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Astrocyte-Derived ATP Induces Vesicle Shedding and IL-1?
Release from Microglia1
Fabio Bianco,*†Elena Pravettoni,*†Alessio Colombo,*†Ursula Schenk,*†Thomas Mo ¨ller,‡
Michela Matteoli,*†and Claudia Verderio2*†
ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By acting on different
purinergic receptors 2, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The
activation of purinergic receptors 2 in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury
or by ATP released from astrocytes, in the absence of cell damage. By the use of a biochemical approach integrated with video
microscopy experiments, we investigated the functional consequences triggered in microglia by ATP released from mechanically
stimulated astrocytes, in mixed glial cocultures. Astrocyte-derived ATP induced in nearby microglia the formation and the
shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X7R antagonists.
Isolation of shed vesicles, followed by IL-1? evaluation by a specific ELISA revealed the presence of the cytokine inside the
vesicular organelles and its subsequent efflux into the extracellular medium. IL-1? efflux from shed vesicles was enhanced by ATP
stimulation and inhibited by pretreatment with the P2X7antagonist oxidized ATP, thus indicating a crucial involvement of the
pore-forming P2X7R in the release of the cytokine. Our data identify astrocyte-derived ATP as the endogenous factor responsible
for microvesicle shedding in microglia and reveal the mechanisms by which astrocyte-derived ATP triggers IL-1? release from
these cells. The Journal of Immunology, 2005, 174: 7268–7277.
ported to promote cytokine release, ATP, acting at high concen-
trations on the ionotropic P2X7purinergic receptors, is one of the
most powerful stimuli for the processing and release of IL-1?, the
key initiator of acute inflammatory response (1). The physiological
source of ATP needed to trigger cytokine release is still debated.
Large amounts of the purine, accumulated extracellularly at the
site of lesion, can directly activate P2X7-mediated cytokine release
(2). In contrast, cytokine secretion at sites far away from damaged
cells could proceed through the spreading of ATP-mediated cal-
cium signals among astrocytes (3, 4). In a previous study we re-
ported that ATP released by astrocytes during calcium wave prop-
agation can activate P2X7Rs on microglial cells (3). Therefore, in
principle, astrocyte-released ATP could be relevant for the control
of cytokine secretion from microglia.
The signaling mechanism by which P2X7activation regulates
IL-1? release in microglia is not completely defined yet, although
it involves activation of caspase-1, which proteolytically cleaves
the biological inactive precursor of the cytokine (pro-IL-1?), gen-
icroglial cells play a major role in the inflammation
processes which take place in the nervous tissue by
secretion of specific cytokines. Among agents re-
erating the mature, active form (2). Caspase-1 activation is pro-
moted both by calcium release from intracellular stores (5) and by
K?efflux through P2X7Rs, which ultimately triggers phospho-
lipase A2activation (6, 7). Release of IL-1? also requires the up-
regulation of pro-IL-1? gene expression, which is triggered by cell
exposure to bacterial LPS (8). However the molecular mechanism
by which bioactive IL-1? is released is still debated. IL-1? lacks
a conventional secretory sequence and is known not to be released
through the classical endoplasmic reticulum (ER)3-Golgi pathway
(9). Alternative pathways of secretion have been proposed in dif-
ferent cell types, including simple cell lysis (10), fusion of endoly-
sosome-related vesicles (9), bleb formation, and shedding of mem-
brane vesicles (11). The latter mechanism, which commonly
occurs in hemopoietic and immune cells (12), represents a process
whereby signal molecules are released via membrane-enclosed
particles into the microenvironment. Based on the finding that
IL-1? is detected inside shed vesicles shortly after P2X7activation
and becomes detectable in the extracellular medium only at later
time, it has been suggested that formation and shedding of mem-
brane vesicles could represent a pathway for the release of the
cytokine (11). More recently it has been suggested in murine mac-
rophages that IL-1? secretion can be dissociated from P2X7-de-
pendent formation of blebs at the plasma membrane, although a
role of ATP-induced shed microvesicle in mediating IL-1? secre-
tion has not been excluded (13). Because microglia has been in-
dicated as the major source of IL-1? in the brain, it is of crucial
importance to define the molecular mechanisms which mediate the
cytokine release from these cells. Aims of the present study were
to investigate 1) whether IL-1? is released from microglia simi-
larly to monocytes, through the shedding of membrane vesicles,
and 2) whether astrocyte-derived ATP can control the cytokine
*Consiglio Nazionale delle Ricerche-Institute of Neuroscience, Cellular and Molec-
ular Pharmacology and Department of Medical Pharmacology, University of Milan
and†Center of Excellence on Neurodegenerative Diseases, Milan, Italy; and‡Depart-
ment Of Neurology, University of Washington, Seattle, WA 98195
Received for publication August 2, 2004. Accepted for publication March 15, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was supported by Fondo per gli Investimenti della Ricerca di Base
Grants RBAUO19 ZEN and RBNE01ERXR_007, European Community Grant QLG
3-2000-01343, Ministero dell'Istruzione dell'Universita ` e della Ricerca-Programmi di
Ricerca Scientifica di Rilevante Interesse Nazionale 2002, and Fondazione Italiana
Sclerosi Multipla Grant 2003/R/35.
2Address correspondence and reprint requests to Dr. Claudia Verderio, Consiglio
Nazionale delle Ricerche-Institute of Neuroscience, Via Vanvitelli 32, 20129 Milano,
Italy. E-mail address: firstname.lastname@example.com
3Abbreviations used in this paper: ER, endoplasmic reticulum; GFAP, glial fibrillar
acidic protein; LDH, lactate dehydrogenase; oATP, oxidized ATP; PS, phosphatidyl-
serine; P2Rs, purinergic receptor 2; FLICA, fluorochrome inhibitor of caspases;
SNAP-23, synaptosomal-associated protein of 23 kDa.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
Page 2
production from microglial cells. Our results indicate that ATP
released from astrocytes modulates the cytokine release, which
proceeds in microglia through the shedding of plasma membrane
vesicles, containing packages of IL-1?. Our data also suggest a
role for the pore-forming P2X7Rs in the processing and release of
the cytokine from shed vesicles into the extracellular medium.
Materials and Methods
Primary cocultures of astrocytes and microglial cells
Primary mixed glial cultures from embryonic rat pups (embryonic days
18–19) were obtained as previously described (14). The dissociated cells
were plated onto glass coverslips at a density of 0.5 ? 106cells/ml, and
grown in MEM (Invitrogen Life Technologies) supplemented with 20%
FCS (Euroclone) and 100 IU/ml penicillin, 10 mg/ml streptomycin, and 5.5
g/L glucose (glial medium). Purified microglial cultures were harvested by
shaking 3-wk-old mixed glial cultures. Detached microglia was seeded on
glass coverslips and cultured in the same medium. To obtain a pure astro-
cyte monolayer, mixed cultures were maintained in a serum-free medium
for 2–3 days, and microglia cells were removed by vigorously shaking the
cultures. Microglial cells present in mixed glial cultures were distinguished
from astrocytes by morphological criteria and retrospective immunostain-
ing for CSF-1R and glial fibrillar acidic protein (GFAP), respectively. The
absence of microglia and astrocyte contamination in primary astrocytes and
purified microglia cultures was assessed by Western blotting for the mi-
croglial marker CSF-1R (Molecular Probes) and the astrocytic marker
GFAP (Sigma-Aldrich).
Microglial N9 cell line
The N9 murine microglial cell line was generated by infecting embryonic
brain cultures with the 3RV retrovirus carrying an activated v-myc onco-
gene (15). N9 cells were maintained in IMDM (Invitrogen Life Technol-
ogies) supplemented with 100 IU/ml penicillin, 10 mg/ml streptomycin, 2
mM L-glutamine, and 50 nM 2-ME (Invitrogen Life Technologies) at 37°C
and 5% CO2. To obtain cocultures of N9 cells and astrocytes, N9 cells were
plated at a density of 1.2 ? 105cells/ml on top of astrocytes previously
cultured for 7–10 days on glass coverslips or 60-mm petri dishes.
N9 cells stably expressing GFP were obtained as described in Ref. 16.
Western blot analysis of IL-1? processing and release
Approximately 4,500,000 cells were primed for 6 h with 100 ng/ml LPS
and stimulated with 1 mM ATP in 1 ml of a Krebs-Ringer solution (KRH)
for 30 min (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4,
2 mM CaCl2, 6 mM D-glucose, and 25 mM HEPES/NaOH, pH 7.4). The
conditioned saline was then collected and concentrated by 2,340 ? g cen-
trifugation at 4°C on centrifugal filters with a 10-kDa cut-off (Millipore),
for ?6 h to a final volume of 150 ?l, run on a 12% polyacrylamide gel and
blotted onto nitrocellulose filters (Millipore). IL-1? was detected with goat
anti-mouse Abs (R&D Systems) followed by secondary HRP-conjugated
anti-goat Abs (Jackson ImmunoResearch Laboratories) and revealed using
an ECL system (Supersignal from Pierce) following the manufacturer's
protocol. Anti- IL-1? Ab recognizes both the 36-kDa precursor (pro-IL-
1?) and the 17-kDa active form of the cytokine.
ELISA analysis of IL-1?
A mouse IL-1? ELISA kit, which does not discriminate between pro- and
mature cytokine forms (Pierce Endogen), was used to quantify the presence
of IL-1? in the supernatant of N9 cells. Approximately 1,000,000 cells
were preactivated with 100 ng/ml LPS for 6 h and stimulated with 1 mM
ATP for different time periods (5, 10, 20, and 30 min) in 1 ml of culture
medium. Conditioned media were then collected, and the assay was com-
pleted following the manufacturer's protocol, adding, in each well, 50 ?l
of collected medium to 50 ?l of biotinylated Ab. Release of IL-1? from
shed vesicles was directly evaluated by assaying the medium (50 ?l) in
which vesicles were maintained for different times (10, 20, and 30 min) at
37°C after isolation. IL-1? content inside shed vesicle was determined
after detergent permeabilization with 2% Triton X-100. Sample absorbance
was measured with a spectrophotometric system (1420 Multilabel Counter
Victor 2; Wallac) at 450 nm at 10 Hz. The actual IL-1? concentration was
estimated on the basis of a standard curve at known concentration of IL-1?,
both in the presence and in the absence of 2% Triton X-100.
Annexin VFITCstaining and microscope counting
N9 cells were either directly plated at a density of 1.2 ? 105cells/ml on
glass coverslips or plated on top of astrocytes previously cultured for 7–10
days. Sixteen hours after plating, coverslips were either maintained in static
condition or exposed to mechanical stimulation for 1 min in KRH with or
without apyrase (30 U/ml). Mechanical stimulation was conducted by
shaking the petri dishes onto an orbital shaker at a rate of 160 per min (17)
(Orbital Shaker SO3; Stuart Scientific). Stimulations were repeated two to
three times, with a 5-min pause in between stimuli. Alternatively, the me-
chanical stimulation was conducted by the gentle mechanical contact of a
patch pipette with the surface of a single astrocyte either in primary astro-
cyte-microglia cocultures or in astrocyte-N9 cocultures. Mechanically
stimulated cultures were incubated with Annexin VFITC(4.2 ?g/ml) for 3
min and the number of annexin V-positive N9 cells in living cultures was
determined by counting positive cells in at least 50 microscope fields (Ax-
iovert 100; Zeiss). In a set of experiments, cultures were fixed after An-
nexin VFITCstaining with 4% paraformaldehyde in 0.12 M phosphate
buffer containing 0.12 M sucrose, and images were acquired using a Bio-
Rad MCR-1024 confocal microscope equipped with LaserSharp 3.2 soft-
ware (Bio-Rad Hercules).
Vesicle isolation
Approximately 4,500,000 cells, primed with LPS, were exposed to 1–3
mM ATP for 10 min under gentle rotation in 1 ml of KRH. The superna-
tant, containing shed vesicles, was withdrawn and incubated for 10 min at
4°C under gentle periodic rotation with streptavidin beads (Sigma-
Aldrich), precoated with biotinylated annexin V (Sigma-Aldrich). Shed
vesicles bound to annexin-coated beads were then separated from the su-
pernatant by gravity sedimentation at 4°C, treated with a reducing sample
buffer and run on a 12% polyacrylamide gel. Alternatively, vesicles bound
to beads were permeabilized with Triton X-100 (2%) in 100 ?l of culture
medium and probed for IL-1? content by ELISA (Pierce Endogen).
Caspase-1 activity in isolated vesicles was quantified by a carboxyfluores-
cein-fluorochrome inhibitor of caspases (FLICA) assay kit (B-Bridge In-
ternational), based on the use of the caspase-1 fluorescent inhibitor FAM-
YVAD-FMK. The FAM-YVAD-FMK FLICA probe enters the cells and
covalently binds to a reactive cysteine residue, thereby inhibiting further
enzymatic activity. The assay was conducted following the manufacturer's
Analysis of vesicle shedding
To visualize shed vesicles present in the supernatant of ATP-stimulated
cells, ?1,000,000 N9 cells were labeled with the fluorescent styryl dye
FM1–43 (2 ?M) for 2 min, extensively washed and exposed for 20 min to
1 mM ATP with or without 500 ?M EDTA or 100 ?M oxidized ATP
(oATP) in 1 ml of KRH. Supernatant was then deposed on glass coverslips
precoated with poly-lysine (Sigma-Aldrich). Images of sedimented vesicles
were acquired 20 min after supernatant addition and analyzed using Meta-
morph Imaging Series 6.1 software (Universal Imaging). Alternatively, to
quantify the amount of shed vesicles present in supernatants under different
experimental conditions, total green fluorescence of collected supernatants
was assayed at 485/535 nm and 10 Hz with a spectrophotometric system
(1420 Multilabel Counter Victor 2; Wallac). A similar spectrophotometric
analysis was performed on supernatants containing GFP vesicles, shed
from N9 cells expressing cytosolic GFP.
Analysis of vesicle integrity
To investigate whether shed vesicles disrupt with time, thus allowing the
release of pro- and active-IL-1? into the extracellular medium, we moni-
tored the presence of GFP in vesicles shed from GFP-mutant N9 cells, and
in vesicle-depleted medium 10, 20, and 30 min after vesicle isolation. GFP
is a 40-kDa cytosolic protein that cannot be released extracellularly
through the P2X7pore, given that the channel-pore is permeable to mol-
ecules up to ?1 kDa. Total green fluorescence in vesicle and vesicle-free
fractions was assayed at 485/535 nm as described above.
Video microscopy
Cells were mounted on an inox cell chamber (Elettrofor) and observed at
37°C, 5% CO2, and 55–60% relative humidity using an inverted Zeiss
Axiovert 200M microscope equipped with a chamber Incubator S (Zeiss)
controlled by a CTI Controller 3700 (Zeiss). Images were acquired with a
digital CCD camera system Micromax 512 BFT (Princeton Instruments)
and analyzed using Metamorph Imaging Series 6.1 software. Cells were
considered as blebbing when at least three blebs per cell were clearly
Cytotoxicity assay
Aliquots of culture medium were collected at different time points (10, 20,
and 30 min) for the evaluation of lactate dehydrogenase (LDH) activity by
7269The Journal of Immunology
Page 3
a cytotoxicity assay (Promega). LDH levels in culture supernatant were
measured via a coupled enzymatic assay, which resulted in the formation
of a colored product, the absorbance of which was recorded at 490 nm and
10 Hz (1420 Multilabel Counter Victor 2; Wallac). Caspase-3 activity in
N9 cells exposed to 1 mM ATP for different time periods was evaluated
using a carboxyfluorescein FLICA assay (B-Bridge International). Briefly,
cells were detached from culture substrate using 0.05% trypsin, 0.02%
EDTA in PBS (Euroclone) and then exposed to ATP in KRH solution.
Sample absorbance was measured with a spectrophotometric system (1420
Multilabel Counter Victor 2; Wallac) at 450 nm at 10 Hz.
Rabbit Abs against synaptosomal-associated protein of 23 kDa (SNAP-23)
were kindly provided by Dr. T. Gally (Institut National de la Sante ´ et de la
Recherche Me ´dicale, Paris, France). Rabbit Abs against P2X7was obtained
from Alomone Labs. Anti-GFAP Abs, Annexin VFITC, biotinylated annexin
V, oATP, apyrase, LPS, ATP were obtained from Sigma-Aldrich. Abs anti-
CSF-1R were purchased from Santa Cruz Biotechnology. Rabbit Abs against
ribophorin were kindly provided by Dr. G. Kreibich (New York University,
New York, NY). FM1–43 was from Molecular Probes. The polyclonal Abs
against human cathepsin D were generously provided by Dr. C. Isidoro
(University of Novara, Novara, Italy). Rabbit Abs against LAMP-1 (lgp120)
were a kind gift of Dr. I. Mellman (Yale University, New Haven, CT). Rabbit
Abs against rab-7 were a kind gift of Dr. R. Jahn (Max Planck Institute,
Go ¨ttingen, Germany).
Data analysis
The data are presented as means ? SE. Statistical significance was eval-
uated by the Student's test or one-way ANOVA. Differences were consid-
ered significant if p ? 0.05 and are indicated by an asterisk in all figures,
whereas those at p ? 0.01 are indicated by double asterisks.
Microglia, but not hippocampal astrocytes, release IL-1? upon
ATP exposure
Several evidences have been reported demonstrating that large
microglia, the cell type which serves as a major source of the cytokine
in the CNS upon injury or inflammation (18). Less clear is the
scenario in astrocytes, given that most of the evidence for IL-1?
release from these cells derives from studies using mixed primary
cultures, containing both astrocytes and microglia (19). To verify, in
our experimental model, the relative contribution of microglia and
pure hippocampal astrocytes were stimulated for 30 min with 1 mM
ATP after 6 h of LPS priming. Although both cell types express
P2X7Rs and respond with intracellular calcium elevations to 100 ?M
BzATP (20, 21), an agonist rather selective for this receptor subtype,
the mature form of IL-1? (17 kDa) was detected, by Western blot
analysis, in the supernatant of N9 cells but not in the supernatant of
primary astrocytes (Fig. 1A). In contrast, the immature form of the
cytokine (36 kDa) was clearly detectable in both astrocyte and N9
homogenates (Fig. 1A). In the absence of ATP treatment, the mature
form of IL-1? fails to be detected in N9 supernatant (data not shown).
These data indicated that, at least in our experimental model, hip-
pocampal astrocytes are not able to process and release IL-1? upon
purinergic receptor 2 (P2R) activation. The ELISA analysis of IL-1?
and supernatants of N9 cells and primary hippocampal astrocytes, primed 6 h with 100 ng/ml LPS and incubated with 1 mM ATP for 30 min. One
representative experiment of three is shown. B, IL-1? detection by ELISA in the supernatants collected from N9 cells, hippocampal microglia, and
hippocampal astrocytes primed 6 h with 100 ng/ml LPS and incubated with 1 mM ATP for 30 min. Values are presented as mean ? SE picograms per
milliliter obtained from three independent experiments and are normalized to protein concentration of cell extracts (51.0 ? 5.56 pg/ml/mg proteins in N9
supernatants; 68.0 ? 11.79 pg/ml/mg proteins in medium collected from purified microglia; 3.5 ? 1.35 pg/ml/mg proteins in medium collected from
purified astrocytes, one-way ANOVA, post hoc Tukey's method, p ? 0.01). C, Representative Western blotting of primary astrocytes for the microglial
marker CSF-1R and the astrocytic marker GFAP, revealing the absence of microglia contamination in astrocytic cultures. The ER marker ribophorin was
used as loading control. D, Kinetics of IL-1? secretion, as measured by ELISA. LPS-activated N9 cells were exposed to 1 mM ATP for different times
in normal cell culture medium or in medium supplemented with 500 ?M EDTA. Chelation of calcium ions in the extracellular medium almost completely
inhibits the release of the cytokine. Values are presented as mean ? SE obtained from three independent experiments. Statistical significance was evaluated
by Student's test (p ? 0.015 at 20 min; p ? 0.005 at 30 min, n ? 3)
ATP induces IL-1? release from microglial cells but not from hippocampal astrocytes. A, Western blot analysis for IL-1? on cell lysates
Page 4
levels in the cell supernatants of N9 cells, primary purified microglia
and primary hippocampal astrocytes further confirmed that ATP
stimulation induces IL-1? release essentially from microglia (Fig.
1B). In the absence of ATP stimulation, IL-1? levels were below the
assay sensitivity. Western blot analysis for CSF-1R and GFAP,
specific markers of microglia and astrocytes (3) ruled out the possible
contamination of astrocytic cultures with primary microglia (Fig. 1C).
The kinetic analysis of IL-1? release from microglial N9 cells indi-
cated that significant levels of the cytokine are present extracellularly
15–20 min after ATP stimulation. In line with previous results,
removal of calcium ions from the extracellular medium by addition of
500 ?M EDTA strongly prevented the release of the cytokine, thus
suggesting a calcium-dependent mechanism of secretion (22) (Fig. 1D).
Exposure to exogenous ATP induces vesicle formation in
microglial cells
To investigate whether ATP stimulation induces vesicle formation
in microglia, video microscopy experiments were conducted upon
1 mM ATP treatment both in primary hippocampal microglia and
in N9 cells. Bright field time-lapse microscopy revealed the for-
mation of plasma membrane structures (blebs) continuously ex-
truding and retracting from the cell membrane (Fig. 2A), ?180 s
after 1 mM ATP addition. As already described for platelets and
monocytes (11, 23), in microglia ATP-induced blebs have phos-
phatidylserine (PS) exposed on the outer leaflet of the plasma
membrane and bind annexin V, a high affinity ligand for the phos-
pholipid. Annexin V-positive vesicle formation was confined to
discrete regions of the cell surface (Fig. 2B) or more evenly dis-
tributed to the entire surface of the cells (see an extreme case in
Fig. 2C). Vesicle formation proceeded even in the absence of ex-
tracellular calcium ions, although with a latency of ?60 s as com-
pared with controls (Fig. 2D); calcium chelation by EDTA did not
prevent annexin V binding to the forming vesicles (number of
annexin-positive cells per field increases by 4.44 ? 0.18-fold upon
ATP treatment, by 2.9 ? 0.086 upon ATP treatment in the pres-
ence of EDTA). Pretreatment of N9 cells with 100 ?M oATP or
100 nM Brilliant Blue G, two antagonists rather selective for the
P2X7Rs (24, 25), quite completely inhibited vesicle formation,
thus suggesting the requirement of P2X7R activation for ATP-
induced bleb formation (Fig. 2D). ATP-induced vesicle formation
also occurred in microglial cells not primed with LPS (data not
After a brief ATP exposure (4–5 min) both N9 and primary
microglia were able to recover the cell shape, indicating that bleb
formation is not necessarily linked to an apoptotic fate of the cell.
Accordingly, a negligible increase of LDH concentration was de-
tected in medium collected from N9 cells exposed to ATP up to 20
min (increase of LDH concentration: 4.83 ? 0.89%). Furthermore,
in line with previous results indicating that blebbing induced by
ATP does not require activity of the apoptotic marker caspase-3
(13), no significant activation of caspase-3 was observed in N9
cells exposed to ATP up to 30 min. Caspase-3 activity became
detectable in N9 cells only after 60 min of chronic ATP treatment
(Fig. 2E), thus excluding that ATP-induced bleb formation, which
times before and after exposure to 1 mM ATP. Images reveal that ATP induces the formation of vesicular structures continuously extruding and retracting
from the cell membrane. B and C, Differential interference contrast and fluorescence images (490 nm) of N9 cells incubated with FITC-conjugated annexin
V after a 5-min treatment with 1 mM ATP. Formation of annexin V-positive blebs can be confined to a discrete region of the plasma membrane, suggesting
a focalized process (B) or localized to the entire cell surface (an extreme case is shown in C). D, The graph shows the percentage of N9 cells displaying
blebs at different time points after continuous ATP stimulation, based on the analysis of time lapse images. Pretreatment of N9 cells with P2X7antagonists
oATP and Brilliant Blue G significantly inhibits bleb formation. Removal of calcium ions from the extracellular medium slows down bleb appearance by
?2 min. (values are compared with ATP-stimulated cells. Statistical significance was evaluated by one-way ANOVA, post hoc Tukey's method, n ? 3).
E, Caspase-3 activity assay on N9 cells exposed to 1 mM ATP for different time periods. A significant level of caspase-3 activity is detected after 60 min
of chronic exposure to ATP. No significant increase in the enzyme activity was observed in cells exposed to ATP for up to 30 min (statistical significance
was evaluated by one-way ANOVA, post hoc Dunnett's method, n ? 3).
ATP causes formation of membrane blebs. A, Series of bright field images of two primary hippocampal microglial cells taken at different
7271 The Journal of Immunology
Page 5
takes place within 4–5 min from ATP addition, is necessarily
linked to apoptotic cell death.
To better follow the dynamics of bleb formation in ATP-stim-
ulated microglia we conducted fluorescence video microscopy on
cells incubated with the fluorescent styryl dye FM1–43, which
labels lipid bilayers. By this approach, we clearly observed not
only the formation but also the shedding from the plasma mem-
brane of vesicular structures of variable size (Fig. 3, A and B) (see
also supplemental movie).4To visualize vesicles shed into the ex-
tracellular medium, the supernatant collected from FM1–43-la-
beled cells exposed to ATP was deposed on glass coverslips. As
shown in Fig. 3C collected supernatants contained a heterogeneous
population of FM1–43-labeled vesicles (mean diameter value 980
nm, n ? 780) (Fig. 3D). Pretreatment with 100 ?M oATP or
removal of calcium ions from the extracellular saline significantly
reduced the number of shed vesicles in the supernatants (Fig. 3C).
Accordingly the amount of fluorescence in N9 collected superna-
tants, due to the presence of FM1–43-labeled vesicles, was sig-
nificantly reduced when cells were exposed to ATP in the absence
of calcium ions and when cells were pretreated with oATP (Fig.
3E). Similar experiments were conducted on a clone of N9 cells,
which stably express high levels of GFP in the cytosol. GFP-ex-
pressing N9 cells shed GFP-vesicles upon ATP stimulation. An
example of GFP-vesicles are shown in Fig. 3F. Quantitative anal-
ysis of total green fluorescence of supernatants containing GFP-
vesicles confirmed that vesicle shedding is markedly inhibited by
calcium chelation (fluorescence intensity, arbitrary units: 18.83 ?
0.4 controls, 105.83 ? 2.35 ATP, 3.67 ? 0.2 ATP ? EDTA, n ?
3, p ? 0.004, one-way ANOVA, post hoc Dunnett's method) dif-
ferently from ATP-induced bleb formation which appeared to be
only slightly delayed by EDTA (Fig. 2D).
Shed vesicles contain IL-1?
To verify whether vesicle formation could mediate IL-1? release
from microglia, shed vesicles were isolated from the supernatant of
LPS-primed N9 cells, 8–10 min after ATP stimulation, when low
levels of IL-1? were detectable in the extracellular medium (Fig.
1D). Shed vesicles were isolated using annexin V-coated beads,
permeabilized with 2% Triton X-100 to release vesicle content and
assayed for the presence of IL-1?. High levels of IL-1? were de-
tected in shed vesicles (14.66 ? 3.5 pg/ml) while minimum
amounts of the cytokine were observed in the vesicle-depleted me-
dium (1.08 ? 0.14 pg/ml, n ? 3, p ? 0.008, Fig. 4A). The amount
of IL-1? measured in Triton X-100-treated supernatant of N9 cells
(i.e., total extracellular IL-1?) at 10 min was ?27% of the total
cellular content, in agreement with data from Sanz and Di Virgilio
(2) who reported a releasable fraction of ?25%. To investigate
whether the cytokine could be released from shed vesicles into the
extracellular medium, isolated vesicles were maintained in KRH at
37°C for different time periods. The evaluation of IL-1? content of
both vesicular and vesicle-free supernatant fractions indicated a
progressive flow of the cytokine from shed vesicles into the ex-
tracellular medium (IL-1? in vesicle-free supernatant: 10 min ?
1.25 ? 0.35 pg/ml corresponding to 9.36 ? 1.97% of total IL-1?
present in the extracellular medium; 20 min ? 11.5 ? 3.5 pg/ml
corresponding to 27.37 ? 9.3% of total IL-1? 30 min ? 22.5 ?
4The online version of this article contains supplemental material.
FM1–43 during ATP exposure. Frames were taken at the indicated times before and after ATP addition. Note that ATP treatment induces formation of
FM1–43-labeled vesicles which appear loosely associated with cell margins. Formation and budding of vesicles from the plasma membrane illustrated in
B can be also seen in the supplementary movie. The arrow points to a vesicle which moves along microglial filopodia and eventually detaches from the
cell membrane. C, Fluorescence images of FM1–43-labeled vesicles collected from supernatant of N9 cells exposed to ATP and then sedimented on glass
coverslips. Note a clear reduction in the number of vesicles shed from N9 cells exposed to ATP either in the absence of extracellular calcium ions or after
culture treatment with 100 ?M oATP. D, Frequency histogram indicating the size distribution of shed vesicles. E, The histogram shows the spectropho-
tometric analysis of supernatants collected from N9 cells containing FM1–43-labeled vesicles. Note a significant reduction in the fluorescence of super-
natants from cells stimulated either in the absence of extracellular calcium ions or after treatment with oATP (fluorescence arbitrary units: 22.5 ? 2.5,
control; 42.99 ? 2.13, ATP; 4.56 ? 0.56, oATP; 6.36 ? 1.16, EDTA, n ? 3, p ? 0.016, one-way ANOVA, post hoc Dunnett's method). F, Fluorescent
image of vesicles shed from GFP-N9 microglia cells.
Vesicle shedding from microglia exposed to exogenous ATP. A and B, Fluorescence images of two different fields of N9 cells labeled with
Page 6
2.5 pg/ml corresponding to 41.6 ? 9.5% of total IL-1?, n ? 3, Fig.
4B). Cytokine release from shed vesicles was enhanced when ves-
icles were stimulated with 1 mM ATP (IL-1? in vesicle-free me-
dium: control ? 22.5 ? 2.5 pg/ml, ATP ? 42.99 ? 2.1 pg/ml)
while it was significantly inhibited when vesicles were pretreated
with 100 ?M oATP (4.56 ? 0.56 pg/ml) or maintained in a cal-
cium-free medium (6.36 ? 1.16 pg/ml), thus indicating the in-
volvement of the pore-forming P2X7R in the release of the cyto-
kine (n ? 3, p ? 0.016, one-way ANOVA, post hoc Dunnett's
method, Fig. 4C). Interestingly, IL-1? concentration in the extra-
cellular medium was reduced even below control level after ves-
icle exposure to oATP. This may suggest a partial activation of
P2X7R on shed vesicles, due to the previous ATP treatment needed
to induce vesicle shedding. The gradual increase of IL-1? in the
extracellular medium does not appear to result from progressive
vesicle disruption, as indicated by the time course analysis of flu-
orescence in GFP-vesicle and GFP-vesicle-free medium, collected
from N9 cells stably expressing cytosolic GFP (Fig. 4B). If GFP-
vesicles broke with time, GFP would gradually flow into the ves-
icle-free supernatant. A similar percentage of GFP fluorescence
was instead detected in vesicle-free fractions at all analyzed time
points (16.8 ? 5.0, 10 min; 10 ? 5.3, 20 min; 12.8 ? 7.22, 30 min;
Fig. 4B). Accordingly, no significant reduction of GFP fluores-
cence was detected at different time points in isolated vesicles
(65.5 ? 4.1, 10 min; 67.5 ? 6.5, 20 min; 62 ? 5.16, 30 min).
Western blot analysis revealed the presence of pro-IL-1? (Fig.
4E), procaspase-1 (Fig. 4F), and P2X7Rs (Fig. 4D) in vesicle frac-
tions, solubilized immediately after isolation. Isolated vesicles
were also stained for the ER marker ribophorin, for the plasma
membrane protein SNAP-23 and for actin (Fig. 4D), in line with
previous reports indicating that microvesicles and apoptotic blebs
could carry a number of ER-derived proteins (26) and could con-
tain cytoskeletal elements. Isolated vesicles were not immunore-
active for the endosomal (45 kDa) and lysosomal (31 kDa) forms
of cathepsin D (Fig. 4D), for the endolysosomal markers rab-7
(Fig. 4D) and LAMP-1 (data not shown). Lack of cathepsin D and
rab-7 staining in isolated vesicles but not in N9 homogenate, aside
from indicating that isolated vesicles do not derive from lysosomal
organelles, ruled out the possible contamination of isolated vesi-
cles with apoptotic cell fragments (Fig. 4D). Interestingly, after 30
min of in vitro exposure to ATP, a processed band of both IL-1?
and caspase-1 appeared in isolated vesicles (Fig. 4, E and F), sug-
gesting that both precursors can be locally activated upon ATP
stimulation (active-/pro-IL-1? ratio: 0.39 after 30 min of stimula-
tion with 1 mM ATP, n ? 3). In line with this finding, quantitative
analysis of active caspase-1 by FLICA assay showed an increase in
the number of active caspase-1 enzymes upon ATP stimulation in
isolated vesicles as compared with vesicles incubated in vitro in
the presence of oATP, after isolation (fluorescence arbitrary units:
601.85 ? 105.95, ATP; 147.1 ? 76.0, ATP ? oATP, p ? 0.01228,
n ? 2).
medium of N9 cells after a 10 min exposure to 1 mM ATP. B, Histogram showing time course of 1) IL-1? content of isolated vesicles (gray bars) and
medium in which vesicles are contained (white bars), and 2) GFP content in vesicle-free medium (dark gray bars). Values represent mean ? SE percentage
of total GFP, i.e., vesicular plus extracellular GFP, obtained from three independent experiments (10 min ? 16.8 ? 5.00%; 20 min ? 10.00 ? 5.3%; 30
min ? 12.8 ? 7.2%). C, Quantitative analysis of IL-1? content in the extracellular medium after a 30-min incubation with shed vesicles. Note that ATP
stimulation enhances IL-1? release from shed vesicles while oATP pretreatment or extracellular calcium chelation reduces IL-1? release below control
condition. D, Western blot analysis performed on extracts from shed vesicles and N9 cells extracts, for P2X7Rs, actin, the ER marker ribophorin (Rib), the
plasma membrane protein SNAP-23 and the endolysosomal markers rab-7 and cathepsin D (CD). Isolated vesicles are not immunoreactive for the
endolysosomal markers. E, Western blot analysis of shed vesicles for IL-1?, revealing the presence of procytokine inside vesicles lysed immediately after
isolation and of both pro- and active-IL-1? in vesicles incubated for 30 min at 37°C with 1 mM ATP after isolation. F, Representative Western blot showing
procaspase-1 in shed vesicles lysed immediately after isolation and the presence of the mature form of the enzyme in vesicles exposed for 30 min to 1 mM
ATP after isolation.
P2X7mediated IL-1? processing and release from shed vesicles. A, IL-1? detection by ELISA in the vesicle fraction and in the vesicle-free
7273The Journal of Immunology
Page 7
Altogether, our results indicate that shed vesicles, isolated from
ATP-stimulated microglia cells, contain pro-IL-1?, which is con-
verted in the active cytokine upon ATP exposure and is progres-
sively released into the external medium via a calcium and P2X7-
dependent mechanism.
Astrocyte-derived ATP stimulates IL-1? release from
microglial cells
To investigate whether ATP derived from astrocytes is sufficient to
induce IL-1? release from adjacent microglial cells, we evaluated
the presence of the cytokine in the supernatant of astrocyte-N9
cocultures. It has been previously shown that release of ATP from
astrocytes is enhanced by mechanical stimulation (17, 27) and that
astrocyte-derived ATP may activate P2Rs present in adjacent mi-
croglial cells (3, 4). To evaluate whether ATP release, induced by
mechanical stimulation of astrocytes, triggers IL-1? secretion from
adjacent microglia cells, astrocyte-N9 cocultures were mechani-
cally stimulated on an orbital shaker, as previously described (17).
ELISA analysis of collected media indicated a 3- to 4-fold increase
of IL-1? concentration in the supernatant of mechanically stimu-
lated cocultures (27.5 ? 0.5 pg/ml), as compared with supernatants
from control cultures maintained under static conditions (7.25 ?
0.75 pg/ml) (Fig. 5A). Interestingly, IL-1? decreased even below
control levels (0.5 ? 0.05 pg/ml) when mechanical stimulation
was performed in the presence of the ATP-degrading enzyme
apyrase (30 U/ml), which is known to significantly prevent the
ATP-mediated astrocyte-to-microglia calcium signaling (3). IL-1?
release from primary astrocyte-microglia cocultures was also
markedly reduced when mechanical stimulation was performed in
high extracellular concentration of K?(IL-1?, 27.5 ? 0.5 pg/ml,
control medium; 4.5 ? 0.375 pg/ml, high K?medium), thereby
indicating that inhibition of P2X7-induced K?efflux (28) by as-
trocyte-derived ATP does repress the paracrine activation of
caspase-1 and of IL-1? release from microglia. IL-1? concentra-
tion in the supernatant of N9 cultures in the absence of astrocytes
was significantly lower as compared with cocultures (1.00 ? 0.3
pg/ml) and the mechanical stimulation of pure N9 cultures only
slightly increased IL-1? concentration in the supernatant (1.92 ?
0.5 pg/ml) (Fig. 5A). These data suggested that astrocyte-derived
ATP, released either spontaneously or upon mechanical stimula-
tion, induces IL-1? secretion from N9 cells (Fig. 5A). An almost
negligible contribution to cytokine release seems to be due to ATP
derived from microglia (Fig. 5A).
Astrocyte-derived ATP induces bleb formation in
microglial cells
To evaluate whether ATP released from astrocytes could be suf-
ficient to induce vesicle formation in microglial cells, bright field
video microscopy experiments were conducted on cocultures of
astrocytes and either primary microglia or N9 cells. Gentle me-
chanical contact between a patch pipette and the surface of a single
astrocyte induced in adjacent microglia the formation of vesicular
structures, which bound Annexin VFITC(Fig. 5B). The quantitative
analysis of annexin V-positive cells was conducted in astrocyte-N9
cocultures, mechanically stimulated on an orbital shaker. Fig. 5C
showed a 3-fold increase in the number of annexin-positive N9
cells upon mechanical stimulation. Binding of annexin V was not
significantly enhanced in pure N9 cultures, upon mechanical stim-
ulus, thus excluding that the mechanical stress per se or ATP se-
cretion from microglial cells play a relevant role in vesicle forma-
tion (number of annexin V-positive cells per field, normalized to
static coculture condition: 3.84 ? 0.28 cocultures, mechanical
stimulation; 1.05 ? 0.28, cocultures, mechanical stimulation plus
apyrase; 0.67 ? 0.06, N9 cells, static conditions; 0.82 ? 0.08, N9
supernatants of N9-astrocyte cocultures and pure N9 cultures maintained in static condition or mechanically stimulated with or without apyrase (one-way
ANOVA, post hoc Dunn's method, p ? 0.003, n ? 3). B, Merged differential interference contrast and fluorescence images from an astrocyte-microglia
coculture incubated with Annexin VFITCfollowing the mechanical stimulation of a single astrocyte present in the field by a patch pipette. Note the presence
of annexin V-positive blebs on the microglial cell adjacent to the stimulated astrocyte. C, Quantitative analysis of annexin V-positive N9 cells present in
N9-astrocyte cocultures maintained in static condition or mechanically stimulated by an orbital shaker in the presence or in the absence of the ATP
degrading enzyme apyrase. The mechanical stimulus significantly increases the number of annexin V-positive N9 cells present in cocultures but not in pure
N9 cultures.
Astrocyte-derived ATP induces vesicle shedding and IL-1? release from microglial cells. A, ELISA evaluation of IL-1? levels in the
Page 8
cells, mechanical stimulation; one-way ANOVA, post hoc Dunn's
method, p ? 0.006, n ? 3 Fig. 5C). Note that the presence of apyrase
during mechanical stimulation almost completely inhibited bleb for-
mation (Fig. 5C), further supporting a key role of astrocyte-derived
ATP in the observed blebbing phenomenon.
It is currently assumed that, in the CNS, IL-1? is mainly expressed
and released from microglia, although there is evidence that also
astrocytes and neurons may contribute to IL-1? production, par-
ticularly in the late phase after excitotoxicity (18, 29).
In the present study we directly evaluated the specific contribution
of astrocytes and microglia to the cytokine release. We demonstrate
that 1) in hippocampal primary astrocyte-microglia cocultures, micro-
glia is indeed the cell type which mainly releases IL-1?, 2) ATP
released from astrocytes is the endogenous stimulus which leads to
the cytokine secretion from microglial cells, and 3) astrocyte-derived
surface of membrane vesicles which contain all the machinery nec-
essary for the cytokine processing, including P2X7Rs.
Pro-IL-1? is also expressed by primary hippocampal astrocytes,
ATP stimulation. Because both astrocytes and microglia bear P2X7Rs
different behavior of hippocampal astrocytes and microglia regarding
IL-1? secretion. Lack of IL-1? release from astrocytes might result
from differences in caspase-1 activation and/or cytokine processing,
possibly due to insufficient intracellular Ca2?concentration increases
and K?drop, following P2X7activation (F. Bianco and C. Verderio,
unpublished observations).
The main finding of this study is that the release of at least a
fraction of IL-1? from microglia takes place, upon ATP stimula-
tion, through the shedding of vesicles from the plasma membrane.
Shed vesicles appear to be the site of IL-1? processing.
Different populations of membrane vesicles which can be re-
leased from cells in the extracellular environment have been de-
scribed so far, including exosomes, microvesicles, and apoptotic
vesicles. Exosomes originate from exocytosis of endolysosome-
related multivesicular bodies and represent a population of vesicles
homogenous in size and shape (40–80 nm) (32). Microvesicles
and apoptotic vesicles represent instead a heterogeneous popula-
tion of vesicles relatively larger than exosomes (100 nm to 1 ?m)
which bud directly from the plasma membrane of either viable
cells or cells undergoing apoptosis (26). Exosomes and mi-
crovesicles/apoptotic vesicles are biochemically distinct; the latter
population have PS exposed to the outer cell membrane (11, 26)
carry some cytoskeletal elements (26) and can bear both nuclear
and ER proteins (33, 34). Exosomes instead do not have PS ex-
posed to the outer membrane leaflet and carry a selected subset of
protein including rab-7 (26, 35).
Our video microscopy data indicate that ATP stimulation in-
duces in microglia the shedding of membrane vesicles of hetero-
geneous diameter, which detach from the membrane in the pres-
ence of extracellular calcium. Because calcium chelation slightly
delayed formation of ATP-induced membrane blebs, but markedly
inhibited vesicle shedding, our results seem to dissociate the P2X7-
dependent formation of membrane blebs from the release of ves-
icles. Bleb formation and vesicle shedding appear to be distinct
parallel pathways both initiated by P2X7activation. Shed vesicles
carry the plasma membrane soluble N-ethylmaleimide-sensitive
fusion protein attachment protein receptor (SNARE) protein
SNAP-23, have PS exposed to the outer cell membrane, do not
express lysosomal markers, including rab-7, lamp-1, and cathepsin
D, but bear the ER marker ribophorin and some actin. Based on
biochemical and morphological analysis, vesicles isolated from the
supernatant of ATP-stimulated microglia have all the features of
microvesicles/apoptotic vesicles. Given that microglia are able to
recover from ATP challenge and do not necessarily undergo apo-
ptosis, and because no caspase-3 activity is detectable inside iso-
lated vesicles (F. Bianco and C. Verderio, unpublished observations),
we exclude these formations to be apoptotic vesicles. Accordingly, it
has been reported that caspase-3 activation is not a necessary prereq-
uisite for vesicle formation induced by ATP, although it plays a nec-
essary role in blebbing induced by apoptosis (13).
The experiments described in this study indicate that shed ves-
icles contain pro-IL-1? which is cleaved in the active form and
released into the extracellular environment in an ATP- and P2X7-
dependent manner. These findings strongly suggest that IL-1? pro-
cessing could proceed in shed vesicles, which indeed contain
caspase-1 both as proenzyme and as active form. The presence of
caspase-1 activity in shed vesicles is in agreement with previous
which can locally process their substrates (38). Our results point to a
crucial involvement of the P2X7Rs in the mechanism mediating the
cytokine processing and release from shed vesicles, although the
and release from shed vesicles and from cell
surface. Two pathways of IL-1? processing
and release may coexist in microglia: one
pathway, mediated by the exocytosis of en-
dolysosome-related vesicles,
place at the cell surface (6); the other takes
place at shed vesicles although the mecha-
nism underlying the efflux of the cytokine
from the vesicles remains to be defined (this
Model for IL-1? cleavage
7275 The Journal of Immunology
Page 9
mechanism underlying the efflux of the cytokine from the vesicles
remains to be defined. Vesicle disruption does not significantly con-
tribute to the release of the cytokine, differently from what is de-
scribed in fibroblasts where vesicle lysis mediates the release of
fibroblast growth factor-2, another protein lacking a conventional
secretory peptide which is released by vesicle shedding (32).
Globally, our data support and complement results obtained in
monocytes by MacKenzie et al. (11), which indicated the existence
of a vesicular pathway for IL-1? release by vesicle shedding.
However, the observation that vesicle shedding can occur in rest-
ing microglia, which does not release IL-1?, supports the idea that
vesicle shedding does not represent a secretory mechanism selec-
tive for the cytokine, but rather a secretory pathway possibly
shared by other releasable factors, which is used by IL-1? when
produced in activated microglia. Given that P2X7R activation in
microglia triggers the synthesis and release of other cytokines like
TNF (39) and proteases as well as their precursors, including plas-
minogen (40), it would be of interest to investigate whether ATP-
induced shed vesicles can also mediate the release of such factors.
Furthermore, vesicle shedding, through the release of cell surface
components, including membrane receptors (41–43), can serve
functions other than secretion of soluble factors. In this regard, we
clearly demonstrate the presence of P2X7Rs on isolated vesicles,
thus indicating that vesicles shedding might represent a mecha-
nism to remove P2X7Rs from the cell surface in response to as-
trocyte-derived ATP signaling. From this point of view the shed-
ding process could represent a defense strategy of microglia
against apoptotic insults, produced by excessive or repetitive stim-
ulation by astrocyte-derived ATP. Removal from the cell surface
of functional P2X7Rs could facilitate microglial survival and avoid
P2X7-mediated microglial apoptosis (3).
Based on the amount of IL-1? detected in Triton X-100-treated
supernatant at 10-min stimulation, when negligible levels of the
cytokine are present in vesicle-free supernatant, we estimated the
fraction releasable by vesicle shedding to be ?27% of total cel-
lular content. This estimation is in agreement with results from a
previous study (2), which reported a releasable IL-1? fraction of
?25% in these cells. Based on these findings, one could speculate
that vesicle shedding may be the predominant mechanism of IL-1?
secretion in microglia. However, different pathways of IL-1? re-
lease might coexist in these cells (Fig. 6). This possibility has been
already described for monocytes, where vesicle shedding (11) and
exocytosis of endolysosome-related vesicles (6) have been shown
to account for ?12 and 30% of total IL-1? cellular content, re-
spectively. Further studies are needed to determine the possible
contribution of the endolysosome pathway to IL-1? secretion in
microglia, although the lack of inhibition of IL-1? release by
AAC0CF3 (F. Bianco and C. Verderio, unpublished observations),
a PLA2blocker which strongly inhibits the exocytosis of secretory
lysosomes (6), plays against a major contribution of the endoly-
sosomal pathway in microglia.
Although vesicle shedding is most likely a nonspecific and non-
unique pathway for IL-1? release, it is extremely important to
understand the physiological relevance of IL-1? release from mi-
croglial cells by this mechanism. Shed vesicles, containing pack-
ages of IL-1?, can deliver the cytokine in possible proximity to
IL-1?Rs present on target cells. Furthermore, given that IL-1?
release is enhanced by ATP, the cytokine might be released from
shed vesicles when they impact local high concentration of the
purine, presumably in close proximity of releasing astrocytes, thus
avoiding dilution of the cytokine in the extracellular environment.
This might also turn out to be of crucial importance for the onset
of a detrimental effect of IL-1? toward degenerating cells, which
are known to release large amounts of ATP. Therefore, IL-1? re-
lease from microglia can represent a backward signal sent by mi-
croglial cells in response to ATP-mediated astrocyte-to-microglia
We thank Dr. Paola Castagnoli-Ricciardi (University of Milano-Bicocca,
Milan, Italy) for generously providing N9 microglial cells. We thank
Dr. Elisabetta Menna (Consiglio Nazionale delle Ricerche-Institute of Neu-
roscience, Milan, Italy) for helpful comments and discussion.
The authors have no financial conflict of interest.
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ceptor is derived from receptor shed in vesicles. Blood 78: 2444–2450.
42. Harding, C., J. Heuser, and P. Stahl. 1983. Receptor-mediated endocytosis of
transferrin and recycling of the transferrin receptor in rat reticulocytes. J. Cell
Biol. 97: 329–339.
43. Tuck, D. P., D. P. Cerretti, A. Hand, A. Guha, S. Sorba, and N. Dainiak. 1994.
Human macrophage colony-stimulating factor is expressed at and shed from the
cell surface. Blood 84: 2182–2188.
7277 The Journal of Immunology


text:
hurd, marvin, loewen, or semco?
im building a pretty large house in new jersey. the foundation is in. my architect called for marvin windows. marvin is quoting a very high price. my GC recommends semco windows. we definitely want to go with the clad window as opposed to wood. does anyone have any suggestions? semco quoted us $100k including install.
It is pointless to compare brand names. They all make many types, models and configurations of windows in many price ranges.
Try pricing the doors separately; there's no reason to get everything from the same manufacturer.
Try a different supplier of the Marvin windows.
I would suggest other options but I don't know what kind of windows you are looking for.
Your architect should be familiar with the design and the local market and should be able to give you better advice than anyone here.
Semco isn't sold in my area so I know nothing about them but they don't seem to be well thought of on the Bob Vila web site.
What type of windows, dbl hungs casements, tilt-n-turns, pushout casements, inswing casements?
What type of doors. sliding, hinged?
Any architectural shapes?
How many units?
Lite Cut, SDL, TDL One lite?
What type of install, vycor wrap, stainless steel pans under doors?
It is impossible for anyone on here to know anymore that what you stated without these questions and many more being answered
I own Loewen windows and would recommend them or the Marvins highly. I'll bet close to 25% of your home's exterior is probably windows. Purchasing good windows now for new construction is much less expensive than having to redo sub-par performing windows again in a few years.
Knowing from what I have learned the hard way is that if my new home contractor was recommending Hurd or Semco I would run far away from him as I quickly as I could. Never will I make that mistake again.
If your plan calls for $100K of the cheap windows you probably have the financial means to purchase the better materials. You will never be disappointed that you purchased the best windows.

subset: commoncrawl


text:
I have this HeaderlessTabControl, which subclasses the classic TabControl.
// From http://social.msdn.microsoft.com/forums/en-US/winforms/thread/c290832f-3b84-4200-aa4a-7a5dc4b8b5bb/
// Author: Hans Passant (nobugz)
public class HeaderlessTabControl : TabControl {
protected override void WndProc(ref Message m) {
// Hide tabs by trapping the TCM_ADJUSTRECT message
if (m.Msg == 0x1328 && !DesignMode) {
m.Result = (IntPtr)1;
} else {
base.WndProc(ref m);
When I run Code Analysis on my project, I get this warning:
Warning 1 CA2122 : Microsoft.Security : 'HeaderlessTabControl.WndProc(ref Message)' calls into 'Message.Msg.get()' which has a LinkDemand. By making this call, 'Message.Msg.get()' is indirectly exposed to user code. Review the following call stack that might expose a way to circumvent security protection: ->'HeaderlessTabControl.WndProc(ref Message)'
->'HeaderlessTabControl.WndProc(ref Message)'
...as well as two similar warnings relating to
Message.Msg.set() and
TabControl.WndProc(). I understand that I've exposed some code by doing this. Can someone explain what kind of security holes I might have opened up here, and possible ways to fix it?


text:
Mischa Barton's Supportive Boyfriend
26th August 2012
Mischa Barton's boyfriend is very supportive of her fashion career.The 26-year-old actress is currently dating Sebastian Knapp, and she admits he is always keen to show how much he admires her talents.He said: ''He's great....
Tagged in this article - Mischa Barton, Ashley Madekwe, Cisco Adler, Luke Pritchard, The Kooks, Whitestarr
Adler Slams Split Reports
8th February 2007
LATEST: Rocker CISCO ADLER has slammed reports he has split with girlfriend MISCHA BARTON. Media reports claimed the actress dumped her boyfriend after a nude photo of him appeared on the Internet last month (JAN07)....
Tagged in this article - Cisco Adler, Mischa Barton, Whitestarr, Paris Hilton
Barton Breaks Up With Adler
7th February 2007
MISCHA BARTON has ended her relationship with musician CISCO ADLER after nude photos of him appeared on the Internet. THE OC star was reportedly shocked when pictures of her boyfriend turned up among PARIS HILTON's...
Tagged in this article - Mischa Barton, Cisco Adler, The Oc, Paris Hilton, Whitestarr, Rod Stewart, Kimberly
Rod Stewart Gives Daughter Tour Advice
22nd October 2006
ROD STEWART gave his wildchild daughter KIMBERLY some sage advice when she was asked to tour with CISCO ADLER's rock band WHITESTARR - get your own hotel room and a return flight ticket. The blonde...
Tagged in this article - Rod Stewart, Kimberly, Cisco Adler, Whitestarr
Barton Has No Interest An Actor
19th September 2006
MISCHA BARTON has no interest in dating an actor, because there would be war over who gets to spend the most time in front of the mirror every morning. The former THE OC star, who...
Tagged in this article - Mischa Barton, The Oc, Whitestarr, Cisco Adler
Adler Sings About Ex-girlfriend
16th May 2006
Rocker CISCO ADLER insists fans are shocked when they find out his band WHITESTARR's first single, SUNSHINE GIRL, is not about his current girlfriend MISCHA BARTON, but ex-girlfriend KIMBERLY STEWART. Adler began dating THE OC...
Tagged in this article - Cisco Adler, Whitestarr, Mischa Barton, Kimberly Stewart, Rod Stewart
Barton And Adler Perfect Match
2nd March 2006
THE OC star MISCHA BARTON knows exactly why she and boyfriend CISCO ADLER are the perfect match - because they're both "boring dorks". The 20-year-old actress is bemused by how fascinated fans are by her...
Tagged in this article - Mischa Barton, Cisco Adler, Whitestarr
Adler Slams Hilton
1st March 2006
CISCO ADLER has lashed out at PARIS HILTON over her petty feud with his girlfriend MISCHA BARTON, insisting the heiress should "butt out and get a job." The WHITESTARR frontman stood up for Barton...
Tagged in this article - Cisco Adler, Paris Hilton, Mischa Barton, Whitestarr, Kimberly Stewart, Nicole Richie
Barton Denies Engagement
18th February 2006
Actress MISCHA BARTON and her boyfriend CISCO ADLER insist they are not engaged, despite recent rumours. THE OC star and her rocker beau have only been dating for five months, but have been plagued...
Tagged in this article - Mischa Barton, Cisco Adler, Whitestarr
Barton's Beau Annoyed By Media Portrayal
16th December 2005
THE OC star MISCHA BARTON's new boyfriend CISCO ADLER is paranoid the media are deliberately printing ugly photos of him. The 19-year-old actress started dating the 26-year-old WHITESTAR rocker in August (05), two...
Tagged in this article - Mischa Barton, Cisco Adler, Whitestar, Brandon Davis
Rod's Girl To Wed
28th October 2004
ROD STEWART has even more reason to celebrate this week - his daughter KIMBERLY has become engaged. Her rocker boyfriend CISCO ADLER, who plays in WHITESTARR, proposed to the socialite in a limousine on...
Tagged in this article - Rod Stewart, Kimberly, Cisco Adler, Whitestarr, Lou Adler


text:
AD Tag – GNN
2AD – grey matter book ad
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New Ad – Wil Jackson
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Interview: Todd Lincoln - Director (V/H/S: Viral, The Apparition, Gorgeous Vortex)March 26th, 2015Interviewed by: Todd Martin
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Book Review: The Pendle Curse - Author Catherine CavendishReviewed by: Dave Gammon
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Book Review: Alternate Movie Posters – Author Matthew Chojnacki
As an avid movie fan myself, it sure puts a smile on my face when I hear of news book releases that pay homage to the artistry of the craft. I have spent many the years dealing with marketers who have grown accustomed to quick turnaround photoshop poster designs, so much… that the art of the craft itself has become almost extinct. Though for every "upper quadrant floating heads" poster or movie monster production shot … Continue reading


text:
PTG Pillar Bedding Columns Ruger 77, 77 Mark II
Product #: 874663 PTG #: ColumnRuger77
Product Information
Columns have been precisely machined with the proper angles for a simple bedding job on the Ruger 77. Aluminum.
Technical Information
Aluminum, front .600" long, .535" diameter. Rear 1.145" long, .535" diameter.
Front pillar is cut to match the receiver screw angle, fits against the bottom of the recoil lug.
Technical Information
Delivery Information
The total weight of the product, including the product's packaging as it sits on our shelf. Since some products ship directly from the Manufacturer, their weight may be unavailable. Because of variations in the manufacturing process, similar products may have differing weights. Unless the product is designated as "Ships Alone", Shipping Weight does not include the shipping box or packing materials associated with your order.
Product ships from MidwayUSA, separate from any other products on your order due to its size. Products that are Shipped Alone incur separate "Per Order" and "Per Pound" shipping charges.
Product is regulated by the US Department of Transportation (DOT) and cannot be shipped via USPS or air shipping methods. DOT-Regulated products must ship UPS Ground.
Product is restricted by shipping carriers and cannot be shipped to certain locations or via certain shipping methods.
Additional shipping charge for HazMat products. Carriers charge extra to ship packages with products they consider hazardous, such as reloading powder and primers.
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text:
Bosnia and Herzegovina
Alberta, Halifax, Hamilton, Montreal, Ottawa, Toronto, Vancouver, Victoria,
Czech Republic
Bengaluru, Chandigarh, Chennai, Delhi, Mumbai, Pathankot,
New Zealand
South Africa
United Kingdom
Belfast, Birmingham, Bristol, Edinburgh, Glasgow, Hull University, London, Nottingham, Sheffield, University of Leicester, University of London,
United States
Appalachian Ohio, Athens GA, Atlanta, Baltimore, Chicago, Cleveland, Columbia MO, Columbus, Denver, Des Moines, Durham & Chapel Hill, East Lansing, Fredericksburgh VA, Houston, Las Vegas, Los Angeles, Lubbock TX, Manhattan KS, Muncie IN, New Orleans, New York City, NYU, Pittsburgh, Plattsburgh, Richmond VA, San Fernando Valley, San Francisco, SUNY Oneonta, Tucson, Twin Cities
A friend and I went to the Pratt art store (Clinton Hill) to buy canvas and there was a well dressed man standing right in the middle of the store. I needed help and he looked like he worked there.
me: Sir do you work here?
creep: No, but I still want to talk to you
me: No, thanks.
As I walk away he grabs my arm and starts caressing it!! I pulled away.
me: DON'T TOUCH ME!!
creep: Oh so it's like that??!!
me: I dont like being touched by strangers!!
creep: Go back to where you came from!! Go back to f*cking Eastern Europe you b*tch, get the f*ck out of here.
(I am from Moscow but I have been here since I was 9 and have had my citizenship for 10 years now.) He kept saying nasty things and I was terrified. So I went to get the store security guard. When we walked over to the racist creep the security guard asked him if there was a problem… creep: "there is no problem, I was just trying to shop and she was talking me up and touching me!!!!"
me:no, you just sexually and racially harassed me.
creep:you don't know what harassment is.. go back to f*cking Eastrern Europe! ect.."
A manager came over and I told her what happened, I was very upset and scared and I couldn't help it when I started to cry. She told me and my friend to wait behind the isles on the other side of the store while she took care of it. My friend who was with me the whole time was very scared too and didn't say much the whole time.
The creep by now was yelling at everyone, saying that "can only Eastern European b*tches shop here???!!" and other nasty things. The manager told him she's going to call the police and he was yelling "yeah call them" Then when she walked away to call the cops he ran out.
The manager was very nice and sympathetic and took my ID to file a report and then let us out the back door. This happened almost a week ago, the first couple of days I didn't think about it because I think I was in shock but now I can't get it out of my head. The store filed a report but I doubt anything will be done about it. I am terrified now because I live 5 minutes away from the store and if I run into him again there will be no security guards to protect me. I decided to go to the police today and file a report. The store has cameras and I'm sure they got the creep on tape.
Hopefully I can get the surveillance video of the creep and post it as well.
Submitted by Tais
Author comments are in a darker gray color for you to easily identify the posts author in the comments


text:
Vote up!-15
Vote down!
delegate strategy chosen, bec they believe they can't win
the honest way thru gaining the popular vote.
so 'delegate strategy' means backing backroom deals to get delegates to vote against the wishes of those who came out and voted. clearly and unfortunately, romney won by a long shot that paul lost his chance last year with the campaign not defending him and not allowing debate coaches.
- Login to post comments


text:
50% OFF! - Summer Vacation Elements
By Caroline B
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Product Information
- Type: Embellishment Pack
- Designer: Caroline B
- Size: 34.1 MB (approximate)
Design Elements
- Page Elements: 20
Coordinating Products
All the design elements of this product are
also included as part of the following products:
Summer Umbrella Pack Pack
Designer Notes
Soft and fruity colors for this Summer Vacation perfect pages. This kit of elements incudes : - 10 stickers (BBQ, hat, parasol, sun, floater, glass, strawberry, wordart, googles...) - 1 flower border - 2 brackets - 1 journaling - 1 bracket page - 1 slide - 1 tag - 1 button - 1 paintchip.
We're sorry, but something went wrong.
We've been notified about this issue and we'll take a look at it shortly.
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text:
Retreat Track-Arm Leather Sectional
Item# 727680G
Starting at $6,797.00
Item#: 727680G
Product Actions
The Details
Retreat Track-Arm Leather Sectional
Starting at $6,797.00
Dimensions: 98"w x 98"w x 36"h x 40"d
Item#: 727680G
Time to relax. Fall into comfort with the deep-cushioned, luxe proportions of the Retreat sectional. The tailored, transitional track arms define the lines of each of these modern pieces with numerous configuration options to choose from. The leather and fabric upholstery choices are many, varied, and exciting. Have fun!
Seating Area By Section:
- 727674 Right Arm Facing: 51" wide x 22" deep
- 727675 Left Arm Facing: 51" wide x 22" deep
- 727680 Square Corner: 22" wide x 22" deep
- Seat Back: 17" high
- Seat Height (floor to top of cushion): 18" high
- Arm Height: 26" high
The Short List
Starting at $899.00
Item# 158721
Item# 139145
Item# 097184
Item# 073020
$599.00 - $3,199.00
Item# 041442


text:
OP Member
Thanks Meter: 1
59 posts
Join Date:Joined: Aug 2011
As the title suggests, I want to know if there's a flash tool for broadcom that will work on BCM 23550 devices. I can't find a way to backup a stock ROM for this device aside from CWM. Is there any flash tool which I could use for creating a ROM and make it flashable through CWM?
Please help me out.. Thanks in advance!


text:
Orange for Animal Cruelty Awareness wants to
Help Puppy Doe's Campaign to Stop Violence Against Animals
Puppy Doe was brutally tortured and in her memory we want to help animals.
Puppy Doe was brutally tortured and in her memory we want to help animals.
* There needs to be changes made in the Nursing Homes for Residents and Patients to have their 100% Resident
New York State's laws protect the rapists of children from prosecution meaning they are free to rape more children.
POUR LA LIBERTÉ DES FEMMES Sahraouis dans les territoires occupés du Sahara Occidental. Les pratiques actuelles de
Our mission is to reform the meat industry and rebuild networks for sustainable farming across the country.
Hello and thankyou for taking action on this most brutal of Animal cruelty crimes.WE WANT JUSTICE FOR BUDDY...let the


text:
Register now for free, or sign in with any of these services:
Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Be the first NJ.com user to add photos or videos of AMITY PLUMBING HEATING AND COOLING
AMITY PLUMBING HEATING AND COOLING provides Plumbing services in Jersey City. Call them at 201-942-2024 for more details.


text:
Roll over image to magnify & Click to Zoom
Why Set & Save?
Just click "Add to Set & Save" when placing items in your cart.
Lightly Fragranced
All Skin Types
Retail price: $10.50
Vitacost price: $7.35
Light and silky facial care with beta-liposomes
Lightly Scented - Moisturize, nourish and protect your skin, every day. This ultra-light, ultra-rich lotion combines the benefits of beta-liposomes (nature's most effective skin softeners, soothers, and moisturizers) with herball emollients and anti-oxidants to help protect skin from premature aging and wrinkling.
Vitacost price: $7.99
Vitacost price: $9.99
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Vitacost price: $7.19
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text:
Open Positions
We are currently recruiting for the following scientific positions at Neuropore Therapies:
Neuropore Therapies In Vivo Research Scientist
Neuropore Therapies, Inc. is a San Diego based biopharmaceutical company utilizing innovative structure-based drug design technology to discover new therapies for Alzheimer's disease, Parkinson's disease and related neurodegenerative disorders
Neuropore Therapies is seeking a research scientist with behavioral pharmacology and biochemistry expertise to support these drug-discovery and development efforts. This is a hands-on technical and scientific position supporting in vivo studies related to Neuropore Therapies drug discovery and development efforts. Working independently the person holding this position will perform all aspects of in vivo experiments with rodents including: compound administration, behavioral assay development and testing, specimen collection, biochemistry and data analysis. The incumbent will maintain documentation related to study execution to industry standards and will be responsible for assuring that protocols and company operating procedures are followed. The person holding this position will also be responsible for generating written study reports and disseminating findings at internal review meetings.
Candidates for this position will be expected to have a Ph.D. degree in pharmacology, pharmaceutical science, biology, neuroscience, or related discipline along with a minimum of 1-2 years relevant postdoctoral experience. Exceptional candidates with a BS/MS degree and at least 5+ years of relevant industry experience conducting animal studies in support of drug discovery will also be considered.
Additional technical experience requirements for this position include:
- A minimum of 3 years recent (within the past 12 months) direct hands on experience working with rodent models (mice or rats) is required.
- A demonstrated ability to administer test article via IP, SC, IV or PO routes to rodents and the ability to collect blood and tissue samples is required.
- Candidates with experience conducting behavioral studies (motor and cognition) in animal models commonly used for CNS drug-discovery research are highly preferred.
- Candidates with experience conducting biochemical evaluations (e.g., Western blots, ELISAs) of in vivo samples are highly preferred.
Click here to submit your resume for the In Vivo Research Scientist position now
Neuropore Therapies, Inc. is an Equal Opportunity Employer.
Thank you for your interest. There are currently no other open positions at Neuropore Therapies, Inc.


text: Warning: fetch_template() calls should be replaced by the vB_Template class. Template name: googlescript in ..../includes/functions.php on line 4591 View Profile: Tumul Ahmed - Pen & Paper Games Register Help Remember Me? Home Forum FAQ Calendar Community Groups Albums Member List Forum Actions Mark Forums Read Quick Links View Site Leaders Contribution Actions Contribute What's New? Blogs Find Players Groups Contribute Downloads Chat [2] Advanced Search Recent Chat Activity (Main Lobby)Join Chat Loading Chat Log... Player Registry Tumul Ahmed Prefer not to see ads? Become a Community Supporter. Welcome to Pen & Paper Games, an online community dedicated to bringing roleplayers together!You are currently viewing our boards as a guest which gives you limited access to view most discussions and access our other features. By joining our free community you will automatically be added to our player registry (unless you opt not to) and will be able to privately find and communicate with other players in your area. You will also be able to post and reply to topics, vote in polls, and many other special features. Registration is fast, simple and absolutely free so please, join our community today! If you have any problems with the registration process or your account login, please contact us. Site News: Pen & Paper Games Gets New Leadership! [Read More] Tumul Ahmed Lurker Find latest posts Find latest started threads View Articles View Blog Entries Mini Statistics Join Date 08-07-2012 Last Activity 08-07-2012 05:34 AM Blog Entries 0 Recent Visitors This page has had 572 visits Badges and Icons My Icon: Player Tab Content Tumul Ahmed's Activity About Me Tab Content All Tumul Ahmed Friends Photos New Activity () Please reload this page to view the 200+ new activity items that have been created. Older Activity More Activity No More Results No Recent Activity About Tumul Ahmed Basic Information Badges and Icons My Icon: Player Location City: dhaka Zipcode: 1217 Country: Bangladesh Willing to Travel: 10 miles Availability LFG/LFP: No Availability (Days): Friday Availability (Times): Afternoons Gaming Preferences Player/GM: Player Preferred Genres: Dark Future About Me Biography: want to be professional.............! Statistics Total Posts Total Posts 1 Posts Per Day 0.00 Downloads Downloads 0 Uploads 0 General Information Last Activity 08-07-2012 05:34 AM Join Date 08-07-2012 Referrals 0 -- P&PG v4 -- Default Mobile Style Contact Us Pen and Paper Games Archive Privacy Statement Top All times are GMT -5. The time now is 06:58 PM. Powered by vBulletinCopyright ©2000 - 2015, Jelsoft Enterprises Ltd. Donation System provided by vBDonate (Lite) - vBulletin Mods & Addons Copyright © 2015 DragonByte Technologies Ltd.Copyright ©2006-2014 - All Additional Rights Reserved - Robert A. Howard


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How to use past exam papers
Five key points to ace your examsIf mark schemes, specifications, Chief Examiner reports and past papers all sound a bit confusing, don't worry. After reading this article you'll be able to use past papers to really get on top of your subject and nail your exams. »
What's hot, what's not
Loved by Students results revealedWhat's the most-wanted mobile phone? Where's the best place to eat? What's the most-loved brand? We asked these questions and more in our big Loved By Students poll: check out the results. »
Thinking about studying abroad?
Here's how to find out more...If you're looking into leaving the UK, you can get answers to these questions and more in a few ways – many of them face-to-face. Here are just some of your options... »
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Watch our interview with Norman Lamb
Check out our videos to hear the answersTSR's miser visited Lib Dem HQ to ask your questions to Norman Lamb MP, the Minister of State for Care and Support, and Parliamentary Private Secretary to the Deputy PM Nick Clegg. Watch and discuss the videos here »
Should you be studying?
Here's ten ways to stop wasting timeAlphabetising your entire DVD collection. Hoovering your room just one more time. Sound familiar? Here's how to get back on track and send procrastination packing »
Enter our Political Party Contest!
Create your own party to win an awesome prizeIn this contest, you will get to form a party, or stand as an independent candidate. You will be asked to write a manifesto, answer questions from the TSR electorate, and face the public vote. If that sounds good, check out the official thread to see how you can take part. »
University guides A-Z
Find the uni that's right for youFind out more about unis across the UK (and beyond), ask questions on the forums and meet other students via Connect. »
Is university the only option?
See what else you could get into...There are many other options to leave you in high spirits about your life and career prospects.»
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Would you change the smoking laws?
We asked the community how they felt.For No Smoking Day 2015, we took a look at the current smoking laws and what our members had to say on the subject. Read our article and join in the debate here »
The facts about apprenticeships
How you could get a headstartAn apprenticeship can lead to a career in all kinds of areas, even within the same company. See more facts »
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UCAS Extra questions answered
Everything you need to knowIf you haven't received any offers, or you've declined all the ones you did get, then the UCAS Extra application route could be for you. Here's everything you need to know, as well as answers to some frequently asked questions. »
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Aixa Diaz is anchor/reporter at WESH-TV in Orlando.
Aixa has covered the 2008 Summer Olympics in Beijing, the 2010 Winter Olympics in Vancouver, and now the 2012 Summer Olympics in London!
Eight people died and 10 injured when the van they were in crashed into a Florida canal near Lake Okeechobee early Monday, officials said.
The Florida Highway Patrol said a vehicle with 18 people was eastbound on State Road 78 at about 12:30 a.m. ET near Moore Haven, Fla. The vehicle ran a ...
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Learn to Knit with Felisia - RSVP Required
Wed, Feb 20, 2013
Times: 2:00pm-3:00pm
Venue: World's Only Curious George Store
1 Jfk Street, Cambridge, MA 02138Contact:
Shoshana Smith
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CG waving 150x150 Learn to Knit with Felisia RSVP Required
For creative kids really into crafts, Felisia will be teaching a beginning knitting class. Parent-child dynamic duos or older kids looking for a new hobby will learn the basics of casting on, casting off, knit stitch, purl stitch, and everything else you need to start your very own project. This hour includes activities perfect for ages 6 and up.
Required materials are:
Acrylic craft yarn about 5-6 in. gauge (will be listed on the packaging)
Size #8 knitting needles
A positive, can-do attitude!
RSVP via e-mail for 2 p.m. Please include the names of the children who will be attending and the date and time of the event in your e-mail. You will receive a confirmation email from us once you have been added to the list!
The Borscht Belt Comes Alive Again in Amherst
Growing up in New York's Catskills region in the 1980s and '90s, Marisa...
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Crackdown on commercial vehicles begins next week in Minn.
ST. PAUL – Commercial drivers in Minnesota can expect stricter enforcement of seatbelts, driver roadside inspections and regulatory compliance next week.
The Minnesota State Patrol Commercial Vehicle Section will be increasing traffic enforcement statewide Oct. 20-26.
Non-commercial motorists can expect an increase in enforcement of safe driving conduct near commercial vehicles.
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