjected to flow cytometry; and in concert
with fluorescent microscopy, most subcellular granule structures
retained the granule dye (Figure 5C). Importantly, in contrast to
apoptotic bodies derived from anti-Fas Ab–treated cells (supplemental Figure 3B), EETosis-induced subcellular structures lacked
PS expression on their surfaces (Figure 5D).
To obtain granule-rich subcellular structures, any shed vesicles,
membranes, and residual contaminating cells were removed by
differential centrifugation. In concert with their identities as
eosinophil granules, as shown in Figure 5E, isolated structures
were positive for their contents of granule MBP. Moreover, there
was bimodal staining for PM-derived major histocompatibility
complex (MHC) class I protein, indicating that some, but not all,
granule-containing structures lacked evidence of an associated PM.
To target single granules (;0.5 to1 mm in diameter), we also gated
for small structures (<1 mm), and similar results were obtained
(Figure 5E, lower panels). Indeed, both PM-bound granules
(supplemental Figure 7B,C) and PM-free granules (data not
shown) were observed microscopically. To assess the intactness of
PM-bound granules, live eosinophils were loaded with LysoTracker
for labeling granules and CellTracker for cytoplasm and were
stimulated with A23187. Intact granules were found both with and
free of CellTracker-positive cytoplasm (supplemental Figure 7D).
Similarly staining structures were observed following immobilized
IgG- or PMA-induced EETosis (data not shown).
Collectively, we conclude that (1) EETosis releases eosinophil
granules, (2) the extracellular granules retain specific granule
proteins even after being released in culture medium, (3) some
released granules may singularly or in clusters be surrounded by
PS-nonexposed intact PM, and (4) some cytolytically released
individual granules were devoid of PM.
EETosis-released granules free of plasma membranes are
secretion competent and release ECP in response to CCL11
We previously demonstrated that human eosinophil granules, as
isolated free of PMs principally by subcellular fractionation,
express functional CCR3 chemokine receptors and secrete ECP in
response to the ligand CCL11 by using their intragranular signaling
and membranotubular network-based secretion systems.12,14 We
evaluated whether EETosis-released granules would possess
similar functional secretory properties. Following the stimulation
of eosinophils with 2 mM A23187 for 90 minutes, ;4% of the total
ECP was recovered within the released granule structures. Isolated
granules were stimulated with A23187 or CCL11 for 60 minutes,
and secreted ECP was measured. Interestingly, the released granules
did not respond to A23187. In contrast, the granules secreted
granule-derived ECP in response to CCL11 in a concentrationdependent manner (Figure 6A).
The activation of AO-loaded eosinophil granules releases,
into nonacidic media, monomeric AO that exhibits a spectral
shift to green, yielding transient green flashes from secretionresponding granules.14 To obtain direct evidence showing that
EETosis-released granules were responsive to CCL11, after
AO-loaded cells were treated with 2 mM A23187, isolated
granules were stimulated with CCL11. Some EETosis-released
free granules exhibited rapid activation in response to CCL11, as
evidenced by fluorescent flashes attributable to the release of AO
(Figure 6B, upper panels). In contrast, some granules, including
both free and those likely clustered within PMs, failed to exhibit
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2080
UEKI et al
BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
Figure 5. Characteristics of EETosis-induced intact eosinophil granule release. (A) Several cell-free granules were released by budding from PMs of eosinophils
undergoing ETosis. Eosinophils were studied with time-lapse, phase-contrast imaging every 3 seconds following 1 mg/mL immobilized IgG or 2 mM A23187 stimulation.
Images were captured from supplemental Video 1 and supplemental Video 2 (Nikon Eclipse TE300, 1003 PlanApo objective with a numerical aperture of 1.40, equipped with
a Qimaging Retiga EXi cooled digital camera in conjunction with iVision image analysis software). Eosinophils that initially showed a typical bilobed nucleus then demonstrated
that peripheral small PM protrusions developed. Arrowheads show releasing/released granules. After nuclear membrane disintegration to release DNA nets within the
cytoplasm, eosinophils gradually turned into a rounded shape, and granule movement slowed with deflected intracellular distribution of the granules. Several extracellular
granules remained attached to the culture plate. Data representative of .3 experiments of independent donors with similar results are shown. (B) EETosis-elicited production
of subcellular structures (#4 mm) was inhibited by DPI. Eosinophils were stimulated with A23187 (2 mM) in the presence of DPI in 0.1% BSA containing RPMI medium for
60 minutes. Subcellular structures in culture medium were counted as described in “Materials and methods” and supplemental Figure 2. Data (triplicate, mean 6 SD) are
representative of 3 experiments from independent donors with similar results. *P , .05. (C) Released cell-free structures retained the lysosomal dye AO. AO-loaded
eosinophils were stimulated with A23187 for 1 hour. The structures (#4 mm) in culture medium from different preparations were subjected for flow cytometry. The graph
shows the structures from AO-loaded cells (black line); fixed (2% PFO) and permeabilized (0.1% saponin) structures from AO-loaded cells (gray line); and structures from
cells without AO staining (filled histogram). Data are representative of 3 experiments from independent donors with similar results. (D) A23187-induced subcellular structures
were not apoptotic bodies. Subcellular structures (#4 mm) from A23187 and anti-Fas Ab–stimulated cells were stained with annexin V, followed by flow cytometric analysis.
Fas-stimulated apoptotic cells produce annexin V–positive subcellular structures (ie, apoptotic bodies, filled histogram), but A23187-stimulated ETosis cells did not (open
histogram). Apoptosis was induced, as described in supplemental Figure 4. Data are representative of 3 experiments from independent donors with similar results. (E) Isolated
granule-rich subcellular structures retained the granule protein MBP; some were variably associated with PM-derived MHC class I proteins, regardless of size. Culture
medium of A23187-stimulated cells was collected, and residual cells were removed by centrifugation (200 3 g for 10 minutes). DNA was removed by DNase treatment, and
buoyant vesicles and membranes were removed by further centrifugation (2500 3 g for 10 minutes). Isolated granule-rich structures were stained for MBP and MHC class I
(open histograms). Total structures (#4 mm) and small structures (#1 mm) were gated. Filled histograms show isotype-matched controls. Data are representative of
3 experiments from independent donors with similar results. Scale bars represent 5 mm (A). NS, not significant.
secretory AO flashes in response to CCL11 (Figure 6B, lower
panels). Stimulation of isolated granules with medium or
A23187 did not induce AO secretion (data not shown). These
results indicate that some granules extruded during A23187mediated EETosis retained their capacity for CCL11-elicited
secretory responses.
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BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
EOSINOPHIL ETOSIS RELEASES GRANULES
2081
Figure 6. Released granules were secretion competent in response to CCL11. (A) Culture medium of
A23187-stimulated cells (2 mM, 90 minutes) was
collected, and granule-rich subcellular structures were
isolated. The cell-free structures were stimulated with
2 mM A23187 or the indicated concentrations of
CCL11 for 60 minutes. Secreted ECP levels, assessed
by enzyme-linked immunosorbent assay, were normalized with spontaneous ECP release (100%), and
data are expressed as means 6 SD, from 3 different
donors. *P , .05 vs nonstimulated controls. The
spontaneous secretion levels were 10.0 6 4.4% of
the total ECP in the structures. (B) AO-loaded
eosinophils were stimulated with 2 mM A23187 to
induce EETosis, followed by the isolation of subcellular
granule structures. Among the 80 single granules or
groups of granules, following CCL11 stimulation,
significant responses with intense transient fluorescent
flashes indicative of the release of monomeric AO were
observed from 14 nonenveloped granules (17.5%). The
secretory response was not observed by likely PMbound clusters of granules (lower panels). Images
were obtained with a Hamamatsu Orca-AG fire-wire
cooled digital camera coupled to a BX62 Olympus
microscope using a 603 PlanApo objective with
a numerical aperture of 1.42. Fluorescence intensity
was analyzed by iVision software and pseudocolored
with red to represent the greatest intensity as indicated
by the scale color. Experiments were repeated with
eosinophils from 8 independent donors. The scale bar
represents 3 mm (B).
Discussion
Eosinophils as granulocytes contain distinct cytoplasmic granules,
notable by their ultrastructurally unique crystalline cores and by their
content of proteins, including specific cationic proteins as well as
preformed cytokines. In diverse human diseases in which eosinophils
may participate, there have been recognitions over many years that
cell-free eosinophil granules, released from eosinophils, are found in
tissues or secretions.4 These findings include ultrastructural studies
recognizing intact free eosinophil granules;8,10 immunofluorescent
demonstrations of eosinophil cationic proteins in extracellular granule
foci; and, especially in asthma, clusters of extracellular eosinophil
granules.7 Disruptions of PMs and nuclear membranes along with
chromatolysis have been observed in eosinophils associated with
allergic and anthelmintic responses.28,29 Our studies aimed to examine
the mechanisms and consequences by which human eosinophils could
cytolytically and extracellularly release their intact granules.
Ultrastructural examination of eosinophils in the sinus tissue of
a patient with allergic sinusitis and the skin of a patient with
a hypereosinophilic syndrome, respectively, (Figure 1) revealed the
liberation of free extracellular granules as well as nuclear changes
(disruption of nuclear membranes and decondensation of nuclear
chromatin) that were distinct from apoptosis or necrosis. Apoptotic
eosinophils exhibit condensed compaction of chromatin against the
nuclear envelope and shrinkage and budding of the whole cell to
form membrane-bound, PS-exposed apoptotic bodies. Neither anti-
Fas–elicited eosinophil apoptosis nor heat-elicited eosinophil
necrosis yielded extracellular release of nuclear DNA or cell-free
eosinophil granules (supplemental Figure 3). To investigate
mechanisms of eosinophil cytolysis, we used immobilized immunoglobulins (IgG, IgA), PAF with either IL-5 or GM-CSF, and
nonphysiologic stimuli, calcium ionophore A23187 and PMA.
Each of these stimuli elicited eosinophil lytic death (EETosis) that
was distinct from apoptosis or necrosis. EETosis occurred progressively over 30 to 120 minutes and was dependent on the
generation of ROS, being inhibitable by DPI. These stimuli are
known to induce/prime ROS production by eosinophils.35-37
Prior studies demonstrated that cytokine-primed human eosinophils, in response to certain agonists such as lipopolysaccharide,
C5a, and CCL11, can rapidly (within seconds) and noncytolytically
release not nuclear, but mitochondrial DNA to form extracellular
DNA nets.21 Our findings identify another means by which
eosinophils can form DNA nets, more akin temporally and
mechanistically to ETosis in neutrophils16 and mast cells.17 As in
neutrophils,18 eosinophil ETosis involves a more delayed, cytolytic
release of nuclear DNA to form extracellular DNA nets. The
temporal sequence of changes within eosinophils undergoing cytolysis
is presented in Figure 7. Following stimuli-elicited initiation of
eosinophil cytolysis, the typically bilobed nuclei of eosinophils lost
their lobulation to coalesce into single nuclear structures. During
this time, some eosinophil granules were released extracellularly as
PM-enveloped structures. Thereafter, nuclei disintegrated intracellularly
to form intracellular DNA nets. Subsequently, the eosinophils’ PMs
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2082
BLOOD, 14 MARCH 2013 x VOLUME 121, NUMBER 11
UEKI et al
Figure 7. EETosis induces the release of intact cell-free granules through budding and cell lysis. The graphic shows the temporal course of the morphologic changes
exhibited by eosinophils undergoing EETosis. Stimuli-elicited NADPH oxidase activation leads to the common process of eosinophil cytolysis, followed by the loss of the
typically bilobed nuclei into a single round nuclei. During this time, some eosinophil granules were released extracellularly as PM-enveloped structures. Thereafter, nuclei
disintegrated to form intracellular DNA nets. Subsequently, the eosinophils’ PMs rupture, releasing both a weblike chromatin structure and free eosinophil granules. EETosismediated cytolysis leads to the extracellular liberation of intact granules, some of which retained their capacity for secretory responses.
rupture, releasing both nuclear DNA-derived (histone-positive) nets
and free eosinophil granules. Eosinophil ETosis-mediated cytolysis
leads to the extracellular liberation of intact eosinophil granules.
In neutrophils undergoing ETosis, granule-contained proteins
can associate intracellularly with nuclear DNA even before PMs
rupture, and granule proteins are bound to extracellular DNA
nets.19,38 Intact neutrophil granules are not recognized as bound to
extracellular DNA nets. In contrast, in eosinophils undergoing
ETosis, granules are released as intact structures. Our findings
indicated that eosinophil granule proteins were not predominantly
free and bound to DNA nets, but rather they remained principally
within granules that themselves could bind to DNA nets. Moreover,
we showed that some released cell-free eosinophil granules exhibited
secretion competency in response the eosinophil-active chemokine
CCL11 and secreted ECP and granule AO (Figure 6). Other granules,
including clusters of likely PM-enveloped granules, did not exhibit
individual CCL11-elicited granule secretory responses, at least in shortterm in vitro assays. Whether the clusters of PM-enveloped granules
might exhibit secretory competence in vivo requires further study.
In conclusion, our results reveal a process by which eosinophils
undergo cytolysis to liberate intact cell-free granules, as is well
recognized in tissues and secretions in association with diverse
eosinophil-associated diseases. The process of ETosis in eosinophils is akin to that in neutrophils18 but notably differs in that intact
eosinophil granules are released extracellularly and still contain the
bulk of their protein contents. At least some EETosis-released
granules remain secretion competent. Thus, EETosis results in the
generation of histone-bearing nuclear DNA extracellular nets and
cell-free granules, both of which may exert biological activities for
eosinophils postmortem.
Acknowledgments
The authors are grateful to Kristen Young and Jason Xenakis for
their outstanding technical assistance and Dr. Praveen Akuthota
and Professor Hélio Chiarini-Garcia for helpful discussions.
This work was supported in part by Uehara Memorial Foundation
(S.U.); Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológicoand Fundação de Amparo à Pesquisa do Estado de
Minas Gerais (R.C.N.M.); National institute of Health: National
Heart, Lung and Blood Institute R01-HL096795 (I.G.); R01HL095699 (L.A.S.); and grants from the National Institutes of
Health: National Institute of Allergy and Infectious Diseases (R37AI020241 and R01-AI051645, P.F.W.).
Authorship
Contribution: S.U. designed and performed experiments and wrote
the manuscript. R.C.N.M. and A.M.D. performed electron microscopic experiments and wrote the manuscript. I.G. provided
technical assistance and edited the manuscript. L.A.S. contributed
scientific advice and discussions. P.F.W. supervised the research
and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Peter F. Weller, MD, 330 Brookline Ave, CLS943, Boston, MA 02215; e-mail: [email protected].
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2013 121: 2074-2083
doi:10.1182/blood-2012-05-432088 originally published
online January 9, 2013
Eosinophil extracellular DNA trap cell death mediates lytic release of
free secretion-competent eosinophil granules in humans
Shigeharu Ueki, Rossana C. N. Melo, Ionita Ghiran, Lisa A. Spencer, Ann M. Dvorak and Peter F.
Weller
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