Subversion of a Lysosomal Pathway Regulating by a Major Bacterial Toxin, Pyocyanin

This information is current as Lynne R. Prince, Stephen M. Bianchi, Kathryn M. Vaughan, of September 23, 2021. Martin A. Bewley, Helen M. Marriott, Sarah R. Walmsley, Graham W. Taylor, David J. Buttle, Ian Sabroe, David H. Dockrell and Moira K. B. Whyte J Immunol 2008; 180:3502-3511; ;

doi: 10.4049/jimmunol.180.5.3502 Downloaded from http://www.jimmunol.org/content/180/5/3502

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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 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 Downloaded from 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 http://www.jimmunol.org/ pathway of cell death to which are particularly susceptible, and could be therapeutically targeted to limit neutrophil death and preserve host responses. The Journal of Immunology, 2008, 180: 3502–3511.

eutrophils are the predominant inflammatory cells re- philic inflammation (6). Although immune defenses in cystic fibrosis cruited during the innate immune response to bacterial may be impaired at multiple levels, an excess of apoptotic neutrophils N infection and are critical for bacterial clearance. Subse- in this setting implies a neutrophil defect may contribute significantly quent resolution of inflammation requires removal of these poten- to unresolved infection (7). The prominence of P. aeruginosa sepsis tially toxic leukocytes to prevent a dysregulated immune response. in neutropenic patients (8) also highlights both the role of the neutro-

Induction of apoptosis is a crucial mechanism of homeostasis, phil in defense against this organism and the clinical importance of by guest on September 23, 2021 down-regulating proinflammatory functions (1) and resulting in understanding how this pathogen subverts the innate immune re- macrophage-mediated clearance of apoptotic cells (2). Some bac- sponse. P. aeruginosa generates highly diffusible toxic secondary me- teria, however, induce inappropriate or premature apoptosis of tabolites known as , which are critical for P. aeruginosa phagocytes, particularly macrophages, depleting cell numbers and virulence and cytotoxicity in and mouse in- function, with associated impairment of host defense (3, 4). fection models (9), and it is the only common organism to produce a is an important human pathogen. specific , named pyocyanin (10). We have shown pyocyanin, Chronic infection with P. aeruginosa is a major cause of pulmonary at concentrations detected in sputum of cystic fibrosis patients (11), in- damage and mortality in cystic fibrosis, and acute infection is ob- duces a rapid, profound, and selective acceleration of neutrophil apoptosis served both in the immunocompromised host and in patients with in vitro (12). In a murine model of pulmonary P. aeruginosa infection, ventilator-associated pneumonia (5). In patients with cystic fibrosis, mice infected with a pyocyanin-producing strain, as compared with a persistent P. aeruginosa colonization of the lung demonstrates inad- pyocyanin-deficient, but otherwise genetically identical strain, also equate mechanisms of bacterial clearance despite profound neutro- showed accelerated neutrophil apoptosis and impaired bacterial clearance (13). Neutrophils are short-lived cells. Two major pathways to apop- *Academic Unit of Respiratory Medicine, †Academic Unit of Biochemical and Mus- tosis are recognized, as follows: one proceeds through death re- ‡ culoskeletal Medicine, and Academic Unit of Infectious Diseases, University of ceptor signaling, via membrane-associated signaling complexes Sheffield, Sheffield, United Kingdom; and §Department of Medicine, Hampstead Campus, Royal Free and University College School of Medicine, London, United and caspase-8 activation, and a second stress pathway, known to Kingdom be regulated by oxidant stress, is mediated by mitochondria and Received for publication March 1, 2007. Accepted for publication December 26, 2007. regulated by bcl-2 family members (14). The mechanisms of pyo- The costs of publication of this article were defrayed in part by the payment of page cyanin-induced acceleration of neutrophil apoptosis are largely un- charges. This article must therefore be hereby marked advertisement in accordance known, but may involve reactive oxygen intermediates (ROI)4 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a Wellcome Clinical Research Fellowship (064997) (to 4 ⌬␺ S.M.B.). I.S. is a Medical Research Council Senior Clinical Fellow (G116/170), and Abbreviations used in this paper: ROI, reactive oxygen intermediates; m, mito- D.H.D. is a Wellcome Trust Senior Clinical Fellow (076945). chondrial membrane potential; Boc-D-fmk, Boc-Asp(OMe)-fmk; BODIPY FL, boron dipyrromethane difluoride; CTSD, cathepsin D; CTSG, cathepsin G; DAME, diazo- 2 L.R.P. and S.M.B. contributed equally to this work and are joint first authors. acetyl-DL-2-aminohexanoic acid-methyl ester; dbcAMP, dibutyryl cAMP; DHR, 3 Address correspondence and reprint requests to Dr. Moira K. B. Whyte, Academic dihydrorhodamine; MFI, mean fluorescence intensity; z-VAD.fmk, N-benzyloxycar- Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, Uni- bonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone; DEVD-AMC, 7-amino-4-methyl- versity of Sheffield, L Floor, Royal Hallamshire Hospital, Glossop Road, Sheffield, coumarin, N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide. S10 2JF, U.K. E-mail address: m.k.whyte@sheffield.ac.uk Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 3503

generation and altered redox status (12). It is also unclear why Enzyme Systems Products, and Boc-Asp(OMe)-fmk (Boc-D-fmk) (50 neutrophils are exquisitely sensitive to pyocyanin. We therefore ␮M) was obtained from Calbiochem. MeOSuc-Ala-Ala-Pro-Ala-chloro- ␮ ␮ investigated the mechanisms of pyocyanin-induced apoptosis in methylketone (Bachem) (10 M) and elastatinal (Sigma-Aldrich) (10 M) were used as neutrophil elastase inhibitors, with optimal inhibitory con- neutrophils, and describe a novel pathway of pathogen-mediated centrations determined by assessment of inhibition of fluorogenic substrate neutrophil apoptosis, characterized by lysosomal acidification and digestion by purified neutrophil elastase (data not shown). BB94 (1 ␮M; activation of cathepsin D (CTSD). 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- Materials and Methods Aldrich) and diazoacetyl-DL-2-aminohexanoic acid-methyl ester (DAME; Neutrophil isolation and culture Bachem) for CTSD, and CK-08 (1 ␮M; Enzyme Systems Products) for cathepsin G (21). Bafilomycin A1 (100 nM) (Sigma-Aldrich) inhibits the Human neutrophils were isolated by dextran sedimentation and plasma- membrane vacuolar ATPase (22). 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. ROI production Ͼ Ͼ Purity of neutrophil populations ( 95%) was assessed by counting 500 ROI production was assessed by incubating 5 ϫ 105 neutrophils in 200 ␮lof ϫ 6 cells on duplicate cytospins. Neutrophils were suspended at 2.5 10 /ml RPMI 1640 with 5 ␮M dihydrorhodamine (DHR; Sigma-Aldrich) for 30 min in RPMI 1640 with 1% penicillin/streptomycin and 10% FCS (all Invitro- at 37°C, and measuring fluorescence in the FL-1 channel by flow cytometry. gen Life Technologies) and cultured in 96-well Flexiwell plates (BD Pharmingen). Hypoxia experiments Preparation and analysis of pyocyanin

Peripheral blood neutrophil culture in hypoxia was performed, as previ- Downloaded from ously described (23). Neutrophils were resuspended at 5 ϫ 106/ml in RPMI Pyocyanin was prepared by photolysis of phenazine methosulfate (Sigma- 1640 plus 10% FCS and incubated in normoxic (19 kPa) or hypoxic (3 kPa) Aldrich) and purified and characterized, as previously described (16). environments in the presence or absence of pyocyanin (50 ␮M) for 6 h. Assessment of viability and apoptosis Normoxia was controlled using a humidified 5% CO2/air incubator, and hypoxia, by pregassing medium for 30 min in a sealed hypoxic work sta-

Nuclear morphology was assessed on Diff-Quik-stained cytospins, with tion with 5% CO2/balance N2 gas mix and subsequent culture in a humid- Ͼ blinded observers counting 300 cells per slide on duplicate cytospins. ified hypoxic (CO2/N2) incubator. Cytospins were prepared and apoptosis was assessed by trypan blue exclusion and was Ͻ2%, unless was scored by light microscopy. http://www.jimmunol.org/ indicated. Alternatively, neutrophils were washed in PBS and stained with PE-labeled annexin V (BD Biosciences) and TOPRO-3 iodide (Molecular Assessment of mitochondrial and lysosomal membrane ϩ ϩ Probes) to identify apoptotic (annexin V ) and necrotic (TOPRO-3 ) cells permeability (17). Samples were analyzed using a FACSCalibur flow cytometer (BD ⌬␺ ␮ Biosciences). Twenty thousand events were recorded, and data were ana- To detect loss of m, neutrophils were incubated with 10 g/ml JC-1 lyzed by CellQuest software (BD Biosciences). (5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethyl-benzimidazolyl-carbocyanine io- ⌬␺ dide; Molecular Probes) at 37°C. Loss of m was assayed by observing Caspase activity assay a shift in fluorescence emission from red (ϳ590 nm) to green (ϳ525 nm) using flow cytometry (24). Neutrophils were treated with valinomycin (100 Caspase-3 activity was determined by measuring enzymatically cleaved ␮M; Sigma-Aldrich) as a positive control. Lysosomal pH was measured by fluorescent substrate 7-amino-4-methylcoumarin, N-acetyl-L-aspartyl-L- by guest on September 23, 2021 incubating neutrophils with 1 mg/ml FITC-dextran 70S (Sigma-Aldrich), a glutamyl-L-valyl-L-aspartic acid amide (DEVD-AMC; Bachem), as pre- pH-sensitive fluorescent probe, for 30 min at 37°C. Increasing pH (i.e., loss viously described (18). Neutrophil lysates were prepared by resuspension of acidification) within the lysosomal compartment is associated with in- of treated cells in lysis buffer (100 mM HEPES (pH 7.5), 10% w/v sucrose, creased green fluorescence detected in the FL-1 channel (25). Loss of ly- 0.1% CHAPS, and 5 mM DTT) at a concentration of 1 ϫ 108/ml. Lysates sosomal acidification was determined by incubating neutrophils with 5 ␮M were frozen at Ϫ80°C until required. Using the FLUSYS software package acridine orange (Sigma-Aldrich) for 30 min at 37°C, and loss of FL-3 for the PerkinElmer LS-50B fluorometer, lysate equivalents of 5 million fluorescence was measured by flow cytometry (21). Cytospins were also neutrophils were coincubated with 20 ␮M Ac-DEVD-AMC in DMSO. prepared and viewed by fluorescence microscopy. Neutrophils treated with Kinetic data were collected for at least 20 min to ensure stability of activity. 100 nM bafilomycin A1, a known inhibitor of vacuolar ATPase (a key A known amount of free AMC was used to calibrate the system and al- regulator of lysosomal pH Ran, 2003 no. 965), were used as a positive lowed calculation of caspase-3 activity. In separate experiments, execu- control in these experiments. tioner caspase (caspases-3 and -7) activity was measured using a Caspase- Glo 3/7 Assay (Promega). Neutrophils were cultured at 5 ϫ 106/ml and treated with medium (control), pyocyanin (50 ␮M), and pyocyanin with CTSD translocation dibutyryl cAMP (dbcAMP; 100 ␮M) for 3 h. Cells were directly trans- Boron dipyrromethane difluoride (BODIPY FL)-pepstatin A (Molecular ferred to a white 96-well flat-bottom plate (Dynex Technologies) at a den- Probes) is a fluorescent pH-sensitive probe used to measure the sub- sity of 62,500 cells/well in a 25-␮l vol. An equivalent volume of Caspase- cellular distribution of CTSD (26). Neutrophils were treated with me- Glo 3/7 buffer mixed with substrate reagent was added to each well. The dium (control), bafilomyin A1 (100 nM), or pyocyanin (50 ␮M) for 30 plate was read using a Lumistar Galaxy Luminometer (BMG Labtechnolo- min. The cells were washed and incubated with 1 ␮M BODIPY FL- gies) at 25°C for 200 cycles. 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 ATP and glucose measurements min to allow endosomal trafficking. Cytospins were prepared and ϫ ATP was measured using a commercially available bioluminescent kit viewed under a fluorescent microscope (Leica AF6000, 63 objective). (Sigma-Aldrich) using a Lumistar Galaxy Luminometer. Glucose was as- CTSD-labeled BODIPY FL-pepstatin A was visible as punctate fluo- sayed by detecting change in glucose concentration in lysates and culture rescent inclusions. supernatants using a commercial kit (Sigma-Aldrich), as previously de- scribed (19). Neutrophils were cultured in RPMI 1640 alone, with a glu- SDS-PAGE and Western immunoblotting cose concentration of 2 mg/ml. Both assays were standardized using known Whole-cell extracts were used for Mcl-1 and actin immunoblots and pre- concentrations of ATP and glucose, respectively (data not shown). pared as described (27). Cytosolic and membrane fractions used in CTSD Modulation of pyocyanin-induced apoptosis and cathepsin G (CTSG) immunoblots were prepared by sonication (three 10-s bursts in HBSS supplemented with protease inhibitor mixture III (Cal- Neutrophils were incubated in the presence and absence of pyocyanin fol- biochem), followed by 25,000 rpm microcentrifugation for 45 min). Pro- lowing preincubation with candidate modulators of pyocyanin-induced teins were separated by 15% v/v SDS-PAGE and blotted onto nitrocellu- apoptosis. Except where indicated, a concentration of 50 ␮M pyocyanin lose membranes (Bio-Rad), and protein transfer was confirmed by Ponceau was used because it significantly accelerates neutrophil apoptosis ϳ5-fold S (BDH) staining. Blots were incubated overnight at 4°C for Mcl-1 (S-19; at 5 h (12). The pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala- Santa Cruz Biotechnology) and at room temperature for 2 h for actin (Sigma- Asp(O-methyl) fluoromethyl ketone (z-VAD.fmk) (18), was obtained from Aldrich), CTSG (Abcam), and CTSD (Calbiochem); protein detection was 3504 MECHANISMS OF PYOCYANIN-INDUCED NEUTROPHIL APOPTOSIS with HRP-conjugated IgG (DakoCytomation) and ECL (Amersham Biosciences). Statistics 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.

Results 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- Downloaded from 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 at5hwas 5.7 Ϯ 1.2% and was not significantly inhibited by zVAD.fmk at any concentration used (data not shown). A second pan-caspase http://www.jimmunol.org/ inhibitor, Boc-D.fmk, also inhibited pyocyanin-induced apoptosis at 5 h (pyocyanin-induced apoptosis in the absence (34.8 Ϯ 3.8%) and presence (21.9 Ϯ 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 FIGURE 1. Pyocyanin-induced neutrophil apoptosis is caspase depen- (Fig. 1C). Thus, pyocyanin-induced neutrophil apoptosis is de- dent. A, Neutrophils were treated with pyocyanin (50 ␮M) in the presence by guest on September 23, 2021 layed by caspase inhibition and associated with caspase-3 or absence of a concentration range of zVAD.fmk for 4 h. Apoptosis was activation. assessed by morphologic criteria, and chart shows mean Ϯ SEM percent- age of apoptosis from three independent experiments. Baseline control Pyocyanin-induced oxidative stress mediates neutrophil apoptosis in these experiments was 5.7 Ϯ 1.2% and was not significantly apoptosis inhibited by any concentration of zVAD.fmk (data not shown). Statistically significant inhibition of pyocyanin-induced apoptosis was observed at con- The cytotoxic effects of pyocyanin on bacteria and eukaryotic cells p Ͻ ,ءء p Ͻ 0.05 and ,ء) centrations of zVAD.fmk of 5 ␮M and greater are linked to its ability to undergo nonenzymatic redox cycling 0.01 ANOVA with Dunnett’s posttest). B, Neutrophils were treated with 50 within cells, with resultant ROI formation, and further loss of re- ␮M pyocyanin in the absence (Ⅺ) or presence (f) of zVAD.fmk (50 ␮M) ducing capacity by direct oxidation of NADH/NADPH and re- for 0, 2, 5, and 10 h. Apoptosis was assessed by flow cytometry, and the duced (29). We found that pyocyanin induces pro- chart shows mean Ϯ SEM percentage of apoptosis for three independent longed generation of ROI in neutrophils as measured by oxidation experiments. Significant inhibition of pyocyanin-induced apoptosis was -p Ͻ 0.001; ANOVA, Bonfer ,ءءء) p Ͻ 0.05) and 10 h ,ء) of DHR (Fig. 2A), in keeping with our previous observations (12). observed at 5 To further corroborate the role of oxidative stress in pyocyanin- roni’s posttest). C, Lysates were prepared from neutrophils treated with Ⅺ ␮ f induced apoptosis, neutrophils were incubated in a hypoxic envi- medium (control, )or50 M pyocyanin ( ) for 1 and 4 h. Caspase-3 ronment in the presence of pyocyanin for 5 h. We hypothesized activity was detected using a specific fluorogenic substrate and measured kinetically (Flusys software). Pyocyanin significantly increased caspase-3 that reduced availability of oxygen would hinder the ability of .(p ϭ 0.0355; Student’s t test ,ء)activity at4h pyocyanin to induce apoptosis. Fig. 2B shows pyocyanin is unable to induce neutrophil apoptosis in hypoxia. showed increased glucose uptake (Fig. 3B) and maintenance of Metabolic activity is maintained in pyocyanin-treated intracellular glucose concentrations in these cells (Fig. 3C). neutrophils ROI are a feature of the stress pathway of apoptosis, and ROI Pyocyanin-induced ROI production is linked to depletion of intra- production leads to loss of mitochondrial membrane potential ⌬␺ cellular ATP in epithelial cells (29, 30). To determine whether ( m) (14). Because pyocyanin can interrupt mitochondrial respi- neutrophil death was associated with cellular ATP depletion in ration in epithelial cells as a result of ROI generation (29), we response to the profound ROI induction, we measured intracellular determined whether pyocyanin-mediated ROI production was in- ⌬␺ ⌬␺ ATP in the neutrophil by a bioluminescence technique. We did not ducing neutrophil apoptosis via loss of m. We measured m in detect a reduction in ATP levels at time points up to 3 h following neutrophils using the mitochondrial dye, JC-1. Fig. 4A shows flow pyocyanin treatment (Fig. 3A). Metabolic pathways in pyocyanin- cytometry dot plots illustrating the distribution of high-FL-1 flu- ⌬␺ treated neutrophils remained viable, as shown by this maintenance orescent neutrophils (associated with a loss of m (24, 31)) in of intracellular ATP, and by our subsequent experiments, which control, pyocyanin-, and valinomycin-treated populations at 4 h. The Journal of Immunology 3505 Downloaded from

FIGURE 3. Pyocyanin leads to metabolic cell stress in the neutrophil. A, http://www.jimmunol.org/ FIGURE 2. Oxidative stress is essential for pyocyanin-induced neutro- Neutrophils were treated with medium (control, Ⅺ)or50␮M pyocyanin phil apoptosis. A, DHR-loaded neutrophils were treated with medium (con- (f) for 1 and 3 h, following which lysates were prepared and intracellular trol, Ⅺ)or50␮M pyocyanin (f) for a range of time points: 30, 60, 120, ATP was measured. No significant differences were observed between the and 180 min. Increases in fluorescence were measured by flow cytometry groups. B and C, Neutrophils were treated with medium (control, Ⅺ)or50 and indicated enhanced ROI production. The chart shows mean Ϯ SEM of ␮M pyocyanin (f) for 2 h, and glucose was measured in extracellular (B) mean fluorescence intensity (MFI) of three independent experiments. Sta- and intracellular (C) compartments. The charts show mean Ϯ SEM from tistically significant differences were seen between control and pyocyanin- four independent experiments. A statistically significant decrease in extra- -p Ͻ 0.05; Stu ,ء) p Ͻ 0.001; ANOVA, Bonferroni’s cellular glucose was detected in pyocyanin-treated cells ,ءءء) treated cells at all time points posttest). B, Neutrophils incubated in normoxic (Ⅺ) or hypoxic (f) con- dent’s t test). by guest on September 23, 2021 ditions were cultured with medium (control) or pyocyanin (50 ␮M) for 6 h. Apoptosis was assessed by light microscopy. Pyocyanin treatment of nor- p Ͻ 0.01; ANOVA, Bonferroni’s mulates in acidic organelles (35). On fluorescence microscopy ,ءء) moxic neutrophils induced apoptosis posttest). Hypoxic neutrophils survived in the presence of pyocyanin, to (Fig. 6A), a punctate staining pattern was seen in the cytosol of levels comparable with the control. 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- Pyocyanin induced only modest changes in the proportions of cells mal acidification (21, 32). Statistically significant losses of fluo- ⌬␺ showing loss of m, and these nonsignificant changes were only rescence were detected at 15 min following pyocyanin treatment observed at later time points (Fig. 4B). These observations were (Fig. 6B). Once again, bafilomycin A1 was used as a positive con- confirmed using a second mitochondrial dye, 3,3-dihexyloxacarbo- trol in these experiments and, at a concentration (100 nM) that ⌬␺ cyanine iodide (data not shown). Thus, although loss of m ap- inhibits V-ATPase function in neutrophils (36), caused loss of ly- pears to occur in pyocyanin-induced neutrophil apoptosis, changes sosomal acidification to an even greater degree than pyocyanin. are not apparent until later time points, when there is already a However, this concentration of bafilomycin A1 was without effect ⌬␺ significant increase in apoptosis. Loss of m was therefore un- on constitutive neutrophil apoptosis (Fig. 6C), in keeping with pre- likely to be an initiating factor in the engagement of apoptosis. vious studies (37), although higher concentrations of bafilomycin are proapoptotic to neutrophils (data not shown). Lysosomal alka- Pyocyanin-induced apoptosis is preceded by changes in linization alone is not, therefore, sufficient to induce neutrophil lysosomal pH, but these are not sufficient to induce death apoptosis. A pathway of cell death is now recognized, activated primarily in pathological rather than homeostatic circumstances, in which ly- CTSD translocation and activation are associated with ⌬␺ pyocyanin-induced apoptosis sosmal dysfunction may precede loss of m (32). Lysosomal pH was measured using a pH-sensitive marker (FITC-conjugated dex- Alterations in lysosomal pH alone do not induce apoptosis, and tran) that is taken up by acidic structures and increases in FL-1 release of lysosomal proteases is critical for completion of the channel fluorescence as pH rises due to loss of protonation (25, apoptotic program in a range of cell types (21, 35). No attenuation 33). Exposure of neutrophils to pyocyanin or bafilomycin A1 (an of pyocyanin-induced neutrophil apoptosis was seen by treatment inhibitor of the vacuolar (Hϩ)-ATPase that maintains normal ly- with elastase inhibitors or a pan matrix metalloproteinase inhibitor sosomal pH gradients (34)) increased lysosomal pH compared (Fig. 7, A and B). In contrast, significant reductions in pyocyanin- with untreated controls (Fig. 5). induced apoptosis were observed with pepstatin A, an inhibitor of We confirmed loss of lysosomal acidification in neutrophils by aspartyl proteases (21), both alone and in combination with inhib- staining with acridine orange, which is lysosomotropic and accu- itors of the cathepsins B, G, and L (Fig. 7C). The latter inhibitors 3506 MECHANISMS OF PYOCYANIN-INDUCED NEUTROPHIL APOPTOSIS Downloaded from

FIGURE 4. Pyocyanin-induced neutrophil apoptosis does not depend on early mitochondrial dysfunction. Neutrophils were treated with medium Ⅺ ␮ u ␮ f (control, ), 50 M pyocyanin ( ), or 1 M valinomycin ( ) for 1, 2, and http://www.jimmunol.org/ ␮ ⌬␺ 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 Ϯ SEM percentage of cells with loss ⌬␺ FIGURE 6. Pyocyanin induces early loss of lysosomal acidification. A, of m (high JC-1) from three independent experiments. Valinomycin Ͻ Representative fluorescent microscopy images illustrating typical punctate ءءء Ͻ ءء ␺⌬ caused significant loss of m at every time point ( , p 0.01; , p 0.001; ANOVA, Bonferroni’s posttest). staining with acridine orange, which is lost after treatment with pyocyanin or baflinomycin A1. B, Neutrophils were treated with medium (control, Ⅺ), 50 ␮M pyocyanin (u), and baflinomycin A1 (100 nM, f) for 15, 30, and were without effect, either alone or in combinations that excluded 60 min. Loss of lysosomal acidification was assessed with the fluorescent by guest on September 23, 2021 lysosomal dye, acridine orange, and fluorescence was measured by flow pepstatin A, on constitutive neutrophil apoptosis (data not shown) cytometry. Decreases in FL-3 reflect loss of acridine orange due to lyso- C or on pyocyanin-induced apoptosis (Fig. 7 ). Although pepstatin somal disruption. The chart shows mean Ϯ SEM MFI from three indepen- dent experiments. Pyocyanin caused significant loss of lysosomal acidifi- ,p Ͻ 0.01; ANOVA, Bonferroni’s posttest). C ,ءء) cation at all time points Neutrophils were incubated with medium (control, Ⅺ) or 100 nM bafilo- mycin A1 for varying times. Apoptosis was assessed by flow cytometry; chart shows mean Ϯ SEM percentage of apoptosis (as defined by annexin V positivity) for three independent experiments.

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). FIGURE 5. Pyocyanin induces loss of lysosomal acidification. A, Illus- BODIPY FL-pepstatin A is a pH-dependent fluorescent probe trative flow cytometry histogram showing right shifts in FL1-H fluores- that binds to CTSD within the acidified lysosomal compartment cence of FITC-dextran-labeled neutrophils treated with either medium (26). We demonstrated the expected pattern of CTSD localization ␮ (control), pyocyanin (50 M), or bafilomycin A1 (100 nM) for 2 h. In- with punctate cytosolic inclusions in control neutrophils (Fig. 8A). creasing fluorescence in FL-1 reflects elevated intralysosomal pH second- Neutrophils treated for 30 min with pyocyanin or bafilomycin A1 ary to reduced protonation. B, FITC-dextran-stained neutrophils were in- cubated with medium (control, Ⅺ)or50␮M pyocyanin (f) for the lost this typical granular staining pattern. To determine whether the indicated times. The chart shows fold change of MFI (mean Ϯ SEM, n ϭ decrease in lysosomal BODIPY FL-pepstatin A staining reflected 3) vs control at 30 min. Pyocyanin treatment significantly elevated lyso- alteration in lysosomal pH alone or lysosomal membrane perme- -p Ͻ 0.01; ANOVA, ability, we assessed translocation of CTSD by Western immuno ,ءء) p Ͻ 0.001) and 240 min ,ءءء) somal pH at 120 Bonferroni’s posttest). blotting. Fig. 8B shows CTSD distribution in neutrophil lysates The Journal of Immunology 3507 Downloaded from

FIGURE 8. CTSD is translocated to the cytosol following pyocyanin treatment. A, Neutrophils were treated with medium (control), pyocyanin http://www.jimmunol.org/ (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- ϫ 6

pared from 4 10 neutrophils treated with medium or pyocyanin (50 by guest on September 23, 2021 ␮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 FIGURE 7. Pyocyanin-induced neutrophil apoptosis is mediated by ac- immunoblots were probed for CTSG and actin (loading control) and tivation of CTSD. A and B, Medium (control) and pyocyanin (50 ␮M)- showed significantly more CTSG in the pyocyanin-treated sample com- treated neutrophils were incubated in the absence (Ⅺ) or presence of in- pared with control or bafilomycin A1. 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)- tion to the cytosolic fraction (Fig. 8C), although as shown in Fig. treated neutrophils were incubated in the presence or absence of a range of 7C, CTSG inhibition did not abrogate pyocyanin-mediated cathepsin inhibitors for 5 h, and apoptosis was assessed by flow cytometry. apoptosis. The inhibitor of CTSD and combinations, including the CTSD inhibitor, p Ͻ A stable cAMP analog retards pyocyanin-mediated neutrophil ,ءء ;p Ͻ 0.05 ,ء) significantly inhibited pyocyanin-induced apoptosis 0.01; ANOVA, Dunnett’s posttest). D, Neutrophils were incubated for 5 h apoptosis by regulating CTSD translocation, caspase activity, with medium (Ⅺ) or pyocyanin (50 ␮M) (f) both alone and in the presence and Mcl-1 expression of a concentration range of the CTSD inhibitor, DAME. Apoptosis was assessed by morphology and is expressed as fold change from either con- We previously reported that the synthetic cAMP analog dbcAMP trol or pyocyanin alone. Five-micromolar DAME significantly inhibited is able to significantly abrogate pyocyanin-induced apoptosis of pyocyanin-induced apoptosis and was without effect on constitutive neutrophils (12). Having identified pathways of pyocyanin-in- Ͻ ء apoptosis ( , p 0.05; ANOVA, Dunnett’s posttest). 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). separated into membrane (upper panel) and cytosolic (lower However, translocation of CTSD to the cytosol in pyocyanin- panel) fractions. The predicted 44- and 31-kDa forms (41) are treated cells was reduced in cells also treated with dbcAMP (Fig. present within the membrane fraction of both control and bafilo- 9C). Pyocyanin-induced caspase activity was also prevented by mycin A1-treated cells, whereas pyocyanin-treated cells show dbcAMP (Fig. 9D). Another potentially important effect of db- translocation of the 31-kDa form into the cytosol. We also studied cAMP was also studied. The antiapoptotic bcl-2 family member, translocation of another cathepsin, CTSG, and demonstrated that Mcl-1, plays a critical role in the regulation of neutrophil apoptosis treatment with pyocyanin, but not bafilomycin, caused transloca- (42), and cAMP analogues have been shown to stabilize Mcl-1 3508 MECHANISMS OF PYOCYANIN-INDUCED NEUTROPHIL APOPTOSIS

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.

Discussion 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- Downloaded from 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 generation (45, 46), and production of pyocyanin is an important determinant of severity in

murine models of sepsis (9). The important potential for pyocyanin http://www.jimmunol.org/ 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 by guest on September 23, 2021

pyocyanin in the presence (f) or absence (Ⅺ) 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 Ϯ 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 Ϯ p Ͻ ,ءءء ;p Ͻ 0.01 ,ءء) SEM MFI from three independent experiments 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 ϫ 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- ;p Ͻ 0.001 ,ءءء) cantly greater than control and is inhibited by dbcAMP 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, FIGURE 9. A stable cAMP analog retards pyocyanin-mediated lower panel). dbcAMP prevented pyocyanin-induced degradation of apoptosis. A, Neutrophils were preloaded with DHR and incubated with Mcl-1. The Journal of Immunology 3509 since it is the major executioner caspase in human neutrophils (47, mal in Chediak-Higashi Syndrome, a disorder of lysosomes and 48). These data provided biochemical confirmation that the cell related structures (62, 63). However, because they lack classical death induced was apoptotic and represented a subversion of im- lysosomal membrane markers such as lysosomal-associated mem- portant normal regulatory pathways controlling neutrophil lifes- brane proteins 1 and 2 (62, 64), they are sometimes described as pan. We then sought the apoptotic pathways upstream of caspase lysosome-related organelles (63). activation to determine the mechanism of pyocyanin-induced neu- Recent studies by Ran et al. (22) provided important insights trophil killing. The dependence upon ROI for pyocyanin-induced into the actions of pyocyanin. Yeast mutants with reduced sensi- death was implied by effects of antioxidants in our previous studies tivity to pyocyanin frequently had mutations in the V-ATPase, an (12), and in this study we further show the proapoptotic effects of enzyme complex involved in mitochondrial electron transport and pyocyanin were prevented by culture in hypoxia. Recent studies in ATP synthesis, but also a major regulator of lysosomal pH (65). epithelial cells found pyocyanin, in addition to causing intracellu- Ran et al. (22) found pyocyanin both induced lysosomal membrane lar ROI generation, can directly oxidize both NADH and NAPDH permeabilization and inhibited V-ATPase function in epithelial (49). This loss of cellular reducing capacity is associated with im- cells, with high concentrations (2 mM) of a V-ATPase inhibitor, paired glycolysis (50) and reduced levels of cyclic nucleotides, bafilomycin A1, having similar effects to pyocyanin. In neutro- particularly ATP (29, 51). Although detailed experiments studying phils, bafilomycin A1, at a concentration (100 nM) that inhibits the energetic status of the cell were not performed, intracellular neutrophil V-ATPase function (36), reduced lysosomal acidifica- levels of ATP and glucose were maintained following pyocyanin tion to an even greater degree than pyocyanin. This concentration treatment, data that are in keeping with the requirement of neu- of bafilomycin A1 was, however, without effect on neutrophil trophils to maintain function in very demanding environments of Downloaded from apoptosis, in keeping with previous studies (37). Two other global low pH, low glucose, low oxygen tension, and high oxidative ϩ ϩ stress such as in purulent secretions (52). Indeed, preservation of regulators of intracellular pH, amiloride (an inhibitor of Na /H ATP is necessary for coordinated execution of apoptotic programs, exchangers) and zinc chloride (an inhibitor of NADPH-oxidase- and ATP depletion favors necrotic cell death rather than classical associated proton channels), were also without effect on pyocya- apoptosis (53), providing further support for our data showing no nin-induced apoptosis (data not shown). Our findings support those

significant loss of intracellular ATP. Neutrophils, again because of of Ran et al. (22) in demonstrating pyocyanin-induced loss of ly- http://www.jimmunol.org/ the environments in which they must be active, are unique in that sosomal acidification in neutrophils that is most likely mediated the majority of ATP generation in these cells occurs via oxygen- via the lysosmal V-ATPase, but also show lysosomal alkaliniza- independent glycolysis (54), and we found intracellular glucose tion alone is not sufficient to induce apoptosis. It is not clear levels were maintained in pyocyanin-treated neutrophils. Glucose whether the effect of pyocyanin upon V-ATPase function is indi- uptake from the extracellular medium was measured and, for un- rect, perhaps resulting from ROI generation, or whether pyocyanin treated neutrophils, was comparable to previous studies using is lysosomotropic and binds and directly inhibits V-ATPase func- the same methodology (19), with an increased uptake following tion, as has been described for other agents, e.g., quinolones (21) pyocyanin treatment that was comparable to that of LPS- or and concanamycin (66). PMA-activated neutrophils (55, 56). Lysosomes contain multiple potent proteases that contribute to by guest on September 23, 2021 We then sought evidence for mitochondrial inner transmem- bacterial killing (67), several of which have been associated with brane permeabilization, which is characteristic of the stress path- onset of apoptosis (32). Using a series of broad and narrow-spec- way of apoptosis characterized by ROI generation (14). Although trum protease inhibitors, we identified a role for CTSD in pyocy- mitochondria have a minimal role in ATP generation in neutro- anin-induced apoptosis. We found that a specific CTSD inhibitor, phils, they do have a critical role in apoptosis induction (24, 57). pepstatin A, delayed pyocyanin-induced death, but also caused ac- Studies in Jurkat T cells have highlighted the ability of ATP de- tivation of neutrophils (38) (our data not shown). We therefore ⌬␺ rived by glycolysis to maintain m for some hours following a used a second specific CTSD inhibitor (DAME) that also delayed metabolic insult, a process that is critically dependent upon en- pyocyanin-induced neutrophil apoptosis. We identified CTSD hanced uptake of extracellular glucose (58). We detected increased staining in neutrophils, with the expected distribution in subcellu- ⌬␺ following pyocyanin treatment, but this occurred in concert m lar organelles, and we detected translocation of a 31-kDa fragment with, rather than preceding, onset of apoptosis. Thus, whereas ⌬␺ m of CTSD from the membrane to the cytosolic fraction of neutro- may be part of the amplification mechanism leading to executioner phils following pyocyanin treatment. These data are in keeping caspase activation, it is unlikely to be part of the program initiating with data showing a role for CTSD in apoptosis of fibroblasts (59, apoptosis. 68) and endothelial cells (69) following oxidant stress. Impor- A number of studies have shown oxidative stress-induced cell tantly, neutrophil primary granules contain significant amounts of death is associated with lysosomal destabilization (32) and that CTSD (40, 70), accounting for 38% of acidic protease activity of ROI can induce lysosomal permeabilization (35, 59). Within 15 min of pyocyanin treatment of neutrophils, there was evidence of neutrophils (71). Although CTSG was also released into the cy- alkalinization of the lysosomal compartment that preceded any de- tosol by pyocyanin treatment, CTSG inhibition did not abrogate ⌬␺ apoptosis, suggesting a particular role for CTSD in this system. tectable changes in m or caspase-3 activity. A lysosomal path- way of apoptosis that precedes mitochondrial changes is recog- Our work showing that pyocyanin destabilizes neutrophil granules nized in other cell types, with critical proteases translocating from and releases CTSD into the cytosol may in part explain the ex- lysosomes and other secretory vesicles into the cytoplasm (21, 60). quisite susceptibility of neutrophils to pyocyanin-induced apopto- This pathway is activated primarily in pathological rather than ho- sis (12), because cells with lower or absent numbers of CTSD- meostatic circumstances (32) and can be activated by death recep- containing granules (such as epithelial cells) are not stimulated to tors or lipid mediators (61) and following accumulation of lyso- die when exposed to pyocyanin. The mechanisms by which CTSD somotropic agents (21). The azurophilic or primary granules are induces apoptosis are uncertain. CTSD release is upstream of generally regarded as the lysosomal structures within neutrophils, caspase activation (72), although a recent overexpression study because they are the major cellular reservoir of acid-dependent suggests that the catalytic activity of CTSD is not essential for its hydrolases, contain lysosomal membrane proteins, and are abnor- proapoptotic role (73). The recent studies of Blomgran et al. (74) 3510 MECHANISMS OF PYOCYANIN-INDUCED NEUTROPHIL APOPTOSIS show a role for cathepsins, including CTSG, in mediating Esche- 14. Green, D. R., and G. Kroemer. 2004. The pathophysiology of mitochondrial cell richia coli-induced neutrophil apoptosis, with evidence of cathe- death. Science 305: 626–629. 15. Haslett, C., L. A. Guthrie, M. M. Kopaniak, R. B. Johnston, Jr., and psin-mediated Bid cleavage and down-regulation of Mcl-1. Our P. M. Henson. 1985. Modulation of multiple neutrophil functions by preparative studies also demonstrate reductions in Mcl-1 protein, and that res- methods or trace concentrations of bacterial lipopolysaccharide. Am. J. Pathol. toration of Mcl-1 protein levels by dbcAMP is associated with 119: 101–110. 16. Knight, M., P. E. Hartman, Z. Hartman, and V. M. Young. 1979. A new method delay of pyocyanin-induced apoptosis. of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity. A number of pathogens such as E. coli (75), Staphylococcus Anal. Biochem. 95: 19–23. 17. Sabroe, I., E. C. Jones, L. R. Usher, M. K. Whyte, and S. K. Dower. 2002. aureus (76), and Streptococcus pyogenes (77) are associated with Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a neutrophil apoptosis following phagocytosis; this is likely to be a critical role for monocytes in leukocyte lipopolysaccharide responses. J. Immu- host-mediated process to prevent intracellular persistence of bac- nol. 168: 4701–4710. 18. Garcia-Calvo, M., E. P. Peterson, B. Leiting, R. Ruel, D. W. Nicholson, and teria (78). However, our studies and those of Blomgran et al. (74) N. A. Thornberry. 1998. Inhibition of human caspases by peptide-based and identify a relationship between lysosome function and apoptosis in macromolecular inhibitors. J. Biol. Chem. 273: 32608–32613. bacterial infection has not previously been recognized, despite the 19. Healy, D. A., R. W. Watson, and P. Newsholme. 2002. Glucose, but not glu- tamine, protects against spontaneous and anti-Fas antibody-induced apoptosis in prominent role of lysosomal proteases in antibacterial responses of human neutrophils. Clin. Sci. 103: 179–189. neutrophils. In these studies, pathogens effectively subvert these 20. Beekman, B., J. W. Drijfhout, H. K. Ronday, and J. M. TeKoppele. 1999. Flu- ␣ processes, with release of granule proteases triggered by bacterial- orogenic MMP activity assay for plasma including MMPs complexed to 2- macroglobulin. Ann. NY Acad. Sci. 878: 150–158. driven ROI generation. 21. Boya, P., K. Andreau, D. Poncet, N. Zamzami, J. L. Perfettini, D. Metivier, In summary, we have demonstrated pyocyanin induces apopto- D. M. Ojcius, M. Jaattela, and G. Kroemer. 2003. Lysosomal membrane perme- sis by engagement of lysosomal pathways of cell death, and we abilization induces cell death in a mitochondrion-dependent fashion. J. Exp. Med. Downloaded from 197: 1323–1334. provide evidence it may be a pathological mechanism of cell death 22. Ran, H., D. J. Hassett, and G. W. Lau. 2003. Human targets of Pseudomonas to which neutrophils are particularly susceptible. Furthermore, this aeruginosa pyocyanin. Proc. Natl. Acad. Sci. USA 100: 14315–14320. is the first description of a bacterial toxin using this pathway of 23. Walmsley, S. R., C. Print, N. Farahi, C. Peyssonnaux, R. S. Johnson, T. Cramer, A. Sobolewski, A. M. Condliffe, A. S. Cowburn, N. Johnson, and E. R. Chilvers. mammalian cell apoptosis to subvert host defense. Our findings are 2005. Hypoxia-induced neutrophil survival is mediated by HIF-1␣-dependent of clinical relevance, because understanding mechanisms to inhibit NF-␬B activity. J. Exp. Med. 201: 105–115. 24. Martin, M. C., I. Dransfield, C. Haslett, and A. G. Rossi. 2001. Cyclic AMP pyocyanin-induced neutrophil death in P. aeruginosa infections http://www.jimmunol.org/ regulation of neutrophil apoptosis occurs via a novel -indepen- may lead to development of therapies that favor an effective im- dent signaling pathway. J. Biol. Chem. 276: 45041–45050. mune response to this major human pathogen. 25. Hishita, T., S. Tada-Oikawa, K. Tohyama, Y. Miura, T. Nishihara, Y. Tohyama, Y. Yoshida, T. Uchiyama, and S. Kawanishi. 2001. 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