INFECTION AND IMMUNITY, Apr. 2011, p. 1671–1679 Vol. 79, No. 40019-9567/11/$12.00 doi:10.1128/IAI.01204-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

In Vivo Leukocyte Changes Induced by Escherichia coliSubtilase Cytotoxin�

Hui Wang, Adrienne W. Paton, Shaun R. McColl, and James C. Paton*Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of

Adelaide, Adelaide, SA 5005, Australia

Received 12 November 2010/Returned for modification 22 December 2010/Accepted 19 January 2011

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5 cytotoxins produced by Shiga-toxigenicEscherichia coli. Its cytotoxicity is due to its capacity to enter cells and specifically cleave the essential endoplasmicreticulum chaperone BiP. Previous studies have shown that intraperitoneal injection of mice with purified SubABcauses a pathology that overlaps with that seen in human cases of hemolytic-uremic syndrome, as well as dramaticsplenic atrophy, suggesting that leukocytes are targeted. Here we investigated SubAB-induced leukocyte changes inthe peritoneal cavity, blood, and spleen. After intraperitoneal injection, SubAB bound peritoneal leukocytes (in-cluding T and B lymphocytes, neutrophils, and macrophages). SubAB elicited marked leukocytosis, which peakedat 24 h, and increased neutrophil activation in the blood and peritoneal cavity. It also induced a marked redistri-bution of leukocytes among the three compartments: increases in leukocyte subpopulations in the blood andperitoneal cavity coincided with a significant decline in splenic cells. SubAB treatment also elicited significantincreases in the apoptosis rates of CD4� T cells, B lymphocytes, and macrophages. These findings indicate thatapart from direct cytotoxic effects, SubAB interacts with cellular components of both the innate and the adaptivearm of the immune system, with potential consequences for disease pathogenesis.

Shiga-toxigenic Escherichia coli (STEC) causes severe gas-trointestinal infections in humans, which may progress tohemorrhagic colitis and the hemolytic-uremic syndrome(HUS), a life-threatening combination of microangiopathichemolytic anemia, thrombocytopenia, and renal failure (14,19). These clinical manifestations have long been consid-ered to be largely attributable to the effects of Shiga toxin(Stx) on microvascular endothelial cells (19). However,some STEC strains produce an additional AB5 cytotoxinnamed subtilase cytotoxin (SubAB), which has the potentialto make a major contribution to the pathogenesis of HUS inits own right. SubAB was initially detected in a locus ofenterocyte effacement-negative O113:H21 STEC strain re-sponsible for a small outbreak of HUS in South Australia(17, 18), but it is also produced by numerous other disease-causing STEC serotypes (8, 16, 17). SubAB is extraordi-narily toxic for eukaryotic cells, and its mechanism of actioninvolves highly specific A-subunit-mediated proteolyticcleavage of the essential endoplasmic reticulum (ER) chap-erone BiP (GRP78) (15). This triggers a massive ER stressresponse, ultimately leading to apoptosis (11, 12, 26).

Our recent in vitro studies have shown that SubAB alsoincreases tissue factor-dependent factor Xa generation bycultured human umbilical vein endothelial cells and humanmacrophages, suggesting a direct procoagulant effect (25).In mice, intraperitoneal (i.p.) injection of purified SubABcauses microangiopathic hemolytic anemia, thrombocytope-nia, and renal impairment, characteristics typical of Stx-induced HUS (24). Histological examination of organs re-

moved from SubAB-treated mice revealed extensivemicrovascular thrombosis and other histological damage inthe brain, kidneys, and liver, as well as dramatic splenicatrophy. Levels of peripheral blood leukocytes were raisedat 24 h; there was also significant neutrophil infiltration inthe liver, kidneys, and spleen, and toxin-induced apoptosisat these sites (24). These findings raise the possibility ofpathologically significant direct interactions between SubABand various leukocyte subsets, including immune cell acti-vation and induction of inflammatory responses. Accord-ingly, we have now conducted a detailed examination of theeffects of SubAB on leukocytes in toxin-treated mice.

MATERIALS AND METHODS

Toxin purification. SubAB holotoxin, its nontoxic derivative SubAA272B, andthe isolated A and B toxin subunits (SubA and SubB) were purified fromrecombinant E. coli lysates by Ni-nitrilotriacetic acid (NTA) chromatography asdescribed previously (17, 21). Purified proteins were fluorescently labeled withOregon green (OG) as described previously (3).

Mice. Animal experimentation was approved by the Animal Ethics Committeeof the University of Adelaide. Male BALB/c mice, 5 to 6 weeks old, were injectedi.p. with 5 �g purified SubAB (dissolved in 200 �l of phosphate-buffered saline[PBS]). Control mice received 200 �l PBS.

Detection of SubAB in peritoneal leukocytes. At various times post-SubABinjection, mice were anesthetized by inhalation of halothane. Peritoneal leuko-cytes were harvested by washing the peritoneal cavity three times with 3 ml ofice-cold PBS. The leukocyte suspensions were washed twice in 10 ml ice-coldPBS and were fixed in 1% paraformaldehyde in PBS overnight at 4°C. The fixedleukocytes were washed, permeabilized with 0.1% Triton X-100 in PBS for 2 min,and then incubated with Fc blocker (5 �l of 10-mg/ml mouse gamma globulin per1 million cells; D609-0100; Rockland) at 4°C for 30 min to minimize any non-specific binding.

For immunofluorescence labeling, 1 million cells were transferred to a fluo-rescence-activated cell sorter (FACS) tube (35-2008; BD Falcon) and wereincubated with polyclonal rabbit anti-SubA or a control rabbit serum, followed bybiotinylated goat anti-rabbit Ig (BA1000; Vector) and R-phycoerythrin (PE)-conjugated streptavidin (016-110-084; Jackson ImmunoResearch). To identifythe leukocyte subpopulation that SubAB attacks, the cells were double labeled

* Corresponding author. Mailing address: School of Molecular andBiomedical Science, University of Adelaide, Adelaide, SA 5005, Aus-tralia. Phone: 61-8-83035929. Fax: 61-8-83033262. E-mail: james.paton@adelaide.edu.au.

� Published ahead of print on 31 January 2011.

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for SubAB and different leukocyte markers. The leukocyte markers were labeledwith rat anti-mouse monoclonal antibodies (see below) that were detected withfluorescein isothiocyanate (FITC)-conjugated donkey anti-rat Ig (712-096-153;Jackson ImmunoResearch). The mouse leukocyte surface markers examinedwere CD4 (helper T lymphocytes), CD8 (cytotoxic T lymphocytes), B220 (Blymphocytes), F4/80 (mainly macrophages), and Ly-6G (granulocyte-restrictedcells, mainly neutrophils). Rat anti-CD4, anti-CD8, and anti-Ly-6G were pur-chased from BD Biosciences, PharMingen (catalog no. 553649, 553039, and551459); rat anti-B220 and anti-F4/80 were in-house hybridoma supernatants(clones RA3-682 and A3-1). The labeled cells were analyzed immediately usinga FACScan flow cytometer (Becton Dickinson) and CellQuest Pro (version 4.0.1)and Weasel (version 2.6) software. SubAB-related fluorescence (PE; excitationat 565 nm and emission at 575 nm) was detected via channel FL2, and leukocytemarker-related fluorescence (FITC; excitation at 490 nm and emission at 525nm) was detected via channel FL1.

Leukocyte quantitation and assessment of apoptosis. At different times post-SubAB injection, mice were anesthetized by inhalation of halothane, and bloodwas drawn via cardiac puncture and was collected into EDTA tubes. Bloodleukocytes were separated by lysing the red blood cells (RBC) with a hypotonicshock. Peritoneal leukocytes were collected as described above. To harvestsplenocytes, spleens were removed, placed on cell strainers (70-�m-pore sizenylon mesh; BD Falcon reference no. 352350) resting in a 35-mm-diameter tissueculture dish containing 1 ml of ice-cold PBS, and cut into small pieces withscissors, followed by gentle homogenization with the plunger of a 3-ml syringe.Splenocytes were collected and were washed in ice-cold PBS. To obtain totalleukocyte counts, blood, peritoneal, and spleen leukocytes were counted using ahemocytometer with a light microscope.

Apoptotic and necrotic cells were differentiated by staining live cells on icewith annexin V-Fluos (FITC-conjugated annexin V; catalog no. 1 828 681;Roche) and propidium iodide (PI) (P4170; Sigma). One million freshly harvestedleukocytes were washed with ice-cold annexin V binding buffer (8.78 g/liter NaCl,

0.38 g/liter KCl, 0.2 g/liter MgCl2, 2 g/liter CaCl2, and 10 mM HEPES [pH 7.4]),resuspended in 50 �l of annexin V working reagent (consisting of 1/50 annexinV-Fluos and 1 �g/ml of PI in annexin V binding buffer), and incubated on ice inthe dark for 30 min. Cells were then washed and were analyzed immediately ona FACscan flow cytometer. Fluorescence related to annexin V-FITC was read viathe FL1 channel, and the fluorescence of PI (excitation at 536 nm; emission at617 nm) was read via the FL3 channel.

Apoptosis in subpopulations of leukocytes was detected by double labeling oflive cells with annexin V and anti-leukocyte markers (as indicated in the figures).Leukocytes were first incubated with monoclonal rat anti-mouse leukocyte mark-ers, followed by biotinylated donkey anti-rat Ig (621-706-120; Rockland), andwere then incubated simultaneously with PE-conjugated streptavidin and an-nexin V-FITC. All incubations were carried out in the dark on ice at 4°C for 30min. Labeled cells were analyzed immediately on a FACscan flow cytometer. Thefluorescence of FITC-annexin V was read via channel FL1, and leukocyte mark-er-related fluorescence (PE) was detected via channel FL2.

In vitro binding of SubAB to leukocytes. Mouse peritoneal or blood leukocyteswere freshly harvested and pooled from 2 normal mice, as described above.Leukocytes were then washed and resuspended in complete leukocyte culturemedium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine,and 100 �g/ml of penicillin and streptomycin). One million peritoneal or bloodleukocytes, resuspended in 500 �l complete leukocyte culture medium contain-ing 1 �g/ml OG-labeled SubAB or labeled control proteins (OG-SubA, OG-SubB, OG-SubAA272B, or OG-ovalbumin [unlabeled ovalbumin was obtainedfrom Sigma]), were cultured at 37°C under 95% air–5% CO2 for 90 min. Cellscultured in medium only (no OG-labeled protein added) were used as negativecontrols. Labeling of cells was stopped by the addition of 500 �l of 4% formal-dehyde, and the cells either were washed with ice-cold PBS and were thenanalyzed by flow cytometry or underwent further leukocyte surface marker la-beling as described above.

Statistical analysis. Statistical analysis was performed using GraphPad Prism(version 3.03) or Microsoft Excel 2003 software. FACS data are presented asmeans � standard errors of the means (SEM), and differences between cellsfrom control and SubAB-treated mice were analyzed using Student’s unpaired ttest. A P value of �0.05 was considered significant.

FIG. 1. Binding of SubAB to peritoneal leukocytes. Mouse peri-toneal leukocytes were harvested at the indicated times post-i.p.injection of 5 �g SubAB, washed, fixed, and then permeabilized.SubAB was labeled with rabbit anti-SubA, followed by biotinylatedanti-rabbit IgG and streptavidin-PE, and leukocytes were analyzedby FACS. SubAB-related immunofluorescence is expressed as therelative fluorescence intensity (RFI). SubAB-positive cells (har-vested from mice treated with SubAB) were defined by the histo-gram mark (W0), which was set with reference to controls (cellsharvested from untreated mice at 0 h and labeled with anti-SubA,and cells harvested from SubAB-treated mice at 6 h and labeledwith nonimmune rabbit serum [not shown]). Data are from a singleexperiment with one mouse per time point; a repeat experimentyielded essentially identical results (not shown).

FIG. 2. SubAB binding by peritoneal leukocyte subpopulations.Mouse peritoneal leukocytes were harvested at 6 h post-SubAB (5 �g)injection, washed, and fixed. Cells were then permeabilized and doublelabeled with rabbit anti-SubA and rat anti-leukocyte markers (as indi-cated), followed by biotinylated anti-rabbit IgG and PE-conjugatedstreptavidin (for SubAB) or FITC-conjugated anti-rat IgG (for leuko-cyte markers). The labeled leukocytes were analyzed by FACS. TheSubAB- or leukocyte marker-related immunofluorescence intensity isexpressed as relative fluorescence intensity (RFI). Double-positivecells (upper right quadrants) were defined by quadrant marks setaccording to the distribution of control cells. The data shown are fromexperiments using cells from a single mouse.

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RESULTS

Binding of SubAB by peritoneal leukocytes in vivo. Initialexperiments used flow cytometry to examine the bindingand/or uptake of SubAB by murine peritoneal leukocytes atvarious times after i.p. injection of 5 �g toxin (Fig. 1). Boundor internalized toxin was detected using a polyclonal rabbitantibody raised against the A subunit of the toxin (SubA).SubAB binding/uptake was maximal at 6 h postinjection, atwhich time approximately 82% of the leukocyte populationwas labeled; at 16 and 24 h, labeling was still substantial (58%and 65% of the total population, respectively). The specificityof the rabbit anti-SubA antibody was confirmed by comparingthe FACS scan of peritoneal leukocytes from control (non-SubAB-treated) mice after labeling with anti-SubA, or from6-h toxin-treated mice after labeling with nonimmune serum,

with that from 6-h toxin-treated mice whose peritoneal leuko-cytes were labeled with anti-SubA (result not shown).

To examine whether the toxin bound preferentially to spe-cific leukocyte subsets, peritoneal leukocytes harvested 6 hpostinjection were double labeled with anti-SubA and variousleukocyte marker-specific monoclonal antibodies (Fig. 2). Thepercentage of labeling for each subpopulation was calculatedfrom the numbers in the upper right quadrant (leukocytemarker-high, SubAB-high cells) as a proportion of the totalnumber of leukocyte marker-high cells (upper right quadrantplus lower right quadrant). SubAB was detected in all sub-populations, labeling 55% of neutrophils (Ly-6G� cells), 66%of macrophages (F4/80� cells), 96% of CD4� T cells, 86% ofCD8� T cells, and 85% of B lymphocytes (B220� cells).

Specificity of binding to leukocytes in vitro. The findingsdiscussed above suggested that SubAB could directly bind all

FIG. 3. Specificity of leukocyte binding. Peritoneal (A) and blood (B) leukocytes from normal mice were first cultured with 1 �g/ml OG-SubAB,OG-SubA, OG-SubB, OG-SubAA272B, or OG-ovalbumin (OVA) and then analyzed by flow cytometry, as described in Materials and Methods.Control cells were cultured without labeled protein. The data shown are from experiments using pooled cells harvested from two mice; a repeatexperiment yielded essentially identical results (not shown).

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leukocyte subsets after i.p. injection. As independent verifica-tion, we examined the capacity of OG-labeled SubAB andsimilarly labeled control proteins to interact with murine leu-kocytes in vitro. Peritoneal and peripheral blood leukocytesharvested from normal mice were cocultured for 90 min with 1�g/ml OG-SubAB, OG-SubA, OG-SubB, OG-SubAA272B, orOG-ovalbumin and were then analyzed by flow cytometry (Fig.3A and B). For both peritoneal and blood leukocytes, strongOG labeling was observed using OG-SubAB, OG-SubB, orOG-SubAA272B. However, there was no detectable labeling ofleukocytes incubated with OG-SubA or OG-ovalbumin. Thisindicates that interaction with leukocytes derived from normalmice is absolutely dependent on the presence of the B subunitof the toxin. OG-SubAB-treated blood leukocytes were alsodouble labeled with the leukocyte marker-specific monoclonalantibodies and were analyzed by flow cytometry (the percent-age of labeling for each subset was calculated as describedabove for Fig. 2). As seen above for peritoneal leukocytesharvested from SubAB-injected mice, there was significantbinding of labeled toxin to CD4� and CD8� T lymphocytes, Blymphocytes, and neutrophils (98.1%, 94.5%, 82.3%, and99.8% of the respective leukocyte subpopulations were positivefor OG-SubAB binding) (Fig. 4).

Effect of injection of SubAB on leukocyte distribution. Thetotal numbers and distribution of leukocyte subpopulations inthe blood, peritoneal cavities, and spleens of mice were thenexamined at various times after SubAB injection (Fig. 5).Toxin treatment significantly increased the total numbers of

peritoneal and blood leukocytes, which peaked at 24 h. How-ever, a significant decrease in splenic leukocytes was evidentfrom 6 h.

Immunofluorescence labeling and FACS analysis (Fig. 6)indicated that the increase in blood leukocytes was a conse-quence of significant increases in the absolute numbers of allfive subpopulations (CD4� and CD8� T lymphocytes, B lym-phocytes, macrophages, and neutrophils) at 24 h (P, �0.05 inall cases). CD4� cells also increased markedly as a percentageof total blood leukocytes, from 4.55% at 0 h to 8.94% at 24 h(P, �0.05). In the spleen, marked drops in the numbers of boththe CD4� and CD8� subpopulations from 6 h (P, �0.01 inboth cases) largely accounted for the decrease in total splenicleukocytes. The number of splenic B lymphocytes also de-creased, although this decrease was statistically significant onlyat 48 h (P � 0.05). In the peritoneal cavity, where SubAB wasinjected, the total numbers of both neutrophils and macro-phages increased swiftly. Significant neutrophil recruitment

FIG. 4. In vitro binding of leukocyte subpopulations by OG-SubAB.Mouse blood leukocytes were first cultured with 1 �g/ml OG-SubABand then incubated, in sequence, with rat anti-mouse leukocyte surfacemarkers (Ly-6G, B220, CD4, and CD8) and with biotinylated donkeyanti-rat Ig and PE-streptavidin. Leukocytes were subsequently ana-lyzed by flow cytometry as described in Materials and Methods. Cellsin the upper right quadrant are double positive. The data shown arefrom experiments using cells pooled from two mice.

FIG. 5. SubAB-induced changes in total leukocyte numbers.Mouse blood, peritoneal, and splenic leukocytes were harvested at theindicated times post-SubAB injection. Total leukocyte counts weredetermined using a hemocytometer. Nine, 6, 6, 8, and 5 mice weretested at 0, 1, 6, 24, and 48 h, respectively. The differences between thenumbers of leukocytes at 0 h and at the various times postinjectionwere analyzed using Student’s t test. **, P � 0.01; *, P � 0.05.

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was evident from 1 h and was highest at 48 h (P � 0.01 for alltime points). Macrophage numbers were significantly elevatedat 6 h and peaked at 24 h (P � 0.01 in both cases). The numberof neutrophils as a percentage of total peritoneal leukocytesalso increased dramatically, from 20% at 0 h to 67% at 6 hpostinjection.

After SubAB injection, blood and peritoneal neutrophilsincreased not only in absolute numbers but also in the meanlevel of expression of the surface protein Ly-6G (Fig. 7), anindication of neutrophil maturation.

Effect of SubAB on leukocyte apoptosis. Double labelingwith annexin V and PI was used to investigate the total rates ofapoptosis and necrosis in the various niches after toxin treat-ment (Fig. 8). For the total leukocyte population, rates of

necrosis remained low (below 7.4%) in all niches throughoutthe experiment. However, apoptosis rates were elevated from1 h after toxin treatment and reached statistical significance fortotal blood leukocytes at 6 h and 24 h (P � 0.05).

Double labeling with annexin V and anti-leukocyte markerswas used to establish apoptosis rates in different subpopula-tions of leukocytes (Fig. 9). The apoptosis rates of blood CD4�

T cells and B220� B cells were significantly elevated abovebaseline at 1 and 6 h. The apoptosis rates of splenic CD4� Tcells and B220� B cells were also elevated at these time points,reaching statistical significance at 6 h and 1 h, respectively. Inblood and the peritoneal cavity, apoptosis of macrophages wassignificantly elevated at 1 h but not thereafter. Interestingly,there was a marked difference in the baseline level of neutro-

FIG. 6. SubAB-induced changes in differential leukocyte counts. Mouse blood, peritoneal, and splenic leukocytes were harvested andwashed at the indicated times post-SubAB injection. They were then labeled with rat anti-leukocyte markers, followed by biotinylatedanti-rat Ig and PE-streptavidin, and were analyzed by FACS. Nine, 6, 6, 8, and 5 mice were tested at 0, 1, 6, 24, and 48 h postinjection,respectively. The differences between the numbers of leukocytes at 0 h and at various times postinjection were analyzed using Student’s ttest. **, P � 0.01; *, P � 0.05.

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phil apoptosis between blood and the peritoneal cavity (1%versus 34%, respectively). However, there was a marked andsustained reduction in the apoptosis rate of peritoneal neutro-phils after toxin treatment, to approximately 8 to 10% at 6, 24,and 48 h (P � 0.05 in all cases). In contrast, the apoptosis rateof blood neutrophils, like that of most of the other leukocytesexamined, was increased by toxin treatment from the low base-line level, reaching statistical significance at 6 h post-SubABinjection (P � 0.05).

DISCUSSION

In this study, we have examined the capacity of SubAB tointeract with murine leukocytes after intraperitoneal injectionof purified toxin. FACS analysis demonstrated specific bindingto peritoneal leukocytes, peaking at 6 h postinjection but withsignificant amounts still detectable at 24 h. Double labelingwith leukocyte markers demonstrated a lack of preference forany given subpopulation, with similar binding to CD4� andCD8� T lymphocytes, B lymphocytes, macrophages, and neu-trophils. The similar uptake by both phagocytic and nonphago-cytic cell types suggested direct recognition of toxin receptorson the cell surface rather than phagocytosis. We have shownpreviously that the pentameric B subunit of SubAB has a highdegree of specificity for sialated glycans terminating in �2-3-linked N-glycolylneuraminic acid (Neu5Gc). Crystallographicanalysis of the toxin-receptor complex showed that the inter-action is driven principally by this terminal sialic acid moiety,with minimal contribution from subterminal sugars (2). This

accounted for a previous observation that SubAB could bind toseveral glycoprotein species, including �2�1 integrin, on thesurfaces of Vero cells (27). Members of the integrin family areknown to be heavily sialated and are expressed in abundanceon the surfaces of all the leukocyte subsets discussed above (7).In the present study, we also confirmed that binding to thevarious leukocyte subpopulations is absolutely dependent onthe presence of the toxin B subunit. The absence of binding oflabeled ovalbumin by blood leukocytes in vitro also eliminatesnonspecific phagocytosis as an explanation for the binding/uptake of toxin.

An important finding of the present study is the markedeffect of toxin treatment on leukocyte distribution betweenhost compartments. Total leukocyte counts in the peritonealcavity increased nearly 3-fold by 24 h; the bulk of this in-crease consisted of macrophages and neutrophils presum-ably recruited to the site of toxin injection. In a separate in

FIG. 7. SubAB-induced changes in Ly-6G expression levels. Mouseblood and peritoneal leukocytes were harvested and washed at theindicated times post SubAB injection, incubated with rat anti-Ly-6Gfollowed by biotinylated anti-rat IgG and PE-streptavidin, and ana-lyzed by FACS. The Ly-6G-related fluorescence intensity is expressedas geometric mean relative fluorescence intensity (GM-RFI). Nine, 6,6, 8, and 5 mice were tested at 0, 1, 6, 24, and 48 h postinjection,respectively. The differences between the values at 0 h and at thevarious times postinjection were analyzed using Student’s t test. **,P � 0.01; *, P � 0.05.

FIG. 8. SubAB-induced changes in the apoptosis/necrosis rates ofleukocytes. Mouse blood, peritoneal, and splenic leukocytes were har-vested and washed at the indicated times post-SubAB injection, doublelabeled with annexin V and propidium iodide, and then analyzed byFACS. Cells positive for annexin V only were classified as apoptotic;those double positive for annexin V and PI were classified as necrotic.Apoptosis and necrosis rates are expressed as percentages of the totalleukocyte population. Nine, 6, 6, 8, and 5 mice were tested at 0, 1, 6,24, and 48 h postinjection, respectively. The differences between theapoptosis rates at 0 h and at the various times postinjection wereanalyzed using Student’s t test. *, P � 0.05.

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vitro study, we have shown that treatment of U937 (humanmacrophage) and Hct-8 (human colonic epithelial) cellswith purified SubAB significantly upregulates the expressionof CXC chemokines, particularly interleukin-8 (IL-8), mac-rophage inflammatory protein 2� (MIP-2�), and MIP-2�, allof which are potent neutrophil chemoattractants (unpub-lished data). An analogous response by murine macro-phages and other resident peritoneal cells would explain theleukocyte influx observed in the present study. The effects oftoxin injection were not confined to the peritoneal cavity;there was significant leukocytosis in the blood at 24 h. Allleukocyte subpopulations measured were elevated at thistime point, although the greatest relative increases over

untreated baseline levels were seen for T and B lympho-cytes. Nevertheless, blood neutrophil numbers were signifi-cantly elevated; moreover, there was a significant increase inmean Ly-6G expression on the surfaces of these cells, as wellas on peritoneal neutrophils. The Ly-6G expression level hasbeen reported to be directly proportional to neutrophil dif-ferentiation, maturation, and IL-1 receptor expression andhence is indicative of activation (1). Indeed, we have shownthat treatment of U937 cells with SubAB upregulates theexpression of tumor necrosis factor alpha (TNF-�) andIL-1� in addition to the CXC chemokines mentioned above(unpublished data). It has long been known that neutrophilleukocytosis correlates with a poor outcome for HUS pa-

FIG. 9. SubAB-induced changes in the apoptosis rates of leukocyte subpopulations. Mouse blood, peritoneal, and splenic leukocytes wereharvested and washed at the indicated times post-SubAB injection, double labeled for annexin V and leukocyte markers (as indicated), and thenanalyzed by FACS. Cells double positive for annexin V and a leukocyte marker were defined as apoptotic. The apoptosis rate is expressed as apercentage of the total number of cells in the respective subpopulation. Nine, 6, 6, 8, and 5 mice were tested at 0, 1, 6, 24, and 48 h postinjection,respectively. Differences between apoptosis rates at 0 h and at the various times postinjection were analyzed using Student’s t test. **, P � 0.01;*, P � 0.05.

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tients, and activated neutrophils play a key role in patho-genesis as mediators of endothelial injury (6).

The toxin-induced leukocytosis observed in the presentstudy was accompanied by a marked reduction in the numbersof splenic lymphocytes, most notably CD4� and CD8� T cells.The total numbers of splenic leukocytes decreased more than2-fold within 6 h of toxin treatment, consistent with our pre-vious histological observation of profound splenic atrophy anddepletion of lymphocytes in the white pulp (24). This reductionwas presumably a consequence of the combination of leuko-cyte migration into the peripheral circulation and significantlyincreased rates of apoptosis of both T and B lymphocytes.Increased apoptosis was a widespread toxin-induced phenom-enon and was seen for all leukocyte subpopulations and at allsites examined. The only apparent exception to this was thesignificant decrease in apoptosis rates in peritoneal neutro-phils, from an initially high baseline (0-h) level of 34% to 8%at 6 h postinjection. However, during this period there wasmassive recruitment of neutrophils from the peripheral circu-lation and presumably also the bone marrow, with total num-bers in the peritoneal cavity increasing roughly 10-fold. Unsur-prisingly, the level of apoptosis in peritoneal neutrophils at 6 hclosely reflects that seen in peripheral blood neutrophils at thesame time point.

Interestingly, injection of purified Stx2 has also been shownto induce neutrophilia and neutrophil activation in a murinemodel of HUS (5). Thus, our findings provide a further exam-ple of the similarities between the effects of the two toxins invivo. In vitro studies have also shown binding of Stx1 and Stx2to peripheral blood monocytes and monocytic cell lines, as wellas Stx1-induced production of proinflammatory cytokines bythese cells and by murine peritoneal macrophages (20, 22, 23).Stx1 has also been shown to block the activation and prolifer-ation of lymphocytes (4, 13). The similarities in the propertiesof SubAB and Stx are remarkable given the fact that the twoAB5 toxins are unrelated and have distinct host receptors,intracellular targets, and molecular mechanisms of action. TheB subunit of SubAB mediates binding to Neu5Gc displayed onsialated glycoproteins (2), triggering clathrin-dependent inter-nalization and retrograde transport via the Golgi complex tothe ER lumen (3), where the proteolytic A subunit cleaves theessential Hsp70 family chaperone BiP (15). BiP is responsiblefor the proper folding of newly synthesized proteins, and as themaster regulator of the ER stress response, it is essential forthe maintenance of ER homeostasis (9). Cleavage of BiP trig-gers ER stress-signaling pathways and eventually leads toapoptosis (11, 12, 26). Stx, on the other hand, binds to the cellsurface glycolipid Gb3 and follows a slightly different retro-grade pathway to the Golgi complex and ER (4), after whichthe A subunit (an RNA-N-glycosidase) is retrotranslocatedinto the cytosol, where it modifies 28S rRNA and inhibits theelongation step of protein synthesis (19). Interestingly, in spiteof this distinct mode of action, Stx1 has also been shown totrigger ER stress-mediated apoptosis in human monocytic cells(10).

The present study has provided the first evidence that leu-kocytes express functional receptors for SubAB and that thetoxin induces in vivo inflammatory responses and leukocytemigration that could contribute to disease pathogenesis. Theprecise nature of the inflammatory responses elicited by

SubAB in specific leukocyte subsets, and the extent to whichthese synergize with those triggered by Stx in the pathogenesisof human disease, remains a fertile field for future investiga-tion.

ACKNOWLEDGMENTS

This work was supported by program grant 565526 from the Na-tional Health and Medical Research Council of Australia (NHMRC)and R01AI-068715 from the U.S. National Institutes of Health. J.C.P.is an NHMRC Australia Fellow.

REFERENCES

1. Bliss, S. K., B. A. Butcher, and E. Y. Denkers. 2000. Rapid recruitment ofneutrophils containing prestored IL-12 during microbial infection. J. Immu-nol. 165:4515–4521.

2. Byres, E., A. W. Paton, J. C. Paton, J. C. Lofling, and D. F. Smith, et al. 2008.Incorporation of a non-human glycan mediates human susceptibility to abacterial toxin. Nature 456:648–652.

3. Chong, D. C., J. C. Paton, C. M. Thorpe, and A. W. Paton. 2008. Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5 toxin that targetsthe ER chaperone BiP. Cell. Microbiol. 10:795–806.

4. Ferens, W. A., and C. J. Hovde. 2000. Antiviral activity of Shiga toxin 1:suppression of bovine leukemia virus-related spontaneous lymphocyte pro-liferation. Infect. Immun. 68:4462–4469.

5. Fernandez, G. C., et al. 2000. Shiga toxin-2 induces neutrophilia and neu-trophil activation in a murine model of hemolytic uremic syndrome. Clin.Immunol. 95:227–234.

6. Forsyth, K. D., A. C. Simpson, M. M. Fitzpatrick, T. M. Barratt, and R. J.Levinsky. 1989. Neutrophil-mediated endothelial injury in haemolytic urae-mic syndrome. Lancet ii:411–414.

7. Harris, E. S., T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 2000.The leukocyte integrins. J. Biol. Chem. 275:23409–23412.

8. Khaitan, A., D. M. Jandhyala, C. M. Thorpe, J. M. Ritchie, and A. W. Paton.2007. The operon encoding SubAB, a novel cytotoxin, is present in Shigatoxin-producing Escherichia coli isolates from the United States. J. Clin.Microbiol. 45:1374–1375.

9. Lee, A. S. 2001. The glucose-regulated proteins: stress induction and clinicalapplications. Trends Biochem. Sci. 26:504–510.

10. Lee, S. Y., M. S. Lee, R. P. Cherla, and V. L. Tesh. 2008. Shiga toxin 1 inducesapoptosis through the endoplasmic reticulum stress response in humanmonocytic cells. Cell. Microbiol. 10:770–780.

11. Matsuura, G., et al. 2009. Novel subtilase cytotoxin produced by Shiga-toxigenic Escherichia coli induces apoptosis in Vero cells via mitochondrialmembrane damage. Infect. Immun. 77:2919–2924.

12. May, K. L., J. C. Paton, and A. W. Paton. 2010. Escherichia coli subtilasecytotoxin induces apoptosis regulated by host Bcl-2 family proteins, Bax/Bak.Infect. Immun. 78:4691–4696.

13. Menge, C., L. H. Wieler, T. Schlapp, and G. Baljer. 1999. Shiga toxin 1 fromEscherichia coli blocks activation and proliferation of bovine lymphocytesubpopulations in vitro. Infect. Immun. 67:2209–2217.

14. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin.Microbiol. Rev. 11:142–201.

15. Paton, A. W., et al. 2006. AB5 subtilase cytotoxin inactivates the endoplasmicreticulum chaperone BiP. Nature 443:548–552.

16. Paton, A. W., and J. C. Paton. 2005. Multiplex PCR for direct detection ofShiga toxigenic Escherichia coli strains producing the novel subtilase cyto-toxin. J. Clin. Microbiol. 43:2944–2947.

17. Paton, A. W., P. Srimanote, U. M. Talbot, H. Wang, and J. C. Paton. 2004.A new family of potent AB5 cytotoxins produced by Shiga toxigenic Esche-richia coli. J. Exp. Med. 200:35–46.

18. Paton, A. W., M. C. Woodrow, R. M. Doyle, J. A. Lanser, and J. C. Paton.1999. Molecular characterization of a Shiga toxigenic Escherichia coli O113:H21 strain lacking eae responsible for a cluster of cases of hemolytic-uremicsyndrome. J. Clin. Microbiol. 37:3357–3361.

19. Paton, J. C., and A. W. Paton. 1998. Pathogenesis and diagnosis of Shigatoxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11:450–479.

20. Ramegowda, B., and V. L. Tesh. 1996. Differentiation-associated toxinreceptor modulation, cytokine production, and sensitivity to Shiga-liketoxins in human monocytes and monocytic cell lines. Infect. Immun.64:1173–1180.

21. Talbot, U. M., J. C. Paton, and A. W. Paton. 2005. Protective immunizationof mice with an active-site mutant of subtilase cytotoxin of Shiga toxin-producing Escherichia coli. Infect. Immun. 73:4432–4436.

22. Tesh, V. L., B. Ramegowda, and J. E. Samuel. 1994. Purified Shiga-like toxinsinduce expression of proinflammatory cytokines from murine peritonealmacrophages. Infect. Immun. 62:5085–5094.

23. van Setten, P. A., L. A. Monnens, R. G. Verstraten, L. P. van den Heuvel, and

1678 WANG ET AL. INFECT. IMMUN.

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V. W. van Hinsbergh. 1996. Effects of verocytotoxin-1 on nonadherent hu-man monocytes: binding characteristics, protein synthesis, and induction ofcytokine release. Blood 88:174–183.

24. Wang, H., J. C. Paton, and A. W. Paton. 2007. Pathologic changes in miceinduced by subtilase cytotoxin, a potent new Escherichia coli AB5 toxin thattargets the endoplasmic reticulum. J. Infect. Dis. 196:1093–1101.

25. Wang, H., et al. 2010. Tissue factor-dependent procoagulant activity of

subtilase cytotoxin, a potent AB5 toxin produced by Shiga toxigenic Esche-richia coli. J. Infect. Dis. 202:1415–1423.

26. Wolfson, J. J., et al. 2008. Subtilase cytotoxin activates PERK, IRE1 andATF6 endoplasmic reticulum stress-signalling pathways. Cell. Microbiol. 10:1775–1786.

27. Yahiro, K., et al. 2006. Identification and characterization of receptors forvacuolating activity of subtilase cytotoxin. Mol. Microbiol. 62:480–490.

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