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Friday, May 27, 2011

[AlternativeAnswers] Borrelia burgdorferi, Host-Derived Proteases, and the Blood-Brain Barrier

 



Borrelia burgdorferi, Host-Derived Proteases, and the Blood-Brain Barrier

ABSTRACT

Neurological manifestations of Lyme disease in humans are attributed in
part to penetration of the blood-brain barrier (BBB) and invasion of the
central nervous system (CNS) by Borrelia burgdorferi. However, how the
spirochetes cross the BBB remains an unresolved issue. We examined the traversal of
B. burgdorferi across the human BBB and systemic endothelial cell barriers
using in vitro model systems constructed of human brain microvascular
endothelial cells (BMEC) and EA.hy 926, a human umbilical vein endothelial cell
(HUVEC) line grown on Costar Transwell inserts. These studies showed that
B. burgdorferi differentially crosses human BMEC and HUVEC and that the
human BMEC form a barrier to traversal. During the transmigration by the
spirochetes, it was found that the integrity of the endothelial cell monolayers
was maintained, as assessed by transendothelial electrical resistance
measurements at the end of the experimental period, and that B. burgdorferi
appeared to bind human BMEC by their tips near or at cell borders, suggesting a
paracellular route of transmigration. Importantly, traversal of B.
burgdorferi across human BMEC induces the expression of plasminogen activators,
plasminogen activator receptors, and matrix metalloproteinases. Thus, the
fibrinolytic system linked by an activation cascade may lead to focal and
transient degradation of tight junction proteins that allows B. burgdorferi to
invade the CNS.

RESULTS

B. burgdorferi crosses the human BMEC in vitro. In two separate
experiments, by 5 h approximately 0.012% of the spirochetes had crossed the human
BMEC barrier (Fig. 2A). In the absence of any human BMEC, approximately 10
times more spirochetes (0.15% of the spirochetes added) migrated into the lower
chamber of a Transwell insert (Fig. 2A, inset). To determine if the
presence of B. burgdorferi compromised the integrity of the human BMEC
monolayers, we determined the TEER of the BMEC monolayers. In the presence of B.
burgdorferi 297 there were no major changes in the TEER (>40 x cm2) compared to
the controls, indicating that the integrity of the BMEC monolayer was
essentially maintained at 5 h (Fig. 2B).

B. burgdorferi differentially crosses HUVEC and human BMEC. We also
evaluated the ability of B. burgdorferi to cross both human BMEC and HUVEC
barriers. Using 10-fold-fewer spirochetes than were used in the experiment shown
in Fig. 2, we were unable to detect by dark-field microscopy spirochete
transmigration across human BMEC (Fig. 3A). Interestingly, far more
spirochetes crossed the HUVEC monolayers; 0.27 and 1.79% of the spirochetes had
crossed HUVEC by 5 and 18 h, respectively. As observed in the experiment
described above, in the presence of B. burgdorferi 297 there were no major changes
in the TEER after 18 h of incubation for human BMEC cells compared to the
uninfected controls (Fig. 3C). This finding indicates that the integrity of
the BMEC monolayers was essentially maintained. However, there was a small
but significant change in the TEER after incubation for 5 h (P = 0.019) and
18 h (P = 0.017) for the EA.hy926 cell line.

Because we were unable to detect spirochetes by dark-field microscopy in
the bottom chamber for the human BMEC samples 5 and 18 h after the
spirochetes were added to the top chamber, we reanalyzed the samples from the
experiment shown in Fig. 3A by quantitative real-time PCR to determine the
quantity of B. burgdorferi in the transmigrated medium (Fig. 3B). No B.
burgdorferi was detected crossing human BMEC at 5 h, while 1.19% of the cells
crossed HUVEC. In contrast, 21-fold more spirochetes (4.23 versus 0.20%) had
crossed the HUVEC than had crossed the human BMEC after 18 h. We hypothesized
that the fact that B. burgdorferi was able to cross HUVEC far more
efficiently than it crossed human BMEC may have been due in part to the relative
differences in the observed initial monolayer tightness, as reflected in the
TEER; i.e., the spirochetes crossed the less tight EA.hy926 barriers (mean
TEER, 11 x cm2) more easily than they crossed the tighter barrier formed by
the human BMEC (mean TEER, 28 x cm2). It is of related interest that
spirochete transendothelial migration across HUVEC is facilitated when the
monolayer integrity is altered with EDTA (88).
B. burgdorferi traversal of human BMEC is facilitated by proteases. It has
been shown with monocytes that B. burgdorferi can induce the expression of
uPA and its receptor (10, 19, 20). Furthermore, several Borrelia species
may utilize the fibrinolytic system to cross vascular endothelium (9) and
for dissemination (8, 25, 64). To understand whether plasminogen can
contribute to invasion of the CNS, we examined the traversal of B. burgdorferi
across our BBB model in the presence and absence of plasminogen under
serum-deprived conditions. B. burgdorferi strain N40 was used for this part of the
study, as this strain survives in low-serum culture conditions. The
spirochetes (107 B. burgdorferi N40 cells) were incubated for 18 h with a confluent
human BMEC monolayer grown in Transwell inserts. While only 0.4% ± 0.1% of
the spirochetes traversed the BBB without added plasminogen, when 1 µg of
plasminogen per ml was added in the top chamber, there was a dramatic
increase in spirochete traversal of human BMEC (5.6% ± 0.7%).

To determine whether the interaction of B. burgdorferi N40 with human BMEC
can also lead to plasminogen activation, human BMEC monolayers were
incubated with B. burgdorferi overnight at 37°C. The supernatant was collected,
and its ability to activate human plasminogen was assessed with spectrozyme
PL as a substrate. The supernatant of B. burgdorferi cultures alone was
unable to activate plasminogen. However, the supernatants from the cocultures
were able to activate plasminogen in a dose-dependent manner up to 3.3
times more efficiently than control human BMEC activated plasminogen (Fig. 4A).
To determine whether the ability to activate plasminogen was present not
only in the supernatant but also on the surface of the human BMEC, the
cultures were washed, exogenous human uPA was added for 30 min, and the cultures
were washed again. After adding exogenous human plasminogen and
spectrozyme PL, we found that cells preincubated with B. burgdorferi were able to
activate plasminogen 6.5-fold more efficiently than control human BMEC were
able to activate plasminogen (Fig. 4B). These results suggest that B.
burgdorferi not only induces the expression of a plasminogen activator but also
induces the expression of a plasminogen activator receptor, presumably uPAR.

To ensure that this increase in spirochete crossing of the BBB was due to
plasminogen, we examined the traversal of B. burgdorferi N40 across the BBB
model in the presence of plasmin and MMP inhibitors. As shown in Fig. 5,
traversal of spirochetes across the human BMEC was inhibited by 63% ± 5.6%
in the presence of 50 nM BB-94, a broad-specificity matrix metalloproteinase
inhibitor. Similarly, EACA (200 µM) and 2-antiplasmin (40 µg/ml), both of
which are plasmin inhibitors, reduced traversal by approximately 60%. No
further increase in inhibition was observed when both inhibitors were present
together, indicating that BB-94 and EACA work through a common pathway. To
ensure that this inhibition was not due to any adverse effect of EACA or
BB-94 on spirochete viability (i.e., motility), we tested whether these two
compounds could inhibit the traversal of B. burgdorferi across the inserts
in the absence of human BMEC. The number of spirochetes incubated with BB-94
and/or EACA that crossed was identical to the number of spirochetes that
crossed in the absence of these inhibitors and in the absence of human BMEC.
These results suggest that the fibrinolytic system and matrix
metalloproteinases participate in B. burgdorferi migration across the BBB model.

Previous experiments with astrocytes (69), chondrocytes (33), and
endothelial cells (Perides, unpublished data) have shown that these cells express
various MMPs in response to interaction with B. burgdorferi. To determine
whether some of the known matrix metalloproteinases (MMP-1, -2, or -9) are
expressed in response to the interaction of human BMEC with B. burgdorferi,
we performed zymography and immunoblot analysis with antibodies raised
against MMPs. No induction or increased expression of MMP-2 or MMP-9 was
detected in the supernatant of human BMEC cultures incubated with B. burgdorferi
N40 (data not shown). However, when we used MMP-1 antibody, we found that
there were significant levels of MMP-1 (interstitial collagenase 1) in the
cocultures (Fig. 6). The induction appeared to be dependent on the
spirochete/BMEC ratio.

DISCUSSION

The neurological manifestations of Lyme disease in humans caused by B.
burgdorferi are attributed in part to penetration of the CNS by spirochetes,
yet how the Lyme disease spirochetes cross the BBB remains an understudied
and unresolved issue. B. burgdorferi freely crosses nonbrain vascular
endothelium (13, 57, 88, 90). Our knowledge concerning how this occurs stems from
in vitro studies that examined the ability of the spirochetes to bind to
and cross confluent vascular endothelial cell monolayers in vitro (13, 57,
88, 90). After the initial binding event, how the spirochetes cross vascular
endothelium (paracellular versus transcellular) remains controversial. By
using electron microscopy, Comstock and Thomas (13) first demonstrated that
B. burgdorferi spirochetes are able to enter and translocate across the
cytoplasm of HUVEC grown on polycarbonate filters (Nuclepore inserts), a
process that requires intact viable cells and bacteria (13, 57). The
spirochetes were first observed to cross by dark-field microscopy as early as 2 h,
and almost 8% of the added bacteria crossed by 4 h (13). Low-passage B.
burgdorferi isolates adhere to HUVEC up to 30-fold more than spirochetes
maintained continuously in culture adhere to HUVEC (88). While adherence to and
transcellular crossing of endothelial cells is both time and inoculum
dependent (13, 57, 90, 91), not all studies have supported a transcellular route
of crossing. For example, Szczepanski et al. (88) cited the presence of B.
burgdorferi in the intercellular junctions between endothelial cells, as
well as beneath the monolayers, as evidence that spirochetes actually pass
between the cells. More spirochetes crossed the barrier when the monolayers
were pretreated with EDTA that was used to lower the TEER of the endothelial
cell barrier (88). It is of related interest that Treponema pallidum, the
causative agent of syphilis, also migrates across endothelial cell monolayers
at intercellular junctions (89).
In spite of these investigations of B. burgdorferi-endothelial cell
interactions, no study has been conducted to examine the interactions of these
bacteria with brain microvascular endothelial cells (the functional unit of
the BBB), an in vitro BBB model that has been used to study the
transmigration of monocytes, neutrophils, bacteria, fungi, and African trypanosomes
(17, 28, 29, 32, 36, 37, 38, 62, 66, 70, 74). Our data show that B.
burgdorferi spirochetes differentially cross human BMEC and HUVEC and that the human
BMEC form a barrier to traversal by B. burgdorferi. If spirochetes are able
to cross human BMEC as easily as they cross systemic nonbrain endothelium,
one might expect an earlier and/or far higher incidence of CNS
involvement, observations not supported by the clinical findings that have been
described. HUVEC lack the tight junctional complex that is key to BMEC's function
as a barrier to pathogen entry into the brain. From a comparative
viewpoint, it is also interesting that while Szczepanski et al. (88) observed that
22-fold more low-passage B. burgdorferi than high-passage spirochetes
adhered and crossed HUVEC, we found that about 21-fold more low-passage Borrelia
crossed HUVEC than crossed human BMEC. This finding also underscores the
concept that one cannot extrapolate data concerning B. burgdorferi
penetration of the BBB from experimental data based on nonbrain vascular endothelial
cell models.

Many different types of cells produce MMPs, including stromal cells,
glandular epithelial cells, and neutrophils, and most of these cells also
express inhibitors of MMPs called tissue inhibitors of metalloproteinases (41,
67, 73, 94). All MMPs (except membrane-type MMPs) are secreted in proenzyme
forms and require proteolytic cleavage at the N terminus for activation. The
activation cascade for MMPs in the healthy host is closely tied to the
fibrinolytic pathway. Plasmin can activate many MMPs, including MMP-1
(interstitial collagenase) and MMP-3 (stromelysin). Activated MMP-3 is believed to
be the major physiological activator of most MMPs (49). While regulation of
MMP production in normal cells is tightly controlled and occurs at many
levels, dysregulation of MMPs (due to increased transcription of MMPs,
up-regulation of plasminogen activators, and often down-regulation of inhibitors
such as tissue inhibitors of metalloproteinases and plasminogen activator
inhibitors [PAI-1 and PAI-2]) is often associated with disease.

It has been shown (68) that the enzymatic activity of human plasmin, which
is highly unstable in solution, can be stabilized by the presence of B.
burgdorferi. It has also been shown that plasminogen bound to the surface of
a spirochete can be activated to plasmin by uPA (34), and binding to B.
burgdorferi appears to protect the enzyme from autodigestion, as well as from
inhibition by its natural inhibitor, 2-antiplasmin (9, 6, 8). The
plasminogen binding protein has been isolated, sequenced, and expressed (35), and it
stabilizes plasmin in animals and humans with Lyme disease (34). In fact,
Benach and coworkers have shown that plasminogen plays a role in the in
vitro migration of B. burgdorferi through HUVEC monolayers and that
plasminogen facilitates infection of mice and ticks by B. burgdorferi (8, 9).
Interestingly, under normal growth conditions, human BMEC express (i) serine or
cysteine proteinase inhibitor clade E (nexin, plasminogen activator inhibitor
type 1), (ii) plasminogen activator urokinase, (iii) urokinase-type
plasminogen activator receptor, and (iv) soluble urokinase plasminogen activator
receptor precursor genes (Francescopaolo Di Cello, Department of
Pediatrics, Johns Hopkins University School of Medicine, personal communication).

B. burgdorferi can also induce MMP expression by neural cells, cartilage
explants, and chondrocytes (33, 69). When such cells are incubated with B.
burgdorferi, they express and secrete in a dose-dependent manner several
MMPs, including MMP-1, MMP-3, and MMP-9, as well as proteolytic activity
associated with ADAM-TS4 and ADAM-TS11 (33, 53, 69). Using actinomycin D to
inhibit RNA transcription, we determined by reverse transcriptase PCR that MMP
induction is transcriptionally regulated (69).

To understand whether proteases play a role in penetration of the BBB, we
examined the roles of plasminogen and MMPs. To minimize the effects of
natural inhibitors of proteolytic activity in serum, the addition of
plasminogen under reduced serum conditions dramatically enhanced spirochete crossing
of the human BMEC. This effect was significantly reduced in the presence of
either MMP or plasmin
inhibitors that appear to function through a common pathway. In addition,
we showed that B. burgdorferi also induces the expression of a plasminogen
activator, as well as the expression of a plasminogen activator receptor,
presumably uPAR. Interestingly, while no induction or increased expression of
MMP-2 or MMP-9 was detected in the supernatants of cultures of BMEC
incubated with B. burgdorferi, there were significant levels of MMP-1 in these
cocultures, and the induction appeared to be dependent on the spirochete/BMEC
ratio. These results suggest that the fibrinolytic system and matrix
metalloproteinases participate in B. burgdorferi migration through the BBB
model.

In summary, B. burgdorferi depends heavily on matrixolytic enzymes
secreted not by the spirochete itself but by its host (8, 33). These enzymes
include the enzymes associated with the fibrinolytic system and
metallopeptidases (e.g., MMPs) in particular. Thus, we hypothesized and showed that B.
burgdorferi induces the expression of plasminogen activators and MMPs. These
enzymes linked by an activation cascade may lead to the focal and transient
degradation of tight junction proteins that allows B. burgdorferi to invade
the CNS, yet our preliminary experiments indicated that B. burgdorferi
cells could bind via their tips prior to crossing the in vitro human BBB model
(data not shown) and that they did so without evidence of breakdown of the
BBB integrity based on endpoint Endohm TEER measurements. The TEER and
permeability data are consistent with what has been observed in vivo; i.e.,
unlike what is seen in purulent bacterial meningitis, B. burgdorferi infection
usually causes aseptic meningitis in which the permeability of the BBB is
not substantially altered (18).

While the failure to see a generalized loss of tight junctional integrity
could indicate that B. burgdorferi enters the brain via transcytosis across
endothelial cells, tight junctions are maintained after paracellular
transendothelial migration of large cells, such as monocytes (28) and
neutrophils (4). A critical investigation of the phenomenon linking morphological
methods (i.e., immunoelectron microscopy) with sensitive real-time TEER
measurements (i.e., electric cell-substrate impedance sensing [42]) during the
course of spirochete interaction with human BMEC is required before a
concrete determination concerning the mechanism of spirochete BBB traversal can be
made.

Our in vitro model of the human BBB mimics many of the important features
of in vivo B. burgdorferi interactions with the BBB. Hence, this model
should be an important tool for identifying the cellular and molecular elements
implicated in B. burgdorferi interactions with the BMEC, as well as for
helping characterize the biochemical mechanisms by which the bacteria cross
the BBB. It may also help identify possible targets for intervening in the
transmigration of the Lyme disease spirochetes into the CNS.

_http://iai.asm.org/cgi/content/full/73/2/1014_
(http://iai.asm.org/cgi/content/full/73/2/1014)


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