In vitro transcriptome analysis of porcine choroid plexus epithelial cells in response to Streptococcus suis: release of pro-inflammatory cytokines and chemokines

The Gram-positive zoonotic bacterium Streptococcus suis (S. suis) is responsible for a wide range of diseases including meningitis in pigs and humans. The blood-cerebrospinal fluid (CSF) barrier is constituted by the epithelial cells of the choroid plexus, which execute barrier function also after bacteria have entered the central nervous system (CNS). We show that the bacterial capsule, a major virulence factor, strongly attenuates adhesion of S. suis to the apical side of porcine choroid plexus epithelial cells (PCPEC). Oligonucleotide microarray analysis and quantitative PCR surprisingly demonstrated that adherent wild-type and capsule-deficient S. suis influenced expression of a pronounced similar pattern of genes in PCPEC. Investigation of purified capsular material provided no evidence for a significant role of the capsule. Enriched among the regulated genes were those involved in “inflammatory response”, “defense response” and “cytokine activity”. These comprised several cytokines and chemokines including the interleukins 6 and 8, which could be detected on protein level. We show that after infection with S. suis the choroid plexus contributes to the immune response by actively producing cytokines and chemokines. Other virulence factors than the bacterial capsule may be relevant in inducing a strong inflammatory response in the CNS during S. suis meningitis.     

Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.     

MMPs contribute to TNF-alpha-induced alteration of the blood-cerebrospinal fluid barrier in vitro

The epithelial cells of the choroid plexus separate the central nervous system from the blood forming the blood-cerebrospinal fluid (CSF) barrier. The choroid plexus is the main source of CSF, whose composition is markedly changed during pathological disorders, for example regarding matrix metalloproteases (MMPs) and tissue inhibitors of matrix metalloproteases (TIMPs). In the present study, we analyzed the impact of the proinflammatory cytokine tumor necrosis factor- (TNF-) on the blood-CSF barrier using an in vitro model based on porcine choroid plexus epithelial cells (PCPEC). TNF- evoked distinct inflammatory processes as shown by mRNA upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. The cytokine caused a drastic decrease in transepithelial electrical resistance within several hours representing an enhanced permeability of PCPEC monolayers. In addition, the distribution of tight junction proteins was altered. Moreover, MMP activity in PCPEC supernatants was significantly increased by TNF-, presumably due to a diminished expression of TIMP-3 that was similarly observed. MMP-2, -3, and -9 as well as TIMP-1 and -2 were also analyzed and found to be differentially regulated by the cytokine. The TNF--induced breakdown of the blood-CSF barrier could partially be blocked by the MMP inhibitor GM-6001. Our results show a contribution of MMPs to the inflammatory breakdown of the blood-CSF barrier in vitro. Thus TNF- may mediate the binding of leukocytes to cellular adhesion molecules and the transmigration across the blood-CSF barrier. choroid plexus; matrix metalloproteases; tight junction; transepithelial electrical resistance; porcine choroid plexus epithelial cells; tumor necrosis factor-     

Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro

Previous experimental studies in a standard Transwell culture system have shown Streptococcus suis ability to compromise barrier function of porcine choroid plexus epithelial cells (PCPEC). The development of an 'inverted' Transwell filter system of PCPEC enables us now for the first time to investigate bacterial invasion and translocation from the physiologically relevant basolateral (blood) to the apical (cerobrospinal fluid) side. Most importantly, we observed specific invasion and translocation of S. suis across the PCPEC exclusively from the basolateral side. During this process, bacterial viability and the presence of a capsule as well as cytoskeletal regulation of PCPEC seemed to play an important role. No loss of barrier function was observed. Bacterial translocation could be significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, but not by its inactive analogue   Ly303511 or dexamethasone. Apotome imaging as well as electron microscopy revealed intracellular bacteria often in cell vacuoles. Thus, possibly regulated by the presence of a capsule, S. suis induces signals that depend on the lipid kinase phosphatidylinositol 3-kinase pathway, which paves the way for cellular uptake during the bacterial transcellular translocation process. Taken together, our data underline the relevance of the blood–cerebrospinal fluid barrier as a gate for bacterial entry into the central nervous system.     

Ultrastructural localization of adhesion molecules in the healthy

The functional expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and MAdCAM-1 in the choroid plexus is indicative of a role of this structure in the communication of the immune system with the central nervous system (CNS). In order to gain further insight into the possible functions of adhesion molecules expressed in the choroid plexus, we investigated the exact ultrastructural localization of VCAM-1, ICAM-1 and MAdCAM-1 on semithin and ultrathin cryosections of the choroid plexus of healthy mice and of mice suffering from experimental autoimmune encephalomyelitis (EAE). In the healthy choroid plexus VCAM-1 and ICAM-1, but not MAdCAM-1, could be detected on the apical surface of the choroid plexus epithelial cells. During EAE, immunoreactivity for VCAM-1 and ICAM-1 was dramatically increased. Additionally, apical expression of MAdCAM-1 was observed on individual choroid plexus epithelial cells during EAE. At the same time, VCAM-1, ICAM-1 or MAdCAM-1 were never present on the endothelial cells of the fenestrated capillaries within the choroid plexus. The polar expression of VCAM-1, ICAM-1 and MAdCAM-1 on the apical surface of choroid plexus epithelial cells, which form the blood-cerebrospinal fluid barrier, implies a previously unappreciated function of this barrier in the immunosurveillance of the CNS.     

Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain

ATP-binding cassette (ABC) transporter A4 is a member of the ABC transporter subfamily A which has been reported to be exclusively expressed in the retina. In contrast, a previous report has suggested a possible relationship between ABCA4 and CNS function. The purpose of the present study was to investigate the localization of ABCA4 mRNA and protein in rat brain. In situ hybridization analysis revealed that ABCA4 mRNA was localized in the lateral ventricles. RT-PCR analysis detected ABCA4 mRNA in isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB). Furthermore, ABCA4 protein was also detected in the isolated rat choroid plexus at about 250 kDa by western blot analysis, and its apparent molecular size was reduced by N-glycosidase F treatment. These results suggest that glycosylated ABCA4 protein is expressed in rat choroid plexus epithelial cells. ABCA4 may play a role in the function of the blood-cerebrospinal fluid barrier and affect CSF conditions.     

Carnosine uptake in rat choroid plexus primary cell cultures and choroid plexus whole tissue from PEPT2 null mice

PEPT2 is functionally active and localized to the apical membrane of rat choroid plexus epithelial cells. However, little is known about the transport mechanisms of endogenous neuropeptides in choroid plexus, and the role of PEPT2 in this process. In the present study, we examined the uptake kinetics of carnosine in rat choroid plexus primary cell cultures and choroid plexus whole tissue from wild-type (PEPT2(+/+)) and null (PEPT2(-/-)) mice. Our results indicate that carnosine is preferentially taken up from the apical as opposed to basolateral membrane of cell monolayers, and that basolateral efflux in limited. Transepithelial flux of carnosine was not distinguishable from that of paracellular diffusion. The apical uptake of carnosine was characterized by a high affinity (K(m) = 34 microM), low capacity (V(max) = 73 pmol/mg protein/min) process, consistent with that of PEPT2. The non-saturable component was small (K(d) = 0.063 microL/mg protein/min) and, under linear conditions, was only 3% of the total uptake. Studies in transgenic mice clearly demonstrated that PEPT2 was responsible for over 90% of carnosine's uptake in choroid plexus whole tissue. These findings elucidate the unique role of PEPT2 in regulating neuropeptide homeostasis at the blood-cerebrospinal fluid interface.     

Physiological concentrations of bile salts inhibit recovery of ischemic-injured porcine ileum

We have previously shown rapid in vitro recovery of barrier function in porcine ischemic-injured ileal mucosa, attributable principally to reductions in paracellular permeability. However, these experiments did not take into account the effects of luminal contents, such as bile salts. Therefore, the objective of this study was to evaluate the role of physiological concentrations of deoxycholic acid in recovery of mucosal barrier function. Porcine ileum was subjected to 45 min of ischemia, after which mucosa was mounted in Ussing chambers and exposed to varying concentrations of deoxycholic acid. The ischemic episode resulted in significant reductions in transepithelial electrical resistance (TER), which recovered to control levels of TER within 120 min, associated with significant reductions in mucosal-to-serosal (3)H-labeled mannitol flux. However, treatment of ischemic-injured tissues with 10(-5) M deoxycholic acid significantly inhibited recovery of TER with significant increases in mucosal-to-serosal (3)H-labeled mannitol flux, whereas 10(-6) M deoxycholic acid had no effect. Histological evaluation at 120 min revealed complete restitution regardless of treatment, indicating that the breakdown in barrier function was due to changes in paracellular permeability. Similar effects were noted with the application of 10(-5) M taurodeoxycholic acid, and the effects of deoxycholic acid were reversed with application of the Ca(2+)-mobilizing agent thapsigargin. Deoxycholic acid at physiological concentrations significantly impairs recovery of epithelial barrier function by an effect on paracellular pathways, and these effects appear to be Ca(2+) dependent.     

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier

Unidirectional flux of 125I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of 125I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternal perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides.     

SILVER DEPOSITION IN THE CENTRAL NERVOUS SYSTEM AND THE HEMATOENCEPHALIC BARRIER STUDIED WITH THE ELECTRON MICROSCOPE

For the purpose of studying the hematoencephalic barrier as it is concerned with silver circulating in the blood stream, silver nitrate was vitally administered to rats in their drinking water over periods of 6 to 8 months. The cerebrum, cerebellum, medulla, area postrema, and choroid plexus were prepared for light and electron microscopy. Silver deposition was found in the perivascular spaces in the choroid plexus, area postrema, in the medulla surrounding the area postrema, and in minute quantities in the cerebrum, cerebellum, and most of the medulla. Two levels of the hematoencephalic barrier were apparently demonstrated in our investigations. The endothelial linings of the vessels in the cerebrum, cerebellum, and medulla constitute the first threshold of the hematoencephalic barrier (specifically here, blood-brain barrier). The cell membranes adjacent to the perivascular spaces form the second threshold, as follows:—the neuroglial cell membranes in the cerebrum, cerebellum, and medulla (blood-brain barrier); the membranes of the neuroglial cells in the area postrema (blood-brain barrier); and the membranes of the epithelial cells of the choroid plexus (blood-cerebrospinal fluid barrier). This study deals with silver deposition and does not infer that the penetration of ionic silver, if present in the blood stream, would necessarily be limited to the regions described. Bleb-like structures were observed to cover the epithelial cell surfaces in the choroid plexus. They may be cellular projections increasing the cell surface area or they may be secretory droplets.     

Silver deposition in the central nervous system and the hematoencephalic barrier studied with the electron microscope

For the purpose of studying the hematoencephalic barrier as it is concerned with silver circulating in the blood stream, silver nitrate was vitally administered to rats in their drinking water over periods of 6 to 8 months. The cerebrum, cerebellum, medulla, area postrema, and choroid plexus were prepared for light and electron microscopy. Silver deposition was found in the perivascular spaces in the choroid plexus, area postrema, in the medulla surrounding the area postrema, and in minute quantities in the cerebrum, cerebellum, and most of the medulla. Two levels of the hematoencephalic barrier were apparently demonstrated in our investigations. The endothelial linings of the vessels in the cerebrum, cerebellum, and medulla constitute the first threshold of the hematoencephalic barrier (specifically here, blood-brain barrier). The cell membranes adjacent to the perivascular spaces form the second threshold, as follows:-the neuroglial cell membranes in the cerebrum, cerebellum, and medulla (blood-brain barrier); the membranes of the neuroglial cells in the area postrema (blood-brain barrier); and the membranes of the epithelial cells of the choroid plexus (blood-cerebrospinal fluid barrier). This study deals with silver deposition and does not infer that the penetration of ionic silver, if present in the blood stream, would necessarily be limited to the regions described. Bleb-like structures were observed to cover the epithelial cell surfaces in the choroid plexus. They may be cellular projections increasing the cell surface area or they may be secretory droplets.     

Deoxycytidine transport and metabolism in choroid plexus

In vitro, the transport into and release of [3H]deoxycytidine from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, were studied separately. By use of the ability of nitrobenzylthioinosine (NBTI) to inhibit deoxycytidine efflux from choroid plexus, the transport of 1 microM [3H]deoxycytidine into choroid plexus at 37 degrees C was measured. Deoxycytidine was transported into choroid plexus against a concentration gradient by a saturable process that depended on intracellular energy production, but not intracellular binding or metabolism. The Michaelis-Menten constant (KT) for the active transport of deoxycytidine into choroid plexus was 15 microM. The active transport system for deoxycytidine was inhibited by naturally occurring nucleosides and deoxynucleosides, but not by 1 mM probenecid and 2-deoxyribose or 100 microM cytosine and cytosine arabinoside. With less than 1 microM [3H]deoxycytidine in the medium, the choroid plexus accumulated [3H]deoxycytidine against a concentration gradient. However, approximately 50% of the [3H]deoxycytidine was phosphorylated to [3H]deoxycytidine nucleotides at a low extracellular [3H]deoxycytidine concentration (6 nM) in 15-min incubations. This accumulation process depended, in part, on saturable intracellular phosphorylation. These studies provide further evidence that the choroid plexus contains an active nucleoside transport system of low specificity for deoxynucleosides and ribonucleosides, and a separate, saturable efflux system for deoxynucleosides which is very sensitive to inhibition by NBTI.     

Differential effects of claudin-3 and claudin-4 on alveolar epithelial barrier function

Alveolar barrier function depends critically on the claudin family tight junction proteins. Of the major claudins expressed by alveolar epithelial cells, claudin (Cldn)-3 and Cldn-4 are the most closely related by amino acid homology, yet they differ dramatically in the pattern of expression. Previously published reports have shown that Cldn-3 is predominantly expressed by type II alveolar epithelial cells; Cldn-4 is expressed throughout the alveolar epithelium and is specifically upregulated in response to acute lung injury. Using primary rat alveolar epithelial cells transduced with yellow fluorescent protein-tagged claudin constructs, we have identified roles for Cldn-3 and Cldn-4 in alveolar epithelial barrier function. Surprisingly, increasing expression of Cldn-3 decreased alveolar epithelial barrier function, as assessed by transepithelial resistance and dye flux measurements. Conversely, increasing Cldn-4 expression improved alveolar epithelial transepithelial resistance compared with control cells. Other alveolar epithelial tight junction proteins were largely unaffected by increased expression of Cldn-3 and Cldn-4. Taken together, these results demonstrate that, in the context of the alveolar epithelium, Cldn-3 and Cldn-4 have different effects on paracellular permeability, despite significant homology in their extracellular loop domains.     

Morphological Ground of Formation of the Brain Choroid Plexus Blood-Cerebrospinal Fluid Barrier in Human Prenatal Ontogenesis

Using light and electron microscopy and immunocytochemistry methods, structural organization of the formed blood-cerebrospinal fluid barrier (BCSFB) of the human brain choroid plexus in embryos of 6–9 weeks of development was studied. The main structures peculiar to the mature BCSFB have been established to appear with formation of the choroid plexus at the end of the 2nd month of the human intrauterine development. Fenestrae in the choroid plexus capillary endothelium are revealed since the 9th week of prenatal development. Characteristic of the human embryonic BCSFB are a poor development of the plexus capillary basal membrane, scanty pericytes, a high activity of interstitial macrophages, which suggests the barrier immaturity. A significant amount of cytoplasmic glycogen inclusions revealed in plexus epitheliocytes seems to be due to peculiar trophic requirements of developing brain cells under conditions of an insufficient development of the local blood supply.     

Evaluation of the value of determining lysozyme levels in cerebrospinal fluid in myeloencephalitis

The study aimed at assessing the value lysozyme assay in CFS as indicator of the damage to blood-cerebrospinal fluid barrier and intensification of inflammatory process in the course of meningitis. The study involved 20 patients with suppurative and 66 with viral meningitis. Control group included 26 patients without nervous system disease. To estimate the degree of blood-cerebrospinal fluid barrier damage albumin and lysozyme indicators were calculated. It was proved, that CSF lysozyme levels are bigger in the suppurative meningitis than in viral meningitis. According to the author, CSF lysozyme levels the value of lysozyme indicator may inform on intensification of the inflammatory process and the degree of blood-cerebrospinal fluid barrier damage in suppurative meningitis, whereas in the viral meningitis they inform on degree of blood-cerebrospinal fluid barrier damage, only.     

Mitogen-activated protein kinases are required for effective infection of human choroid plexus epithelial cells by Listeria monocytogenes

Listeria monocytogenes, a Gram-positive bacterium, can cause meningitis after invading the human central nervous system. The blood-cerebrospinal fluid barrier (BCSFB), located at the epithelium of the choroid plexus, is a possible entry site for L. monocytogenes into the brain, and in vitro L. monocytogenes invades human choroid plexus epithelial papilloma (HIBCPP) cells. Although host cell signal transduction subsequent to infection by L. monocytogenes has been investigated, the role of mitogen-activated protein kinases (MAPK) is not clarified yet. We show that infection with L. monocytogenes causes activation of the MAPKs Erk1/2 and p38 preferentially when bacteria are added to the physiologically more relevant basolateral side of HIBCPP cells. Deletion of the listerial virulence factors Internalin (InlA) and InlB reduces MAPK activation. Whereas inhibition of either Erk1/2 or p38 signaling significantly attenuates infection of HIBCPP cells with L. monocytogenes, simultaneous inhibition of both MAPK pathways shows an additive effect, and Erk1/2 and p38 are involved in regulation of cytokine and chemokine expression following infection. Blocking of endocytosis with the synthetic dynamin inhibitor dynasore strongly abrogates infection of HIBCPP cells with L. monocytogenes. Concurrent inhibition of MAPK signaling further reduces infection, suggesting MAPKs mediate infection with L. monocytogenes during inhibition of dynamin-mediated endocytosis.     

Expression and possible role of creatine transporter in the brain and at the blood-cerebrospinal fluid barrier as a transporting protein of guanidinoacetate, an endogenous convulsant

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a K(m) value of 269 microM/412 microM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of d-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.