Bb,Host-Derived Proteases, and the Blood-Brain Barrier

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Yvonne
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Bb,Host-Derived Proteases, and the Blood-Brain Barrier

Post by Yvonne » Thu 27 Dec 2007 16:22

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
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Yvonne
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Re: Bb,Host-Derived Proteases, and the Blood-Brain Barrier

Post by Yvonne » Fri 13 Aug 2010 10:13

FEMS Immunol Med Microbiol. 2009 Dec;57(3):203-13. Epub 2009 Aug 6.

Pathogen translocation across the blood-brain barrier.

Pulzova L, Bhide MR, Andrej K.

Department of Microbiology and Immunology, University of Veterinary Medicine, Kosice, Slovakia.

Abstract
Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the blood-brain barrier (BBB) and invasion of the central nervous system. However, the mechanisms used by many pathogens (such as Borrelia) to traverse the BBB are still unclear. Recent studies revealed that microbial translocation across the BBB must involve a repertoire of microbial-host interactions (receptor-ligand interactions). However, the array of interacting molecules responsible for the borrelial translocation is not yet clearly known. Pathogens bind several host molecules (plasminogen, glycosaminoglycans, factor H, etc.) that might mediate endothelial interactions in vivo. This review summarizes our current understanding of the pathogenic mechanisms involved in the translocation of the BBB by neuroinvasive pathogens.

PMID: 19732140
Listen to all,
plucking a feather from every passing goose,
but follow no one absolutely

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