of June 13, 2013.
This information is current as
Bacterial Toxin, Pyocyanin
Regulating Neutrophil Apoptosis by a Major
Subversion of a Lysosomal Pathway
Dockrell and Moira K. B. Whyte
Graham W. Taylor, David J. Buttle, Ian Sabroe, David H.
Martin A. Bewley, Helen M. Marriott, Sarah R. Walmsley,
Lynne R. Prince, Stephen M. Bianchi, Kathryn M. Vaughan,
2008; 180:3502-3511; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2008 by The American Association of
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The Journal of Immunology
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Subversion of a Lysosomal Pathway Regulating Neutrophil
Apoptosis by a Major Bacterial Toxin, Pyocyanin1
Lynne R. Prince,2* Stephen M. Bianchi,2* Kathryn M. Vaughan,* Martin A. Bewley,‡
Helen M. Marriott,* Sarah R. Walmsley,* Graham W. Taylor,§David J. Buttle,†
Ian Sabroe,* David H. Dockrell,‡and Moira K. B. Whyte3*
Neutrophils undergo rapid constitutive apoptosis that is accelerated following bacterial ingestion as part of effective immunity, but
is also accelerated by bacterial exotoxins as a mechanism of immune evasion. The paradigm of pathogen-driven neutrophil
apoptosis is exemplified by the Pseudomonas aeruginosa toxic metabolite, pyocyanin. We previously showed pyocyanin dramat-
ically accelerates neutrophil apoptosis both in vitro and in vivo, impairs host defenses, and favors bacterial persistence. In this
study, we investigated the mechanisms of pyocyanin-induced neutrophil apoptosis. Pyocyanin induced early lysosomal dysfunc-
tion, shown by altered lysosomal pH, within 15 min of exposure. Lysosomal disruption was followed by mitochondrial membrane
permeabilization, caspase activation, and destabilization of Mcl-1. Pharmacological inhibitors of a lysosomal protease, cathepsin
D (CTSD), abrogated pyocyanin-induced apoptosis, and translocation of CTSD to the cytosol followed pyocyanin treatment and
lysosomal disruption. A stable analog of cAMP (dibutyryl cAMP) impeded the translocation of CTSD and prevented the desta-
bilization of Mcl-1 by pyocyanin. Thus, pyocyanin activated a coordinated series of events dependent upon lysosomal dysfunction
and protease release, the first description of a bacterial toxin using a lysosomal cell death pathway. This may be a pathological
pathway of cell death to which neutrophils are particularly susceptible, and could be therapeutically targeted to limit neutrophil
death and preserve host responses. The Journal of Immunology, 2008, 180: 3502–3511.
quent resolution of inflammation requires removal of these poten-
tially toxic leukocytes to prevent a dysregulated immune response.
Induction of apoptosis is a crucial mechanism of homeostasis,
down-regulating proinflammatory functions (1) and resulting in
macrophage-mediated clearance of apoptotic cells (2). Some bac-
teria, however, induce inappropriate or premature apoptosis of
phagocytes, particularly macrophages, depleting cell numbers and
function, with associated impairment of host defense (3, 4).
Pseudomonas aeruginosa is an important human pathogen.
Chronic infection with P. aeruginosa is a major cause of pulmonary
damage and mortality in cystic fibrosis, and acute infection is ob-
served both in the immunocompromised host and in patients with
ventilator-associated pneumonia (5). In patients with cystic fibrosis,
persistent P. aeruginosa colonization of the lung demonstrates inad-
equate mechanisms of bacterial clearance despite profound neutro-
eutrophils are the predominant inflammatory cells re-
cruited during the innate immune response to bacterial
infection and are critical for bacterial clearance. Subse-
philic inflammation (6). Although immune defenses in cystic fibrosis
may be impaired at multiple levels, an excess of apoptotic neutrophils
in this setting implies a neutrophil defect may contribute significantly
to unresolved infection (7). The prominence of P. aeruginosa sepsis
in neutropenic patients (8) also highlights both the role of the neutro-
phil in defense against this organism and the clinical importance of
understanding how this pathogen subverts the innate immune re-
sponse. P. aeruginosa generates highly diffusible toxic secondary me-
tabolites known as phenazines, which are critical for P. aeruginosa
virulence and cytotoxicity in Caenorhabditis elegans and mouse in-
fection models (9), and it is the only common organism to produce a
specific phenazine, named pyocyanin (10). We have shown pyocyanin,
at concentrations detected in sputum of cystic fibrosis patients (11), in-
in vitro (12). In a murine model of pulmonary P. aeruginosa infection,
mice infected with a pyocyanin-producing strain, as compared with a
pyocyanin-deficient, but otherwise genetically identical strain, also
Neutrophils are short-lived cells. Two major pathways to apop-
tosis are recognized, as follows: one proceeds through death re-
ceptor signaling, via membrane-associated signaling complexes
and caspase-8 activation, and a second stress pathway, known to
be regulated by oxidant stress, is mediated by mitochondria and
regulated by bcl-2 family members (14). The mechanisms of pyo-
cyanin-induced acceleration of neutrophil apoptosis are largely un-
known, but may involve reactive oxygen intermediates (ROI)4
*Academic Unit of Respiratory Medicine,†Academic Unit of Biochemical and Mus-
culoskeletal Medicine, and‡Academic Unit of Infectious Diseases, University of
Sheffield, Sheffield, United Kingdom; and
Campus, Royal Free and University College School of Medicine, London, United
§Department of Medicine, Hampstead
Received for publication March 1, 2007. Accepted for publication December 26, 2007.
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 work was supported by a Wellcome Clinical Research Fellowship (064997) (to
S.M.B.). I.S. is a Medical Research Council Senior Clinical Fellow (G116/170), and
D.H.D. is a Wellcome Trust Senior Clinical Fellow (076945).
2L.R.P. and S.M.B. contributed equally to this work and are joint first authors.
3Address correspondence and reprint requests to Dr. Moira K. B. Whyte, Academic
Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, Uni-
versity of Sheffield, L Floor, Royal Hallamshire Hospital, Glossop Road, Sheffield,
S10 2JF, U.K. E-mail address: email@example.com
4Abbreviations used in this paper: ROI, reactive oxygen intermediates; ??m, mito-
chondrial membrane potential; Boc-D-fmk, Boc-Asp(OMe)-fmk; BODIPY FL, boron
dipyrromethane difluoride; CTSD, cathepsin D; CTSG, cathepsin G; DAME, diazo-
acetyl-DL-2-aminohexanoic acid-methyl ester; dbcAMP, dibutyryl cAMP; DHR,
dihydrorhodamine; MFI, mean fluorescence intensity; z-VAD.fmk, N-benzyloxycar-
bonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone; DEVD-AMC, 7-amino-4-methyl-
coumarin, N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
by guest on June 13, 2013
generation and altered redox status (12). It is also unclear why
neutrophils are exquisitely sensitive to pyocyanin. We therefore
investigated the mechanisms of pyocyanin-induced apoptosis in
neutrophils, and describe a novel pathway of pathogen-mediated
neutrophil apoptosis, characterized by lysosomal acidification and
activation of cathepsin D (CTSD).
Materials and Methods
Neutrophil isolation and culture
Human neutrophils were isolated by dextran sedimentation and plasma-
Percoll (Sigma-Aldrich) gradient centrifugation from whole blood of nor-
mal volunteers (15). The studies were approved by the South Sheffield
Research Ethics Committee, and subjects gave written, informed consent.
Purity of neutrophil populations (?95%) was assessed by counting ?500
cells on duplicate cytospins. Neutrophils were suspended at 2.5 ? 106/ml
in RPMI 1640 with 1% penicillin/streptomycin and 10% FCS (all Invitro-
gen Life Technologies) and cultured in 96-well Flexiwell plates (BD
Preparation and analysis of pyocyanin
Pyocyanin was prepared by photolysis of phenazine methosulfate (Sigma-
Aldrich) and purified and characterized, as previously described (16).
Assessment of viability and apoptosis
Nuclear morphology was assessed on Diff-Quik-stained cytospins, with
blinded observers counting ?300 cells per slide on duplicate cytospins.
Necrosis was assessed by trypan blue exclusion and was ?2%, unless
indicated. Alternatively, neutrophils were washed in PBS and stained with
PE-labeled annexin V (BD Biosciences) and TOPRO-3 iodide (Molecular
Probes) to identify apoptotic (annexin V?) and necrotic (TOPRO-3?) cells
(17). Samples were analyzed using a FACSCalibur flow cytometer (BD
Biosciences). Twenty thousand events were recorded, and data were ana-
lyzed by CellQuest software (BD Biosciences).
Caspase activity assay
Caspase-3 activity was determined by measuring enzymatically cleaved
fluorescent substrate 7-amino-4-methylcoumarin, N-acetyl-L-aspartyl-L-
glutamyl-L-valyl-L-aspartic acid amide (DEVD-AMC; Bachem), as pre-
viously described (18). Neutrophil lysates were prepared by resuspension
of treated cells in lysis buffer (100 mM HEPES (pH 7.5), 10% w/v sucrose,
0.1% CHAPS, and 5 mM DTT) at a concentration of 1 ? 108/ml. Lysates
were frozen at ?80°C until required. Using the FLUSYS software package
for the PerkinElmer LS-50B fluorometer, lysate equivalents of 5 million
neutrophils were coincubated with 20 ?M Ac-DEVD-AMC in DMSO.
Kinetic data were collected for at least 20 min to ensure stability of activity.
A known amount of free AMC was used to calibrate the system and al-
lowed calculation of caspase-3 activity. In separate experiments, execu-
tioner caspase (caspases-3 and -7) activity was measured using a Caspase-
Glo 3/7 Assay (Promega). Neutrophils were cultured at 5 ? 106/ml and
treated with medium (control), pyocyanin (50 ?M), and pyocyanin with
dibutyryl cAMP (dbcAMP; 100 ?M) for 3 h. Cells were directly trans-
ferred to a white 96-well flat-bottom plate (Dynex Technologies) at a den-
sity of 62,500 cells/well in a 25-?l vol. An equivalent volume of Caspase-
Glo 3/7 buffer mixed with substrate reagent was added to each well. The
plate was read using a Lumistar Galaxy Luminometer (BMG Labtechnolo-
gies) at 25°C for 200 cycles.
ATP and glucose measurements
ATP was measured using a commercially available bioluminescent kit
(Sigma-Aldrich) using a Lumistar Galaxy Luminometer. Glucose was as-
sayed by detecting change in glucose concentration in lysates and culture
supernatants using a commercial kit (Sigma-Aldrich), as previously de-
scribed (19). Neutrophils were cultured in RPMI 1640 alone, with a glu-
cose concentration of 2 mg/ml. Both assays were standardized using known
concentrations of ATP and glucose, respectively (data not shown).
Modulation of pyocyanin-induced apoptosis
Neutrophils were incubated in the presence and absence of pyocyanin fol-
lowing preincubation with candidate modulators of pyocyanin-induced
apoptosis. Except where indicated, a concentration of 50 ?M pyocyanin
was used because it significantly accelerates neutrophil apoptosis ?5-fold
at 5 h (12). The pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-
Asp(O-methyl) fluoromethyl ketone (z-VAD.fmk) (18), was obtained from
Enzyme Systems Products, and Boc-Asp(OMe)-fmk (Boc-D-fmk) (50
?M) was obtained from Calbiochem. MeOSuc-Ala-Ala-Pro-Ala-chloro-
methylketone (Bachem) (10 ?M) and elastatinal (Sigma-Aldrich) (10 ?M)
were used as neutrophil elastase inhibitors, with optimal inhibitory con-
centrations determined by assessment of inhibition of fluorogenic substrate
digestion by purified neutrophil elastase (data not shown). BB94 (1 ?M;
gift from British Biotechnology, Oxford) was used as a pan matrix metal-
loproteinase inhibitor (20). Specific cathepsin inhibitors were CA-074Me
(25 ?M) for cathepsin B (Calbiochem), pepstatin A (10 ?M; Sigma-
Aldrich) and diazoacetyl-DL-2-aminohexanoic acid-methyl ester (DAME;
Bachem) for CTSD, and CK-08 (1 ?M; Enzyme Systems Products) for
cathepsin G (21). Bafilomycin A1 (100 nM) (Sigma-Aldrich) inhibits the
membrane vacuolar ATPase (22).
ROI production was assessed by incubating 5 ? 105neutrophils in 200 ?l of
RPMI 1640 with 5 ?M dihydrorhodamine (DHR; Sigma-Aldrich) for 30 min
at 37°C, and measuring fluorescence in the FL-1 channel by flow cytometry.
Peripheral blood neutrophil culture in hypoxia was performed, as previ-
ously described (23). Neutrophils were resuspended at 5 ? 106/ml in RPMI
1640 plus 10% FCS and incubated in normoxic (19 kPa) or hypoxic (3 kPa)
environments in the presence or absence of pyocyanin (50 ?M) for 6 h.
Normoxia was controlled using a humidified 5% CO2/air incubator, and
hypoxia, by pregassing medium for 30 min in a sealed hypoxic work sta-
tion with 5% CO2/balance N2gas mix and subsequent culture in a humid-
ified hypoxic (CO2/N2) incubator. Cytospins were prepared and apoptosis
was scored by light microscopy.
Assessment of mitochondrial and lysosomal membrane
To detect loss of ??m, neutrophils were incubated with 10 ?g/ml JC-1
dide; Molecular Probes) at 37°C. Loss of ??mwas assayed by observing
a shift in fluorescence emission from red (?590 nm) to green (?525 nm)
using flow cytometry (24). Neutrophils were treated with valinomycin (100
?M; Sigma-Aldrich) as a positive control. Lysosomal pH was measured by
incubating neutrophils with 1 mg/ml FITC-dextran 70S (Sigma-Aldrich), a
pH-sensitive fluorescent probe, for 30 min at 37°C. Increasing pH (i.e., loss
of acidification) within the lysosomal compartment is associated with in-
creased green fluorescence detected in the FL-1 channel (25). Loss of ly-
sosomal acidification was determined by incubating neutrophils with 5 ?M
acridine orange (Sigma-Aldrich) for 30 min at 37°C, and loss of FL-3
fluorescence was measured by flow cytometry (21). Cytospins were also
prepared and viewed by fluorescence microscopy. Neutrophils treated with
100 nM bafilomycin A1, a known inhibitor of vacuolar ATPase (a key
regulator of lysosomal pH Ran, 2003 no. 965), were used as a positive
control in these experiments.
Boron dipyrromethane difluoride (BODIPY FL)-pepstatin A (Molecular
Probes) is a fluorescent pH-sensitive probe used to measure the sub-
cellular distribution of CTSD (26). Neutrophils were treated with me-
dium (control), bafilomyin A1 (100 nM), or pyocyanin (50 ?M) for 30
min. The cells were washed and incubated with 1 ?M BODIPY FL-
pepstatin A (in medium) at 37°C for 30 min, after which they were
washed, resupended in medium, and incubated at 37°C for a further 60
min to allow endosomal trafficking. Cytospins were prepared and
viewed under a fluorescent microscope (Leica AF6000, ?63 objective).
CTSD-labeled BODIPY FL-pepstatin A was visible as punctate fluo-
SDS-PAGE and Western immunoblotting
Whole-cell extracts were used for Mcl-1 and actin immunoblots and pre-
pared as described (27). Cytosolic and membrane fractions used in CTSD
and cathepsin G (CTSG) immunoblots were prepared by sonication (three
10-s bursts in HBSS supplemented with protease inhibitor mixture III (Cal-
biochem), followed by 25,000 rpm microcentrifugation for 45 min). Pro-
teins were separated by 15% v/v SDS-PAGE and blotted onto nitrocellu-
lose membranes (Bio-Rad), and protein transfer was confirmed by Ponceau
S (BDH) staining. Blots were incubated overnight at 4°C for Mcl-1 (S-19;
Santa Cruz Biotechnology) and at room temperature for 2 h for actin (Sigma-
Aldrich), CTSG (Abcam), and CTSD (Calbiochem); protein detection was
3503 The Journal of Immunology
by guest on June 13, 2013
with HRP-conjugated IgG (DakoCytomation) and ECL (Amersham
For multiple comparisons, means and SEM were analyzed by ANOVA
with posttest, as indicated (GraphPad). For comparison of two sample
means, paired Student’s t tests were used.
Pyocyanin-induced neutrophil apoptosis is caspase dependent
Pyocyanin-induced cell death in neutrophils displays both mor-
phological features (nuclear condensation, cell shrinkage) and cell
surface changes (annexin V binding to exposed phosphatidylser-
ine) of apoptosis (12). We therefore investigated whether pyocy-
anin-induced neutrophil death was also associated with caspase
activation, using both morphology and annexin V binding to quan-
tify neutrophil apoptosis. A pan-caspase inhibitor, zVAD.fmk, in-
hibited pyocyanin-induced apoptosis in a concentration-dependent
manner, with significant reduction in neutrophil death at concen-
trations from 5 ?M (Fig. 1A). A total of 50 ?M zVAD.fmk de-
layed pyocyanin-induced death up to 10 h (Fig. 1B); thereafter,
secondary necrosis in pyocyanin-treated cells made estimations of
apoptosis unreliable. Apoptosis of control neutrophils at 5 h was
5.7 ? 1.2% and was not significantly inhibited by zVAD.fmk at
any concentration used (data not shown). A second pan-caspase
inhibitor, Boc-D.fmk, also inhibited pyocyanin-induced apoptosis
at 5 h (pyocyanin-induced apoptosis in the absence (34.8 ? 3.8%)
and presence (21.9 ? 5.5%) of 50 ?M Boc-D.fmk). Because
zVAD-fmk has some caspase-independent effects in neutrophils
(28), we confirmed caspase activation by demonstrating cleavage
of a caspase-3-specific fluorescent substrate, DEVD-AMC. Pyo-
cyanin treatment (4 h) caused a significant increase in neutrophil
intracellular caspase-3 activity compared with untreated controls
(Fig. 1C). Thus, pyocyanin-induced neutrophil apoptosis is de-
layed by caspase inhibition and associated with caspase-3
Pyocyanin-induced oxidative stress mediates neutrophil
The cytotoxic effects of pyocyanin on bacteria and eukaryotic cells
are linked to its ability to undergo nonenzymatic redox cycling
within cells, with resultant ROI formation, and further loss of re-
ducing capacity by direct oxidation of NADH/NADPH and re-
duced glutathione (29). We found that pyocyanin induces pro-
longed generation of ROI in neutrophils as measured by oxidation
of DHR (Fig. 2A), in keeping with our previous observations (12).
To further corroborate the role of oxidative stress in pyocyanin-
induced apoptosis, neutrophils were incubated in a hypoxic envi-
ronment in the presence of pyocyanin for 5 h. We hypothesized
that reduced availability of oxygen would hinder the ability of
pyocyanin to induce apoptosis. Fig. 2B shows pyocyanin is unable
to induce neutrophil apoptosis in hypoxia.
Metabolic activity is maintained in pyocyanin-treated
Pyocyanin-induced ROI production is linked to depletion of intra-
cellular ATP in epithelial cells (29, 30). To determine whether
neutrophil death was associated with cellular ATP depletion in
response to the profound ROI induction, we measured intracellular
ATP in the neutrophil by a bioluminescence technique. We did not
detect a reduction in ATP levels at time points up to 3 h following
pyocyanin treatment (Fig. 3A). Metabolic pathways in pyocyanin-
treated neutrophils remained viable, as shown by this maintenance
of intracellular ATP, and by our subsequent experiments, which
showed increased glucose uptake (Fig. 3B) and maintenance of
intracellular glucose concentrations in these cells (Fig. 3C).
ROI are a feature of the stress pathway of apoptosis, and ROI
production leads to loss of mitochondrial membrane potential
(??m) (14). Because pyocyanin can interrupt mitochondrial respi-
ration in epithelial cells as a result of ROI generation (29), we
determined whether pyocyanin-mediated ROI production was in-
ducing neutrophil apoptosis via loss of ??m. We measured ??min
neutrophils using the mitochondrial dye, JC-1. Fig. 4A shows flow
cytometry dot plots illustrating the distribution of high-FL-1 flu-
orescent neutrophils (associated with a loss of ??m(24, 31)) in
control, pyocyanin-, and valinomycin-treated populations at 4 h.
dent. A, Neutrophils were treated with pyocyanin (50 ?M) in the presence
or absence of a concentration range of zVAD.fmk for 4 h. Apoptosis was
assessed by morphologic criteria, and chart shows mean ? SEM percent-
age of apoptosis from three independent experiments. Baseline control
apoptosis in these experiments was 5.7 ? 1.2% and was not significantly
inhibited by any concentration of zVAD.fmk (data not shown). Statistically
significant inhibition of pyocyanin-induced apoptosis was observed at con-
centrations of zVAD.fmk of 5 ?M and greater (?, p ? 0.05 and ??, p ?
0.01 ANOVA with Dunnett’s posttest). B, Neutrophils were treated with 50
?M pyocyanin in the absence (?) or presence (f) of zVAD.fmk (50 ?M)
for 0, 2, 5, and 10 h. Apoptosis was assessed by flow cytometry, and the
chart shows mean ? SEM percentage of apoptosis for three independent
experiments. Significant inhibition of pyocyanin-induced apoptosis was
observed at 5 (?, p ? 0.05) and 10 h (???, p ? 0.001; ANOVA, Bonfer-
roni’s posttest). C, Lysates were prepared from neutrophils treated with
medium (control, ?) or 50 ?M pyocyanin (f) for 1 and 4 h. Caspase-3
activity was detected using a specific fluorogenic substrate and measured
kinetically (Flusys software). Pyocyanin significantly increased caspase-3
activity at 4 h (?, p ? 0.0355; Student’s t test).
Pyocyanin-induced neutrophil apoptosis is caspase depen-
3504 MECHANISMS OF PYOCYANIN-INDUCED NEUTROPHIL APOPTOSIS
by guest on June 13, 2013
Pyocyanin induced only modest changes in the proportions of cells
showing loss of ??m, and these nonsignificant changes were only
observed at later time points (Fig. 4B). These observations were
confirmed using a second mitochondrial dye, 3,3-dihexyloxacarbo-
cyanine iodide (data not shown). Thus, although loss of ??map-
pears to occur in pyocyanin-induced neutrophil apoptosis, changes
are not apparent until later time points, when there is already a
significant increase in apoptosis. Loss of ??mwas therefore un-
likely to be an initiating factor in the engagement of apoptosis.
Pyocyanin-induced apoptosis is preceded by changes in
lysosomal pH, but these are not sufficient to induce death
A pathway of cell death is now recognized, activated primarily in
pathological rather than homeostatic circumstances, in which ly-
sosmal dysfunction may precede loss of ??m(32). Lysosomal pH
was measured using a pH-sensitive marker (FITC-conjugated dex-
tran) that is taken up by acidic structures and increases in FL-1
channel fluorescence as pH rises due to loss of protonation (25,
33). Exposure of neutrophils to pyocyanin or bafilomycin A1 (an
inhibitor of the vacuolar (H?)-ATPase that maintains normal ly-
sosomal pH gradients (34)) increased lysosomal pH compared
with untreated controls (Fig. 5).
We confirmed loss of lysosomal acidification in neutrophils by
staining with acridine orange, which is lysosomotropic and accu-
mulates in acidic organelles (35). On fluorescence microscopy
(Fig. 6A), a punctate staining pattern was seen in the cytosol of
control neutrophils, consistent with lysosomal accumulation of the
stain. In neutrophils treated with pyocyanin or bafilomycin A1, the
punctate staining pattern was lost, in keeping with loss of lysoso-
mal acidification (21, 32). Statistically significant losses of fluo-
rescence were detected at 15 min following pyocyanin treatment
(Fig. 6B). Once again, bafilomycin A1 was used as a positive con-
trol in these experiments and, at a concentration (100 nM) that
inhibits V-ATPase function in neutrophils (36), caused loss of ly-
sosomal acidification to an even greater degree than pyocyanin.
However, this concentration of bafilomycin A1 was without effect
on constitutive neutrophil apoptosis (Fig. 6C), in keeping with pre-
vious studies (37), although higher concentrations of bafilomycin
are proapoptotic to neutrophils (data not shown). Lysosomal alka-
linization alone is not, therefore, sufficient to induce neutrophil
CTSD translocation and activation are associated with
Alterations in lysosomal pH alone do not induce apoptosis, and
release of lysosomal proteases is critical for completion of the
apoptotic program in a range of cell types (21, 35). No attenuation
of pyocyanin-induced neutrophil apoptosis was seen by treatment
with elastase inhibitors or a pan matrix metalloproteinase inhibitor
(Fig. 7, A and B). In contrast, significant reductions in pyocyanin-
induced apoptosis were observed with pepstatin A, an inhibitor of
aspartyl proteases (21), both alone and in combination with inhib-
itors of the cathepsins B, G, and L (Fig. 7C). The latter inhibitors
phil apoptosis. A, DHR-loaded neutrophils were treated with medium (con-
trol, ?) or 50 ?M pyocyanin (f) for a range of time points: 30, 60, 120,
and 180 min. Increases in fluorescence were measured by flow cytometry
and indicated enhanced ROI production. The chart shows mean ? SEM of
mean fluorescence intensity (MFI) of three independent experiments. Sta-
tistically significant differences were seen between control and pyocyanin-
treated cells at all time points (???, p ? 0.001; ANOVA, Bonferroni’s
posttest). B, Neutrophils incubated in normoxic (?) or hypoxic (f) con-
ditions were cultured with medium (control) or pyocyanin (50 ?M) for 6 h.
Apoptosis was assessed by light microscopy. Pyocyanin treatment of nor-
moxic neutrophils induced apoptosis (??, p ? 0.01; ANOVA, Bonferroni’s
posttest). Hypoxic neutrophils survived in the presence of pyocyanin, to
levels comparable with the control.
Oxidative stress is essential for pyocyanin-induced neutro-
Neutrophils were treated with medium (control, ?) or 50 ?M pyocyanin
(f) for 1 and 3 h, following which lysates were prepared and intracellular
ATP was measured. No significant differences were observed between the
groups. B and C, Neutrophils were treated with medium (control, ?) or 50
?M pyocyanin (f) for 2 h, and glucose was measured in extracellular (B)
and intracellular (C) compartments. The charts show mean ? SEM from
four independent experiments. A statistically significant decrease in extra-
cellular glucose was detected in pyocyanin-treated cells (?, p ? 0.05; Stu-
dent’s t test).
Pyocyanin leads to metabolic cell stress in the neutrophil. A,
3505The Journal of Immunology
by guest on June 13, 2013
were without effect, either alone or in combinations that excluded
pepstatin A, on constitutive neutrophil apoptosis (data not shown)
or on pyocyanin-induced apoptosis (Fig. 7C). Although pepstatin
A is a specific inhibitor of CTSD, it has been reported to cause
neutrophil activation similar to changes induced by FMLP treat-
ment (38), and we found it also had an antiapoptotic effect upon
neutrophils in the absence of pyocyanin (data not shown). We
therefore investigated the potential of a second and unrelated
CTSD inhibitor, DAME (39), to modulate pyocyanin-induced neu-
trophil cell death. DAME treatment significantly abrogated pyo-
cyanin-induced neutrophil apoptosis (Fig. 7D) at concentrations
that were without effect upon constitutive neutrophil death. CTSD
is bound within the matrix of neutrophil azurophilic granules, but
on activation is released to the cytosol (40).
BODIPY FL-pepstatin A is a pH-dependent fluorescent probe
that binds to CTSD within the acidified lysosomal compartment
(26). We demonstrated the expected pattern of CTSD localization
with punctate cytosolic inclusions in control neutrophils (Fig. 8A).
Neutrophils treated for 30 min with pyocyanin or bafilomycin A1
lost this typical granular staining pattern. To determine whether the
decrease in lysosomal BODIPY FL-pepstatin A staining reflected
alteration in lysosomal pH alone or lysosomal membrane perme-
ability, we assessed translocation of CTSD by Western immuno-
blotting. Fig. 8B shows CTSD distribution in neutrophil lysates
on early mitochondrial dysfunction. Neutrophils were treated with medium
(control, ?), 50 ?M pyocyanin (u), or 1 ?M valinomycin (f) for 1, 2, and
4 h. The fluorescent dye, JC-1 (10 ?g/ml), was used to determine ??m, and
changes in FL-1 fluorescence were measured by flow cytometry. A, Rep-
resentative dot plots showing distribution of cells staining high and low for
JC-1 at 4 h. B, The charts show mean ? SEM percentage of cells with loss
of ??m(high JC-1) from three independent experiments. Valinomycin
caused significant loss of ??mat every time point (??, p ? 0.01; ???, p ?
0.001; ANOVA, Bonferroni’s posttest).
Pyocyanin-induced neutrophil apoptosis does not depend
trative flow cytometry histogram showing right shifts in FL1-H fluores-
cence of FITC-dextran-labeled neutrophils treated with either medium
(control), pyocyanin (50 ?M), or bafilomycin A1 (100 nM) for 2 h. In-
creasing fluorescence in FL-1 reflects elevated intralysosomal pH second-
ary to reduced protonation. B, FITC-dextran-stained neutrophils were in-
cubated with medium (control, ?) or 50 ?M pyocyanin (f) for the
indicated times. The chart shows fold change of MFI (mean ? SEM, n ?
3) vs control at 30 min. Pyocyanin treatment significantly elevated lyso-
somal pH at 120 (???, p ? 0.001) and 240 min (??, p ? 0.01; ANOVA,
Pyocyanin induces loss of lysosomal acidification. A, Illus-
Representative fluorescent microscopy images illustrating typical punctate
staining with acridine orange, which is lost after treatment with pyocyanin
or baflinomycin A1. B, Neutrophils were treated with medium (control, ?),
50 ?M pyocyanin (u), and baflinomycin A1 (100 nM, f) for 15, 30, and
60 min. Loss of lysosomal acidification was assessed with the fluorescent
lysosomal dye, acridine orange, and fluorescence was measured by flow
cytometry. Decreases in FL-3 reflect loss of acridine orange due to lyso-
somal disruption. The chart shows mean ? SEM MFI from three indepen-
dent experiments. Pyocyanin caused significant loss of lysosomal acidifi-
cation at all time points (??, p ? 0.01; ANOVA, Bonferroni’s posttest). C,
Neutrophils were incubated with medium (control, ?) or 100 nM bafilo-
mycin A1 for varying times. Apoptosis was assessed by flow cytometry;
chart shows mean ? SEM percentage of apoptosis (as defined by annexin
V positivity) for three independent experiments.
Pyocyanin induces early loss of lysosomal acidification. A,
3506MECHANISMS OF PYOCYANIN-INDUCED NEUTROPHIL APOPTOSIS
by guest on June 13, 2013
separated into membrane (upper panel) and cytosolic (lower
panel) fractions. The predicted 44- and 31-kDa forms (41) are
present within the membrane fraction of both control and bafilo-
mycin A1-treated cells, whereas pyocyanin-treated cells show
translocation of the 31-kDa form into the cytosol. We also studied
translocation of another cathepsin, CTSG, and demonstrated that
treatment with pyocyanin, but not bafilomycin, caused transloca-
tion to the cytosolic fraction (Fig. 8C), although as shown in Fig.
7C, CTSG inhibition did not abrogate pyocyanin-mediated
A stable cAMP analog retards pyocyanin-mediated neutrophil
apoptosis by regulating CTSD translocation, caspase activity,
and Mcl-1 expression
We previously reported that the synthetic cAMP analog dbcAMP
is able to significantly abrogate pyocyanin-induced apoptosis of
neutrophils (12). Having identified pathways of pyocyanin-in-
duced apoptosis, we asked how dbcAMP might inhibit pyocyanin-
induced death. dbcAMP was unable to inhibit pyocyanin-induced
ROI generation or loss of lysosomal acidification (Fig. 9, A and B).
However, translocation of CTSD to the cytosol in pyocyanin-
treated cells was reduced in cells also treated with dbcAMP (Fig.
9C). Pyocyanin-induced caspase activity was also prevented by
dbcAMP (Fig. 9D). Another potentially important effect of db-
cAMP was also studied. The antiapoptotic bcl-2 family member,
Mcl-1, plays a critical role in the regulation of neutrophil apoptosis
(42), and cAMP analogues have been shown to stabilize Mcl-1
tivation of CTSD. A and B, Medium (control) and pyocyanin (50 ?M)-
treated neutrophils were incubated in the absence (?) or presence of in-
hibitors of elastase (A, elastatinal (10 ?M), u and Me-O-Suc (10 ?M), f)
or matrix metalloproteinases (B, BB94 (1 ?M), u) for 5 h, and apoptosis
was assessed morphologically. None of the inhibitors significantly inhib-
ited apoptosis (ANOVA, Bonferroni’s posttest). C, Pyocyanin (50 ?M)-
treated neutrophils were incubated in the presence or absence of a range of
cathepsin inhibitors for 5 h, and apoptosis was assessed by flow cytometry.
The inhibitor of CTSD and combinations, including the CTSD inhibitor,
significantly inhibited pyocyanin-induced apoptosis (?, p ? 0.05; ??, p ?
0.01; ANOVA, Dunnett’s posttest). D, Neutrophils were incubated for 5 h
with medium (?) or pyocyanin (50 ?M) (f) both alone and in the presence
of a concentration range of the CTSD inhibitor, DAME. Apoptosis was
assessed by morphology and is expressed as fold change from either con-
trol or pyocyanin alone. Five-micromolar DAME significantly inhibited
pyocyanin-induced apoptosis and was without effect on constitutive
apoptosis (?, p ? 0.05; ANOVA, Dunnett’s posttest).
Pyocyanin-induced neutrophil apoptosis is mediated by ac-
treatment. A, Neutrophils were treated with medium (control), pyocyanin
(50 ?M), or bafilomycin A1 (100 nM) for 30 min before staining with the
CTSD probe, BODIPY FL-pepstatin A (1 ?M). Cytospins were prepared,
and the cells were viewed by fluorescence microscopy. Cells without
BODIPY FL-pepstatin A were used to measure background fluorescence
(data not shown). Cells treated with pyocyanin or bafilomycin A1 lost the
punctate fluorescent inclusions that are typical of granular localization,
indicating translocation of CTSD and/or loss of lysosomal acidifcation. B,
Membrane (upper panel) and cytosolic (lower panel) fractions were pre-
pared from 4 ? 106neutrophils treated with medium or pyocyanin (50
?M) for 30 min and subjected to SDS-PAGE immunoblotting. Blots were
probed for CTSD and showed the appearance of the 31-kDa fragment of
CTSD in the pyocyanin-treated cytosolic fractions that was not present in
the cytosol of control or bafilomycin A1-treated cells. C, Cytosolic frac-
tions were prepared from neutrophils treated with medium (control), pyo-
cyanin (50 ?M), or bafilomycin A1 (100 nM) for 30 min. SDS-PAGE
immunoblots were probed for CTSG and actin (loading control) and
showed significantly more CTSG in the pyocyanin-treated sample com-
pared with control or bafilomycin A1.
CTSD is translocated to the cytosol following pyocyanin
3507 The Journal of Immunology
by guest on June 13, 2013
protein levels in neutrophils (43). We showed pyocyanin treatment
of neutrophils reduced Mcl-1 protein levels, as did cycloheximide
and sodium salicylate treatment, as previously described (44), but
this effect of pyocyanin was reversed by coincubation with db-
cAMP (Fig. 9E). These data suggest dbcAMP modulates pyocy-
anin-induced neutrophil apoptosis via multiple downstream mech-
anisms that include reduced CTSD translocation, caspase
activation, and stabilization of Mcl-1.
In these studies, we describe a novel mechanism of pathogen-in-
duced subversion of neutrophil apoptosis that is critically depen-
dent upon disruption of intracellular organelles and protease ac-
tivity. This pathway is highly analogous to the recently described
lysosomal death pathway, used in the regulation of cell survival in
a range of pathological processes (reviewed by Guicciardi et al.
(32)), providing additional insights into the pathways capable of
regulating neutrophil survival.
Pyocyanin is a low m.w., bluish pigment secreted by P. aerugi-
nosa that determines the characteristic color of infected pus and
sputum. It is a major factor responsible for oxidant-dependent kill-
ing of C. elegans by P. aeruginosa through its ability to undergo
redox cycling and to cause superoxide generation (45, 46), and
production of pyocyanin is an important determinant of severity in
murine models of sepsis (9). The important potential for pyocyanin
to be an agent of immune subversion by prevention of neutrophil-
mediated bacterial clearance has only recently been realized. To
this end, we have shown pyocyanin both acclerates neutrophil
apoptosis in vitro (12) and in vivo (13) and impairs clearance of P.
aeruginosa from the lung (13).
In this work, we show that pyocyanin-induced neutrophil death
is caspase dependent and associated with increased executioner
caspase activity, which is likely to be attributable to caspase-3
apoptosis. A, Neutrophils were preloaded with DHR and incubated with
A stable cAMP analog retards pyocyanin-mediated
pyocyanin in the presence (f) or absence (?) of dbcAMP (100 ?M) for
3 h. Increases in fluorescence were measured by flow cytometry (FL-1) and
indicated enhanced ROI production. Chart shows mean ? SEM MFI from
four individual experiments. Pyocyanin-induced ROI production was un-
affected by dbcAMP (ANOVA, Bonferroni’s posttest). B, Neutrophils were
treated with medium (control), bafilomycin A1 (100 nM), or pyocyanin (50
?M) alone or in combination with dbcAMP (100 ?M) for 30 min. Loss of
lysosomal acidification was assessed with acridine orange, and fluores-
cence was measured by flow cytometry. Decreases in FL-3 reflect loss of
acridine orange due to lysosomal disruption. The chart shows mean ?
SEM MFI from three independent experiments (??, p ? 0.01; ???, p ?
0.001; ANOVA, Bonferroni’s posttest). Pyocyanin-induced loss of lyso-
somal acidification was not prevented by dbcAMP. C, Membrane (upper
panel) and cytosolic (lower panel) fractions were prepared from 4 ? 106
neutrophils treated for 30 min with medium (control), bafilomycin A1 (100
nM), or pyocyanin (50 ?M) alone or in combination with dbcAMP (100
?M). SDS-PAGE immunoblots were probed for CTSD and show the trans-
location of the 31-kDa form of CTSD to the cytosol in pyocyanin-treated
lysates, which was reduced in the presence of dbcAMP. D, Neutrophils
were incubated for 3 h with medium (control) or pyocyanin (50 ?M) alone
or in combination with dbcAMP (100 ?M). Executioner caspase (3 and 7)
activity was measured using a Caspase-Glo 3/7 assay. Chart shows fold
change from control. Caspase activity induced by pyocyanin is signifi-
cantly greater than control and is inhibited by dbcAMP (???, p ? 0.001;
ANOVA, Bonferroni’s posttest). E, Whole cell protein lysates were pre-
pared from neutrophils treated for 2 h with pyocyanin (50 ?M), cyclohex-
imide (CHX, 10 ?g/ml) plus sodium salicylate (sal, 10 ?M) or pyocyanin
plus dbcAMP (100 ?M) and subjected to SDS-PAGE immunoblotting.
Blots were probed for Mcl-1 (upper panel) and actin (loading control,
lower panel). dbcAMP prevented pyocyanin-induced degradation of
3508 MECHANISMS OF PYOCYANIN-INDUCED NEUTROPHIL APOPTOSIS
by guest on June 13, 2013
since it is the major executioner caspase in human neutrophils (47,
48). These data provided biochemical confirmation that the cell
death induced was apoptotic and represented a subversion of im-
portant normal regulatory pathways controlling neutrophil lifes-
pan. We then sought the apoptotic pathways upstream of caspase
activation to determine the mechanism of pyocyanin-induced neu-
trophil killing. The dependence upon ROI for pyocyanin-induced
death was implied by effects of antioxidants in our previous studies
(12), and in this study we further show the proapoptotic effects of
pyocyanin were prevented by culture in hypoxia. Recent studies in
epithelial cells found pyocyanin, in addition to causing intracellu-
lar ROI generation, can directly oxidize both NADH and NAPDH
(49). This loss of cellular reducing capacity is associated with im-
paired glycolysis (50) and reduced levels of cyclic nucleotides,
particularly ATP (29, 51). Although detailed experiments studying
the energetic status of the cell were not performed, intracellular
levels of ATP and glucose were maintained following pyocyanin
treatment, data that are in keeping with the requirement of neu-
trophils to maintain function in very demanding environments of
low pH, low glucose, low oxygen tension, and high oxidative
stress such as in purulent secretions (52). Indeed, preservation of
ATP is necessary for coordinated execution of apoptotic programs,
and ATP depletion favors necrotic cell death rather than classical
apoptosis (53), providing further support for our data showing no
significant loss of intracellular ATP. Neutrophils, again because of
the environments in which they must be active, are unique in that
the majority of ATP generation in these cells occurs via oxygen-
independent glycolysis (54), and we found intracellular glucose
levels were maintained in pyocyanin-treated neutrophils. Glucose
uptake from the extracellular medium was measured and, for un-
treated neutrophils, was comparable to previous studies using
the same methodology (19), with an increased uptake following
pyocyanin treatment that was comparable to that of LPS- or
PMA-activated neutrophils (55, 56).
We then sought evidence for mitochondrial inner transmem-
brane permeabilization, which is characteristic of the stress path-
way of apoptosis characterized by ROI generation (14). Although
mitochondria have a minimal role in ATP generation in neutro-
phils, they do have a critical role in apoptosis induction (24, 57).
Studies in Jurkat T cells have highlighted the ability of ATP de-
rived by glycolysis to maintain ??mfor some hours following a
metabolic insult, a process that is critically dependent upon en-
hanced uptake of extracellular glucose (58). We detected increased
??mfollowing pyocyanin treatment, but this occurred in concert
with, rather than preceding, onset of apoptosis. Thus, whereas ??m
may be part of the amplification mechanism leading to executioner
caspase activation, it is unlikely to be part of the program initiating
A number of studies have shown oxidative stress-induced cell
death is associated with lysosomal destabilization (32) and that
ROI can induce lysosomal permeabilization (35, 59). Within 15
min of pyocyanin treatment of neutrophils, there was evidence of
alkalinization of the lysosomal compartment that preceded any de-
tectable changes in ??mor caspase-3 activity. A lysosomal path-
way of apoptosis that precedes mitochondrial changes is recog-
nized in other cell types, with critical proteases translocating from
lysosomes and other secretory vesicles into the cytoplasm (21, 60).
This pathway is activated primarily in pathological rather than ho-
meostatic circumstances (32) and can be activated by death recep-
tors or lipid mediators (61) and following accumulation of lyso-
somotropic agents (21). The azurophilic or primary granules are
generally regarded as the lysosomal structures within neutrophils,
because they are the major cellular reservoir of acid-dependent
hydrolases, contain lysosomal membrane proteins, and are abnor-
mal in Chediak-Higashi Syndrome, a disorder of lysosomes and
related structures (62, 63). However, because they lack classical
lysosomal membrane markers such as lysosomal-associated mem-
brane proteins 1 and 2 (62, 64), they are sometimes described as
lysosome-related organelles (63).
Recent studies by Ran et al. (22) provided important insights
into the actions of pyocyanin. Yeast mutants with reduced sensi-
tivity to pyocyanin frequently had mutations in the V-ATPase, an
enzyme complex involved in mitochondrial electron transport and
ATP synthesis, but also a major regulator of lysosomal pH (65).
Ran et al. (22) found pyocyanin both induced lysosomal membrane
permeabilization and inhibited V-ATPase function in epithelial
cells, with high concentrations (2 mM) of a V-ATPase inhibitor,
bafilomycin A1, having similar effects to pyocyanin. In neutro-
phils, bafilomycin A1, at a concentration (100 nM) that inhibits
neutrophil V-ATPase function (36), reduced lysosomal acidifica-
tion to an even greater degree than pyocyanin. This concentration
of bafilomycin A1 was, however, without effect on neutrophil
apoptosis, in keeping with previous studies (37). Two other global
regulators of intracellular pH, amiloride (an inhibitor of Na?/H?
exchangers) and zinc chloride (an inhibitor of NADPH-oxidase-
associated proton channels), were also without effect on pyocya-
nin-induced apoptosis (data not shown). Our findings support those
of Ran et al. (22) in demonstrating pyocyanin-induced loss of ly-
sosomal acidification in neutrophils that is most likely mediated
via the lysosmal V-ATPase, but also show lysosomal alkaliniza-
tion alone is not sufficient to induce apoptosis. It is not clear
whether the effect of pyocyanin upon V-ATPase function is indi-
rect, perhaps resulting from ROI generation, or whether pyocyanin
is lysosomotropic and binds and directly inhibits V-ATPase func-
tion, as has been described for other agents, e.g., quinolones (21)
and concanamycin (66).
Lysosomes contain multiple potent proteases that contribute to
bacterial killing (67), several of which have been associated with
onset of apoptosis (32). Using a series of broad and narrow-spec-
trum protease inhibitors, we identified a role for CTSD in pyocy-
anin-induced apoptosis. We found that a specific CTSD inhibitor,
pepstatin A, delayed pyocyanin-induced death, but also caused ac-
tivation of neutrophils (38) (our data not shown). We therefore
used a second specific CTSD inhibitor (DAME) that also delayed
pyocyanin-induced neutrophil apoptosis. We identified CTSD
staining in neutrophils, with the expected distribution in subcellu-
lar organelles, and we detected translocation of a 31-kDa fragment
of CTSD from the membrane to the cytosolic fraction of neutro-
phils following pyocyanin treatment. These data are in keeping
with data showing a role for CTSD in apoptosis of fibroblasts (59,
68) and endothelial cells (69) following oxidant stress. Impor-
tantly, neutrophil primary granules contain significant amounts of
CTSD (40, 70), accounting for 38% of acidic protease activity of
neutrophils (71). Although CTSG was also released into the cy-
tosol by pyocyanin treatment, CTSG inhibition did not abrogate
apoptosis, suggesting a particular role for CTSD in this system.
Our work showing that pyocyanin destabilizes neutrophil granules
and releases CTSD into the cytosol may in part explain the ex-
quisite susceptibility of neutrophils to pyocyanin-induced apopto-
sis (12), because cells with lower or absent numbers of CTSD-
containing granules (such as epithelial cells) are not stimulated to
die when exposed to pyocyanin. The mechanisms by which CTSD
induces apoptosis are uncertain. CTSD release is upstream of
caspase activation (72), although a recent overexpression study
suggests that the catalytic activity of CTSD is not essential for its
proapoptotic role (73). The recent studies of Blomgran et al. (74)
3509 The Journal of Immunology
by guest on June 13, 2013
show a role for cathepsins, including CTSG, in mediating Esche-
richia coli-induced neutrophil apoptosis, with evidence of cathe-
psin-mediated Bid cleavage and down-regulation of Mcl-1. Our
studies also demonstrate reductions in Mcl-1 protein, and that res-
toration of Mcl-1 protein levels by dbcAMP is associated with
delay of pyocyanin-induced apoptosis.
A number of pathogens such as E. coli (75), Staphylococcus
aureus (76), and Streptococcus pyogenes (77) are associated with
neutrophil apoptosis following phagocytosis; this is likely to be a
host-mediated process to prevent intracellular persistence of bac-
teria (78). However, our studies and those of Blomgran et al. (74)
identify a relationship between lysosome function and apoptosis in
bacterial infection has not previously been recognized, despite the
prominent role of lysosomal proteases in antibacterial responses of
neutrophils. In these studies, pathogens effectively subvert these
processes, with release of granule proteases triggered by bacterial-
driven ROI generation.
In summary, we have demonstrated pyocyanin induces apopto-
sis by engagement of lysosomal pathways of cell death, and we
provide evidence it may be a pathological mechanism of cell death
to which neutrophils are particularly susceptible. Furthermore, this
is the first description of a bacterial toxin using this pathway of
mammalian cell apoptosis to subvert host defense. Our findings are
of clinical relevance, because understanding mechanisms to inhibit
pyocyanin-induced neutrophil death in P. aeruginosa infections
may lead to development of therapies that favor an effective im-
mune response to this major human pathogen.
M. K. Whyte has received a small research grant from GlaxoSmithKline
relating to a multi-centre asthma genetics study. There is no overlap with
this study in any way.
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3511The Journal of Immunology
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