Address correspondence and reprint requests to Dr William M. Pardridge, UCLA Warren Hall 13–164, 900 Veteran Avenue, Los Angeles, CA 90024, USA. E-mail: email@example.com
The blood–brain barrier (BBB) restricts transport of immunoglobulin G (IgG) in the blood to brain direction. However, IgG undergoes rapid efflux in the brain to blood direction via reverse transcytosis across the BBB after direct intracerebral injection. This BBB IgG transport system has the characteristics of an Fc receptor (FcR), but there is no molecular information on the putative BBB FcR. The present study uses confocal microscopy and an antibody to the rat neonatal FcR (FcRn), and demonstrates the expression of the FcRn at the brain microvasculature and choroid plexus epithelium. Co-localization with the Glut1 glucose transporter indicates the brain microvascular FcRn is expressed in the capillary endothelium. The capillary endothelial FcRn may mediate the ‘reverse transcytosis’ of IgG in the brain to blood direction.
Immunoglobulin G (IgG) molecules are generally excluded from the CNS because these large molecules do not readily traverse the brain capillary endothelial wall, which forms the blood–brain barrier (BBB) in vivo. However, IgGs are produced by activated B lymphocytes, and these cells are able to enter the CNS via lymphocyte homing mechanisms (Vrethem et al. 1992). The fate of IgG molecules produced in the brain is uncertain. One possibility is that IgG may be actively effluxed from brain back to blood across the BBB. This hypothesis is supported by the recent observation that IgG, but not other large molecules such as albumin or 75-kDa dextran, are actively effluxed from brain to blood across the BBB via a saturable transport system (Zhang and Pardridge 2001a). The IgG efflux system at the BBB was competitively inhibited by IgG Fc fragments, but not by F(ab′)2 fragments, which suggested an Fc receptor (FcR) mediates the asymmetric transcytosis of IgG across the BBB in the brain–blood direction only (Zhang and Pardridge 2001a). The BBB transferrin receptor (TfR) also mediates the reverse transcytosis of transferrin (Tf) in the brain–blood direction (Zhang and Pardridge 2001b).
The BBB TfR receptor is characterized at the molecular level (Li et al. 2001), but there is no information on the identity of the putative BBB FcR. There are at least four types of FcR, including FcR-I, FcR-II, FcR-III, and the neonatal FcR, also called FcRn (Hulett and Hogarth 1994) or FcRB (Junghans 1997). Prior work failed to demonstrate the expression of FcR-I, or FcR-II at the brain microvasculature, although the FcR-III was observed in some venular endothelium in human brain (Ulvestad et al. 1994). There is preliminary information that brain endothelial cells grown in tissue culture express an FcR (Nag and Gupta 1981), and that the choroid plexus epithelium of human brain expresses the FcRn (Kristoffersen 1997). There has apparently been no investigation as to whether the FcRn is expressed at the brain microvasculature in vivo. The FcRn is the most likely candidate for a BBB IgG transcytosis system, as this receptor is expressed on endothelium in peripheral tissues (Borvak et al. 1998), and mediates the transcytosis of IgG molecules across epithelial barriers in peripheral tissues (McCarthy et al. 2000; Telleman and Junghans 2000). The 1G3 mouse monoclonal antibody (MAb) to the rat FcRn is available (Raghavan et al. 1994), so the present studies use confocal microscopy of frozen sections of rat brain to examine the expression of the FcRn in rat brain. An antibody to the Glut1 glucose transporter, which is selectively expressed in brain at the capillary endothelium (Pardridge et al. 1990), is used in parallel with the FcRn antibody to localize the BBB in the brain sections.
Materials and methods
Mouse hybridoma cell line CRL-2434 that produces the 1G3 MAb against the heavy chain of rat FcRn heterodimers was purchased from the American Type Culture Collection and the HL-1 complete serum-free medium for this line was obtained from BioWhittaker. The rabbit anti-Glut1 glucose transporter polyclonal antiserum was prepared against a synthetic peptide encoding the 13 amino acids at the carboxy terminus of the rat or human Glut1 isoform as described previously (Pardridge et al. 1990), and this antiserum is designated rabbit anti-Glut1. Secondary antibodies Alexa Fluor® 488 goat anti-mouse IgG (fluorescein channel) and Alexa Fluor® 594 goat anti-rabbit IgG (rhodamine channel) were purchased from Molecular Probes; goat serum and Vectashield® were from Vector Laboratories. Mouse IgG1, which is the isotype control IgG for the 1G3 MAb, and all other reagents were obtained from Sigma.
HL-1 medium was supplemented with 4 mm l-glutamine, 1 mm sodium-pyruvate, penicillin, streptomycin, bicarbonate, and 1% fetal bovine serum. Cells were incubated in a 37°C humidified chamber with 5% CO2 until cells were confluent and almost overgrown. Cells and medium were aspirated and centrifuged at 300 g for 5 min. The supernatant, designated mouse anti-FcRn was aliquoted and frozen at −20°C until use.
Brain, skeletal muscle, kidney and liver tissues were rapidly removed from three-month-old healthy male Sprague–Dawley rats and frozen in Tissue Tek OCT embedding medium (Sakura). Blocks were sectioned at 20-µm thickness at −16°C with a microtome cryostat (HM 503E; Microm) and mounted on microscope slides. Tissue sections were fixed with at −20°C with 100% acetone for 20 min and air dried for 60 min followed by washing in 0.01 m phosphate-buffered saline (PBS) buffer (pH 7.4). Slides were blocked with 10% goat serum in PBS for 60 min at room temperature (23°C), followed by washing in PBS. Primary antibodies were added in the following concentrations: anti-FcRn conditioned medium (undiluted), anti-Glut1 (1 : 1000). Control sections consisted of rabbit pre-immune serum (1 : 1000) and mouse-IgG1 (10 µg/mL). Sections were incubated overnight in a humidified chamber and washed with PBS. The secondary antibodies added were Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG, both in a concentration of 10 µg/mL. After 30 min, slides were rinsed with PBS thoroughly and coverslipped with Vectashield®. Confocal imaging was performed employing a Zeiss LSM 5 PASCAL confocal microscope with dual argon and helium/neon lasers equipped with Zeiss lsm software for image reconstruction. All sections were scanned in multitrack mode to avoid overlap of the fluorescein and rhodamine channels.
Confocal microscopy revealed expression of FcRn throughout the rat cerebral microvasculature, including the brain capillary bed (Fig. 1a), and pre-capillary arterioles (not shown). The expression of immunoreactive Glut1 in cerebral endothelial cells is shown Fig. 1(b and e), and the yellow signal in Fig. 1(c and f) demonstrated immunolabeling of FcRn and Glut1 restricted to the endothelial cells. Arterioles and the elongated capillary network in the endomysium of skeletal muscle showed strong FcRn labeling (Fig. 1g), and no Glut1 staining (Figs 1h and i). However, in spaces between the fasciculi, filled with loose connective tissues, a transverse section of a peripheral nerve shows strong FcRn and Glut1 colabeling of the perineurium (Figs 1g–i). The choroid plexus ependymal cells also coexpress Glut1 and FcRn (Figs 1j–l). FcRn expression in liver and kidney was weak (not shown) and control staining with mIgG1 and rabbit IgG was negative (not shown).
This confocal microscopy study provides evidence for the expression of the FcRn at the brain microvascular endothelium, and corroborates physiologic studies which demonstrated that IgG molecules are actively transported across the BBB in the brain–blood direction via an FcR (Zhang and Pardridge 2001a), although IgG generally has restricted transport across the BBB in the blood to brain direction (Pardridge 1991). The combined results support the hypothesis that IgG molecules secreted within the brain are actively exported from brain back to blood via reverse transcytosis on the BBB FcRn.
The FcRn was first described by Brambell more than 30 years ago (Junghans 1997), and is structurally related to the class-I major histocompatibility complex (MHC) antigen (Simister and Mostov 1989). The FcRn mediates passive immunity from the mother to the young by transcytosis of maternal IgGs across both the placental and the neonatal intestinal epithelial barriers (Hobbs et al. 1987; Hulett and Hogarth 1994; Ghetie and Ward 2000). The FcRn is a type-I membrane glycoprotein (Raghavan and Bjorkman 1996), and is expressed in peripheral tissues of the adult, including endothelial cells of skin, muscle and adipose tissue, and kidney glomerular endothelial cells in humans, reflecting its role in serum IgG homeostasis (Borvak et al. 1998; Haymann et al. 2000). However, expression within the CNS has not yet been reported for the FcRn. The experimental results in Fig. 1(g) show the immunoreactive FcRn is expressed in the capillary endothelium of skeletal muscle. Muscle capillaries do not express Glut1, although this glucose transporter is expressed at the blood–nerve barrier in skeletal muscle (Fig. 1h). In constrast, the microvasculature in brain coexpresses both the FcRn and the Glut1 glucose transporter (Figs 1c and f). The coexpression of FcRn and Glut1 in rat brain microvasculature is indicative of a capillary endothelial origin in brain, as the Glut1 glucose transporter is expressed in this cell in the brain (Pardridge et al. 1990).
The finding that the FcRn is expressed at the BBB is consistent with other work showing that multiple components of the immune systems are expressed at the brain microvasculature. The class-I MHC is expressed at the brain microvasculature (Li et al. 2001), and the class-II DR antigen is expressed at the arteriolar smooth muscle cell in control brain, and in capillary pericytes in multiple sclerosis brain (Pardridge et al. 1989). The complement receptor, CD46, is expressed at the BBB, as demonstrated by recent expression cloning investigations (Shusta et al. submitted). There is homing of both B and T lymphocytes to the brain (Wekerle 1993; Vrethem et al. 1994), and these pathways involve direct transport of circulating activated lymphocytes through the brain capillary endothelium (Wekerle 1993). These immune system pathways are likely to play a role in the interaction between brain and the immune system in autoimmune diseases (Ulvestad et al. 1994; Hickey 2001; Vedeler et al. 2001; Williams et al. 2001; Zameer and Hoffman 2001). In particular, the BBB FcRn may play a role in the development of antibody-based neurotherapeutics. Local radiotherapy with antibody radiopharmaceuticals may have limited success following direct intracerebral injection (Riva et al. 1997) if the IgG molecules are readily eliminated from the brain via reverse transytosis on the BBB FcRn. Conversely, the BBB export of IgG may be beneficial in certain cases such as Alzheimer's disease (AD). The development of an AD vaccine is based on the immunization against Aβ1−40 neuropeptide, which is a principal component of the amyloid plaque of AD (Bard et al. 2000; Frenkel et al. 2001). It is not clear how circulating anti-Aβ antibodies gain access to the brain (Lee 2001), because the BBB FcRn only transports IgG in the brain to blood direction (Zhang and Pardridge 2001a). However, once in brain these antibodies may bind the Aβ peptide in amyloid plaque, and the BBB FcRn may facilitate export of the MAb/Aβ complex from brain back to blood.
The subcellular localization of the FcRn within the brain capillary endothelium, the differential distribution of the FcRn within the cytoplasm, and the endothelial luminal and abluminal membranes is not known at present. The FcRn has an intracellular localization in some cells (Ghetie and Ward 2000), and there is diffuse cytoplasmic FcRn immunoreactivity in the choroid plexus epithelium (Fig. 1j). However, the pattern of diffuse cellular expression of the immunoreactivity in the choroid plexus epithelium is also seen with the Glut1 glucose transporter (Fig. 1k). The diffuse cellular immunoreactivity is likely to be an artifact of acetone fixation (Materials and methods), as immunoreactive Glut1 glucose transporter is selectively expressed at the basolateral membrane of choroid plexus epithelium (Farrell et al. 1992). The FcRn may also be expressed on the plasma membrane of cells that transcytose IgG, as the FcRn mediates the transcytosis across intestinal or renal epithelial cell barriers (McCarthy et al. 2000; Telleman and Junghans 2000), and the initial endocytosis across the membrane must be receptor-mediated. The BBB TfR mediates transferrin trancytosis in both blood–brain and brain–blood directions (Zhang and Pardridge 2001b), and the BBB TfR is localized on both luminal and abluminal endothelial membranes (Huwyler and Pardridge 1998). The BBB FcRn may be localized to either the endothelial abluminal and/or luminal membrane so as to allow for reverse transcytosis of IgG in the brain to blood direction.
This work was supported by a grant form the US Department of Energy ER62655. FS was supported by a grant from the Ernst Schering Research Foundation (Berlin, Germany).