1 Institut für Infektiologie Zentrum für Molekularbiologie der
Entzündung (ZMBE), Universitätsklinikum Münster,
Von-Esmarch-Str. 56, 48149 Münster, Germany
2 Institut für Biochemie, Westfälische Wilhelms-Universität
Münster, Von-Esmarch-Str. 56, 48149 Münster, Germany
* Author for correspondence (e-mail: infekt{at}uni-muenster.de)
Accepted 21 January 2003
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Summary |
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Key words: Cerebral endothelial barriers, Pertussis toxin, Transient permeabilization, PKC, cAMP
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Introduction |
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Although the structure-function of PT has been thoroughly investigated, and
the toxin is frequently applied in biochemical and pharmacological studies,
its role in the onset of systemic disease is still not completely understood.
Especially, whether PT might be instrumental in the development of
neurological complications that are occasionally observed as a sequelae of
pertussis disease has not been elucidated. In the pathogenesis of
pertussis-related neurologic disorders an important step might affect the
integrity of cerebral barriers represented either by the Plexus
chorioideum epithelium or the cerebral capillary endothelium. The
presence of tight junctions in both barriers severely limits the passage of
even small solutes from blood into the brain and the central nervous system
(Franke et al., 1999;
Wegener and Galla, 1996
).
Previous studies by Amiel (Amiel,
1976
) already showed an increase in cerebral vascular permeability
of mice 24 hours after i.v. injection of killed pertussis organisms.
Furthermore, PT is frequently used in immunological studies to enhance the
onset of autoimmune disease in experimental animals
(Munoz, 1985
). Experimental
autoimmune encephalomyelitis (EAE), an accepted model for multiple sclerosis,
can be induced in genetically susceptible laboratory animals by a single
injection of a central nervous system tissue homogenate or purified myelin
antigens. The mechanism by which PT might enhance the development of EAE has
not been elucidated but it appears to involve an increase of the vascular
permeability of the blood-brain-barrier
(Linthicum et al., 1982
;
Yong et al., 1993
;
Ben-Nun et al., 1997
).
To investigate the influence of PT on cerebral barriers we employed tissue
culture systems to model cerebral barriers using epithelial Plexus
chorioideus-derived as well as cerebral endothelial monolayers. Although
in both cell types -Gi proteins were nearly completely
ADP-ribosylated by PT, in epithelial monolayers the barrier function was not
affected. In contrast, in endothelial monolayers PT substantially enhanced
their permeability for the marker protein horse radish peroxidase (HRP). In
parallel the transendothelial resistance decreased. Raising cAMP levels with
cholera toxin (CT) as well as forskolin proved to be inhibitory for the
PT-induced permeabilization. Thus, in cerebral endothelial cells PT appears to
act via a different signalling pathway. Activating or inhibiting central
effectors with various well established cellular drugs indicated protein
kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-kinase) to be involved
in the PT-mediated permeabilization of endothelial monolayers. Recent studies
already indicated PKC as a downstream enzyme of PI3-kinase and a connection
between PT-sensitive G proteins and PI 3-kinase activity has been suggested
(Vanhaesebroeck et al., 1997
;
Takeda et al., 1999
). This
study shows that the PT-induced permeability of cerebral endothelial barriers
is mediated through the PKC and PI3-kinase pathways and is abrogated by high
cAMP levels. Furthermore, this study implies a potential mechanism for the
onset of neurological disorders associated with pertussis disease due to the
effect of PT on the integrity of the blood-brain-barrier.
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Materials and Methods |
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Cell lines and tissue culture
Experiments were performed on brain capillary endothelial cells (BCECs)
isolated from pig brains in primary and secondary culture. Cells were cultured
at 37°C in a humidified atmosphere in DMEM medium with 10% CO2
supplemented with 10% fetal calf serum, 1 mM L-glutamine, 100 units
penicillin/ml and 100 µg streptomycin/ml. SCP, ESP and SP-R cell lines
derived from sheep Plexus chorioideus were obtained as a kind gift
from R. Riebe (BFA für Viruskrankheiten der Tiere, Insel Riems, Germany)
and were cultured on laminin-coated Transwell-clear filters (Corning Costar,
Bodenheim, Germany).
Isolation of porcine brain capillary endothelial cells (BCECs)
Brain capillary endothelial cells were isolated from pig brains essentially
as described by Mischek et al. (Mischek et al., 1989) with minor modifications
(Tewes et al., 1997). Pig
brains were obtained fresh from a slaughterhouse and transported on ice cold
Dulbecco's PBS containing penicillin and streptomycin. Cerebra were cleared of
meninges and homogenized mechanically. The brain homogenate was digested
enzymatically using 1% (w/v) dispase II from Achromobacter
iophagus/Bacillus polymyxa in BCEC preparation medium (Medium 199 Earle
supplemented with 0.7 mM L-glutamine, 100 µg/ml gentamycin, 100 U/ml
penicillin and 100 µg/ml streptomycin; 100 ml medium/brain). After a 2.5
hour incubation at 37°C with continuos stirring, brain capillaries were
separated from myelin and cell debris by dextrane density centrifugation. For
this, aliquots of the suspension (100 ml) were mixed with 230 ml 15% (w/v)
dextrane solution and centrifuged at 5800 g for 10 minutes at
4°C. Each pellet was resuspended in plating medium (BCEC preparation
medium containing 10% v/v ox serum) and triturated using a glass pipette. A
second enzymatic digestion with 0.1% (w/v) collagenase/dispase II for 60
minutes at 37°C was performed to remove the capillary basement membrane.
The suspension was centrifuged at 100 g for 10 minutes and the
pellet was resuspended in plating medium (10 ml/brain). The endothelial cells
released were further purified using a discontinous percoll density gradient.
Endothelial cells were collected from the percoll gradient with a pipette,
resuspended in plating medium and seeded on collagen G-coated culture flasks
at a density of approx. 2x105 cells/cm2.
Twenty-four hours after initial plating the cells were washed with Dulbecco's
PBS and supplied with BCEC culture medium (BCEC plating medium without
gentamycin).
Preparation of cell extracts
Extracts of BCECs were prepared according to Brennan et al.
(Brennan et al., 1988).
Briefly, cells were grown to confluency, scraped from the tissue culture
flasks, centrifuged (200 g, 5 minutes, 4°C) and washed
with phosphate buffered saline (PBS). After extraction with 0.2 M
n-octylglucoside containing 2 mM phenylmethylsulfonyl fluoride (PMSF) and 50
mM K3PO4 with shaking at 4°C for 30 minutes, the
extracts were centrifuged at 15,000 g for 20 minutes in an
Eppendorf centrifuge (Model 5403) and the supernatant was collected. For
measuring the uptake of PT cells were preincubated in culture with pertussis
toxin for various intervals. In some experiments extracted proteins were
treated with 40 µg sialidase from Clostridium perfringens/ml 50 mM
sodium acetate pH 5.0 for 2 hours at 37°C in order to remove terminal
sialic acid residues.
In vitro ADP-ribosylation assay
The ADP-ribosylation assay was performed essentially as described by Xu and
Barbieri (Xu and Barbieri,
1995) and more recently by el Bayâ et al.
(el Bayâ et al., 1995
)
with minor modifications. PT uptake and intoxication of various cells were
investigated by an indirect ADP-ribosylation assay. If PT were routed to its
target
-Gi proteins resulting in successful ADP-ribosylation
during the incubation period, the target proteins would have been blocked for
further PT-mediated modification. Thus, incorporation of 32P-ADP
ribose in vitro serves as a measure of the residual
-Gi
subunits available compared with controls without prior PT incubation
(`subtractive modification'). Cells were extracted as described above and
incubated for 2 hours at 20°C with 100 ng activated PT (activated by
preincubation for 1 hour at 20°C with 50 mM dithiothreitol in 100 mM
Tris-HCl, pH 8.2) and 1 µCi 32P-NAD in 100 mM Tris-HCl pH 8.2,
25 mM dithiothreitol and 2 mM ATP in a final volume of 10 µl. Sample buffer
was added (final concentration: 5% glycerol, 0.75% SDS, 2%
ß-mercaptoethanol, 16 mM Tris-HCl, pH 6.8) and the mixture was heated at
95°C for 10 minutes. After separation by SDS-PAGE (15% polyacrylamide),
labeled proteins were visualized and quantified using a bioimager
(Phosphoimager Fuji BAS 1000). All assays were performed in at least four
independent experiments.
SDS-PAGE and western blotting
Cell extracts were separated by SDS-PAGE with 10% polyacrylamide in buffer
containing 5% glycerol, 0.75% SDS, 2% ß-mercaptoethanol, 16 mM Tris-HCl
(pH 6.8). After protein transfer to nitrocellulose membranes by
electroblotting the blots were blocked in 5% BSA/PBS, washed with 0.05% Tween
20/PBS and incubated with the monoclonal antibody 6FX1 (1:100 dilution of a
hybridoma supernatant), washed again, and then incubated with goat anti-mouse
antibodies coupled to alkaline phosphatase [1:7500; 30 minutes at room
temperature (RT)]. Bound antibodies were visualized by enzyme reaction using
nitroblue tetrazolium chloride
(NBT)/5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine (BCIP) as
substrate.
Measurement of endothelial barrier function
To determine the permeability of cell monolayers endothelial cells were
subcultured on polycarbonate filters (10 mm in diameter, 0.4 µm pore size;
Transwell, Costar) mounted in Transwell inserts. For subculture, endothelial
cells grown on flasks (75 cm2) were washed twice with Dulbecco's
PBS and incubated with 4 ml of a trypsin (0.25%)/ ethylene diamine tetraacetic
acid (EDTA; 0.1%) solution until the cells had detached. The suspension was
added to 2 ml ox serum to inactivate trypsin and was subsequently centrifuged
at 100 g for 10 minutes at RT. The pellet was resuspended in culture
medium (described above) and seeded onto rat tail collagen coated Transwell
filters. 24 hours later, the cell layers were washed with PBS and incubated in
serum-free medium (DMEM/Ham's F12 supplemented with 550 nM hydrocortisone, 865
nM insulin, 6.5 nM L-glutamine, 100 units penicillin/ml and 100 µg
streptomycin/ml). Confluent monolayers formed within 2-4 days after plating at
a density of 2x105 cells/cm2.
For measurements of monolayer permeability for horse radish peroxidase (HRP) a solution of serum-free medium containing HRP (30 µg/ml) was added to the medium in the upper or luminal compartment for 1 hour at day 4 of subculture. The transendothelial HRP transport was then assessed by measuring HRP in the medium of the abluminal compartment.
Application of cellular drugs
For the investigation of effector pathways cellular monolayers subcultured
for 4 days were treated with different established pharmacological agents at
the concentrations given in Table
1. The drugs were applied either alone or in combination with PT.
All reagents were incubated for 4 hours before and for one additional hour
throughout the permeability assay (total incubation time 5 hours). All
reagents had been evaluated for potentially toxic activities at the
concentration applied during the time course of the experiment. The analysis
were performed in at least four independent experiments.
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Measurements of transendothelial resistance (TER)
The TER was measured using a custom made epithelial tissue voltohmmeter
(Evom, World Precision Instruments, Sarasota, FL), where the chambers for
measurements are designed specifically for use with Transwell filters.
Confluency of the monolayers and barrier properties were documented by
measuring transendothelial resistance (200-1000 Ohm cm2). TER
values of cell layers obtained from different BCEC preparations varied from
200 to
1000
cm2 and reached their maxima on day 4
of subculture.
Measurements of horse radish peroxidase (HRP)
HRP concentrations were determined spectrophotometrically by assaying
peroxidase activity using the chromogen ABTS
[2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid)] as substrate.
For ABTS-buffer preparation 50 mg ABTS were dissolved in 500 ml of 100 mM
citric acid, pH 5.0. Immediately before performing the assay 28 µl
H2O2 were added to 100 ml of ABTS-buffer. Samples
containing an unknown HRP concentration (60 µl) were mixed with 200 µl
ABTS-buffer and after 15 minutes of incubation at RT in the dark
the OD was measured at 405 nm. The HRP that had traversed through the
monolayer as indicated by the OD values was taken as a measure of the level of
relative permeability.
Measurements of cyclic AMP (cAMP)
Intracellular cAMP levels were determined using a cAMP enzyme immunoassay
system (Amersham Pharmacia Biotech, Braunschweig, Germany). Endothelial cells
were subcultured on 24-well tissue culture plates for 24 hours. After
incubation with medium alone or medium containing PT (200 ng/ml or 1000
ng/ml), CT (1 µg/ml) or forskolin (50 µM) for 2 hours, the medium was
removed by aspiration and cells were washed three times with Dulbecco's PBS
before cell-lysis was performed using the reagent provided with the cAMP
enzyme immunoassay system. An aliquot of the supernatant was transfered to the
assay-plate. The assay is based on the competition between unlabeled cAMP and
a fixed quantity of peroxidase-labeled cAMP for a limited number of binding
sites of a cAMP specific antibody
Distribution of cellular components by immunofluorescence
For the localization of cytoskeletal proteins, endothelial cells were
seeded on tissue culture chamber slides (Labtek; Nalge Nunc Int., Wiesbaden,
Germany) and grown to confluency. After overnight incubation with medium alone
or medium containing PT (200 ng/ml), cells were washed with Dulbecco's PBS
three times. Cells were fixed with 4% (w/v) paraformaldehyde in water for 15
minutes followed by a 5 minute incubation of 0.2% (v/v) Triton X-100 solution.
Actin microfilaments were stained with FITC-phalloidin a specific stain for
F-actin. The distribution of vinculin, vimentin, -tubulin,
- and
ß-catenin and zonula occludens-1 proteins was visualized by a 60 minutes
incubation with specific monoclonal IgG antibodies directed against vinculin
(Sigma, Deisenhofen, Germany), vimentin (Sigma, Deisenhofen, Germany),
-tubulin (Sigma, Deisenhofen, Germany), ZO-1 (Chemicon Int., Single Oak
Dr, CA), and
- and ß-catenin followed by a 60 minute incubation of
FITC-labeled secondary antibodies (goat anti-mouse IgG or goat anti-rabbit
IgG; Nordic Immunological Antibodies, Tilburg, The Netherlands). The chamber
slides were then separated and the cells were mounted in Mowiol. After 24
hours the cytoskeletal proteins were visualized by immunofluorescence
microscopy.
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Results |
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Morphological changes of endothelial cells treated with PT
As in some cells, such as CHO cells
(Hewlett et al., 1983), PT
induces profound changes in cell morphology; therefore, we investigated its
effect in sub-confluent and confluent BCEC monolayers. In sub-confluent BCEC
monolayers PT was found to cause elongations of the cell body in a time and
dose dependent manner (data not shown). BCECs in confluent and non-confluent
cultures were treated with PT concentrations ranging from 1 pg/ml to 2
µg/ml. Already after 2.5 hours, PT-induced changes could be detected in
sub-confluent BCECs when the cells were incubated with 100 pg/ml. Incubation
of BCECs with up to 1 µg PT/ml for several days did not produce obviously
detrimental or toxic effects. Concentrations of PT exceeding 2 µg/ml proved
to be lethal for BCECs. However, the distribution of cytoskeletal proteins,
such as actin and
-tubulin, or of proteins involved in cell-cell
contacts, such as vinculin, vimentin,
-catenin, ß-catenin and
ZO-1, in confluent BCEC monolayers was not obviously rearranged.
Changes in permeability and transendothelial resistance
Although in confluent monolayers no obvious changes in the investigated
cytoskeletal proteins could be observed the effect of PT on cell morphology in
sub-confluent monolayers raised the question whether PT might, nevertheless,
affect barrier formation by enhancing the permeability of endothelial cell
monolayers. To perform permeability studies BCECs were subcultured on
Transwell polycarbonate filters. We measured the transendothelial resistance
(TER) to monitor the generation and to assess the quality of the barrier
formed by the cell monolayers. The resistance of the BCEC monolayers varied
with time in culture, and on Transwell filters reached its maximum usually on
day 4. To address the influence of PT on the BCEC monolayers the cells were
incubated with different concentrations of PT for 12 hours starting on day 3
of subculture. For these experiments only BCEC monolayers exhibiting a
transendothelial resistance at least between 250 and 300
cm2 were used. Measurements of TER and permeability were always
performed in at least four independent experiments. The permeability of
monolayers induced by the incubation with PT was assessed by the translocation
of the 44 kDa horse radish peroxidase (HRP) protein as an enzymatic marker
protein. The permeability of BCEC monolayers started to increase with a PT
concentration of 0.1 ng/ml, reaching a plateau at 10-200 ng/ml
(Fig. 2). Interestingly, when
the concentration of PT was increased further to 1 µg/ml, the TER increased
and permeability decreased indicating a reversal of the PT-mediated response.
As expected, transendothelial permeability and TER exhibit an inverse
relationship (Fig. 2). The
PT-induced effect on BCEC monolayers were followed for several hours. The time
course of PT-enhanced permeability and the concomitant reduction of TER is
shown for a PT concentration of 200 ng/ml
(Fig. 3).
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To address the question whether the S1-subunit or the B-oligomer of PT is responsible for the PT effect, cell monolayers were incubated with the isolated B-oligomer alone instead of whole PT. No changes in permeability or TER were observed (data not shown). This clearly indicated that the PT effect on cerebral endothelial cells is due to the activity of the S1 subunit.
The PT-mediated increase in permeability is not due to elevated
intracellular cAMP levels
Cellular intoxication by PT is thought to proceed via the ADP-ribosylation
of heterotrimeric G proteins that interferes with the homeostatic inhibitory
regulation of adenylate cyclase, potentially leading to non-physiological
levels of intracellular cAMP. In most cells, including the BCECs in this
study, however, PT itself only induces an incremental increase in cAMP (e.g.
Weiss and Hewlett, 1986;
Glineur and Locht, 1994
). To
investigate whether the observed increase in permeability might be affected by
elevated cAMP levels we employed cholera toxin (CT) and forskolin for the
direct stimulation of the adenylate cyclase. Both reagents efficiently raised
the level of cAMP in BCECs (Fig.
4). CT and forskolin alone had no effect on the permeability of
BCEC monolayers. However, when CT or forskolin were applied in combination
with PT, also no increase in permeability was found
(Fig. 5). This demonstrates
that elevated intracellular cAMP levels completely abolish the PT-induced
permeability of BCEC monolayers. Comparing intracellular cAMP levels after
treatment with PT (200 ng/ml or 1000 ng/ml), CT (1000 ng/ml) or forskolin (50
µM), by using a cAMP enzyme immunoassay system, showed that even at a
concentration of 1000 ng/ml PT induced only a minor increase in cAMP in BCECs.
Incidentally, at this PT concentration the permeability induced at lower
concentrations was reversed (Fig.
2). This indicates that higher levels of cAMP stabilize the
barrier function of BCEC monolayers.
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Identification of effector pathways for PT-mediated
permeabilization
PT-mediated intoxication of target cells has been thought to involve
elevated levels of the second messenger cAMP. However, PT-induced
permeabilization of cerebral endothelial barriers has been shown to be
reversed by elevated cAMP levels. Therefore, PT activity in cerebral
endothelia must involve other messengers and/or signal transduction pathways.
To identify potential pathways involved in steps leading to permeabilization
we employed various cellular drugs that are well established in their effect
on the activation and inhibition of central effectors
(Table 1). For this, BCEC
monolayers were incubated with medium, medium containing the drug to be tested
or PT (200 ng/ml), or medium containing the drug and PT (200 ng/ml) for 4
hours and for 1 additional hour while performing the permeability assay. Most
of the cellular drugs investigated had no effect on the PT-induced
permeability (data not shown). Interestingly, however, treatment of BCEC
monolayers with staurosporine (100 nM) or H-7 (50 µM), which are both used
as inhibitors of protein kinase C (PKC), in combination with 200 ng/ml PT
enhanced the permeability for HRP substantially
(Fig. 6). Control experiments
performed with staurosporine or H-7 alone by incubating BCEC monolayers had no
effect on permeability. If PKC is crucial in the PT-induced pathway leading to
permeabilization this would mean that activation of PKC should significantly
reduce the PT-induced permeability increase. This is exactly what we found
when we used PMA (500 nM) and dioctanoyl-sn-glycerol (100 µg/ml) as
activators of PKC (Fig. 7).
Analogous results were obtained by inhibiting PI3-kinase with wortmannin or
LY294002 (Fig. 8). Both
compounds represent potent and selective inhibitors of PI3-kinase, which
enhanced the PT-induced permeability response. Because to our knowledge no
biochemical activators of PI3-kinase have been reported the opposite effect
could not be investigated. These results strongly suggest that protein kinase
C and PI3-kinase are involved in the PT-induced permeability response, where
PKC would act as a downstream enzyme of PI3-kinase.
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Discussion |
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Interactions of pertussis toxin with brain barrier cells
To address this issue in more detail we investigated the influence of PT on
cells involved in cerebral barriers, such as isolated Plexus
chorioideus-derived epithelial cells, Plexus chorioideus cell lines, and
porcine BCECs.
PT uptake and intoxication efficiency clearly demonstrated that
brain-derived epithelial and endothelial cells are target cells for PT. PT
uptake is followed by retrograde transport and ADP-ribosylation of
-Gi subunits (e.g. el
Bayâ et al., 1997
). However, we found that in our model
systems the barrier function was compromised only in endothelial cell
monolayers. Therefore, in this study we employed tight monolayers of BCECs as
an in vitro model system for the blood-brain barrier and focussed on the
investigation of the effect of PT on monolayer integrity. Changes in cell
morphology observed in sub-confluent BCECs were not apparent in confluent and
tight monolayers. No lethal effect was observed for PT concentrations up to
1000 ng/ml even upon incubation for several days. Changes of cell shape and
the maintenance of cell-cell contacts are complex processes where many
components of the cytoskeleton, such as microtubules or actin filaments, have
to participate. However, in this study no apparent changes in the arrangement
and localization of the investigated marker proteins, such as F-actin,
-tubulin,
-catenins, ß-catenins, ZO-1, vimentin and
vinculin, could be identified by immunofluorescence.
Influence of PT on barrier integrity and the effect of cAMP
levels
To further investigate potential consequences of changes in cell morphology
we performed permeability assays and measured electrical resistance across
endothelial as well as in epithelial cell monolayers. While our results
clearly showed that PT had no influence on the performance of Plexus
chorioideus-derived epithelial cell monolayers we found that PT
substantially enhanced the permeability of BCEC monolayers for HRP used as a
translocation marker. Using HRP as a marker protein, a contribution to the
translocation of HRP by transcytosis cannot be ruled out and previous studies
by Karnovsky and co-workers (Reese and
Karnovsky, 1967; Karnovsky,
1967
) have indeed demonstrated the uptake of HRP in
micropinocytotic vesicles. However, HRP-containing vesicles were few in number
and appeared not to be involved in peroxidase transport. These early studies
already indicated that the main translocation of HRP across endothelial
barriers proceeds by way of an intercellular passage. In addition, we have
investigated HRP translocation with a stable human brain microvascular
endothelial cell line (HBMEC) by electronmicroscopy and rarely found the HRP
label intracellularly. However, a contribution of transcytosis to the
translocation of HRP cannot be ruled out with certainty and has to be analyzed
in more detail in future studies. Our conclusion that intercellular
translocation represents the main passageway is further supported by the
finding that, in parallel to the translocation of HRP, also the
transendothelial resistance (TER) was reduced in a time and dose-dependent
manner. Higher concentrations of PT accelerated the effect and maximum effect
on TER and permeability was observed after about 5 hours
(Fig. 3). However, by raising
the PT concentration to 1000 ng/ml the increase in permeability in BCEC
monolayers was less pronounced (Fig.
2). This corresponds to the increased cAMP levels generated by
this concentration compared with the 200 ng/ml of PT usually employed in the
HRP-translocation assays (Fig.
4) and already suggested that the PT-induced permeabilization of
BCEC monolayers is not mediated by an increase in cAMP.
To address the influence of elevated cAMP levels on the PT-mediated
permeabilization forskolin and cholera toxin have been used in combination
with PT. Both cholera toxin and forskolin induced high levels of intracellular
cAMP (Fig. 4). However, both
reagents also reduced or even completely abolished the PT-mediated increase in
permeability in the BCEC monolayers (Fig.
5). This clearly shows that elevated cAMP levels actually abrogate
the PT-mediated reduction of the barrier function. This is in accordance with
results reported by Raub (Raub,
1996), who found in bovine endothelial cells co-cultured with rat
C6 glioma cells that PT irreversibly obliterates TER.
Potential signalling pathways involved in PT-mediated
permeabilization
To identify potential effector pathways we employed a number of established
cellular drugs activating or inhibiting central effectors, such as PI3-kinase,
adenylate cyclase, PLC, myosin light chain kinase, PKA, PKC, and different
phosphatases (PP) such as PP1, PP2A and PP2B
(Table 1). BCEC monolayers were
incubated either with the respective compounds, with PT, with the drug in
combination with PT, or with medium alone. The control experiments excluded
the possibility that the drugs themselves had any effects on the integrity of
the monolayer. Cellular drugs used for the inhibition of protein kinase C
(PKC), such as H-7 and staurosporine, dramatically enhanced the PT effect and,
in parallel, activators of PKC, such as PMA and dioctanoyl-sn-glycerol,
reduced the PT enhanced permeability of BCEC monolayers. Similar results were
obtained using inhibitors selective for PI3-kinase such as wortmannin and
LY294002 (Table 1). The
PT-mediated permeability of BCEC monolayers increased upon PI3-kinase
inhibition. Because no drugs are available that could activate PI3-kinase the
effect of PI3 kinase activation could not be examined. All other drugs that
would inhibit or activate signal transduction pathways involving effectors
distinct from PKC or PI3-kinase had no effect whatsoever on the PT-induced
increase in permeability.
The results of these studies strongly suggest that PI3-kinase and PKC are
involved in the PT-induced enhancement of the endothelial barrier
permeability. Regarding the known signal transduction pathways in eukaryotic
cells, PKC is a potential downstream enzyme of PI3-kinase
(Duronio et al., 1998). Among
the presently known PI3-kinases (PI3K
, PI3Kß, PI3K
and
PI3K
) the PI3-kinase isoform
has been described recently
(Stephens et al., 1997
). This
particular isoform of the PI3-kinases consists of a novel noncatalytic subunit
that has been named p101 and was found to be unrelated to p85
(Vanhaesebroeck et al., 1997
).
Gi-protein (ß
-subunit)-coupled receptors were shown to
activate this PI3-kinase isoform
(Lopez-Ilasaca et al., 1998
;
Murga et al., 1998
;
Vanhaesebroeck et al., 1997
).
PI3-kinases in turn phosphorylate phosphatidylinositol (PtdIns),
PtdIns(4)P, and PtdIns(4,5)P2 to generate
PtdIns(3)P, PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 representing substrates for specific
isoforms of PKC (Duronio et al.,
1998
; Wagey et al.,
1998
; Ettinger et al.,
1996
). Recently, it has been shown that PI3-kinase
products such as PtdIns(3,4,5)P3 are substrates and
activators of a number of isoforms of PKC such as PKC
, PKC
, PKC
and PKC
(Ettinger et al.,
1996
; Quest,
1996
). The isoforms PKC
, PKC
and PKC
have
been detected in endothelial cells
(Wellner et al., 1999
) and in
the rat brain (Shin et al.,
1998
). The isoforms PKC
and PKC
could be activated
by diacylglycerol and phorbolesters as well. Previous studies have shown that
activation of MAP kinase as a potentially downstream target of PKC via
Gi-coupled receptor, could be blocked by inhibitors of PI3-kinase
(wortmannin and LY294002) and PT (Takeda
et al., 1999
). Among the several cellular phosphorylation systems
that potentially regulate cytoskeletal protein phosphorylation, PKC appears as
an apparently central regulator (Hazel and
Malik, 1996
; Stasek et al.,
1992
). Protein phosphorylation mediated by PKC might also be
responsible for the reduced barrier function and altered cell morphology in
subconfluent monolayers.
Based on the results described in this study we conclude that PT
permeabilizes cerebral endothelial cells via a G protein
ß-subunit-coupled effector involving PI3-kinase and PKC. PLC is
not necessary for this pathway because PI3-kinase
lipid products such
as PtdIns(3,4,5)P3 are direct activators of PKC isoforms.
The mechanism by which the inhibition of PKC effects endothelial cells remains
to be resolved. The involvement of the PKC isoforms PKC
and PKC
in this pathway seems very likely as these isoforms have been detected in
endothelial cells and can activated by DAG/PMA as well as
PtdIns(3,4,5)P3. Further studies will identify the
specific PKC isoform involved in the pathway of PT-induced barrier
dysfunction, and which mechanism is involved in PKC-mediated alteration of
cell shape and permeability response.
Potential dual role of PT for blood-brain barrier integrity
This study provides a molecular explanation for the frequently performed
enhancement of experimental autoimmune encephalomyelitis (EAE) by the
injection of myelin basic protein (MBP) with B. pertussis or PT, or
with PT alone. While the enzymatic activity of the S1 subunit is clearly
needed (Ben-Nun et al., 1997)
for the induction of the disease conflicting reports implicate the B-oligomer
subunits (Lehmann and Ben-Nun,
1992
) as well as the S1 subunit
(Robbinson et al., 1996
) of PT
to be involved in the protective effect of PT against EAE. The dual role of PT
observed in vivo might be due to the particular PT concentration available
locally as at higher PT concentrations elevated cAMP levels proved to reduce
the permeability. Furthermore, as the protective effect of the B-oligomer of
PT in EAE might be due to the S2/S3-mediated inhibition of leukocyte adherence
to selectins on inflamed endothelial cells
(Rozdzinski et al., 1993a
),
elevated PT concentrations might enhance this effect. Thus, with regard to the
induction of encephalopathies as a potential consequence of pertussis
infection, which has recently been discussed by Donnelly et al.
(Donnelly et al., 2001
), PT
might exert a dual effect in permeabilizing cerebral endothelial barriers
mediated by the activity of the ADP-ribosyltransferase and, by contrast,
mediating an anti-inflammatory effect
(Rozdzinski et al., 1993b
) by
competitively blocking leukocyte adherence and recruitment.
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Acknowledgments |
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References |
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