Skip Navigation

Anthrax Lethal Toxin Paralyzes Neutrophil Actin-Based Motility

  1. Russell L. During1,
  2. Wei Li1,
  3. Binghua Hao1,
  4. Joyce M. Koenig2,
  5. David S. Stephens3,
  6. Conrad P. Quinn4 and
  7. Frederick S. Southwick1
  1. Departments of
  2. 1Medicine/Infectious Diseases and
  3. 2Pediatrics, University of Florida, Gainesville;
  4. 3Department of Medicine, Division of Infectious Diseases, Emory University, and
  5. 4Centers for Disease Control and Prevention, Atlanta, Georgia
  1. Reprints or correspondence: Dr. Frederick Southwick, Division of Infectious Diseases, Box 100277, University of Florida College of Medicine, Gainesville, FL 32610 (southfs{at}medicine.ufl.edu)
 
Next Section

Abstract

Bacillus anthracis causes high-level bacteremia, strongly suggesting paralysis of the innate immune system. We have examined the effects of anthrax lethal toxin (LT) on human neutrophil chemotaxis, a process that requires actin filament assembly. Polymorphonuclear neutrophils (PMNs) treated with a sublethal concentration of LT (50 ng/mL) for 2 h demonstrated insignificant apoptosis or necrosis. However, this same concentration slowed human PMN formylmethionylleucylphenylalanine (FMLP)–stimulated chemokinesis by >60%, markedly reduced polar morphology, and rendered PMNs incapable of responding to a chemotactic gradient. These changes were accompanied by a >50% reduction in FMLP-induced actin filament assembly. One hour of exposure to LT failed to impair polarity or actin assembly, and the effects of LT were independent of mitogen-activated protein kinase kinase 1 inhibition. We conclude that 2 h of exposure to LT markedly impairs PMN actin assembly, and reductions in actin filament content are accompanied by a profound paralysis of PMN chemotaxis

As illustrated by the index case of the 2001 bioterrorist attack in the United States, Bacillus anthracis can cause fulminant sepsis, meningitis, and death within hours [1]. The high number of bacteria in the blood stream in association with a normal initial peripheral polymorphonuclear neutrophil (PMN) count suggests that B. anthracis may impair the migration of PMNs from the bone marrow to the peripheral blood stream. Similarly, the peripheral PMN counts in the other 10 cases of inhalation anthrax associated with this attack were normal or mildly elevated at the time of hospital admission [2, 3]. In addition to defective PMN migration, the normal initial peripheral PMN counts could be the consequence of defective chemokine signaling and increased PMN turnover. Another characteristic feature of these cases was the development of hemorrhagic pleural effusions containing a paucity of white blood cells, a finding indicative of poor PMN delivery to a primary site of infection [4]. These clinical findings suggest an early paralysis of the innate immune system, particularly of the PMNs

These effects on the immune system are likely to be mediated by anthrax toxin, an exotoxin that consists of 3 components: protective antigen (PA), edema factor (EF), and lethal factor (LF). PA is responsible for binding target cell receptors and ushering LF or EF into the cytoplasm [5]. LF and EF in the absence of PA are not toxic to intact cells. In recognition of this fact, the combination of PA plus LF is called “lethal toxin” (LT), and the combination of PA plus EF is called “edema toxin” (ET). EF has been previously shown to be an adenylate cyclase that increases the intracellular levels of cAMP [6]. An increase in cAMP levels inhibits human PMN phagocytosis [7] and modestly impairs chemoattractant-mediated lamellipodia extension [8]. However, animal studies suggest that, as its name implies, LF is primarily responsible for the fatal outcome of B. anthracis sepsis [9, 10]. LF is a Zn2+-dependant metalloprotease, which has been previously shown to cleave mitogen-activated protein kinase kinases (MAPKKs, or MEKs) [11]. The cleavage of MEKs results in activation-induced apoptosis of macrophages [12], and the toxic effects of LT on macrophages have been emphasized [9]. On the basis of the recent clinical data described above, we hypothesized that one mechanism by which LT could impair innate immunity is by paralyzing PMN actin-base motility. The role of PMNs in LF-mediated toxicity was last addressed 2 decades ago. In those experiments, (1) PA plus LF, (2) PA plus EF, and (3) PA plus LF plus EF all increased PMN-directed migration, as measured using an agarose gel system [13]. These observations contradict our hypothesis; however, the recent clinical findings prompted us to reexamine the effects of LT on PMN chemotaxis, as well as to examine its effects on PMN actin filament assembly, the primary biochemical event responsible for the shape changes associated with ameboid movement [14]. We find that relatively low concentrations of PA plus LF (50 ng/mL) profoundly impair chemotaxis and the ability of PMNs to form polar structures. A slowing of the rate and extent of FMLP-induced PMN actin filament assembly accompanies these defects in chemotaxis. Furthermore, we have uncovered an explanation for the contradictory findings in earlier studies, and, finally, we demonstrate that the effects of LT on PMN actin assembly are independent of MEK1 inhibition

Previous SectionNext Section

Materials and Methods

PMN isolation and treatment with anthrax toxins Whole blood was collected by venous puncture from healthy volunteers and isolated using a Ficoll-Hypaque gradient medium (ICN Biomedical) as described elsewhere [15]; this procedure yielded 99% PMNs. Informed consent was obtained from all subjects. The study followed US Department of Health and Human Services guidelines and was approved by the University of Florida Institutional Review Board. Purified cells were resuspended in 5 mL of RPMI without l-glutamine (Mediatech), and the volume was adjusted to a concentration of 1.0×106 cells/mL. PA and LF were purified as described elsewhere [16], resulting in proteins of >99% purity as assessed by Coomassie blue–stained SDS-PAGE. In most experiments, unless otherwise noted, PMNs were incubated with (1) PA plus LF (50 ng/mL) in RPMI without l-glutamine (LT treated) or (2) RPMI without l-glutamine (control), for 2 h at 37°C in 1.5-mL tubes that were gently rotated to prevent cell clumping. Cells were studied immediately after incubation, and all experiments were completed within 3–4 h of phlebotomy. To assess LT activity, in multiple experiments, PMN extracts were subjected to Western blot analysis using an antibody directed against the amino-terminus of MEK, as described elsewhere [17]. This antibody identifies epitopes on the first 7 amino acids of MEK, the region cleaved by LF, and loss of antibody cross-reactivity indicates proteolysis by LF. Before cell lysis, PMNs were treated with a final concentration of 1 mmol/L diisoproplyfluorophosphate (Sigma), a cell-permeable, potent serine protease inhibitor that greatly reduces proteolysis in PMN extracts [18]. After cell lysis, an antiprotease cocktail (complete Mini protease inhibitor; Roche) was also added to extracts, to further block nonspecific proteolysis

Annexin V staining, propidium iodide (PI) staining, and nitroblue tetrazolium (NBT) test PMN annexin V staining was performed using the Annexin-V-FLUOS Staining Kit (Roche), in accordance with the manufacturer’s protocol, and stained cells were subjected to flow cytometry (FACScan; BD Biosciences) using an excitation wavelength of 488 nm and emission spectrums of 518 nm to detect anti–annexin antibody and 617 nm to detect PI. For Fas-mediated apoptosis, PMNs were incubated with mouse anti–human Fas IgM (500 ng/mL) for 2 h or with nonspecific IgG (500 ng/mL), as described elsewhere [19, 20]. For LT-treated cells, anti–Fas IgM was added at the same time as LT (50 ng/mL). The NBT test was performed before and after stimulation with a final concentration of 200 ng/mL of phorbol myristate acetate (Sigma), in accordance with the manufacturer’s protocol. One hundred cells were analyzed for each condition

PMN chemokinesis, chemotaxis, and polarization  Untreated and LT-treated PMNs (1×105 cells in 2 mL of RPMI) were added to 35-mm glass-bottom microwell dishes (MatTek Cultureware) coated with 0.1% fibronectin (Sigma). After the addition of a final concentration of 1 μmol/L FMLP, time-lapse phase-contrast images were captured at 20-s intervals using an inverted microscope (Nikon) and a cooled charge-coupled–device camera (model C5985; Hamamatsu). All images were processed using Metamorph image software (version 4.0; Universal Imaging). Velocities of PMNs were determined using the Metamorph track image program, as described elsewhere [21]. The percentage of polarized PMNs (defined as having a distinct lamellipod, or leading vale of cytoplasm, and a narrow tail, or uropod) after 4–12 min of exposure to FMLP was assessed by a blinded observer. To explore directed chemotaxis toward a gradient, a FemtoJet needle (0.5-μm tip diameter; Eppendorf) containing a needle concentration of 10 μmol/L FMLP was introduced at one corner of the coverslip and dispensed at 15 psi by use of an Eppendorf micromanipulator [22]

PMN phalloidin staining and fluorescence-activated cell sorting (FACS) analysis Immediately after 2 h of incubation with LT or buffer, PMNs were exposed to a final concentration of 1 μmol/L FMLP for 0, 5, 10, 15, 30, 60, or 120 s, and the reaction was stopped by the addition of a final concentration of 3.7% formalin, followed by 0.2% Triton and phalloidin Alexa 488 stain, as described elsewhere [23]. FACS analysis was performed using an excitation wavelength of 488 nm and an emission wavelength of 518 nm

Triton X-100 insoluble cytoskeleton For the Triton X-100 insoluble cytoskeleton experiments, higher concentrations of PMNs were required; 1.0×107 cells/mL were purified, and the assay was performed exactly as described elsewhere [24]. Cells were stimulated with 1 μmol/L FMLP for 0, 20, or 40 s, and the reaction was stopped by the addition of a 1:1 volume of a 2% triton X-100 stop solution (2% triton, 160 mmol/L KCl, 40 mmol/L imidazole HCl, 20 mmol/L EGTA, and 8 mmol/L sodium azide [pH 7.0] containing complete Mini protease inhibitor [Roche]). Cells were then centrifuged, and the resulting pellet was subjected to SDS-PAGE and Coomassie blue staining. The density of the 43-kDa polypeptide (actin) was integrated using AlphaImager (version 5.5; Alpha Innotech) for each sample

Statistical analysis The Wilcoxon 2-tailed nonparametric test and Fischer’s exact test were used to determine statistical significance. In all experiments except for FACS analysis, n refers to the number of cells analyzed. In most experiments, a minimum of 3 separate experiments were performed, unless otherwise noted

Previous SectionNext Section

Results

PMN apoptosis and necrosis after LT treatment There is considerable evidence that LT can induce apoptosis in macrophages [25]. Therefore, we first utilized annexin V staining in conjunction with PI to assess PMN apoptosis and necrosis after LT treatment. As shown in figure 1, no significant increase in apoptosis or necrosis was observed in PMNs treated with 50 ng/mL LT for 2 h at 37°C. Both control and LT-treated cells exhibited 0.5%–5% PI staining, indicating that this concentration of LT did not induce necrosis (figure 1 and table 1). These concentrations of LT also had minimal effects on apoptosis as assessed by annexin V staining (control cells vs. LT-treated cells, 2.5%–8.4% vs. 4%–8.2%, respectively) (figure 1 and table 1). In the experiment shown in figure 1, control PMNs exhibited 2.5% annexin V staining, whereas LT-treated cells exhibited 4% staining. In 2 subsequent experiments, control cells showed slightly higher levels of annexin V staining than did LT-treated cells. To further explore apoptosis, we also compared the effects of stimulating apoptosis in control and LT-treated PMNs by the addition of anti–Fas IgM antibody. This antibody cross-links Fas and accelerates apoptosis in PMNs [19]. As shown in table 1, we detected no significant difference in anti–Fas antibody–stimulated apoptosis between control and LT-treated PMNs. We also compared the morphology of Wright-stained control and LT-treated PMNs and found no changes characteristic of apoptosis (nuclear condensation or vacuolization of the cytoplasm) in either group during the time frame of our experiments [26]. The final concentration of toxin used in our experiments, 50 ng/mL, was sufficient to efficiently cleave MEK, as assessed by Western blot analysis (figure 1C ). These experiments indicate that human PMNs do not undergo significant apoptosis or necrosis in response to low concentrations of active LT

Figure 1
View larger version:
Figure 1

Effects of protective antigen plus lethal factor (lethal toxin [LT]) (50 ng/mL) on apoptosis and necrosis of polymorphonuclear neutrophils (PMNs). A Fluorescence-activated cell sorting (FACS) analysis of anti–annexin V antibody– and propidium iodide (PI)–stained PMNs after incubation in buffer for 2 h at 37°C. The vertical axis shows PI staining. Cells with staining intensities above the horizontal line were considered to be necrotic (0.5% of the cells). The horizontal axis represents the intensity of anti–annexin V staining. Cells to the right of the vertical line were considered to be apoptotic, and 2.5% of the cells were above this staining intensity. B FACS analysis of identically stained PMNs incubated with LT (50 ng/mL) for 2h at 37°C. The percentage of necrotic cells was also 0.5%, and that of apoptotic cells was 4%. C Western blot analysis of extracts from control and LT-treated PMNs using anti–amino-terminal mitogen-activated protein kinase kinase (MEK) antibody. The arrow points to expected molecular weight of MEK. Under the conditions of our experiments, MEK was fully cleaved by LT

To further ensure that other PMN functions unrelated to actin-based motility were intact, we also compared the ability of control and LT-treated PMNs to reduce NBT. The reduction of this dye to a blue precipitate reflects the generation of superoxide. We found no significant differences in the percentage of NBT+ cells in resting (control, 6.5%; LT-treated, 6%) or phorbol myristate acetate–stimulated (control, 92%; LT-treated, 90%) PMNs (P=1.0, Fisher’s exact test)

PMN chemokinesis and chemotaxis As described above, PMNs were treated with a final concentration of 50 ng/mL LT for 2 h and compared with PMNs incubated in buffer. The ability of these cells to crawl on a fibronectin-coated surface was assessed by video microscopy. First, cells were stimulated with a homogeneous solution of 1 μmol/L FMLP. As shown in figure 2A the mean random migration velocity of LT-treated PMNs was significantly decreased, compared with that of control PMNs, and this decrease was observed during the entire 12 min of observation (figure 2B ). These differences were highly significant (P<.001). After 5 min of stimulation with 1 μmol/L FMLP, a high percentage of control PMNs formed a broad lamellipod at their front and a narrow tail or uropod at their back, which is termed a “polarized” morphology (figure 2C and 2E ), although a high percentage of LT-treated PMNs remained rounded and symmetric in their morphology (figure 2D and 2E ). Quantitation of these morphologic differences revealed a marked reduction in the percentage of polarized cells after LT treatment, and this difference was highly significant (P < .0001) in 2 independent experiments (figure 2E ). The degree of spreading on the fibronectin-coated surface was also quantified by measuring the 2-dimensional area of control and LT-treated cells. A statistically significant reduction in 2-dimensional area was observed in LT-treated cells (mean ± SE area, 21±1.2 μm2; n=39), compared with control cells (mean ± SE area, 30±2.6 μm2; n=39) (P=.013), indicating a reduced ability to spread (compare figure 2C with 2D )

Figure 2
View larger version:
Figure 2

Effects of protective antigen plus lethal factor (lethal toxin [LT]) (50 ng/mL) on polymorphonuclear neutrophil (PMN) chemokinesis and polarity. A Graph showing PMN migration speeds over time. The paths of PMNs were tracked every 20 s, beginning immediately after the addition of FMLP (final concentration, 1 μmol/L), and the distance traveled per second was determined for each interval. PMNs treated with LT (50 ng/mL) (white circles, dashed line) consistently demonstrated slower speeds of ameboid movement than did control cells (black circles, solid line). Bars represent the SE of 11–14 PMNs. B Bar graph showing PMN mean migration speeds during various periods after the addition of FMLP. At time 0, FMLP (final concentration, 1 μmol/L) was added to the tissue culture dishes containing adherent PMNs. The left frame shows PMNs treated with LT for 2 h. Brackets represent the SE of n=135–210 measurements of 14 cells/period. In all instances, the mean speeds of migration were significantly slower for LT-treated PMNs (P<.0001, Wilcoxon nonparametric analysis). Results are representative of 3 separate experiments. The right frame shows the effects of 1 h of treatment with the same concentration of LT. Brackets represent the SE of n=230–240 measurements of 30 cells over 0–12 min. Differences were also statistically significant (P<.001); however, 1 h of treatment resulted in a 33% reduction in speed, vs. a 66% reduction in PMNs treated with LT for 2 h. Control values were normalized for experiments performed on different days, to allow meaningful comparisons. C Phase contrast micrograph of control PMNs, 5 min after the addition of 1 μmol/L FMLP. PMNs show broad lamellipodia at the head and narrow uropods at their back. The arrow points to the direction of polarity and movement of each cell. Bar, 10 μm. D Phase contrast micrograph of LT-treated PMNs, 5 min after the addition of 1 μmol/L FMLP. Formation of clear polar morphology is seen in only 1 cell (arrow) and the cells in this field demonstrated minimal movement. E Bar graph showing the percentage of polarized PMNs under conditions C and D in 2 separate experiments with PMNs treated with LT for 2 h (left frame). The black bars represent control cells, and the light gray bars represent LT-treated cells. Differences were highly significant (P<.0001, Fischer’s exact test). The right frame shows the effects of treating PMNs with the same concentration of LT for 1 h rather than 2 h. The difference in the percentage of polarized cells in control and LT-treated PMNs was not statistically significant (P=.66, Fischer’s exact test). For each condition, 37–112 cells were analyzed

To explore the ability of PMNs to move toward a chemoattractant gradient, we used a microinjection needle, as described elsewhere [22]. Control PMNs extended large lamellipodia and crawled toward the needle (figure 3A , left), whereas LT-treated PMNs remained rounded and demonstrated only minimal movement. (figure 3A , right). Control PMNs moved at a mean ± SE speed of 0.090±0.003 μm/s, whereas LT-treated cells demonstrated minimal directional movement (0.004±0.001 μm/s) (P<.0001) (figure 3B )

Figure 3
View larger version:
Figure 3

Effects of protective antigen plus lethal factor (lethal toxin [LT]) (50 ng/mL) on polymorphonuclear neutrophil (PMN) chemotaxis. A Human PMNs incubated with buffer or LT for 2 h at 37°C were placed in glass-bottomed Petri dishes coated with 0.1% fibronectin. After 5 min, a microinjection needle containing 10 μmol/L FMLP was positioned at the left side of the image. Sample was dispensed at 15 psi. Note the control PMN forming a lamellipod (arrow) and crawling toward the needle. The LT-treated PMNs remain rounded and fail to crawl toward the needle. Upper left-hand numbers represent the time in seconds. Bar, 10 μm. B Bar graph showing the mean speeds of control (black) and LT-treated (light gray) cells. Brackets represent the SE of n=131 measurements for control PMNs and n=105 for LT-treated PMNs (30 cells; 5 experiments)

PMN actin assembly PMN chemotaxis requires rapid assembly and disassembly of actin filaments, and the profound defects in chemotaxis that are induced by treatment with relatively low concentrations of LT encouraged us to examine chemoattractant-induced PMN actin polymerization. We used phalloidin conjugated to Alexa 488 to measure the relative increase in F-actin content on stimulation with 1 μmol/L FMLP. Phalloidin binds F-actin, and fluorescence intensity has been shown to be proportional to the F-actin content of PMNs [14]. After exposure to FMLP, the F-actin content of control PMNs rapidly increased; a significant increase was observed at the earliest time point that could be measured, 5 s (figure 4A ). A peak in F-actin content was observed at 30 s. The kinetics of actin assembly was delayed in LT-treated PMNs, with no increase in actin filament content being observed at 5 s (10 separate experiments), and the peak F-actin content was significantly lower than in control PMNs. Incubation of PMNs with PA or LF alone had no effect on chemoattractant-associated actin filament assembly (data not shown). A concentration of 5 ng/mL of LT also caused significant impairment of PMN actin assembly, at both 5 and 30 s. Increasing the toxin concentration to 500 ng/mL failed to further inhibit the increase in F-actin content (figure 4B ). However, a reduction in the time that cells were incubated with LT, from 2 to 1 h, profoundly affected LT impairment of PMN actin assembly. Under these incubation conditions, LT treatment caused minimal impairment of FMLP-induced actin assembly (figure 4C ). This shorter incubation period also reduced toxin inhibition of chemokinesis (figure 2B , right) and abrogated toxin-induced blockade of cell polarity (figure 2E , right)

Figure 4
View larger version:
Figure 4

Effects of protective antigen plus lethal factor (lethal toxin [LT]) on FMLP-induced polymorphonuclear neutrophil (PMN) actin assembly. A Graph showing the median fluorescence intensity as determined by fluorescence-activated cell sorting analysis of 5000–10,000 Alexa-phalloidin–stained PMNs over time. FMLP (1 μmol/L) was added at time 0 s. Cells were incubated with buffer (black circles) or LT (50 ng/mL) (white circles) for 2 h, as described in figure 1, before the addition of FMLP. Brackets represent the SE of 3 separate experiments. Note the delay in initiation of actin assembly, and the lower peak F-actin content in the LT-treated PMNs. B Bar graph comparing the effects of varying the concentrations of LT on the inhibition of FMLP-induced PMN actin assembly. Cells were treated with final LT concentrations of 5, 50, and 500 ng/mL (gray bars) and were compared with PMNs incubated in buffer alone (black bars) after stimulation with 1 μmol/L FMLP at time 0 s. Concentrations of LT in nanograms are shown above each bar. Because of some variation in the fluorescence intensities at time 0 s, the 5 and 30 s time points were divided by the intensity value at time 0 s. In control PMNs, the relative F-actin content increased 1.2-fold after 5 s of exposure to 1 μmol/L FMLP, whereas PMNs exposed to both 5 and 50 ng/mL demonstrated values below that of unstimulated PMNs, suggesting a slight decrease in F-actin content (left set of bars). At 30 s, >50% inhibition of actin assembly was observed at all 3 concentrations of LT (right set of bars). C Graph showing the median PMN F-actin content over time under experimental conditions identical to those described in A, except PMNs were treated with LT for 1 h rather than 2 h. Brackets represent the SE of 3 separate experiments. D Graph showing the relative increase in F-actin content over time of control (black circles) and LT-treated (50 ng/mL) (white circles) PMNs after FMLP stimulation (1 μmol/L), as assessed by measuring the actin associated with the Triton-insoluble cytoskeleton. At times 0, 20, and 40 s after exposure to FMLP, a Triton-stop solution was added, and the cells were centrifuged. The resulting pellet, representing the Triton-insoluble fraction, was then subjected to SDS-PAGE and Coomassie blue stained, and the density of the 43-kDa polypeptide, representing actin, was determined by densitometry. The area under the density curve (integration units) at time 0 s was compared to that at time 20 s and 40 s. Note the marked reduction in FMLP-induced Triton-associated actin content in the LT-treated PMNs. Values represent the mean of 2 experiments

To confirm the results of our phalloidin staining experiments, PMN actin filament content in cells incubated with LT for 2 h was compared with that in control cells by measuring the relative actin concentrations associated with the PMN Triton-insoluble cytoskeleton before and after exposure to FMLP. As shown in figure 4D after FMLP exposure for 20 and 40 s, the F-actin content of LT-treated PMNs exhibited a marked reduction, compared with that of control PMNs

One mechanism by which LT is known to impair cell function is by cleaving and inactivating MEK. To determine whether impairment of MEK mediates LT inhibition of PMN actin assembly, we examined the effects of the specific MEK1 inhibitor PD98059. Under conditions previously shown to inhibit MEK1 in PMNs [27], we observed no inhibition of FMLP-induced PMN actin assembly. In fact, in 2 of 2 experiments, we observed a slight increase in the peak F-actin content (figure 5)

Figure 5
View larger version:
Figure 5

Effects of treatment with the mitogen-activated protein kinase kinase inhibitor PD98059 (100 μmol/L) on FMLP-induced polymorphonuclear neutrophil (PMN) actin assembly. The graph shows the median PMN F-actin content over time after exposure to FMLP. Instead of being treated with lethal toxin, PMNs were treated with a final concentration of 100 μmol/L PD98059 for 10–15 min before exposure to FMLP. Subsequent steps were identical to those described in figure 4A . The graph is the mean of 2 experiments, both demonstrating slightly higher F-actin content in PD98059-treated PMNs than in control PMNs

Table 1
View larger version:
Table 1

Effects of anthrax lethal toxin (LT) on Fas-mediated apoptosis

Previous SectionNext Section

Discussion

We have discovered that low concentrations of LT cause near-complete paralysis of directed migration by human PMNs (figure 3) and also significantly impair chemokinesis and cell polarity (figure 2). Paralysis of PMNs, a vital component of the innate immune system, would help B. anthracis to establish a systemic infection before an adaptive immune response could be mounted. This profound PMN chemotactic defect is not accompanied by significant apoptosis or necrosis, indicating that the effects of anthrax toxin on cell motility are not a secondary consequence of impending cell death (figure 1). The ability of PMNs to resist apoptosis differs from that of macrophages, which, depending on the species, often undergo rapid cell death on exposure to LT. Unlike in macrophages, exposure to many inflammatory stimuli, including lipopolysaccharide, delays PMN apoptosis, and exposure to FMLP has no effect on this process [28]. In the absence of external stimuli, once they fully mature, PMNs do undergo relatively rapid programmed cell death; however, significant apoptosis does not begin for 3–4 h after cells are removed by phlebotomy [26]. Therefore, to minimize any contribution of this ongoing process, all of our experiments were performed within this time frame

Our findings differ from those of the 1 previous study on PMN chemotaxis, performed 2 decades ago. In those experiments, PMNs were treated for 1 h (one-half the duration used in our experiments). This treatment had no significant effect on random migration (chemokinesis); however, it did result in a doubling of the speed of directed migration (chemotaxis) [13]. The shorter incubation period used in those experiments may account for these contradictory findings. The duration of LT incubation in our experiments, 2 h, was sufficient to fully cleave MEK, as assessed by Western blot analysis, which is an accepted criterion for LF activity (figure 1C ) [11]. Previous studies have shown that 1.5–2 h is required for LF to fully cleave MEK [17]. Our experiments now document that incubation with LT for 1 h, compared with 2 h, markedly reduces the toxin’s ability to inhibit chemoattractant-induced PMN actin assembly (figure 4C ), cell polarity (figure 2E ), and chemokinesis (figure 2B )

A major biochemical event that accompanies the shape changes required for chemotaxis and chemokinesis is the assembly of new actin filaments [29]. Normally, on being exposed to the chemoattractant FMLP, PMNs double their F-actin content within 30 s, and the increase in F-actin content is observed within 5 s of stimulation. We observed this behavior in our control PMNs. However, treatment with LT resulted in a reproducible delay in the initial increase in F-actin content, with no increase in F-actin content being observed after 5 s. Furthermore, the peak in F-actin content was consistently <50% of that observed in control PMNs (figure 4A and 4B ). The inhibition of actin filament formation was even more profound as measured by the actin associated with the Triton insoluble cytoskeleton (figure 4D ). This assay has been shown to detect longer actin filaments and, therefore, is a more rigorous test for new actin assembly [30]

The mechanism by which LT impairs actin assembly is unclear. To date, 3 mechanisms have been discovered that can bring about the assembly of new actin filaments. Preformed actin filaments can be uncapped, creating free filament ends for new actin assembly; actin nucleating proteins, such as Arp2/3 complex, can become activated and initiate de novo actin filament assembly; and severing proteins can break apart old actin filaments, and these actin fragments can serve as the sites for new actin filament assembly [29]. The inability of higher concentrations of LF to completely block actin assembly suggests that this metalloprotease acts specifically on a limited number of these pathways (figure 4B ). LF is thought to act by cleaving MEK and inhibiting its function; however, to date, MEK has not been shown to mediate actin assembly, and we show that a specific MEK1 inhibitor does not impair the extent or time course of chemoattractant-induced PMN actin assembly (figure 5). Our findings are in agreement with those of a previous study demonstrating that MEK1 inhibition does not impair PMN chemotaxis or actin filament assembly [27]. The links between the cleavage of MEK by LF and the pathogenesis of anthrax are poorly understood [31], and recent studies suggest that some effects of LF are independent of MEK cleavage [32, 33]. Our experiments suggest that, in addition to MEK, LF could alter the function of actin or 1 or more actin regulatory proteins, and we are presently exploring these possibilities

In conclusion, we found that LT paralyzes human PMN chemotaxis, and this paralysis is associated with a marked inhibition in chemoattractant-induced PMN actin assembly. These effects are likely to contribute to the poor initial PMN response in the peripheral blood and pleural effusions of patients with overwhelming inhalation anthrax

Previous SectionNext Section

Acknowledgments

We thank Stephen H. Leppla of the National Institutes of Health for providing lethal factor. We also thank Gurjit Sidhu of the University of Florida for helpful discussions

Previous SectionNext Section

Footnotes

  • ↵Potential conflicts of interest: none reported

    Financial support: National Institutes of Health (grants RO1AI34276, RO1AI-23262, and RO1 HD-47401); Southeastern Center for Emerging Biological Threats (start-up grant)

  • Received January 10, 2005.
  • Accepted April 6, 2005.
Previous Section
 

References

  1. 1.↵
    1. Bush LM,
    2. Abrams BH,
    3. Beall A,
    4. Johnson CC
    . Index case of fatal inhalational anthrax due to bioterrorism in the United States. N Engl J Med 2001;345:1607-10.
    CrossRefMedlineWeb of Science
  2. 2.↵
    1. Jernigan JA,
    2. Stephens DS,
    3. Ashford DA,
    4. et al
    . Bioterrorism‐related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis 2001;7:933-44.
    MedlineWeb of Science
  3. 3.↵
    1. Barakat LA,
    2. Quentzel HL,
    3. Jernigan JA,
    4. et al
    . Fatal inhalational anthrax in a 94‐year‐old Connecticut woman. JAMA 2002;287:863-8.
    Abstract/FREE Full Text
  4. 4.↵
    1. Guarner J,
    2. Jernigan JA,
    3. Shieh WJ,
    4. et al
    . Pathology and pathogenesis of bioterrorism‐related inhalational anthrax. Am J Pathol 2003;163:701-9.
    MedlineWeb of Science
  5. 5.↵
    1. Collier RJ,
    2. Young JA
    . Anthrax toxin. Annu Rev Cell Dev Biol 2003;19:45-70.
    CrossRefMedlineWeb of Science
  6. 6.↵
    1. Leppla SH
    . Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci USA 1982;79:3162-6.
    Abstract/FREE Full Text
  7. 7.↵
    1. O’Brien J,
    2. Friedlander A,
    3. Dreier T,
    4. Ezzell J,
    5. Leppla S
    . Effects of anthrax toxin components on human neutrophils. Infect Immun 1985;47:306-10.
    Abstract/FREE Full Text
  8. 8.↵
    1. Chodniewicz D,
    2. Zhelev DV
    . Chemoattractant receptor‐stimulated F‐actin polymerization in the human neutrophil is signaled by 2 distinct pathways. Blood 2003;101:1181-4.
    Abstract/FREE Full Text
  9. 9.↵
    1. Hanna PC,
    2. Acosta D,
    3. Collier RJ
    . On the role of macrophages in anthrax. Proc Natl Acad Sci USA 1993;90:10198-201.
    Abstract/FREE Full Text
  10. 10.↵
    1. Beall FA,
    2. Taylor MJ,
    3. Thorne CB
    . Rapid lethal effect in rats of a third component found upon fractionating the toxin of Bacillus anthracis . J Bacteriol 1962;83:1274-80.
    Abstract/FREE Full Text
  11. 11.↵
    1. Duesbery NS,
    2. Webb CP,
    3. Leppla SH,
    4. et al
    . Proteolytic inactivation of MAP‐kinase‐kinase by anthrax lethal factor. Science 1998;280:734-7.
    Abstract/FREE Full Text
  12. 12.↵
    1. Park JM,
    2. Greten FR,
    3. Li ZW,
    4. Karin M
    . Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 2002;297:2048-51.
    Abstract/FREE Full Text
  13. 13.↵
    1. Wade BH,
    2. Wright GG,
    3. Hewlett EL,
    4. Leppla SH,
    5. Mandell GL
    . Anthrax toxin components stimulate chemotaxis of human polymorphonuclear neutrophils. Proc Soc Exp Biol Med 1985;179:159-62.
    Abstract/FREE Full Text
  14. 14.↵
    1. Howard TH,
    2. Oresajo CO
    . The kinetics of chemotactic peptide‐induced change in F‐actin content, F‐actin distribution, and the shape of neutrophils. J Cell Biol 1985;101:1078-85.
    Abstract/FREE Full Text
  15. 15.↵
    1. Southwick FS,
    2. Young CL
    . The actin released from profilin‐actin complexes is insufficient to account for the increase in F‐actin in chemoattractant‐stimulated polymorphonuclear leukocytes. J Cell Biol 1990;110:1965-73.
    Abstract/FREE Full Text
  16. 16.↵
    1. Quinn CP,
    2. Shone CC,
    3. Turnbull PC,
    4. Melling J
    . Purification of anthrax‐toxin components by high‐performance anion‐exchange, gel‐filtration and hydrophobic‐interaction chromatography. Biochem J 1988;252:753-8.
    MedlineWeb of Science
  17. 17.↵
    1. Chopra AP,
    2. Boone SA,
    3. Liang X,
    4. Duesbery NS
    . Anthrax lethal factor proteolysis and inactivation of MAPK kinase. J Biol Chem 2003;278:9402-6.
    Abstract/FREE Full Text
  18. 18.↵
    1. Amrein PC,
    2. Stossel TP
    . Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate. Blood 1980;56:442-7.
    Abstract/FREE Full Text
  19. 19.↵
    1. Allgaier B,
    2. Shi M,
    3. Luo D,
    4. Koenig JM
    . Spontaneous and Fas‐mediated apoptosis are diminished in umbilical cord blood neutrophils compared with adult neutrophils. J Leukoc Biol 1998;64:331-6.
    Abstract
  20. 20.↵
    1. Luo D,
    2. Schowengerdt KO Jr,
    3. Stegner JJ,
    4. May WS Jr,
    5. Koenig JM
    . Decreased functional caspase‐3 expression in umbilical cord blood neutrophils is linked to delayed apoptosis. Pediatr Res 2003;53:859-64.
    CrossRefMedlineWeb of Science
  21. 21.↵
    1. Zeile WL,
    2. Purich DL,
    3. Southwick FS
    . Recognition of two classes of oligoproline sequences in profilin‐mediated acceleration of actin‐based Shigella motility. J Cell Biol 1996;133:49-59.
    Abstract/FREE Full Text
  22. 22.↵
    1. Srinivasan S,
    2. Wang F,
    3. Glavas S,
    4. et al
    . Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. J Cell Biol 2003;160:375-85.
    Abstract/FREE Full Text
  23. 23.↵
    1. Shalit M,
    2. Dabiri GA,
    3. Southwick FS
    . Platelet‐activating factor both stimulates and “primes” human polymorphonuclear leukocyte actin filament assembly. Blood 1987;70:1921-7.
    Abstract/FREE Full Text
  24. 24.↵
    1. Southwick FS,
    2. Dabiri GA,
    3. Stossel TP
    . Neutrophil actin dysfunction is a genetic disorder associated with partial impairment of neutrophil actin assembly in three family members. J Clin Invest 1988;82:1525-31.
    MedlineWeb of Science
  25. 25.↵
    1. Hanna P
    . How anthrax kills. Science 1998;280:1671. 1673–4.
    Medline
  26. 26.↵
    1. Savill JS,
    2. Henson PM,
    3. Haslett C
    . Phagocytosis of aged human neutrophils by macrophages is mediated by a novel “charge‐sensitive” recognition mechanism. J Clin Invest 1989;84:1518-27.
    MedlineWeb of Science
  27. 27.↵
    1. Downey GP,
    2. Butler JR,
    3. Tapper H,
    4. et al
    . Importance of MEK in neutrophil microbicidal responsiveness. J Immunol 1998;160:434-43.
    Abstract/FREE Full Text
  28. 28.↵
    1. Colotta F,
    2. Re F,
    3. Polentarutti N,
    4. Sozzani S,
    5. Mantovani A
    . Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 1992;80:2012-20.
    Abstract/FREE Full Text
  29. 29.↵
    1. Condeelis J
    . Life at the leading edge: the formation of cell protrusions. Annu Rev Cell Biol 1993;9:411-44.
    CrossRefMedlineWeb of Science
  30. 30.↵
    1. Watts RG,
    2. Howard TH
    . Evidence for a gelsolin‐rich, labile F‐actin pool in human polymorphonuclear leukocytes. Cell Motil Cytoskeleton 1992;21:25-37.
    CrossRefMedlineWeb of Science
  31. 31.↵
    1. Mock M,
    2. Fouet A
    . Anthrax. Annu Rev Microbiol 2001;55:647-71.
    CrossRefMedlineWeb of Science
  32. 32.↵
    1. Roberts JE,
    2. Watters JW,
    3. Ballard JD,
    4. Dietrich WF
    . Ltx1, a mouse locus that influences the susceptibility of macrophages to cytolysis caused by intoxication with Bacillus anthracis lethal factor, maps to chromosome 11. Mol Microbiol 1998;29:581-91.
    CrossRefMedlineWeb of Science
  33. 33.↵
    1. Pellizzari R,
    2. Guidi‐Rontani C,
    3. Vitale G,
    4. Mock M,
    5. Montecucco C
    . Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNγ‐induced release of NO and TNFα. FEBS Lett 1999;462:199-204.
    CrossRefMedlineWeb of Science
| Table of Contents

This Article

  1. J Infect Dis. (2005) 192 (5): 837-845. doi: 10.1086/432516
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most