Tumor necrosis factor-alpha increases lactoferrin transcytosis through the blood-brain barrier

Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood-brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor-alpha (TNF-alpha). As TNF-alpha can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF-alpha, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune-mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up-regulation of the Lf receptor but rather an increase in the rate of transendothelial transport.     

Tyrphostin A8 stimulates a novel trafficking pathway of apically endocytosed transferrin through Rab11-enriched compartments in Caco-2 cells

The potential application of transferrin receptors as delivery vehicles for transport of macromolecular drugs across intestinal epithelial cells is limited by several factors, including the low level of transferrin receptor-mediated transcytosis, particularly in the apical-to-basolateral direction. The GTPase inhibitor, AG10 (tyrphostin A8), has been shown previously to increase the apical-to-basolateral transcytosis of transferrin in Caco-2 cells. However, the mechanism of the increased transcytosis has not been established. In this report, the effect of AG10 on the trafficking of endocytosed transferrin among different endosomal compartments as well as the involvement of Rab11 in the intracellular trafficking of transferrin was investigated. Confocal microscopy studies showed a high level of colocalization of FITC-transferrin with Rab5 and Rab11 in Caco-2 cells pulsed at 16 degrees C and 37 degrees C, which indicated the presence of apically endocytosed FITC-transferrin in early endosomes and apical recycling endosomes at 16 degrees C and 37 degrees C, respectively. The effect of AG10 on the accumulation of transferrin within different endosomal compartment was studied, and an increase in the transcytosis and recycling of internalized (125)I-labeled transferrin, as well as a decrease in cell-associated (125)I-labeled transferrin, was observed in AG10-treated Caco-2 cells pulsed at 37 degrees C for 30 min and chased for 30 min. Moreover, confocal microscopy showed that FITC-transferrin exhibited an increased level of colocalization with Rab11, but not with Rab5, in the presence of AG10. These results suggest an effect of AG10 on the later steps of transferrin receptor trafficking, which are involved in subsequent recycling, and possibly transcytosis, of endocytosed transferrin in Caco-2 cells.     

Transferrin and Transferrin Receptor Function in Brain Barrier Systems

1. Iron (Fe) is an essential component of virtually all types of cells and organisms. In plasma and interstitial fluids, Fe is carried by transferrin. Iron-containing transferrin has a high affinity for the transferrin receptor, which is present on all cells with a requirement for Fe. The degree of expression of transferrin receptors on most types of cells is determined by the level of Fe supply and their rate of proliferation.2. The brain, like other organs, requires Fe for metabolic processes and suffers from disturbed function when a Fe deficiency or excess occurs. Hence, the transport of Fe across brain barrier systems must be regulated. The interaction between transferrin and transferrin receptor appears to serve this function in the blood–brain, blood–CSF, and cellular–plasmalemma barriers. Transferrin is present in blood plasma and brain extracellular fluids, and the transferrin receptor is present on brain capillary endothelial cells, choroid plexus epithelial cells, neurons, and probably also glial cells.3. The rate of Fe transport from plasma to brain is developmentally regulated, peaking in the first few weeks of postnatal life in the rat, after which it decreases rapidly to low values. Two mechanisms for Fe transport across the blood–brain barrier have been proposed. One is that the Fe–transferrin complex is transported intact across the capillary wall by receptor-mediated transcytosis. In the second, Fe transport is the result of receptor-mediated endocytosis of Fe–transferrin by capillary endothelial cells, followed by release of Fe from transferrin within the cell, recycling of transferrin to the blood, and transport of Fe into the brain. Current evidence indicates that although some transcytosis of transferrin does occur, the amount is quantitatively insufficient to account for the rate of Fe transport, and the majority of Fe transport probably occurs by the second of the above mechanisms.4. An additional route of Fe and transferrin transport from the blood to the brain is via the blood–CSF barrier and from the CSF into the brain. Iron-containing transferrin is transported through the blood–CSF barrier by a mechanism that appears to be regulated by developmental stage and iron status. The transfer of transferrin from blood to CSF is higher than that of albumin, which may be due to the presence of transferrin receptors on choroid plexus epithelial cells so that transferrin can be transported across the cells by a receptor-mediated process as well as by nonselective mechanisms.5. Transferrin receptors have been detected in neurons in vivo and in cultured glial cells. Transferrin is present in the brain interstitial fluid, and it is generally assumed that Fe which transverses the blood–brain barrier is rapidly bound by brain transferrin and can then be taken up by receptor-mediated endocytosis in brain cells. The uptake of transferrin-bound Fe by neurons and glial cells is probably regulated by the number of transferrin receptors present on cells, which changes during development and in conditions with an altered iron status.6. This review focuses on the information available on the functions of transferrin and transferrin receptor with respect to Fe transport across the blood–brain and blood–CSF barriers and the cell membranes of neurons and glial cells.     

Bidirectional transcytosis determines the steady state distribution of the transferrin receptor at opposite plasma membrane domains of BeWo cells

Trophoblast-like BeWo cells form well-polarized epithelial monolayers, when cultured on permeable supports. Contrary to other polarized cell systems, in which the transferrin receptor is found predominantly on the basolateral cell surface, BeWo cells express the transferrin receptor at both apical and basolateral cell surfaces (Cerneus, D.P., and A. van der Ende. 1991. J. Cell Biol. 114: 1149-1158). In the present study we have addressed the question whether BeWo cells use a different sorting mechanism to target transferrin receptors to the cell surface, by examining the biosynthetic and transcytotic pathways of the transferrin receptor in BeWo cells. Using trypsin and antibodies to detect transferrin receptors at the cell surface of filter-grown BeWo cells, we show that at least 80% of newly synthesized transferrin receptor follows a direct pathway to the basolateral surface, demonstrating that the transferrin receptor is efficiently intracellularly sorted. After surface arrival, pulse-labeled transferrin receptor equilibrates between apical and basolateral cell surfaces, due to ongoing transcytotic transport in both directions. The subsequent redistribution takes over 120 min and results in a steady state distribution with 1.5-2.0 times more transferrin receptors at the basolateral surface than at the apical surface. By monitoring the fate of surface-bound 125I-transferrin, internalized either from the apical or basolateral surface transcytosis of the transferrin receptor was studied. About 15% of 125I-transferrin is transcytosed in the basolateral to apical direction, whereas 25% is transcytosed in the opposite direction, indicated that the fraction of receptors involved in transcytosis is roughly twofold higher for the apical receptor pool, as compared to the basolateral pool. Upon internalization, both apical and basolateral receptor pools become redistributed on both surfaces, resulting in a twofold higher number of transferrin receptors at the basolateral surface. Our results indicate that in BeWo cells bidirectional transcytosis is the main factor in surface distribution of transferrin receptors on apical and basolateral surfaces, which may represent a cell type-specific, post-endocytic, sorting mechanism.     

Involvement of OCTN2 and B0,+ in the transport of carnitine through an in vitro model of the blood-brain barrier

Carnitine is known to accumulate in brain, therefore transport of carnitine through the blood-brain barrier was studied in an in vitro system using bovine brain capillary endothelial cells (BBCEC) grown on filter inserts in a co-culture system with glial cells. Long-term exposure of BBCEC to carnitine resulted in a high accumulation of long-chain acyl carnitines, which decreased dramatically upon removal of carnitine. Kinetic analysis of carnitine accumulation indicated a possibility of functioning of more than one transporter. BBCEC were incubated in the presence of substrates and inhibitors of known carnitine transporters added from either apical or basolateral side. Inhibition by replacement of sodium and expression of OCTN2 (RT-PCR) were in agreement with earlier reports on the functioning of OCTN2 in apical membrane. For the first time, functioning of OCTN2 was demonstrated in the basolateral membrane, as well as functioning in both membranes of a low affinity carnitine transporter B(0,+). Expression of B(0,+) in BBCEC was confirmed by RT-PCR. These results suggest that OCTN2 and B(0,+) could be involved in carnitine transport in both the apical and basolateral membrane.     

The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor-mediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K+ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-beta-cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, alpha(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes alpha(2,3)-, but not with Sambucus nigra agglutinin that recognizes alpha(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal alpha(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.     

A Human Blood-Brain Barrier Transcytosis Assay Reveals Antibody Transcytosis Influenced by pH-Dependent Receptor Binding

We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3), to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R) was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR) was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation.     

Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2

The blood–brain barrier (BBB) restricts the entry of proteins as well as potential drugs to cerebral tissues. We previously reported that a family of Kunitz domain-derived peptides called Angiopeps can be used as a drug delivery system for the brain. Here, we further characterize the transcytosis ability of these peptides using an in vitro model of the BBB and in situ brain perfusion. These peptides, and in particular Angiopep-2, exhibited higher transcytosis capacity and parenchymal accumulation than do transferrin, lactoferrin, and avidin. Angiopep-2 transport and accumulation in brain endothelial cells were unaffected by the P-glycoprotein inhibitor, cyclosporin A, indicating that this peptide is not a substrate for the efflux pump P-glycoprotein. However, competition studies show that activated α₂-macroglobulin, a specific ligand for the low-density lipoprotein receptor-related protein-1 (LRP1) and Angiopep-2 can share the same receptor. In addition, LRP1 was detected in glioblastomas and brain metastases from lung and skin cancers. Fluorescent microscopy also revealed that Alexa488-Angiopep-2 co-localized with LRP1 in brain endothelial cell monolayers. Overall, these results suggest that Angiopep-2 transport across the BBB is, in part, mediated by LRP1.     

Genetically engineered brain drug delivery vectors: cloning, expression and in vivo application of an anti-transferrin receptor single chain antibody-streptavidin fusion gene and protein.

A single chain Fv antibody-streptavidin fusion protein was expressed and purified from bacterial inclusion bodies following cloning of the genes encoding the variable region of the heavy chain and light chain of the murine OX26 monoclonal antibody to the rat transferrin receptor. The latter undergoes receptor mediated transcytosis through the brain capillary endothelial wall in vivo, which makes up the blood-brain barrier (BBB); therefore, the OX26 monoclonal antibody and its single chain Fv analog may act as brain drug delivery vectors in vivo. Attachment of biotinylated drugs to the antibody vector is facilitated by production of the streptavidin fusion protein. The bi-functionality of the OX26 single chain Fv antibody-streptavidin fusion protein was retained, as the product both bound biotin and the rat transferrin receptor in vitro and in vivo, based on pharmacokinetic and brain uptake analyses in anesthetized rats. The attachment of biotin-polyethyleneglycol-fluorescein to the OX26 single chain Fv antibody-streptavidin fusion protein resulted in illumination of isolated rat brain capillaries in confocal fluorescent microscopy. In conclusion, these studies demonstrate that genetically engineered single chain Fv antibody-streptavidin fusion proteins may be used for non-invasive neurotherapeutic delivery to the brain using endogenous BBB transport systems such as the transferrin receptor.     

Brain uptake of multivalent and multi-specific DVD-Ig proteins after systemic administration

Therapeutic monoclonal antibodies and endogenous IgG antibodies show limited uptake into the central nervous system (CNS) due to the blood-brain barrier (BBB), which regulates and controls the selective and specific transport of both exogenous and endogenous materials to the brain. The use of natural transport mechanisms, such as receptor-mediated transcytosis (RMT), to deliver antibody therapeutics into the brain have been studied in rodents and monkeys. Recent successful examples include monovalent bispecific antibodies and mono- or bivalent fusion proteins; however, these formats do not have the capability to bind to both the CNS target and the BBB transport receptor in a bivalent fashion as a canonical antibody would. Dual-variable-domain immunoglobulin (DVD-Ig) proteins offer a bispecific format where monoclonal antibody-like bivalency to both the BBB receptor and the therapeutic target is preserved, enabling independent engineering of binding affinity, potency, valency, epitope and conformation, essential for successful generation of clinical candidates for CNS applications with desired drug-like properties. Each of these parameters can affect the binding and transcytosis ability mediated by different receptors on the brain endothelium differentially, allowing exploration of diverse properties. Here, we describe generation and characterization of several different DVD-Ig proteins, specific for four different CNS targets, capable of crossing the BBB through transcytosis mediated by the transferrin receptor 1 (TfR1). After systemic administration of each DVD-Ig, we used two independent methods in parallel to observe specific uptake into the brain. An electrochemiluminescent-based sensitive quantitative assay and a semi-quantitative immunohistochemistry technique were used for brain concentration determination and biodistribution/localization in brain, respectively. Significantly enhanced brain uptake and retention was observed for all TfR1 DVD-Ig proteins regardless of the CNS target or the systemic administration route selected.     

Rapid transferrin efflux from brain to blood across the blood-brain barrier

The brain efflux index method is used to examine the extent to which transferrin effluxes from brain to blood across the blood-brain barrier (BBB) following intracerebral injection. Whereas high-molecular-weight dextran is nearly 100% retained in brain for up to 90 min after intracerebral injection in the Par2 region of the parietal cortex of brain, there is rapid efflux of transferrin from brain to blood across the BBB. The efflux of apotransferrin is 3.5-fold faster than the efflux of holo-transferrin. The brain to blood efflux of apotransferrin is completely saturable by unlabeled transferrin, but is not inhibited by other plasma proteins. These studies provide evidence for reverse transcytosis of transferrin from brain to blood across the BBB. As circulating transferrin is known to undergo transcytosis across the BBB in the blood-to-brain direction, these studies support the model of bidirectional transcytosis of transferrin through the BBB in vivo.     

Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat

Anti-transferrin receptor IgG2a (OX26) transport into the brain was studied in rats. Uptake of OX26 in brain capillary endothelial cells (BCECs) was > 10-fold higher than isotypic, non-immune IgG2a (Ni-IgG2a) when expressed as % ID/g. Accumulation of OX26 in the brain was higher in 15 postnatal (P)-day-old rats than in P0 and adult (P70) rats. Iron-deficiency did not increase OX26 uptake in P15 rats. Three attempts were made to investigate transport from BCECs further into the brain. (i) Using a brain capillary depletion technique, 6-9% of OX26 was identified in the post-capillary compartment consisting of brain parenchyma minus BCECs. (ii) In cisternal CSF, the volume of distribution of OX26 was higher than for Ni-IgG2a when corrected for plasma concentration. (iii) Immunohistochemical mapping revealed the presence of OX26 almost exclusively in BCECs; extravascular staining was observed only in neurons situated periventricularly. The data support the hypothesis of facilitated uptake of OX26 due to the presence of transferrin receptors at the blood-brain barrier (BBB). However, OX26 accumulation in the post-capillary compartment was too small to justify a conclusion of receptor-mediated transcytosis of OX26 occurring in BCECs. Accumulation of OX26 in the post-capillary component may result from a diphasic transport that involves high-affinity accumulation of OX26 by the BCECs, clearly exceeding that of Ni-IgG2a, followed by a second transport mechanism that releases OX26 non-specifically further into the brain. The periventricular localization suggests that OX26 probably also derives from transport across the blood-CSF barrier.     

Brain iron homeostasis.

The anatomical and cellular distribution of non-haem iron, ferritin, transferrin, and the transferrin receptor have been studied in postmortem human brain and these studies, together with data on the uptake and transport of labeled iron, by the rat brain, have been used to elucidate the role of iron and other metal ions in certain neurological disorders. High levels of non-haem iron, mainly in the form of ferritin, are found in the extrapyramidal system, associated predominantly with glial cells. In contrast to non-haem iron, the density of transferrin receptors is highest in cortical and brainstem structures and appears to relate to the iron requirement of neurones for mitochondrial respiratory activity. Transferrin is synthesized within the brain by oligodendrocytes and the choroid plexus, and is present in neurones, consistent with receptor mediated uptake. The uptake of iron into the brain appears to be by a two-stage process involving initial deposition of iron in the brain capillary endothelium by serum transferrin, and subsequent transfer of iron to brain-derived transferrin and transport within the brain to sites with a high transferrin receptor density. A second, as yet unidentified mechanism, may be involved in the transfer of iron from neurones possessing transferrin receptors to sites of storage in glial cells in the extrapyramidal system. The distribution of iron and the transferrin receptor may be of relevance to iron-induced free radical formation and selective neuronal vulnerability in neurodegenerative disorders.     

Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers.

The effect of albumin binding to cultured bovine pulmonary artery endothelial cell (BPAEC) monolayers on the transendothelial flux of 125I-labelled bovine serum albumin (BSA) was examined to determine its possible role on albumin transcytosis. The transport of 125I-BSA tracer across BPAEC grown on gelatin- and fibronectin-coated filters (0.8 microns pore diam.) was affected by the presence of unlabelled BSA in the medium in that transendothelial 125I-BSA permeability decreased, reaching a 40% reduction at BSA concentrations equal to or greater than 5 mg/ml. BSA binding to BPAEC monolayers was saturated at concentration of 10 mg/ml with an apparent binding affinity of 6 x 10(-7) M. In contrast, gelatin added to the medium altered neither 125I-BSA binding nor transport. Several lectins were tested for their ability to inhibit 125I-BSA binding and transport. One lectin, Ricinus communis (RCA), reduced 125I-BSA binding by 70% and transport by 40%. Other lectins, Ulex europaeus, Triticum vulgare, and Glycine max decreased neither 125I-BSA binding nor transport. The reduction of 125I-BSA transport by RCA was not observed in the presence of saturating levels of BSA, indicating that RCA influenced only the albumin-dependent component of transport. RCA, but not other lectins, precipitated a 60 kDa plasmalemmal glycoprotein from cell lysates of surface radioiodinated BPAEC monolayers. This 60 kDa glycoprotein appears to be the equivalent of gp60 identified previously as an albumin binding glycoprotein in rat microvascular endothelium. In summary, approximately 40% of albumin transport across BPAEC monolayers is dependent on albumin binding. This component of albumin transport is inhibited by 80% by the binding of RCA to gp60. These results suggest that binding of albumin to gp60 on pulmonary artery endothelial cell membrane is a critical determinant of transendothelial albumin flux involving mechanisms such as plasmalemmal vesicular transcytosis.     

Transport of arylsulfatase A across the blood-brain barrier in vitro

Enzyme replacement therapy is an option to treat lysosomal storage diseases caused by functional deficiencies of lysosomal hydrolases as intravenous injection of therapeutic enzymes can correct the catabolic defect within many organ systems. However, beneficial effects on central nervous system manifestations are very limited because the blood-brain barrier (BBB) prevents the transfer of enzyme from the circulation to the brain parenchyma. Preclinical studies in mouse models of metachromatic leukodystrophy, however, showed that arylsulfatase A (ASA) is able to cross the BBB to some extent, thus reducing lysosomal storage in brain microglial cells. The present study aims to investigate the routing of ASA across the BBB and to improve the transfer in vitro using a well established cell culture model consisting of primary porcine brain capillary endothelial cells cultured on Transwell filter inserts. Passive apical-to-basolateral ASA transfer was observed, which was not saturable up to high ASA concentrations. No active transport could be determined. The passive transendothelial transfer was, however, charge-dependent as reduced concentrations of negatively charged monosaccharides in the N-glycans of ASA or the addition of polycations increased basolateral ASA levels. Adsorptive transcytosis is therefore considered to be the major transport pathway. Partial inhibition of the transcellular ASA transfer by mannose 6-phosphate indicated a second route depending on the insulin-like growth factor II/mannose 6-phosphate receptor, MPR300. We conclude that cationization of ASA and an increase of the mannose 6-phosphate content of the enzyme may promote blood-to-brain transfer of ASA, thus leading to an improved therapeutic efficacy of enzyme replacement therapy behind the BBB.     

Functional and morphological studies of protein transcytosis in continuous endothelia

Continuous microvascular endothelium constitutively transfers protein from vessel lumen to interstitial space. Compelling recent biochemical, ultrastructural, and physiological evidence reviewed herein demonstrates that protein transport is not the result of barrier "leakiness" but, rather, is an active process occurring primarily in a transendothelial vesicular pathway. Protein accesses the vesicular pathway by means of caveolae open to the vessel lumen. Vascular tracer proteins appear in free cytoplasmic vesicles within minutes; contents of transport vesicles are rapidly deposited into the subendothelial matrix by exocytosis. Caveolin-1 deficiency eliminates caveolae and abolishes vesicular protein transport; interestingly, exchange vessels develop a compensatory transport mode through the opening of a paracellular permeability pathway. The evidence supports the transcytosis hypothesis and the concept that transcytosis is a fundamental component of transendothelial permeability of macromolecules.     

Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Brain capillary endothelial cells mediate iron transport into the brain by segregating iron from transferrin without the involvement of divalent metal transporter 1

Rats were studied for [(59)Fe-(125)I]transferrin uptake in total brain, and fractions containing brain capillary endothelial cells (BCECs) or neurons and glia. (59)Fe was transported through BCECs, whereas evidence of similar transport of transferrin was questionable. Intravenously injected transferrin localized to BCECs and failed to accumulate within neurons, except near the ventricles. No significant difference in [(125)I]transferrin distribution was observed between Belgrade b/b rats with a mutation in divalent metal transporter I (DMT1), and Belgrade +/b rats with regard to accumulation in vascular and postvascular compartments. (59)Fe occurred in significantly lower amounts in the postvascular compartment in Belgrade b/b rats, indicating impaired iron uptake by transferrin receptor and DMT1-expressing neurons. Immunoprecipitation with transferrin antibodies on brains from Belgrade rats revealed lower uptake of transferrin-bound (59)Fe. In postnatal (P)0 rats, less (59)Fe was transported into the postvascular compartment than at later ages, suggesting that BCECs accumulate iron at P0. Supporting this notion, an in situ perfusion technique revealed that BCECs accumulated ferrous and ferric iron only at P0. However, BCECs at P0 together with those of older age lacked DMT1. In conclusion, BCECs probably mediate iron transport into the brain by segregating iron from transferrin without involvement of DMT1.     

Comparison of FcRn‐ and pIgR‐Mediated Transport in MDCK Cells by Fluorescence Confocal Microscopy

Protein delivery across polarized epithelia is controlled by receptor‐mediated transcytosis. Many studies have examined basolateral‐to‐apical trafficking of polymeric IgA (pIgA) by the polymeric immunoglobulin receptor (pIgR). Less is known about apical‐to‐basolateral transcytosis, the direction the neonatal Fc receptor (FcRn) transports maternal IgGs across intestinal epithelia. To compare apical‐to‐basolateral and basolateral‐to‐apical transcytosis, we co‐expressed FcRn and pIgR in Madin‐Darby canine kidney (MDCK) cells and used pulse‐chase experiments with confocal microscopy to examine transport of apically applied IgG Fcγ and basolaterally applied pIgA. Fcγ and pIgA trafficking routes were initially separate but intermixed at later chase times. Fcγ was first localized near the apical surface, but became more equally distributed across the cell, consistent with concomitant transcytosis and recycling. By contrast, pIgA transport was strongly unidirectional: pIgA shifted from near the basolateral surface to an apical location with increasing time. Some Fcγ and pIgA fluorescence colocalized in early (EEA1‐positive), recycling (Rab11a‐positive), and transferrin (Tf)‐positive common/basolateral recycling endosomes. Fcγ became more enriched in Tf‐positive endosomes with time, whereas pIgA was sorted from these compartments. Live‐cell imaging revealed that vesicles containing Fcγ or pIgA shared similar mobility characteristics and were equivalently affected by depolymerizing microtubules, indicating that both trafficking routes depended to roughly the same extent on intact microtubules.     

A New Function for the LDL Receptor: Transcytosis of LDL across the Blood–Brain Barrier

Lipoprotein transport across the blood–brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.The maintenance of the homeostasis of brain interstitial fluid, which constitutes the special microenvironment for neurons, is established by the presence of the blood–brain barrier (BBB)¹ at the transition area from endothelial cells (ECs) to brain tissue. Of primary importance in the formation of a permeability barrier by these cells is the presence of continuous tight junctions that seal together the margins of the ECs and restrict the passage of substances from the blood to the brain. Furthermore, in contrast to ECs in many other organs, the brain capillary ECs contain no direct transendothelial passageways such as fenestrations or channels. But obviously, the BBB cannot be absolute. The brain is dependent upon the blood to deliver metabolic substrates and remove metabolic waste, and the BBB therefore facilitates the exchange of selected solutes. Carrier-mediated transport systems that facilitate the uptake of hexoses, amino acids, purine compounds, and mono-carboxylic acids have been revealed in the cerebral endothelium (Betz and Goldstein, 1978), but until now little information has come to light regarding the cerebral uptake of lipids.There is growing evidence that the brain is equipped with a relatively self-sufficient transport system for maintaining cholesterol and lipid homeostasis. The presence of a low density lipoprotein (LDL) receptor has been demonstrated by immunocytochemistry in rat and monkey brains; and apolipoprotein (apo) E and apo AI-containing particles have been detected in human cerebrospinal fluid (Pitas et al., 1987). Furthermore, enzymes involved in lipid metabolism have been located within the brain: LCAT mRNA has been shown to be expressed in rat brains and cholesteryl ester transfer protein, which plays a key role in cholesterol homeostasis, has been detected in human cerebrospinal fluid and seems to be synthesized in the brain (Albers et al., 1992). The distribution of the LDL receptor-related protein, a multifunctional receptor that binds apoE, is highly restricted and limited to the gray matter, primarily associated with neuronal cell population (Wolf et al., 1992). The difference in cellular expression of ligand (apoE) and receptor (LDL receptor-related protein) may provide a pathway for intracellular transport of apoE-containing lipoproteins in the central nervous system. All these data leave little doubt that the brain is equipped with a relatively self-sufficient transport system for cholesterol.Cholesterol could be derived from de novo synthesis within the brain and from plasma via the BBB. Malavolti et al. (1991) indicate the presence of unexpectedly close communications between extracerebral and brain cholesterol. Changes in the extracerebral cholesterol levels are readily sensed by the LDL receptor in the brain and promptly provoke appropriate modifications in its activity. Méresse et al. (1989a) provided direct evidence for the occurrence in vivo of an LDL receptor on the endothelium of brain capillaries. Furthermore, the fact that enzymes involved in the lipoprotein metabolism are present in the brain microvasculature (Brecher and Kuan, 1979) and that the entire fraction of the drug bound to lipoproteins is available for entry into the brain strongly suggest that this cerebral endothelial receptor plays a role in the interaction of plasma lipoproteins with brain capillaries. These results pinpoint the critical importance of the interactions between brain capillary ECs and lipoproteins. Owing to the fact that the neurological abnormalities that result from the inadequate absorption of dietary vitamin E can be improved by the oral administration of pharmacological doses of vitamin E, Traber and Kayden (1984) have suggested that LDL functions as a transport system for tocopherol to the brain. Furthermore, the trace amounts of apolipoprotein B that were detected by Salem et al. (1987) in cerebrospinal fluid from healthy patients using a very sensitive immunoblot technique confirm that, at most, small amounts of apolipoprotein B normally pass through the BBB. However, whether LDL is involved in the exchange is not known.Using an in vitro model of the BBB that imitates an in vivo situation by culturing capillary ECs and astrocytes on opposite sides of a filter (Dehouck et al., 1990a , 1992), we have demonstrated that in culture, like in vivo, in contrast to peripheral endothelium and in spite of the tight apposition of ECs and their contact with physiological concentrations of lipoproteins, brain capillary ECs express an LDL receptor (Méresse et al., 1991; Dehouck et al., 1994). The capacity of ECs to bind LDLs is greater when cocultured with astrocytes than in their absence. Futhermore, we have shown that the lipid requirement of astrocytes increases the expression of the LDL receptor on brain capillary ECs. Taken together, the presence of LDL receptors on brain capillary ECs and the modulation of the expression of these receptors by the lipid composition of astrocytes suggest that cholesterol used by cells in the central nervous system may be derived, at least in part, from the periphery via transport across the BBB.In the present study, we provide direct evidence that after binding to brain capillary ECs, there is a specific mechanism for the transport of LDL across the endothelial monolayer from the apical to the abluminal surface. This mechanism might be best explained by a process of receptor-mediated transcytosis. Preliminary results pinpoint the role of caveolae in the transcellular transport of LDL across the brain endothelium.