Effects of anthrax lethal toxin on human primary keratinocytes

ORIGINAL ARTICLE

Effects of anthrax lethal toxin on human primarykeratinocytesS.S. Kocer1,2, M. Matic1,2, M. Ingrassia3, S.G. Walker4, E. Roemer2, G. Licul3 and S.R. Simon1,2,3,4

1 Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, New York, USA

2 Department of Pathology, State University of New York at Stony Brook, New York, USA

3 Program in Cellular and Developmental Biology, State University of New York at Stony Brook, New York, USA

4 Department of Oral Biology & Pathology, State University of New York at Stony Brook, New York, USA

Introduction

Cutaneous infection by Bacillus anthracis, while resulting in

the least serious form of the disease anthrax, is the most

common route of human infection (Mock and Fouet

2001). The risk of human cutaneous anthrax is the greatest

in developing nations where zoonotic anthrax has not been

eliminated, such as India (Vijaikumar et al. 2001), Turkey

(Oncul et al. 2002; Demirdag et al. 2003; Irmak et al. 2003;

Yetkin et al. 2006), and other countries (Shiferaw 2004;

Woods et al. 2004; Maguina et al. 2005). Although mortal-

ity rates as high as 20% have been reported for untreated

cutaneous anthrax [Centers for Disease Control and

Prevention (CDCP) 2000; Bravata et al. 2007], very little is

known about the molecular details of the course of the dis-

ease. Cutaneous infection begins when B. anthracis spores

gain access to tissues via an abrasion of the skin. The spores

then germinate into vegetative forms of the bacterium,

typically after having been ingested by macrophages. The

vegetative bacteria finally escape and multiply extracellu-

larly (Mock and Fouet 2001; Godyn et al. 2005). Neutroph-

ils are recruited to the cutaneous infection site (Wade et al.

1985; Mayer-Scholl et al. 2005), which progresses from a

papule to a malignant pustule with localized swelling.

Eventually, a characteristic black necrotic eschar forms over

the pustule (Roche et al. 2001; Karakas et al. 2006). In

cutaneous anthrax, keratinocytes are among the primary

targets of lethal toxin (LeTx), the form of anthrax toxin

Keywords

anthrax, cutaneous, keratinocytes, lethal

toxin, proteasome, RANTES.

Correspondence

Sanford R. Simon, Department of Pathology,

BST-9 Room #151 HSC, State University of

NY at Stony Brook, NY, 11794-8691, USA.

E-mail: [email protected]

2007 ⁄ 1750: received 1 November 2007,

revised 14 January 2008 and accepted 24

January 2008

doi:10.1111/j.1365-2672.2008.03806.x

Abstract

Aims: To investigate the effects of anthrax lethal toxin (LeTx) on human pri-

mary keratinocytes.

Methods and Results: We show here that human primary keratinocytes are

resistant to LeTx-triggered cytotoxicity. All but one of the MEKs (mitogen-acti-

vated protein kinase kinases) are cleaved within 3 h, and the cleavage of MEKs

in keratinocytes leads to their subsequent proteasome-mediated degradation at

different rates. Moreover, LeTx reduced the concentration of several cytokines

except RANTES in culture.

Conclusions: Our results indicate that primary keratinocytes are resistant to

LeTx cytotoxicity, and MEK cleavage does not correlate with LeTx cytotoxicity.

Although LeTx is considered as an anti-inflammatory agent, it upregulates

RANTES.

Significance and Impact of the Study: According to a current view, the action

of LeTx results in downregulation of the inflammatory response, as evidenced

by diminished expression of several inflammatory biomarkers. Paradoxically,

LeTx has been reported to attract neutrophils to cutaneous infection sites. This

paper, which shows that RANTES, a chemoattractant for immune cells, is

upregulated after exposure of keratinocytes to LeTx, although a number of

other markers of the inflammatory response are downregulated. Our results

might explain why the exposure of keratinocytes to LeTx results in the recruit-

ment of neutrophils to cutaneous infection sites, while the expression of several

inflammatory biomarkers is diminished.

Journal of Applied Microbiology ISSN 1364-5072

1756 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1756–1767

ª 2008 The Authors

that is produced when protective antigen (PA) associates

with the bacterial metalloproteinase lethal factor (LF)

(Klimpel et al. 1994).

Although there have been significant advances in

anthrax research in recent years, the majority of investiga-

tive efforts have focussed on inhalation anthrax, which is

the most deadly form. This report presents our studies on

the molecular events associated with cutaneous anthrax,

using human cell-based in vitro models. In this report, we

focus on the effects of anthrax toxin on human keratino-

cytes. We show that primary human keratinocytes

obtained from foreskin are resistant to LeTx-initiated

cytotoxicity, even though all the MEK (MEK1 through 7),

except MEK5, are cleaved in these cells, as we previously

observed in mononuclear phagocytes exposed to LeTx.

Furthermore, we demonstrate that in keratinocytes, the

cleaved products of the MEK are targeted for destruction

by the proteasome at different rates. Although keratino-

cytes are resistant to LeTx cytotoxicity over the 24 h time

course of our study, we observed that within this time

interval, the levels of two pro-inflammatory molecules,

interleukin-6 (IL-6) and granulocyte-macrophage colony

stimulating factor (GM-CSF), decline, but unexpectedly,

the production of RANTES, a known chemoattractant for

multiple types of immune cells, increases. Overall, our

data suggest that in human keratinocytes, LF-mediated

MEK cleavage can be correlated with LeTx-triggered dim-

inutions in levels of biomarkers of the innate immune

response but not with cell death. Such diminished levels

of pro-inflammatory cytokines and growth factors would

compromise recruitment and activation of professional

phagocytes and might contribute to the systemic bactere-

mia and toxemia that can occur in untreated cutaneous

anthrax, even while focal recruitment of neutrophils may

occur at the initial site of cutaneous infection.

Materials and methods

Materials

Recombinant LF and PA of B. anthracis were purchased

from List Biological Laboratories, Inc. (Campbell, CA,

USA). The biological and enzymatic activities of LF

and PA from this source have been discussed previously

(Kocer et al. 2005). Unless otherwise specified, we have

generated LeTx in situ by adding 1 lg ml)1 each of LF

and PA to the appropriate cell cultures. Acetyl-leu-leu-

norleucinal (ALLN) was purchased from Calbiochem

(San Diego, CA, USA), and was dissolved in dimethylsulf-

oxide (DMSO) to provide a 20 mmol ml)1 stock solution

which was stored at )20�C. Escherichia coli lipopolysac-

charide (LPS) was purchased from Sigma (St. Louis, MO,

USA) and was dissolved in DMSO to provide a

1 mg ml)1 stock solution which was stored at )20�C. A

purified preparation of B. anthracis cell wall components

(CWC) was purchased from List Biological Laboratories,

Inc., and was dissolved in Dulbecco’s phosphate-buffered

saline (PBS) to provide a 1 mg ml)1 stock solution. Tri-

ton X-100 was purchased from Sigma.

Cells

The isolation and maintenance of keratinocytes from

human foreskin has been described elsewhere (Matic et al.

2002). Organotypic models of human skin prior to and

subsequent to the formation of a complete basement

membrane at the dermal–epidermal junction (EFT-100

and EFT-200) were purchased from MatTek Corporation

(Ashland, MA, USA). Human peripheral blood mono-

cytes were isolated according to a protocol previously

published (Kocer et al. 2005).

Cell viability assay

The CellTiter 96� AQueous One Solution Cell Proliferation

Assay (Promega, Madison, WI, USA), an assay of tetrazo-

lium salt reductase activity which reflects the presence of

dehydrogenases only within viable cells, was employed as

previously described (Kocer et al. 2005) and used to eval-

uate cytotoxicity after 24 h of exposure to the agents

employed in this study.

In our studies, 500 ll fresh serum-free medium was

added to adherent keratinocytes, followed by the addition

of 100 ll of a solution of the tetrazolium salt MTS (3-(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethonylphenol)-2-(4-

sulfophenyl)-2H-tetrazolium, inner salt) as provided by the

manufacturer. Cells were incubated at 37�C for 1–4 h and

absorbance was recorded at 490 nm at hourly intervals for

up to 4 h. Cells treated with 3% Triton X-100 were used as

a control representing 100% cytotoxicity.

LF proteolytic activity in viable cells

Human keratinocytes were plated on 24-well microplates

at a density of 4 · 105 ml)1 per well. Cells were incubated

overnight at 37�C in a humidified atmosphere containing

5% CO2 before initiating experiments to allow them to

attach to the wells. The cells were washed once with

warm Hank’s balanced salt solution (HBSS) (Hyclone,

Logan, UT, USA), which was then replaced with serum-

free medium.

The organotypic human skin models, EFT-100 and

EFT-200, were prepared according to the manufacturer’s

protocol. These models are formed in porous membrane-

bottomed cell culture inserts for multiwell microplates.

Each unit is composed of a stratified epidermis containing

S.S. Kocer et al. Cutaneous anthrax

ª 2008 The Authors

Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1756–1767 1757

multiple layers of human keratinocytes, complete with

stratum corneum, and an underlying dermis composed of

a fibrillar collagen gel containing human dermal fibro-

blasts. The surrounding medium in the wells contacts

the ‘dermis’ at the bottom of each unit, but the apical

‘epidermal’ surface contacts only air. Upon arrival, the

shipping medium was replaced with fresh warm medium

supplied by the manufacturer, and the units were held at

37�C for 1 h. Then 1 lg ml)1 of recombinant PA and LF

was added to the medium contacting the basal surface

units. At various time points, the cells were lysed with

a buffer containing 0Æ1% Nonidet P-40 (NP40),

150 mmol ml)1 NaCl, 40 mmol ml)1 Tris (pH 7Æ2), 10%

glycerol, 5 mmol ml)1 NaF, 1 mmol ml)1 Na pyrophos-

phate, 1 mmol ml)1 Na o-vanadate, 10 mmol ml)1 o-phe-

nanthroline and 100 ng ml)1 phenylmethyl sulfonyl

fluoride (PMSF) (Kocer et al. 2005).

Effects of proteasome inhibitor ALLN

The calpain inhibitor ALLN, which is also an inhibitor of

proteasome proteolytic activity, has been reported to

prevent LeTx-triggered cytotoxicity in the murine macro-

phage line RAW 264Æ7 (Tang and Leppla 1999). Accord-

ingly, we evaluated ALLN for its capacity to inhibit

degradation of MEKs cleaved by LF in human keratino-

cytes. The keratinocytes were plated without NIH/3T3

feeder layers on 24-well microplates at a density of

1 · 106 ml)1 per well and were incubated overnight at

37�C in a humidified atmosphere containing 5% CO2

before initiating experiments to allow them to attach to

the wells. The cells were then incubated with LeTx, i.e. a

mixture of 1 lg ml)1 each of LF and PA, for 3 h in the

absence or presence of 20 lmol ml)1 ALLN. Cells were

exposed to ALLN + LeTx or to LeTx alone. As controls

other wells were exposed to LF alone or to LF + ALLN in

the absence of PA. The cells were lysed as described ear-

lier and the lysates were analysed by SDS-PAGE and

immunoblotting. It must be noted that electrophoresis of

the samples shown in the immunoblots in Fig. 3 was

extended for a longer duration than was employed for the

samples shown in the immunoblots in Fig. 2 to enhance

detection of any changes in electophoretic mobilities of

the bands recognized by all the anti-MEK antibodies.

Western immunoblotting and antibodies

Westen blot analyses for MEK were performed as previ-

ously described (Kocer et al. 2005). The primary antibodies

used for blotting were anti-MEK-1 (C-18), anti-MEK-2

(N-20), anti-MEK-2 (C-16), anti-MEK-3 (C-19), anti-

MEK-4 (C-20), anti-MEK-7 (H-160) monoclonal antibod-

ies (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA, USA);

and anti-MEK-6 and anti-MEK-5 rabbit polyclonal

antibodies with specificity for the C-terminus (Stressgen

Biotechnologies Corp., Victoria, BC, Canada), all of

which were employed at a dilution of 1 lg ml)1. Horse-

radish peroxidase-conjugated goat anti-mouse or anti-

rabbit IgG (KPL, Gaithersburg, MD, USA) was used at a

1 : 1000 dilution as the secondary antibody. Blots were

washed and processed by enhanced chemiluminescence

(ECL) detection (Amersham Pharmacia, Piscataway, NJ,

USA) in accordance with the protocol suggested by the

manufacturer.

Biomarker immunoassays

Human keratinocytes at a density of 1 · 106 ml)1 per

well were plated on 24-well microplates either directly

onto the plastic surface or over a feeder layer established

by previously plating and irradiating 5 · 104 NIH 3T3

cells per ml per well. Wells containing irradiated 3T3 cells

alone served as controls. The cells were maintained for

1 h, either in serum-free medium alone or in medium

containing LPS or CWC. Selected wells were then treated

with 1 lg ml)1 each of LF plus PA for an additional

23 h. The supernatant medium was then collected from

all the wells for quantization of inflammatory biomarkers

by enzyme-linked immunosorbant assay (ELISA). ELISA

kits for human GM-CSF, IL-6, RANTES, and tumour

necrosis factor alpha (TNF-a) (ultra sensitive) were pur-

chased from Biosource (Camarillo, CA, USA).

Transfections of plasmid DNA

AP1 and NF-jB luciferase reporter plasmids were pur-

chased from Stratagene (La Jolla, CA, USA). pFC-MEKK,

a plasmid that expresses a constitutively active form of

MAP kinase, was purchased from Stratagene. The b-galac-

tosidase expression plasmid pSV-bgal was purchased from

Promega (Madison, WI, USA). Dr Antonella Casola, from

University of Texas at Galveston, kindly provided the

RANTES luciferase reporter plasmid (Casola et al. 2001).

Human keratinocytes were plated in 48-well plates

(100 000 cells per well) and were allowed to adhere over-

night before transfection. Transient transfections were per-

formed with Lipofectamine reagent, which was purchased

from Invitrogen (Carlsbad, CA, USA), and was employed

according to the manufacturer’s instructions.

Reporter assays

A luciferase assay kit was purchased from Promega and a

b-galactosidase assay kit was obtained from Stratagene;

these kits were used according to the manufacturers’ pro-

tocols. Cells were transfected in duplicate or triplicate as

Cutaneous anthrax S.S. Kocer et al.


ª 2008 The Authors

mentioned earlier with constant amounts of plasmid

DNA, including luciferase reporter plasmids, a b-galacto-

sidase expression plasmid (pSV-bgal), and experimental

and reporter plasmids. After lysis, luciferase reporter

assays were performed in duplicate and read in a Lumat

LB 9501 luminometer (Berthold Technologies, Oak Ridge,

TN, USA). Ten microlitres of lysate was added to 50 ll of

assay buffer (Promega) and luminescence quantitated for

15 sn. Luciferase activity was normalized to relative

amounts of b-galactosidase activity present within each

lysate. Error bars represent the range of normalized lucif-

erase responses among replicate samples, and representa-

tive experiments are presented.

Transcription factor assays

Assays of the active forms of the transciption factors

NF-jB, c-jun and MEF2 were performed on lysates of

EFT-100 organotypic units, which had been exposed to

LF, PA, or LeTx, using TransAm� kits (Active Motif Inc.,

Carlsbad, CA) which recognize only those forms of the

transcription factors which bind to their target nucleotide

sequences. The organotypic models were freeze-thawed in

lysis buffer prior to homogenization using a Dremel tissue

disintegrator. Protein was quantitated with BCA reagent

from Pierce, and 10 lg aliquots of protein were added to

a microplate with immobilized consensus oligonucleotides

specific for binding transcription factors controlled by the

MAPK pathways (MAPK Family TransAm kit, Active

Motif). The levels of each bound transcription factor were

quantitated with a specific monoclonal antibody as per the

kit manufacturer’s instructions. Statistical significance was

determined by anova analysis, using a Dunnet’s post test.

Results

Human keratinocytes are resistant to LeTx cytotoxicity

Figure 1a demonstrates that the viability, as estimated by

MTS reduction, of human keratinocytes alone, irradiated

3T3 cells alone, or keratinocytes on a 3T3 feeder layer

was unaffected after being incubated for 24 h in the

presence of LF and PA. In 80% of our experiments with

irradiated NIH 3T3 cells, we found that these cells were

resistant to LeTx cytotoxicity as shown in Fig. 1. Our

results may indicate that the gene(s) regulating LeTx

cytotoxicity in this murine fibroblast cell line may be

sensitive to the effects of irradiation. Overall, even though

the concentration of LeTx used in this experiment is suf-

ficient to cause cytotoxicity in other cell types (Tang and

Leppla 1999; Popov et al. 2002a;b), including human

peripheral blood monocytes (Fig. 1b), human primary

keratinocytes are resistant to LeTx.

LeTx cleaves six out of seven MEKs in human

keratinocytes

The resistance of human keratinocytes to LeTx cytotoxi-

city might be hypothesized to result from the failure of

25(a)

(b)

20

15

10

5

00 1 10 100 1000 TX-100

LeTx–

– + + + + –

LF ng ml–1

PA

50

40

30

Rel

ativ

e M

TS

red

ucta

se a

ctiv

ityR

elat

ive

MT

S r

educ

tase

act

ivity

(ar

bitr

ary

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20

10

0

Figure 1 (a) Keratinocyte viability is unaffected by exposure to lethal

toxin (LeTx) for 24 h. Keratinocytes with or without NIH 3T3 feeder

layers and feeder layers alone (black, cross-hatched and hatched bars,

respectively) were exposed to protective antigen (PA) (1 lg ml)1) and

indicated the amount of lethal factor (LF) for 24 h. Cells treated with

3% Triton-X were used as a control for 100% cytotoxicity. Cytotoxic-

ity was measured by MTS reduction. (b) Freshly isolated human

peripheral blood monocytes exposed to LeTx (100 ng ml)1 LF and

200 ng ml)1 PA) for 24 h. At the end of 24 h, MTS assay was per-

formed. Figure indicates that �50% of reduction in MTS reductase

activity in LeTx exposed cells. These experiments were repeated at

least three times and similar results were obtained each time. A repre-

sentative experiment is shown. Error bars represent standard deviation

(n = 3, 4).


ª 2008 The Authors


LF to enter the cytosol of these cells, either owing to the

absence of PA receptors or the presence of unidentified

inhibitors of LF in the culture system. To determine if

incubation of cultures of primary human keratinocytes

or the EFT-100 and EFT-200 organotypic human skin

models with LeTx results in any alterations in the known

intracellular targets of LeTx, the MEKs, we evaluated the

immunoreactivity of seven MEKs before and after incuba-

tion of the cells with LeTx (Fig. 2). The anti-MEK anti-

bodies employed in the studies illustrated in Fig. 2 all

bind to the C-terminal domains of these elements of the

MAP-Kinase cascade, with the exception of the antibody

raised against MEK-2, which reacts with the N-terminal

domain of MEK-2 (antibody N-20). The immunoblots

shown in Fig. 2a reveal that the immunoreactivities of

MEK-1, MEK-2, MEK-4, MEK-6 and MEK-7 in human

keratinocytes were all altered in the presence of LeTx, as

evidenced by progressive declines in immunoreactivity

with minimal changes in electrophoretic mobility of the

immunoreactive bands over time. Immunoblots obtained

with anti-MEK-3 antibody revealed declines in immuno-

reactivity of this MEK along with significantly increased

electrophoretic mobility of the immunoreactive band.

Immunoblots probed with anti-MEK-5 antibody however

showed neither alterations in intensity nor electrophoretic

mobility of the immunoreactive bands, consistent with

observations of other researchers using other cell types

that MEK-5 is not susceptible to LeTx cleavage and thus

effectively serves as a control for protein loading (Vitale

et al. 2000).

Qualitatively similar observations were seen when lysates

were prepared from EFT-100 organotypic units, which had

been first exposed on their basal surface to LeTx, and were

analysed by SDS-PAGE and immunoblotting using anti-

MEK primary antibody (Fig. 2b). Note that the rate at

which the MEKs decreased in immunoreactivity was slower

than that seen for keratinocyte monolayers exposed to

LeTx (Fig. 2a). This may reflect the slow diffusion of LeTx

through the dermal layer of the models, containing sparse

numbers of fibroblasts, into the lower layers of keratino-

cytes in the model epidermis which is not yet separated

from the dermis by a fully developed basement membrane.

In contrast to the EFT-100 organotypic units, we did not

observe any significant MEK cleavage in EFT-200 organo-

typic units (data not shown). This may reflect the establish-

ment of a fully functional basement membrane at the

dermal–epidermal junction of the EFT-200 units, which

may act as a barrier to LeTx diffusion.

These results indicate that LF cleaves six of seven MEKs

in human keratinocytes and in an organotypic model of

skin. Therefore, the resistance of human keratinocytes to

LeTx-mediated cytotoxicity, shown in Fig. 1, is not the

result of the loss of LF proteolytic activity or the failure

of PA to facilitate entry of LF into the cytosol of these

cells. This suggests that LF-mediated MEK cleavage dur-

ing the 24 h duration of our observations is not sufficient

to decrease the viability of these cells.

The immunoblots in Fig. 2 show that after incubation

of cells with LeTx, the intensities of the bands for all the

MEK except for MEK-5 decline over time. The loss of

immunoreactivities of MEKs when visualized by antibod-

ies to the N-terminal domains of these components of the

MAP-kinase pathway can be ascribed to the removal of

the immunoreactive domains by proteolysis mediated by

LF, which has been reported to cleave at the N-terminal

domains of the MEKs. However, the loss of immunoreac-

tivities when visualized by antibodies to the C-terminal

domains may reflect a secondary event occurring within

the target cells. Kirby (2004) has also described the disap-

pearance of the bands recognized by antibodies directed

to the C-termini of MEKs in human umbilical vein endo-

thelial cells, which he attributed to ‘unknown reasons’

(Kirby 2004). We hypothesized that this loss of immuno-

reactivity of the cleaved products of the MEKs may be

the result of proteasome-mediated destruction following

proteolytic cleavage of the MEKs by LeTx.

Cleavage of MEKs by LeTx is followed by degradation

of the cleavage products at different rates by the

proteasome

To evaluate the role of proteasome-mediated MEK degra-

dation subsequent to LF-mediated proteolysis, human

0 1·5 3 h 0 1·5 3 h

MEK1

MEK2

MEK3

MEK4

MEK5

MEK6

MEK7

(a) (b)

Figure 2 Susceptibility of MEKs to lethal factor (LF) proteolytic activ-

ity in viable cells. Keratinocytes (a) and EFT-100 organotypic skin

model units (b) were treated with LF plus protective antigen (PA)

(1 lg ml)1 each) for different periods of time, and after lysis of the

cells, all seven MEK proteins were analysed by SDS-PAGE (35 min) fol-

lowed by immunoblotting using antibodies raised against the N-termi-

nal domain of MEK-2 and the C-terminal domains of the other six

proteins. This experiment was repeated three times and similar results

were obtained each time. A representative experiment is shown.



ª 2008 The Authors

keratinocytes were incubated with LeTx in the presence

or absence of the proteasome inhibitor ALLN for 3 h

(Fig. 3). Lysates of these cells were then analysed by SDS-

PAGE, followed by immunoblotting with antibodies

directed against the C-terminal epitopes of MEK-1,

MEK-2 and MEK-4. To see any alterations in the electro-

phoretic mobilities of bands clearly, samples were electro-

phoresed for twice the duration employed with the

samples shown in Fig. 2. Incubation with LeTx alone

resulted in significant increases in the electrophoretic

mobilities of bands recognized by all three anti-MEK

antibodies, but the degree to which the bands decreased in

immunoreactivity varied. We observed marked decreases

in immunoreactivity of bands recognized by anti-MEK-1

and anti-MEK-4 antibodies subsequent to intracellular

cleavage, but the band recognized by the anti-MEK-2

C-terminus antibody showed only a minimal decrease in

immunoreactivity over the incubation interval employed.

Lysates of cells treated simultaneously with LeTx and

ALLN were then analysed by SDS-PAGE and immuno-

blotting with anti-MEK-1 and -4 antibodies. The immu-

noblots of ALLN-treated cell lysates did not reveal

significant declines in the immunoreactivity of any bands,

although the electrophoretic mobility of the bands still

increased. The band recognized by MEK-2 did not

decrease in immunoreactivity in the presence of LeTx with

or without the addition of ALLN over the duration of the

incubation employed in this study, but its increased elec-

trophoretic mobility indicated that it had been cleaved by

LeTx. The relative resistance of MEK-2 to proteasome-

mediated degradation, observed as sustained immunoreac-

tivity in the absence or presence of ALLN, justifies the use

of MEK-2 as a loading control in immunoblots.

As the immunoreactivities of the bands recognized by

antibodies directed against the C-terminal epitopes of the

different MEKs appear to decline at quite different rates

(for instance, the loss of immunoreactivity of MEK-3 in

Fig. 2 and MEK-2 in Fig. 3 is slower than loss of immu-

noreactivity of the other MEKs), these results suggest that

intracellular LF-mediated cleavage of MEKs directs the

cleaved products to proteasome-mediated destruction at

different rates.

LeTx treatment diminishes release of several

pro-inflammatory proteins from human keratinocytes

but upregulates RANTES

Figure 4a shows that the levels of IL-6 released by human

keratinocytes after stimulation with LPS were lower in

cells exposed to LeTx than control cells. In contrast, the

levels of IL-6 released by keratinocytes after incubtion

with CWC were lower than those released after stimula-

tion with LPS, and these lower levels were not altered

significantly after exposure of the cells to LeTx. Keratino-

cytes, which were not stimulated with LPS or CWC, also

produced similar levels of IL-6 in the presence or absence

of LeTx.

We further observed that LPS was a more potent stim-

ulus than CWC for production of GM-CSF by keratino-

cytes, as shown in Fig. 4b, but unlike the effect of

exposure to LeTx on IL-6 release, exposure of keratino-

cytes to the toxin almost completely obliterated release of

GM-CSF, regardless of whether they had been stimulated

with CWC or LPS.

In contrast to the effects of LeTx on the levels of secre-

tion of GM-CSF and IL-6, the levels of RANTES secreted

from keratinocytes were observed to increase in the pres-

ence of LeTx (Fig. 4c). LeTx-induced increases in RAN-

TES were observed in control cells, in cells stimulated

with CWC, and in cells stimulated with LPS. Significant

stimulation of RANTES release was also observed after

exposure of cells to CWC alone, and even more marked

stimulation was observed after exposure of the cells to

LPS alone. These increases in RANTES levels induced by

the pro-inflammatory stimuli of CWC and LPS appeared

to be additive to the increases induced by exposure to

LeTx, an observation which distinguishes the response of

this cytokine to the response of other biomarkers of

inflammation and innate immunity.

We conducted a control experiment to determine if

supernatant medium from HIH/3T3 murine fibroblasts,

which are used as feeder layers for the keratinocyte cul-

tures, contained significant amounts of IL-6, GM-CSF

MEK1

MEK2

MEK4

LF

ALLN

PA

+ + + +

+ +

+ +

Figure 3 Proteasome inhibitor acetyl-leu-leu-norleucinal (ALLN) pro-

tects MEKs cleaved by lethal factor (LF) from subsequent proteasome-

mediated degradation. Keratinocytes were exposed to LF plus

protective antigen (PA) (1 lg ml)1 each) alone or together with

20 lmol ml)1 ALLN for 3 h, and after lysis of the cells, MEK-1, MEK-2

and MEK-4 proteins were analysed by SDS-PAGE (until the blue

indicator dye ran out of the gel) followed by immunoblotting using

antibodies raised against the C-termini of each of the three proteins.

This experiment was repeated three times and similar results were

obtained each time. A representative experiment is shown.


ª 2008 The Authors


or RANTES (data not shown). We determined that the

mouse cells do not produce cytokines in amounts that

can be detected by the ELISA kits used in this

research.

These observations agree in part with other research

that cleavage of MEKs by LF reduces their ability to

bind and activate downstream effector proteins in the

MAPKinase cascade (Ho et al. 2003; Bardwell et al. 2004),

and that this disruption results in decreases in the levels

of mRNA encoding proinflammatory cytokines and the

levels of the proteins that are secreted into the culture

supernatants (Pellizzari et al. 1999; Erwin et al. 2001;

Popov et al. 2002a; Baldari et al. 2006; Batty et al. 2006).

Effects of proinflammatory bacterial cell surface

components on LeTx cytotoxicity

To determine whether pre-exposure to bacterial compo-

nents affected the response of human keratinocytes to

LeTx, the cells were incubated with medium alone, LPS

or CWC for 1 h prior to the addition of LeTx, and incu-

bation was then continued for an additional 23 h. As

shown in Fig. 5, a 1-hour pre-exposure to LPS or CWC

did not compromise the viability of the keratinocytes

after addition of LeTx. These results are in contrast to the

observations of Kim et al. (2003), who reported that

LeTx-resistant murine macrophages undergo a conversion

to LeTx sensitivity after exposure to a mixture of LPS or

CWC, which was associated with the production of TNF-

a after exposure to the bacterial components induction of

TNF-a in the mouse cells. This was postulated by Kim

25(a)

(b)

(c)

20

15

10

IL-6

pg

ml–1

GM

-CS

F p

g m

l–1R

AN

TE

S p

g m

l–1

5

0

LeTx

LeTx

LeTx

SFM CWC LPSSFM+

CWC+

LPS+

SFM CWC LPSSFM+

CWC+

LPS+

SFM CWC LPSSFM+

CWC+

LPS+

6·00

4·80

3·60

2·40

1·20

0·00

80

7525

20

15

10

5

0

Figure 4 Modulation of the released cytokine levels in human kerati-

nocytes by lethal toxin (LeTx). Human keratinocytes were either main-

tained in serum-free medium (SFM) alone or were incubated with

lipopolysaccharide (LPS) (1 lg ml)1) or cell wall components (CWC)

(1 lg ml)1) for 1 h; lethal factor (LF) plus protective antigen (PA)

(1 lg ml)1 each) were then added and the cultures were incubated

for an additional 23 h. The concentrations of interleukin-6 (IL-6) (a),

granulocyte-macrophage colony stimulating factor (GM-CSF) (b) and

RANTES (c) in the supernatant medium were determined by enzyme-

linked immunosorbant assay (ELISA). This experiment was repeated

three times and similar results were obtained each time. A representa-

tive experiment is shown.

50

40

30

20

10

0– LeTx – LeTx

CWC LPSSFM

– LeTx

Rel

ativ

e M

TS

red

ucta

se a

ctiv

ity(a

rbitr

ary

units

)

Figure 5 Effects of bacterial components on lethal toxin (LeTx) cyto-

toxicity. Primary keratinocyte cultures with NIH 3T3 feeder layers or

NIH 3T3 feeder layers alone were either maintained in serum-free

medium alone or were incubated with lipopolysaccharides (LPS)

(1 lg ml)1) or CWC (1 lg ml)1) for 1 h. Lethal factor (LF) plus protec-

tive antigen (PA) (1 lg ml)1 each) were then added to the cultures

for an additional 23 h of incubation. Cytotoxicity was measured by

MTS reduction. Black bars show MTS reductase activity of the kerati-

nocytes with their feeder layers while the cross-hatched bars show

MTS reductase activity from the feeder layers alone. This experiment

was repeated three times and similar results were obtained each time.

A representative experiment is shown.



ª 2008 The Authors

et al. (2003) to be critical for acquisition of LeTx sensitiv-

ity. Accordingly, we assayed the supernatant medium

collected after 24 h from the cultured keratinocytes

employed in the experiments shown in Fig. 5 for TNF-aby ELISA, but none was detected (data not shown). Our

results from Fig. 5 also indicate that the decreases in IL-6

and GM-CFS following exposure of the cells to LeTx, as

shown in Fig. 4, were not attributed to any LeTx-medi-

ated cytotoxicity.

Effects of LeTx on MEKK-directed AP1 transcriptional

responses

Several viral and bacterial factors, in addition to the

presence of other cytokines and chemokines, have been

shown to induce the release of RANTES from cells.

Induction of RANTES release from cells might be

achieved either by increasing gene transcription or by

stabilizing RNA transcripts. Most noteworthy, activation

of the members of the c-Jun family of transcription fac-

tors consequent to MEK activation has been shown to

play an important role in the upregulation of RANTES

(Casola et al. 2001; Kudo et al. 2005). In contrast,

B. anthracis LeTx, which inactivates MEKs, has been

reported to compromise the function of the MAPK

pathway components downstream of the MEKs, includ-

ing the c-Jun family (Bardwell et al. 2004; Baldari et al.

2006). As we have demonstrated (Fig. 4) that, despite

the destruction of MEKs, the release of RANTES from

keratinocytes increases, we sought to investigate whether

downstream targets of MEK activation in the MAPK

pathway are compromised after exposure to LeTx as a

consequence of destruction of the MEKs themselves. To

evaluate the function of one of the downstream compo-

nents of the MAPK pathway, keratinocytes were trans-

fected with an AP1 luciferase reporter plasmid and a

b-galactosidase expressing plasmid and 6 h after initiat-

ing transfection, cells were incubated with LPS, CWC or

medium containing no bacterial components, in the

absence or presence of LeTx. After 24 h incubation, the

luciferase activity was measured (Fig. 6). The addition of

CWC or LPS to cultures without LeTx stimulated lucif-

erase activity. However, co-incubation of CWC or LPS

with LeTx reduced luciferase activity down to that of

control cultures which had been incubated without bac-

terial components. AP1-directed transcription of lucifer-

ase could be augmented by exposure of human

keratinocytes to bacterial CWC or to bacterial LPS, but

was reduced to baseline levels by subsequent treatment

of the cells with LeTx. These results suggested that in

LeTx-treated kerotinocytes, cleavage of MEKs by the

toxin does indeed compromise the function of at least

one MAPK pathway product downstream of the MEKs.

Effects of LeTx on MEKK-mediated transcriptional

activation of AP1, NFjb, and RANTES

As AP1 has been implicated in the induction of RANTES

transcription (Casola et al. 2001; Kudo et al. 2005), the

upregulation of RANTES release from LeTx-exposed

human keratinocytes in spite of the marked diminution

in AP1-directed transcription of the message for a repor-

ter protein after toxin exposure was unexpected. We

therefore evaluated the effects of LeTx on RANTES

expression in addition to the activity of both AP1 and

NF-jB, another transcription factor which was previously

shown to be responsive to the activation of the MEK in

other systems, in a construct in which MEK activation

could be maintained constitutively high through intro-

duction of a pc-MEKK plasmid.

Transfection with the pc-MEKK plasmid along with

AP1, NF-jB and RANTES luciferase reporter plasmids

induced AP1- and NF-jB-mediated transcription signifi-

cantly compared with the basal levels of the reporter

protein activity (Fig. 7a,b). It has been shown that NF-jB

and AP1 play an important role in the induction of RAN-

TES (Casola et al. 2001; Kudo et al. 2005); thus, it would

be predicted that the activation of these factors should

upregulate RANTES expression in a construct in which

an exogenous MEKK plasmid had been introduced.

Indeed, we found that constitutively active MEKK also

induced luciferase activity under the control of the

RANTES promoter (Fig. 7c).

LeTxCWC

+LPS

+SFM

+CWC LPSSFM

0

130

260

Rel

ativ

e lu

cife

rase

act

ivity

390

520

650

Figure 6 Effects of bacterial components and lethal toxin (LeTx) on

AP1-directed transcription. Human keratinocytes transfected with

equal amount of AP1 luciferase reporter plasmid and b-galactosidase

expressing plasmid (0Æ5 lg each). Six hours after the transfection, cells

were either left alone or incubated with bacterial cell wall compo-

nents for additional 24 h. As controls, some of the samples were trea-

ted with LeTx at the same time as the exposure to the bacterial cell

wall preparation.


ª 2008 The Authors


When we treated pc-MEKK-transfected cells with LeTx,

we observed that the transcriptional activation mediated

by AP1 and NF-jB, in addition to the expression of repor-

ter protein under the control of the RANTES promoter,

were all still reduced dramatically (Fig. 7). This result is in

contrast to the effects of LeTx on the endogenous pathway

for RANTES expression in keratinocytes which we have

described (Fig. 4c). Our results support the hypothesis

that, although at least two MEK-directed transcription fac-

tors can contribute to enhanced expression of RANTES in

the absence of LeTx, the increased levels of RANTES in

human keratinocytes exposed to LeTx, which destroys and

thereby inhibits MEKs, may indicate that the control of

the expression of this cytokine is mediated, at least in part,

by another, possibly MEK-independent, pathway which is

itself upregulated by the toxin.

We also investigated the effects of LeTx on levels of the

active forms of c-jun, NF-jB and MEF2 in organotypic

EFT-100 models, because these transcription factors

have previously been implicated in RANTES upregulation

(Casola et al. 2001; Kudo et al. 2005). We found that the

application of LeTx to both the apical and basal surfaces

resulted in decreases in active c-jun levels, but did not

affect active NF-jB levels (Fig. 8). Previously, Park et al.

(2002) showed that LeTx treatment does not affect the

NF-jB pathway induced by bacterial CWC. Thus, our

results using organotypic EFT-100 models are in agree-

ment with the observations of Park et al. (2002). The fail-

ure to detect increased levels of the active form of MEF2

is also consistent with the pivotal role of MEK-5, which is

resistant to cleavage by LeTx, in activating this transcrip-

tion factor.

Discussion

How LeTx kills cells is still a puzzle. It has been demon-

strated that the MEKs are cleaved by LF in a wide range

of target cells. When the MEKs were first identified as cel-

lular substrates for LF, these signal transduction pathway

components were considered as necessary and sufficient

mediators of all the effects of LF, including cytotoxicity

(Park et al. 2002). However, it was subsequently shown

that the MEKs are still cleaved in LeTx-resistant immor-

talized macrophage-like cell lines and in macrophages iso-

lated from LeTx-resistant mice (Mock and Fouet 2001;

Watters et al. 2001; Kim et al. 2003; Kocer et al. 2005).

These results provided convincing evidence that MEK

cleavage alone may not be the cause of LeTx-triggered

cytotoxicity.

In this investigation on the effects of LeTx on human

keratinocytes, we found that these cells are resistant to

LeTx-directed cytotoxicity over 24 h of continuous expo-

sure to the toxin. First, we considered the possibility that

750(a)

(b)

(c)

600

450

300

Rel

ativ

e lu

cife

rase

act

ivity

Rel

ativ

e lu

cife

rase

act

ivity

Rel

ativ

e lu

cife

rase

act

ivity

150

0–

– –

pFCMEKK pFCMEKK

LeTx

–

– –

pFCMEKK pFCMEKK

LeTx

–

– –

pFCMEKK pFCMEKK

LeTx

6000

4800

3600

2400

1200

0

650

520

390

260

130

0

Figure 7 Effects of lethal toxin (LeTx) on MEKK-mediated transcrip-

tional activation of AP1, NF-jB and RANTES. Human keratinocytes

transfected with equal amount of luciferase reporter plasmid (0Æ5 lg),

b-galactosidase expressing plasmid (0Æ5 lg) and a vector expressing

constitutively active MEKK (0Æ5 lg). Cells were transfected separately

with AP1 (a), with NF-jB (b) or with RANTES luciferase reporter plas-

mids (c). Six hours after transfection, cells were treated with LeTx for

additional 24 h. Cells transfected with luciferase reporter plasmid alone

were used as negative control, and cells transfected with luciferase

reporter plasmid together with MEKK were used as positive control.



ª 2008 The Authors

the resistance of human keratinocytes to LeTx-triggered

cytotoxicity might reflect the absence of anthrax toxin

receptors on these cells. We found that LF can enter

human keratinocytes and cleave six of the seven MEKs,

with the exception of MEK-5, indicating that human

keratinocytes express anthrax toxin receptors and that

LF-mediated MEK cleavage alone is indeed not sufficient

for LeTx-triggered cytotoxicity over 24 h of continuous

exposure to the toxin. All these results indicate that

LF-directed MEK cleavage does not correlate with LeTx-

induced cell death and brings to mind the possibility of

another target.

Moreover, we found that LF cleavage directs the clea-

vaged products to proteasome degradation. It appears

that proteasome inhibitors, which do not target and inhi-

bit LF directly (Fig. 3), can prevent LeTx-induced cell

death (Tang and Leppla 1999; Kocer and Simon 2007).

Recently, one group observed that the activation of cas-

pase-1 LeTx-induced necrosis in murine macrophages

and proteasome inhibitors, which do not affect caspase-1,

can block LeTx-induced caspase-1 activation and cell

death, indicating that proteasome inhibitors control an

upstream event in LeTx-treated mouse macrophages lead-

ing to caspase-1 activation (Squires et al. 2007). These

results point out a possibility of an LF target that may

function at the upstream of pathway, which leads to cas-

pase-1 activation, and as long as that target, regardless of

whether or not it is cleaved, may still function as long as

that target is not destroyed by proteasome.

Another distinctive effect of LeTx we observed is the

induction of RANTES release from human primary kerat-

inocytes. According to a current view, LeTx is considered

mainly as an anti-inflamatory agent, which downregulates

the expression of several inflammatory biomarkers (Bal-

dari et al. 2006). On the other hand, paradoxically, LeTx

has been reported to attract neutrophils to cutaneous

infection sites (Wade et al. 1985; Mayer-Scholl et al.

2005). Here, we showed that RANTES, a chemoattractant

for several immune cells, including neutrophils, is upreg-

ulated after exposure of keratinocytes to LeTx, even

though a number of other markers of the inflammatory

response are downregulated. It seems that LeTx selectively

downregulates the inflammatory response. Our results

might explain why the exposure of keratinocytes to LeTx

results in the recruitment of neutrophils to cutaneous

infection sites, while the expression of a panel of inflam-

matory biomarkers is diminished.

How LeTx induces RANTES is not clear. We investi-

gated the effects of LeTx on several transcription factors

which might be involved in LeTx-mediated RANTES

induction. First, we transfected cells with a plasmid

expressing the active form of MEKK1 and treated these

cells with LeTx. We found that LeTx treatment signifi-

cantly reduced MEKK1-directed transcription of reporter

protein under the control of the RANTES promoter and

AP1- and NF-jB-directed transcription (Fig. 7). As

MEKK1 activates the MEKs, which in turn, activate these

transcription factors, it is not surprising to observe that

LeTx, an agent which cleaves and directs most of the

MEKs to proteasome degradation, inhibits MEKK1-direc-

ted transcriptional effects. However, when we evaluated

the effects of LeTx on the levels of the active forms of

multiple transcription factors using the EFT-100 organo-

typic skin model, we found that the exposure of the tissue

models to LeTx did not change the levels of active NF-jB

(a)

(b)

(c)

0·4500·4000·3500·3000·2500·200

0·400

0·600

0·700

0·500

0·300

0·200

0·1500·100

0·100

0·050

Abs

orba

nce

450

nmA

bsor

banc

e 45

0 nm

Abs

orba

nce

450

nm

–0·050–0·100

Control LF PA LF + PA



**0·000

0·000

0·1600·1400·1200·1000·0800·0600·0400·0200·000

Figure 8 Effects of lethal toxin (LeTx) on levels of active forms of

c-jun, MEF2 and NF-jB in organotypic models. EFT-100 models were

incubated with 1 lg ml)1 LeTx added to both the apical and basal

surfaces for 6 h. The medium containing LeTx was then removed and

replaced with fresh growth medium and the units were then incu-

bated further for 24 h. The active forms of the transcription factors

were assayed using TransAm� immunoassay kits (active motif), as

described in Materials and Methods. The figure shows levels of active

c-jun (a), active NF-jB (p50 subunit) (b) and active MEF2 (c) after

exposure of the organotypic models to Lethal factor (LF) alone, pro-

tective antigen (PA) alone, and LeTx. **P < 0Æ01.


ª 2008 The Authors


(p50 subunit) or MEF2 but reduced the levels of active

c-jun, a component of AP1 (Fig. 8). Our result with the

organotypic EFT-100 models is in agreement with the

observations of Park et al. (2002), who showed that LeTx

treatment does not affect the NF-jB pathway induced by

bacterial CWC. Our data suggest that LeTx-mediated

RANTES induction might not be the result of the cleav-

age of MEKs with LF, but possibly could be achieved via

another pathway controlling RANTES production which

has not yet been identified.

Our data further suggest that LeTx reduces the activa-

tion of NF-jB directed by a constitutively active form of

MEKK1, but does not affect the activation of NF-jB in

EFT-100 organotypic models. Similarly, Park et al. (2002)

did not observe any significant change in NF-jB activa-

tion which had been induced by the treatment of cells

with bacterial cell walls after the cells were exposed to

LeTx. Bacterial CWC activate NF-jB in several ways,

including, but not restricted to, an MEK-directed activa-

tion pathway (Dauphinee and Karsan 2006; Dziarski and

Gupta 2006). The effects of an exogenous constitutively

active MAPK cascade component, MEKK1, on the NF-jB

activation pathway may be diminished by LeTx, but the

levels of active NF-jB in unstimulated keratinocytes or in

cells exposed to bacterial CWC are clearly less sensitive to

the toxin, reinforcing the notion that NF-jB activation is

modulated by multiple pathways.

Acknowledgements

We are grateful to our colleagues in Dr S.R. Simon’s lab-

oratory who have provided assistance in this project. We

are grateful to Dr Antonella Casola from University of

Texas at Galveston, for kindly providing us RANTES

luciferase reporter plasmid and to Dr Stella Tsirka for use

of her lab facilities. We would like to thank Dr Jorge

Benach, Director of the Center for Infectious Diseases at

SUNY at Stony Brook, for his helpful suggestions. This

work was supported by NIH (NIAID) R21-AI53524.

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Effects of anthrax lethal toxin on human primary keratinocytes

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