Pediatric Infectious Diseases Revisited - part 5 pps

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Role of the blood-brain barrier and blood-CSF barrier in the pathogenesis of bacterial meningitis

Rüdiger Adam1, Kwang Sik Kim2 and Horst Schroten1

1 Pediatric Infectious Diseases, Klinik für Allgemeine Pädiatrie, Universitätsklinikum, Düsseldorf, Germany; 2 Pediatric Infectious Diseases, Johns Hopkins Hospital, Baltimore, Maryland, USA

Abstract

Despite significant progress in prevention, diagnosis and therapy acute bacterial gitis remains an important cause of high morbidity and mortality in the pediatric popula- tion with no significant improvement in the outcome in recent years Further ameliora- tion in treatment can only result from a better understanding of the pathophysiological events that occur after activation of the host’s inflammatory pathways secondary to initial bacterial invasion The need for improved management strategies is highlighted by the observed increase in antibiotic resistance of microbial pathogens and recent develop- ments in the pharmacological treatment of meningitis patients with dexamethasone, which might adversely influence delivery of drugs to the central nervous system (CNS)

menin-In this respect the cellular and molecular events at the blood-CNS barriers come to the focus of attention It has become evident that these anatomical and functional barriers with their differentiated functionality and vast surface area centrally contribute to the development of bacterial meningitis This holds true not only for their role as a port of entry into the CNS but also as key players in the pathophysiological cascade following bacterial invasion into the brain Important aspects that have to be considered are the unique anatomical and functional features of the blood-brain barrier and the blood- cerebrospinal fluid barrier, and their distinct interactions with the variety of pathogens responsible for the development of bacterial meningitis.

Introduction

In spite of marked progress in diagnostic procedures, improvement in intensive care and introduction of new antimicrobials, bacterial meningitis still remains a serious, sometimes life-threatening disease in children A high number of survivors are left with persistent neurological or neuro-psychological sequelae To improve present strategies and to develop new options in diagnostic, prevention and therapy, knowledge and understand-ing of pathogenesis and pathophysiology of bacterial meningitis is of utmost importance It is well established that most cases of bacterial meningitis

develop through hematogenous spread of bacteria after crossing peripheral mucosal barriers.

Even though major insights in pathophysiological events have been

derived from experimental animal and in vitro models in recent years, many

aspects of the subsequent invasion of the central nervous system (CNS), the role of the blood-brain barrier (BBB) and even more the blood-cerebrospi-nal fluid (CSF) barrier, remain incompletely understood

It has become clear that these anatomical and functional barriers play

a central role as a port of entry into the CNS but also as key players in the pathophysiological cascade following bacterial invasion into the brain They are involved in the often deleterious events secondary to the host immune response and are also important for therapeutic issues

Bacterial meningitis

Bacterial meningitis as the most common serious infection of the CNS tinues to be an important cause of morbidity and mortality in children The causative organism varies with age, immune function and immunization sta-

con-tus The majority of cases are associated with an infection with Streptococcus

pneumoniae and Neisseria meningitidis, whereas Haemophilus influenzae

type b (Hib) infections have been virtually eradicated as a result of routine

vaccination policies Streptococcus agalactiae, Escherichia coli and Listeria

monocytogenes are the most common meningitis pathogens in neonates

[1–3] Bacterial meningitis typically presents with the triad of headache, fever and meningism in adolescents, but the clinical picture can vary widely

in younger children [3] Despite the development of highly effective otics, improvement of early diagnosis and intensive care management, the disease is fatal in 5–40% of the cases depending on the etiological agent and the patient’s age [2, 4]

antibi-Neurological sequelae develop in up to one third of children and adults who survive an episode of bacterial meningitis [5] These sequelae can be related to direct damage of neuroacoustic structures with following hearing impairment, and to disturbances of CSF dynamics and cerebral blood flow with consequent hydrocephalus, brain edema and intracranial pressure They can also be caused by direct damage of brain parenchymal tissue leading to focal sensory-motor deficits, neuropsychological impairment, or seizures [6]

Despite all improvements in early detection and antibiotic treatment, the rate of sequelae has proven to be rather unchanged in recent years [2, 7] One main reason for this unacceptable rate of complications is the incomplete knowledge about the pathogenesis of this disease, even though experimental studies with cell cultures and animal models have substantially contributed to our understanding of the interactions of bacterial pathogens with mammalian cells and their entry into the CNS

The pathogenetic cascade

Apart from external protection by the skull and the leptomeninges, the CNS

is protected against blood-borne pathogen invasion by effective cellular barriers Thus, a meningitis pathogen can gain access to the CNS through a defect within the external barriers, be it a congenital malformation such as

a dermal sinus or a myelomeningocele, accidentally acquired or iatrogenic,

e.g., after a neurosurgical procedure An infection per continuitatem from

purulent mastoiditis or sinusitis is also possible In the vast majority of cases, however, a pathogen reaches the CNS by hematogenous seeding, after run-ning “a biological gauntlet of host defenses” [8]

It has become an accepted pathogenetic concept that the disease typically progresses through several interconnected phases of interactions between the pathogen and the host (Fig 1)

Mucosal colonization and invasion

Initially, mucosal surfaces of the host’s upper respiratory and nal tract are colonized by bacterial pathogens The bacteria must attach to the mucosal epithelium and resist clearance by mechanical and immuno-logical mechanisms All meningeal bacterial pathogens seem to express a Figure 1 Pathogenetic cascade of bacterial meningitis [9] With friendly permission of Springer.

gastrointesti-range of surface proteins that facilitates pathogen-host cell interaction This event is followed by bacterial penetration of the mucosal epithelium either transcellularly or paracellularly, depending on the organism Many patho-gens niftily use host-specific transport mechanisms to safely transverse this epithelial barrier.

Survival within the bloodstream

Once the bacteria gain access to the bloodstream, they must overcome the host defense to survive, disseminate and replicate to a sufficiently high den-sity within the blood Several studies have suggested that a threshold level

of bacteremia is necessary for a successful invasion into the CNS To remain viable, bacterial phase variable switching of surface elements, such as the polysaccharide capsule, seems to be a prerequisite to counteract opsono-phagocytosis and complement-mediated cell lysis [10] The population of organisms recovered from blood or CSF in the acute phase of bacteremia or meningitis is the believed to be the progeny of a few founder bacteria, often

a single clone, mostly suited to survival within the bloodstream [11]

Breaching of blood-CNS barriers and replication in the CSF

Reaching the blood-CNS barriers, the bacteria then attach to and transgress them through mechanisms that will be outlined in more detail below It became evident that the host defense mechanisms within the brain are nota-bly ineffective in eliminating invading bacterial pathogens Bacterial multi-plication within the subarachnoid space is facilitated by the virtual absence

of host defensive factors such as complement and immunoglobulins, the limited number of endogenous antigen-presenting cells and the limited exchange of immune cells and mediators due to restrictive barriers [12] As these bacterial compounds are formidable immunological stimuli, various cells within the CNS [e.g., resident leptomeningeal phagocytes, microglia, choroid plexus (CP) epithelia, endothelial cells, astrocytes] are activated to produce a wide array of proinflammatory cytokines There is a substantial body of evidence that tumor necrosis factor-_ (TNF-_), interleukin-1` (IL-1`) and interleukin-6 (IL-6) play a central role in this setting [13, 14]

Local intraventricular inflammation

After reaching a critical bacterial concentration and subsequent stationary growth phases or after treatment with antibiotics, a number of bacterial cell wall products, toxins and DNA are released into the CSF compartment [2, 15] In gram-positive pneumococci for example, peptidoglycans, lipoteichoic

acid and pneumolysin are liberated after activation of autolytic hydrolases (Lyt A-C) [8] In gram-negative infections such as meningococci, lipopoly-saccharide (LPS) and non-LPS compounds are released during growth and lysis [15, 16].

“Maximal CNS inflammation”

In this critical phase of meningitis a sequence of parallel and dependent eterious events leads to maximal leptomeningeal inflammation A substan-

del-tial body of evidence mainly derived from animal and in vitro models shows

that cytokines, chemokines, proteolytic enzymes, and oxidants together with

an influx of leukocytes are essentially involved in the inflammatory cascade that leads to tissue destruction and brain dysfunction during bacterial men-ingitis [17] (Fig 2)

The blood-CNS barriers

The homeostasis in the brain is an unconditional prerequisite for correct ron function Thus, several barrier systems are present in the brain regulating the distribution of substances between the blood stream and the CNS

neu-Of all these CNS interfaces the BBB is not only dominant with regard to the surface area available for interchange with the CNS compartment but also with regard to coverage by scientific examinations Neglecting other Figure 2 Concept of “maximal inflammation” [9] With friendly permission of Springer.

anatomical sites of interchange, it is yet infrequently regarded as the only blood-CNS-barrier.

Theoretically, blood-derived substances can gain access to the CNS access at various different anatomical sites [18]:

1 the CP with high perfusion, a wide surface area and tight barrier ties despite fenestrated capillaries due to tight junctions at the epithelial lining

proper-2 the circumventricular organs with fenestrated capillaries but a tight ependymal cell lineage of so-called tanycytes [19]

3 the ependymal lining covering the surface of intracerebral ventricles with a less tight cellular layer (gap junctions) and correspondingly less restrictions to extracellular fluid to communicate with CSF

4 the whole subarachnoid space with a network of tight capillaries in the pia mater and arachnoid mater

5 dural venous sinuses, pial and intracerebral veins or postcapillary venules

Whether these barriers function as a port of entry during bacterial gitis is most likely dependent on the nature of the invading microorgamism The major barriers are described below

menin-The blood-brain barrier

The BBB is a dynamic membranous interface between the systemic culation and the brain, protecting it and maintaining its homeostasis Its anatomical base constitutes a complex system of brain microvascular endo-thelial cells (BMECs) (Fig 3A) These cells are ensheathed by astrocytic outgrowths, which are referred to as astrocytic end-feet, necessary to main-tain barrier properties, and associated pericytes, important for structural support and vasodynamic capacity [20] The BMECs are unique insofar as their cellular clefts are sealed by tight junctions that closely join adjacent cells, resulting in a transendothelial electrical resistance of 1000–2000 1·cm2

cir-[21] Paracellular diffusion of molecules larger than Mr 200–400 and the formation of extracellular fluid is thus inhibited [22] Transcellular passage

of solutes is also impeded, as the endothelial cells have only a limited cytotic capacity and lack endothelial fenestrations [12]

pino-The BBB eliminates (toxic) substances from the endothelial ment and supplies the brain with nutrients and other (endogenous) com-pounds, while restricting the entrance of potentially harmful substances, e.g., bacteria and circulating toxins It does so by specific ionic channels, transporters, energy-dependent pumps and limited receptor-mediated endocytosis [20, 23]

compart-However, during infectious diseases of the CNS, the BBB integrity may

be lost and permeability may be increased

The blood-CSF barrier

The second system that prevents the free passage of substrates between blood and brain is the blood-CSF barrier, represented by the CP epithelium The CP is comprised of a vascularized stromal core surrounded by epithelial cells that are aligned in villi The CP capillaries have a much bigger diameter than cerebral microvasculature (~50 +m vs 8 +m, respectively) [25], the per-fusion of about 5 mL/min/g is about tenfold faster than the average cerebral blood flow [24]

The cell surface is greatly increased due to an array of microvilli on the CSF side and basolateral interdigitations directed towards the basal membrane [26] The CP surface area calculated from animal experiments

is believed to be much bigger than previously appreciated, especially when put into relation to the BBB interface [27, 28] The epithelial cells are sealed

by tight junctions, which become indispensable since the endothelium of CP capillaries is fenestrated, non-continuous and has ‘window’-like openings being highly permeable to hydrophilic substrates Thus, it is the CP epithe-lial cells welded by tight junctions that constitute the anatomical basis of the blood-CSF barrier [24] (Fig 3B)

The CPs are located throughout the fourth ventricle near the base of the brain and in the lateral ventricles inside the right and left cerebral hemisphere They are known to be centrally involved in CSF formation and

Figure 3 Parenchymal cells of the blood-brain barrier (BBB) and blood-CSF barrier (A) Schema for the components of the BBB The endothelial cells of the cerebral capillaries lack fenestrations and are tightly joined by zonulae occludentes (see arrows) Astrocyte foot proc- esses extensively abut the outside surface of the endothelium The darkened area is the inter- stitial space surrounding the capillary wall (N, neuron) (B) Cross-section of a choroidal villus

A ring of choroid epithelial cells surround the interstitial fluid and adjacent vascular core The basolateral surface of the cells has interdigitations, whereas the outer CSF-facing apical mem- brane has an extensive microvilli system Arrows point to the tight junctions between cells at their apical ends [24].

actively regulate the concentration of molecules within the CSF by ous transport mechanisms [29] Whereas a number of molecular carriers are responsible for controlling the influx of nutrients into the CSF, a potent efflux apparatus promotes the discarding of noxious substances in the CNS-to-blood direction [28].

numer-Circumventricular organs

Circumventricular organs (CVOs) are situated at several strategic locations around the ventricles of the brain They are midline structures within the ependymal lining bordering the 3rd and 4th ventricle The most outstanding morphological characteristic of CVOs is a dense and intricate network of mostly fenestrated capillaries, making it readily accessible to blood-borne substances, some of which effect the functions of the subfornical organ [30]

With the exception of the subcommisural organ, the fenestration of blood vessels makes the CVOs part of the blood-CSF barrier CVOs are rec-ognized as important sites for blood-brain communication as neurosecreto-

ry products gain access to the bloodstream and blood-borne substances can

be detected by neuronal structures The term CVOs comprises the following organs: pineal gland, median eminence, neurohypophysis, subfornical organ, area postrema, subcommissural organ, organum vasculosum of the lamina terminalis (Fig 4) Sometimes the CP is also included as well as the interme-diate and neural lobes of the pituitary [26]

Collectively, the ependymal and capillary surface areas of the CVOs are relatively small, likely accounting for less than 1% of the ventricles and brain capillary bed, respectively Despite these diminutive transport interfaces, a possible role in microbial CNS invasion is conjectural, since involvement of these areas during inflammation and leucocyte invasion has been reported [31]

Gateways into the brain

It is still unclear why many pathogens principally have the potential to ate meningitis, but only a relatively small number of them account for the vast majority of cases The crucial step for all microorganisms after invasion

initi-of the host is the attachment and subsequent penetration initi-of the structures that separate the CNS from the periphery For most pathogens, however, the exact port of entry into the brain remains unclear

Nonetheless, observations on cell culture and animal models as well as histological experiments allow conclusions about the primary site of inva-sion to be drawn It has to be kept in mind, though, that multiple routes into the CNS compartment may be used simultaneously

Importance of threshold bacteremia

Even though the exact sites of entry might not be exactly known, eral studies suggest the probability of developing meningitis to be directly related to the concentration of bacteria in the blood and to their exceeding

sev-a criticsev-al threshold For exsev-ample, Dietzmsev-an et sev-al [32] reported sev-a higher

inci-dence of E coli meningitis in neonates who had bacterial counts in blood

> 103 CFU/mL (6 out of 11 cases, 60%) compared to those with bacterial counts less than 103 CFU/mL (1 out of 19 cases, 5%) Such associations between a certain degree of bacteremia and subsequent disease have also been described for all other pathogens relevant for meningeal infections

such as Hib [33, 34], S agalactiae [35], S pneumoniae [36, 37], E coli [32, 38] and, with some conflicting data, N meningitidis [37, 39] The infection of the

CSF compartment possibly appears as a kinetic process with bacteria ing from blood and being cleared into the cerebral venous sinuses within the CSF flow Bacteria have been shown to exit from the CSF to the venous blood through the arachnoid villi [40] The balance of bacterial ingress and egress is proposed to be important in the establishment of meningitis and its severity [41]

enter-Figure 4 Sagittal view of the anatomical relationship among the circumventricular organs (CVOs), which are located on the midline of the brain (AP, Area postrema; SFO, subfornical organ; ME, median eminence; PI, pineal gland; OVLT, organum vasculosum of the lamina terminalis) [24].

The blood-brain barrier

Many investigations on meningitis pathogenesis focus on the BBB or its morphological correlate, the endothelium of the cerebral microvasculature

As outlined below, a respectable number of both in vitro and in vivo

mod-els are available One reason for this emphasis on the BBB is its assumed preponderance regarding surface area in comparison with the other blood-CNS barriers As one researcher puts it, “the cerebral microvasculature was chosen for morphological assessment in this study because it represents the dominant site of the BBB The surface area of the cerebral microvas-culature is 5000-fold greater than the surface area of capillaries supplying the circumventricular organs, rendering the former more pertinent for this investigation” [42] This view, however, has been seriously questioned by other researchers who believe the ratio to be more in the area of 1:10 tak-ing into account more recent data derived from calculations on neonatal rat CP extrapolated to conditions in humans [27, 28] Others have put their emphasis on the cerebral vasculature because it plays a dominant role in the pathophysiology of bacterial meningitis after the initial stages of blood-CNS barrier breakdown [43]

In an infant rat model of S pneumoniae meningitis, brain tissue

exami-nations from animals with positive CSF cultures revealed histopathological signs of inflammation predominantly within the meningeal region [44]

In cryostat sections of infant rat brain cortical slices, S-fimbriated E coli

strains have been shown to bind specifically to the luminal surfaces of bral endothelial cells besides binding to CP epithelial cells and ependymal cells [45] In contrast, gram-negative rods were present in the subarachnoid space predominantly around the perivascular areas not in the CP, pointing towards the BBB as being the major gateway into the CSF [38]

cere-In a mouse model, the animals that developed pneumococcal meningitis after intranasal inoculation and treatment with hyaluronidase, showed a sig-nificant inflammatory infiltrate predominantly composed of polymorpho-nuclear leukocytes preferentially around the leptomeningeal blood vessels, suggesting them to be the area of blood-CNS barrier breaching [46]

Challenge of mice with S agalactiae by intraperitoneal injection led to

bacteremia and subsequent meningitis Histopathological studies of brain and meninges of animals with positive CSF cultures principally revealed that bacteria and leukocytic infiltrate distributed surrounding the menin-geal vessels and the perivascular spaces within the cerebral cortex [35].Histological examination of brain tissue from a fatal case of meningococ-

cal disease revealed attachment of N meningitidis on the CP and

microvas-cular endothelium, indicating that both loci may be used by meningococci for invasion of the meninges [47]

Despite decades of investigation on microbial interactions at the CNS barriers, there remains a distinct paucity of studies clearly pointing towards the cerebral vasculature as the primary site of CNS invasion for

blood-certain pathogens Probably due to this dilemma, some authors dently cite a reviewing feature in the News of the American Society of Microbiology (ASM News) [48] as the only reference for microbial BBB invasion [49–51].

indepen-However, abundant experimental studies have demonstrated that tors for various meningeal pathogens are present on cells of cerebral capil-laries potentially mediating attachment or penetration of the BBB [52, 53]

recep-The blood-CSF barrier

For many important meningitis pathogens certain experimental data gests the CPs to be involved in bacterial entry into the brain Whether the blood-CSF barriers represent the primary sites of invasion or one of several

sug-ports of entry remains to be clarified Insufficient availability of suitable in

vitro models throughout recent decades may be in part responsible for the

lack of supportive data

In the fatal case of an infant having succumbed to fulminant infection

with N meningitidis, histopathological investigations of brain sections at

autopsy revealed the greatest number of bacteria attaching to CP laries (68% in CP vs 7% in meningeal capillaries) No meningococci were found to be adhered to the plexus epithelial cells Interestingly the bacteria isolated from the CSF expressed significantly more PilC protein than blood isolates, suggesting this adhesin plays an important role in attachment and invasion of meningococci [47]

capil-In Hib meningitis, early studies on infant rats suggested that invasion

from the bloodstream occurred via the dural sinus veins, while other

stud-ies favored the cribriform plate or the CPs to be the main site of entry into the brain The latter notion was supported by infant rat models with serial CSF sampling from infected animals Here, at least in the early phases of infection before an assumed equilibrium within the CSF compartments has occurred, the highest density of bacteria was found in the CSF of the lateral ventricles in comparison to the lumbar and cortical subarachnoid space or the cisterna magna, respectively, suggesting an entry of bacteria primarily

via the CPs [54].

This observation is supported by studies on primates, in which the CP has been found to be the site of earliest histopathological changes during Hib infections [55] Another line of evidence favoring the CP to be the main site of bacterial entry is derived from the observation that, in experimen-tal meningitis of infant primates, a concordance of bacterial density in the CSF between the lumbar subarachnoid space and the cisterna magna was observed even at low bacterial concentrations (i.e., in early stages of the disease) [56] Since the CSF flow is unidirectionally circulating from the ventricles down to the lumbar region, the presence of bacteria in the ven-

tricular fluid suggests entry via the CPs.

Another pathogen, Streptococcus suis, which accounts for both human

and porcine meningitis cases, is also suspected of entering the CNS

primar-ily via the blood-CSF barrier In a porcine animal model, infected pigs were

killed at the earliest clinical signs of meningitis In these cases, in which a low bacterial density within the CSF can be assumed, streptococci were almost exclusively detected in the CP epithelium [57] The lack of diffuse

parenchymal lesions in most S suis cases of meningitis suggests access to the CNS via the CPs.

Experiments with E coli strains possessing S-fimbriae demonstrated

specific binding sites on CP epithelial cells, to a lesser extent also to thelial cells of the CP core besides vascular endothelial cells and ependymal cells In this work, which was performed on cryostat brain sections of neo-natal rats, pre-treatment of the slices with neuraminidase or a fimbrial ana-

endo-logue abolished attachment of E coli, demonstrating the specificity of these

interactions [45] In contrast, in infant rats with experimental hematogenous

E coli meningitis, gram-negative rods were demonstrated around the

peri-vascular area, not in the CP [38] Thus, entry of E coli into the CNS via the

CP may be unlikely and additional studies are needed to clarify this issue

A study on experimental listeriosis in mice showed that after taneous injection the animals developed meningitis displaying a mixed inflammatory infiltration in the ventricular system, especially in the CPs

subcu-Inflammatory lesions were associated with the presence of L

monocyto-genes within phagocytic cells It is suggested that choroiditis and meningitis

developed as a consequence of hematogenous dissemination of L

monocy-togenes within mononuclear phagocytes and penetration of these cells into

the ventricular system through the CP [58]

In addition, invasion of the CNS via the blood-CSF barrier may also be

facilitated by the high blood flow in the CPs of up to 500 mL/g/min [24], which allows putative delivery of a relatively high number of pathogens to

this site via blood stream.

Experimental models for blood-CNS-barrier observations

Animal models

A number of animal models have been successfully established to study cellular and molecular mechanisms of microbial invasion into the brain Apart from bacterial species used or animals selected as a host, the informa-tion obtained from these models is very much dependent on the mode of inoculation Intranasal, orogastral, intravenous/intracardial, subcutaneous

or intraperitoneal inoculation primarily focus on events on “the blood side”, e.g., bacterial and host factors that determine the pathogen’s fate within the bloodstream and the potential of CNS invasion In contrast, experimental models using direct inoculation into the CSF rather highlight pathogenetic

events on “the brain side” Notwithstanding bypassing the microbial ation of blood-CNS barriers artificially, these models have the advantage of reliably inducing lethal infections with reproducible bacterial inocula over

perme-a predictperme-able time course [59, 60]

These animal models have contributed considerably to the study of pathogen and host factors such as bacterial virulence traits, microbial inva-sion genes, intracellular signaling cascades and modes of cellular perme-ation Furthermore, they have helped in understanding the complications

of meningeal inflammation and evaluating potentially useful agents for treatment therapy [61, 62]

An infant rat model has been widely used to mimic human neonatal bacterial meningitis An important advantage of this model lies in the devel-opment of meningitis after bacterial hematogenous spread similar to human newborn meningitis

The pathogenesis of meningitis has been studied essentially with two

major pathogens, Hib [63, 64] or E coli [65–68] using many different routes

of inoculation (nasopharyngeal, orogastric, subcutaneous, intraperitoneal

or intracardial) For other purposes an infant rat model with intracisternal

inoculation of S pneumoniae has been used [69, 70] Other important

men-ingitis models are performed with adult animals by direct systemic or cerebral inoculation mostly in rabbits [71, 72], rats [73] or mice [74]

intra-In recent years, knockout mice with targeted deletion of specific genes have become a powerful tool in investigating the roles of the different adhesins, cytokines, proteases, and oxidants involved in the inflammatory cascade during bacterial meningitis [75]

Cell culture models

To identify and study cellular and molecular mechanisms of microbial meation of the blood-CNS barriers, it has become important to model the

per-blood-CNS barriers in vitro [76, 77] Both primary and immortalized cell

culture systems have been established One of the major potential benefits of

these in vitro systems in comparison to animal models lies in the possibilities

to measure cellular responses to a variety of stimuli without the risk of ference by possible contributions of other cell types such as neuroglia or resi-dent macrophages Furthermore, no experimental bias is risked by changes

inter-in functional and structural characteristics of the blood-CNS barriers

Blood brain barrier

As outlined above the BBB principally consists of a tight microvascular endothelium, a basal membrane and the pericytic sheath that have to be crossed by bacteria when entering the CNS The central component of all

models is the BMEC BMECs are usually harvested from brain enates, purified on dextran gradients and cultured alone or together with supporting glial cells Many mammal BMECs have been used: rat, mouse, dog, dogs, cattle and human [78–84] Models using peripheral endothelial cell such as human umbilical vein endothelial cells (HUVECs) have also been introduced, but these systemic endothelial cells are likely not appro-priate targets for meningitic bacteria [52].

homog-Extending the potential of cell monolayers, several coculture systems have been developed Bilayer systems consisting of endothelial and epithe-lial cocultures separated by a porous membrane offer added complexity of

multiple layers that might more closely resemble the in vivo situation and

allow examination of microbial penetration and associated effects [85].Multiple studies have indicated that coculturing of BMECs with astro-cytes or neuroglia on opposing sides of a permeable support has mutual benefits as endothelial cells facilitate astrocyte differentiation but, more importantly, astrocytic metabolism contributes to the formation of BBB properties in BMECs (reviewed in [86]) These culture systems were employed in studies on bacterial interactions with cerebral endothelium,

e.g., using S pneumoniae [83, 87] or E coli [88, 89].

Primary BMEC isolation is laborious, time consuming and the cells are difficult to maintain in native tissue culture and suffer from contamination

In addition, these cells often lose their typical features such as Factor VIII Rag or a-GTP upon subcultivation Immortalizations and spontaneous transformations have been reported for mouse, rat, cow and human-derived brain endothelial cells

The best-studied system so far is a human brain microvascular thelial cell line (HBMEC) that has been derived from a brain biopsy of an adult female with epilepsy The HBMEC were immortalized by transfection with simian virus 40 large-T antigen [90] This cell line has proven invalu-able in multiple experiments on bacterial interaction with the BBB Many

endo-different bacterial species have been examined, e.g S agalactiae [91], S suis [92], S pneumoniae [83], N meningitidis [93], Staphylococcus aureus [94], and H influenzae [95].

In addition to a bovine cell line [90], a porcine counterpart of HBMEC,

an immortalized porcine brain microvascular endothelial cell line (PBMEC/C1-2) has recently been established by lipofection with simian virus 40 small and large T-antigens [96] It was shown to maintain its morphological and

functional characteristics and was used in several investigations with S suis [49, 97] and Haemophilus parasuis [98].

BMEC cells in vitro as models of the BBB should exhibit substantial

properties of cerebral microvascular endothelium At best they should express tight junction proteins (such as claudins, occludin and ZO-1 and 2) and adherens junction proteins (such as VE-cadherin and `-catenin)spatially separated to morphologically demonstrate features of a polarized monolayer Functionally, this should translate to a limited permeability to

paracellular tracers (e.g., inulin, sucrose, mannitol or dextran) and to ions, resulting in low permeability coefficients and high transendothelial electri-cal resistance, respectively [99, 100].

Blood-CSF barrier

As mentioned earlier, the tight CP epithelial lining constitutes the structural

correlate of the blood-CSF barrier The establishment of in vitro models of

CP epithelial cells has been a challenge for many years Several tion methods of primary cells have been established, all based on the initial experiments with rat and cow cells [101, 102] Subsequently, other working groups have been successful in culturing primary CP epithelial cells includ-ing other species: rabbit [103, 104], rat [105–107], cow [102] and swine [108] However, many primary cultures have been problematic regarding contami-nating fibroblasts

prepara-The CP epithelial cells are principally isolated with enzymatic digestion after mechanical pre-treatment and cultured either on flat bottom culture dishes or in permeable filter inserts, where they are able to maintain a hydrostatic pressure difference between apical and basolateral compart-ment and, thus, are able to establish an effective hydrodynamic barrier.Just recently our working group has adopted a primary porcine CP cell model [108] for studies of bacterial interactions at the blood-CSF barrier

We were the first to demonstrate a bacteria-CP interaction in vitro using S.

suis [109–111] (see also p 216).

Several CP cell lines have been established from rat [112], mouse [113]

or sheep [114] with varying quality regarding typical markers, phenotypes and especially barrier function Therefore, their impact regarding questions

on CNS infections has been limited so far

Microbial translocation across the blood-CNS barrier

Recent studies on E coli have elegantly shown that successful crossing of

the BBB by circulating bacteria requires, as mentioned above, a certain degree of bacteremia, a direct attachment of the microbe to and subsequent invasion of the endothelial cells, a rearrangement of the BMEC actin cyto-skeleton and the traversal of the cells alive [1, 53, 115]

Once attachment to the tight blood-CNS barriers has occurred, several pathogen-specific strategies can be employed to migrate across and gain access to the CSF space (Fig 5):

– disruption of tight cell-to-cell contacts and passage between the cells (paracellular route)

– direct or indirect invasion of the endothelial cells, permeation and release in

a vital state on the contralateral side of the barrier (transcellular passage)

– penetration of the barrier attached to or phagocytosed by leukocytes during their diapedesis (direct or ‘modified’ Trojan horse mechanism)– destruction of the barrier by cellular injury, e.g., due to release of cytotoxic enzymes or bacterial fragments.

Transcellular passage

Just like overcoming the nasopharyngeal barriers, pathogens use several

host transport systems to breach the blood-CNS barrier For N

meningiti-dis, interaction between surface proteins (Opc) with endothelial integrin

receptors is important [116] S pneumoniae utilizes the internalization of

platelet-activation factor (PAF) receptor via binding of phosphorylcholine and is likewise incorporated While a fraction of the internalized pneumo-cocci dies, others transverse the cells via transcytosis [117] A similar mode

of action is known for S agalactiae They are also internalized by “induced

transcytosis” after attachment to fibrinogen, even though in higher densities they might also damage the cellular barrier by release of toxins (see also below) [52, 118, 119]

E coli displays attachment and invasion characteristics specific for

cere-bral endothelial cells They adhere to and invade HBMEC using several capsular and fimbrial epitopes and can be found within intracellular vacu-oles of HBMEC [67] Bacterial proteins necessary for bacterial invasion have been identified, i.e., IbeA, IbeB, YijP and CNF1 [52, 65, 67] Using the Figure 5 Possible strategies of microbial penetration of blood-CNS barriers [9] With friendly permission of Springer.

host cytoskeleton they are able to transverse the BBB and reach the CNS

in a vital state

Other pathogens believed to breach the BBB by transcellular passage

are L monocytogenes [120], Mycobacterium tuberculosis [121], and fungal pathogens such as Candida albicans [122] and Cryptococcus neoformans

[123] Figure 5 illustrates possible strategies of microbial penetration of blood-CNS barriers [9]

Paracellular/intercellular passage

If cerebral endothelial cells are confronted with high bacterial loads, other factors besides the transcellular passage supposedly become relevant Both the `-hemolysin production of S agalactiae and the pneumolysin of S.

pneumoniae are capable of damaging the endothelial layer integrity, thus

possibly allowing direct paracellular passage of bacteria [52, 124] In studies

on Hib, it has been suspected that the bacteria cross the BBB paracellularly

[125] Borrelia burgdorferi is also suspected of reaching the subarachnoid

space after paracellular penetration, although some aspects point at a

trans-cellular route as well [126] Protozoans such as Trypanosoma brucei at least

partly penetrate endothelial linings via a paracellular mechanism, although recently transcellular permeation has been documented [127]

Transmigration via leucocytes (Trojan horse mechanism)

Pathogens with the ability to survive within phagocytes can take advantage

of being phagocytosed and reach the brain when their “Trojan horses” migrate through blood-CNS barriers Such mechanisms have been suggest-

ed for Brucella spp., M tuberculosis and L monocytogenes [128, 129] It is of interest that, in some events, Listeria are even able to spread by retrograde

neuronal transport from the periphery to the CNS [130] Whether this axonal movement is pathogenetically relevant in humans is not known yet

intra-Intracellular survival in macrophages has also been demonstrated for S.

agalactiae [131] and E coli [132], but it is unclear whether this property has

any relevance to transversal of blood-CNS barriers In S suis, a “modified

Trojan horse” mechanism, in which bacteria transverse blood-CNS barriers

by adhering to diapeding macrophages, rather than residing in phagosomes within them, was discussed [133, 134]

Interactions between bacteria and blood-CNS barrier cells

As stated earlier, one key factor for microbial entry into the subarachnoid space is the ability to reach a critical and sustained bacterial concentration

in the bloodstream Consequently, the ability of a pathogen to escape the host defenses is crucial for meningeal invasion.

However, high level of bacteremia per se is not sufficient for the

devel-opment of meningitis Bacterial adhesins and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are believed to be centrally involved in binding to blood-CNS barrier cellular receptors or interactions with extracellular matrix proteins [1, 51] Such interactions can then promote attachment to and invasion of BMEC or CP epithelial cells, a prerequisite for bacterial penetration of the blood-CNS barriers

E coli

In extensive studies with E coli K1 and HBMEC, it has been shown that

several microbial determinants contribute to a successful traversal of the BBB (Fig 6)

Fimbrial proteins such as FimH or membrane proteins such as OmpA

mediate attachment to the cerebral endothelium via ligand-receptor

inter-action and contribute to subsequent invasion [67, 135] Other structures such as S-fimbriae, previously shown to facilitate bacterial adhesion, failed

to demonstrate a pivotal role for invasion in ensuing experiments [45, 136,

137] Components of K1 E coli, identified as Ibe proteins, AslA, TraJ, and

cytotoxic necrotizing factor 1 are believed to contribute to HBMEC

inva-sion, even though the exact mechanisms why these E coli determinants are

required for invasion yet remain incompletely understood (summarized in [1])

Several signal transduction pathways, e.g., phosphatidylinositol 3-kinase, focal adhesion kinase, Rho GTPases and others, have been shown to be involved in bacterial invasion of human BMEC, most likely through their effects on actin cytoskeleton rearrangements [53] (Fig 6)

S pneumoniae

Initial attachment of S pneumoniae involves the recognition of host cell

receptor glycoconjugates [138] Subsequently, the bacteria invade BMEC in

part via interaction between pneumococcal surface component

phosphoryl-choline and the BMEC PAF receptor [117] This has been shown by partial inhibition of pneumococcal invasion of BMEC by a PAF receptor antago-nist Phosphorylcholine decoration was found to be up-regulated in pneu-mococci retrieved from CSF samples of experimentally infected rodents [139] Choline-binding protein SpsA mediates pneumococcal adherence to and invasion of mucosal epithelial cells by a human-specific interaction with the polymeric immunoglobulin receptor (pIgR) [140] and might be involved

in crossing the blood-CSF barrier

In addition, the PavA protein, which shows a close relationship to nectin-binding proteins of other streptococcal species, was identified as a pneumococcal adhesin for fibronectin In an experimental mouse menin-gitis model, pneumococcal strains deficient in PavA showed substantially reduced adherence to and internalization of HBMEC [141].

fibro-Pneumolysin, a major virulence factor of S pneumoniae, was shown to

damage endothelial cells and to be an important component for mising the BBB [83] Ependymal cells were shown to be damaged in a rat meningitis model by pneumolysin and hydrogen peroxide [142] Infection

compro-with S pneumoniae led to a loss of ciliae, decrease in their beat frequency

and damage to their ultrastructure [143]

N meningitidis

Several groups had previously reported that encapsulation of N

menin-gitidis impedes interaction with epithelial or endothelial cells preventing

their invasion or transversal [144] It was reasoned that relevant binding sites such as the bacterial outer membrane proteins Opa and Opc proteins were masked by the capsule [145] It has recently been shown in a study with mutants unable to inactivate capsule expression that fully encapsu-lated meningococci are well capable of adhering to HBMEC Invasion of

N meningitidis in HBMEC was mediated by Opc binding to fibronectin,

thus anchoring the bacteria to the _5`1-integrin receptor on human BMEC surface [116]

Invasion of N meningitidis into HBMEC has been shown to involve

c-Jun kinases 1 and 2 (JNK1 and JNK2) as their inhibition significantly

Figure 6 Microbial and host factors that contribute to successful crossing of E coli across brain

microvascular endothelial cells (BMECs) (O-LPS, O-lipopolysaccharide) [1].

reduced meningococcal invasion in HBMEC [93] Another factor essential

for meningeal invasion by N meningitidis seems to be an adhesin located at

the tip of type IV pili, PilC Meningeal invasion of meningococci was ated with an increase in the expression of this adhesin [146]

associ-S agalactiae (Group B streptococci)

Invasion of HBMEC by S agalactiae was shown to require active bacterial

DNA, RNA, and protein synthesis, as well as microfilament and bule elements of the eukaryotic cytoskeleton The streptococcal polysaccha-ride capsule reduced the invasive ability of the organism [119] The bacteria were found inside membrane-bound vacuoles within the cells, suggesting the bacteria might induce their own uptake

microtu-A streptococcal adhesin just recently identified for HBMEC is the fibrinogen-binding protein fbsA, which mediated attachment to the BBB but failed to support invasion of the cells [118] Using microarray systems and knockout bacteria a recent study determined the `-hemolysin of S

agalactiae to be the principal provocative factor for activation of HBMEC

It was found that streptococcal infection induced a highly specific and dinate set of genes known to orchestrate neutrophil recruitment, activation and enhanced survival (e.g., CXC family chemokines IL-8, Gro-_ and `, IL-

coor-6, granulocyte-macrophage colony stimulating factor (GM-CSF), myeloid cell leukemia sequence 1 and intercellular adhesion molecule 1)

The bacterial capsule, in contrast, was believed to rather conceal the pathogen’s surface to diminish host recognition The authors concluded

that the innate immune response of the BBB endothelium to S agalactiae

is to activate circulating neutrophils under modulation by specific bacterial virulence determinants [91]

S suis

In studies using a porcine microvascular endothelial cell line, S suis was

shown to adhere to the cells [49, 97] In addition, intracellular survival and some degree of invasion were observed A cytolysin was noted to be mainly responsible for endothelial damage [49] Besides damaging a cellular barrier

with the help of suilysin, S suis was shown to bind to porcine and human

plasminogens on its surface; this could then be activated into an endogenous plasminogen activator As acquisition of plasmin activity is a mechanism by which invasive bacteria can enhance their capabilities to destroy cell integ-rity this capacity may affect blood-CNS barrier permeability and contribute

to the invasive potential of S suis [147].

Experiments using porcine CP epithelial cells highlighted that S suis is

also able to markedly affect the barrier function and cell integrity of the

CP epithelium [110] Further investigations revealed that the infection with

S suis induced cell death both by apoptosis, indicated by strain-dependent

DNA fragmentation and caspase activation, and by necrosis, shown by the increase of cell membrane permeability and release of nuclear high mobility group box 1 protein [111]

BBB disruption and pleocytosis

After bacteria have accomplished invasion into the CNS, they multiply and induce the release of a multitude of proinflammatory and toxic compounds, leading to the hallmarks of bacterial meningitis, the disintegration of blood-CNS barriers and the infiltration of leukocytes with subsequent pleocytosis.Animal experiments failed to demonstrate a close association between blood-CNS barrier breakdown and CSF pleocytosis [148–150], and clinical observations have shown that either pleocytosis without significant CNS barrier dysfunction [151, 152], apurulent courses of bacterial meningitis [153] or blood-CNS barrier dysfunction in neutropenic patients [154] do occur This has led to the conclusion that initial bacterial entry into the CNS

per se takes place without pleocytosis and blood-CNS barrier breakdown,

and that bacteria can then induce inflammation or other alterations such as pleocytosis or increased BBB permeability [1] Although not in the focus of this review, it is of note that cerebral edema, increased intracranial pressure and altered cerebral blood flow occur in bacterial meningitis, resulting in neuronal injury

Leukocyte recruitment

CSF pleocytosis is a result of leukocyte extravasation from the circulation into the extravascular space after chemotactic attraction [155, 156] It occurs through a tightly controlled multistep process governed by the sequential activation of adhesion receptors and their ligands on both leukocytes and the endothelium [157, 158] The multistep paradigm postulates that four sequential steps (capture, activation, adhesion strengthening, transmigra-tion) are involved in this cascade

The initial capture, the ‘tethering’ of leukocytes as well as subsequent ing are mediated by adhesion molecules such as P-, E-, and L-selectin, and their corresponding carbohydrate ligands Firm adhesion of leukocytes to the endothelium is subsequently mediated by a family of integrins, which have to

roll-be ‘activated’ by a proinflammatory cytokine (e.g., IL-1`), chemokines (e.g., IL-8), complement products, or bacterial cell wall components to reach ade-quate avidity [159] Macrophage antigen 1 (MAC-1; CD11b/CD18) from the

Ig superfamily of adhesion receptors is the predominant integrin involved

in neutrophil binding to their endothelial ligands Intercellular adhesion

molecule (ICAM)-1 exhibits low constitutive levels on the cell surface of the resting endothelium but is markedly induced by exposure to inflammatory stimuli and is the most important endothelial ligand for MAC-1.

In experiments with HBMEC, challenge with S agalactiae led to the

up-regulation of a number of CXC chemokines for recruitment of phils, GM-CSF for bone marrow stimulation of neutrophils, ICAM-1 for adhesion of neutrophils, and Mcl-1 for prevention of neutrophil apoptosis, demonstrating the interconnection between microbial infection and leuko-

neutro-cyte activation [91] Infection of HBMEC with L monocytogenes led to a

significant expression of ICAM-1 [160] as well as HBMEC challenge with

Plasmodium falciparum-infected erythrocytes [161].

Antibodies directed against the adhesion molecules MAC-1 or ICAM-1 profoundly attenuated invasion of neutrophils during experimental menin-gitis and led to significant reductions in intracranial complications such as brain edema formation [162]

An animal model of experimental autoimmune encephalomyelitis (EAE) demonstrates the involvement of the CP in leukocyte recruitment

Using immunohistochemistry and in situ hybridization, expression of

VCAM-1, ICAM-1 and MAdCAM-1 has been observed on the CP lial cells in combination with a complete absence of these structures on the fenestrated endothelium [163]

epithe-Immunological properties of the blood-CNS barrier

Induction of inflammation

Cells of the intracerebral microvasculature and the CP epithelium are, among many other cells of the CNS, capable of expressing several cytokines and other proinflammatory molecules [164] In humans, the classic proin-flammatory cytokines such as TNF-_, IL-1`, and IL-6, as well as a great variety of other cytokines, are present in CSF during meningitis In addition, CXC and CC chemokines have been found in the CSF of these patients [13, 165] Concentrations of IL-1`, but not IL-6 and TNF-_, are associated with significantly worse disease outcome or disease severity [14]

Chemokine production at the blood-CNS-barrier

Numerous observations highlight that the cerebral endothelium is capable of releasing an array of factors for leukocyte attraction In experimental stud-ies it was shown that HBMEC are capable of secreting IL-8 in response to

challenge with S agalactiae [118] After infection of HBMEC with N

menin-gitidis, the endothelial cells were also shown to respond with IL-8 production

with the p38 mitogen-activated (MAP) kinase being centrally involved [93]