Impairment of Apoptotic Cell Engulfment by Pyocyanin,
a Toxic Metabolite of Pseudomonas aeruginosa
Stephen M. Bianchi1*, Lynne R. Prince1*, Kathleen McPhillips2, Lucy Allen3, Helen M. Marriott1, Graham W. Taylor4,
Paul G. Hellewell3, Ian Sabroe1, David H. Dockrell5, Peter W. Henson2, and Moira K. B. Whyte1
1Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom;
2Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado;3Academic Unit of Cardiovascular Research, School of
Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom;4Department of Medicine, Hampstead Campus, Royal Free
and University College School of Medicine, London, United Kingdom; and5Academic Unit of Infectious Diseases, School of Medicine and
Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom
Rationale: Cystic fibrosis lung disease is characterized by accumula-
cells. Pseudomonas aeruginosa, the principal microbial pathogen in
cystic fibrosis, manipulates apoptosis induction via production of
toxic metabolites. Whether these metabolites, particularly pyocya-
nin, can also modulate apoptotic cell engulfment is unknown.
Objectives: To assess the effects of pyocyanin on apoptotic cell
engulfment by macrophages in vitro and in vivo and to investigate
potential mechanisms of the observed effects.
pyocyanin before challenge with apoptotic neutrophils, apoptotic
Jurkat cells, or latex beads, and phagocytosis was assessed by light
microscopy and flow cytometry. Effects of pyocyanin production on
apoptotic cell clearance in vivo were assessed in a murine model,
comparing infection by wild-type or pyocyanin-deficient P. aerugi-
and pharmacologic inhibition and Rho GTPase signaling by immu-
noblotting and inhibitor studies.
Measurements and Main Results: Pyocyanin treatment impaired mac-
rophage engulfment of apoptotic cells in vitro, without inducing
significant macrophage apoptosis, whereas latex bead uptake was
preserved. Macrophage ingestion of apoptotic cells was reduced
and late apoptotic/necrotic cells were increased in mice infected
with pyocyanin-producingP. aeruginosa comparedwith the pyocya-
nin-deficient strain. Inhibition of apoptotic cell uptake involved
on Rho GTPase signaling. Antioxidants or blockade of Rho signaling
substantially restored apoptotic cell engulfment.
Conclusions: These studies demonstrate that P. aeruginosa can ma-
nipulate the inflammatory microenvironment through inhibition of
apoptotic cell engulfment, and suggest potential strategies to limit
pulmonary inflammation in cystic fibrosis.
Keywords: macrophages; phagocytosis; apoptosis; inflammation;
Resolution of inflammation involves apoptosis of recruited
inflammatory cells and their recognition and clearance by pro-
fessional phagocytes (1). These mechanisms can clear substan-
tial inflammatory infiltrates without significant inflammatory
cell necrosis or bystander tissue injury (2), but dysregulation of
these efficient clearance systems is now widely described in
inflammatory disease (3). The pathology of cystic fibrosis (CF)
lung disease is characterized by a massive chronic neutrophilic
inflammation of the airways, with the infiltrate containing
excessive numbers of both apoptotic and necrotic neutrophils
on a scale not seen in other inflammatory lung diseases (4).
These findings could reflect increased neutrophil apoptosis,
or impairment of apoptotic cell clearance, or a combination of
these processes. This accumulation of effete neutrophils has
important functional consequences, particularly the liberation
of granule proteases (4, 5), and there is evidence in CF that
neutrophil elastase can impair apoptotic cell clearance (4).
Pseudomonas aeruginosa is an opportunistic pathogen that
causes a range of infections in the immunocompromised host
and is the principal cause of mortality in CF lung disease (6).
P. aeruginosa produces a range of factors that modify host
immune responses and contribute to its pathogenicity (7). We
have begun to dissect the impact of P. aeruginosa on both
induction of apoptosis and clearance of apoptotic cells. In host–
pathogen interactions, pathogen-driven neutrophil apoptosis is a
well-recognized mechanism of immune evasion used by a num-
ber of bacteria (8), and we and others have shown P. aeruginosa
can induce neutrophil apoptosis (9, 10). There are, however,
no reports of a microbial factor modulating the engulfment of
apoptotic cells by professional phagocytes such as macrophages.
P. aeruginosa produces highly diffusible, pigmented toxic sec-
ondary metabolites, known as phenazines, that play a major role
in killing infected organisms such as Caenorhabditis elegans and
mice (11) and, in pneumonia models, cause extensive tissue
damage (12). We have shown that pyocyanin, the principal phen-
azine generated by P. aeruginosa, induces rapid apoptosis of
neutrophils (9, 13). Excess apoptotic neutrophils are detected in
mice infected with a pyocyanin-producing, wild-type P. aeruginosa
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Engulfment of apoptotic cells plays a crucial role in the
resolution of inflammation and infection.
What This Study Adds to the Field
This study reports that a bacterial toxin modulates the
ingestion of apoptotic cells, and identifies an additional
mechanism by which pathogens subvert the host response
to favor their own survival.
Supported by a Wellcome Trust Clinical Training Fellowship to S.M.B. (ref.
064997) and the Sheffield Hospitals Special Trustees. D.H.D. is a Wellcome
Senior Clinical Fellow and I.S. holds a Medical Research Council Senior Clinical
*These authors contributed equally to this article and are joint first authors.
Correspondence and requests for reprints should be addressed to Prof. Moira
Whyte, F.R.C.P., Academic Unit of Respiratory Medicine, School of Medicine and
Biomedical Sciences, M Floor, Royal Hallamshire Hospital, Sheffield S10 2JF, UK.
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.200612-1804OC on October 4, 2007
Internet address: www.atsjournals.org
Vol 177. pp 35–43, 2008
as compared with pyocyanin-deficient strains (13), confirming
acceleration of apoptosis but also raising the possibility that
pyocyanin might alter clearance of apoptotic neutrophils.
We therefore examined whether pyocyanin modulated up-
take of apoptotic neutrophils by macrophages. We show signifi-
cant reductions in apoptotic cell engulfment in vitro that are
confirmed in a murine pneumonia model in vivo and are not due
to reduced macrophage viability. We further show that impair-
ment of apoptotic cell engulfment is dependent on reactive
oxygen species (ROS) generation by macrophages and modu-
lation of GTPase activity. To our knowledge, this is the first
description of a microbial factor modulating apoptotic cell en-
gulfment and identifies a potentially important mechanism of
host tissue damage in infection.
Pyocyanin was prepared by photolysis of phenazine methosulphate
(Sigma, Poole, UK) and purified and characterized as described (14).
Dihydroethidium (DHE), dihydrorhodamine (DHR), carboxyl-modi-
fied green fluorescent latex beads were from Sigma. Hoechst 33342,
MnTBAP, and the Rho-kinase inhibitor (Y-27632) were from Calbio-
chem (San Diego, CA), C3 transferase (C3T) protein from Cytoskeleton
(Denver, CO), and Rho/Rac activity assay from Upstate (Charlottesville,
VA). All media, antibiotics and sera were from Life Technologies
(Glasgow, UK). The terminal deoxynucleotidyl transferase biotin-dUTP
nick end labeling (TUNEL) ApopTag Plus Peroxidase In Situ Apo-
ptosis Detection kit used for in vivo studies was from the Intergen Co.
(Oxford, UK), the TUNEL ApopTag Fluorescein Direct In Situ
Apoptosis Detection kit used for assessment of human monocyte–
derived macrophage (HMDM) apoptosis from Chemicon (Hampshire,
UK), the Caspase-Glo 3/7 assay was from Promega (Southampton,
UK), annexin V was from BD PharMingen (Oxford, UK), and To-Pro3
was from Invitrogen (Paisley, UK).
Cell Isolation and Culture
Human peripheral blood cells, neutrophils and PBMCs, were isolated
from whole blood of healthy volunteers by dextran sedimentation and
from the South Sheffield Research Ethics Committee (Sheffield, UK),
and all subjects gave informed consent. Resulting neutrophil populations
were more than 97% pure, with the majority of contaminating cells being
eosinophils. PBMCs were plated and matured to human monocyte-
derived macrophages (HMDM) as previously described (1) and studied
at Days 6–8 in culture. Jurkat cells were obtained from American Type
Induction and Assessment of Apoptosis
Polymorphonuclear leukocytes (PMNs) (.95% purity) were cultured
at 2.5 3 106/ml in RPMI with 1% penicillin/streptomycin and 10% fetal
calf serum in 96-well Flexiwell plates (BD PharMingen) for 24 hours.
At the 24-hour time point, cells were washed and shown to be typically
60 to 70% apoptotic by assessment of nuclear condensation on Giemsa-
stained cytospins and necrosis was less than 2% by trypan blue
exclusion. Jurkat cells were exposed to ultraviolet irradiation at 254
nm for 10 minutes, with similar levels of apoptosis. HMDM numbers
and apoptosis were assessed by identification of characteristic nuclear
morphology of apoptosis in Hoechst 33342–stained cells on fluores-
cence microscopy (15). In further experiments, HMDMs grown on
coverslips were assessed for apoptosis by TUNEL, using an ApopTag
Fluorescein Direct In Situ Apoptosis Detection kit following the
manufacturer’s recommended protocol. Coverslips were mounted on
slides using VectaShield mounting media (Vector Labs, Peterborough,
UK) containing 49-6-diamidino-2-phenylindole (DAPI) (nuclear coun-
terstain). Caspase 3/7 activity of HMDMs was measured using a Cas-
pase-Glo 3/7 assay, following the manufacturer’s recommended pro-
tocol. Briefly, 24 hours post-treatment, HMDMs were incubated with
Caspase-Glo reagent for 1 hour in the dark, and the luminescence was
measured with a Lumistar Galaxy luminometer (BMG Lab Technol-
ogies Ltd, Offenburg, Germany).
HMDMs were cocultured for 1 hour with ‘‘aged’’ neutrophils (range,
60–70% apoptotic) suspended in 500 ml of Iscove’s medium (without
serum) in 24-well plates. Uningested cells were removed by washing
with Hanks balanced salt solution (HBSS), then plates were fixed and
stained for myeloperoxidase (1). Phagocytosis was determined by visual
counting of 500 HMDMs in duplicate wells and was scored both as per-
centage of HMDM ingesting and as phagocytic index, generated by mul-
tiplying the percentage of macrophages that had phagocytosed cells by
the average number of apoptotic cells ingested per macrophage (16).
Uptake of fluorescent Cell Tracker (Invitrogen)–loaded apoptotic neu-
trophils or Jurkat cells was measured by flow cytometry (FACSCalibur;
Becton Dickinson, San Jose, CA) using CellQuest software (Becton
Dickinson), as previously described (17). Uptake of mixed serum-
opsonized carboxyl-modified green fluorescent latex beads was scored
by fluorescence microscopy, and we confirmed there were no differ-
ences in ingested rather than adherent cells by confocal microscopy
(data not shown). In experiments using pharmacologic inhibitors,
HMDMs were treated with the inhibitor before the addition of
pyocyanin for the following times: MnTBAP (1–100 mM), 30 minutes;
Y-27632 (10 mM), 30 minutes; C3T (1 mg/ml), 24 hours. The inhibitors
also remained present throughout the pyocyanin pretreatment period.
Murine Model of P. aeruginosa Infection
This model of acute P. aeruginosa infection has been previously
described (13). Briefly, C57BL6 mice (8–12 wk) were instilled with
1 3 107cfu of bacteria via the trachea, either a pyocyanin-producing
wild-type strain, PA14, or a pyocyanin-deficient but otherwise genet-
ically identical strain, DphnAB (11). At the time points indicated, mice
were killed by overdose and bronchoalveolar lavage (BAL) performed
to obtain total and differential cell counts, including the proportion of
neutrophils that were apoptotic (18). Macrophage ingestion of apopto-
tic cells was identified by morphologic criteria (16) and by TUNEL
staining of BAL cytospins. Apoptotic and necrotic macrophages and
neutrophils in BAL were identified by flow cytometry using annexin V
and ToPro-3 staining, respectively (19).
Assessment of ROS Production
Production of ROS by HMDMs was assessed using cell-permeable
DHR staining (5 mM for 30 min) and cells were analyzed by flow
cytometry (20), with each sample run in triplicate. HMDMs were also
stained with the oxidant-sensitive fluorescent probe (DHE), and ROS
production assessed by nuclear fluorescence via microscopy (21).
Rho/Rac Activity Assays
Rho/Rac activity assays were performed according to the manufac-
turer’s instructions. Briefly, 2.5 3 107HMDMs were stimulated for
time points as indicated. Active Rho and Rac were isolated by
incubating lysates with sepharose beads bound to Rhotekin or p-21
activated kinase (PAK), respectively, which were run using a sodium
dodecyl sulfate–polyacrylamide gel electrophoresis. Whole cell lysates
were run on separate gels and total Rho/Rac levels were evaluated
using mouse anti-Rho (Cytoskeleton) or mouse anti-Rac (Upstate).
The densitometry was measured using Image J software (National
Institutes of Health, Bethesda, MD) and expressed as a percentage of
the untreated control.
Results are expressed as mean 6 SEM. Analysis of variance (ANOVA)
was applied for multiple comparisons, and posttests were applied
where appropriate. Student’s t tests were used for comparison of two
sample means. Where important data comparisons did not reveal
significant differences, this is indicated by NSD (no significant differ-
ence). All data were analyzed using GraphPad Prism version 4.0b (San
36 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1772008
Pyocyanin Impairs Engulfment of Apoptotic Cells
To determine whether pyocyanin could inhibit apoptotic cell
engulfment, we assayed phagocytosis of apoptotic PMNs
(APMNs) by HMDMs in vitro. Pyocyanin caused a concentra-
tion- and time-dependent reduction in phagocytosis of APMNs.
Of note, pyocyanin reduced both the proportion of HMDMs
that ingested APMNs and the average number of APMNs
ingested (Figure 1). In three further experiments, we confirmed
that the defect was of ingestion rather than adherence of
apoptotic cells to HMDMs using a flow-based assay to detect
engulfment of Cell Tracker–labeled APMNs (see Table E1 in
online supplement). Because pyocyanin causes acceleration of
neutrophil apoptosis (9) and loss of macrophage viability would
lead to loss of phagocytic capacity, we confirmed that pyocyanin
was not causing macrophage death using three independent
methods. First, pyocyanin-treated HMDMs were stained with
a fluorescent nuclear dye, Hoechst 33342, and no significant loss
of cell numbers was detected in pyocyanin-treated HMDMs
compared with untreated control cells (Figures 2A and 2B).
Second, TUNEL staining showed there was minimal, if any,
increase in nuclear changes of apoptosis (Figure 2C), and third
a caspase 3/7 activity assay detected no increase in caspase
activation after pyocyanin treatment, in contrast to a positive
control, staurosporine (Figure 2D).
We addressed whether the phagocytic defect was specific for
apoptotic cell uptake or more generalized. Engulfment of
fluorescent-labeled apoptotic Jurkat cells was assessed by flow
cytometry. Ingestion of these cells by control macrophages was
significantly greater than ingestion of apoptotic neutrophils,
80% compared with 35%, but was significantly reduced after
pyocyanin pretreatment (Figure 3A). In contrast, uptake of
an assay of Fc-receptor–mediated phagocytosis, did not differ
between pyocyanin-treated and control HMDMs (Figure 3B).
Mice Infected with Pyocyanin-producing P. aeruginosa Have
Reduced Clearance of Apoptotic Cells
To determine whether pyocyanin impaired clearance of apopto-
tic cells in an acute P. aeruginosa pneumonia model (13), mice
infected with a wild-type, phenazine-producing strain were
compared with mice infected with a phenazine-deficient strain
that has only 10% of wild-type pyocyanin production (11). The
model is associated with rapid neutrophil influx and, in mice
infected with phenazine-producing bacteria, with accelerated
neutrophil apoptosis by 18 hours after bacterial instillation
(13). To determine macrophage engulfment of apoptotic cells,
we assessed apoptotic cell uptake by alveolar macrophages using
TUNEL staining (Figure 4A) at 18 and 30 hours after instillation
of bacteria. At 18 hours, total neutrophil counts were similar in
mice infected with both strains (PA14, 1.92 6 0.23 3 106;
DphnAB, 1.80 6 0.12 3 106) but were increased in the pyocya-
nin-deficient infection at 30 hours (PA14, 2.02 6 0.14 3 106;
DphnAB 3.90 6 0.54 3 106; P , 0.05). There were increased
numbers of apoptotic PMNs in BAL from mice infected with the
wild-type strain at 18 hours (PA14, 8.20 6 0.91 3 105; DphnAB,
105; DphnAB, 1.57 6 0.37 3 105; P , 0.05), in keeping with our
previous findings(13).Despitetheexcessof apoptoticcellsinthe
infected PA14 mice, the proportion of macrophages containing
apoptotic cells was lower in these mice (Figures 4B and 4C) and
ingesting macrophages also contained reduced numbers of
apoptotic bodies (Figures 4D and 4E) in wild-type infection
compared with mice infected with the phenazine-deficient strain.
Importantly, increased numbers of late apoptotic/necrotic (To-
Pro3 positive) cells were detected at 30 hours in mice infected
with the pyocyanin-producing strain (Figures 4F and 4G),
suggesting the wave of early apoptotic cells detected at 18 hours
had not been efficiently cleared. Moreover, these data support
the view that the reduced numbers of apoptotic cells detected
within macrophages in wild-type P. aeruginosa infection reflect
Figure 1. Pyocyanin impairs macrophage uptake of apoptotic neutro-
phils. Human monocyte–derived macrophages (HMDMs) were ex-
posed to pyocyanin for up to 24 hours before incubation with
apoptotic polymorphonuclear leukocytes (APMNs) for 1 hour and
phagocytosis of APMNs was quantified by staining for myeloperoxidase
(MPO). (A) Pyocyanin inhibition of apoptotic neutrophil engulfment is
concentration dependent. Phagocytosis is expressed as mean 6 SEM
phagocytic index (% HMDMs phagocytosing 3 mean number APMNs
per phagocytosing HMDM) after exposure to varying concentrations of
pyocyanin for 24 hours. Data shown are from three independent
experiments and significant differences from untreated cells are in-
dicated for each concentration of pyocyanin (significance calculated by
analysis of variance [ANOVA] with Dunnet’s posttest). (B) The percent-
age of HMDMs ingesting APMNs after varying lengths of preincubation
with pyocyanin (50 mM) in five independent experiments and signif-
icant differences from cells harvested at time 0 are indicated (signifi-
cance calculated by ANOVA with Dunnet’s posttest). (C, D) Ingestion of
apoptotic cells is quantified by counting MPO-stained inclusions in
HMDMs by light microscopy. Arrowheads highlight the presence of
MPO-stained apoptotic neutrophils within untreated HMDMs (control;
C), whereas apoptotic neutrophils are largely extracellular in HMDMs
pretreated with pyocyanin for 24 hours (D). PYO 5 pyocyanin.
Statistical significance is illustrated by *P , 0.05, **P , 0.01.
Bianchi, Prince, McPhillips, et al.: Pyocyanin Reduces Cell Clearance37
impaired engulfment rather than increased efficiency of apopto-
tic body degradation by macrophages. In keeping with our
previous data (13), there was no difference in BAL macrophage
numbers in the two groups of mice, and we detected no differ-
ences in macrophage death, assessed by To-Pro3 staining,
between mice infected with the different strains (see Figure
E1). There was a trend toward increased bacterial numbers in
mice infected with wild-type P. aeruginosa (log10cfu: PA14, 5.82
6 0.47; DphnAB, 4.09 6 0.55 at 18 h; and PA14, 4.98 6 0.16;
DphnAB, 4.02 6 0.54 at 30 h). These colony numbers were not
significantly different, perhaps reflecting the sample size, al-
though significant differences were found only at 48 hours and
beyond in our previous study (13).
ROS Production Mediates Reduced Apoptotic Cell Uptake by
The cytotoxic effects of pyocyanin on bacteria and eukaryotic
cells are linked to its ability to undergo nonenzymatic redox
cycling within cells, with resulting ROS generation (22). P.
aeruginosa–induced killing of C. elegans and in a murine model
of sepsis is dependent on both pyocyanin production and ROS
generation (11), and pyocyanin-mediated neutrophil apoptosis
viable. Human monocyte–derived macrophage (HMDM) viability was
assessed using Hoechst 33342 staining and fluorescence microscopy.
Cell counts were performed for cell number and cells were also assessed
for morphologic changes of apoptosis. (A) There was no loss of cell
number in HMDMs pretreated with pyocyanin (50 mM) for 24 hours
(solid bar) relative to controls (open bar) in three independent experi-
ments. NSD signifies no significant difference. Chart shows data
normalized to 100% in controls. (B) Photomicrographs illustrating
typical morphology of viable cells in both control and pyocyanin-
treated populations. (C) HMDM apoptosis was assessed by TUNEL
staining in cells pretreated with pyocyanin (50 mM; solid bar) or
staurosporine (10 mg/ml; shaded bar) for 24 hours relative to controls
(open bar) in four independent experiments. The percentage of
apoptotic cells was not significantly increased in pyocyanin-treated
cells. (D) Caspase 3/7 activity was measured in HMDMs pretreated with
pyocyanin (50 mM; solid bar) or staurosporine (10 mg/ml; shaded bar)
for 24 hours compared with controls (open bar) (n 5 4).
Pyocyanin-treated monocyte-derived macrophages remain
apoptotic cells. (A) Apoptotic Jurkat cells were labeled with Cell Tracker
(Invitrogen) and coincubated with human monocyte–derived macro-
phages (HMDMs) for 1 hour after pretreatment of HMDMs with 50 mM
pyocyanin (solid bar) or media control (open bar) for 24 hours.
Engulfment of apoptotic cells was measured by flow cytometry in
three independent experiments and was significantly reduced by
pyocyanin treatment. (B) HMDMs were pretreated with media (open
bar) or 50 mM pyocyanin (solid bar) for 24 hours before being
coincubated with carboxyl-modified green fluorescent latex beads at
a ratio of 1:5 (HMDM:bead) for 1 hour. Phagocytosis was scored by
fluorescence microscopy based on the percentage of macrophages
containing at least one latex bead and did not differ significantly
between the groups.
The pyocyanin-mediated phagocytic defect is specific for
38 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1772008
is associated with massive and sustained generation of ROS (9).
We measured ROS production in HMDMs using a fluorescent
probe, DHR. We observed a significant increase in ROS 30
minutes after pyocyanin treatment (Figures 5A and 5B) that
remained elevated to 24 hours. The magnitude of ROS gener-
ation by macrophages appeared less than that of neutrophils
(data not shown), possibly reflecting their greater antioxidant
levels (23), which may also explain their resistance to pyocya-
nin-induced apoptosis. An antioxidant and superoxide dismu-
tase-mimetic, MnTBAP, significantly reduced ROS levels at
6 hours (Figure 5C) at concentrations of 1 mM and above. We
confirmed these findings using a second fluorescent probe,
DHE, demonstrating nuclear fluorescence after pyocyanin
treatment that was inhibited by MnTBAP (Figure 5C). A
Figure 4. Pyocyanin production is associated with impaired apoptotic
cell clearance in vivo. C57BL6 mice were instilled with 107cfu live wild-
type strain (PA14; open bars) or phenazine-deficient strain (DphnAB;
solid bars) of P. aeruginosa, and bronchoalveolar lavage (BAL) was
performed after 18 (B, D, F) or 30 (C, E, G) hours. Total and differential
cell counts were obtained by hemocytometer and cytospin counts.
Data shown were obtained from three independent experiments. (A)
Cytospins were TUNEL stained and macrophage phagocytosis of apo-
ptotic bodies (TUNEL positive inclusions, indicated by arrowheads)
visualized by microscopy. (B, C) Phagocytosis of APMNs by macro-
phages was assessed and represented as percentage of macrophages
engulfing APMNs (D, E). Phagocytic index was similarly calculated from
TUNEL-stained cytospins. (F, G) The presence of necrotic cells in BAL
was assessed by ToPro-3 uptake on flow cytometry. Statistical signifi-
cance was calculated by Student’s t test and is illustrated by†P , 0.05,
††P , 0.01.
Figure 5. Pyocyanin impairment of macrophage apoptotic cell uptake
is mediated via reactive oxygen species (ROS) generation. (A) ROS
production by human monocyte–derived macrophages (HMDMs) was
measured by flow cytometry. Dihydrorhodamine-loaded HMDMs were
treated with 50 mM pyocyanin over a range of time points (30, 60, 240,
and 360 min). Representative histograms show increases in fluores-
cence (oxidant production) as right shifts on the x axis (FL-1). (B) Mean
data for ROS production, expressed as geometric mean fluorescence
intensity (MFI) over time, in HMDMs treated with 50 mM pyocyanin in
the presence (solid bars) or absence (open bars) of an antioxidant,
MnTBAP (1 mM). Data were from three independent experiments and
significant differences are indicated for pyocyanin-treated cells (open
bars) for each time point compared with cells at time 0. Results for cells
incubated with MnTBAP and pyocyanin compared with pyocyanin
alone were significantly different only at 360 minutes as indicated by
the bar (significance calculated by analysis of variance with Bonferroni’s
posttest). (C) HMDMs were treated for 6 or 24 hours with media
(control), pyocyanin (50 mM), MnTBAP (100 mM), or pyocyanin and
MnTBAP in combination. Nuclear fluorescence was visualized by
dihydroethidium staining and fluorescence microscopy. Representative
images are shown from a single experiment of a set of three. (D)
Media- and pyocyanin-treated HMDMs were cultured in the presence
(solid bars) or absence (open bars) of MnTBAP (1 mM). HMDMs were
subsequently incubated with APMNs for 1 hour and ingestion scored
by myeloperoxidase staining and microscopy. Data from three in-
dependent experiments are shown as phagocytic index. Statistical
significance was calculated by ANOVA and is illustrated by *P , 0.05,
**P , 0.01, ***P , 0.001.
Bianchi, Prince, McPhillips, et al.: Pyocyanin Reduces Cell Clearance39
previous study showed that some fluorescent probes, including
DHR, can be directly oxidized by redox-active compounds,
including pyocyanin, but the increased fluorescence signal that
results is not prevented by antioxidants (24). Our data show that
the pyocyanin-induced increase in macrophage fluorescence is
significantly inhibited by antioxidant treatment (Figure 5C).
Crucially, MnTBAP, at a concentration that inhibited ROS
production, also reversed pyocyanin-mediated impairment of
apoptotic cell uptake, demonstrating dependence on ROS
generation (Figure 5D).
Pyocyanin Inhibits Apoptotic Cell Uptake by Effects
on Small GTPases
Pathways originally defined in C. elegans involve the Rho family
of low-molecular-weight GTPases in signaling pathways medi-
ating apoptotic cell engulfment (25). The Rho GTPase Rac
facilitates engulfment of apoptotic cells, whereas Rho inhibits
uptake (25). Rho-kinase is a downstream mediator of RhoA
inhibition of uptake of apoptotic cells, and blockade of Rho-
kinase enhances apoptotic cell uptake (26). We therefore
investigated whether pyocyanin-induced ROS might mediate
its actions by alterations in Rho signaling. Rho activity was
measured using Rhotekin to pull down active Rho, and we
analyzed both total and active Rho by Western blotting. We
found that Rho activity was significantly increased after pyo-
cyanin treatment (Figures 6A and 6B), whereas Rac-1 activity,
measured using PAK to pull down active Rac, was significantly
reduced by pyocyanin treatment (Figures 6C and 6D). Thus,
pyocyanin treatment of HMDMs inhibited apoptotic cell up-
take, inhibited Rac, and activated Rho.
We next examined the ability of C3 transferase (from
Clostridium botulinum), which inactivates RhoA, as well as Y-
26732, a specific inhibitor of Rho-kinase, to reverse pyocyanin-
impaired macrophage engulfment of apoptotic neutrophils. We
found that both compounds, at concentrations previously shown
to inhibit their respective targets (26), substantially restored
engulfment of APMNs by pyocyanin-treated macrophages
(Figure 6E). These data place regulation of GTPase activity
downstream of induction of ROS in macrophages and are
consistent with other recent data from one of our labs that
identified a role for ROS production and inactivation of small
GTPases in impairment of apoptotic cell uptake after tumor
necrosis factor-a treatment of macrophages (21).
In this study, we show that pyocyanin, a major secondary
metabolite of P. aeruginosa, impairs macrophage engulfment
of apoptotic cells as a result of intracellular ROS generation and
modulation of small GTPase signaling. This finding identifies
a novel and potentially important mechanism by which patho-
gens could disrupt efficient clearance of inflammatory cells,
increasing host tissue injury.
Chronic infection with P. aeruginosa is a major cause of
pulmonary damage and mortality in patients with CF (27), and
a number of different P. aeruginosa products have been shown
to modify host immune responses (7). A central feature of CF
lung disease is abnormal neutrophil recruitment and persistence
in the airway, often beginning early in childhood (28). There is
also evidence for aberrant neutrophil death in the CF lung,
leading to DNA release and sputum hyperviscosity (29), and
leakage of major proteases such as neutrophil elastase that
exacerbate lung injury (30). The proportion of neutrophils in
CF sputum that are apoptotic is in the region of 30 to 40% (4),
vastly in excess of the levels of less than 1% found in patients
with community-acquired pneumonia (31). This in turn suggests
delayed clearance of apoptotic cells in CF airways, which could
reflect either macrophage capacity for phagocytosis being over-
whelmed by vast numbers of effete neutrophils or a more
specific defect of macrophage engulfment. Against the former
possibility is the observation of effective clearance of apoptotic
cells and subsequent resolution of acute lobar pneumonia where
the burden of neutrophils is also very substantial (32). Vandiv-
ier and colleagues showed that the presence of neutrophil
elastase in CF sputum specifically impairs engulfment of
apoptotic neutrophils, demonstrating a ‘‘vicious circle’’ in which
the presence of large numbers of effete neutrophils will further
impair apoptotic cell clearance (4).
We investigated the possibility that P. aeruginosa might
directly impair apoptotic cell engulfment. There is some evi-
dence that dysregulation of clearance mechanisms may be
pathogen dependent. Watt and colleagues (34) analyzed sputum
samples from patients with CF and identified a large excess of
late apoptotic or secondarily necrotic neutrophils in patients
infected with P. aeruginosa or Burkholderia cenocepacia com-
pared with those infected with other gram-negative pathogens.
The findings suggest these pathogens are causing accelerated
neutrophil apoptosis, as previously described for both P.
aeruginosa (9) and B. cenocepacia (33), as well as other
bacterial pathogens (8), but could also imply that phagocytosis
of effete neutrophils by macrophages was impaired in these
infections (34). The patients with CF studied by Vandivier and
coworkers, who showed evidence both of profoundly impaired
apoptotic cell clearance and of neutrophil necrosis, were also all
infected with P. aeruginosa (4). We studied the major toxic
metabolite of P. aeruginosa, pyocyanin, because it has been
shown to be a key agent of P. aeruginosa pathogenicity in
multiple experimental models and to cause massive oxidant
stress by intracellular redox generation (11, 35). We found that
pyocyanin impaired macrophage engulfment of apoptotic cells
in vitro at concentrations that have been reported in sputum
from patients chronically colonized with P. aeruginosa (36).
Although there are reports of P. aeruginosa inducing apoptosis
of a macrophage-like cell line in vitro (37), pyocyanin treatment
did not cause significant macrophage apoptosis. Using a murine
model of acute resolving P. aeruginosa, we showed that in-
fection with a pyocyanin-producing strain of P. aeruginosa was
associated with reduced numbers of apoptotic cells within
airway macrophages and with increased numbers of necrotic
neutrophils that had not been engulfed. There were no differ-
ences in macrophage numbers or evidence of increased macro-
phage apoptosis in mice infected with the pyocyanin-producing
strain of P. aeruginosa, compared with a phenazine-deficient
strain in which pyocyanin production was shown to be 10% of
that in wild-type strains (11). There is a previous report of P.
aeruginosa inducing apoptosis of a macrophage-like cell line via
type III secretion-dependent mechanisms (37). We cannot
exclude effects of other P. aeruginosa virulence factors on
macrophage numbers, but there were no differences in macro-
phage numbers between the two strains, so the impact of any
such factors was similar between wild-type and phenazine-
deficient bacteria. The reduction in apoptotic cell engulfment
in mice infected with the wild-type strain was observed at both
time points studied, but was more marked at 30 hours. Because
we were unable to measure pyocyanin production in vivo in the
murine model, we cannot determine whether this reflects
a delayed pyocyanin effect or that an increase in ‘‘available’’
apoptotic cells ‘‘unmasks’’ the defect at 30 hours. However, it is
notable that pyocyanin produces long-lasting effects in vitro:
oxidant production persisted 6 hours after pyocyanin treatment
(the latest time point at which it was measured), down-
40 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1772008
regulation of Rac activity was significant only at 24 hours after
pyocyanin treatment, and maximal inhibition of apoptotic cell
engulfment was also detected after 24 hours. These data identify
a pathologic process that could exacerbate host tissue injury,
both directly by release of proteases and other toxic products
from secondarily necrotic neutrophils (4) and indirectly by
failure of apoptotic cell uptake to generate macrophage release
of antiinflammatory cytokines (38). A more chronic model of P.
aeruginosa infection, as opposed to the acute, resolving model
used in these experiments, could further address effects on host
As predicted from other cell types, including neutrophils (9,
35) and pulmonary epithelial cells (22), pyocyanin treatment
caused generation of ROS in macrophages, using pyocyanin
concentrations detected in clinical samples. Pyocyanin-induced
ROS generation induces apoptosis in neutrophils (9) and in
pulmonary epithelial cells, although over a longer time course
(9, 22, 39). We did not detect significant macrophage apoptosis
sensitivity of neutrophils to pyocyanin-induced apoptosis reflects
their low basal activity of antioxidant enzymes, in contrast to
macrophages which have much higher levels, particularly of
glutathione (GSH) and glutathione peroxidase (23). The mac-
rophage thus exhibits a more subtle phenotype of impaired
apoptotic cell engulfment, without loss of viability. Engulfment
was partially restored by treatment with an antioxidant,
MnTBAP, confirming that the effects of pyocyanin were ROS
dependent. A previous study had shown that monocyte-derived
Figure 6. Pyocyanin inhibits apoptotic cell up-
take via effects on Rho/Rac GTPases. Human
were treated with pyocyanin for the indicated
times and analyzed for active and total Rho (A,
B) and Rac (C, D). Representative gels are shown
from a single experiment of a set of four and the
mean 6 SEM amounts of active Rho/Rac were
quantified using Image J densitometry software
(n 5 4; National Institutes of Health). *Amounts
of Rho/Rac significantly different from levels in
untreated cells. (E) HMDMs were treated with
media (control) or pyocyanin (50 mM) (open
bars) for 24 hours in the presence or absence of
Y-27632 (10 mM) (solid bars) or C3 transferase
(1 mg/ml) (shaded bars). HMDMs were sub-
sequently incubated with APMNs and ingestion
scored by microscopy (times indicated above
represent incubation periods before addition of
APMNs) and expressed as phagocytic index.
Data were obtained from seven independent
experiments (significance calculated by analysis
of variance with Bonferroni posttest). PYO 5
pyocyanin. Statistical significance was calcu-
lated by ANOVA and is illustrated by *P , 0.05,
**P , 0.01, ***P , 0.001
Bianchi, Prince, McPhillips, et al.: Pyocyanin Reduces Cell Clearance41
macrophage engulfment of apoptotic cells was impaired by
hydrogen peroxide treatment, supporting a specific role for
by the signaling pathway implicated, and also by preserved
Fc-mediated uptake of fluorescent beads. The latter is in keeping
with studies by Muller and colleagues (41), who showed macro-
phage uptake of bacteria was preserved in the presence of
A large number of receptors for surface changes on apopto-
tic cells have been implicated in the tethering, engulfment, and
signaling uptake of apoptotic cells in mammalian systems, with
evidence for considerable redundancy (42). Studies of deficien-
cies of engulfment in C. elegans, in contrast, have identified
a number of genes in signaling pathways for cell clearance,
including evidence the Rho-family GTPases are involved in
engulfment of apoptotic cells (43) and in regulation of sub-
sequent maturation of the phagosome (44). We found that
pyocyanin treatment of HMDMs increased Rho activity and
reduced Rac-1 activity, a pattern previously associated with
impaired apoptotic cell engulfment (25, 26). There is evidence
the Rho/Rac balance within cells might determine the rate of
phagosome maturation and that, in monocyte-derived macro-
phages, inhibition of Rho signaling pathways delays intracellu-
lar disposal of ingested apoptotic cells (44). Theoretically,
therefore, activation of these pathways by pyocyanin might
accelerate degradation of ingested apoptotic cells and thus
contribute to the finding of smaller numbers of apoptotic cells
detected within macrophages after pyocyanin treatment or in
the P. aeruginosa infection model. However, the increased
numbers of uningested late apoptotic/necrotic neutrophils ob-
served in the mice infected with pyocyanin-producing P.
aeruginosa support the view that there is impaired engulfment
of apoptotic cells. Moreover, inhibition of Rho signaling path-
ways does not alter the numbers of ingested apoptotic neutro-
phils detected within monocyte-derived macrophages in vitro,
as shown in Figure 6E, showing accelerated degradation of
apoptotic cells is not an explanation for our findings.
We showed that the effects of pyocyanin on apoptotic cell
engulfment could be reversed by treatment with an antioxidant,
MnTBAP, or by Rho-kinase inhibition, and thus are amenable
to therapeutic targeting. The specific defect of apoptotic cell
engulfment could be abrogated by specific Rho-kinase inhib-
itors, novel compounds which have been used as vasodilators in
clinical trials in patients with stable angina (45) and ischemic
stroke (46). Recent evidence also suggests that statins, which
inhibit prenylation of Rho GTPases, can also enhance apoptotic
cell engulfment by both monocyte-derived macrophages and,
importantly, human alveolar macrophages (47). More generally,
there is evidence of increased oxidative stress in the airways in
CF, which is a major factor in lung damage and is believed to be
caused by excessive neutrophil activation and oxidant release
(48).Oxidativestress is associatedwith airwayinflammation(49)
and is increased in infection with P. aeruginosa or B. cenocepacia
(50). Treatment with antioxidants may be of clinical benefit (48)
and has specifically been shown to reduce neutrophilic inflam-
mation and neutrophil elastase activity in the airways (48). Our
data suggest that one important effect of antioxidant treatment
might be restoration of apoptotic cell engulfment that has been
impaired by P. aeruginosa infection, in addition to other benefi-
cial effects of these compounds.
In conclusion, we show that pyocyanin, a major toxin pro-
duced by P. aeruginosa, impairs macrophage engulfment of
apoptotic cells both in vivo and in vitro and that these effects
are substantially reversed by antioxidants or by blockade of
Rho signaling pathways. Because more than 95% of those with
significant CF lung disease are infected with P. aeruginosa and
pyocyanin also accelerates neutrophil apoptosis, therapies di-
rected at the toxic effects of pyocyanin could abrogate both
cell clearance, with the potential to reduce chronic tissue injury.
Conflict of Interest Statement: S.M.B. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript. L.R.P.
does not have a financial relationship with a commercial entity that has an
interest in the subject of this manuscript. K.M. does not have a financial
relationship with a commercial entity that has an interest in the subject of this
manuscript. L.A. does not have a financial relationship with a commercial entity
that has an interest in the subject of this manuscript. H.M.M. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. G.W.T. does not have a financial relationship with a commer-
cial entity that has an interest in the subject of this manuscript. P.G.H. does not
have a financial relationship with a commercial entity that has an interest in the
subject of this manuscript. I.S. has received support from GlaxoSmithKline and
AstraZeneca for conference attendance, and has received lecture fees from
GlaxoSmithKline, Boehringer Ingelheim, and AstraZeneca. He has also received
an unrestricted research fellowship from GlaxoSmithKline. D.H.D. has received
support from GlaxoSmithKline, Gilead, Boehringer Ingelheim, Abbott, and Roche
for conference attendance and has received lecture fees from GlaxoSmithKline to
facilitate conference attendance. P.W.H. does not have a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
M.K.B.W. has received a research grant from GlaxoSmithKline, relating to
a multicenter asthma genetics study. She has received support from Boehringer
Ingelheim for conference attendance and lecture fees from AstraZeneca.
Acknowledgment: The authors thank Dr. Frederick Ausubel (Harvard Medical
School) for the gift of PA14 and DphnAB strains of P. aeruginosa and Professor
John Savill and Dr. Simon Brown (University of Edinburgh) for very helpful
1. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C.
Macrophage phagocytosis of aging neutrophils in inflammation.
Programmed cell death in the neutrophil leads to its recognition by
macrophages. J Clin Invest 1989;83:865–875.
2. Haslett C. Granulocyte apoptosis and inflammatory disease. Br Med
3. Bianchi SM, Dockrell DH, Renshaw SA, Sabroe I, Whyte MK.
Granulocyte apoptosis in the pathogenesis and resolution of lung
disease. Clin Sci (Lond) 2006;110:293–304.
4. Vandivier RW, Fadok VA, Hoffmann PR, Bratton DL, Penvari C,
Brown KK, Brain JD, Accurso FJ, Henson PM. Elastase-mediated
phosphatidylserine receptor cleavage impairs apoptotic cell clearance
in cystic fibrosis and bronchiectasis. J Clin Invest 2002;109:661–670.
5. Birrer P, McElvaney NG, Rudeberg A, Sommer CW, Liechti-Gallati S,
Kraemer R, Hubbard R, Crystal RG. Protease–antiprotease imbal-
ance in the lungs of children with cystic fibrosis. Am J Respir Crit Care
6. Elkin S, Geddes D. Pseudomonal infection in cystic fibrosis: the battle
continues. Expert Rev Anti Infect Ther 2003;1:609–618.
7. Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic
fibrosis. Clin Microbiol Rev 2002;15:194–222.
8. DeLeo FR. Modulation of phagocyte apoptosis by bacterial pathogens.
9. Usher LR, Lawson RA, Geary I, Taylor CJ, Bingle CD, Taylor GW,
Whyte MK. Induction of neutrophil apoptosis by the Pseudomonas
aeruginosa exotoxin pyocyanin: a potential mechanism of persistent
infection. J Immunol 2002;168:1861–1868.
10. Dacheux D, Attree I, Schneider C, Toussaint B. Cell death of human
polymorphonuclear neutrophils induced by a Pseudomonas aerugi-
nosa cystic fibrosis isolate requires a functional type III secretion
system. Infect Immun 1999;67:6164–6167.
11. Mahajan-Miklos S, Tan MW, Rahme LG, Ausubel FM. Molecular
mechanisms of bacterial virulence elucidated using a Pseudomonas
aeruginosa–Caenorhabditis elegans pathogenesis model. Cell 1999;96:
12. Lau GW, Ran H, Kong F, Hassett DJ, Mavrodi D. Pseudomonas
aeruginosa pyocyanin is critical for lung infection in mice. Infect
13. Allen L, Dockrell DH, Pattery T, Lee DG, Cornelis P, Hellewell PG,
Whyte MK. Pyocyanin production by Pseudomonas aeruginosa
induces neutrophil apoptosis and impairs neutrophil-mediated host
defenses in vivo. J Immunol 2005;174:3643–3649.
42 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 177 2008
14. Knight M, Hartman PE, Hartman Z, Young VM. A new method of Download full-text
preparation of pyocyanin and demonstration of an unusual bacterial
sensitivity. Anal Biochem 1979;95:19–23.
15. Ferri KF, Jacotot E, Blanco J, Este JA, Zamzami N, Susin SA, Xie Z,
Brothers G, Reed JC, Penninger JM, et al. Apoptosis control in
syncytia induced by the HIV type 1-envelope glycoprotein complex:
role of mitochondria and caspases. J Exp Med 2000;192:1081–1092.
16. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson
PM. Exposure of phosphatidylserine on the surface of apoptotic
lymphocytes triggers specific recognition and removal by macro-
phages. J Immunol 1992;148:2207–2216.
17. Brown S, Heinisch I, Ross E, Shaw K, Buckley CD, Savill J. Apoptosis
disables CD31-mediated cell detachment from phagocytes promoting
binding and engulfment. Nature 2002;418:200–203.
18. Rowe SJ, Allen L, Ridger VC, Hellewell PG, Whyte MK. Caspase-1-
deficient mice have delayed neutrophil apoptosis and a prolonged
inflammatory response to lipopolysaccharide-induced acute lung in-
jury. J Immunol 2002;169:6401–6407.
19. Dockrell DH, Marriott HM, Prince LR, Ridger VC, Ince PG, Hellewell
PG, Whyte MK. Alveolar macrophage apoptosis contributes to
pneumococcal clearance in a resolving model of pulmonary infection.
J Immunol 2003;171:5380–5388.
20. Emmendorffer A, Hecht M, Lohmann-Matthes ML, Roesler J. A fast
and easy method to determine the production of reactive oxygen
intermediates by human and murine phagocytes using dihydrorhod-
amine 123. J Immunol Methods 1990;131:269–275.
21. McPhillips K, Janssen WJ, Ghosh M, Byrne A, Gardai S, Remigio L,
Bratton DL, Kang JL, Henson P. TNF-alpha inhibits macrophage
clearance of apoptotic cells via cytosolic phospholipase A2 and
oxidant-dependent mechanisms. J Immunol 2007;178:8117–8126.
22. O’Malley YQ, Abdalla MY, McCormick ML, Reszka KJ, Denning GM,
Britigan BE. Subcellular localization of Pseudomonas pyocyanin
cytotoxicity in human lung epithelial cells. Am J Physiol Lung Cell
Mol Physiol 2003;284:L420–L430.
23. Pietarinen-Runtti P, Lakari E, Raivio KO, Kinnula VL. Expression of
antioxidant enzymes in human inflammatory cells. Am J Physiol Cell
24. O’Malley YQ, Reszka KJ, Britigan BE. Direct oxidation of 29,79-
dichlorodihydrofluorescein by pyocyanin and other redox-active com-
pounds independent of reactive oxygen species production. Free Radic
Biol Med 2004;36:90–100.
25. Leverrier Y, Ridley AJ. Requirement for Rho GTPases and PI 3-kinases
during apoptotic cell phagocytosis by macrophages. Curr Biol 2001;
26. Tosello-Trampont AC, Nakada-Tsukui K, Ravichandran KS. Engulf-
ment of apoptotic cells is negatively regulated by Rho-mediated
signaling. J Biol Chem 2003;278:49911–49919.
27. Wilson R, Dowling RB. Lung infections. 3. Pseudomonas aeruginosa
and other related species. Thorax 1998;53:213–219.
28. Muhlebach MS, Stewart PW, Leigh MW, Noah TL. Quantitation of
inflammatory responses to bacteria in young cystic fibrosis and control
patients. Am J Respir Crit Care Med 1999;160:186–191.
29. Kirchner KK, Wagener JS, Khan TZ, Copenhaver SC, Accurso FJ. In-
creased DNA levels in bronchoalveolar lavage fluid obtained from in-
fants with cystic fibrosis. Am J Respir Crit Care Med 1996;154:1426–1429.
30. Rees DD, Brain JD. Effects of cystic fibrosis airway secretions on rat
lung: role of neutrophil elastase. Am J Physiol 1995;269:L195–L202.
31. Droemann D, Aries SP, Hansen F, Moellers M, Braun J, Katus HA,
Dalhoff K. Decreased apoptosis and increased activation of alveolar
neutrophils in bacterial pneumonia. Chest 2000;117:1679–1684.
32. Boutten A, Dehoux MS, Seta N, Ostinelli J, Venembre P, Crestani B,
Dombret MC, Durand G, Aubier M. Compartmentalized IL-8 and
elastase release within the human lung in unilateral pneumonia. Am J
Respir Crit Care Med 1996;153:336–342.
33. Hutchison ML, Poxton IR, Govan JR. Burkholderia cepacia produces
a hemolysin that is capable of inducing apoptosis and degranulation
of mammalian phagocytes. Infect Immun 1998;66:2033–2039.
34. Watt AP, Courtney J, Moore J, Ennis M, Elborn JS. Neutrophil cell
death, activation and bacterial infection in cystic fibrosis. Thorax
35. Ras GJ, Anderson R, Taylor GW, Savage JE, Van Niekerk E, Wilson R,
Cole PJ. Proinflammatory interactions of pyocyanin and 1-hydrox-
yphenazine with human neutrophils in vitro. J Infect Dis 1990;162:
36. Wilson R, Sykes DA, Watson D, Rutman A, Taylor GW, Cole PJ.
Measurement of Pseudomonas aeruginosa phenazine pigments in
sputum and assessment of their contribution to sputum sol toxicity for
respiratory epithelium. Infect Immun 1988;56:2515–2517.
37. Hauser AR, Engel JN. Pseudomonas aeruginosa induces type-III-
secretion-mediated apoptosis of macrophages and epithelial cells.
Infect Immun 1999;67:5530–5537.
38. Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson
PM. Macrophages that have ingested apoptotic cells in vitro inhibit
proinflammatory cytokine production through autocrine/paracrine
mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest 1998;
39. Muller M. Premature cellular senescence induced by pyocyanin, a redox-
active Pseudomonas aeruginosa toxin. Free Radic Biol Med 2006;41:
40. Anderson HA, Englert R, Gursel I, Shacter E. Oxidative stress inhibits
the phagocytosis of apoptotic cells that have externalized phosphati-
dylserine. Cell Death Differ 2002;9:616–625.
41. Muller PK, Krohn K, Muhlradt PF. Effects of pyocyanine, a phenazine
dye from Pseudomonas aeruginosa, on oxidative burst and bacterial
killing in human neutrophils. Infect Immun 1989;57:2591–2596.
42. Savill J, Fadok V. Corpse clearance defines the meaning of cell death.
43. Henson PM. Engulfment: ingestion and migration with Rac, Rho and
TRIO. Curr Biol 2005;15:R29–R30.
44. Erwig LP, McPhilips KA, Wynes MW, Ivetic A, Ridley AJ, Henson
PM. Differential regulation of phagosome maturation in macrophages
and dendritic cells mediated by Rho GTPases and ezrin-radixin-
moesin (ERM) proteins. Proc Natl Acad Sci USA 2006;103:12825–
45. Vicari RM, Chaitman B, Keefe D, Smith WB, Chrysant SG, Tonkon MJ,
Bittar N, Weiss RJ, Morales-Ballejo H, Thadani U. Efficacy and safety
of fasudil in patients with stable angina: a double-blind, placebo-
controlled, phase 2 trial. J Am Coll Cardiol 2005;46:1803–1811.
46. Shibuya M, Hirai S, Seto M, Satoh S, Ohtomo E. Effects of fasudil in
acute ischemic stroke: results of a prospective placebo-controlled
double-blind trial. J Neurol Sci 2005;238:31–39.
47. Morimoto K, Janssen WJ, Fessler MB, McPhillips KA, Borges VM,
Bowler RP, Xiao YQ, Kench JA, Henson PM, Vandivier RW.
Lovastatin enhances clearance of apoptotic cells (efferocytosis) with
implications for chronic obstructive pulmonary disease. J Immunol
48. Tirouvanziam R, Conrad CK, Bottiglieri T, Herzenberg LA, Moss RB,
Herzenberg LA. High-dose oral N-acetylcysteine, a glutathione pro-
drug, modulates inflammation in cystic fibrosis. Proc Natl Acad Sci
49. Hull J, Vervaart P, Grimwood K, Phelan P. Pulmonary oxidative stress
response in young children with cystic fibrosis. Thorax 1997;52:557–
50. McGrath LT, Mallon P, Dowey L, Silke B, McClean E, McDonnell M,
Devine A, Copeland S, Elborn S. Oxidative stress during acute
respiratory exacerbations in cystic fibrosis. Thorax 1999;54:518–
Bianchi, Prince, McPhillips, et al.: Pyocyanin Reduces Cell Clearance 43