Published Ahead of Print 6 April 2009. 2009, 77(6):2455. DOI: 10.1128/IAI.00839-08. Infect. Immun. 

Quinn, Wei-Jen Tang and Frederick S. SouthwickSarah E. Szarowicz, Russell L. During, Wei Li, Conrad P. Neutrophil Actin-Based Motility

Edema Toxin ImpairsBacillus anthracis

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INFECTION AND IMMUNITY, June 2009, p. 2455–2464 Vol. 77, No. 60019-9567/09/$08.00�0 doi:10.1128/IAI.00839-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Bacillus anthracis Edema Toxin Impairs Neutrophil Actin-Based Motility�

Sarah E. Szarowicz,1 Russell L. During,1 Wei Li,1 Conrad P. Quinn,2Wei-Jen Tang,3 and Frederick S. Southwick1*

Division of Infectious Diseases, College of Medicine, University of Florida, Gainesville, Florida 326101; NCIRD, DBD,Centers for Disease Control and Prevention, Atlanta, Georgia 303332; and Ben May Department for Cancer Research,

University of Chicago, Chicago, Illinois 606373

Received 7 July 2008/Returned for modification 8 July 2008/Accepted 26 March 2009

Inhalation anthrax results in high-grade bacteremia and is accompanied by a delay in the rise of theperipheral polymorphonuclear neutrophil (PMN) count and a paucity of PMNs in the infected pleural fluidand mediastinum. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of theadenylate cyclase edema factor (EF) and protective antigen (PA). Relatively low concentrations of ET (100 to500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize.These changes are accompanied by a reduction in chemoattractant-stimulated PMN actin assembly. ET alsocauses a significant decrease in Listeria monocytogenes intracellular actin-based motility within HeLa cells.These defects in actin assembly are accompanied by a >50-fold increase in intracellular cyclic AMP and a>4-fold increase in the phosphorylation of protein kinase A. We have previously shown that anthrax lethaltoxin (LT) also impairs neutrophil actin-based motility (R. L. During, W. Li, B. Hao, J. M. Koenig, D. S.Stephens, C. P. Quinn, and F. S. Southwick, J. Infect. Dis. 192:837-845, 2005), and we now find that LTcombined with ET causes an additive inhibition of PMN chemokinesis, polarization, chemotaxis, and FMLP(N-formyl-met-leu-phe)-induced actin assembly. We conclude that ET alone or combined with LT impairsPMN actin assembly, resulting in paralysis of PMN chemotaxis.

Inhalation anthrax can lead to sepsis and death within daysif not diagnosed early and treated effectively (21). Epidemio-logical analyses of the anthrax bioterrorist attacks in 2001 in-dicated a mean duration of 4.5 days between exposure andsymptom onset in the six inhalation anthrax cases for whom theexposure dates could be determined. Analyses of the clinicalfindings from 10 of the 11 inhalation anthrax cases revealednormal or minimally elevated peripheral polymorphonuclearneutrophil (PMN) counts at the time of hospital admission,despite high-level Bacillus anthracis bacteremia (22). Further-more, heavily infected pleural fluid demonstrated a paucity ofwhite blood cells. In the fatal cases, mediastinal infection wasassociated with marked edema and hemorrhage but minimalinfiltration by acute inflammatory cells (16). Similarly, experi-mental inhalation anthrax in monkeys was associated withedema and hemorrhage of the mediastinum and pulmonaryinterstitium, with absent or modest infiltration by neutrophils(37). These findings suggest impaired delivery of neutrophils tothe sites of infection during the early stages of systemic B.anthracis infection.

B. anthracis produces three exotoxins, protective antigen(PA), edema factor (EF) and lethal factor (LF), that accountfor many of the clinical manifestations of this deadly pathogen.PA binds to the widely distributed host cell receptors and thenself-associates into heptamers and ushers LF and EF into thecytoplasm of cells (4). The anthrax toxins have been termedAB toxins, PA combined with LF being called lethal toxin

(LT), and PA combined with EF termed edema toxin (ET). LFis a Zn2�-dependent metalloprotease that cleaves mitogen-activated protein kinase kinases (12). EF is a calcium calmod-ulin-dependent adenylate cyclase, an enzyme that convertsATP to cyclic AMP (cAMP) and pyrophosphate (17) and in-creases intracellular cAMP levels (26).

Neutrophils constitute the first line of defense against bac-terial infections. These phagocytic cells are able to quicklycrawl, or chemotax, to the site of infection, and defects inneutrophil chemotaxis compromise the innate immune re-sponse. Chemotaxis is accompanied by shape changes that aremediated by rapid assembly and disassembly of actin filaments.We have previously shown that anthrax LT impairs neutrophilchemotaxis and chemokinesis by reducing the formation ofactin filaments in response to the chemoattractant N-formyl-met-leu-phe (FMLP) (14). To better understand the full con-sequences of B. anthracis infection on neutrophil motile func-tion, we have now focused on the effects of ET alone and incombination with LT. Although a prior study suggested thatET enhanced chemotaxis (38), we find that ET impairs neu-trophil chemotaxis and when combined with LT has additiveinhibitory effects. The findings demonstrate that anthrax toxinscan induce near-complete paralysis of neutrophil actin-basedmotility, and these effects may explain the meager neutrophilresponse that accompanies early stages of systemic anthrax.

MATERIALS AND METHODS

Toxin purification. EF was expressed and purified from Escherichia coli aspreviously described (33). PA and LF were purified from Bacillus anthracis aspreviously described (28).

PMN isolation and treatment with ET. Human neutrophils were purified byusing a Ficoll-Hypaque gradient as previously described (14). The study followedU.S. Department of Health and Human Services guidelines and was approved bythe Institutional Review Board at the University of Florida. Healthy volunteer

* Corresponding author. Mailing address: Division of InfectiousDiseases, Box 100277, University of Florida College of Medicine,Gainesville, FL 32610. Phone: (352) 392-4058. Fax: (352) 392-6481.E-mail: Southfs@medicine.ufl.edu.

� Published ahead of print on 6 April 2009.

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donors (total of seven subjects) ranged in age from 24 to 58 years and includedboth males and females of Caucasian and Asian descent. Purified neutrophilswere resuspended in RPMI medium with L-glutamine (Mediatech) and adjustedfor 1 � 106 cells/ml. Neutrophils were treated with various concentrations of EFplus PA, LF plus PA, or PA plus LF plus EF for 2 h at 37°C while being gentlyrotated to prevent clumping or cell activation. For experiments with ET and LT,a 1:1 weight ratio of PA to EF was used. We found that a weight ratio for PA toEF of 2:1 had effects identical to those of a 1:1 ratio. For experiments combiningall three toxins, the PA concentration was increased to 1 �g/ml to assure suffi-cient binding sites for both EF and LF. For the majority of experiments, controlcells were incubated with buffer alone. In addition, to assure the specificity of ourfindings, for each experimental condition cells were incubated with EF, LF, andPA alone. Neutrophils were studied immediately after the 2 h of incubation, andexperiments were completed within 4 to 5 h of blood drawing.

Cell culture. HeLa cells were grown in Dulbecco’s modified Eagle’s mediumwith 4.5 g/liter glucose supplemented with 10% fetal bovine serum and 5%penicillin and streptomycin (Cellgro.) Cells were grown to 70 to 80% confluence.

Annexin V staining, analysis for necrosis, and NBT assay. Annexin V stainingwas performed on neutrophils by using an annexin V-Fluos staining kit (Roche)combined with propidium iodide to assess necrosis, and 10,000 cells were ana-lyzed by fluorescence-activated cell sorting (FACS) as previously described (14).To further assess necrosis, in separate experiments cells were mixed in a 1:1 ratiowith 0.4% trypan blue (Sigma). Samples were then loaded onto a hemacytometerand allowed to sit for 2 min, and the intracellular content of trypan blue deter-mined by light microscopy. Two hundred cells were analyzed for each condition.The nitroblue tetrazolium (NBT) assay was performed before and after stimu-lation with a final concentration of 200 ng/ml of phorbol myristate acetate(Sigma), in accordance with the manufacturer’s protocol. One hundred cellswere analyzed for each condition.

Neutrophil chemokinesis, chemotaxis, and polarization. Neutrophil chemoki-nesis, chemotaxis, and polarization assays were performed as previously de-scribed (14). Briefly untreated and ET-, LT-, and ET-plus-LT-treated PMNs(1 � 105 cells in 2 ml of RPMI medium) were added to a 35-mm glass-bottommicrowell dish (Matek) coated in 0.1% fibronectin (Sigma). For chemokinesisexperiments, 1 �M FMLP was added to each plate 5 min prior to time-lapsephase-contrast video imaging. Images were captured at 10-s intervals using aninverted Zeiss microscope and a cooled charge-coupled-device camera (modelC5985; Hamamatsu). The velocity of PMNs was determined by using the Meta-Morph software Track Objects application (Universal Imaging). The percentageof polarized PMNs (having a distinct lamellipod and uropod) was assessed 15min after the addition of FMLP. Chemotaxis toward a gradient was assessed byusing a FemtoJet needle (0.5-�m tip; Eppendorf) containing a concentration of10 �M FMLP. The tip of the needle was placed just inside the visual field, andchemoattractant was infused into buffer solution at a pressure of 15 lb/in2 usingan Eppendorf micromanipulator. The velocity of movement toward the needlewas measured by time-lapse video microscopy at 10-s intervals. In addition toanthrax toxins, in selected experiments, cells were treated with 10 �M of fors-kolin (Sigma-Aldrich) and 100 �M 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich) for 15 min at 37°C.

Whole-cell cAMP levels. cAMP levels in human neutrophils and HeLa cellswere determined by using an enzyme-linked immunoassay (Amersham Bio-sciences) as previously described (6).

PMN phalloidin and CD11/CD18 staining and FACS analysis. Phalloidinstaining was performed as previously described (14). Briefly, after incubation for2 h at 37°C with PA plus EF (ET), PA plus LF (LT), or both toxins (PA plus LFplus EF), neutrophils were exposed to 1 �M FMLP (Sigma-Aldrich) for 0, 5, 10,15, 30, 60, and 120 s. Cells were fixed at these time points with a final concen-tration of 3.7% formaldehyde, followed by permeabilization with 0.2% Tritonand staining with Alexa 488-phalloidin stain (Invitrogen-Molecular Probes). Inseparate experiments, live PMNs were stained using CD11/CD18 primary anti-body (Abcam) at 2.5 �g/ml, followed by secondary antibody conjugated withhorseradish peroxidase at a 1:100 dilution (Invitrogen). Immediately followingstaining, cells were subjected to FACS analysis.

Measurement of phosphorylated PKA. Protein kinase A (PKA) phosphoryla-tion was determined by using a PKA assay kit (Upstate Cell Signaling Solutions)with [�-32P]ATP (PerkinElmer). Neutrophils were treated with 500 ng/ml of ETand incubated at 37°C for 2 h or treated with the positive control 6-dibutyryl–cAMP at 100 �M for 1 h (Biolog Life Science Institute.) Cells were lysed usingnondenaturing lysis buffer (1% Triton X-100, 50 mM Tris-Cl, 150 mM KCl, 50mM EDTA, 0.2% NaA2, 200 mM imidazole, 100 mM NaFl, 100 mM Na3VO4)and a complete mini-protease inhibitor cocktail tablet (Roche). As previouslydescribed (3), phosphorylation of PKA was determined by using 25 �g of radio-

labeled protein lysate and measuring the transfer of radioactive phosphate fromATP into Kemptide.

Listeria monocytogenes and Shigella flexneri infection and phalloidin staining.Listeria monocytogenes infection and phalloidin staining were performed as pre-viously described (7). Listeria motility and tail lengths were determined 4 to 6 hafter the initiation of Listeria infection, as previously described (13). Shigellaflexneri infection was performed as previously described (41), and velocitiesdetermined two and a half hours after infection.

Statistical analysis. The Kruskal-Wallis test for multiple comparisons and theFischer’s exact test were used to determine statistical significance. Results are theaverages from three separate experiments unless otherwise noted.

RESULTS

Anthrax ET is active in human neutrophils. ET enteredboth human neutrophils and HeLa cells, as evidenced by aconcentration-dependent rise in cAMP levels. Neutrophils andHeLa cells exposed to increasing concentrations of ET (50 to1,000 ng/ml of a 1:1 weight ratio of EF and PA; Fig. 1A legend)for 2 h demonstrated a progressive rise in intracellular cAMP,reaching a maximum rise of �50-fold the level in untreatedcells (Fig. 1A to D). In neutrophils, this marked increase incAMP was similar in magnitude to that induced by the com-bined cAMP agonists forskolin and IBMX (Fig. 1A). In HeLacells, ET caused a greater rise in cAMP levels than theseagonists (Fig. 1C). These effects were shown to be time depen-dent in neutrophils and HeLa cells, prolonged exposure beingassociated with a progressive accumulation of cAMP (Fig. 1Band D). Exposure of human neutrophils and HeLa cells to PA,EF, or LF alone had no effect on cAMP levels, the resultingconcentrations being identical to those in cells exposed tobuffer alone (data not shown).

Cellular cAMP’s main downstream effector protein is PKA(20). For assessment of PKA phosphorylation, cell extractsfrom untreated neutrophils were compared to extracts fromcells treated with 6-db-cAMP (positive control) or 500 ng/ml ofET. ET treatment of neutrophils resulted in a fourfold increasein 32P incorporation into PKA (Fig. 1E). This is the first timethat ET-induced 32P incorporation in PKA has been demon-strated in neutrophils.

Effects of ET on apoptosis, necrosis, and NBT reduction. Toensure that ET’s effects on motility were not the result ofapoptosis, we compared annexin V staining in ET-treated cellsto the staining in those exposed to buffer. Propidium iodideexclusion was also measured to assess necrosis. As observedpreviously with LT (14), exposure to concentrations of up to500 ng/ml of ET did not significantly increase neutrophil apop-tosis and resulted in only low levels of necrosis compared toexposure to buffer alone (Table 1). Similarly, these concentra-tions of ET had minimal effects on HeLa cell apoptosis ornecrosis (Table 1). To further assess cell viability, the ability ofcontrol, ET, and ET-plus-LT-treated cells to exclude trypanblue was assessed. Under control conditions, 99% of PMNsexcluded trypan blue. After ET treatment (100 to 500 ng/ml,1:1 weight ratio), 96 to 97% of cells excluded trypan blue, andfollowing ET-plus-LT treatment (50 ng to 500 ng/ml of ET andLF combined with 1 �g of PA), 93 to 94% of cells excluded thedye. To further assure that PMN functions unrelated to actin-based motility remained intact, we compared the ability ofcontrol and ET-treated, as well as ET-plus-LT-treated, PMNsto reduce NBT. The reduction of this dye to a blue precipitatereflects the generation of superoxide. We found comparable

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percentages of NBT-positive cells in phorbol myristate acetate-stimulated PMNs, as follows: control, 91%; ET treated, 89%(500 ng/ml PA plus 500 ng/ml EF); and ET plus LT treated,91% (1 �g/ml PA plus 500 ng/ml EF plus 500 ng/ml LF).

PMN chemokinesis and chemotaxis. We have previouslyshown that LT exposure significantly impairs neutrophil che-motaxis and chemokinesis (14). However, during systemicanthrax infection, neutrophils are exposed to both LT and ET.Therefore, we have now examined the effects of ET alone, aswell as the combination of ET and LT, on human neutrophilmotility. The velocity of chemokinesis was significantly reducedby treatment with ET, a reduction in velocity being observed atconcentrations of 50 ng/ml, with maximal reduction in velocityof 40% being observed at 300 to 500 ng/ml (Fig. 2A). Dualtreatment with ET and LT had additive inhibitory effects onthe velocity of chemokinesis, causing a maximal inhibition of80% at a concentration of 250 ng/ml for both toxins (Fig. 2B).When the concentration of ET combined with LT was raised to300 ng/ml, neutrophils no longer adhered to the surface.Therefore, for our chemokinesis and chemotaxis experimentscombining both toxins, the individual concentrations of EF andLF did not exceed 250 ng/ml. ET and LT alone at concentra-tions of up to 1,000 ng/ml had no measurable effect on adher-ence, indicating that impaired adherence required the activi-ties of both toxins (data not shown).

In control neutrophils, exposure to FMLP resulted in a dis-tinctly polarized morphology, a high percentage of cells form-ing broad lamellipodia at the leading edge and small uropodiaat the rear (Fig. 2C and E). Exposure to ET alone markedlyimpaired the ability of neutrophils to polarize, maximum inhi-bition being seen at the same concentrations that maximallyslowed chemokinesis, 300 to 500 ng/ml (Fig. 2E). Exposure ofneutrophils to dual toxin treatment resulted in greater inhibi-tion than for either toxin alone, maximum reductions in thepercentage of polarized cells being observed at 250 ng/ml (Fig.2D and F). ET treatment also reduced the speed of neutrophil-directed migration, or chemotaxis, treatment with 300-ng/ml-or-higher concentrations of ET reducing mean chemotacticvelocity by 50% (Fig. 2G). Under these same conditions, LTalso caused a concentration-dependent reduction in chemo-taxis velocity, and a maximum inhibition of nearly 50% was

FIG. 1. Effects of ET on intracellular cAMP levels and PKAphosphorylation. (A) Bar graph showing the concentration depen-dence of ET-induced intracellular cAMP levels in human neutro-phils. Cells incubated with ET (PA and EF in a 1:1 weight ratio, e.g.,50 ng/ml ET � 50 ng/ml PA � 50 ng/ml EF) for 2 h at 37°C werecompared to cells incubated in buffer. The far right bar shows theresults for neutrophils treated with the cAMP agonists forskolin (10mM) and IBMX (100 mM) for 15 min at 37°C. Error bars indicatethe SEMs of the results of three separate experiments. PA aloneand EF alone (final concentrations, 500 ng/ml) failed to alter cAMPlevels, the resulting concentrations being identical to those in cellsincubated in buffer. (B) Bar graph showing the effects of incubationtime on the ET-induced rise in intracellular cAMP levels. Neutro-phils were treated with 500 ng/ml of ET (500 ng/ml PA plus 500ng/ml EF) and incubated at 37°C. Error bars show the SEMs of theresults of three experiments. (C) Bar graph showing the concentra-tion dependence of ET-induced intracellular cAMP levels in HeLacells. Experimental conditions were identical to those described forpanel A. Error bars indicate the SEMs of the results of threeexperiments. As observed with neutrophils, PA alone and EF alone(500 ng/ml) had no effect on cAMP levels. (D) Bar graph showingthe effects of incubation time on the ET-induced rise in intracellularcAMP levels in HeLa cells. Conditions were identical to thosedescribed for panel B. Error bars indicate the SEMs of the results ofthree experiments. (E) Bar graph showing the effects of ET on PKAphosphorylation (P-PKA). Neutrophils were exposed to buffer, treatedwith 6-db-cAMP (100 mM) for 1 h, or treated with 500 ng/ml ET for 2.5 h.A �4-fold increase in phosphorylated PKA was observed after ET treat-ment. Error bars indicate the SEMs of the results of three experiments.Ctl, control (buffer treated); Fsk, forskolin.

TABLE 1. Effects of ET on cell necrosis and apoptosisa

Cell type and treatmentNo. (%) of cells

Viable Necrotic Apoptotic

NeutrophilsControl 8,502 (96.7) 291 (3.3) 0 (0)50 ng/ml ET 8,324 (89.9) 936 (10.1) 2 (�1)300 ng/ml ET 8,541 (95.8) 375 (4.2) 0 (0)500 ng/ml ET 8,558 (92.0) 741 (8.0) 1 (�1)

HeLa cellsControl 8,604 (93.9) 557 (6.1) 36 (�1)50 ng/ml ET 7,476 (98.5) 111 (1.5) 41 (�1)300 ng/ml ET 7,270 (96.3) 277 (3.7) 61 (�1)500 ng/ml ET 7,274 (96.0) 303 (4.0) 41 (�1)

a Cells were treated with ET for 2 h and stained with annexin V and propidiumiodide, followed by FACS analysis (see Materials and Methods). HeLa cells werescraped from tissue culture dishes, explaining the relatively high percentage ofnecrosis in the control HeLa cells.

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FIG. 2. Effects of anthrax toxins on neutrophil chemokinesis, polarity, and chemotaxis. (A) Bar graph showing the mean relative velocity ofhuman neutrophil chemokinesis after treatment with buffer, increasing concentrations of ET (PA plus EF, 1:1 weight ratio; see Fig. 1), orforskolin-IBMX (10 nM/100 nM). A final concentration of 1 �M FMLP was added to the solution, and velocities measured between 5 and 15 minafter addition. Error bars indicate the SEMs of the results of seven experiments. Incubation with PA, EF, or LF alone (500 ng/ml) resulted invelocities that were identical to those of neutrophils incubated in buffer. Differences were highly significant (P � 0.0001). (B) Bar graph showingthe effects of the combination of PA, EF, and LF on neutrophil chemokinesis. Conditions were identical to those described for panel A except thatcells were incubated with 1 �g of PA and increasing concentrations of 1:1 weight ratios of EF and LF (e.g., 50 ng � 1 �g PA � 50 ng/ml EF �50 ng/ml LF). Error bars indicate the SEMs of the results of three experiments. Differences were highly significant (P � 0.0001). (C) Phase-contrastmicrograph of control PMNs 5 min after the addition of 1 �M FMLP. PMNs show broad lamellipodia at the head and narrow uropods at the back.The arrows point in the direction of polarity and movement of each cell. Bar � 10 �m. (D) Phase-contrast micrograph of PMNs treated with PA,

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observed at concentrations of between 250 and 500 ng/ml (Fig.2H). Dual toxin treatment also inhibited chemotaxis, a maxi-mal inhibition of 80% being observed at 250 ng/ml of thecombination. (Fig. 2I). Thus, as observed for chemokinesis andpolarity, the combination of LT and ET had additive inhibitoryeffects on chemotaxis. PA, EF, or LF alone at a concentrationof 500 ng/ml had no significant effect on chemokinesis or che-motaxis, the resulting velocities being identical to those ofcontrol cells (data not shown).

CD11/CD18 expression in neutrophils. Signaling via the ad-hesion molecules of the �2 integrin family, CD11b/CD18, playsan essential role in PMN recruitment and activation duringinflammation. ET-induced reductions in chemotaxis and che-mokinesis could in part be mediated by a change in the surfaceexpression of these adherence molecules. Therefore, we exam-ined the effects of treatment with 500 ng/ml ET and forskolin-IBMX on the PMN surface marker expression of CD11b/CD18. No significant differences in surface expression wereobserved compared to the level of expression in control neu-trophils (Fig. 3A). Similarly, LT at concentrations of up to 500ng/ml had no effect on the surface expression of CD11b/CD18(Fig. 3A). However, when EF, LF, and PA were combined, aconcentration-dependent decrease in receptor surface expres-sion was observed (Fig. 3B). Minimal effects were seen at 100ng/ml; however, a 50% reduction in expression was observed at300 ng/ml. PA, LF, and EF alone (500 ng/ml) had no effect onsurface expression (data not shown).

Effects of ET alone and ET combined with LT on neutrophilactin assembly. Neutrophil chemotaxis requires rapid assem-bly of actin filaments at the leading edge, and ET-inducedimpairment of chemotaxis could be mediated by inhibitionof neutrophil actin assembly. As assessed by Alexa-phalloi-din staining of filamentous actin and by FACS analysis, ETtreatment resulted in a delay in FMLP-stimulated actin as-sembly and a reduction in the maximum actin filament con-tent (Fig. 4A), and the extent of inhibition was comparableto that observed for treatment with 500 ng/ml of LT (Fig.4A) (14). The combination of PA plus EF plus LF (ET plusLT) resulted in an additive reduction in F-actin content,suggesting that EF and LF impair neutrophil actin assembly

by different signaling pathways (Fig. 4A). PA, EF, or LFalone at 500 ng/ml had no significant effect on FMLP-stim-ulated actin assembly, the resulting F-actin content beingnearly identical to that of neutrophils incubated in buffer(data not shown).

To further explore the effects of anthrax toxins on neutrophilactin assembly, we compared fluorescence micrographs of Al-exa-phalloidin-stained adherent neutrophils following expo-sure to 1 �M FMLP. Control neutrophils were polarized anddemonstrated a high content of F-actin at the leading edge inlamellipodia (Fig. 4B). LT-treated adherent neutrophils ap-peared rounded, with considerably reduced F-actin content(Fig. 4C). ET- and ET-plus-LT-treated neutrophils demon-strated a distribution of filamentous actin that was distinctlydifferent from that in control cells or cells treated with LTalone (Fig. 4D and E). Small discrete regions of increased

EF, and LF (1 �g/ml PA, 250 ng/ml EF, and 250 ng/ml LF for 2 h) 5 min after the addition of 1 �M FMLP. The number of polarized cells (arrows)is markedly decreased. (E) Bar graph showing the percentage of polarized PMNs after FMLP exposure as described for panels A to D above.Control cells were incubated in buffer for 2 h, and other bars represent exposure to increasing concentrations of ET (1:1 weight ratio of PA plusEF, e.g., 50 ng/ml ET � 50 ng/ml PA � 50 ng/ml EF) for 2 h, as well as forskolin-IBMX (10 nM/100 nM) for 15 min. Error bars show SEMs ofthe results of seven experiments. The differences were highly significant (P � 0.001), except for the 50-ng/ml concentration (P � 0.05). For eachcondition, 130 cells were analyzed per experiment. (F) Bar graph showing the effects of the combination of PA, EF, and LF on neutrophil polarity.Experimental conditions were identical to those described for panels A to E. Cells were incubated with 1 �g/ml PA plus increasing concentrationsof a 1:1 weight ratio of EF and LF (e.g., 50 ng/ml � 1 �g/ml PA � 50 ng/ml EF � 50 ng/ml LF). Error bars indicate the SEMs of the results ofthree experiments. Compared to the results shown in panel E, there was increased inhibition from combining both LF and EF with PA (ET plusLT). (G) Bar graph showing the relative velocity of human neutrophil chemotaxis after treatment with buffer, increasing concentrations of ET (1:1weight ratio of PA plus EF), or forskolin-IBMX (10 nM/100 nM). The chemotactic gradient was generated by using a microinjection needlecontaining a needle concentration of 10 �M FMLP. Error bars indicate the SEMs of the results of three experiments. Differences were highlysignificant (P � 0.0001). (H) Bar graph showing the relative velocity of human neutrophil chemotaxis after treatment with buffer or increasingconcentrations of LT (1:1 weight ratio of PA plus LF). Error bars show the SEMs of the results of three experiments. Differences were highlysignificant (P � 0.0001). (I) Bar graph showing the effects of the combination of PA, EF, and LF on neutrophil chemotaxis. Experimentalconditions were identical to those described for panel G except that cells were incubated with the three exotoxins as described for panel F. Errorbars show the SEMs of the results of three experiments. Note the additive inhibition of chemotaxis compared to the levels of inhibition with ETand LT alone (G and H). Differences were highly significant (P � 0.0001). Ctl, control (buffer treated); Fsk, forskolin. To allow comparisons fromseparate experiments, mean velocities of toxin and forskolin-IBMX-treated cells were divided by the mean velocity of buffer-treated cells for eachexperiment.

FIG. 3. Effects of anthrax toxins on CD11/CD18 surface expressionby neutrophils. (A) Neutrophils were treated with buffer or 500 ng/mlET or LT (500 ng/ml PA plus 500 ng/ml EF or LF) for 2 h or forskolin(Fsk)-IBMX (10 mM/100 mM) for 15 min, followed by surface stainingand FACS analysis of 10,000 cells. In comparison to the level ofexpression in cells in buffer alone, no significant differences in receptorexpression were observed in toxin- or forskolin-IBMX-treated cells.(B) Neutrophils were treated with 1 �g/ml of PA plus increasingconcentrations of a 1:1 weight ratio of EF and LF (identical to condi-tions described for Fig. 2F) for 2 h, followed by staining and sorting asdescribed above. A 50% reduction in the CD11/CD18 surface receptorexpression was observed with 300 ng/ml (1 �g/ml PA plus 300 ng/ml EFplus 300 ng/ml LF). Error bars indicate the SEMs of the results ofthree experiments (P � 0.001). y axis, relative fluorescence intensity.

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FIG. 4. Effects of anthrax toxins on FMLP-induced neutrophil actin assembly. (A) Graph showing the effects of ET (500 ng/ml PA plus 500ng/ml EF; open circles) and LT (500 ng/ml PA plus 500 ng/ml LF; open squares), as well as the combination of ET and LT (1 �g PA plus 500 ng/mlEF plus 500 ng/ml of LF; closed squares), on actin filament content of neutrophils measured as relative fluorescence (see Materials and Methods)compared to that of cells incubated in buffer (Control; closed circles). Cells were treated for 2 h with toxin or buffer and then exposed to 1�mol/liter of FMLP at time zero. Cells were formalin fixed at the times indicated, permeabilized, and stained with Alexa-phalloidin. The medianfluorescence intensity was determined by FACS analysis of 10,000 cells for each time point. ET and LT slowed the onset of actin assembly bothalone and in combination, and the combination resulted in an additive reduction in peak F-actin content of 34%, compared to the reduction withET (15% reduction) and LT (15% reduction) alone (P � 0.001). Error bars show the SEMs of the results of three experiments. (B) Fluorescencemicrograph of human neutrophils incubated with buffer for 2 h, allowed to attach to fibronectin-coated glass slides, and then exposed to 1 �MFMLP for 10 min, followed by formalin fixation, permeabilization, and staining with Alexa-phalloidin. Note the high concentrations of filamentousactin at the leading edge of the polarized neutrophils. Bar � 10 �m. (C) Fluorescence micrograph of human neutrophils incubated with 500 ng/mlLT (500 ng/ml PA plus 500 ng/ml LF) for 2 h and then treated as described for panel B. Note the reduction in fluorescence intensity, indicative

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F-actin content were noted throughout the horizontal plane ofthe cells. By shifting the Z-plane of focus in 0.5-�m stepsbeginning at the top of the cell, these F-actin structures werefound to be in the lowest focal plane, adjacent to the slidesurface (Fig. 4F).

Effects of ET alone and combined with LT on Listeria mono-cytogenes and Shigella flexneri actin-based motility. Listeriamonocytogenes (7, 36) and Shigella flexneri (2) both highjack theactin-regulating system of host cells to induce the assembly ofactin filaments. Both organisms bypass many of the signaltransduction mechanisms required for receptor-mediated actinassembly, and we have used these model systems to furtherassess the effects of ET and the combination of PA plus EFplus LF on in vivo actin assembly. The velocity of bacterialmovement directly correlates with the rate of actin assemblyand, assuming that the rate of actin disassembly is constant, thelength of each actin filament tail also directly correlates withthe assembly rate of actin filaments within the tail (30, 35).Therefore, we examined the effects of these toxins on bacterialintracellular velocities and on actin tail lengths. Exposure ofHeLa cells to 500 ng/ml of ET resulted in a reduction of nearly50% in Listeria velocity, and treatment with forskolin-IBMXalso reduced Listeria velocity (Fig. 5A). Listeria actin taillengths were reduced comparably (Fig. 5B to D). The combi-nation of PA plus EF plus LF (ET plus LT) also impairedListeria actin-based motility, maximum inhibition occurring ata concentration of 50 to 100 ng/ml (Fig. 5E and F). The max-imal inhibition of the combined toxins was similar to that of ETor LT alone. PA, EF, or LF alone at 500 ng/ml had no signif-icant effect on Listeria intracellular actin-based motility, theresulting velocities and tail lengths being identical to those incells treated with buffer alone (data not shown).

Treatment of Shigella flexneri-infected HeLa cells with 500ng/ml of ET minimally slowed the velocity of intracellularmovement (mean control velocity standard error of themean [SEM], 0.125 0.007 �m/s, n � 270, versus 0.109 0.007 �m/s for ET-treated cells, n � 315; P � 0.07). Thisfinding is consistent with previous observations that Shigellaand Listeria actin-based motilities utilize different actin-regu-lating proteins and signal transduction pathways (5).

DISCUSSION

Inhalation anthrax is a rapidly progressive disease that isassociated with a level of mortality of 85%, death occurring onaverage 4 to 5 days after the onset of symptoms (19). Thefulminant nature of this illness, combined with autopsy findingsof hemorrhagic necrosis and a general paucity of acute inflam-matory cells, indicates that systemic anthrax is accompanied by

profound impairment of the innate immune response (1, 16).The toxins produced by virulent B. anthracis are likely to playa critical role in paralyzing the innate immune system. Neu-trophils are a primary component of the innate immune re-sponse and are the earliest responders to invasion by bacterialpathogens. Investigations of the anthrax toxins’ effects on neu-trophil function promise to provide insight into the pathogen-esis of systemic, as well as cutaneous, anthrax. Recently, weinvestigated the effects of LT on neutrophil motile functionand discovered that relatively low concentrations of LT (50 to100 ng/ml) impair neutrophil chemotaxis and chemoattractant-induced actin assembly (14). Both LT and ET have been shownto markedly impair the activation of neutrophil NADPH oxi-dase activity, thus disarming the powerful superoxide bacteri-cidal system (6).

Less is known about the effects of ET on cell motility. Morethan 2 decades have passed since the effects of ET on humanneutrophil chemotaxis were last examined (38). Recently, inves-tigators have been able to express ET in E. coli and have shownthat this purified recombinant protein has binding affinity andbiological activity comparable to those of toxin purified from B.anthracis (24, 33). This advance has allowed us to reexamine thebiological effects of this calcium-sensitive, calmodulin-dependentadenylate cyclase on neutrophil motility. Unlike the original studythat noted a doubling of directed neutrophil migration in re-sponse to ET, as well as to the combination of PA, EF, and LF(38), we find that ET treatment results in a concentration-depen-dent reduction in neutrophil chemotaxis (Fig. 2G). We utilized adifferent assay for chemotaxis, video microscopy of neutrophilsadherent to a fibronectin-coated surface rather than migrationthrough agarose, and our utilization of this assay may account forour contradictory findings. As further support for ET-mediatedimpairment of chemotaxis, we find that ET treatment also re-duces chemokinesis (Fig. 2A) and the ability of neutrophils topolarize in response to the chemoattractant FMLP (Fig. 2E).These findings suggested that ET may globally impair neutrophilactin assembly, and our assessment of filament assembly kineticsusing Alexa-phalloidin staining revealed that ET slows both theonset and extent of chemoattractant-stimulated neutrophil actinassembly (Fig. 4A). The ET-mediated reduction in actin filamentcontent is accompanied by a distinct change in the actin filamentlocalization. Rather than homogeneously concentrating at theleading edge in lamellipodia as observed in untreated neutrophilsexposed to FMLP, actin filaments in ET-treated neutrophils con-centrate in discrete small foci dispersed throughout the cell nearthe adherent membrane surface. This staining pattern is reminis-cent of actin filament clusters associated with focal contacts inneutrophils adhered to uncoated plastic surfaces, a condition

of reduced filamentous actin. The cells failed to polarize, remaining rounded. (D) Fluorescence micrograph of human neutrophils incubated with500 ng/ml ET (500 ng/ml PA plus 500 ng/ml EF). Note the reduction in fluorescence intensity, as well as punctate staining. Based on varying thefocus (Z-stack; see the legend for panel F), these discrete concentrations of filamentous actin localize near the adherent membrane surface. Asobserved after treatment with LT, the cells have a rounded morphology. (E) Fluorescence micrograph of human neutrophils incubated with ETplus LT (1 �g/ml PA plus 500 ng/ml LF plus 500 ng/ml EF). Note the reduction in fluorescence intensity, as well as the punctate staining androunded morphology, similar to cells treated with ET alone. (F) Fluorescence micrographs (Z-stack) showing different focal planes of threeadjacent neutrophils that were treated with ET, exposed to FMLP, and stained as described above. The far left image shows the top of the cells.Each subsequent image represents a 0.5-�m step toward the slide surface, as indicated. The far right image focuses within 0.5 �m of the slidesurface and shows the discrete foci of filamentous actin staining. Images are at a higher magnification than those in panels B to E (bar � 10 �m).

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shown to stimulate actin assembly in the absence of FMLP (25,34), and suggests that ET may act through similar signal trans-duction pathways.

Because systemic anthrax infection would be expected toexpose neutrophils to the combination of LF, EF, and PA, wealso examined the effects of this combination. We find that

inhibition of chemotaxis, chemokinesis, and polarization, aswell as FMLP-induced actin assembly, are additive. These find-ings suggest that LF and EF act by different pathways to impairactin-based motility. In addition, the combination of both tox-ins can reduce the surface expression of the adherence recep-tor CD11b/CD18 (Fig. 3), and this effect may contribute to the

FIG. 5. Effects of anthrax toxins on Listeria actin-based motility. (A) Bar graph showing the relative mean velocities of intracellular Listeria in HeLacells after treatment for 2 h with buffer or increasing concentrations of ET (1:1 weight ratio of PA plus EF; see Materials and Methods) or for 15 minwith forskolin-IBMX (see Materials and Methods). Error bars indicate the SEMs (n � 730; P � 0.001). Measurements were made 5 to 6 h after the initialListeria infection. (B) Bar graph comparing the mean lengths of Listeria-induced actin filament tails. Cells were formalin fixed, Triton permeabilized, andstained with Alexa-phalloidin 6 h after infection was initiated. Error bars indicate the SEMs (n � 100; P � 0.001). (C) Fluorescent micrograph ofbuffer-treated HeLa cells infected with Listeria for 6 h, fixed, and stained with Alexa 488-phalloidin. Note the long Listeria actin rocket tails (arrows pointto the beginning and end of selected tails). Bar � 10 �m. (D) Fluorescent micrograph of ET-treated (500 ng/ml PA plus 500 ng/ml EF) HeLa cellsinfected with Listeria. Cells were fixed and stained as described for panel C. Note the shorter Listeria actin tails. (E) Bar graph showing the effects ofincreasing concentrations of LF, EF, and PA in combination on the velocity of Listeria. Cells were incubated with 1 �g/ml of PA plus increasingconcentrations of a 1:1 weight ratio of EF and LF (e.g., 500 ng/ml ET � LT � 1 �g/ml PA � 500 ng/ml EF � 500 ng/ml LF). Experimental conditionswere identical to those described for panel A. Error bars show the SEMs (n � 100). (F) Bar graph showing the mean Listeria tail lengths under the sameconditions as described for panel E. Error bars show the SEMs (n � 75). Ctl, control (buffer treated); Fsk, forskolin.

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poor delivery of neutrophils to the sites of infection. Given theclose association between the cytoskeleton and integrins, it islikely that the marked reduction in actin filament assemblyinduced by dual toxin treatment may contribute to the reduc-tion in CD11b/CD18 surface expression. ET-mediated activa-tion of PKA would be expected to phosphorylate and inacti-vate the actin regulatory protein VASP, as well as to activatethe G proteins CDC42 and Rac (10), while LT would beexpected to block the activation of extracellular signal-regu-lated kinase, a necessary step in early focal contact formation(15). Our future experiments will focus on assessing the con-tributions of these pathways to adherence and actin assembly.It is of interest that in HeLa cells, this same combination doesnot result in an additive reduction in Listeria actin-based mo-tility, each individual toxin, as well as the combination, result-ing in a similar level of inhibition (Fig. 5). However, Listeriabypasses many of the signal transduction pathways required forFMLP-induced actin assembly, and therefore, the pathways bywhich these two toxins inhibit Listeria may differ from those ofreceptor-induced actin assembly.

What are the mechanisms underlying anthrax toxin-medi-ated inhibition of host cell actin assembly? We have recentlydiscovered that one of the primary downstream targets for LTis the actin monomer sequestering heat shock protein 27(Hsp27). The inability of LT-treated cells to phosphorylateHsp27 prevents the shuttling of actin monomers to the leadingedge of motile cells (13). We are presently beginning to ex-plore the pathway or pathways by which ET interferes withactin assembly. As previously reported (6, 38), we find that ETinduces a rise in cAMP levels, and under the conditions of ourexperiments, we observe a �50-fold rise of cAMP in neutro-phils. Our findings are consistent with previous observations ofneutrophils and T lymphocytes showing that agents inducing arise in cAMP can impair actin assembly and chemotaxis (11,18, 29, 31, 39, 40). However, a simple quantitative relationshipbetween cAMP levels and alterations in chemotaxis has notbeen observed, suggesting that additional signal transductionpathways can modify the effects of cAMP (18, 29, 40).

We document for the first time that the ET-induced rise incAMP is accompanied by the phosphorylation of PKA in hu-man neutrophils, a condition that would be expected to acti-vate this kinase. Our findings are consistent with previousobservations that ET treatment increases cAMP levels andactivates PKA in many other cell types (8, 9, 23, 27). Ourexperiments utilizing the intracellular bacterium Listeriamonocytogenes help to narrow the potential downstream tar-gets for ET and activated PKA. Listeria requires the bacterialsurface protein ActA, and this protein directly activates thehost cell Arp2/3 complex. ActA also attracts the actin-regulat-ing protein VASP. On the other hand, Shigella flexneri requiresthe bacterial surface protein IcsA. IcsA directly attracts andactivates the host cell protein N-WASP, and this protein inturn activates the Arp2/3 complex. Also Shigella, unlike Liste-ria, does not require VASP for intracellular actin-based motil-ity (5). Finally, Listeria requires phosphatidylinositol 3-kinaseactivity, while Shigella does not (32). Thus, the ability of ET toimpair Listeria but not Shigella actin-based motility points tothree potential mechanisms of ET action: (i) impairment ofActA-induced activation of the Arp2/3 complex, (ii) inhibitionof VASP binding or activation by ActA, and (iii) inhibition of

phosphatidylinositol 3-kinase activity. It is also possible that anadditional previously unappreciated difference in the pathwaysby which Listeria and Shigella utilize the actin-regulatory pro-tein pathways of the host cell accounts for our observations.We are presently exploring all three of these possibilities. Al-though many of the actin-regulatory proteins and pathways forListeria-induced actin assembly have proven to be applicable tonon-muscle cell actin-based motility, it will be important torelate our specific findings for Listeria to the regulation of actinassembly in neutrophils. Further investigations will also berequired to determine if ET specifically affects Listeria andneutrophil function independently of its ability to raise cAMPlevels.

Understanding the mechanisms by which anthrax toxins im-pair actin assembly promises to not only provide a new under-standing of anthrax pathogenesis, but also provide new insightsinto how immune cells regulate actin filament assembly inorder to change shape and crawl toward the sites of activeinfection.

ACKNOWLEDGMENTS

These studies were funded by NIH grants R01AI064891 andRO1AI034276.

The findings and conclusions in this report are those of the authorsand do not necessarily represent the views of the Centers for DiseaseControl and Prevention.

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