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of July 31, 2017.
NADPH Oxidase NOX2 Mediates Rapid
Cellular Oxidation following ATP
Stimulation of Endotoxin-Primed
Macrophages
Samantha F. Moore and Amanda B. MacKenzie
J Immunol 2009; 183:3302-3308; ;
doi: 10.4049/jimmunol.0900394
http://www.jimmunol.org/content/183/5/3302
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Supplementary
Material
The Journal of Immunology
NADPH Oxidase NOX2 Mediates Rapid Cellular Oxidation
following ATP Stimulation of Endotoxin-Primed Macrophages1
Samantha F. Moore and Amanda B. MacKenzie2
A
denosine triphosphate is an extracellular signaling molecule that binds to a number of membrane proteins, including ionotropic P2X receptors (1). Secreted ATP
plays a fundamental role in numerous events from CNS transmission to platelet aggregation. Cells of the innate immune system
express P2X7 receptors that bind ATP to trigger proinflammatory
signaling cascades, including secretion of proinflammatory cytokines and the generation of reactive oxygen species (ROS)3 (2–5).
P2X7-deficient mice have demonstrated a role for P2X7 in the
progression of collagen-induced rheumatoid arthritis, inflammatory and neuropathic pain, as well as the development of bone
(6 – 8). From these studies, it is proposed that P2X7 receptors play
a key role in chronic inflammatory signaling pathways in vivo and
provides a promising therapeutic target in the management of
chronic inflammatory conditions. Indeed, several selective P2X7
receptor antagonists have been developed in the last 5 years
(9 –11).
P2X7 receptors are reported to trigger rapid oxidative stress via
activation of NADPH oxidase (2, 4, 12). Aberrant NADPH oxidase signaling can contribute to a range of chronic inflammatory
conditions, including inflammatory pain and vascular atheroscle-
rosis (13, 14). For example, macrophage NADPH oxidase activity
contributes to the oxidation of lipids in atherosclerosis and may
contribute to the progression of vascular injury (13, 15). Activation
of phagocytic NADPH oxidase (NOX2) requires assembly of a
multiprotein complex consisting of two membrane components
gp91phox/NOX2 and p22phox that recruit several regulatory proteins from the cytoplasm (p67phox, p47phox, p40phox, and p21Rac)
(16). The potential role for NADPH oxidase in ATP signaling is
currently largely based primarily upon pharmacological inhibitors
(2, 4), although NOX2 knockdown via small interfering RNA has
demonstrated partial involvement in ATP-mediated oxidation in a
RAW246.7 macrophage cell line (12). We have extended these
studies to investigate the role of phagocyte NOX2 isoform in ROS
generation following nucleotide stimulation of endotoxin-exposed
primary murine macrophages. Using NOX2-deficient macrophages, we have demonstrated the critical role of NOX2 in ATPmediated oxidation. Moreover, we have compared nucleotidestimulated oxidation in primary macrophages with J774.2 cells, a
commonly used murine macrophage cell line.
Materials and Methods
Reagents and solutions
Department of Pharmacy and Pharmacology, University of Bath, Bath, United
Kingdom
Received for publication February 5, 2009. Accepted for publication June 24, 2009.
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.
1
This work was supported by grants from the University of Bath and Heart Research UK.
2
Address correspondence and reprint requests to Dr. Amanda B. MacKenzie, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath
BA2 7AY, U.K. E-mail address: [email protected]
3
Abbreviations used in this paper: ROS, reactive oxygen species; ASC, apoptosisassociated speck-like protein containing a caspase-recruitment domain; BMDM, bone
marrow-derived macrophage; DPI, diphenyleneiodonium; EtBr, ethidium bromide;
H2DCFDA, 2⬘,7⬘-dichlorodihydrofluorescein diacetate; LDH, lactate dehydrogenase;
NAC, N-acetylcysteine; P2X7R, P2X7 receptor; SOD, superoxide dismutase;
Z-YVAD-FMK, Tyr-Val-Ala-Asp-fluoromethyl ketone.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900394
All reagents were obtained from Sigma-Aldrich unless stated otherwise.
Cell stimulations were conducted using an external physiological salt solution containing 147 mM NaCl, 2 mM KCl, 10 mM HEPES, 12 mM
glucose, 2 mM CaCl2, and 1 mM MgCl2 (pH 7.3 with NaOH).
Cell culture
The J774.2 mouse macrophage cell line (European Collection of Cell Cultures) was maintained in complete medium consisting of DMEM/F12 media containing 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100
U/ml penicillin, and 100 ␮g/ml streptomycin (Invitrogen) at 37°C in a
humidified atmosphere of 5% CO2 and 95% air. Cells were detached by
mechanical scraping. For LPS priming, J774.2 cells plated overnight in
complete medium were incubated for 4 – 6 h at 37°C with 1 ␮g/ml LPS
from Escherichia coli 055:B5.
Isolation, generation, and culture of murine bone
marrow-derived macrophages (BMDMs)
Mouse bone marrow cells were collected from femurs of control group
C57BL/6 mice (8 –12 wk old) or NOX2⫺/⫺ mice (C57BL6/J background,
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The phagocytic NADPH oxidase (NOX2) plays a fundamental role in host defense and innate immunity. Here we demonstrate
that external ATP triggers rapid cellular oxidation inhibited by diphenyleneiodonium in endotoxin-primed J774 macrophages and primary murine bone marrow-derived macrophages. To identify the source of reactive oxygen species (ROS), we
compared responses between wild-type and NOX2-deficient macrophages. ATP-mediated ROS production was strongly
attenuated in NOX2-deficient macrophages where responses were comparable to inhibition with diphenyleneiodonium. Notably, spatial differences in superoxide anion formation were observed where ROS formation was partially antagonized by
extracellular superoxide dismutase in primary bone marrow-derived macrophages but unaffected in J774 macrophages. Loss
of NOX2 was not observed to affect ATP-induced cell death. However, ATP-evoked cell death was found to be partially
dependent on caspase-1 and cathepsin B activation. In conclusion, NOX2 plays a fundamental role in conferring macrophages with the ability to respond to extracellular ATP stimulation with robust changes in cellular oxidation. The Journal
of Immunology, 2009, 183: 3302–3308.
The Journal of Immunology
3303
8 –12 wk old) by flushing with bone marrow medium consisting of RPMI
1640 containing 15% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100
U/ml penicillin, and 100 ␮g/ml streptomycin. Cell suspensions were centrifuged at 240 ⫻ g (S4108 rotor, Beckman GS-15R) for 10 min at room
temperature. Cells were resuspended in bone marrow medium and cell
clumps were dispersed. Adherent bone marrow cells were removed by
incubation at 37°C for 4 – 6 h in T25 flasks. The nonadherent bone marrow
cells were placed in fresh T25 flasks and M-CSF was added to give a final
concentration of 10 ng/ml (17). Cells were maintained in culture at 37°C in
a humidified atmosphere of 5% CO2 and 95% air. Media and M-CSF were
completely changed on day 5 and experimental assays performed on day 7.
For LPS priming, cells plated overnight in bone marrow medium with
M-CSF were incubated for 4 – 6 h at 37°C with 1 ␮g/ml LPS from E. coli
055:B5.
Ethidium bromide influx
Assays of ethidium bromide influx, as a measure of membrane pore formation, are as previously described (18).
ROS generation
Cells were plated overnight in complete medium in 96-well clear plates at
a seeding density of 1.5 ⫻ 106/ml. Complete medium was removed, and
cells were incubated at 37°C in physiological salt solution containing the
test substance. Supernatants (50 ␮l) were removed and added to a cytotoxicity detection kit (Roche Applied Science) reaction mixture (50 ␮l).
Absorbance of samples at 490 nm was measured using a multidetection
plate reader (FLUOstar Optima; BMG Labtech). Total cellular lactate dehydrogenase (LDH) was determined by the addition of 1% Triton X-100 to
untreated cells and then the amount of LDH released expressed as a percentage of the total LDH release.
FIGURE 1. ATP evokes ROS generation in murine macrophages. A,
Representative trace demonstrating that 1 mM ATP evokes an increase
in DCF fluorescence in J774 macrophages. B, Representative trace demonstrating that 5 mM ATP evokes an increase in DCF fluorescence in
BMDMs. C, Concentration-response curve of ATP evoked ROS generation in J774 macrophages (pEC50 of 3.24 ⫾ 0.04 and pEC50 of
2.56 ⫾ 0.05) (n ⫽ 10 ⫾ SEM). D, Histogram demonstrating concentration-dependent ATP-evoked ROS generation in BMDMs (n ⫽ 3 ⫾
SEM). E, Concentration-response curve of ATP evoked EtBr influx in
J774 macrophages (pEC50 of 2.82 ⫾ 0.03) (n ⫽ 3 ⫾ SEM). F, Histogram demonstrating concentration-dependent ATP-evoked EtBr influx
in BMDMs (n ⫽ 3 ⫾ SEM).
Western blot analysis
Results
Cell viability
LPS-primed (1 ␮g/ml, 4 – 6 h) BMDMs (1.5 ⫻ 10 ) were washed once in
physiological salt solution and stimulated in physiological salt solution in
the absence or presence of 5 mM ATP for 30 min at 37°C. Physiological
salt solution above the cells was collected for analysis of secreted proteins
and, following a wash step in ice-cold physiological salt solution, cells
were lysed on ice in 75 ␮l of protease inhibitor cocktail plus 1% Triton
X-100. Lysates and secreted protein fractions were cleared at 15,000 ⫻ g
for 10 min at 4°C. Secreted protein fractions were concentrated (⫻6) using
3K Nanosep cut-off filters (VWR International) according to the manufacturer’s protocol. Solubilized proteins were resolved on a 12% SDS-PAGE
gel and transferred onto nitrocellulose membrane. Nitrocellulose membrane was probed with mouse IL-1␤ mAb (Thermo Fisher Scientific) followed by rabbit anti-mouse IgG/HRP (Dako), and immune complexes were
detected using the Amersham ECL Advance Western blotting detection
agent.
6
Data analyses and statistics
All data points shown are the means ⫾ SEM from a minimum of three
experiments performed in triplicate. Data were analyzed by one-way
ANOVA with Dunnett’s post hoc or unpaired two-tailed Student’s t test
using GraphPad Prism version 4.0. Dose-response curves were fit using
Equation 1, where x is the logarithm of the molar concentration of the drug,
␣ is the maxima (maximum response-baseline), log A50 is the 50% maximal response, and nH is the Hill slope.
y ⫽ baseline ⫹
␣
1 ⫹ 10共logA50⫺x兲nH
Extracellular ATP triggers sustained generation of ROS in
murine macrophages and J774 macrophages
Addition of extracellular ATP triggers a sustained formation of
ROS in endotoxin-primed J774.2 macrophages and BMDMs, detected by loading with H2DCFDA (Fig. 1, A and B). In J774.2
macrophages, ATP initiates ROS generation with a bell-shaped
concentration response curve with a peak response detected at 1
mM ATP (Fig. 1C). The up-phase of the concentration response
curve had an apparent EC50 value of 575 ␮M (pEC50 of 3.24 ⫾
0.04) and the down-phase an apparent IC50 value of 2.75 mM
(2.56 ⫾ 0.05) (n ⫽ 10 ⫾ SEM) (Fig. 1C). In contrast, ATP-mediated ethidium bromide (EtBr) influx displayed an EC50 value of
1.51 mM (pEC50 of 2.82 ⫾ 0.03) (n ⫽ 3 ⫾ SEM) (Fig. 1E).
Extracellular ATP initiated a slower generation of ROS in
BMDMs with a peak response detected with 2 mM ATP, while
EtBr influx is triggered with a peak response at 3 mM ATP (Fig.
1, B, D, and F). Priming with LPS potentiated ATP-mediated ROS
formation in J774.2 macrophages but not in BMDMs after subtracting the elevated background ROS formation resulting from
LPS stimulation (Fig. 2A–D). In contrast, ATP-mediated EtBr influx was unaltered in J774.2 macrophages following ATP stimulation, while it was elevated in LPS-primed BMDMs (Fig. 2, E
and F).
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Cells were plated overnight in complete medium in a 96-well black plate
at a seeding density of 1.5 ⫻ 106/ml. Complete medium was removed and
cells incubated with 10 ␮M 2⬘,7⬘-dichlorodihydrofluorescein diacetate
(H2DCFDA; Invitrogen) in DMEM/F12 with 2.5 mM probenecid for 40
min at 21 ⫾ 3°C, under limited light conditions. Cells were washed in
physiological salt solution containing 2.5 mM probenecid (37°C) 5 min
before agonist application. Fluorescence was monitored every 10 –25 s
using a multidetection plate reader (FLUOstar Optima; BMG Labtech; excitation, 485 nm; emission, 520 nm). Data obtained were of three wells per
experimental group. Data were analyzed using the formula FX ⫺ F0, where
FX is the DCF fluorescence measured at the indicated time, and F0 is the
DCF fluorescence measured at the beginning of analysis. Linear regression
was performed on analyzed data to determine the rate of ROS generation.
3304
For both J774.2 and BMDMs, preincubation with 100 ␮M DPI
or 20 mM N-acetylcysteine (NAC) blocked ATP-mediated ROS
generation (Figs. 3, A and B, and 4, A and B). DPI is an inhibitor
of flavin-containing enzymes that include NADPH oxidase, mitochondrial complex I, xanthine oxidase, and NO synthase. We also
tested inhibitors of each enzyme complex. Preincubation of J774.2
cells with either 100 ␮M allopurinol, a xanthine oxidase inhibitor,
or 1 mM L-NAME (NG-nitro-L-arginine methyl ester), a broadspectrum NO synthase inhibitor, did not inhibit ATP-mediated
ROS formation (Fig. 3, C and D). Addition of 5 ␮M rotenone (an
inhibitor of mitochondrial complex I) to J774.2 macrophages did
not inhibit ROS formation, but it did partially antagonize ATPevoked ROS formation detected in BMDMs (Figs. 3E and 4C). For
each of these inhibitors, ATP-mediated EtBr influx was measured
as an indicator of receptor stimulation and subsequent pore formation (data not shown). None of the above inhibitors affected
ATP-induced EtBr influx in J774.2 or BMDMs, demonstrating that
any observed inhibition was not a direct action at the receptor, as
well as suggesting that no redox modulation of pore formation
occurs.
FIGURE 3. Effect of ROS inhibitors on ATP-evoked cellular oxidation
in J774 macrophages. A, Histogram demonstrating inhibition of ATPevoked ROS generation by preincubating cells with NAC (20 mM, 2 h)
(n ⫽ 3 ⫾ SEM). B, Histogram demonstrating inhibition of ATP-evoked
ROS generation by preincubating cells with DPI (100 ␮M, 1.5 h) (n ⫽ 3 ⫾
SEM). C, Histogram demonstrating that the xanthine oxidase inhibitor allopurinol (100 ␮M, 30 min) does not inhibit ATP-evoked ROS generation
(n ⫽ 3 ⫾ SEM). D, Histogram demonstrating that the NO synthase inhibitor L-NAME (1 mM, 1 h) does not inhibit ATP-evoked ROS generation
(n ⫽ 3 ⫾ SEM). E, Histogram demonstrating that the complex I inhibitor
rotenone (5 ␮M, 30 min) does not inhibit ATP-evoked ROS generation
(n ⫽ 3 ⫾ SEM). ⴱⴱ, p ⬍ 0.01 by Student’s two-tailed t test.
ATP stimulation activates NOX2 isoform of NADPH oxidase
To analyze the involvement of the phagocytic NOX isoform
NOX2, BMDMs were isolated from wild-type and NOX2
(gp91phox)-deficient mice. Macrophages were generated from bone
marrow cultured in the presence of M-CSF. No difference was
observed in final cell number in cells differentiated in M-CSF from
wild-type or NOX2⫺/⫺ bone marrow (data not shown). ATP-stimulated ROS generation was strongly attenuated in NOX2⫺/⫺ macrophages compared with wild-type macrophages (Fig. 5, A and B).
In contrast, ATP-mediated EtBr influx and IL-1␤ processing was
comparable between NOX2⫺/⫺ and wild-type macrophages (Fig.
5C and supplemental Fig. 1).4 We conclude that NOX2 plays a
central role in extracellular ATP-mediated ROS generation in primary macrophages.
Spatial localization of superoxide anion formation
NADPH oxidase activation results in the liberation of electrons
that combine with oxygen to form superoxide anions. Previously
we have reported that exposure of human monocytes to extracellular ATP triggers the extracellular generation of superoxide anions (2). To investigate whether ATP stimulation of macrophages
4
The online version of this article contains supplemental material.
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FIGURE 2. Effect of endotoxin priming on ROS generation and EtBr
uptake. A, Histogram demonstrating that ROS generation is enhanced when
J774.2 macrophages are primed with 1 ␮g/ml for 4 – 6 h (n ⫽ 3 ⫾ SEM).
B, Histogram demonstrating that ROS generation is enhanced when
BMDMs are primed with 1 ␮g/ml for 4 – 6 h (n ⫽ 3 ⫾ SEM). C, Histogram
demonstrating that LPS priming of J774.2 macrophages leads to an increase in ATP-evoked ROS generation once basal ROS generation is taken
into account (n ⫽ 3 ⫾ SEM). D, Histogram demonstrating that LPS priming of BMDMs does not lead to an increase in ATP-evoked ROS generation once basal ROS generation is taken into account (n ⫽ 3 ⫾ SEM). E,
Histogram demonstrating that LPS priming does not lead to increases in
basal or ATP-evoked uptake of EtBr in J774.2 macrophages (n ⫽ 3 ⫾
SEM). F, Histogram demonstrating that LPS priming leads to increases in
ATP-evoked but not basal uptake of EtBr in BMDMs (n ⫽ 3 ⫾ SEM). ⴱ,
p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001 by Student’s two-tailed t test.
ROLE OF NOX2 IN ATP-EVOKED CELLULAR OXIDATION
The Journal of Immunology
3305
FIGURE 6. Spatial localization of superoxide anion formation. A, Histogram demonstrating that extracellular SOD (100 U/ml) does not affect
ATP-induced ROS generation in J774 macrophages (n ⫽ 3 ⫾ SEM). B,
Histogram demonstrating that a cell-permeant SOD mimetic (Mn-cpx 3)
significantly increases ATP-evoked ROS generation in J774 macrophages
(n ⫽ 3 ⫾ SEM). C, Histogram demonstrating that extracellular SOD (100
U/ml) significantly decreases BMDM cellular oxidation induced by ATP
(n ⫽ 3 ⫾ SEM). ⴱ, p ⬍ 0.05 by Student’s two-tailed t test.
results in the formation of superoxide anions, we have investigated
the effect of extracellular superoxide dismutase (SOD) and a cellpermeant SOD mimetic. Superoxide anions will react with SOD to
generate H2O2. The addition of extracellular SOD to J774.2 macrophages did not alter ATP-mediated oxidation. However preincubation with a membrane-permeant SOD mimetic (Mn-cpx 3;
Calbiochem) augmented ATP-triggered ROS formation presum-
ably due to the intracellular generation of H2O2 (Fig. 6, A and B).
These data suggest that ATP triggers the formation of superoxide
anions at internal membranes in J774.2 macrophages. In contrast,
addition of extracellular SOD to BMDMs partially antagonized
ATP-mediated ROS formation (Fig. 6C), indicating extracellular
generation of superoxide anions.
Cell necrosis
FIGURE 5. Evidence for ATP-evoked activation of NADPH oxidase in
generating ROS. A, Representative trace demonstrating that 5 mM ATP
evokes an increase in DCF fluorescence in wild-type (WT) but not
NOX2⫺/⫺ BMDMs. B, Histogram demonstrating that ATP does not evoke
ROS generation in BMDMs from NOX2⫺/⫺ mice above control (n ⫽ 5 ⫾
SEM). C, Histogram demonstrating that ATP evokes EtBr influx at similar
levels in WT and NOX2⫺/⫺ BMDMs (n ⫽ 5 ⫾ SEM). ⴱⴱⴱ, p ⬍ 0.001 by
Student’s two-tailed t test.
We have investigated the redox dependence of P2X7 receptor-mediated necrosis in J774.2 macrophages and BMDMs. ATP stimulation of J774.2 macrophages triggers rapid cell necrosis detectable
by the release of LDH following a 30-min agonist application (Fig.
7A). ATP-dependent cell lysis was concentration-dependent, with
a variable peak response from 1 to 3 mM ATP followed by a
decline at higher concentrations (Fig. 7, B and F). This pharmacological profile is comparable to ATP-induced ROS generation. Treatment of J774.2 cells with 100 ␮M DPI attenuated
LDH release, while 5 ␮M rotenone had no affect on cell necrosis (Fig. 7, C and D). Preincubation with Tyr-Val-Ala-Aspfluoromethyl ketone (Z-YVAD-FMK; Cambridge Bioscience)
(10 ␮M, 30 min), a caspase-1 peptide inhibitor, partially attenuated ATP-mediated LDH release (Fig. 7E). In contrast, ATPmediated IL-1␤ processing was strongly blocked in J744.2 macrophages treated with 10 ␮M Z-YVAD-FMK (data not shown).
Preincubating cells with Ca-074 Me (Calbiochem) (100 ␮M, 30
min), a cathepsin B inhibitor, also partially inhibited ATP-mediated cell necrosis, suggesting the involvement of multiple
proteases (Fig. 7F).
Extracellular ATP stimulation of BMDMs also resulted in rapid
cell necrosis where LDH release was detected following 30 min
stimulation with 5 mM ATP (Fig. 8A). However, in contrast to
J774.2 macrophages, 100 ␮M DPI did not inhibit ATP-mediated
membrane necrosis (Fig. 8B). Moreover, ATP stimulation of
NOX2⫺/⫺ macrophages also resulted in release of LDH comparable to wild-type macrophages (Fig. 8C). Finally, 5 ␮M rotenone
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FIGURE 4. Effect of ROS inhibitors on ATP-evoked cellular oxidation
in BMDMs. A, Histogram demonstrating inhibition of ATP-evoked ROS
generation by preincubating cells with NAC (20 mM, 2 h) (n ⫽ 3 ⫾ SEM).
B, Histogram demonstrating inhibition of ATP-evoked ROS generation by
preincubating cells with DPI (100 ␮M, 1.5 h) (n ⫽ 3 ⫾ SEM). C, Histogram demonstrating that the complex I inhibitor rotenone (5 ␮M, 30 min)
inhibits ATP-evoked ROS generation in J774.2 cells (n ⫽ 3 ⫾ SEM). ⴱⴱ,
p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001 by Student’s two-tailed t test.
ROLE OF NOX2 IN ATP-EVOKED CELLULAR OXIDATION
FIGURE 7. Role of ROS in ATP-induced cell death in J774 macrophages. A, Histogram demonstrating that 1 mM ATP evokes LDH release
after a 30- to 60-min stimulation (n ⫽ 3 ⫾ SEM). B, Histogram demonstrating that only 1 mM ATP evoked a significant release of LDH (n ⫽ 3 ⫾
SEM). C, Histogram demonstrating that DPI significantly inhibited LDH
release from cells stimulated with 1 mM ATP (n ⫽ 3 ⫾ SEM). D, Histogram demonstrating that rotenone did not significantly effect LDH release
from cells stimulated with 1 mM ATP (n ⫽ 3 ⫾ SEM). E, Histogram
demonstrating that Z-YVAD-FMK significantly inhibited LDH release
from cells stimulated with 1 mM ATP for 60 min (n ⫽ 3 ⫾ SEM). F,
Histogram demonstrating that Ca-074 Me significantly inhibited LDH release from ATP-stimulated cells (n ⫽ 3 ⫾ SEM). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍
0.001; ⴱⴱⴱ, p ⬍ 0.001 by Student’s two-tailed t test.
FIGURE 8. ROS appear to not be involved in ATP-induced cell death in
BMDMs. A, Histogram demonstrating that 5 mM ATP evokes LDH release
from BMDMs after a 60-min stimulation (n ⫽ 3 ⫾ SEM). B, Histogram
demonstrating that DPI failed to significantly inhibit LDH release from
BMDMs stimulated with 5 mM ATP for 60 min (n ⫽ 3 ⫾ SEM). C, Histogram demonstrating that ATP-evoked LDH release from NOX2⫺/⫺ BMDMs
did not significantly differ from wild-type (WT) BMDMs (n ⫽ 3 ⫾ SEM). D,
Histogram demonstrating that rotenone did not significantly effect LDH release from BMDMs stimulated with 5 mM ATP for 60 min (n ⫽ 3 ⫾ SEM).
E, Histogram demonstrating that Z-YVAD-FMK significantly inhibited LDH
release from cells stimulated with 5 mM ATP for 60 min (n ⫽ 3 ⫾ SEM). ⴱⴱⴱ,
p ⬍ 0.001 by Student’s two-tailed t test.
had no affect on ATP-mediated LDH release, while Z-YVADFMK partially antagonized cell necrosis (Fig. 8, D and E). ATPmediated IL-1␤ processing, reflecting caspase-1 activation, was
attenuated in BMDMs treated with 10 ␮M Z-YVAD-FMK (data
not shown). In summary, cell necrosis was DPI-sensitive in J774.2
macrophages, but both DPI-insensitive and NOX2-independent in
BMDMs.
ATP triggers an increase in ROS formation where J774.2 macrophages had an apparent EC50 of 575 ␮M that contrasts with the
EtBr influx EC50 value of 1.5 mM. This leftward shift in the concentration response curve for ATP-mediated oxidation may reflect
the contribution of other P2 receptors in addition to a potential
underestimation of the EC50 value due to the bell-shaped concentration response curve. In contrast, in BMDMs, ATP triggered
ROS formation with a peak response at 2 mM ATP and EtBr at a
peak response at 3 mM. The high concentrations of ATP that are
required to trigger ROS formation suggests the contribution of
P2X7 receptors but does not eliminate additional contribution from
other P2 receptors.
We confirm that exposure of LPS-primed macrophages to extracellular ATP leads to a rapid burst in cellular oxidation blocked
by an inhibitor of flavoproteins, DPI. This study demonstrates that
ATP stimulation of endotoxin-primed macrophages triggers rapid
activation of NOX2 where ATP-mediated oxidation is abrogated
in NOX2-deficient macrophages. We have examined the action of
the pathogen-associated molecular pattern LPS on both basal levels of cellular oxidation and ATP-stimulated bursts in oxidation.
LPS stimulation of primary macrophages and J774.2 macrophages
Discussion
NOX2 plays an important role in bactericidal activity of phagocytes and in supporting innate immunity. Controlled NOX2 activation is fundamental for antimicrobial defense and phagocytosis.
However, deregulated NOX2 activity could lead to adjacent tissue
damage and propagation of an inflammatory response. Patients
lacking functional NADPH oxidase and NOX2 develop chronic
granulomatous disease characterized by increased susceptibility to
microbial infections, particularly aspergillus (19). Several reports
have implicated NADPH oxidase in P2X7 receptor-mediated ROS
generation in a range of phagocytic cells, including human monocytes, murine macrophages, and microglial cells (2, 12, 20, 21).
We have demonstrated that high concentrations of extracellular
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3306
The Journal of Immunology
plasma membrane localization of NOX2 prompts future studies
to investigate subcellular distribution and whether NOX2
maybe be co-secreted with other membrane proteins within microvesicles and/or exosomes (3, 29).
Finally, cell death is an important component of the innate immune
system, leading to either resolution or propagation of inflammation.
Apoptosis is a caspase-dependent form of cell death, which is defined
by a range of biochemical and morphological hallmarks that will ultimately result in the clearance of host cells by phagocytic cells before
the release of their inflammatory contents. Necrotic forms of cell
death, however, are characterized by a breakdown of the plasma
membrane, leading to the release of cellular constituents that could
provoke an inflammatory response (30, 31). For example, macrophages may release cytokines, heat shock proteins, and high-mobility
group box 1 (HMGB1) that are all potently proinflammatory and will
make an important contribution to the innate immune response. Necrosis is a caspase-independent pathway that occurs in the absence of
apoptotic hallmarks. However, the molecular signaling pathways
leading to cell necrosis are not completely understood. Recently, two
novel forms of cell death have been described, pyroptosis and pyronecrosis, both of which are also characterized by a breakdown of the
plasma membrane leading to the release of cellular constituents. Pyroptosis has been demonstrated to be dependent on the activation of
caspase-1 through the use of caspase-1 peptide blockers and caspase1-deficient macrophages and will result in cell lysis leading to the
release of inflammatory proteins (32, 33). Pyroptosis requires the assembly of the apoptosis-associated speck-like protein containing a
caspase-recruitment domain (ASC) into a supramolecular complex
that triggers caspase-1 activation, a protease that leads not only to cell
death via pyroptosis but also the generation of the inflammatory cytokine IL-1␤. In contrast, pyronecrosis is caspase-1-independent and
results from cathepsin B activation leading to cell lysis. Pyronecrosis
has been observed in macrophages infected with Shigella flexneri
(34).
Endotoxin-primed BMDMs are reported to undergo rapid
caspase-1-dependent pyroptosis upon stimulation with extracellular
ATP where cell lysis is completely abolished in caspase-1-deficient
BMDMs (32). This study highlights a key role for the ASC pyroptosome in caspase-1-dependent cell lysis in endotoxin-stimulated
monocytes and macrophages. However, a second study reports a
P2X7 receptor-dependent cell necrosis that is only partially dependent
on caspase-1 activation in LPS-primed peritoneal macrophages (35).
Moreover, we have reported ATP-mediated necrosis of RAW264.7
murine macrophages that do not express the adaptor protein ASC
required for pyroptosis (18, 36). Finally, several studies have reported
ATP stimulation that triggers robust caspase-1-dependent IL-1␤ processing in the absence of detectable cell necrosis (2, 3, 36, 37). Few
studies have investigated the role of oxidation in P2X7 receptor-mediated cell death. Noguchi and coworkers (12) reported that redoxdependent activation of ASK1 is partially responsible for ATP-mediated apoptosis (6 h) in spleen-derived murine macrophages, while
NOX2 is partially responsible for ATP-mediated ROS generation and
apoptosis in RAW264.7 macrophages. In contrast, Pfeiffer et al. (4)
reported that ATP stimulation of RAW264.7 macrophages leads to a
loss of cell viability (detected after 4 h with a MTT metabolic assay)
that is unaffected by antioxidants. Neither study directly investigated
the role of ROS, NOX2, and caspase-1 activation in rapid (⬍1 h)
ATP-mediated cell necrosis in both primary macrophages and a murine macrophage cell line. In the present study, ATP-induced necrosis
of LPS-primed J774.2 macrophages and BMDMs was partially
blocked with the caspase-1 inhibitor Z-YVAD-FMK. Furthermore,
ATP-evoked cell death in LPS-primed J774.2 macrophages was also
partially blocked with the cathepsin B inhibitor Ca-074 Me, thus demonstrating that multiple proteases are involved in ATP-evoked cell
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
led to an increase in basal oxidation. In J774.2 macrophages, ATPmediated oxidation was also increased; however, EtBr influx was
unchanged, suggesting an increase in NOX2-associated proteins or
signaling pathways. In contrast, ATP-mediated oxidation above
basal oxidation was unchanged in primary BMDMs, but ATPmediated EtBr influx was potentiated. We hypothesize that the
increase in EtBr may reflect an increased expression of P2X7 receptors or pannexin-1, but the P2X7-NOX2 signaling pathway is
unchanged.
This is the first demonstration in endotoxin-primed primary
macrophages that ATP stimulation leads to the activation of the
NOX2 isoform. The ability of ATP to stimulate NADPH oxidase
appears to be tissue-specific, as a recent study in peritoneal macrophages reported no change in ATP-mediated oxidation in
NOX2-deficient cells (22). P2X7 receptors are associated with a
number of pathophysiological responses, including inflammatory
and neuropathic pain. Several studies have implicated superoxide
anions in the pathology of hyperalgesia (clinically described as
augmented sensitivity to painful stimuli) associated with inflammation. Indeed, treatment with either a SOD mimetic or radical
scavenging agents attenuated hyperalgesia associated with carrageenan-induced peripheral inflammation (23, 24). NOX1 NADPH
oxidase has been shown to be expressed by dorsal root ganglia
neurons and to mediate superoxide anion generation (14). Thermal
and mechanical hyperalgesia was significantly reduced in NOX1deficient mice, suggesting a role of the NOX1 NADPH oxidase
isoform (14). With the known role of ATP signaling in the pathogenesis of inflammatory pain, a role for macrophage NOX2 should
also be investigated.
Notably, we and other groups have reported an inhibitory effect
of DPI and antioxidants on ATP-, silica-, and monosodium urate
crystal-mediated IL-1␤ processing in human THP-1 monocytes (2,
25, 26). Indeed, small hairpin RNA knockdown of p22phox expression leads to an inhibition of MSU-mediated IL-1␤ processing in
THP-1 monocytes (25). However, monosodium urate-, silica-, and
ATP-mediated IL-1␤ processing and secretion are not inhibited in
NOX2-deficient murine macrophages (22, 27) (supplemental Fig.
1). These data would suggest either a redundancy for NADPH
oxidase in the ATP-mediated IL-1␤ processing pathway or a fundamental difference in human vs mouse signaling pathways.
NADPH oxidase activity depends on spatial regulation of the
cytosolic components and the recruitment of NOX subunits to either cell surface or internal organelle membranes. We predict that
additional extracellular SOD will react with superoxide anions liberated by plasma membrane-associated NADPH oxidase. Indeed,
extracellular SOD attenuated ATP-mediated ROS formation in primary BMDMs comparable to previously reported responses in primary human monocytes and a THP-1 human monocyte cell line
(2). These data would suggest partial localization of NADPH oxidase to the plasma membrane of the primary BMDMs. In contrast,
ATP-mediated ROS formation in J774.2 macrophages was potentiated by a cell-permeable SOD mimetic but not by external SOD.
Detection of a fluorescent signal in J774.2 macrophages, but not in
BMDMs, following the formation of membrane-permeable H2O2
suggests an additional reaction in J774.2 macrophages. We hypothesize that SOD mimetic-formed H2O2 is converted into hydroxyl radicals that are readily detected by the DCF dye (28). The
differential action of extracellular SOD suggests that, at least in
J774.2 macrophages, superoxide anions could be being generated within membrane-bound compartments in the cell. The nature of ROS means they are diffusible, short-lived molecules,
and therefore localizing ROS production to a specific subcellular compartment could be of importance for activating certain
redox signaling events. In primary macrophages, the apparent
3307
ROLE OF NOX2 IN ATP-EVOKED CELLULAR OXIDATION
death and therefore may not be primarily dependent on either a pyroptotic or pyronecrotic pathway. In J774.2 macrophages, ATP-induced cell death was also inhibited by DPI; however, cell death in
BMDMs was found to still occur in the presence of DPI and was not
dependent on the expression of NOX2. These results demonstrate that
ATP stimulation of endotoxin-primed murine macrophages results in
rapid cell necrosis, but further studies are required to elucidate the
signaling pathways underlying this inflammatory form of cell death
and why differences are observed between different monocyte/macrophage models.
The differences in spatial localization of ROS generation between
the two cell types and the role of mitochondrial complex I in BMDM
ROS generation may explain the differential redox dependence of
ATP-evoked cell death between J774.2 macrophages and BMDMs.
The effects of oxidants on redox signaling are largely cell- and stimulus-specific (38). Therefore, differences in the redox properties of
different macrophages should be expected; for example, alveolar macrophages are observed to have high basal NADPH oxidase activity
(39).
Here we have demonstrated that ATP-evoked generation of ROS
in endotoxin-primed macrophages occurs primarily by activation of
NOX2. Our observations have also indicated that extracellular ATP
can evoke ROS generation at different locations and via different generators depending on the macrophage type potentially leading to the
activation of different cellular signaling pathways. This therefore
demonstrates that observations from one macrophage type may not be
simply extrapolated to different types and sources of macrophages.
13. Cathcart, M. 2004. Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages: contributions to atherosclerosis. Arterioscler.
Thromb. Vasc. Biol. 24: 23–28.
14. Ibi, M., K. Matsuno, D. Shiba, M. Katsuyama, K. Iwata, T. Kakehi, T. Nakagawa,
K. Sango, Y. Shirai, T. Yokoyama, et al. 2008. Reactive oxygen species derived
from NOX1/NADPH oxidase enhance inflammatory pain. J. Neurosci. 28:
9486 –9494.
15. Barry-Lane, P., C. Patterson, M. van der Merwe, Z. Hu, S. Holland, E. Yeh, and
M. Runge. 2001. p47phox is required for atherosclerotic lesion progression in
ApoE⫺/⫺ mice. J. Clin. Invest. 108: 1513–1522.
16. Bedard, K., and K. Krause. 2007. The NOX family of ROS-generating NADPH
oxidases: physiology and pathophysiology. Physiol. Rev. 87: 245–313.
17. Lin, H., C. Chen, and B. Chen. 2001. Resistance of bone marrow-derived macrophages to apoptosis is associated with the expression of X-linked inhibitor of apoptosis protein in primary cultures of bone marrow cells. Biochem. J. 353: 299 –306.
18. Moore, S. F., and A. B. MacKenzie. 2007. Murine macrophage P2X7 receptors
support rapid prothrombotic responses. Cell. Signal. 19: 855– 866.
19. Dinauer, M., S. Orkin, R. Brown, A. Jesaitis, and C. Parkos. 1987. The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. Nature 327: 717–720.
20. Parvathenani, L., S. Tertyshnikova, C. Greco, S. Roberts, B. Robertson, and
R. Posmantur. 2003. P2X7 mediates superoxide production in primary microglia
and is up-regulated in a transgenic mouse model of Alzheimer’s disease. J. Biol.
Chem. 278: 13309 –13317.
21. Kim, S. Y., J. H. Moon, H. G. Lee, S. U. Kim, and Y. B. Lee. 2007. ATP released
from ␤-amyloid-stimulated microglia induces reactive oxygen species production
in an autocrine fashion. Exp. Mol. Med. 39: 820 – 827.
22. Meissner, F., K. Molawi, and A. Zychlinsky. 2008. Superoxide dismutase 1 regulates caspase-1 and endotoxic shock. Nat. Immunol. 9: 866 – 872.
23. Khattab, M. 2006. TEMPOL, a membrane-permeable radical scavenger, attenuates
peroxynitrite- and superoxide anion-enhanced carrageenan-induced paw edema and
hyperalgesia: a key role for superoxide anion. Eur. J. Pharmacol. 548: 167–173.
24. Wang, Z., F. Porreca, S. Cuzzocrea, K. Galen, R. Lightfoot, E. Masini,
C. Muscoli, V. Mollace, M. Ndengele, H. Ischiropoulos, and D. Salvemini. 2004.
A newly identified role for superoxide in inflammatory pain. J. Pharmacol. Exp.
Ther. 309: 869 – 878.
25. Dostert, C., V. Pétrilli, R. Van Bruggen, C. Steele, B. T. Mossman, and
J. Tschopp. 2008. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320: 674 – 677.
26. Cassel, S. L., S. C. Eisenbarth, S. S. Iyer, J. J. Sadler, O. R. Colegio, L. A. Tephly,
A. B. Carter, P. B. Rothman, R. A. Flavell, and F. S. Sutterwala. 2008. The Nalp3
inflammasome is essential for the development of silicosis. Proc. Natl. Acad. Sci.
USA 105: 9035–9040.
27. Hornung, V., F. Bauernfeind, A. Halle, E. O. Samstad, H. Kono, K. L. Rock,
K. A. Fitzgerald, and E. Latz. 2008. Silica crystals and aluminum salts activate the
NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9: 847–856.
28. Setsukinai, K., Y. Urano, K. Kakinuma, H. J. Majima, and T. Nagano. 2003.
Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J. Biol. Chem. 278: 3170 –3175.
29. Qu, Y., L. Ramachandra, S. Mohr, L. Franchi, C. V. Harding, G. Nunez, and
G. R. Dubyak. 2009. P2X7 receptor-stimulated secretion of MHC class II-containing exosomes requires the ASC/NLRP3 inflammasome but is independent of
caspase-1. J. Immunol. 182: 5052–5062.
30. Fink, S., and B. Cookson. 2005. Apoptosis, pyroptosis, and necrosis: mechanistic
description of dead and dying eukaryotic cells. Infect. Immun. 73: 1907–1916.
31. Labbé, K., and M. Saleh. 2008. Cell death in the host response to infection. Cell
Death Differ. 15: 1339 –1349.
32. Fernandes-Alnemri, T., J. Wu, J. W. Yu, P. Datta, B. Miller, W. Jankowski,
S. Rosenberg, J. Zhang, and E. S. Alnemri. 2007. The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1
activation. Cell Death Differ. 14: 1590 –1604.
33. Kroemer, G., L. Galluzzi, P. Vandenabeele, J. Abrams, E. S. Alnemri,
E. H. Baehrecke, M. V. Blagosklonny, W. S. El-Deiry, P. Golstein, D. R. Green,
et al. 2009. Classification of cell death: recommendations of the Nomenclature
Committee on Cell Death 2009. Cell Death Differ. 16: 3–11.
34. Willingham, S. B., D. T. Bergstralh, W. O’Connor, A. C. Morrison,
D. J. Taxman, J. A. Duncan, S. Barnoy, M. M. Venkatesan, R. A. Flavell,
M. Deshmukh, et al. 2007. Microbial pathogen-induced necrotic cell death mediated by the inflammasome components CIAS1/cryopyrin/NLRP3 and ASC.
Cell Host Microbe 2: 147–159.
35. Le Feuvre, R., D. Brough, Y. Iwakura, K. Takeda, and N. Rothwell. 2002. Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell
death via a caspase-1-dependent mechanism, independently of cytokine production. J. Biol. Chem. 277: 3210 –3218.
36. Pelegrin, P., C. Barroso-Gutierrez, and A. Surprenant. 2008. P2X7 receptor differentially couples to distinct release pathways for IL-1␤ in mouse macrophage.
J. Immunol. 180: 7147–7157.
37. Grahames, C. B., A. D. Michel, I. P. Chessell, and P. P. Humphrey. 1999. Pharmacological characterization of ATP- and LPS-induced IL-1␤ release in human
monocytes. Br. J. Pharmacol. 127: 1915–1921.
38. Forman, H., and M. Torres. 2001. Redox signaling in macrophages. Mol. Aspects
Med. 22: 189 –216.
39. Tephly, L., and A. Carter. 2007. Constitutive NADPH oxidase and increased
mitochondrial respiratory chain activity regulate chemokine gene expression.
Am. J. Physiol. 293: L1143–L1155.
Acknowledgments
We thank Professor Ajay Shah and Dr. Alison Cave (Kings College, London, U.K.) for providing tissue from NOX2 knockout animals.
Disclosures
The authors have no financial conflicts of interest.
References
1. Surprenant, A., and R. A. North. 2008. Signaling at purinergic P2X receptors.
Annu. Rev. Physiol. 71: 333–359.
2. Hewinson, J., S. Moore, C. Glover, A. Watts, and A. MacKenzie. 2008. A key
role for redox signaling in rapid P2X7 receptor-induced IL-1␤ processing in
human monocytes. J. Immunol. 180: 8410 – 8420.
3. MacKenzie, A., H. Wilson, E. Kiss-Toth, S. Dower, R. North, and A. Surprenant. 2001.
Rapid secretion of interleukin-1␤ by microvesicle shedding. Immunity 15: 825–835.
4. Pfeiffer, Z., A. Guerra, L. Hill, M. Gavala, U. Prabhu, M. Aga, D. Hall, and
P. Bertics. 2007. Nucleotide receptor signaling in murine macrophages is linked
to reactive oxygen species generation. Free Radical Biol. Med. 42: 1506 –1516.
5. Solle, M., J. Labasi, D. Perregaux, E. Stam, N. Petrushova, B. Koller, R. Griffiths,
and C. Gabel. 2001. Altered cytokine production in mice lacking P2X7 receptors.
J. Biol. Chem. 276: 125–132.
6. Chessell, I., J. Hatcher, C. Bountra, A. Michel, J. Hughes, P. Green, J. Egerton,
M. Murfin, J. Richardson, W. Peck, et al. 2005. Disruption of the P2X7 purinoceptor
gene abolishes chronic inflammatory and neuropathic pain. Pain 114: 386 –396.
7. Ke, H., H. Qi, A. Weidema, Q. Zhang, N. Panupinthu, D. Crawford, W. Grasser,
V. Paralkar, M. Li, L. Audoly, et al. 2003. Deletion of the P2X7 nucleotide
receptor reveals its regulatory roles in bone formation and resorption. Mol. Endocrinol. 17: 1356 –1367.
8. Labasi, J., N. Petrushova, C. Donovan, S. McCurdy, P. Lira, M. Payette,
W. Brissette, J. Wicks, L. Audoly, and C. Gabel. 2002. Absence of the P2X7
receptor alters leukocyte function and attenuates an inflammatory response. J. Immunol. 168: 6436 – 6445.
9. Broom, D., D. Matson, E. Bradshaw, M. Buck, R. Meade, S. Coombs, M. Matchett,
K. Ford, W. Yu, J. Yuan, et al. 2008. Characterization of N-(adamantan-1-ylmethyl)5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, a P2X7 antagonist in animal
models of pain and inflammation. J. Pharmacol. Exp. Ther. 327: 620–633.
10. Donnelly-Roberts, D., and M. Jarvis. 2007. Discovery of P2X7 receptor-selective
antagonists offers new insights into P2X7 receptor function and indicates a role
in chronic pain states. Br. J. Pharmacol. 151: 571–579.
11. Stokes, L., L. Jiang, L. Alcaraz, J. Bent, K. Bowers, M. Fagura, M. Furber, M. Mortimore,
M. Lawson, J. Theaker, et al. 2006. Characterization of a selective and potent antagonist
of human P2X7 receptors, AZ11645373. Br. J. Pharmacol. 149: 880–887.
12. Noguchi, T., K. Ishii, H. Fukutomi, I. Naguro, A. Matsuzawa, K. Takeda, and
H. Ichijo. 2008. Requirement of reactive oxygen species-dependent activation of
ASK1–p38 MAPK pathway for extracellular ATP-induced apoptosis in macrophage. J. Biol. Chem. 283: 7657–7665.
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3308
Supplementary Fig.1. BMDMs deficient in NOX2 still process IL-1β in response to
ATP. Representative western blot demonstrating that ATP (5 mM, 30 minutes) evoked
the secretion and maturation of IL-1β in WT and NOX2-/- BMDMs (n = 3 ± s.e.m).