Molecular anatomy of the blood-brain barrier as defined by immunocytochemistry.

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 127

Molecular Anatomy of the Blood-Brain Barrier as Defined by Immunocytochemistry ROLFDERMIETZEL AND DOROTHEE KRAUSE Department of Anatomy and Morphology, University of Regensburg, 0-8400 Regensburg, Federal Republic of Germany

I. Introduction This review deals with immunocytochemical aspects of the specific molecular setup of cerebral microvessels and the perivascular apparatus. The primary intention of this article is not to provide an overview of the physiological and biochemical properties of the blood-brain barrier (BBB). [For this purpose, a number of recent monographs and reviews are cited (Cserr, 1986; Suckling et al., 1986; Strand, 1988).] Rather, a comprehensive presentation of the molecular characteristics of cerebral blood vessels which are relevant to the described immunocytochemicalcharacterization of the BBB is made. Recent developments and improvement of methods for immunolabeling and the production of specific probes, e.g., affinity-purified polyclonal antibodies and monoclonal antibodies, have contributed to new discoveries about the molecular composition and morphogenesis of the BBB. The formulation of other investigative strategies to better define the BBB has also resulted in an improved selection of methods. Future implications of a more molecularly oriented morphology will also be explored. A brief historical synopsis of the development of the BBB concept is included. 11. Concept of Blood-Brain Barrier

A. SHORT HISTORICAL OUTLINE The BBB concept was based on the classic experimentation of Ehrlich

(1885) who showed that the intravenous injection of dyes, e.g., a series of

aniline derivates, results in the penetration of most tissues except in the brain. Lewandowski (1900) introduced the term “blood-brain barrier” to describe this phenomenon. Goldrnan (1913) expanded the original BBB concept by demonstrating that intrathecal administration of Trypan Blue results in a generalized staining of brain tissue, while intravenous application does not. It was assumed that in order for the stain to reach the brain tissue, it must diffuse from the ependyma and glialpia mater interface. 57 Copyright 8 1991 by Academic Press, Inc. All rights of reproductionin any form RSeNed.

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ROLF DERMIETZEL AND DOROTHEE KRAUSE

Goldman (1913) hypothesized that the vehicle for substance transport was the cerebrospinal fluid (CSF) which allegedly gained access to the brain tissue via the choroid plexuses. This theory of ubiquitous material transport in the central nervous system (CNS) by means of the CSF is also referred to in literature as the concept of the “way of the spinal fluid” (von Monakow and Kitabayaski, 1918). This concept was systematically reformulated through the investigations of Stern and Gautier (1921, 1922) in which the intracerebral distribution of various substances was observed. Their “barriere hematoencephalique” provides a considerably different version of the original hypothesis. They defined the barrier to be, as a whole, the cerebral blood vessel compartment in which the choroid plexus was semipermeable, facilitating the flow of substances from the blood into the CSF. As a spinal fluid-producing organ, the plexus was regarded as a central exchange surface for establishingthe homeostatic balance of the cerebrospinal fluid. The aforementioned investigations are of significant historical meaning in the development of the BBB concept. Nevertheless, numerous morphological and physiological investigations have led to an extensive modification of the original concept. It was discovered, for example, that other regions of the brain also possess incomplete barriers similar to those in the region of the plexus, e.g., the neural pituitary gland, the median eminence, the pineal gland, the area postrema, the organum vasculosum of the lamina terminalis, and, in some animal species, the subfornical organ (Wislocki and Leduc, 1952; Dempsy, 1955; Dempsy and Wislocki, 1955; RiveraPomar, 1966; Brightman, 1977; Dermietzel and Leibstein, 1978; van Deurs, 1980; Oksche, 1984). These are structures which are considered to be involved in neuroendocrine feedback mechanisms and/or receptive functions and which are generally termed circumventricular organs (CVOs) (Brightman, 1977). Behnsen (1926) and Stern and Peyrot (1927) observed that when Trypan Blue is intravenously introduced in young animals, it tends to penetrate the brain tissue, indicating that the presence of the BBB is dependent upon the phase of ontogenetic development. This assumption was later supported by the findings of Spatz (1934) in postpartum icterus (for further discussion of the developmental aspects of the BBB, see Article 4, this volume).

B. COMPARTMENT CONCEPT Developmentally related changes in the permeability of the BBB represent only one aspect of the relative barrier phenomenon. Physiological investigation of the dynamics of the blood-brain transfer of electrolytes and nutrients such as glucose and amino acids, as well as morphological

MOLECULAR ANATOMY OF THE BLOOD-BRAIN BARRIER

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investigation through light and electromicroscopic tracing methods have made significant contributionsto the modern concept of the BBB (Davson, 1967; Bradbury, 1979, 1984; Pardridge, 1983). In essence, the BBB concept is a positive reformulation of the original barrier paradigm. The barrier is defined as the sum of all bidirectional exchange processes which occur on the morphological blood-brain interfaces (Davson, 1967), presumably the brain endothelium. Most of the exchange processes are specific to brain endothelium and, in contrast to the extracerebral microvascular segments, are highly selective, permitting or restricting the passage of substances from blood to brain and vice versa. Thus, the cerebral endothelium assists in the integration of brain and body functions by transmitting metabolic and homeostatic information bidirectionally. In this respect, the BBB endothelium can be regarded as a polarized “tight epithelium” that is capable of creating and sustaining ionic and metabolite gradients (Betz and Goldstein, 1981; Crone, 1971, 1986a,b; Crone and Levitt, 1984). This particularly applies to sodium, potassium, glucose, and a number of amino acids and nucleotides whose passage is facilitated by carrier-mediated transport. The selectivity of the transport processes, on one hand, and the restrictive mechanism, on the other, are expressions of the specific molecular and structural properties of the cerebral endothelium and its morphological adjuncts. In essence, the BBB acts as a homeostat between the brain-body interface. SUBSTRATES OF BBB C. STRUCTURAL The endothelium of the intracerebral capillaries plays a crucial role in separating the blood from the two major fluid compartments of the brain. Functioning as a border, the endothelium separates the intravascularcomparment (IVC) from the interstitial cerebral compartment (ICC) and the cerebrospinal compartment (CSC) (Crone and Levitt, 1984; Fenstermacher and Rapoport, 1984; Fenstermacher, 1985). In contrast to most non-BBB microvessels, the endothelium of brain capillaries possesses several unique structural features. (I) Ultrathin sections have revealed the existence of minor endothelial vesicles in the BBB capillaries (Reese and Karnovsky, 1967; Wolff and B k , 1972). (11) The interendothelial space is sealed by continuous tight junctions displaying high resistance (Reese and Karnovsky, 1%7; Brightman and Reese, 1969; Dermietzel, 1975a; Crone and Olesen, 1981).(111) The capillary walls of BBB vessels are thinner than the endothelium of non-BBB microvessels (Wolff, 1963). The tightness of interendothelial contacts can be observed at the electron microscopic level through the intravenous introduction of electrondense tracer substances or exogenous proteins. The brain capillaries are

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typically impermeable to substances such as albumin (MW of 60,000), myofer (MW of 78,000), horseradish peroxidase (MW of 40,000), and microperoxidase (MW of 1,800) (Brightman et al., 1970; Feder, 1971). They are also impermeable to micromolecular substances such as thorium dioxide and lanthanum hydroxide (Bundgaard, 1982). In a number of electron microscopic investigations, silver nitrate was also used as a tracer (Dempsy, 1955; Dempsy and Wislocki, 1955). In these cases, though, the extremely long exposition time, i.e., up to one year, hardly permits discussion of veritable tracer passage. In contrast to the typical BBB capillaries (which will be designated as the “tight” segment of the cerebral microvasculature), endothelial fenestration and “leaky” interendothelialjunctions are prevalent in the capillaries of select CVOs (Brightman, 1977; Dermietzel and Leibstein, 1978; Krisch and Leonhard, 1978; van Deurs, 1980). With respect to the above introduced tracers, including silver nitrate, these brain capillaries may be classified as ‘‘leaky.’’ Here, the BBB appears to be relatively permeable. In addition, the vascular adjuncts of the “tight” and ‘‘leaky’’ segments of brain microvessels differ considerably as the “tight” segments exhibit a typical relationship with the neighboring perivascular glia. These astroglia essentially envelop the vessel wall by means of astroglial endfeet (Wolff, 1963; Brightman and Reese, 1969; Peters et al., 1976; Goldstein and Betz, 1986) . An extensive perivascular space exists only within the CVOs. Here, the perivascular spaces are wide, containing connective tissue elements such as collagen, leptomeningeal cells, and matrix proteins (Leonhard, 1980). The role of the glia in the barrier mechanism was once viewed as purely mechanical. This thinking has been abandoned as the result of tracer experimentation which showed no restriction of tracer substances at the astrocyte-endothelial interface (Brightman, 1968; Brightman and Reese, 1969). Consequent findings regarding the constitution of the interglial membrane contacts at the perivascular astroglial ensheathement have revealed the absence of tight junctions between the perivascular glia (Brightman and Reese, 1%9; Dermietzel, 1974b). The closely linked morphological relationships between the astroglia and vessel walls of BBB capillaries led to early speculation about a feasible functional synergism between these two tissue components (DeBault and Cancilla, 1980; Beck et al., 1984, 1986; Janzer and R a , 1987). The view that the astroglia are actively involved in the functional establishment of the BBB and control interstitial cellular fluid (ICF) homeostasis has assumed increasing credibility (Abbott ef al., 1986; Goldstein and Betz, 1986; Goldstein, 1988). The presence of extensive, close oppositions between astrocytic processes and “tight” BBB capillaries is, therefore, considered to be a decisive criterium for determining the existence of a


61

barrier effect. Figure 1 summarizes the structural features of the “tight” and “leaky” segments of brain capillaries.

D. METABOLIC SUBSTRATES OF THE BBB: THEPRIMARY COMPONENTS As mentioned above, essential metabolites and ions cannot freely pass across “tight” cerebral endothelium: they must first overcome the structural and metabolic obstacle of the BBB. For this to occur, various physiochemical properties of the substances as well as the specific transporter complement of BBB endothelium can favorably enhance their movement from the blood, through the endothelial cells, and into the brain interstitial tissue. Substrate movement can occur in four forms: (1) passive diffusion; (2) facilitated diffusion; (3) active transport; and (4) receptor-mediated endocytosis. Many lipophilicmolecules can freely diffuse through the lipid bilayer of the cerebral endothelium (Dhopeshwarekar and Mead, 1973; Brightman, 1977). Examples of such molecules include fat-soluble forms of vitamins, drugs, alcohol, nicotin, and lipophilic, cyclic peptides (Oldendorf, 1974; Rapoport, 1976; Pratt and Greenwood, 1986; Pardridge, 1986, 1988). Members of the neuropeptide family such as encephalin analogs (von Graffenried et al., 1978), endorphins (Houghten et al., 1980), as well as the delta sleep-inducing peptide (DSIP) (Monnier et al., 1977) are also assumed to passively penetrate the plasma membranes of the cerebral endothelial cells (Zlokovic et af.,1985), although the existence of a specific transporter for neuropeptides has been suggested (Pardridge and Mietus, 1981). The passage of these substances into the nervous tissue is primarily limited by the degree of hydrolyzation in the endothelial cytoplasm (Begley and Zlokovic, 1986). Water also possesses the capability to diffuse from blood to brain by means of solvent drag accompanying ionic transport (Rapoport, 1976), although some authors have even suggested the existence of neural and humoral control of water transport across the BBB (Raichle et al., 1975; Grubb et al., 1978). In contrast, hydrophilic substances including most of the organic nonelectrolytes, i.e., protein, saccharides, ions, cannot simply diffuse through the endothelial cell membrane: for these substances, specific transport mechanisms must be available (Crone, 1%5a,b, 1984a,b; Pardridge, 1983). In actuality, the endothelial cells lining the brain capillaries constitute a polarized epithelium whose individual plasma membranes contain specific sets of carrier proteins and receptor molecules. Thus far, eight independent transport systems governing the blood-brain exchange have been identified on the basis of physiological and biochemical studies (Pardridge, 1986, 1988). Through these integrated systems, carriermediated transport of nutrients across endothelial plasma membranes


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from blood to brain tissue is facilitated; furthermore, the transport of wastes from the brain tissue into the blood stream is also made possible. In the following section, we will discuss some of the most important carrier/ receptor systems. 1. Glucose Transporter

One prime example of a specific transport system involves the transporter which provides for the major energy source of the brain: glucose (Sokoloff, 1981). Crone (1965b) suggested that the glucose transport system is stereospecific as well as carrier mediated, able to distinguish between the stereoisomers D- and L-glucose. As a result, L-glucose does not reach the brain. The brain glucose transporter (GT) does, however, display a slight affinity to other hexose molecules such as D-mannose, Dgalactose, and D-xylose (Lund-Andersen, 1979). When bound to a carrier protein, concurrent inhibition of glucose transport results. Yudilevich and De Rose (1971) as well as Betz et al. (1973) discovered that the transport system localized within the cerebral endothelium resembles the glucose transport system in the erythrocytic membrane. In both systems, the rate of glucose transport is dependent upon the glucose concentration of the blood, increasing until saturation is reached (Pardridge and Oldendorf, 1975a; Lund-Andersen, 1979). Furthermore, the glucose transport rate across the endothelial membrane is essentially identical in both directions (Pardridge and Oldendorf, 1975a; Raichle et al., 1975). Thus, transport does not appear to actively occur against a concentration gradient. Dick et al. (1984) employed cytochalasin B, a metabolite extracted from the fungus Helminthisporium dematoiderum, which can influence numerous transport processes at the membrane level (Carter-Su et al., 1982; Shanahan, 1982),as a blocking substance to determine the number of GTs in brain endothelial cells. It was revealed that “tightness” in glucose transport domains was more prevalent in endothelial cells than in the erythrocytic membrane. Simpson and Cushman (1986) also reported that glucose transport may be stimulated by insulin in some cell types, e.g., adipocytes and skeletal muscle cells. It is known from the erythrocytic glucose transporter that a glycosylated, intrinsic membrane protein (MW of 55,000) is involved which locks onto the glucose molecule and carries it across the plasma membrane (Kasahara and Hinkle, 1977; Baldwin et al., 1979). Still, the exact structure and localization of the GT within the endothelial cells of the brain remained unknown at that time. We will discuss the ultrastructural localization of GT in BBB endothelium in further detail.

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2 . Amino Acid Transporters Three different carrier systems were thought to exist for amino acid transport which, just as for the glucose transport system, are dependent upon concentration, are stereospecific, and may be competitively inhibited (Lajtha and Toth, 1963; Oldendorf, 1971, 1973a; Richter and Wainer, 1971; Pardridge and Oldendorf, 1975b). Large, neutral amino acids (Lsystem) (Christensen, 1979) such as phenylalanine, isoleucine, valine, and tryptophan provide the essential basis for brain synthesis of neurotransmitters and peptides. The L-system was successfully identified at the BBB by Wade and Katzmann (1975; see also Pardridge, 1977). Furthermore, transport systems for both basic (lysine, arginine, ornithine) (Banos et af.,1974) and acidic amino acids (glutamate, aspartate) (Oldendorf and Szabo, 1976) were localized. Select smaller amino acids are synthesized in the brain, acting as inhibitive neurotransmitters, e.g., glycine, y-aminobutyric acid (GABA). For this reason, their concentration in the brain must be constantly controlled. In the abluminal endathelial cell membrane, a carrier system exists explicitly for this purpose, transporting small amino acids out of the extracellular space of the brain (A-system). Along with this system, sodium ions are transported from the brain tissue into the endothelial cell cytoplasm according to the concentration gradient (Betz and Goldstein, 1978). Small amino acids are then surrendered to the blood by transport systems localized in the luminal endothelial cell membranes (presumably the L-system) (Christensen, 1979). 3 . BBB-Specific Enzymes and Ionic Transporter

y-glutamyl transpeptidase (y-GT) catalyzes amino acid transfer in the membranes of various organs, e.g., the cystic duct, bile canaliculi, plasma membrane of hepatocytes, brush border of the renal proximal tubules, and the small intestine. More specifically, y-GT catalyzes the transfer of yglutamyl residue of glutathione to amino acids (Orlowski and Meister, 1970).The significant association of y-GT with cell membranes (especially in tissue where extensive amino acid transport is suspected) supported this hypothesis (Orlowski, 1%3). The reaction chain catalyzed by y-GT was integrated into a cycle (y-glutamyl cyle; Orlowski and Meister, 1970) which is assumed to represent a system for amino acid transport. This transpeptidase has been cytochemically localized in the brain capillaries and choroid plexus epithelia (Albert er al., 1966), apparently serving as par? of the amino acid transport systems within the brain endothelium. One or more of these systems is thought to be associated with y-GT and the y-glutamyl cycle (Orlowski er a f . , 1974). The rapid transport of methio-


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nine, a favorable substrate for the transpeptidase, and the presence of three intermediates of the y-glutamyl cycle, namely, glutathione (Martin and McIlwain, 1959), pyrroline carboxylic acid (Wilk and Orlowski, 1973), and y-glutamyl acid, are also consistent with the concept that y-GT is a major part of the amino acid transport system. It has also been assumed to be involved in the synthesis of extracellularmatrix components by forming y-glutamyl bonds (Mischek et al., 1988). Tissue culture experiments using a coculture of y-GT negative, cerebral endothelial cells require a glial induction factor in order to maintain high y-GTlevels (DeBault and Cancilla, 1980; Mischek et al., 1988). Another example of a brain-specific enzyme is monoamineoxidase (MAO)which provides an enzymatic barrier, impeding the traffic of monoamine precursors into the brain (Bertler et al., 1966; Bjorklund et al., 1969; Hardebo and Owman, 1980). After assimilation of monoamines into the endothelial cells, cytoplasmic MA0 decarboxylizes the monoamines, thereby effectively preventing a flood of monoaminergic peripheral neurotransmitters into the neuronal fluid environment. A list of enzymes and receptors prevalent in the endothelial plasma membrane of the brain is presented in Table I. The ionic homeostasis of the brain and CSF can only be maintained if specific transport systems exist for electrolyte transfer between the blood and brain compartments (Goldstein and Betz, 1983). Since the cerebral endothelium displays a high resistance (>2000Wcm2)due to the presence TABLE I ENZYMES AND RECEPTOR/TRANSPORTER SYSTEMS LOCALIZED IN THE CEREBRAL ENDOTHELIUM Enzyme

Reference

Alkaline phosphatase Aminopeptidase Na+-K+ ATPase Adenyl cyclase Cholinesterase DOPA decarboxylase Guanylate cyclase Glucose transporter y-glutamyl-transpeptidase Monoamine oxidase 5' nucleotidase Phosphoprotein phosphatase Serotonin receptor Transfenin receptor

Landers et al. (1%2) Pardridge and Mietus (1981) Firth (1977) J06 (1979) Kreutzberg and Toth (1983) Bjorklund et al. (1%9) Karanushina et al. (1980) Lidinsky and Drewes (1983) Albert et al. (1966) Bertler et al. (1966) Lidinsky and Drewes (1983) Weber et al. (1987) Olesen (1985) Jefferies et al. (1984)

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of tight junctions (as found in frog skin, for instance), its permeability for ions is minimized (Crone and Christensen, 1981; Crone and Olesen, 1981). The concentrations of sodium and potassium ions in the brain have been found to be controlled by sodium-potassium ATPase, localized in the abluminal endothelial cell membrane of the brain capillaries (Firth, 1977; Betz et al., 1980). This ionic pump permits upstream movement of sodium against the concentration gradient from the endothelial cytoplasm into the brain compartment and successive potassium movement in the reverse direction (Bradbury, 1979). Additional specific carrier systems reside in the cytoplasmic membrane of the endothelial cells, specific for monocarboxylic acids such as acetate, lactate, and pyruvate (Oldendorf, 1973b) as well as nucleic acid precursors (Cornford and Oldendorf, 1975; JoO, 1979; Karnushina er al., 1980). 4. Peptide and Protein Transcytosis

Most of the above described carrier systems for hydrophilic substances transverse the phospholipid bilayer of the luminal endothelial plasma membrane, liberating their substrates into hydrophilic cytoplasm. From the cytoplasm, substrates diffuse to the abluminal side of the BBB endothelium for further discharge into the ICC. Compelling evidence indicates, however, that in addition to the unidirectional andlor bidirectional transport of small molecules, numerous macromolecules are also able to enter brain tissue from the blood. Although no direct structural evidence of endocytotic uptake and transcytotic carriage of physiologically relevant macromolecules exists (as has been generated for the transcytotic traffic of ligands in the small intestine as well as for albumin in peripheral endothelia) (Simionescu et al., 1981a,b, 1982), a vesicular pathway for blood-borne macromolecules, e.g., insulin, is highly likely as has been suggested by radiolabeling kinetic studies (Pardridge et a / ., I985;Duffy and Pardridge, 1987). The transport of transferrin across the BBB is one prime example of a receptor-mediated transfer of blood-borne protein across brain endothelium. Brain cells, including neurons as well as glial cells, require a constant supply of iron in order to maintain their normal function (Fishman et a l . , 1987). Even cultured oligodendrocytes and neurons require a transferrin receptor supplement in their supporting medium for survival (Szuchet et al., 1980; Aizenman et al., 1985). In uiuo perfusion of rat brain with '"-SI-transfemn resulted in a receptor-mediated uptake of transfenin into the endothelium, followed by similar uptake in brain tissue (Fishman et al., 1987). This internalization of transferrin into brain endothelium in a receptor-mediated manner suggests that the brain may substitute its iron through transcytosis of iron-loaded transferrin across the brain microves-


67

sels (Fishman et al., 1987). Furthermore, it appears possible that brain endothelium has developed a specialized means of acquiring iron via the endothelial transcytotic mechanism. The actual localization of transfemn receptors (TFR) in BBB endothelia and TFR expression during development was achieved recently. These results indicate that supplement containing TFR is specific for cerebral microvessels (Jefferies et al., 1984; Risau et al., 1986a). The transcytotic passage of blood-borne protein, however, is not unique for transferrin. Considerable evidence suggests that a number of biologically active proteins, including immunoproteins such as IgG (Zlokovic et al., 1990), hormones, e.g., insulin (Duffy and Pardridge, 1987), N-tyrosinated peptides (Banks and Kastin, 1984) are actively transcytosed through BBB endothelia with receptor/canier mechanisms. More recently, aided by wheat germ agglutinin (WGA) conjugated with the enzymatic tracer horseradish peroxidase (HRP), Broadwell et al. (1988) successfully documented luminal absorptive endocytosis of this blood-borne complex (albeit nonphysiological).Its transport occurred via the Golgi saccule to the abluminal side of nonfenestrated brain capillaries, with subsequent discharge into the perivascular space. Transcytotic processes utilizing different vesicular routes through the BBB endothelium may, therefore, serve as efficient pathways for blood-brain transport of macromolecules. The aforementioned paucity of BBB endothelium in vesicular profiles does not necessarily contradict the concept of select vesicular transport, since the reported transendothelial tramcking velocity (Broadwell et al., 1988) seems to be slow (passage of WGA-HRP >6 hours); furthermore, only small quantities of substrates may be necessary to physiologically influence brain tissue. Figure 2 gives a schematic depiction of the different transendothelial transport mechanisms discussed thus far.

E. SECONDARY COMPONENTS OF THE BBB In addition to the specific structure of the endothelial cell barrier with its various selective transport mechanisms, the cerebral vascular bed also contains other structural components which act as a secondary barrier, contributing to the blood-brain barrier phenomenon (see Fig. 1). 1, Pericytes

A dense distribution of perivascular cells is spirally wrapped around the endothelial cells of the brain capillaries, separated only by the basement membrane (which also surrounds both the pericytes and the endothelial cells). Kristensson and Olsson (1973) as well as van Deurs (1976) desig-

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MEANS OF SUBSTRATE MOVEMENT 1 Diffusion 2 Facilitated diffusion 3 Active transport 4 Receptor-mediated endocytosis

FIG. 2. Diagram summarizing the different forms of substrate movement through the BBB. Movement of substrates occurs via the following: (1) paracellular or transcellular diffusion; (2) transendothelal facilitated diffusion which is dependent upon carrier/receptor complexes but is not energy dependent; (3) active transport which is both camer/receptormediated and energy consuming; and (4) endocytosis which may utilize an intermediate cytoplasmic target, e.g., Golgi complex (Broadwellet al., 1988), or overcome the endothelial barrier by direct transcytosis.

nated these perivascular pericytes as phagocytic microglial cells which, in part, are responsible for maintaining the homeostasis between blood and brain. Through induced hypertonia, van Deurs (1976) was able to show that HRP penetrates the cerebral endothelial cells and, thereafter, is actively phagocytized by cerebral pericytes. Under a number of other experimental and pathological conditions inducing permeability of the BBB, this control function of the pericytes in reaction to blood-borne substances invading the brain was also observed (Torack, 1961; Baker et al., 1971; Cancilla et al., 1972; Sumner, 1982). To date, clear definition of the different hypothetical classes of perivascular cells has not been achieved; however, their involvement in the phagocytotic uptake of blood-borne substances, including immunoglobulins, is apparent. Hickey and Kimura (1987) identified these cerebral perivascular cells as immunocompetent antigen-presenting cells. In this respect, cerebral pericytes resemble professional macrophages outside of the brain. Antigenpresenting cells interact with B lymphocytes and T lymphocytes, thereby triggering an immune cascade of cytokine expression and lymphocytic proliferation. The existence of antigen-presenting cells in the brain indicates that the brain is not as immunologically privileged as once thought. A characterization of the perivascular cellular apparatus is, therefore, of


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considerable interest, particularly to immunologists (Fontana et al., 1987; Streit et af., 1988). Pericytes have also been localized in the retina, exhibiting an unusually high content of actin and myosin. These pericytes are in the position to perform in a contractile capacity, thereby assisting in the regulation of the capillary blood flow (Le Beux and Willemot, 1980).

2. Basement Membrane The basement membrane is a specialized, extracellular matrix which separates the endothelial cells as well as the pericytes from the surrounding extracellular space. The membrane is synthesized by adjoining cells which are connected with the basement membrane via fine filaments. By means of heavy metal salt contrast methods, various areas of the basement membrane may be distinguished at the electron microscopic level: an inner, electron-dense layer, the lamina densa; a bordering lamina densa; and less electron-dense layers, the laminae rarae. In brief, the basement membrane is composed of the following molecular components: 1. Laminin (Timpl et al., 1979, 1982), a glycoprotein, is present in its

2.

3.

4.

5.

pure form and complexed with other components of the basement membrane. Collagen IV [al(IV) and a2(IV)] (Crouch et al., 1980; Tryggvason et al., 1979), a microfibrillary molecule, functions as a support apparatus for other basement membrane components and is particularly prevalent in the lamina densa (Timpl et al., 1981). Proteoglycans, notably heparan sulphate, possess an affinity to collagen IV resembling that of laminin. Proteoglycans are commonly found in the outer basement membrane regions, the laminae rarae, and selectively filter-charged macromolecules (Del Risso et al., 1981; Farquhar, 1981). Fibronectins, a group of glycoproteins, function as adhesion molecules which bind to collagen fibrils, thus contributing to the reticulation of the extracellular matrix. Fibronectins are prevalent in the basement membrane, on cell surfaces, and as aggregations in the extracellular space (Yamada and Olden, 1978; Hynes, 1986). Nidogen and entactin are two glycoproteins whose functions remain unknown (Carlin et al., 1981; Timpl et al., 1983); they may be found only in minute concentrations in the basement membrane. The chemical composition of these individual basement membrane components differs among various organs (Kefalides et al., 1979).

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In addition to playing a protective mechanical role, e.g., elastic support of the capillary walls, the basement membrane of the vascular wall also functions as a highly selective filter which is essential to transport of highly charged molecules. This transport capability has been intensively investigated in conjunction with the kidney glomeruli (Farquhar, 1981). As the only continuous structure surroundingthe glomerular capillaries, the basement membrane contributes to the formation of the glomerular filtrate by preventing the passage of macromolecular plasma components such as albumin (Ryan and Karnovsky, 1976). Moreover, the function of the basement membrane in cell differentiation, cell metabolism, the organization of the plasma membranes, as well as cell migration has been discussed (Kefalides et d.,1979). Charge selectivity has been demonstrated in the basement membrane of fenestrated capillaries within the choroid plexuses (Thurauf ef a / ., 1983) and the choriocapillary of the eye (Pino, 1986a,b). In many respects, the charge distribution pattern in these “leaky” segmentsof the brain’s microcirculation resembles that found in kidney glomeruli. Charge selectivity, however, also seems to be an important feature for transendothelial transport within the brain. Within the choroid capillaries, a high positive net charge of fenitin (pi > 9.3) has been shown to trigger endocytotic uptake (Dermietzel et al., 1983), followed by transcytotic trafficking and abluminal exocytosis. Charge selectivity is not unique to the “leaky” segments of brain microvessels. This phenomenon is also present within the “tight” BBB segments, as has been documented by the selective uptake of cationic albumin (PI > 8.5) via absorptive endocytosis (Pardridge, 1986). Whether the basement membrane in the “tight” BBB endothelium also acts as an electrostatic filter after the escape of highly positively charged macromolecules from the endothelium remains to be investigated.

HI. Immuno-approach to BBB Definition The described specific structural and molecular properties of EBB endothelium and its morphological adjuncts have generated considerable efforts to better define the biological properties of this unique blood-tissue interface. Progress toward better understanding the regulative mechanisms which underlie the functional qualities of the EBB has been realized by adopting separation techniques which permit the isolation of metabolically “active” brain capillaries (J06 and Karnushina, 1973; Brendel et al., 1974; Goldstein er a)., 1975; Mrsulja et al., 1976).The current state of knowledge regarding the physiological and biochemical results provided by this approach have been reviewed extensively (Joo, 1985). From a


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morphological point of view, however, biochemical evaluation of isolated BBB endothelia has contributed little to our understanding of the structural constituents and trafficking units serving specific functions in the BBB. A breakthrough was made in the analysis of the molecular components which account for the specific function of the BBB through the employment of immunocytochemical techniques at the light and electron microscopic levels. This strategy involves (1) the application of specific antibodies produced for antigens of non-BBB origin which, however, approach being part of the BBB molecular complement; and (2) the isolation of brain microvessels with the aim of obtaining direct access to the assembly of the BBB’s proteinaceous constituents. Isolated microvessels can be utilized as a source of BBB-specific proteins to produce antibodies specific for BBB antigens (Krause et al., 1988). Such antibodies can concomitantly be used as immunocytological probes to detect antigenic determinants in the BBB and to further define the biochemical nature of the antigen. The latter approach is considerably advantageous when monoclonal antibodies (mAbs) are propagated. Since the mAb technique is highly standardized, it allows rapid generation of sets of antibodies directed to epitopes of brain microvessels. The specificity of antibodies to BBBrelated antigens can be tested by systematically screening nervous and nonnervous tissue by indirect immunofluorescence or alternative immunocytochemical techniques. With this method, mAbs possessing selective specificity to brain microvessels can be generated as immunoprobes for defining and analyzing the molecular architecture of the BBB (Michalak et al., 1986; Risau et al., 1986b;Sternberger and Sternberger, 1987; Krause et al., 1988). In addition, the utilization of antibodies is instrumental in further molecular biological techniques such as cloning and sequencing of the antigens determined (Seulberger et al., 1990). A summary of the described scheme for mAb production to BBB antigens is given in Fig. 3. A. DETERMINATION OF BBB-RELATED ANTIGENS OF KNOWN AND FUNCTION BIOCHEMISTRY Most data on the immunocytochernical characterization of BBB-related proteins (including enzymes as well as receptor/transporter complexes) was obtained by introducing antibodies to well-characterized proteins of non-BBB origin. This approach is advantageous in that it deals with substrates of known biochemical composition and function; thus, it can also be utilized to obtain direct information regarding substrate localization, function, and developmental expression in brain microvessels. The following survey of BBB-related antigens with known function(s) is presented in chronological order, without respect to functional priority.

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ROLF DERMIETZEL AND DOROTHEE KRAUSE Propagation of mAbs to BBB Antigens

0 Immunization Injection of antigen = isolated BBB capillaries

e

Lymphoblasts

Myeloma Cells

G+c

0 Hybridoma

Cultivation

Selection of fused hybridoma (. lymphoblasts myeloma cells) with HAT medium +

0 lmmunofluorescence Check for BBB positivity of Supernatant on cryostat sections

0 Cloning Cloning of individual hybridoma cell lines

FIG. 3. Strategy for monoclonal antibody (mAb) propagation to BBB-related antigens. Isolated brain capillariescan be utilized as a collective immunogen.The Row chart depicts the different steps of mAb production. PEG, polyethylene glycol; HAT medium, selection medium containing hypoxantine, aminopterin, and thymidine.

1. Function and Characterization of Transferrin Receptor

The functional significance of the transferrin receptor (TFR) and its receptor-mediated transcytosis is already discussed. Recently, the expression of TFR has been documented in rat and human brain capillaries (Jefferies et al., 1984). Using mouse mAb (OX-26) against rat and human TFR, Jefferies et a / . (1984) successfully demonstrated the presence of the TFR molecule in adult rat. Injection of this antibody into the blood indi-


73

cates that TFR is accessible from the luminal side of the brain microvessels, as extensive labeling occurred after systemic application ( Jefferies et al., 1984). Studies on developing mouse brain show that TFR expression begins at embryonic day 15 (E15)in mouse and embryonic day 11 (El 1) in chick (Risau et al., 1986a) (Fig. 4). One drawback to exact immunocytochemical determination occurs especially when developmental expression is taken into consideration (Risau et al., 1986a). The presence of TFR at nonendothelial sites, i.e., erythrocytes and in the tissue of the thymus, lymph nodes, spleen, heart, liver, kidney, pancreas, and small intestine (Jefferies et al., 1984). However, no labeling in any other endothelial species has been reported. Therefore, in spite of these limitations, TFRdirected antibodies may be regarded as suitable markers for BBB definition.

2. Glucose Transporter A constant supply of blood-borne glucose is vital to cerebral metabolism. As indicated above, a brain glucose transporter (GT) has been implicated by physiological and biochemical means which effectively trans-

FIG. 4. Immunofluorescence labeling of transfenin receptors (TFR) in mouse cerebral microvessels. A monoclonalanti-TFRantibody was used as a specific immunoprobe. (Micrograph courtesy of W. Risau.)

74



75

ports glucose from blood to the ICF and vice versa. Because of the functional significanceof glucose transport through the BBB endothelium, the exact localization of the transporter as well as its regulation and developmentalexpression have presented considerable challenges to morphologists (Crone, 1986b). Glucose transporters are expressed in virtually all mammalian plasma membranes, generally functioning as transmembranous carriers for hydrophilic monosaccharides. The high concentration of glucose transporter in the erythrocytic membrane and its generally easy accessibility have resulted in the early characterization of the human erythrocytic GT (hEGT) (Jones and Nickson, 1981; Wheeler and Hinkle, 1985). Since most of the data on the localization of brain GT derive from antibodies to hEGT, we will describe this protein in more detail. The use of hEGT is justified in that our data and other data indicate that brain and endothelial GT is immunologically homologous to hEGT. Sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and successive immunoblotting indicate that the relative molecular weight of hEGT approximates 55,000 (Baldwin and Lienhard, 1980; Sogin and Hinkle, 1980; Allard and Lienhard, 1985), appearing in the immunoreaction as a broad, somewhat diffuse band. Following carbohydrate extraction via endoglycosidase F, the SDS-PAGE yields a discrete band, indicating a molecular weight of 46,000 (Lienhard et al., 1984). Proteolysis experimentation was also performed utilizing hEGT (Cairns et al., 1984; Klip et al., 1984).The results of these investigations indicate that hEGT is a transmembranous protein composed of two domains: (1) an extracellular domain which is proteolysis resistent; and (2) a cytoplasmic domain which may be removed via trypsin. Possessing a molecular weight of 19,000 and a binding affinity for cytochalasin B, the tryptic fragment is a potent, competitive inhibitor of GT. Moreover, this fragment contains an epitope for antibodies developed against hEGT (Haspel et al., 1985). Aided by an anti-hEGT antibody, our data shows that antibodies to hEGT cross-react with endothelial cells (Fig. 5a). No staining, however, was achieved in the ‘‘leaky’’ segments of brain microvessels, i.e., in the circumventricular organs (CVO) and the choroid plexuses (Fig. 5b) (Young and Wang, 1990). These results suggest that GT proteins in the

-

FIG. 5. (a) Glucose transporter (GT) immunolabeling with an anti-human glucose transporter antibody (indirect immunofluorescence)in adult rat microvessels. lmmunoreactivityis confined to the endothelium. (b) The “leaky” microvessels of the area postrema (AP) lack GT immunoreactivity,indicative of the lack of GT expression. The area postrema is outlined by a white dashed line. (c) Electron micrograph of GT distribution in cerebral endothelium. Immunogold label is concentrated at the luminal (L) and abluminal (abL) plasma membranes. (d) Detail of an abluminal endothelial plasma membrane exhibiting GT immunoreactivity. The plasma membrane and subplasmalemmal compartment depict a high concentration of immunogold particles.

76


brain endothelial cells of the rat and hEGT possess common immunogenetic determinants (Gerhart ef al., 1989). Using a cDNA clone coding for mRNA of a GT protein in rat brain, Birnbaum ef al. (1986) found a 97% correlation between the amino acid sequences in rat brain and those of hEGT and the human hepatoma cell line (HepG2), respectively. In contrast, the primary structures of human liver and kidney GT proteins exhibit a homology of merely 55% (Fukumoto ef al., 1988). However, the successful cloning of rat brain GT did not contribute to its morphological localization; instead, electron microscopic investigation allowed the ultrastructural identification of the GT protein in brain endothelial cells (Fig. 5c,d). Quantitative analysis of gold-labeled antibody distribution indicated that 88% of the GT protein was situated along the plasma membrane of the cerebral endothelial cells and in a subplasmalemma1 pool within a distance of up to 0.15 p m from the membrane. Thereby, the density of anti-GT labeling was distributed asymmetrically both luminally and abluminally: 36% of the gold particles were identified within the luminal plasma membrane, whereas 50% were associated with the abluminal membrane of the endothelium. The subplasmalemmal pool of GT proteins constitute approximately 38% of membrane-associated labeling. These observations are indicative of the polarized organization of the cerebral endothelial cells. In the perinuclear cytoplasma, only isolated anti-GT labeling ( 15%,, frequently in conjunction with vesicle-like structures, was observed. The distribution pattern of GT proteins in the cerebral endothelial cells is significantly different from the pattern of noncerebral organs. Both the distribution pattern as well as the primary structure of the GT protein have not only been described as tissue-specific (Wang, 1987; Wang and Brennan. 1988) but also species-specific (Allard and Lienhard, 1985; Mueckler et al., 1985). In this manner, a form of tissue adaptation according to the significance of glucose to the tissue’s functional role is feasible. In peripheral tissue, GT regulates the passage of glucose into the cytoplasm where glucose, then, is made available for cellular metabolism. In contrast, the endothelial cells of the brain do not transport glucose in order to sustain metabolism. Instead, they direct glucose molecules via transcellular transport into the brain tissue proper where glycolysis is used to satisfy the energy requirements of the nerve and glial cells. Little is known about GT trafficking in BBB endothelium. In nonnervous tissue, however. trafficking routes have been partially elucidated. Karniele et al. (1981) describe the following hormonally regulated model for glucose transport in adipocytes and muscle cells: stimulated by the binding of insulin on a specific membrane receptor, the translocation rate of intracytoplasmatic glucose transporters stored within vesicles is accelerated. At the plasma membrane, the glucose-loaded vesicle fuses with the


77

cell membrane in a manner resembling exocytosis. With the dislocation of insulin on the membrane receptor, an endocytosis-like mechanism similar to receptor-mediated endocytosis ensues: the GT reenters the cytoplasm, stored in vesicular form until needed for further trafficking. Erythrocytes also transport glucose across their plasma membranes via facilitated diffusion; however, this glucose transport mechanism does not respond to insulin (Czech et al., 1978). In this respect, erythrocytic glucose transport virtually resembles that of the BBB. In kidney and small intestinal epithelia, glucose transport occurs in conjunction with an energy-dependent, Na+ system (Simpson and Cushman, 1986): when a favorable, inwardly directed Na+ concentration gradient exists, glucose is transported solely in the presence of Na+ by means of a common passive carrier. The high external Na+ concentration, in turn, is maintained by active Na+ export against the concentration and energy gradient via membrane-integrated Na+-K+ATPase . Blok et al. (1988) described the primary binding location of GT in the tubulovesicular structures of cultured 3T3-adipocyteson the trans side of the Golgi apparatus, the area where membrane components are “packaged” for further transport (Griffiths and Simons, 1986). Glucose transporter is also present in relatively minute concentrations in small vesicles distributed within the cytoplasm. Only the tubulo-vesicular GT, however, is sensitive to insulin stimulation, resulting in vesicular migration toward the plasma membrane. The above described storage of GT, bound by cytoplasmic vesicles, is characteristic for peripheral cells and organs. In the cerebral endothelial cells, GT occurs primarily in plasmalemmal and subplasmalemmal pools which are not stored in vesicles within the cytoplasm (Fig. Sc,d). It is, however, not clear in which form the GT protein is transported into the cytosol; furthermore, the molecular changes which are necessary for this protein to pass from the hydrophilic cytosol to the hydrophobic plasma membrane have not been defined. The initial results of Mueckler and Lodish (1986) indicate that the posttranslational membrane insertion of GT is apparently ATP-dependent. The results presented in this review discuss the storage of GT as a subplasmalemmal pool in cerebral endothelial cells. This pool enables rapid and effective translocation in the endothelial cell membrane without activating vesicular transport, a mechanism which also ensures the rapid transfer of glucose across the BBB. Yet unexplained is whether additional receptor molecules are involved in the stimulation of canier-mediated glucose transport. The immunocytochemical application of anti-GT antibodies to cerebral microvessels subjected to different physiological stress factors may, therefore, yield definitive answers to this important issue.

78


3 . Tight Junction-related Protein ZOI

One intrinsic feature of the BBB endothelium is its ability to form complex tight junctions which effectively seal the interendothelial spaces (Reese and Karnovsky, 1967). The BBB properties of cerebral endothelium, therefore, rely significantly on the presence of tight junctions. The physiological correlate of tightness in epithelial membranes is transepithelial resistance. Leaky epithelia generally exhibit electrical resistances below 100-200 R-cm'. Cerebral endothelium may be classified as very tight endothelium according to its electrical resistance of about 2000 Qcm2 (Crone and Olesen, 1981). In this respect, it resembles the high resistance epithelia of frog skin (Crone and Levitt, 1984) and toad urinary bladder (Fromter and Diamond, 1972). A common morphological technique to determine the tightness of BBB endothelium is the intravenous application of exogenous protein tracers and visualization by subsequent cytochemical techniques (see section 11,C). The freeze-fracture technique has also been successfully employed to evaluate BBB endothelial tight junctions in sitir (Fig. 6a) (Dermietzel, 1975b) under both normal and pathological conditions (Nagy et a f . , 1984). Despite the prime significance of tight junctions in compartmentalizing spaces within the body at their epithelial boundaries, the biochemical analysis of tight junctions is still in its infancy. Progress was made, however, with the successful production of a mAb that recognizes a tight junction protein in mouse liver with an approximate molecular weight of 225,000 on SDS gel. This protein is associated with the cytoplasmic membrane side of the tight junction domain and is primarily an extramembranous, asymmetrical phosphoprotein (Stevenson et af., 1988a,b). Stevenson et al. (1986), who first characterized the zonulae occludensassociated protein (ZO1) in liver plasma membranes, discovered crossreactions between ZO, antibodies in the intercellular zonulae occludentes of epithelia in rodent kidney, colon, testis, and arterial endothelium, as well as in tight junctions of confluent monolayers from culture of rabbit kidney epithelium (Madin-Darby, MDCK cells). These cross-reactions indicate that the antigen determinants which are recognized by the antiZO, antibody are, most likely, identical in many mammalian tissues. FIG. 6. (a) Freeze-fractured cerebral endothelium showing a fibrillar tight junction component at the protoplasmic P-face. The inset shows the extracytoplasmic E-face in detail. (b) Through anti-ZOI immunoreactivity at an endothelial cerebellar plasma membrane, a fibrillar orientation of tight junction-associated phosphoprotein ZOI becomes apparent. Double immunolabelingusing anti-ZOI(c) and anti-factor VIII-associated antigen (d) antibodies. The latter antibody has been used to conclusively prove endothelial labeling in conjunction with the anti-ZO1antibody.

80


Electron microscopic investigations performed by Stevenson et al. (1986) on isolated plasma membrane sections from mouse liver yielded a labeling pattern for anti-ZO, on the cytoplasmic side of tight junctions. The Z01 protein may also be extracted through the induction of high salt and/or urea concentrations, confirming the protein’s peripheral association with tight junctions (Anderson et al., 1988). With regard to tight junctions in epithelia, it is assumed that the basolateral epithelial plasma membranes interact with cytoskeleton components, resulting in a control mechanism for the permeability of the paracellular, epithelial transport (Madara and Dharmsathaphorn, 1985; Stevenson et al., 1988b). We successfully demonstrated the ultrastructural localization of a-actinin at the lateral plasma membrane of enterocytes and at sites closely linked to the tight junction domain. A regulative influence of the cytoskeleton on tight junction permeability appears feasible in association with actin, concentrated at the apical lateral plasma membrane of small intestinal epithelium (Drenckhahn and Dermietzel, 1988). Physiological evidence indicates that the ionic permeability of BBB endothelium may change in conjunction with transendothelial osmotic gradients (Rapoport and Robinson, 1986). Furthermore, freeze-fracture findings indicate that opening probably occurs at the tight junctions of brain capillaries (Nagy et al., 1984). The cytoskeleton may also be involved in the reversible opening and closing mechanism of the BBB. Anti-ZOI immunoreactivity was successfully demonstrated in cryostat sections of adult brain tissue (R.Dermietzel et al., unpublished observations). By indirect immunofluorescence, sections along the endothelia of brain microvessels exhibited fibrillar fluorescence, which appeared to be localized at the lateral aspects of brain microvessel (Fig. 6b,c,d). The fibrils identified by immunofluorescence apparently constitute interendothelial tight junction domains, not corresponding to the single strands of intermembranous particles obtained by freeze-fracture (Fig. 6a). The ZOI protein occurs in approximately the same quantities (molecules per micron) as the intramembranous particles that constitute the junctional fibrils in freeze-fracture preparations (Anderson et al., 1988; Stevenson et af., 1988b). However, junctional permeability cannot be estimated by the amount of ZO1 or immunofluorescencepattern as has been shown recently by Stevenson et al. (1988b). Rather, permeability depends on biochemical properties, e.g., the degree of phosphorylation (Stevenson et al., 1989), which cannot be distinguished by structural criteria (Stevenson et af., 1988b). Other individual junctional elements such as the recently described tight junction-related protein, cingulin (Citi et al., 1988), may also account for the actual transjunctional resistance of a given epithelium. Therefore, ZO1 content and its staining pattern in BBB endothelium are


81

not necessarily indicative of BBB tightness. In spite of these restrictions, the presence of ZO1 is a reliable indicator for the existence of tight junctions in the central nervous system (CNS). We have obtained evidence that ZO1 immunoreactivity occurs at ail known sites of tight junction expression in the cell layers constituting permeability barriers in the CNS. These include the leptomeningeallayer (Dermietzel, 1975b) which effectively seals the subarachnoid space from the dural compartment (Fig. 7a), the choroid plexus with its tight epithelium (Fig. 7b) (Dermietzel et al., 1977;van Deurs, 1980),and the ependyma which lines the inner ventricular space (Fig. 7c) (Brightman and Palay, 1963). The only CNS tight junctions in which no immunoreactivity for anti-ZO1 has been detected are the interlamellar tight junctions of the central myelin (Dermietzel, 1974a; Mugnaini and Schnapp, 1974). The ubiquitous occurrence of ZOI in the CNS makes the application of this protein unreliable as a quantitative means for estimating BBB tightness. In combination with tracer studies, however, 201 can be used as a tool for the direct detection of de novo tight junction assembly (Fleming et al., 1989), e.g., in developing central nervous tissue. B. DETERMINATION OF BBB-RELATED ANTIGENS BY PROPAGATION OF ANTIBODIES AGAINST ISOLATED BRAINENDOTHELIA OR BRAINHOMOGENATES

Isolated brain microvessels can be used as a collective irnmunogen to produce antibodies to BBB endothelium and/or its structural adjuncts. This approach principally differs from that which was described above, as the nature of the immunogen recognized by the prospective antibody is unknown. The antibody obtained, however, can be further used as an immunoprobe to better define the antigen by immunochemical and/or molecular biological techniques. A variety of monoclonal and polyclonal antibodies have been generated, thus far, that react with brain microvessels. A list of these “BBB-specific” antibody immunoprobes is presented in Table 11. A brief description of the immunochemical and imrnunocytochemical characteristics of these antibodies follows. 1. Zmmunomarkers Specac for BBB Endothelia

Hart et al. (1981) described a polyclonal antiserum generated to plasma membranes from cultured mouse brain endothelial cells. The antibody reacted with brain microvessel in cryostat sections, binding to the luminal side of the endothelium after intravascular injection. The rationale for the production of this antiserum was primarily to prove the in uiuo effect of brain endothelium on an anti-endothelial antibody. The antigens which


83

TABLE I1 ANTI-BBBANTIBODIES Source of antigen

Type of antibody

Antigen MW (kDa)

Onset of expression

Mouse brain endothelium Ratlbovine brain capillaries Rat brain microvessels Chick retina

Poly clonal serum Polyclonal serum mAb

n.d.*

n.d.

45

n.d.

n.d.

n.d.

mAb (HT7)

74

El0 (chick)

Rat brain homogenate

mAb (antiEBA)

Rat brain microvessels Chick retina

mAb

30 2s 23 140

mAb ___

~

43 (neurothelin)

P3 (rat) El8 (rat) E9 (chick)

Reference

Hart et al.

(1981) Pardridge et al. (1986) Michalak e t a / . (1986) Risau et al. (1986b) Sternberger and Sternberger (1987) Krause et al. (1988) Schlosshauer and Herzog (1990)

(' n.d.; not done

were recognized by this antiserum were not further specified. Therefore, this serum did not contribute to the molecular characterizationof the BBB. Rather, it contributed information on the brain endothelial reactivity which is allegedly involved in the autoimmune response of brain microvasculature (see below). A polyclonal antiserum directed to a 45 kDa protein of isolated bovine and rat capillaries has been described by Pardridge et al. (1986). The antigen appears to be localized in the lateral membranes of cultured bovine brain endothelial cells, displaying excessive labeling of brain microvessels in paraffin-embedded sections. From light microscopic data on cultured brain endothelial cells, Pardridge et al. (1986) deduced an asymmetrical distribution of the antigen; furthermore, they suggested that the 46-kDa protein is a component of the interendothelial tight junction complex in FIG. 7. Set of immunofluorescence micrographs showing anti-ZOI immunoreactivity in different brain-CSF border regions. (a) Labeling of tight junctions by the anti-ZOl antibody in leptomeningeal neurothelium which effectively separates the CSF compartment from the subdural space. (b) Intensive anti-ZO1 immunoreactivity expression in the epithelium of the choroid plexus (CP).(c) Tangentially sectioned and immunolabeled ependyma. A honeycomb pattern outlines the ependymal cells at their apical junctional domains.

84


brain capillaries. From the decribed data for Z O , , it appears unlikely that the 46-kDa protein is identical or homologous with this tight junction protein, as suggested by the authors. However, as the tight junction complex seems to be composed of an assembly of different proteins (Dermietzel et al., 1980; Stevenson et al., 1988a) and presumably associated lipid components (Dermietzelet al., 1980; Kachar and Reese, 1982),the 46-kDa protein might well represent a constituent of the brain interendothelial junctional complex. Further electron microscopic data on the localization of anti-46-kDa immunoreactivity is necessary to conclusively address this issue. To date, five mAbs have been characterized which show BBB specificity. Four of them are immunoreactive to BBB endothelium (see Table II), while the fifth labels an antigen on the plasma membrane of cerebral pericytes (Krause et nl., 1988). The four mAbs that apparently react with the BBB endothelium will be reviewed collectively; the latter will be discussed separately. Michalak et a / . (1986) reported on a mAb that labeled the cytoplasm of cerebral microvascular endothelial cells, their luminal membranes, and an extracellular layer which may represent the endocapillary coat. The biochemical nature of the antigen, however, has not been established. From their electron microscopic data, it seems most likely that the antigen is a specific component of the protein complement which coats the luminal wall of the cerebral endothelium and is not part of the endocapillary coat associated with other vessels. Similar immunostaining of BBB endothelia was obtained with a mAb propagated in response to rat brain homogenate (Sternberger and Sternberger, 1987). This mAb reacted with a protein triplet of MWs of 30,000,25,000, and 23,000, respectively. Because of its immunoreactivity to BBB endothelia possessing permeability barriers, the antibody was named anti-endothelial barrier antigen (anti-EBA). In addition, anti-EBA recognized epitopes outside of the nervous system: in select spleen blood vessels, nonvascular spleen cells, and minute cells in the skin (tentatively designated as Langerhans’ cells). A correlation between EBA expression in BBB endothelia and in cells associated with the immune system led the authors to speculate that EBA may represent a class I1 major histocompatibility antigen (Ia). The apparent loss of antiEBA immunoreactivity in vessels through experimentally induced allergic encephalitis, a situation in which Ia antigen presentation of endothelial brain cells has been demonstrated, however, rendered this interpretation less likely (Sternberger et al., 1989). One common feature of anti-EBA and the mAbs discussed below is the lack of immunoreactivity in microvessels located in ‘‘leaky’’ microvascular segments, i.e., the blood vessels of the CVOs and the choroid plexus.


85

These findings also support the hypothesis that the EBA antigen is a BBB-specific protein. A mAb (HT7) has been recently described by Risau et al. (1986b), HT7, which specifically reacts with chick brain endothelium and a plasma membrane antigen present in embryonic blood cells (Fig. 8a). The corresponding antigen appears to be expressed on the luminal surface of brain endothelial cells. Its presence in other vascular cells such as pericytes and perivascular astrocytic endfeet remain to be determined by electron microscopic immunocytochemistry. Like anti-EBA, the HT7 antibody also failed to stain blood vessels outside of the brain, suggesting its specificity to a BBB-related antigen. Interestingly, this protein is also expressed in choroid plexus epithelial cells which define the blood-CSF barrier. On Western blots, the HT7 antibody recognizes a 74-kDa protein in isolated chick brain capillary extract. Using chorio-allantoic membrane as host tissue, Risau et al. (1986b) further demonstrated that avascular mouse brain is capable of inducing HT7 antigen in chick chorio-allantoic blood vessels which were in the process of invading the brain tissue and normally do not express this BBB-related antigen. In other words, brain tissue can induce the expression of BBB-related antigens in endothelial tissue of foreign origin. This finding coincides. with earlier transplantation data from Steward and Wiley (1981) who showed that the transplantation of embryonic quail brain into the coelomic cavity of chick embryos results in vascularization of the transplant; the invading vessels assumed features of the BBB. Although inductive factor(s) have not yet been considered, considerable evidence suggests that the astroglia play a decisive role in the BBB differentiation processes (Goldstein, 1988). Thus, immunoprobes provide a useful means for biochemically describing BBB differentiation and/or induction processes. The most recent probe that recognizes a BBB-specific 43-kDa glycoprotein, neurothelin, is a mouse mAb (1W5) which was originally raised against lentil-lectin-binding proteins (Schlosshauer and Herzog, 1990). In many respects, this antibody shows the same staining pattern as the antiEBA and Risau’s HT7 mAb. Unfortunately, a scrutinous, in situ labeling of different brain tissue, including CVOs and the choroid plexus, has not been performed; thus, the differential properties of this mAb within the brain microvascular segments still require elucidation. Systemic application of the mAb indicates that neurothelin is expressed on the luminal side of BBB endothelium and, like the HT7 epitope, has been shown to be induced by mouse brain in chick microvessels generating from the chorioallantoic membrane after tissue transplantation. In addition to BBB endothelium, other neural tissue or neuroepithelial derivatives express neuro-


87

thelin, e.g., the neurons of nonvascularized chick retina and epithelial pigment cells. Cell culture experiments utilizing epithelial pigment cells show preferential expression of neurothelin at cell-cell contact sites. The association of neurothelin with tight junctions, however, is considered less likely due to the coinciding expression of this antigen in neurons and erythrocytes. The expression of neurothelin outside the brain has not been further evaluated, but the authors (Schlosshauer and Herzog, 1990) do indicate that neurothelin is present at the transport interfaces of kidney tubuli, although its location has not been pinpointed within the tubuli. 2 . A Monoclonal Antibody Specificfor Cerebral Pericytes In addition to the perivascular glial ensheathement, it has been suggested that the pericytes of the cerebral microvasculature also cooperatively participate in the regulation of biood-brain barrier homeostasis (Baker et al., 1971; Cancilla et al., 1972; Kristensson and Olsson, 1973; van Deurs, 1976).Cerebral blood vessel pericytes have been implicated in a “second line of defense” that operates beyond the endothelial front when it becomes impaired (Farrell et al., 1987). We have propagated a mAb (Krause ef a f . , 1988) that recognizes a 140-kDa glycoprotein on cerebral pericytes (Fig. 8b,c). This antibody reached, in particular, arterioles, capillaries, and postcapillary segments; the “leaky” segments of the area postrema and choroid plexuses remained negative for the antibody. The ultrastructural localization of the 140-kDa antigen has been carefully analyzed by the immunogold method: the 140-kDaglycoprotein was scattered in groups throughout the endothelial and ab-endothelial plasma membranes of pericytes (Fig. 9a). The antigen appears to be an extracytoplasmic, peripheral protein possessing no homolog at the endothelial front. Gold particles were often found to be aligned in chains, suggesting that the antigenic target may be composed of a filamentous protein (Fig. 9b). The 140-kDa glycoprotein seems to be specific for cerebral pericytes and not shared by the same cells in nonnervous counterparts. Since the pericytes are thought to perform a type of permanent, “backup” role in BBB regulation via endocytosis of bloodborne proteins under normal (Broadwell et al., 1988) and pathological

FIG. 8. (a) Immunofluorescence of chick cerebral microvessels using a BBB-specific monoclonal antibody (HT7) (Micrograph courtesy of W. Risau). (b) Double immunofluorescence with a cerebral pericyte-specific monoclonal antibody (anti-140-kDa). (c) Corresponding immunofluorescence with anti-Factor VIII-related antigen. Immunoreactivity performed to define blood vessels as immunotargets.


89

conditions (Camilla et al., 1972; van Deurs, 1976),this protein may represent a favorable marker of this particular adsorptive or transportive functions. 3 . What Does BBB-specgc Mean?

The diverse polyclonal and monclonal antibodies described thus far have been reported as brain endothelial or BBB-specific immunoprobes. This, however, is only true if one considers their endothelial or perivascular immunotargetingability. Interestingly, most of the antibodies do recognize one or more targets outside the BBB. Regardless of whether they are polyclonal or monoclonal in nature, the antibodies react with at least one additional target of non-BBB origin. The recognized non-BBB targets have one common feature, namely, they belong to the class of transporting epithelia. Only the anti-EBA described by Sternberger and Sternberger (1987) is an exception as it recognizes an epitope on some cells and tissues associated with the immune system. The anti-140-kDa mAb possesses the widest range of reaction sites outside the BBB, including plasma membranes of polarized epithelia such as the bile canaliculi front of hepatocytes, the brush border of enterocytes of the small intestine, and the proximal epithelial cells of the kidney. The mAb propagated by Michalak et al. (1986) showed a similar pattern of immunoreactivity, although the topology of the recognition sites differs from those of anti-140-kDa. One conclusion which can be drawn is that the specific protein constitution of the BBB consists of a collection of molecules which also account for the molecular setup of polarized epithelia. In terms of BBB specificity, this means that particular selection as well as topological expression of the BBB protein assembly comprises its phenotypical character rather than the expression of BBB-unique molecular species. The molecular fingerprint of the BBB is, thus, designed according to a quantitative and qualitative selection of molecules which are also common to the family of transporting epithelia. In this respect, the data collected on BBB immunospecificity corroborate the physiological hypothesis that the BBB possesses the character of a polarized epithelium (Betz et al., 1980; Crone, 1986b).

FIG. 9. (a) Electron micrograph of anti-140-kDa immunogold-labeled cerebral pericytes. The antigen is clustered on the extracytoplasmic side of the plasma membrane. (b) High resolution electron micrograph of an anti-140-kDa, immunogold-labeledplasmalemmal domain. The arrows point to a chainlike arrangement of some gold particles, implying the presence of a fibrillar conformation of this antigen.

90


IV. Ontogenetic Differentiation of the BBB A. PRENATAL A N D POSTNATAL DEVELOPMENT OF THE BBB

Already in embryonic day 1 1 (E 11) of rat development, the first blood vessels appear in the brain anlage. In this stage, initially a penneural plexus appears which covers the brain vesicles and neural tube (Evans. 1909; Lierse, 1963). At embryonic day 12-14, the first intracerebral blood vessels become visible. In general, these vessels are sporadically distributed within broad extracellular spaces, possessing endothelial walls which vary in thickness; fenestrae and intracellular slits are also present. Perivascular glia have not yet appeared (Caley and Maxwell, 1970; Bar and Wolff, 1972). On approximately embryonic day 15 and thereafter, the existent blood vessels gradually penetrate and branch within the differentiating brain tissue. During the ensuing vascular development continuing through postnatal week 2-3, penvascular pericytes and astroglial elements differentiate in coordination with the development of the brain parenchyma; the astrocytic endfeet wrap about the vessels. First in postnatal week 3-4 is the growth, in thickness, of the basement membrane completed: differentiation of the lamina rara and laminae densae follows.

B. IMMUNOPROBES AS MARKERS OF BBB DIFFERENTIATION The maturation of the BBB during the above described process seems to be developmentally regulated. The most common approach to visualizing maturation is the monitoring of the development of tightness in the BBB endothelium by intravascular injection of HRP with successive cytochemical determination. Developmental studies (Delorrne et al., 1970; Wakai and Hirokawa, 1978; Latker and Beebe, 1984; Risau ef al., 1986a) show that BBB tightness toward HRP develops in a certain spatiotemporal pattern and is species specific. The usefulness of exogenous tracers such as HRP for this purpose has been questioned explicitly by M8llgard and Saunders (1986). According to their data, BBB in chick, rat, and monkey exists from the earliest stages of development. The high concentration of plasma protein in the CSF of immature animals and human beings is regarded by these authors to be plasma protein gene expression by the developing brain and choroid plexus with subsequent release into the CSF. With respect to this controversy, the availability of a collection of BBB-specific markers has led to considerable efforts in the evalulation of the maturation process. The following conclusions can be drawn from the


91

data on the differential expression of BBB-related antigens: (1) BBB differentiation is a sequential event with a particular spatio-temporal pattern (Risau et al., 1986a); (2) Tightness of the microvessels in the embryonic brain does not necessarily indicate complete maturity of the BBB (Krause et al., 1988); (3) The maturation of the BBB is revealed by the sequential expression of BBB-related antigens (see section IV,C). From the above described markers, numerous antibodies have been used to trace their antigens’ developmental expression. In addition, enzymes including alkaline phosphatase, cholinesterase, and y-GT (Risau et al., 1986a) have been utilized for studying BBB maturation. Table I1 lists the sequential expression of the different antigens and enzymes. Although species-specific patterns of BBB-related antigen expression exist, there appears to be a clear correlation between the expression of BBB-specific markers and tightness in the embryonic cerebral endothelium. In rodent brains (for which more data is collectively available), the following scheme can be applied (Fig. 10). Apparently two chronological peaks exist for the onset of gene expression of BBB-related antigens. The first antigens assemble at approximately embryonic day 15 (ElYE16). This assembly essentially coincides with the point in time in which brain cerebral blood vessels become impermeable to HRP in murine. The second peak occurs around E17/E18. It would be premature to speculate about possible regulative mechanisms that might account for this sequence of differential expression of BBB antigens. It may be useful, however, to tentatively classify the BBB-related proteins into three groups according to their developmental appearance: phase E (early BBB) markers are expressed before BBB closure, phase I (intermediate BBB) markers are exhibited around the time of closure, and phase L (late BBB) markers are expressed after BBB “tightness” has appeared. We are fully aware of the inherent limitations of such a phasic classification, particularly when complex developmental phenomena such as the differentiation of the BBB are considered. The following rationale, however, favors this classification scheme. 1. Since transcellular transport via facilitated diffusion or receptor/

carrier-mediated transcytosis becomes obligatory when paracellular traffic is impeded, e.g., by blockage of the intercellular space by tight junctions, it is apparent that this process inevitably determines the pattern of receptor/transporter expression in the BBB. 2. The closure of the endothelium can be experimentally assayed so that a species-specific determination of the particular BBB-related proteins can be obtained. The fact that the closure of the BBB endothelium is a hallmark of BBB maturation becomes apparent when one

92


Birth

20

18

Phase L

16

Phase I BBB closure 14

Phase E

DAY EO

DevelopmentalSequence of BBB Marker Expression

FIG.10. Diagram summarizing the developmental sequence of BBB marker expression in rodent brain. BBB-related proteins are classified according to their time of appearance: phase E (early), phase I (intermediate), and phase L (late). The closure of the BBB is the pivotal event which determines the sequence of marker expression. ZO1, Zonulae occludensassociated protein; FSRA, factor VI11-related antigen; GT, glucose transporter; y-GT, y-glutamyl transpeptidase; TFR, transfemn receptor; 140 kDa. pencytic 140-kDa protein; CEA, cholinesterase; AP, alkaline phosphatase; EBA, endothelial barrier-related antigen.

considers the developmental expression of the BBB constituents discussed thus far. 3. If one considers the BBB as a homeostat that provides for specific, blood-borne substrates which are necessary for maintaining brain metabolism, the closure of the BBB during development is, conse-


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quently, indicative of the differentation processes occurring in individual brain cells. The latter aspect implies that the differentiation of the BBB is also reflected in the brain itself. We do not know exactly which particular functions of the brain require the barrier. Qualitative comparisons regarding the tightness of different barrier areas of the central and peripheral nervous systems (CNS and PNS, respectively) suggest that the “higher” functions of neuronal tissue are protected by a tighter bamer than the “lower” functions (Abbott et al., 1986). The internal milieu of the CNS apparently demands a high degree of protection from the remainder of the body in order to preserve its integrative function. Thus, through correlating the developmental pattern of expression of BBB-specificantigens with other brain differentiation processes, an insight as to exactly which brain functions are dependent upon the BBB phenomenon could be gained.

C. INTERRELATION OF BBB WITH BRAIN CELLDIFFERENTIATION According to our classification scheme, phase E markers apparently do not contribute directly to the barrier function. Early expression of the ZO1 antigen in embryonic brain vessels (around El3 in rat) (Fig. lla) does not necessarily contradict this assertion; rather, it indicates that the early endothelium may assemble “tight” junctions which are not, however, “tight” in aphysiological sense. Instead, ZOI may be involved in mechanical linking andlor assembly of the interendothelial contact zone. This hypothesis is in agreement with observations on different cell lines discussed above, indicating that ZOI expression does not necessarily coincide with the degree of “tightness” (Stevenson er al., 1988b). Our own observations on the expression of ZO1in the ependymal layer at El5 in rat are consistent with this idea: freeze-fracture preparations did not exhibit junctions with a high degree of continuity (Fig. 1la,b) in spite of considerable expression of ZOI. Another marker which may be classified within phase E is neurothelin. This novel BBB-related glycoprotein has been reported to be expressed in chick brain before E9 (Schlosshauerand Herzog, 1990). In contrast, BBB closure in chick embryo as judged from HRP experiments does not occur before E12-13 (Wakai and Hirokawa, 1978). The function of this antigen is unknown: a transport function has been suggested, but the topological distribution of neurothelin on cell surfaces of, for example, retinal neurons in situ, pigment epithelial cells and fascicular axons in uitro, implies that cell-to-cell contact may also play a critical role in the expression of this protein. A similar pattern has been demonstrated for a number of cell

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FIG. 1 1 . (a) Anti-ZO, immunoreactivity of rat embryonic ependyma at El5 revealing a high degree of immunofluorescence of this tight junction-associated protein. (b) Freezefracture obtained from the ependymal cell layer at the same embryonic stage as indicated above (El 5). Occasionally, condensed tight junction elements (arrows) are apparent.

adhesion molecules (CAMS). The direct involvement of neurothelin in a BBB carrier function remains to be clarified. One interesting observation, however, is the lack of neurothelin in differentiated brain neurons as well as its consistent expression in nonvascularized chick retinal cells. In this respect, the pattern of GT expression in developing rat brain is similar. Glucose transporter expression in the BBB shows a characteristic shift at the time of BBB closure; thus, this form of GT can be viewed as a phase I antigen. While the avascularized neuroepithelium at El2 and El3 shows a high degreee of GT expression, its prevalence is considerably reduced within the neuronal cells after the intracerebral vessels become “tight” to HRP (Fig. 12a,b). “Tightness” of the vessel wall obviously exercises an inhibitory effect on GT expression in neuroepithelial cells. Moreover, a form of triangular relationship may &en exist for the regulation of GT expression, as has been suggested for the neurothelin antigen (Schlosshauer and Herzog, IW),including a differentiation effect of neurons on astrocytes and astrocytes on endothelial cells.


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FIG. 12. (a) Glucose transporter (GT) immunoreactivity in embryonic brain (E14).The section exhibits a pattern of GT immunoreactivity at the mesencephalic-rhombencephalic fissure. (b) The corresponding sections show horseradish peroxidase (HRP) reactivity after intravascular HRP application and five minutes of circulation. On the mesencephalic portion, HRP-leaky vessels (arrows) are prevalent. In contrast, no HRP leakage occurs in the lower rhombencephalic region. Leakiness of blood vessels for HRP corresponds with GT expression in neuroepithelium [upper mesencephalic part in (a) and (b)] and tightness of blood vessels for HRP with exclusive expression of GT in the BBB endothelium [lower rhombencephalic part in (a) and (b)].

The “tightness” of BBB endothelium is apparently affected by astrocytic factors (see section V,B). This crucial step in BBB differentiation can exert a feedback effect on neuronal differentiation by inhibiting GT expression, in order that at least this type of GT is “down regulated” in subsequent neuroepithelial derivatives (Fig. 13). The described interrelationship between BBB closure and alteration in the expression of endothelial and/or neuronal antigens is a probable example of the interdependence of neuronal and BBB differentiation. Appropriately designed experiments which take into account this interrelationship may be advantageous in yielding further information on the morphogenetic processes underlying brain development. Two additional BBB-related antigens exhibit expression profiles around the time of BBB closure in rodent brains as well as possessing carrier

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Fiti. 13. Hypothetical model of the triangular interrelationship between neurons, glial cells, and brain endothelium during BBB development. The neurons provide a differentiating intluence on astrocytes (Hatten and Mason, 1986) which is assumed to trigger inductive astrocytic factors that initiate closure of the brain endothelium (Janzer and RafT, 1987). As a consequence, the tightening of the endothelium acts as a signal which down-regulates the expression of neural proteins prevalent only in immature neurons. A similar relationship may also account for the maintenance of BBB properties in the adult brain.

characteristics: y-GT and TFR (Risau et al., 1986a). Both appear around El5 in mouse brain. The onset of y-GT and TFR expression at this time permits their classification as phase I markers; furthermore, it supports the hypothesis that the transportive capacity of BBB endothelia is primarily generated at the time of closure. According to our hypothesis that the differing sequence of BBB marker appearance can be utilized as an indicator of transient metabolic requirements, the catalytic and/or transport role($ of these proteins may be crucial to brain function, even at an early stage of development. The theory that y-GT may play a central role in amino acid transport at the BBB is supported by this finding, as constant supply of hydrophilic amino acids is vital to brain metabolism, especially when the proliferative capacity of the germinal layer is at its peak. A correlation between the developmental appearance of y-GT in BBB endothelium with other amino transport systems may be revealing in exploring y-GTs functional capacity. The expression of TFR at the time of closure also indicates that a constant supply of iron is essential for the developing brain. The importance of iron to motor and behavioral functions has been well established (Pollitt and Leibel, 1982). Its mechanism within the brain, however, remains unclear (Connor and Fine, 1986). It appears unlikely that iron


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supplements administered to the brain during development are crucial to myelination, as may be inferred from the findings of Connor and Fine (1986) which indicated the presence of transferrin storage sites predominantly in oligodendrocytes. Oligodendrocytic differentiation and myelination are relatively late events in the differentiation of the CNS, continuing well into postpartum. The dependence of the developing brain on a constant level of iron, supplied via the TFR, may be indicative of the general importance of this element as an essential cofactor in numerous ironcontaining enzymes. A specific trophic role of iron in differentiating neurons and glial cells, well exceeding a general metabolic demand, appears feasible, as neurons and oligodendrocytes in culture require the addition of transfemn in support medium in order to survive. In contrast, most nonneuronal cells are not dependent on the presence of transferrin. Phase L markers appear after the “tightening” of the BBB. Enzymes (alkaline phosphatase and cholinesterase, for instance) have been detected by cytochemical means at this phase. Their specific function in BBB metabolism is virtually unknown. In addition, no specific substrates exist which could account for this phase L expression. Rather, their expression may reflect the general enhancement of BBB and brain tissue metabolism which inevitably results in increased phosphatase and cholinesterase activity. Of the above described proteins, the 140-kDa pericytic glycoprotein and the EBA antigen also belong to the phase L group. Notably, at least one of these antigens is not associated with the endothelium but rather with a secondary component of the BBB. Although we do not have direct evidence of a functional relationship between brain endothelium and brain pericytes, they may perform a scavenger function (Sturrock, 1987). This function is, most likely, not vital to brain metabolism; rather, it could represent a type of cleansing or defense mechanism which is integrated late in the development of the brain. Interestingly, it has been suggested that the EBA antigen is involved in the immune capabilities of the BBB, as implied from its co-expression in spleen and Langerhans’ cells. In this respect, phase L markers may be indicators of the emergence of secondary functions in the BBB, including the brains’ specific complement to the innate and acquired immune system.

V. Arguments for Better Definition of BBB Properties under Pathological and Experimental Conditions In general, cerebral malfunction, despite its origin, tends to cause opening of the BBB (Bradbury, 1986). It is not within the scope of this chapter to further discuss the pathology of cerebral microvessels (Suckling et al.,

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ROLF DERMIETZEL AND DOKOTHEE KKAUSE

1986; Cervos-Navarro and Ferszt, 1987);rather, we will discuss the possibilities offered by the implementation of BBB-specific markers in the study of BBB functional breakdown. The phenotypical appearance of brain microvessels is severely altered under pathological stress. However, the structural and functional changes that accompany malfunction are, by far, uniform. A . INFLAMMATORY REACTIONS

Subtle structural changes in the BBB are prevalent under inflammatory conditions. The most common model used to induce an inflammatory reaction at the cerebral endothelium is experimental allergic encephalitis (EAE). This induced form of encephalitis frequently serves as a model for studying autoimmune disease of the CNS and is structurally characterized by focal accumulations of inflammatory perivascular cells (Lassmann, 1983). Perivascular infiltration by blood-borne cells provides an indication that the endothelial bamer which normally restricts extravasation of leucocytes has become susceptible to endothelial cellular passage. Barrier breakdown is normally accompanied by an increase in permeability for blood-borne proteins (Reiber, 1980; Suckling et al., 1983). The pathogenic mechanisms which underlie EAE are still only vaguely understood. Recent experimental evidence suggests that an early event may account for promoting lymphocyte (mostly T-lymphocyte)migration, namely, the expression of Ia antigen of the major histocompatibility complex (MHC) (a class I1 MHC derivate in humans) (Sobel er al., 1984; Traugott et al., 1985a; Risau et al., 1990). The onset of Ta antigen expression obviously transforms the cerebral endothelium into an immuno-target for activated T lymphocytes which then leave the blood stream via “emperipolesis” (Astrom et a l . , 1968).The most striking feature of this form of leukocytic extravasation is that it follows a route through the endothelial cytoplasm, leaving the interendothelialjunctions unaffected. This kind of leucocytic migration has been described for lymphocytes in chronically relapsing experimental encephalitis (&trom er af., 1968) as well as for polymorphonuclear leukocytes (PMN) in acute meningitis (Faustmann and Dermietzel, 1985).During the acute phase of EAE, the BBB becomes permeable to serum proteins. Whether this phenomenon precedes or coincides with cellular infiltration is still subject to discussion (Suckling et af., 1983). The most obvious finding pertinent to our discussion of changes in BBB markers is the active involvement of endothelium in the promotion of leukocytic emigration. Two primary aspects seem to be involved in the stimulation of migration: ( I ) change of the molecular endothelial complement, i.e., expression of class Ia antigens; and (2) intensive leukocyte-


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endothelial interaction, first promoting leukocytic adhesion and then transendothelial emperipolesis. A substantial body of evidence suggests that the perivascular astroglia are also involved in initiation of the immune response, as they are competent in expressing Ia antigens after EAE induction (Fontana et al., 1984, 1987; Traugott et al., 1985b; Fierz and Fontana, 1986) and viral infection (Massa et al., 1986). B. TUMORS AND TRANSPLANTS Intracerebral tumors show a loss of interendothelial tightness (Long, 1970), an increase in vesicular activity, and fenestration in the endothelial wall. Moreover, they become permeable to proteins and are also often accompanied by the appearance of collagen-filled perivascular spaces (Waggener and Beggs, 1976). The degree of barrier breakdown appears to correlate with tumor malignancy; even in isolated tumors, BBB deficiencies appear to be heterogeneous (Butler et al., 1978;Greig, 1984).Since the blood vessels vascularizing the tumor derive from BBB-competent, peritumoral brain tissue, it seems likely that the tumorous tissue lacks a certain factor which normally maintains barrier function. In particular, astrocytic engulfment has been implicated in providing the inductive factor(s1 (DeBault and Cancilla, 1980; Janzer and Raff, 1987). As discussed by Bradbury (1986), simple extrapolation based on cumulative evidence presents certain dangers. DeBault and Cancilla’s (1980) in uitro experiment which showed a re-induction of y-GT in cerebral endothelial cells, when cocultivated with C6 glioma cells, could not be corroborated by in uiuo experiments. Astrocytomas induced by C6 glioma cells in rat brain exhibited no indications of tightness in their vascular complement (Shivers et al., 1984) (associated with a high frequency of endothelial vesicles and assemblies of vesiculo-tubular channels). Therefore, the inductive effect of C6 cells in uitro,indicating the reexpression of some amino acid transport capacity (Beck et al., 1984), cannot be taken as a reconstitution of general BBB properties. A similar situation arises in transplantation experiments. The experiments of Steward and Wiley (1981) suggest that avascular tissue from 3 day old quail brains is capable of inducing a competent BBB within blood vessels of noncerebral origin when transplanted into the coelomic cavity of chick embryos (see above). In contrast, when avascular grafts of quail coelomic tissue (mesenchymal in nature) are transplanted into embryonic chick brain, the invading vessels originating from chick brain do not show BBB characteristics. This data has been interpreted as supportive of the general concept that the BBB properties are governed by tissue instead of vessel origin. Janzer and Raff (1987)further showed that type I astrocytes

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can induce bamer properties in newly formed microvessels, sprouting from the iris into astrocytic aggregates. By transplanting fetal neocortex which already possessed certain blood -brain characteristics into the fourth ventricle or directly into neocortex of rat recipients, however, Rosenstein (1987)was unable to induce the barrier characteristic toward blood-borne proteins in transplant vessels: the vessels exhibited permanent barrier dysfunction, probably due to microvascular changes. As invading capillariesbudded from intact brain tissue, one would have expected, according to the Steward and Wiley (1981) paradigm, that the newly formed vessels exhibit bamer properties including, at the very least, restriction of blood-borne proteins. No simple explanation exists for these discrepancies other than that the inductive/suppressive events which generatehhibit the BBB phenotype of cerebral blood vessels are highly dependent on the microevironmental conditions prevailing in the nervous tissue. One critique of most of the described experiments is that, in general, only one factor, e.g., tightness to protein tracers or expression of a single BBB-related enzyme, was used as a criteria for determining the presence of BBB properties. As already discussed, "tightness" does not necessarily mean the achievement of complete BBB capability. The complex functional interrelationship between the vascular bed and the surrounding neural tissue (including neurons as well as glial cells) which interact during embryonic development may also account for the constant maintainance of actual barrier functions in adult brain (Fig. 13). Indeed, deficiencies of BBB function under pathological and experimental conditions may result from an imbalance in the regulative loops among neurons, glia cells, and the cerebral endothelium. Although endothelial tightness seems to be crucial in the establishment of BBB properties, better definition of the specific molecular setups, including the primary and secondary structures, would be advantageous. For instance, the multiple factors which account for the formation and maintainance of the BBB can best be monitored with a collection of molecular probes which cover the spectrum of BBB functions. This is particularly relevant to the consideration of pathogenic mechanisms. Since pathogenic factors may involve only a particular set of functions (leaving the other functions unaffected), this strategy could provide a better insight into BBB malfunction. Moreover, by employing antibodies to specific antigens of BBB components, a more precise diagnosis could be obtained in cases of tumor malignancy, and profiles of soluble serum proteins in inflammatory diseases. Relevant to this suggestion is the research of Sternberger et al. (1989) who demonstrated a lack of EBA expression in animals with induced EAE. Our own data, obtained via the anti-140-kDa antibody, yielded similar results, as this marker was also


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absent in EAE animals (R. Meyermann and R. Dermietzel, unpublished observations). The determination of molecular complements by the immuno-approach reviewed in this article may, therefore, provide promise for future basic research as well as clinical application.

VI. Summary and Conclusion This review outlines the recent developments and improvements of our knowledge concerning the molecular composition of the BBB as revealed by immunocytochemistry. Data have been accumulated which show that the BBB exhibits a specific collection of structural and metabolic properties which are also found in tight transporting epithelia. This conclusion is substantiated by (i) the implementation of antibodies which recognize proteins of non-BBB origin, to show that these biochemical markers and the functions that they represent are localized in the BBB endothelium; and (ii) the characterization of target molecules to which polyclonal or monoclonal antibodies which have been generated to epitopes of the BBB endothelium or brain homogenates. According to these data the protein assemblies comprising the phenotypical appearance of the BBB can therefore be defined by the particular selection as well as topological expression of common epithelial antigens, rather than the expression of BBB-unique molecular species. In this respect the immunocytochemical data corroborate the physiological assumption that the BBB possesses the character of a specific polarized epithelium. Attention is also given to the description of developmental expression of BBB-related immunomarkers. By collecting the data from different sources we introduce a classification of the BBB marker proteins according to their developmental appearance. Three groups of proteins are classified with respect to their sequential expression around the time of BBB closure: Phase E (early) markers which appear before BBB closure, phase I (intermediate) markers which are expressed at the time of BBB tightening, and phase L (late) markers which are detectable after the closure of the BBB. Such a scheme may to be useful in better defining the maturation process of BBB, which apparently is not a momentary event in brain development, but rather consists of a temporally sequenced process of hierarchically structured gene expression which finally define the molecular properties of the BBB. This process continues even after parturition, especially with regard to the achievement of immunological properties of the mature BBB.

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By examining the developmental spatio-temporal expression of different BBB markers we conclude that the mechanisms governing the pattern of BBB maturation are not limited to the interactions occuring between glial and endothelial cells. We therefore suggest a heuristic model in a triangular interrelationship that includes differentiation effects of neurons on glia and of glia cells on the BBB endothelium. The third side of the triangle is considered to involve feedback from the maturation of the BBB endothelium on the neuronal differentiation process. Thus, further studies correlating the developmental pattern of expression of BBB-related antigens with the differentiation process of the neuronal and glial constituents should provide insights into exactly which brain functions are dependent upon BBB phenomenon. Finally, we concentrate on the potential utility of BBB-specific immunoprobes for a better understanding of pathophysiological mechanisms that lead to a breakdown of the BBB. Under normal conditions, subtle changes in the metabolic balance that maintain the BBB might alter BBB properties. In addition, under different pathological conditions, i.e., inflammatory reaction versus tumor induced dysfunctions, different patterns of BBB disintegration might be manifested. Application of highly selective sets of BBB immunoprobes to pathological tissues could prove to be considerably useful tools in achieving further insight into the pathophysiological events underlying BBB disturbances. The determination of the molecular components of the BBB and their changes in development, health, and disease by the immunological approach reviewed in this article may provide the impetus for future basic research as well as promise for clinical application. ACKNOWLEDGMENTS Laboratory research was supported by grants from the Nordrhein-Westfalen Ministry of Sciences (Dusseldorf) and the Deutsche Forschungsgemeinschaft (SFB 43). We gratefully acknowledge the technical assistance of Petra Altenhoff and Dorothee Schiinke. Special thanks to Barbara Bergdolt for her invaluable help with the manuscript and illustrations. Antibodies were donated by Dr. B. Stevenson (anti-ZO,) and Dr. Ch. Wang (anti-glucose transporter).

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