Bacillus anthracis-derived edema toxin (ET) counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway.

0.36 MB·English

by  Nguyen, Chinh

Nguyenetal.BMCMicrobiology2012,12:2
http://www.biomedcentral.com/1471-2180/12/2
RESEARCH ARTICLE Open Access
Bacillus anthracis-derived edema toxin (ET)
counter-regulates movement of neutrophils and
macromolecules through the endothelial
paracellular pathway
Chinh Nguyen1*, Chiguang Feng2, Min Zhan3, Alan S Cross2 and Simeon E Goldblum4
Abstract
Background: A common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear
leukocytes (PMNs) in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of
anthrax is edema toxin (ET), which is formed by the combination of two proteins produced by the organism,
edema factor (EF), which is an adenyl cyclase, and protective antigen (PA). Since cAMP, a product of adenyl
cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of
PMNs into tissues through closure of the paracellular pathway through which PMNs traverse.
Results: Pretreatment of human microvascular endothelial cell(EC)s of the lung (HMVEC-L) with ET decreased
interleukin (IL)-8-driven transendothelial migration (TEM) of PMNs with a maximal reduction of nearly 60%. This
effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free
system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared
to medium controls. Coadministration of ET with either tumor necrosis factor-a or bacterial lipopolysaccharide,
each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The
inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA) activity, but could not be blocked by
inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either
forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP.
Conclusions: We conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of
cAMP/PKA activity.
Background the organism: protective antigen (PA), edema factor
Anthrax refers to those clinical syndromes caused by the (EF), and lethal factor (LF) [1]. PA combines with either
spore-forming, Gram-positive organism, Bacillus anthra- LF to form lethal toxin (LT), or with EF to form edema
cis [1]. Classically, anthrax presents as one of three syn- toxin (ET) [1]. LT received its name as it was thought
dromes: cutaneous, gastrointestinal, and pulmonary [1]. to be the principal virulence determinant responsible for
Pulmonary anthrax is among the most feared of infec- the most deleterious sequelae of anthrax infection [1].
tious diseases; once clinical symptoms have developed, ET was so named as it caused localized edema, in vivo,
mortality remains high even with appropriate treatment. upon subcutaneous injection [1].
Much of the pathogenesis of anthrax is currently attrib- The mechanisms through which ET elicits host cell
uted to two toxins, each of which is produced from two responses are incompletely understood. PA is the recep-
of three proteins synthesized by the bacillary form of tor binding moiety of the toxin complex. After binding
to one of two surface receptors, endothelial marker-8
*Correspondence:[email protected] (TEM-8)/anthrax receptor 1 (ANTXR1) or capillary
1SouthernArizonaVeteransAffairsHealthCareSystems,3601S6thAve,Mail morphogenesis protein-2 (CMG-2)/anthrax receptor 2
Code111-1,Building2,4thfloor,TucsonAZ85723,USA
(ANTXR2), PA is cleaved into a 63 kDa fragment by
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©2011Nguyen;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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surface proteases, such as furin [2,3]. ANTXR1 is pre- alveolar inflammatory infiltrates [25]. The pleural fluid
sent in the epithelial cells lining the respiratory pathway, of several patients contained scant PMNs. Similarly, in
skin, and gastrointestinal tract, as well as being selec- African Green Monkeys exposed to anthrax spores, the
tively upregulated in endothelial cell(EC)s during angio- pulmonary interstitium was expanded by fibrin and
genesis and tumorigenesis [4]. In contrast, ANTXR2 is edema, but contained few PMNs [26]. These combined
ubiquitously expressed in most human tissues [5]. These data suggest an impaired delivery of circulating PMNs
PA fragments oligomerize into ring-shaped heptamers, to extravascular sites of infection. Since PMNs are an
to which EF binds [2]. The entire complex then under- essential host defense against bacterial infection, a survi-
goes receptor-mediated endocytosis [2]. This endosome val advantage would be conferred to any infecting
is acidified, resulting in conformational changes, which organism that could disable these phagocytic cells.
in turn, permit insertion of the multiprotein complex From its name, most observers would intuit that ET
comprised of EF and the PA cleavage product into the increases edema formation, i.e., the paracellular passage
endosomal membrane [2]. EF is then translocated to the of fluid and macromolecules. However, agents that
cytosol, where it exerts its biological effects [2]. EF is increase intracellular cAMP are known to enhance EC-
one of four known bacterial products that are intrinsic EC adhesion, tighten the paracellular pathway, and pro-
adenyl cyclases [6]. Its catalytic rate is 100-fold higher mote barrier integrity [11,27-32]. He et al found that
than any mammalian equivalent [6]. The current under- basal levels of cAMP are necessary to maintain barrier
standing is that most of the effects of EF are due to ele- function under resting conditions [30]. Multiple investi-
vated levels of mislocalized cAMP [1]. ET has been gators have demonstrated that pharmacologic agents
demonstrated to increase cAMP in a variety of cell which increase cAMP or behave as cAMP analogues in
types, including Jurkat cells, Hela cells, monocytes, and ECs enhance barrier function [11,27,28,31-33]. Prior stu-
most relevant to the current studies, ECs and polymor- dies which have looked at ET effects on PMN chemo-
phonuclear leukocyte(PMN)s [3,7-9]. cAMP is a ubiqui- taxis have done so in EC-free systems, and therefore did
tous secondary messenger with multiple downstream not examine whether ET might impair PMN migration
effectors, including protein kinase A (PKA) and protein to target tissues through an effect on the endothelium.
activated by cAMP (EPAC), a guanine nucleotide In the current study, we have defined a novel mechan-
exchange factor (GEF) for Ras-related protein 1 (RAP1) ism through which a bacteria-derived toxin, ET, may
[10]. There are two EPAC variants, EPAC1 and EPAC2, indirectly, through the counter-regulation of the
each of which has a distinct domain structure and tis- endothelial paracellular pathway, impair extravasation of
sue-specific expression [10]. The EPAC1-RAP1 pathway PMNs into tissues.
has been implicated in such cellular processes as vascu-
lar endothelial (VE)-cadherin-mediated cell-cell adhesion Results
[11-13], integrin mediated adhesion [14], monocyte che- ET protects against IL-8-stimulated transendothelial
motaxis [15], Ca2+-induced exocytosis [16], and Fcg- migration (TEM) of PMNs
receptor mediated phagocytosis [17]. Whether ET might Since ET directly stimulates ECs to increase cAMP [7],
also exert biological effects independent of cAMP is which in turn, enhances endothelial barrier integrity
unknown. [11,27-32], we asked whether ET might decrease TEM
Highly purified, recombinant ET is lethal to mice [18] of PMNs. Pretreatment of monolayers of human micro-
at lower doses than is LT [19]. Curiously, edema was vascular endothelial cells of the lung (HMVEC-Ls) with
absent in these mice at the microscopic level [18]. ET ET decreased IL-8-stimulated TEM by ~ 60% (Figure
suppresses the T-lymphocyte secretion of the PMN che- 1A). Neither EF nor PA alone were able to reproduce
moattractant, interleukin (IL)-8 [20]. ET also impairs the ET effect (Figure 1B). For these calculations, total
PMN phagocytosis and superoxide production [21]. In fluorescence associated with PMNs placed in each upper
EC-free systems, investigators have demonstrated that compartment represented 100% migration while %
ET increases PMN chemotaxis [22], whereas others migration was calculated as fluorescence in the lower
have shown an inhibitory effect [9]. Of relevance to the compartment/fluorescence in the upper compartment ×
current report, ET also decreases EC chemotaxis [7]. 100%.
In 2001, renewed interest in pulmonary anthrax was
generated when 11 bioterrorism-related cases were ET acts at the level of the EC to decrease IL-8-drivenTEM
described [23,24]. A unifying feature of these cases was of PMNs
a normal to slightly elevated circulating leukocyte count Since ET decreased the TEM of PMNs (Figure 1A), we
in the face of relatively high levels of bacteremia [24]. asked whether it acted directly on PMNs or indirectly
Although circulating PMNs were abundant, lung tissues via the EC response. When PMNs were co-incubated
from these patients were notable for a lack of intra- with ET in the absence of ECs, ET at the same
Nguyenetal.BMCMicrobiology2012,12:2 Page3of13
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(1000ng/mL: (10ng/mL) (1000ng/mL) (1000ng/mL)
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Figure1EffectofETontheTEMofPMNs.(A)Humanmicrovascularendothelialcellsfromthelung(HMVEC-Ls)culturedtoconfluencein
assaychamberswereexposedfor4htoeitherincreasingconcentrationsofETattheindicateddoseseachofEFandPA(EF:PA)ormedium
alone.(B)HMVEC-Lmonolayersculturedtoconfluenceinassaychamberswereexposedfor4htomedium,ET(1000ng/mL:1000ng/mL),EF
(1000ng/mL),orPA(1000ng/mL).ThesesameHMVEC-Lmonolayersweretheninsertedintothewellsof24-wellplatescontainingeitherIL-8
(10ng/mL)ormediumalone,afterwhichcalcein-AM-labeledPMNswereaddedtotheuppercompartmentofeachchamber.After2h,each
lowercompartmentwasfluorometricallyassayed.Eachverticalbarrepresentsmean(+/-SEM)TEMofPMNs(%).Thenforeachgroupis
indicatedineachbar.*indicatessignificantlyincreasedcomparedtothesimultaneousmediumcontrolsatp<0.05.**indicatessignificantly
decreasedcomparedtotheIL-8stimulusaloneatp<0.05.
concentration that impaired TEM (1000 ng/mL:1000 ng/ Since these PMNs were preloaded with the fluoroprobe,
L) did not decrease IL-8-driven PMN chemotaxis com- calcein-AM, a known intracellular Ca2+-binder [34], and
pared to medium controls (Figure 2A). These data indi- the host response to ET is calmodulin- and Ca2 +
cate that the ability of ET to diminish TEM of PMNs -dependent [1,2,8,22], we asked whether calcein-AM
cannot be explained through a direct effect on PMNs. might diminish PMN responsiveness to ET. The impact
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0.16 *
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1000ng/mL ) (1000ng/mL:1000ng/mL)
14-)C flux (pmol/hsendothelial 000000000....0000.....000001234123455555 C ** ** ** 14)ndothelial-C BSA flux (pmol/h 000000......000000234567 D * *** +* IL-8 ***
Tran 0.0005 12 12 12 12 Transe 0.001 12 6 6 5 6 6
Medium ET (100ng/mL: ET (500ng/mL: ET (1000ng/mL: Baseline Medium LPS (100ng/mL) LPS + ET TNF-(cid:302) TNF-(cid:302)+ ET
100ng/mL) 500ng/mL) 1000ng/mL) (1000ng/mL: (100ng/mL) (1000ng/mL:
200ng/mL) 200ng/mL)
Figure2ETeffectonIL-8-drivenTEMofPMNsisduetoadirecteffectonECs.(A)Nakedfiltersmountedonchemotaxischamberswere
placedintowellscontainingeithermediumorIL-8(10ng/mL),afterwhichcalcein-AM-labledPMNs,suspendedinmediumcontainingET(1000
ng/mL:1000ng/mL)ormediumalone,wereaddedtoeachuppercompartment.After2h,thecontentsofeachlowercompartmentwere
fluorometricallyassayed.Eachverticalbarrepresentsmean(+/-SEM)chemotaxisofPMNs(%).(B)Nakedfiltersweremountedinmodified
BoydenchemotaxischambersinwhichthelowercompartmentcontainedeithermediumorIL-8(10ng/mL).PMNs,suspendedinmedium
containingETormediumalone,wereaddedtoeachuppercompartment.After0.5h,thefilterwasremoved,fixed,washed,stainedwithcrystal
violet,washed,andthetopsurfaceofeachfilterscrapedfreeofcells.Thecrystalvioletwasthenextractedandabsorbancemeasuredat560
nm.Eachverticalbarrepresentsmean(+/-SEM)absorbanceat560nm.(C)HMVEC-Lswereseededatadensityof1.0×105cells/assaychamber
andculturedovernightpriortotreatmentfor6hwitheithermediumorincreasingconcentrationsofET.Eachverticalbarrepresentsmean(+/-
SE)transendothelial14C-BSAflux.(D)HMVEC-Lsculturedtoconfluenceinassaychambersweretreatedfor6hwithmedium,TNF-a(100ng/
mL),TNF-ainthepresenceofET(1000ng/mL:200ng/mL),LPS(100ng/mL),orLPS+ET(1000ng/mL:200ng/mL).Eachverticalbarrepresents
mean(+/-SEM)transendothelialfluxof14C-BSA.Thenforeachgroupisindicatedineachbar.*indicatessignificantlyincreasedcomparedto
thesimultaneousmediumcontrolsatp<0.05.**indicatessignificantlydecreasedcomparedtothesimultaneousmediumcontrolatp<0.05.
***indicatessignificantlydecreasedcomparedtoeitherTNF-aorLPSaloneatp<0.05.
of ET on IL-8 driven chemotaxis of unlabeled PMNs transendothelial 14 C-albumin flux compared to the
was assessed. In these studies, IL-8 increased PMN che- simultaneous medium controls (Figure 2C). ET concen-
motaxis ~ 1.4-fold compared to the simultaneous med- trations as low as 100 ng/mL:100 ng/mL diminished
ium controls (Figure 2B). The addition of ET did not transendothelial 14 C-albumin flux. These data indicate
alter PMN chemotaxis compared to PMNs incubated that ET restricts passage of macromolecules through the
with IL-8 alone. These data confirm those generated in same endothelial paracellular pathway through which
our studies with calcein-AM-labeled PMNs (Figure 2A) PMNs migrate. To provide additional evidence that ET
and further support exclusion of a direct ET effect on decreases IL-8-driven TEM of PMNs through the EC
PMNs. response, we tested whether ET might protect against
To establish whether the ability of ET to decrease IL- agonist-induced barrier disruption. In confluent
8-driven TEM of PMNs was mediated indirectly through HMVEC-Ls where the mean (+/- SEM) baseline trans-
the EC response, we measured the effect of ET on endothelial 14 C-albumin flux was 0.01 (+/- 0.006)
movement of a permeability tracer across the endothelia. pmol/h, both human recombinant tumor necrosis factor
In a subconfluent HMVEC-L monolayers, where the (TNF)-a and bacterial lipopolysaccharide (LPS), each at
average baseline transendothelial 14 C-albumin flux was 100 ng/mL, increased 14 C-albumin flux > 2-fold com-
0.0256 (+/- 0.0147) pmol/h, ET, at increasing concentra- pared to the simultaneous medium controls (Figure 2D).
tions, dose-dependently decreased mean (+/- SEM) When LPS and TNF-a were coadministered with ET at
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1000 ng/mL:200 ng/mL, the increase in transendothelial PMNs might be mediated through EC-generated PKA.
14 C-albumin flux in response to either LPS or TNF-a First, ET was tested for its ability to increase PKA activ-
was decreased by ≥ 60% and ~ 45%, respectively, com- ity in HMVEC-Ls. ET at 1000 ng/mL:1000 ng/mL,
pared to albumin flux in response to each respective increased PKA activity (Figure 3A). When ECs were
agonist alone (Figure 2D). These data indicate that ET exposed for increasing times (0-24 h) to a fixed concen-
provides partial protection against both endogenous tration of ET (1000 ng/mL:1000 ng/mL), PKA activity
host and exogenous bacteria-derived mediators of was increased at 6 h, returning to basal levels at ≤ 24 h
endothelial barrier disruption through its action on ECs. (Figure 3B). Two structurally dissimilar PKA inhibitors,
H-89 and KT-5720, were then tested for their ability to
The effect of ET on IL-8 driven TEM of PMNs is PKA- counteract the ET effect on TEM. To confirm that H-89
independent and KT-5720 impaired PKA activity in HMVEC-Ls, we
Since ET is an adenyl cyclase that increases cAMP, we examined ET-induced phosphorylation of cAMP
asked whether the ability of ET to diminish TEM of response element-binding protein (CREB), a direct PKA
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Figure3ETactivatesPKAinHMVEC-Ls.HMVEC-Lswereseededonto10cmplatesandallowedtoreach80-90%confluencepriorto(A)6h
exposuretoincreasingdosesofET,or(B)increasingexposuretimeswithET(1000ng/mL:1000ng/mL).LysateswerecollectedandPKAactivity
assayedbyELISA.Eachverticalbarrepresentsmean(+/-SEM)absorbanceat450nm.Thenforeachgroupisindicatedineachbar.*indicates
significantlyincreasedcomparedtothesimultaneousmediumcontrolsatp<0.05.
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substrate [35]. Initially, phospho-CREB (pCREB) signal Pretreatment of ECs with either H-89 (10 μM) or KT-
was normalized to total CREB. However, stripping of 5720 (10 μM) alone had no effect on TEM in the pre-
the anti-pCREB antibody was incomplete and inconsis- sence or absence of IL-8 (data not shown). Pretreatment
tent. Consequently, pCREB was normalized to b-tubulin. of ECs with ET (1000 ng/mL:1000 ng/mL) decreased IL-
H-89 and KT-5720 each diminished ET-induced CREB 8-driven TEM of PMNs by ~ 45%. H-89 and KT-5720
phosphorylation (Figure 4A, lanes 3 vs 2, 6 vs 5). Quan- each failed to reverse the ET effect; i.e., the effect of
titative densitometry was performed on each of these either agent co-administered with ET was not signifi-
same blots. H-89 and KT-5720 both completely blocked cantly different than ET alone (Figure 4C). Although H-
phosphorylation of CREB normalized to b-tubulin com- 89 and KT-5720 each completely blocked ET-induced
pared to the simultaneous medium controls (Figure 4B), increments in PKA activity as measured by pCREB/b-
indicating their effectiveness as inhibitors of PKA in tubulin ratios (Figure 4B), these same inhibitors had no
HMVEC-Ls. In these experiments, IL-8 (10 ng/mL) impact on the ET-induced reduction of TEM (Figure
increased TEM of PMNs ~ 4-fold when compared to 4C). Taken together, these data do not support a PKA-
simultaneous medium controls (Figure 4C). mediated ET effect on TEM.
A 1.2 B
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55 kDa 1 2 3 4 5 6 etry ubuli 1
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KT-5720 - - - - - + Den CR 0.6 **
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KT-5720 (10uM) - - - - - +
Figure4ETInhibitionofTEMinthePresenceofPKAInhibitors.(A)HMVEC-Lswerepreincubatedinthepresence(+)orabsence(-)ofH-89
(10μM)orKT-5720(10μM),respectively,beforebeingtreatedwithET(1000ng/mL:1000ng/mL)for6handlysed.Thelysateswereprocessed
forpCREBimmunoblotting.Tocontrolforproteinloadingandtransfer,blotswerestrippedandreprobedforb-tubulin.IB,immunoblot,IB*,
immunoblotafterstripping.(B)ThepCREBsignalsineachblotdescribedin(A)werequantifiedbydensitometryofpCREBandnormalizedtob-
tubulinsignalinthesamelaneinthesameblot.(C)HMVEC-Lsculturedtoconfluenceinassaychamberswerepretreatedwithmedium,H-89
(10μM)orKT-5720(10μM),afterwhichtheyweretreatedfor4hwithmedium,ET,ETwithH-89,orETwithKT-5720.TheHMVEC-Lmonolayers
weretheninsertedintowellscontainingeithermediumorIL-8(10ng/mL),afterwhichcalcein-AM-labeledPMNswereaddedtotheupper
compartmentofeachchamber.After2h,thecontentsofeachlowercompartmentwerefluorometricallyassayed.Eachverticalbarrepresents
mean(+/-SEM)TEMofPMNs(%).Thenforeachgroupisindicatedineachbar.*indicatessignificantlyincreasedcomparedtothe
simultaneousmediumonlycontrolsatp<0.05.**indicatessignificantlydecreasedcomparedtoIL-8aloneatp<0.05.
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Forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) medium control (Figures 5B). Previous investigators
fail to reproduce the ET effect on IL-8-driven TEM of have demonstrated that FSK and IBMX cause maximal
PMNs increases of cAMP at 0.5 h with a decrease by 4 h [36];
To provide further evidence that ET does not decrease in our studies, phosphorylation of CREB normalized to
TEM of PMNs through cAMP or PKA activity, two dis- b-tubulin was elevated but not significantly different
tinct interventions known to increase cAMP, FSK and from the effect at the later time point (Additional File 1:
IBMX, each were introduced. To confirm that FSK and Figure S1A, B). Next, we investigated the effects of FSK
IBMX increased PKA activity in HMVEC-Ls, we first and IBMX on IL-8-driven TEM. In these experiments,
examined FSK- and IBMX- stimulated phosphorylation IL-8 (10 ng/mL) increased TEM of PMNs ~ 6-fold com-
of CREB at 6 h (Figure 5A). FSK (10 μM) and IBMX (1 pared to simultaneous medium control (Figure 5C). Pre-
mM) each increased phosphorylation of CREB normal- treatment of ECs with ET decreased TEM of PMNs by
ized to b-tubulin when compared to the simultaneous ~ 50%. Neither FSK nor IBMX could reconstitute the
)3.5
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1000ng/mL)
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(10ng/mL)(1000ng/mL: (10uM) IL-8 (1mM) IL-8
1000ng/mL)
Figure 5Agents that increase intracellular cAMP do notreproduce the ET effect on IL-8-driven TEMof PMNs. (A) HMVEC-Lswere
treatedfor6hwithET(1000ng/mL:1000ng/mL),FSK(10μM),IBMX(1mM),ormediumalone,andlysed.Thelysateswereprocessedfor
pCREBimmunoblotting.Tocontrolforproteinloadingandtransfer,blotswerestrippedandreprobedforb-tubulin.IB,immunoblot,IB*,
immunoblotafterstripping.(B)ThepCREBsignalsineachblotdescribedin(A)werequantifiedbydensitometryandnormalizedtob-tubulin
signalinthesamelaneinthesameblot.(C)HMVEC-Lsculturedtoconfluenceinassaychambersweretreatedfor4hwithmedium,ET,FSK,or
IBMX.Thesesamechambersweretheninsertedintowellsof24-wellplatescontainingeithermediumorIL-8(10ng/mL),afterwhichcalcein-
AM-labeledPMNswereaddedtotheuppercompartmentofeachchamber.After2h,thecontentsofeachlowercompartmentwere
fluorometricallyassayed.Eachverticalbarrepresentsmean(+/-SEM)TEMofPMNs(%).Thenforeachgroupisindicatedineachbar.*indicates
significantlyincreasedcomparedtothesimultaneousmediumcontrolsatp<0.05.**indicatessignificantlydecreasedcomparedtoIL-8aloneat
p<0.05.
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ET effect on IL-8 driven TEM of PMNs, either at 0.5 h in methodology; mainly the use of a modified Boyden
(Additional File 1: Figure S1C) or at 4 h (Figure 5C). chambers, a shorter incubation time, as well as a dif-
Although FSK and IBMX each upregulated PKA activity ferent means of measuring PMN migration.
comparable to that seen after ET treatment (Figure 5B), Wade et al found that ET stimulated directed neutro-
none could decrease TEM (Figure 5C). Again, these phil migration without having any effect on unstimu-
combined data do not support a cAMP/PKA-dependent lated random migration [22]. They also found that
mechanism through which ET inhibits TEM of PMNs. although ET increased cAMP in PMNs, the absolute
level of that increase was < 1% of that caused by the
Discussion Bordetella pertussis toxin. In contrast, Szarowicz et al
In our studies, we have found that ET decreases IL-8- found that ET reduces chemoattractant-stimulated PMN
driven TEM of PMNs across human lung microvascular actin assembly, chemokinesis, chemotaxis and polariza-
endothelia. We asked whether the observed ET effect tion [9]. In PMNs, ET provoked a > 50-fold increase in
could be attributed to action on either the PMN and/or cAMP and a 4-fold increase in PKA phosphorylation.
endothelium. We found that ET blocked TEM even The differences between our findings and these other
when PMNs were not directly exposed to ET (Figure reports may be attributed to dissimilar techniques. For
1A) and required the presence of both EF and PA (Fig- instance, Wade et al measured chemotaxis of PMNs
ure 1B). At the same concentrations, ET did not inhibit preincubated for 1 h with ET in an agarose-gel based
PMN chemotaxis in an EC-free system (Figure 2A, B). system, both of which were EC-free [22], whereas Szaro-
In contrast, we found that ET decreased 14 C-albumin wicz’s group utilized video microscopy to study adher-
flux across preconfluent endothelia (Figure 2C). Further, ence of PMNs preincubated for 2 h with ET to a
ET attenuated the increase in 14 C-albumin flux pro- fibronectin-coated surface [9]. To our knowledge, none
voked by both endogenous (TNF-a) and exogenous of these previous reports studied PMN migration in the
(LPS) mediators of barrier disruption (Figure 2D). Prior context of the endothelial paracellular pathway. Another
inhibition of PKA with H-89 or KT-5720 did not reverse potential explanation for these disparities may be due to
the ET effect on TEM (Figure 4C), and agents demon- differences in potency of various EF preparations and
strated to elevate intracellular levels of cAMP in their abilities to generate cAMP. Of note, the EF pre-
HMVEC-Ls (Figure 5A, B, Additional File 1: Figure paration offered by List Biologics is the least potent
S1A, B) could not reconstitute the ET effect (Figure 5C, (personal communication, Dr. Erik Hewlett, University
and Additional File 1: Figure S1C). These combined of Virginia, Charlottesville).
data indicate that ET diminishes TEM of PMNs at the Far less is known about the direct effect of ET on ECs.
level of the endothelial paracellular pathway and does so Hong et al demonstrated that ET reorganizes the cytos-
independent of via cAMP/PKA activity. keleton and inhibits chemotaxis of human microvascular
Several studies have examined the direct effect of ET ECs [7]. Tessier’s group found that ET induces a gradual
on in vitro PMN function. O’Brien et al found that ET increase in transendothelial electrical resistance (TEER)
inhibited PMN phagocytosis of opsonized B. anthracis across human umbilical vein EC monolayers cultured on
[21]. Pretreatment of PMNs with ET profoundly collagen-coated inserts. They concluded that ET-
reduced superoxide production in response to either induced edema could not be accounted for by the direct
LPS or muramyl dipeptide. Crawford et al demon- effect of ET on the endothelium [38]. Of interest, in our
strated that ET impaired PMN NADPH oxidase activa- experimental systems for both TEM of PMNs and trans-
tion and downstream N-formyl-methionine-leucine- endothelial 14 C-albumin flux, the ECs were similarly
phenylalanine (fMLP)-induced superoxide production cultured on collagen-impregnated filters. Although Tes-
[37]. Taken together, these studies indicate that ET sier et al studied TEER, their experiments did not
down-regulates PMN phagocytic and oxidative func- include transendothelial flux of a permeability tracer or
tions. Other studies have focused on the impact of ET TEM of PMNs.
on PMN chemotaxis and migration [9,22]. In the cur- ET is an intrinsic adenyl cyclase that increases cAMP
rent studies, ET did not alter the PMN chemotactic [1]. Data exists to support a cAMP-mediated mechanism
response to IL-8 in an EC-free system (Figure 2A). To underlying the ET effect on TEM of PMNs. Moy et al
address concerns that calcein is a Ca2+-binder and found that cAMP agonists attenuated the ability of
would interfere with any Ca2+-mediated ET effect, thrombin to increase permeability [27]. Similarly, Fuku-
these experiments were performed in the absence of hara et al found that cAMP agonists decreased cell per-
the fluoroprobe. Even in the absence of calcein, ET meability and enhanced vascular EC-EC adhesion [11].
had no effect on IL-8 chemotaxis of PMNs (Figure In ECs, cAMP targets multiple downstream signaling
2B). Chemotaxis was not as vigorous in the latter molecules that might promote endothelial barrier integ-
experiment, and this may be secondary to differences rity, including PKA [39] and EPAC1 [40,41].
Nguyenetal.BMCMicrobiology2012,12:2 Page9of13
http://www.biomedcentral.com/1471-2180/12/2
One key effector of cAMP is PKA [10]. PKA has been endothelium. Further support of this concept is offered
shown to inhibit myosin-based contractility through by Wittchen et al, who reported direct activation of
phosphorylation of myosin-light-chain-kinase, thereby RAP1 in EC monolayers decreased both their permeabil-
decreasing its activity [10]. PKA also inhibits RhoA ity as well as TEM of leukocytes [43].
activity, stabilizes microtubules, reorganizes cortical
actin and strengthens tight junctions through phosphor- Conclusions
ylation of vasodilator stimulated protein (VASP) [10]. In In conclusion, we have found that anthrax-derived ET
our studies, we found that ET activates PKA in impedes IL-8 driven movement of PMNs across an EC
HMVEC-Ls in a dose- and time- dependent manner monolayer, as well as attenuates the increase of transen-
(Figure 3A, B). Although ET increases EC PKA activity, dothelial 14 C albumin flux induced by TNF-a and LPS,
its inhibitory effect on TEM could not be ascribed to likely as a direct effect of ET on EC-EC adhesion. This
PKA activity. Two structurally dissimilar pharmacologic ability to counter-regulate paracellular pathway function
inhibitors of PKA, H-89 and KT-5720, each failed to could not be ascribed to cAMP/PKA activity. Whether
attenuate the ET-induced decrease in IL-8-driven TEM this novel pathophysiology for anthrax can be extended
of PMNs (Figure 4C). Further, we were unable to repro- to other pathogenic bacteria and their toxins requires
duce the ET effect on TEM with either of two structu- further study.
rally and functionally distinct pharmacologic agents each
known to increase cAMP, FSK or IBMX (Figure 5C). Methods
Taken together, these data indicate that the mechanism Reagents
through which ET counter-regulates IL-8-driven TEM H-89 and KT-5720 in-solution were purchased from
of PMNs cannot be explained solely through cAMP/ Calbiochem (Gibbstown, NJ). LPS derived from E. coli
PKA activation. 0111:B4, FSK, and IBMX were purchased from Sigma
Another downstream target for cAMP is EPAC1, (St. Louis, MO). EF and PA were purchased from List
which is a GEF for the ras GTPase, RAP1 [10]. Like Biologics (Campbell, CA). Human TNF-a was pur-
PKA activity, the EPAC1-RAP1 pathway also enhances chased from R&D Systems, Inc. (Minneapolis, MN). Bio-
endothelial barrier function [11,12,42-44]. The EPAC1- tinylated rabbit monoclonal anti-pCREB, murine
specific analog 8CPT-2’O-Me-cAMP, which directly monoclonal anti-CREB antibodies, horseradish peroxi-
activates EPAC1 while bypassing PKA, has been shown dase (HRP)-conjugated streptavidin, HRP-conjugated
to decrease permeability of endothelial cell monolayers, goat anti-rabbit IgG, and HRP-conjugated horse anti-
an effect which is ablated by prior siRNA-induced murine IgG antibodies were purchased from Cell Signal-
EPAC1 knockdown [12]. Birukova et al [44] and Fuku- ing Technology (Danvers, MA). Unconjugated murine
hara et al [11] both demonstrated that activation of monoclonal anti-b-tubulin was purchased from Invitro-
EPAC1 attenuated thrombin-induced increases in per- gen (Carlsbad, CA).
meability. As in the case of PKA, the mechanism(s) by
which EPAC1 improves barrier function is still being EC culture
elucidated. Potential EPAC1 targets include activation of Human microvascular endothelial cells from the lung
VASP, as well as activation of ARAP3, which in turn is (HMVEC-Ls), purchased from Promocell (Heidelberg,
a GEF for RhoA, and vinculin, which supports EC-EC Germany) were cultured in EC growth medium MV-2
adherens junctions through association with a-catenin (Promocell) containing 5% fetal bovine serum, human
[10]. As PKA inhibition did not impair the ET effect on recombinant epidermal growth factor (5 ng/mL), human
TEM (Figure 4C), one potential pathway is through recombinant insulin-like growth-factor-1 (20 ng/mL),
EPAC1-RAP1 and its effectors. human basic fibroblast growth factor (10 ng/mL), vascu-
Since ET evokes biological responses in both PMNs lar endothelial growth factor (0.5 ng/mL), hydrocorti-
and ECs, it was unclear as to whether the ability of ET sone (0.2 μg/mL), ascorbic acid (1 μg/mL), gentamicin
to regulate TEM of PMNs could be ascribed to its (30 μg/mL), and amphotericin B (15 ng/mL) [45]. Only
impact on PMNs, ECs, or both. Although prior studies ECs in passages 6-8 were studied.
had demonstrated that ET directly influenced PMN che-
motaxis, in our experiments, it did not (Figure 2A). Preparation and fluorescent labeling of PMNs
Further, ET diminished TEM of PMNs never exposed to Whole peripheral blood from healthy human volunteers
ET (Figure 1A). Finally, not only did ET decrease the was collected under a protocol approved by the Univer-
paracellular movement of PMNs (Figure 1A), but of a sity of Maryland, Baltimore, Institutional Review Board,
permeability tracer as well (Figure 2B, C). These com- into acid citrate dextran (Sigma) solution, and PMNs
bined data indicate that ET counter-regulates PMN dia- were isolated by dextran erythrocyte sedimentation and
pedesis exclusively through its effects on the density gradient centrifugation through Ficoll-Hypaque
Nguyenetal.BMCMicrobiology2012,12:2 Page10of13
http://www.biomedcentral.com/1471-2180/12/2
(Sigma) as previously described [46]. PMNs were resus- labeled PMNs was used to generate total fluorescence.
pended in Hank’s balanced salt solution (HBSS) without % chemotaxis was then expressed as fluorescence sig-
divalent cations (HBSS-) at 5 × 105 PMNs/ml and were nal in the lower chamber/total fluorescence signal in
incubated with 5 μM calcein-AM (Invitrogen) for 30 the upper compartment × 100%. In other experiments,
min at 37°C [46]. PMNs were washed three times with unlabeled PMNs were introduced into the upper com-
HBSS- after which their purity was > 95% and viability partment of a modified Boyden chemotaxis chamber
98% by trypan blue dye exclusion. PMNs were resus- (Neuroprobe Inc., Gaithersburg, MD) while each lower
pended in HBSS with divalent cations (HBSS+) immedi- compartment contained IL-8 (10 ng/mL). After 0.5 h,
ately prior to use. filters were removed, fixed, and washed. PMNs adher-
ent to filters were stained with crystal violet, washed
Assay for TEM of PMNs again, and the top surface of each filter scraped free of
TEM of PMNs was assayed as previously described [46]. stained PMNs. The crystal violet was then extracted
Briefly, gelatin-impregnated polycarbonate filters (13 from each filter with 0.1 M citric acid in 50% ethanol
mm diameter, 3 μm pore size; Nucleopore, Pleasanton, for 5 min and the A of extracts measured, as
560 nm
CA) were mounted in polysterene chemotactic cham- described [48].
bers (ADAPS, Dedham, MA), and sterilized overnight
with UV irradiation. These chambers, which serve as the Assay of transendothelial albumin flux
upper compartment for each assay chamber, were Transendothelial 14 C-bovine serum albumin (BSA)
inserted into the wells of 24-well plates, each well ser- flux was assayed as described [45], with minor modifi-
ving as the lower compartment of the assay chamber cations. Briefly, gelatin-impregnated polycarbonate fil-
and containing 1.5 mL of medium. Each upper compart- ters (13 mm diameter, 0.4 μm pore size) were
ment was seeded with 2.0 × 105 HMVEC-Ls/chamber in mounted on chemotactic chambers, sterilized, and
0.5 mL and cultured to confluence (48 h, 37°C, 5% inserted into the wells of 24-well plates. HMVEC-Ls
CO ). The EC monolayers cultured on filter supports were cultured in the upper compartment of each assay
2
were treated for 4 h with either ET at increasing con- chamber. The baseline barrier function of each mono-
centrations or medium alone. In other experiments, the layer was established by introducing an equivalent con-
EC monolayers were treated for either 0.5 h or 4 h with centration of the permeability tracer, 14 C-BSA (1.1
either FSK (10 μM), IBMX (1 mM), or medium alone. pmol, i.e., 4800-6200 dpm/0.5 ml) (Sigma; St. Louis,
These same chambers were then inserted into wells con- MO), to each upper compartment for 1 h, after which
taining IL-8 (10 ng/mL) or medium alone. Calcein-AM- 0.5 ml from the lower compartment was mixed with
labeled PMNs (5 × 105 cells/well) were introduced into 4.5 ml of Optifluor Scintillation fluid (Packard Instru-
the upper compartments of assay chambers, incubated ments, Downers Grove, IL) and counted in a liquid
for 2 h at 37°C, after which time the contents of each scintillation counter (Beckman, Fullerton, CA). In
lower compartment were fluorometrically assayed in a selected experiments, ECs were seeded at 1 × 105
Thermo Scientific Fluoroskan Ascent fluorometer (exci- cells/chamber and cultured overnight to 80-90% con-
tation 485 nm, emission 530 nm). The fluorescence of 5 fluence. Here, monolayers were cultured to subconflu-
× 105 calcein-AM labeled PMNs was used to generate ence because baseline permeability in postconfluent
total fluorescence. % TEM was expressed as fluorescence monolayers was so low as to make detection of any
signal in the lower chamber/total fluorescence signal in further decreases difficult to measure in our assay sys-
the upper compartment × 100%. tem. The monolayers were then exposed for 6 h to
increasing concentrations of ET, each with a fixed
Chemotaxis of PMNs ratio of EF to PA of 1 ng/mL:1 ng/mL, or medium
Chemotaxis of PMNs was assayed as described [47]. alone, after which transendothelial 14 C-BSA flux was
Briefly, gelatin-impregnated polycarbonate filters were assayed. In other experiments, ECs were seeded at 2 ×
mounted in chemotactic chambers, and the chambers 105 cells/chamber and cultured to confluence over 48
inserted into the wells of 24-well plates containing IL- h. The baseline barrier function of each monolayer was
8 (10 ng/mL) or medium alone, as described above. established and only those chambers which retained ≥
Calcein-AM-labeled PMNs (5 × 105 cells/well) were 97% of the permeability tracer were studied. The
suspended in either medium alone versus medium monolayers were then exposed for 6 h to LPS (100 ng/
containing increasing concentrations of ET before mL), TNF-a (100 ng/mL), either LPS or TNF-a in the
being placed into the upper compartment of assay presence of increasing concentrations of ET, with a
chambers and incubated for 2 h at 37°C. The lower fixed ratio of EF to PA of 5 ng/mL:1 ng/mL, or med-
compartment was then sampled and fluorometrically ium alone. Transendothelial 14 C-BSA flux was again
assayed. The fluorescence of 5 × 105 calcein-AM- assayed and was expressed in pmol/h.