Table 1 documents that transcytosis is widespread. As expected, epithelial cells forming barriers between the outside world and the interstitium or between the internal world (circulation) and the interstitium are the major cells participating in transcytosis. However, the question of whether transcytosis occurs in all adult epithelia (e.g., kidney and skin) is open. While proximal tubule cells are endocytically active, only micronutrients seem to be "transcytosed" by them in vivo. Other segments of the nephron, e.g., the collecting tubule, are more difficult to assess. Transcytosis certainly occurs in the most obvious fetal organs, the yolk sac and placenta, and it probably operates elsewhere in the developing fetus. Further examination of Table 1 reveals that the transport of iron, vitamin B12, and the immunoglobulins IgA and IgG occurs in several organs. However, the routes and fates of the molecules are not always the same. Curiously, the routes and mechanisms by which circulating hormones gain access to their target tissues have not been extensively explored (181). Finally, although not yet examined in all polarized cells, the biogenesis of apical plasma membrane proteins is an example of endogenous molecules using transcytosis to attain their destination.
A. Transcytosis in the Vasculature
The most extensive exchange in vivo is that of plasma constituents across the endothelium that lines the inner surface of the blood vasculature. Of the three types of endothelium, continuous, fenestrated, and discontinuous (sinusoidal), only the first two form selective cellular barriers to the passage of macromolecules between the circulation and the underlying interstitium. All continuous and fenestrated endothelia throughout the vascular system are capable of the rapid and extensive bidirectional exchange of small and large molecules, but those of the capillaries and postcapillary venules are the major players in this activity (535, 536, 539). These two parts of the vascular tree constitute what is often called the microvascular exchange system, whose surface area is enormous (600 m2) (Fig. 1A). While fenestrated endothelia are more permeable to small solutes and water than are continuous endothelia, their relative permeability to macromolecules, and hence participation in transcytosis, is controversial (534). Although obvious, it is nonetheless important to state that transcytosis is but one of many important functions carried out by vascular endothelial cells, which are dynamic and capable of rapid responses to local changes in the environment.
1. Structural features of continuous endothelium
The simple, squamous epithelial cells of continuous endothelium are quite distinctive morphologically (Fig. 1B). They are remarkably thin (0.2–0.5 µm) in regions not including nuclei. A defining feature of these and all epithelial cells is a basement membrane that underlies their basal surface (Fig. 1C). It is made collaboratively by the endothelial and underlying interstitial cells. The most prominent intracellular feature is a population of smooth-surfaced vesicles of 50–70 nm diameter, some of which are in continuity with the plasma membrane facing the circulation (the apical or luminal surface), others in continuity with the opposite surface (the basolateral or abluminal surface), and still others apparently free in the cytoplasm (Fig. 1C). These vesicles, which have an 35-nm-diameter opening with a thin diaphragm across it, were originally termed plasmalemma vesicles but are now called caveolae ("small caves") because of their characteristic flask shape (Fig. 1D) (10). In continuous endothelial cells, the frequency of caveolae varies widely depending on the organ. For example, in endothelium of skeletal muscle (the diaphragm), the estimate is 1,200/µm3, whereas in pulmonary capillaries it is only 130/µm3 (539). This variation does not correlate with permeability, suggesting other functions for caveolae. Caveolae are also found in other cell types where their functions and compositions are actively being investigated (reviewed in Refs. 9, 546).
An important feature of endothelial cells is their tight junctions, which represent a barrier to paracellular diffusion (219). While there is good experimental evidence that the permeability of endothelial cell tight junctions changes depending on local conditions (336), the molecular basis has yet to be elucidated. The discovery of a large family of tight junction membrane proteins, the claudins, and their capacity to form heteroligomeric complexes with distinct permeability properties (580), will undoubtedly provide insights into the dynamic regulation of tight junction permeability in capillaries.
Important to an understanding of transcytosis in endothelial cells is the endocytic system, including clathrin-coated vesicles, endosomes, and lysosomes. These organelles are present in all capillary endothelium but are not abundant, and they are usually located in the thicker, perinuclear regions of cells. The endocytic system is clearly functional, as attested by the delivery of modified albumins and oxidized low-density lipoproteins (LDLs) to lysosomes (296, 506). But how do these cells distinguish between cargo destined for transcytosis versus that for degradation? The simplest explanation is that different cargoes use different receptors that are localized to different entry sites in the plasma membrane (PM). However, how do endothelial cells themselves utilize the same cargo that they transport for use by other cells; that is, how is the apportionment of cargo for self versus others regulated? As we shall see in section III, at least one cargo molecule (e.g., native LDL) may use different entry ports (i.e., caveolae versus clathrin-coated vesicles), offering the interesting possibility that the point of entry determines the subsequent fate of a particular internalized cargo molecule.
2. Microvascular permeability and transcytosis
Microvessels are approximately two orders of magnitude more permeable than other epithelia, making them leaky to the passage of circulating proteins into the interstitium. Most macromolecules move across capillary endothelium by bulk-phase not receptor-mediated mechanisms. Nonetheless, there is selectivity to the process, with the size and charge of cargo being important factors. Furthermore, although transport is bidirectional, the concentration gradient extending from the blood (apical side) to the interstitium (basal side) dictates that the bulk of transport is in an apical-to-basolateral direction. Finally, different continuous capillary beds have distinctive permselectivities, as evidenced by the varied compositions of the lymph draining from them.
The basis for high capillary permeability has been a controversy between physiologists and morphologists for over 50 years. Numerous reviews documenting the history, experimental details, and different interpretations have appeared in this and other journals (377, 467, 539, 571, 608, 614). Because the controversy centers on whether tight junctions or caveolae serve as the major (only) conduit for transported cargo, we will briefly recap this story.
On the basis of experiments in which he compared the compositions of the blood and lymph in the hindleg muscle of cats injected with various size tracers, Pappenheimer et al. (428) postulated in 1951 that plasma components (ions, small solutes, and proteins) were transported through two types of rigid pores: a small, 3- to 5-nm-diameter pore present at a frequency of 100/µm3 and a large, 20- to 40-nm-diameter pore present at 1% the frequency of the small ones (428). When the endothelium was seen at the ultrastructural level, no structures corresponding to the postulated pores were found. Instead, caveolae were observed, leading Bruns and Palade (65, 66) to suggest that they performed the function ascribed to the rigid pores. In the early years of this controversy, differences in the cell systems, approaches, and tracers used by researchers in the two camps often yielded conflicting results with differences in interpretations. However, both sides progressively refined their experimental approaches and have arrived at an apparent consensus: caveolae do play a role in transport across endothelia, either as fused channels (physiologists) or bonafide transport vesicles (cell biologists). Our position is that caveolae contribute to the high permeability of continuous endothelium. However, because their number exceeds the number of functional pores predicted by Pappenheimer's results, there must be other functions for caveolae. In fact, they have been proposed to harbor signal transduction components in both active and inactive states (9, 546). The recent reports of mice genetically engineered to lack the protein caveolin-1, a major component of caveolae, are particularly relevant and are discussed in section IV.
Although the controversy has focused on bulk-phase transcytosis, receptor-mediated transcytosis of specific macromolecules also takes place across the endothelia of the microvasculature (Table 1). Albumin and orosomucoid are both transcytosed in competable, saturable, and temperature-dependent fashions, and caveolae mediate their transport. A putative receptor for albumin of 60 kDa that is present only on continuous capillary endothelia has been identified by several groups (Table 1), but it has not yet been cloned and sequenced. The transcytosis of IgG across continuous endothelial cells is particularly interesting, in light of the expression of the neonatal Fc receptor (FcRn) by these cells (47) and the receptor's role in maintaining high serum IgG in the adult (Table 1). Does the receptor work in both the apical-to-basal and reverse directions? Are clathrin-coated vesicles used, as they are to carry maternal IgG from the gut to the interstitium (apical to basolateral) in neonatal rodents (Table 1)? Is excess IgG degraded when the endothelial FcRn receptor is saturated with its ligand, absent, or dysfunctional? If so, how? This area of endothelial cell biology deserves further study, because it might reveal the mechanism(s) used to selectively deliver a ligand (IgG) from the interstitum back to the circulation (basolateral to apical). We will return to several of these issues below. Finally, one polypeptide hormone, human luteinizing hormone/chorionic gonadotrophin (hLH/CG), is reportedly transcytosed via clathrin-coated pits and vesicles across the continuous endothelium of the testis (Table 1). The transport is apparently mediated by the same receptor present on Leydig cells, the target of the hormone in the testis. This system deserves further study, because it is one of two documented examples in which clathrin-coated vesicles of an endothelium transcytose cargo; the other is brain endothelia and transferrin-Fe (see sect. IIB1C).
B. Transcytosis in the Brain
Since the 19th century dye experiments of Ehrlich, the brain has been known as a "privileged" organ where access is tightly regulated so that the environment remains chemically stable. The brain's fluid is different from either the blood or noncerebral tissue. The two principal gatekeepers of the brain are the cerebral capillary endothelium and the epithelial cells of the choroid plexus (Fig. 2A). These cellular barriers are specialized for the passage of different nutrients from the blood (132, 552). The capillaries move nutrients that are required rapidly and in large quantities, such as glucose and amino acids. These small molecules are transported by membrane carriers using facilitated diffusion. The choroid plexus supplies nutrients that are required less acutely and in lower quantities. These are folate and other vitamins, ascorbate, and deoxyribonucleotides. Their transport requires energy since the blood concentrations of these nutrients are extremely low. Of relevance to this review is experimental evidence that transcytosis of a limited set of macromolecules occurs across brain capillaries from blood to the interstitium (the blood-brain barrier) but does not occur across the epithelial cells of the choroid plexus into the cerebrospinal fluid [the blood-cerebrospinal fluid (CSF) barrier].
1. Cerebral capillaries
Compared with other organs, the abundance of capillary endothelium in brain is very high (35). At the same time, permeability is about two orders of magnitude lower than that of endothelia in peripheral organs, giving rise to the designation of this endothelium as "tight continuous" (201). Two features of brain endothelia are different from endothelia in the periphery. Brain endothelial cells have the lowest frequency of caveolae (3), and the character of their tight junctions is influenced by underlying astrocytes through the actions of soluble factors, including cytokines (111, 263). Claudins 1 and 5 as well as occludin appear to be relevant players in providing a particularly tight junction (229, 289, 319, 385). Very little macromolecular cargo is transcytosed across the cerebral capillary endothelium. The three best-studied ligands are insulin, LDL-cholesterol, and iron; questions and controversy surround each.
- INSULIN. The finding that insulin-sensitive glucose transporters (GLUT 4) were present in the brain (312, 369) led to the search for insulin receptors on endothelium and hormonal effects on the cells. Although receptors were found (431, 592), the current status of insulin's transport by brain capillaries is not resolved.
- ) LSC Cells in the brain require cholesterol, which is synthesized endogenously (127), but can also be provided by the transcytosis of plasma LDL intact across brain endothelium (112). There is good evidence for the presence of LDL receptors on the luminal PM of cerebral endothelium (376). Such expression is unusual, since cells that are constantly exposed to the high LDL levels in plasma normally downregulate their LDL receptors. Experimental evidence from in vitro studies indicates that cholesterol levels in the underlying astrocytes play a role in regulating LDL receptor expression levels in the overlying endothelial cells (111). The puzzle here is how a cell distinguishes between LDL for its own needs and LDL for use by cells behind the permeability barrier it forms. In the periphery, this seems to have been solved by receptor-mediated endocytosis via clathrin-coated pits/vesicles for internal use versus fluid-phase (non-receptor-mediated) transcytosis via caveolae for use by interstitial cells (600). However, in the brain, there is virtually no fluid-phase (i.e., nonselective) transcytosis. Thus it will be important to localize LDL receptors in brain endothelium at the ultrastructural level; are they in caveolae or clathrin-coated pits? Another important issue is how transcytosed cholesterol is presented at the abluminal surface of endothelial cells, since apoprotein B, which is the apoprotein-carrying cholesterol in the circulation, is not present in CSF. Apoproteins A1 and E are the principal cholesterol-carrying molecules in the brain (127, 629). Underlying pericytes of the brain endothelium have been characterized as phagocytic; perhaps they participate in the degradation of apoprotein B and release of cholesterol into the CSF. Cholesterol dynamics in the brain have been reviewed recently (624).
- IRON. Iron is also transported across the blood-brain barrier, but there is conflicting data as to whether it is delivered with or without transferrin (Tf), the principal iron-carrying protein of plasma (52). (Iron and Tf are discussed in more detail in sect. IID2.) While several in vivo studies have reported that injected 125I-Tf does not accumulate to the same extent as 59Fe administered similarly (104), others report equivalent accumulations (152, 635). Tf receptors are definitely present on brain endothelium (248), and Tf is internalized by brain endothelium in vivo via clathrin-coated vesicles (476), leading some to speculate that plasma Tf may be carrying Fe across and then recycling back unloaded (52, 384, 429, 430, 545, 591, 635). Such a scenario would require a milieu on the basal side of sufficiently low pH to effect iron's release. Because the pH of the underlying interstitium in brain is not known to be acidic, there must be novel dissociation mechanisms not yet discovered. Whatever the mechanism, iron is not free in the brain interstitium but is complexed to Tf. Again, there is conflicting data about the source of this protein. Tf is synthesized and secreted by the epithelial cells of the choroid plexus (384). However, hypotransferrinemic mice have been shown to accumulate substantial amounts of intraperitoneally administered human transferrin intracranially, indicating that the endogenous source could also be derived from the serum (126).
The brain endothelial insulin and Tf-Fe transport systems have received attention from researchers working on therapeutic drug delivery systems (96, 161, 165). An anti-Tf-receptor antibody, OX26, is transcytosed into the brain mass, but the amounts are extremely low, Although this amount may be sufficient for drug delivery, it is not definitive evidence for quantitative transcytosis of the receptor along with its cargo. Nonetheless, this approach is being combined with toxins that bind to specific claudins and transiently open tight junctions, to deliver macromolecular drugs (100).
- IMMUNOGLOBULIN G. It turns out that brain endothelial cells express the FcRn and transport intracranially delivered IgG out of the brain in a receptor-mediated fashion (Table 1). The question is how circulating IgG initially crosses into the brain. As for the peripheral endothelium, the vesicular carrier and molecular mechanisms responsible for IgG transport are as yet unknown.
2. Choroid plexus
The choroid plexus is composed of a highly convoluted sheet of cuboidal ependymal epithelium that sits on a closely apposed basal lamina. Both morphological and biochemical tracers have provided good experimental evidence that the apical tight junctions of these epithelial cells are the blood-CSF barrier in the choroid plexus (see Fig. 2A). To date, no obvious ultrastructural or molecular features distinguish these junctions from neighboring ependymal cells, but we would predict that specific claudins are responsible for this difference (323). Interestingly, the basal lamina may act as an inducer of the tight junction specializations that make this cell type highly impermeable to macromolecules (reviewed in Ref. 590). The epithelial cells make much of the CSF that nourishes and cushions the brain. The protein content of CSF (25 mg/100 ml) is low relative to that of plasma (6,500 mg/100 ml), and the composition is different. Transthyretin, which binds thyroxine, and Tf are made and secreted by the epithelial cells, while apoproteins E and A1, which are present in lipoprotein particles in the CSF, are made by astrocytes. The presence of these proteins in CSF raises the obvious questions of where and how their ligands, presumably from the blood, reach them? Furthermore, are the components of CSF fluid functionally accessible to brain tissue or part of a drainage system much like the lymphatics? To our knowledge, there are no definitive answers.
What about transcytosis in the choroid plexus? Interestingly, although endocytosis is robust at the basal surface of the epithelial cells, transcytosis across to the apical environment is minimal to nonexistent; rather, virtually all tracers internalized from the basal side end up in lysosomes (590). The endocytic activity may reflect the high permeability of the fenestrated capillaries that supply the choroid plexus and hence the abundance of plasma proteins bathing the basal side of these cells. Van Deurs (589) examined transcytosis in the apical to basolateral direction as a possible route for elimination of waste from the CSF. Intraventricular injection of soluble horseradish peroxidase and cationized ferritin resulted in their overwhelming delivery to lysosomes; very small amounts appeared in coated pits along the lateral surface (589). The conclusion that apical-to-basolateral transcytosis was not an active pathway has been confirmed by others (24) using additional electron microscopic (EM) tracers.
C. Immunological Protection and Transcytosis
At several stages in the intricate choreography of the vertebrate immune response, transcytosis is used to move antigens and protective antibodies across epithelial barriers (Table 1). "Antigen sampling" is the first step in the mucosal immune response and entails the apical-to-basolateral delivery of soluble and particulate antigens to underlying mucosal-associated lymphoid tissue. This transcytotic event is carried out principally by M cells that are located in lymphoid follicle-associated epithelium throughout the gastrointestinal and urogenital tracts (175, 401, 402, 579, 621). Later in the mucosal immune response, polymeric IgA, secreted by appropriately activated plasma cells, is transcytosed along the basolateralto-apical axis by epithelial cells in the digestive tract, liver, and mammary gland and is released as secretory IgA into the gut lumen, bile, and milk, respectively (245, 302, 393). The third use of transcytosis occurs in a form of systemic immune protection, termed "passive immunity," which is the transport of maternal IgG to the developing fetus or neonate. Species differences dictate whether maternal blood or milk is the source of the IgG and whether the placenta or the intestine is the site of this transfer (178, 245). Certainly, the last two transcytotic processes start with uptake of their cargo through clathrin-coated pits/vesicles and may transit through parts of the endosomal system. Thus the mechanisms regulating these itineraries most likely differ from those used by endothelial cells, where a caveolar pathway predominates.
1. Structural features of intestinal epithelial cells
Figure 3 shows the tissue organization and ultra-structural appearances of M cells and enterocytes (adsorptive columnar cells), the two epithelial cells participating in transcytosis in the intestine. These cells are very different from one another and the capillary endothelial cell. Depending on the species, M cells comprise a variable but small percentage of the epithelia overlying organized mucosal-associated lymphoid tissue, making them a very minor cell population in the gastrointestinal tract. Being epithelial cells, their basal extensions sit on a basal lamina, but much of their basal membrane lines an extracellular "pocket" in which migrating monocytes and lymphocytes accumulate. As can be seen in Figure 3B, the pocket is a short distance from the apical surface. Thus these cells have evolved a short transcellular pathway much like the endothelial cells, but in contrast they have few to no caveolae; rather, coated pits are present on the apical PM. Figure 3B also shows that M cells do not have the luxuriant brush border that is present on adjacent absorptive enterocytes. They have short microvilli, or microfolds, hence the name M cells. In contrast, absorptive enterocytes are simple columnar cells with several apical features in addition to their brush borders (Fig. 3C). Clathrin-coated pits are present at the base of microvilli, and a thick glycocalyx composed of integral membrane proteins with glycosaminoglycan side chains emanates from the microvillar membrane. This latter structural feature as well as the rigidity of the microvilli are thought to prohibit microorganisms from attaching and invading enterocytes. The intracellular organization of these columnar epithelial cells is also polarized, with basally located nuclei, supranuclear Golgi, and an abundance of pleiomorphic membrane compartments underlying the terminal web of the brush border (Fig. 3C). The basolateral-to-apical length of this cell is 20 versus 0.2 µm for a capillary endothelial cell, making the transcytotic route across enterocytes potentially much longer. Furthermore, microtubules are an important structural element of the transcytotic pathway in enterocytes, but not in M or endothelial cells.
2. M cells, transcytosis, and antigen sampling
Quite early, researchers studying the routes of pathogen invasion discerned that specific regions of the intestine collected adherent particulate material present in the gut lumen; when the regions were visualized at the EM level, M cells were identified as the invasion route (72, 579, 621). The transcytotic route across M cells is thought to be part of the mechanism by which antigens are routinely sampled along the entire mucosal surface. Not surprisingly, numerous pathogens have evolved mechanisms to exploit the transcytotic process as a means to invade and disseminate before a strong enough immune response can be mounted (403, 464). In recent years, this route of entry has been studied intensively in the hopes of understanding the basic mechanisms of antigen sampling and developing effective vaccine delivery systems against stealthy invaders. Because adherence is an essential first step in invasion, researchers have focused on identifying the molecular basis for the selective adherence of antigens and pathogens to M cells and not adjacent enterocytes. Lectin staining in situ has been used in attempts to identify particular glycosidic moieties that might be differentially expressed by M cells (160, 186, 187). Recently, 1-integrin was localized to the M cell apical surface and proposed as the receptor for several pathogens (257–259). This membrane protein, which is expressed on the basolateral surface of neighboring absorptive cells, has a cytoplasmic tail that could mediate the endocytosis of particles bound to its extracellular domain. In support of this notion, M cells are avidly endocytic and their apical membrane is much more dynamic than the rigid and stable brush border of the enterocyte. In fact, both phagocytic and pinocytic mechanisms appear to operate at the apical surface of these cells. Adsorbed macromolecules are endocytosed via clathrin-coated vesicles (404) and delivered to a prelysosomal/lysosomal compartment from which they are released into the underlying pocket for subsequent uptake by lymphocytes and macrophages (404, 425, 616). Thus, unlike endothelial cell transcytosis, lysosomes appear to play a role in M cell transcytosis. Whether the cargo in this compartment is modified by acid hydrolases present in it (6) is not currently known.
3. Transcytosis of IgA
The large amount of mucosa-associated lymphoid tissue and its specialization for the production of IgA make IgA the major immunoglobulin in humans (301, 389). Given that it is synthesized and secreted by plasma cells located in the lamina propria of the digestive, respiratory, and urogenital tracts yet functions in external secretions, IgA must be delivered across an epithelial barrier. This requirement is accomplished by the polymeric IgA-receptor (pIgA-R), a single transmembrane protein synthesized by the epithelial cells. As discussed in detail in section IV, this receptor has a long (100 amino acid) cytoplasmic tail that contains most of the signals necessary to direct it through its cellular itinerary. However, unlike most other endocytic receptors that perform repeated rounds of cargo uptake, delivery and recycling, the extracellular domain of pIgA-R is cleaved upon delivery to the apical surface and released into the lumen with its ligand. The presence of the added "secretory component" stabilizes IgA in the gut lumen. This unique transcytotic system is expressed in many epithelia throughout the body, including kidney, trachea, and the digestive tract, including the liver (Table 1). Interestingly, some pathogens appear to have exploited the small percentage of uncleaved pIgA-R present in the apical membrane of nasopharyngeal epithelial cells to gain entry into the underlying interstitium (Table 1). This result suggests that the receptor is able or can be coerced to transcytose in an apical-to-basolateral direction. The mechanism, whether normal or pathogen induced, may have therapeutic potential.
4. Transcytosis of IgG
The transfer of maternal immunoglobulins to fetal or neonatal offspring provides the latter with systemic immunity until their immune system matures. Several organs transport IgG-type immunoglobulins (245). As with IgA, maternal IgG must be transcytosed across an epithelial barrier. In all mammalian species, it is transcytosed in an apical-to-basal direction. Thus in humans, IgG in the maternal blood is transported across the placenta (Fig. 2B), while in rodents, maternal IgG is first delivered into milk, a basal-to-apical route, and secondarily across the absorptive epithelial cells of the small intestine, an apical-to-basolateral route.
The receptor mediating the apical-to-basal transport of IgG is FcRn, a distant member of the major histocompatibility complex (MHC) I family (542). As for other MHC I proteins, the FcRn is a heterodimer, with a transmembrane heavy chain and 2-microglobulin light chain. The heavy chain has a cytoplasmic tail containing an internalization motif that mediates endocytosis of maternal IgG via clathrin-coated pits and vesicles present at the base of the apical brush border of neonatal rodent enterocytes. The receptor and ligand are transported through an endosomal compartment to the lateral surface of these cells (1). Thus this transport system shares the property with M cells of using a prelysosomal compartment to deliver its cargo. However, the unique pH dependence of binding allows the ligand to remain associated with the receptor at low pH (in the gut lumen and through slightly acidic endosomes) and be released at neutral pH (in the interstitial space) without apparent modification. The FcRn recycles back to the apical PM in neonatal intestine.
Although the finding that 2-microglobulin (2-M) knock-out mice lacked the apical-to-basal IgG transport system in the neonatal intestine was expected, it was initially surprising that circulating IgG in 2-M-null adults exhibited a much shorter half-life than in wild-type mice (177). This result suggested that FcRn played a role in IgG homeostasis (541), confirming a long-standing hypothesis by Brambell (54) that the prolonged circulation of IgG in plasma was due to a receptor capable of protecting IgG from degradation. Given the pH dependence of IgG binding to FcRn, the current view is that intracellular FcRn binds nonspecifically endocytosed IgG within an endosome-like compartment and returns it to the circulation (177). The tissues and cells performing the protective function may be hepatocytes or endothelial cells, since both express FcRn at the PM. Quantitative studies are needed.
An important finding in the 2-M knock-out mouse studies was that the levels of maternal IgG in colostrum and milk were normal, indicating that FcRn does not play a role in the transcytosis of IgG in the rodent mammary gland. This is not so surprising, considering that the direction of transport is opposite to that in the placenta or intestine, although in endothelial cells, the FcRn presumably carries IgG in the basal-to-apical direction (Table 1). The identity of the mammary gland receptor system will be important to determine.
D. Role for Transcytosis in the Homeostasis of Micronutrients
Most vitamins, essential minerals, and trace elements, collectively called micronutrients, come from the diet. Thus they must cross an epithelial barrier somewhere along the digestive tract; this occurs primarily at the level of the intestine. However, transcytosis is the least used route for micronutrient absorption. Lipid-soluble vitamins associate with bile acid micelles in the gut lumen and are thought to then partition progressively and passively across absorptive cells, associating with chylomicra somewhere before or at the basal side of the cells. Dietary vitamin B12 (cobalamin) is an exception, because it uses vesicle-mediated steps, in part, to cross intestinal cells. Many minerals are assumed to be absorbed paracellularly (61). This assumption is based on calculations of transit times and absorption rates. However, dietary iron is transported across the intestinal epithelium via multiple membrane transporters; once in the circulation, its delivery to the brain and fetus requires transcytosis. Additionally, Cu and Zn, as well as other heavy metals, appear to be transported into intestinal absorptive cells via membrane transporters at the apical plasma membrane. Finally, the kidney proximal tubule cells provide an important function in vitamin homeostasis by avidly scavenging several vitamins (Table 1) from the urine using a modified type of transcytosis.
1. Vitamin B12
All cells require vitamin B12 as a coenzyme in one-carbon transfers. Methyl malonyl CoA mutase uses it in the adenosyl-B12 form to convert methyl malonyl CoA to succincyl CoA in the mitochondria; methionine synthetase uses it in the methyl-B12 form to convert homo-cysteine to methionine in the cytoplasm. B12's journey to the cytoplasm of all cells is a fascinating and curiously convoluted process. The players so far identified are listed in Table 2 and placed in cellular context in Figure 4A. Several reviews cover this topic in more detail (276, 488, 514).
- UPTAKE FROM THE INTESTINAL LUMEN. In carnivores, B12 is present in ingested meat as the cofactors mentioned above. Upon digestion by pancreatic enzymes in the small intestine, free B12 is bound by intrinsic factor (IF), a 27-kDa glycoprotein secreted by parietal cells of the stomach (317). In the terminal ileum of the small intestine, the luminal B12-IF complex binds to its receptor, cubilin, a large membrane-associated glycoprotein that is located in the microvillar brush border of absorptive enterocytes (515). Cubilin's association with megalin, a member of the LDL receptor-related (LRP) family of endocytic receptors (reviewed in Refs. 192, 619), leads to the internalization via clathrin-coated vesicles of the entire cubilin-IF-B12 complex. After delivery of the IF-B12 complex to endosomes, cubilin and megalin recycle for further rounds of endocytosis; since cubulin's association with megalin is stable at pH 5, it is thought to stay associated throughout. Meanwhile, dissociated B12-IF is delivered to lysosomes, where the protein is degraded by leupeptin-inhibitable acid hydrolases (196) and B12 is transported out of lysosomes. This last step is mediated by a yet-to-be-discovered transporter(s). Interestingly, in this pathway there is no avoidance of lysosomes; to the contrary, lysosomal function is essential, since failure to degrade IF within the intestine results in a B12 deficiency in all subsequent tissues and cells. Some investigators are exploring the B12 entry pathway as a means to deliver drugs orally (487).
- TRANSFER TO CIRCULATING TRANSCOBALAMIN II AND TRANSCYTO SIS. A puzzle is the mechanism by which cytoplasmic B12 is subsequently transported into the basal milieu surrounding the enterocyte. Extracellular transcobalamin (TC) II, a 40-kDa protein in the interstitium/circulation, serves as the major functional carrier of B12 (Table 2). (TCI and TCIII are also B12 carriers, but their functions remain unknown.) TCII is synthesized and secreted principally by the liver (206, 495). Evidence that enterocytes express a TCII transcript (460) suggests that B12 may be loaded onto newly synthesized protein as it transits the secretory pathway (463). The B12-TCII complex would then be released at the basolateral surface of the enterocyte. Of course, this scenario requires the presence of a B12 membrane transporter in the secretory pathway.
Once in the circulation, how does TCII-B12 reach cells? All cells express a TCII receptor, which mediates the endocytosis of the complex via a clathrin-mediated mechanism. The TCII receptor is a single transmembrane glycoprotein of 62 kDa (49) that functions as a homodimer at the plasma membrane (Table 2). After internalization, the TCII-B12 complex is delivered to lysosomes, where TCII is degraded and B12 is again transported into the cytoplasm for subsequent use as a cofactor (249). But how does B12 reach cells behind a selective barrier, for example, the brain or testis? Although a B12-TCII receptor on brain or testicular endothelial cells has not been reported, we predict that it must be there. Could it be the same receptor as that found on the basolateral PM of most epithelial cells, even though it would be on the apical PM of these endothelia? What is the mode of transcytosis and the subsequent fate of the TCII protein and B12 in brain endothelial cells? Are the fates different from those in other cells? Clearly, this system would be interesting to explore further.
- INVOLVEMENT OF ADDITIONAL EPITHELIA IN B12 HOMEOSTASIS. There are additional aspects of B12 homeostasis that deserve comment. The kidney, yolk sac, and placenta express the protein components involved in intestinal B12 absorption (294). For example, cubilin is very abundant in the kidney proximal tubules, where it can bind and internalize B12-IF, again via megalin (91, 517). This is strange, since IF is not normally found in the circulation. Not unexpectedly, given the small size of TCII and thus its filtration by the glomerulus, there is a scavenger of B12-TCII in the kidney, and it is megalin itself on the apical PM of proximal tubule cells. Similar to its behavior in the intestine, megalin internalizes the protein-B12 complex and delivers it to lysosomes, where TCII is degraded and B12 is stored in a form that is retrievable into the circulation upon demand. This last step, storage of B12, points to possible differences between B12 dynamics in intestinal and kidney epithelial cells and is worth following up. Finally, antibodies to cubilin cause severe defects in developing fetuses, possibly due to a failure to deliver B12 and/or other essential components to the developing central nervous system via the yolk sac (516). As can be seen in Table 1, the cubilin-megalin system has the capacity to bind and move many different cargo molecules.
An essential cofactor in the homeostasis of every cell, iron in mammals exists in three predominant forms: bound to the circulating plasma protein transferrin; as heme in intracellular proteins such as mitochondrial cytochromes, hemoglobin, and myoglobin; and in ferritin, the storage form of iron (5). Iron is avidly reutilized by mammals, and thus the daily requirement for it is small. Of 3–4 g total body iron, only 1–2 mg are lost per day through desquamation. However, because there is no mechanism for its disposal, excess iron leads to disease (525, 623). Furthermore, the insolubility of both valence states at neutral pH and the toxicity of Fe2+ in the presence of oxygen makes control of this essential micronutrient especially vital. Studies of both iron deficiency and overload in human disease and animal models have helped to elucidate the mechanisms of iron homeostasis. There are many excellent reviews covering different aspects of iron metabolism (5, 11, 203, 277, 278, 485).
- TRANSPORT ACROSS THE INTESTINE APPEARS NOT TO REQUIRE VESICULAR CARRIERS. Dietary iron is initially absorbed in the duodenum in either the heme and nonheme (free or chelated) form; much more is known about uptake of the latter. The current consensus is that free Fe2+ is transported directly across the apical membrane of absorptive epithelial cells via a multistep process (203) involving the divalent metal transporter DMT-1 (Fig. 4B) (202). The protein accepts a broad range of divalent ions. Older reports in the literature implicated a vesicular process for iron's uptake, consisting of a mucin at the apical surface binding free iron, followed by its import via an integrin and transfer to a molecule termed "mobilferrin," which was later identified as calreticulin, an endoplasmic reticulum lumen chaperone. With the identification of DMT-1, the "mobilferrin" hypothesis is now open to question (12).
After transport into the epithelial cell, cytoplasmic Fe2+ is subsequently directed across the intestinal basolateral membrane via another newly identified transporter, called ferroportin (372). In intestine, a membrane form of the ferro-oxidase ceruloplasmin, called hephaestin (607), is thought to aid both in the oxidation of Fe2+ and the loading of Fe3+ onto Tf. How this occurs is still unknown, although Caco-2 cells are reportedly capable of transferring apically derived iron onto apo-Tf in the basal medium (342). Interestingly, the livers of "atransferrinemic" mice become iron overloaded, implying that a non-Tf-bound transport mechanism must operate to deliver iron from the intestine into the hepatocyte. Possible carriers have recently been reported (reviewed in Ref. 277).
- UPTAKE OF TF-BOUND IRON. Once in the circulation, iron is carried principally by hepatocyte-derived plasma Tf. Cells directly accessible to the circulation take up iron via the well-studied process of Tf receptor-mediated endocytosis (Fig. 4B); that is, iron bound to Tf is internalized in clathrin-coated vesicles, dissociated from Tf by the low pH in endosomes, and transported across the membrane, most likely by a DMT-like transporter. Apo-Tf bound to its receptor recycles to the same cell surface for subsequent rounds of uptake, delivery, and recycling.
How do cells located behind a selective barrier obtain this essential mineral? And how do cells responsible for transcytosing it obtain sufficient iron for their own metabolic needs? In the adult, iron reaches cells in peripheral tissues by nonselective caveolar-mediated transcytosis of Tf-bound iron across the endothelium; there is no dissociation in transit.
- TRANSCYTOSIS OF IRON IN THE PLACENTA AND BRAIN. As described in section IIB1 for brain, transcytosis of iron across cerebral capillaries is receptor mediated. Also, as described earlier, the extent to which plasma-derived Tf moves across brain endothelial cells is controversial and presently unresolved (52). There is a similar uncertainty
in the transcytosis of iron across the human placenta. As for brain endothelium, the direction across the syncytiotrophoblasts is apical to basolateral (Fig. 2A). Is maternal or fetal Tf the carrier? Finally, the fetal endothelial capillary is an additional cellular barrier that must be crossed and the mechanism is currently unresolved.
E. Additional Transcytosis Systems
Transcytosis occurs in the upper regions of the respiratory tract and involves two receptor systems already described, pIgA-R and FcRn (Table 1). Secretory IgA is a known constituent of the lung's immune defense system, with bronchial epithelial cells carrying out basolateral-to-apical transport of dIgA, which is secreted by local plasma cells in underlying lymphoid tissue (reviewed in Refs. 444, 491). A recent study using a clever biological read-out has documented the efficient apical-to-basolateral transcytosis of intact IgG across bronchial epithelium via the FcRn (553). This latter pathway could possibly be exploited to deliver genes systemically. Finally, albumin, which is found in lung fluid, is endocytosed specifically at the apical surface of airway epithelia but is then subsequently degraded. At the alveolar level, the question of whether albumin is transcytosed intact is uncertain (see Ref. 437 for review). Malik and colleagues (267) have recently reported the presence and function in type II epithelial cells of a gp60 membrane protein related to that found in endothelial cells.
2. Mammary gland
Milk is composed largely of locally synthesized nutrients that are secreted by alveolar epithelial cells in the lactating gland (523). However, a substantial fraction of milk proteins is thought to be derived from the serum, meaning that transcytosis must be used to deliver these "exogenous" molecules. To date, distinguishing between the two sources has not been systematically done. Local plasma cells secrete IgA, which is transcytosed by luminal epithelial cells using the pIgA-R (Table 1). As already described, rodents but not humans transcytose maternal IgG into milk for several days after pups are born. The receptor and pathway are unknown. Micronutrients in milk are supplied from the maternal circulation, but neither their transcellular path nor the source of binding protein (e.g., for iron, B12, or vitamin D) is clear. Iron is transcytosed, but milk transferrin is synthesized in the gland, potentially necessitating an intracellular transfer. Ca2+ is also derived from the maternal circulation, but its concentration in milk is 100-fold higher than that in serum. The Golgi Ca2+-ATPase has been proposed as the pump that sequesters Ca+2 in vesicles, which are subsequently delivered to the apical membrane with release of Ca2+ via exocytosis (523).
The thyroid hormones triiodothyronine (T3) and thyroxine (T4) are produced from their iodinated precursor protein, thyroglobulin, which is stored in the lumen of a thyroid follicle. Upon stimulation by thyroid stimulating hormone at the basolateral surface, apical endocytosis increases and thyroglobulin is internalized. The mechanism of uptake is not yet known and may be nonspecific (143, 226, 483). Endocytic vesicles fuse with lysosomes, and the cathepsins act on thyroglobulin to release 20-kDa fragments containing the hormonogenic regions, which are further cleaved to T3 and T4 by endo- and exopeptidases. It is a mystery how the hormones reach the circulation, since T3 receptors are present in the thyroid cell, yet net movement of the hormones is toward the basolateral surface. Once in the circulation, T3 and T4 are bound to their protein carrier, transthyretin, which is synthesized in the liver. The complex is transcytosed across continuous endothelium via caveolae. It is presently unclear how these hormones reach the brain. Perhaps the choroid plexus, which synthesizes transthyretin, plays a role.
Approximately 10% of the thyroglobulin protein internalized from the thyroid follicular lumen is not processed in lysosomes, but is transcytosed intact into the circulation. In the 1980s, Herzog (227) found that this amount did not represent spillover from saturation of a putative lysosomal delivery system, since lysosomes continued to fill as increasing amounts of thyroglobulin were internalized. More recently, researchers have identified megalin as the apical membrane receptor mediating thyroglobulin's apical-to-basolateral transcytosis across the follicular cell (348, 350, 351). This finding raises several questions. Does megalin also cross the cell and reach the basolateral PM? How does megalin's cargo avoid lysosomes in the thyroid, since in the intestine, its cargo, vitamin B12 bound to intrinsic factor, is delivered to lysosomes for degradation? The in vitro cell system models described in section III could be useful for answering these questions.
F. Role of Transcytosis in Plasma Membrane Biogenesis In Vivo
Of the in vivo transcytosis systems described so far, a common feature is that the cargo is exogenous. However, in two epithelial cell types, the hepatocyte and the absorptive enterocyte of the intestinal villus, endogenous membrane proteins are themselves the cargo! These cells use basolateral-to-apical transcytosis exclusively (hepatocytes) or in part (enterocytes) as a pathway for the delivery of specific classes of newly synthesized apical plasma membrane proteins. In hepatocytes we determined some years ago that three single transmembrane domain (TMD) apical proteins and one glycosyl-phosphatidylinositol (GPI)-anchored apical protein followed this route (32, 499). Similar observations were made by Maroux and colleagues (148, 355) in the rabbit intestine for aminopeptidase N. Thus the question is whether such an "indirect" pathway of apical PM biogenesis exists in other epithelia in vivo. Unfortunately, we may never have a definitive answer, largely because other epithelial tissues are not sufficiently homogeneous nor as amenable as the liver and intestine to the biochemical approaches that were used. In these studies, animals were first administered radiolabeled amino acids in a "pulse-chase" fashion, then enriched populations of apical and basolateral membranes were isolated, the specific membrane proteins immunoprecipitated and their radioactive content determined. Because hepatocytes constitute >70% of the total cells in the liver, subcellular fractionation methods could be used that yielded preparations of highly enriched hepatocyte organelles. Similarly, intestinal mucosa could be scraped from the surface of everted intestines, thus enriching for epithelial cells and allowing subsequent subcellular fractionation of membranes into apical- and basolateral-enriched fractions.
Why are these in vivo studies important? First, they represent "reality," that is, the physiological situation. Second, the results were different from those reported earlier using in vitro polarized cell models, specifically MDCK cells derived from dog kidney (360, 479). In the latter cells, newly synthesized apical PM proteins were shown to be delivered directly from the trans-Golgi network (TGN) to the apical surface. Gradually, as different epithelial cells and more membrane proteins have been studied, the plasticity in the routes and mechanisms for the delivery and retention of PM proteins in epithelial cells has become apparent. Such a realization reinforces the importance of studying a variety of epithelial cells to learn the full repertoire of mechanisms. It also points to the possibility that transcytosis is an "ancient" route, since all epithelial cells express this transport system.