Secretory component: The polymeric immunoglobulin receptor

Secretory component: The polymeric immunoglobulin receptor

GASTROENTEROLOGY PROGRESS 1985:89:667-82 ARTICLE Secretory Component: The Polymeric Immunoglobulin Receptor What’s in It for the Gastroenterologis...

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GASTROENTEROLOGY

PROGRESS

1985:89:667-82

ARTICLE

Secretory Component: The Polymeric Immunoglobulin Receptor What’s in It for the Gastroenterologist Hepatologist?

and

DENNIS J. AHNEN, WILLIAM R. BROWN, and THOMAS M. KLOPPEL Veterans Administration University of Colorado

Medical Center and Departments of Medicine School of Medicine, Denver, Colorado

Secretory component (SC) is the receptor on glandular epithelial cells and hepatocytes (of some species) that mediates the transport of immunoglobulin A and M polymers (pIgA and pIgM) into external body fluids. Originally the purview of immunologists, SC in recent years has intensely interested cell biologists-for good reason: The SC-pIgA system is a unique type of receptor-ligand interaction in that the receptor is neither recycled nor degraded subsequent to binding of its ligand, but is secreted by the cell in complex with its ligand. Secretory component is synthesized and expressed by glandular epithelial cells throughout the entire small intestine and colon as well as by biliary and pancreatic ductular epithelium and, under certain circumstances, the gastric epithelium. A most important, recent observation is that SC is expressed by hepatocytes of certain species and that there are marked differences in the amount of IgA transported from the circulation into the bile that correlate with the presence or absence of SC in hepatocytes. Another important observation is that SC in the hepatocyte behaves much differently from many other receptors that also are expressed on the sinusoidal plasma membrane. These are among several aspects of SC that make it an important molecule

Received January 25, 1985. Accepted March 25, 1985. Address requests for reprints to: Dennis J. Ahnen, M.D., Gastroenterology Section, lllE, Veterans Administration Medical Center, 1055 Clermont Street, Denver, Colorado 80220. This study was supported in part by the Veterans Administration and grants from the National Institutes of Health (AM 33664 and CA 17342) through the National Large Bowel Cancer Project. The authors thank Jean Gilbert for expert secretarial assistance. 0 1985 by the American Gastroenterological Association 0016-5085/85/$3.30

and Biochemistry,

for gastroenterologists and hepatologists to appreciate. In this review we have summarized the wealth of information that has accumulated during the past 20 yr or so about SC, with emphasis on its activities in the gastrointestinal tract and liver. Participation the Mucosal

of Secretory Component in Immune System and Liver

The history of how SC achieved recognition as a bona fide cell membrane receptor began with appreciation that the molecular form of IgA in mammalian external body fluids [secretory IgA (sIgA)] is different from that of IgA in the circulation: sIgA consists mainly of dimeric molecules, whereas 80%95% of serum IgA is in the 7S monomeric form. Moreover, sIgA was found to possess an additional glycoprotein moiety, which eventually came to be called SC (1). A variable but sizable proportion of IgM in secretions, usually pentameric molecules, also was found to be linked to SC (z-7). Later, dimeric IgA as well as pentameric IgM were found to often contain yet another moiety, the J or joining daltons that is linked to chain, a peptide of -15,000 the immunoglobulin heavy chains by covalent bonds (8,9). In its classic 11s form, sIgA consists of two pairs of immunoglobulin heavy chains and light chains, one SC chain, and one J chain [Figure 1); its total molecular weight is -385,000. In this review, pIgA will refer to the form of IgA Abbreviations used in this paper: mSC, membrane component; pig, polymeric immunoglobulin; pIgA, immunoglobulin A; pIgM, polymeric immunoglobulin secretory component: sIgA, secretory immunoglobulin

secretory polymeric M; SC, A.

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secretory

component

Jchain -?

Figure

1. Molecular structure of 11s secretory immunoglobulin A (IgAl. Two IgA molecules, with their (Yheavy chains and light chains are linked by disulfide bonds to the glycoprotein J chain to form the IgA dimer. The blackened portion of the molecule is a part of the antigenbinding fragment (Fab). The secretory component is disulfide bonded to the heavy chains in the Fc region of one of the IgA molecules. The branched figures indicate carbohydrate chains on secretory component and J chain (not necessarily the precise number of chains). Reprinted with permission from Kuhn and Kraehenbuhl, Trends Biochem Sci 1982;7:299.

consisting of two or more molecules of IgA and one molecule of J chain; sIgA will refer to the complex of pIgA and SC that is present in secretions (Figure 1). Early on, immunohistochemical studies of intestinal mucosa revealed that IgA was present in plasma cells located in the lamina propria, whereas SC was present only in the epithelium (Figure 2) (l,lO-12). Thus, it was evident that sIgA is the product of at least two different kinds of cells. In support of this concept were the subsequent findings that SC in external fluids is present not only in complex with pig but also in a free state (free SC] (10,13). Indeed, in the total absence of IgA and IgM, free SC may be present in increased amounts (10). Although SC initially was suspected of playing a role in the transport of IgA across the epithelium to the exterior (1,10,14), certain evidence cast doubt on that possibility (3,4): Some immunohistochemical studies were interpreted as favoring an independent secretion of IgA and SC, with the two moieties combining after secretion (11,12,15,16); moreover, some authors doubted that SC is an IgA receptor because there was [and still is) no other known situation in which a receptor becomes permanently attached to its ligand (17). Eventually, though, a large body of immunohistochemical and immunochemical data conclusively established the crucial role of SC in the translocation of polymeric immunoglobulins (pigs), and SC now rightfully can be called the pig receptor (18-20). Among the key immunohistochemical studies that resolved this issue was the electron microscopic demonstration that both SC and IgA (and IgM) were associated with the

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laterobasal plasma membrane and endocytic vesicles in columnar crypt cells in human intestinal epithelium (21). These similarities in distribution of SC and IgA on the surface and within the epithelial cells could be interpreted most logically as IgA and SC combining with one another on the cell surface and being transported in complex through the cell by an endocytic process. The next critical observation was that dimeric IgA was shown to be capable of binding to SC on the surfaces of epithelial cells in vitro (19,22-24). Finally, using a cultured line of human colonic carcinoma cells (HT-29) bearing SC on their surfaces, Crago et al. (25) and Nagura et al. (26) showed that pIgA and pIgM could bind to surfaces of the cells. Immunoelectron microscopic studies (26) also showed that the bound IgA was internalized in endocytic vesicles on the basolateral surface and transported through the cells to their apical surfaces, where the IgA was released to the exterior. Thus, it was established in living epithelial cells that the binding of dimeric IgA to SC on the basolateral cell surface is essential for the subsequent endocytosis

Figure

2. Immunohistochemical localization of secretory component (SC) and immunoglobulin A (IgA) in human jejunal mucosa. a. SC: staining for SC is most prominent in epithelial cells in the crypt. b. IgA: staining is prominent in epithelial cells in the crypt along the lateral cell surface. Numerous IgA immunocytes are present in the lamina propria. Reprinted with permission from Brown et al., Gastroenterology 1976;71: 985-95.

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and transcellular transport of the immunoglobulin. The above-cited immunohistochemical observations were congruent with much immunochemical evidence that, among various immunoglobulin isotypes, only pIgA and pIgM have strong affinity for free SC (27-33). Through its essential role in the transport of pig, SC is inextricably involved in the mucosal immune system. The initial evidence suggesting the existence of such a distinct immune system came largely from the discovery that IgA is the major immunoglobulin in external body fluids. It is now recognized, though, that the mucosal immune system has many additional characteristics that distinguish it from the systemic immune system: (a) a cell trafficking pattern which insures that immunoglobulin-producing cells produced within mucosal lymphoid follicles are, to a considerable extent, retained within mucosal areas; (b) the presence of mucosa-specific cellular elements, such as the mucosal mast cell; (c) the occurrence in mucosal tissues of regulatory T lymphocytes. These unique characteristics of the mucosal immune system undoubtedly evolved because the mucosal immune system must respond vigorously to pathogens while at the same time remaining unresponsive to the antigens present in gut commensals and foods. In addition, the mucosal immune system must exclude exogenous antigens from the internal milieu and regulate the response of the systemic immune system to common environmental materials. The concentration ratio of IgA to other immunoglobulin isotypes is higher in external secretions than in plasma, and the concentrations of sIgA in several external body fluids greatly exceeds that of other immunoglobulins. Usually, IgM concentrations are second to IgA concentrations in external fluids, and the IgM concentrations may be increased when IgA is absent (34,35). Despite its predominence in external fluids, the biological actions of sIgA still are not well understood. Clearly, however, sIgA does not effectively carry out several functions that are characteristic of some other classes of immunoglobulins, e.g., opsonization or complement activation (36). Perhaps because of these characteristics of sIgA, selective deficiency of IgA from serum and secretions is not ordinarily accompanied by an increased frequency of microbial infections (37,38). Most likely, one of the principal biological actions of sIgA is to prevent the attachment of injurious microorganisms or their toxins to mucous membranes (39,40). Whatever the function of IgA in external fluids, its active transport into external fluids is dependent on the presence of SC in the secreting cells. In addition, SC may contribute to the biological effectiveness of

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IgA in secretions by stabilizing the molecule and thereby increasing its resistance to proteolytic degradation (41); sIgA is considerably more resistant to either purified pancreatic proteases or whole intestinal fluid than is either monomeric IgA or IgG. In recent years, an exciting advance in our understanding of the mucosal immune system has been the discovery of the involvement of the liver in this system. Although it was known for years that sIgA is present in human bile, the source of the IgA was presumed to be the gallbladder, which contains numerous IgA-forming plasma cells as well as SC in the mucosa (42), whereas the normal liver contains few plasma cells. In the late 197Os, however, several laboratories showed that the rat hepatocyte actually transports IgA from the circulation to the bile (43-45). The critical role of SC in this hepatic clearance of IgA was soon established (46-49). The biological significance of the transport of pigs from the plasma into bile is not yet defined, but it might be one mechanism for enhancing the immune protection of the biliary tract and intestine. In addition, this pathway may constitute a mechanism for clearing harmful antigens from the body in the form of immune complexes with pigs (50). In the ensuing portions of this review, we shall discuss the synthesis and secretion of sIgA as it occurs stepwise: (a) the synthesis of IgA-J chain polymers in mucosal plasma cells and migration of the pigs to SC-containing secretory cells; (b) the synthesis and expression of SC by epithelial cells and hepatocytes; (c) the formation of SC-IgA complexes and transport of the complexes to the external environment by epithelial cells and hepatocytes.

Synthesis of Immunoglobulin A-J Chain Polymers and Their Migration to Secretory Component-Containing Secretory Cells (Epithelial Cells and Hepatocytes) It is convenient to think of the events that lead to the synthesis and external secretion of IgA as beginning in aggregated lymphoid tissues in the intestinal tract or respiratory tree, the so-called gutassociated or bronchus-associated lymphoid tissue. The major components of the gut-associated lymphoid tissues are Peyer’s patches and the appendix. Immunoglobulin A-containing lymphocytes migrate from gut-associated lymphoid tissues and bronchusassociated lymphoid tissues to eventually populate the various mucous membranes of the body, where they undergo further differentiation to become fully functioning IgA-secreting plasma cells (51-54). Immunoglobulin A-containing cells of course are the predominant immunocyte in intestinal tissues, with

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IgM cells usually being second most numerous (55). The ratio of IgA to IgM cells in the intestinal mucosa is generally stated to be about 15: 1 or 20: 1. Two antigenic subclasses of IgA (IgAl and IgA2) can be identified in human serum and secretions. The subclass IgAl accounts for about 90% of the total IgA in serum, whereas IgA2 comprises as much as 60% of the total IgA in secretions (27,56-58). Despite their predominance in mucosal secretions, the IgA2 proteins do not appear to be superior to IgAl proteins in forming polymers with J chain or in complexing with SC; nor is there evidence of specific biological functions characteristic of each of the IgA subclasses. The IgA-producing plasma cells in mucosal sites synthesize and secrete predominantly dimers or higher polymers of IgA, as documented by these observations: (a) IgA immunocytes in tissue sections can bind SC in vitro (59), which accords with the fact that SC has strong affinity only for the polymeric forms of immunoglobulin molecules; (b) the J chain is present in the cytoplasm of a high proportion (80%-100%) of IgA immunocytes in normal glandular sites (59-61). Moreover, the J chain in these cells appears to be in complex with IgA, as treatment of the tissue with acid-urea raised the J chain positivity by about twofold (59-61); this observation is consistent with data indicating that in the polymerization of IgA, J chain becomes immunologically obscured but can be “exposed” by treatment with denaturing agents, such as acid-urea (5,62). The J chain reportedly enhances the polymerization of immunoglobulins (63), although it is not essential. The mucosal IgA plasma cell thus is responsible for the synthesis of the component proteins of pIgA (the IgA heavy and light chains and the J chain) as well as for the assembly of the components into a functional pIgA molecule before secretion. The secreted pIgA molecule can be considered to have two functional regions, the antigen-combining site located in the Fab region of the molecule and the SC-combining site located in the Fc region (Figure 1). From its synthesis in mucosal plasma cells, pIgA theoretically can take two routes to reach SCcontaining secretory cells in the epithelium or liver. It may diffuse directly through connective tissue and across the basement membrane to the basolateral plasma membrane of epithelial cells. Alternatively, it might enter the blood, either via mucosal capillaries or lymphatics; circulating pIgA could then be cleared by the SC-containing epithelia of various glandular tissues, particularly the liver. The relative proportions of IgA polymers that take either of these two routes vary among species and among glandular tissues (64-67). In humans, the bulk of pIgA in external secretions appears to be derived from local

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synthesis and transepithelial transport rather than by clearance from the circulation. The final, common pathway for transport of pig to the exterior is an SC-mediated transcellular transport across epithelial cells or hepatocytes.

Molecular Structure and Biosynthesis of Secretory Component Before a description of the biochemical structure and synthesis of SC, its molecular forms require definition. It is now well established that SC is initially synthesized and inserted into the plasmalemma as an integral membrane protein. Subsequently, membrane SC is proteolytically processed to a soluble protein before secretion into external fluids. This proteolytic processing and secretion occurs whether or not pIgA is bound to SC. Thus, there are two basic molecular forms of SC; they will be referred to as membrane SC (mSC) and soluble SC (Figure 3). Either of these molecular forms of SC may be found in complex with pig or in an unbound state. We will refer to SC that is not complexed with pig as free SC. In addition to the fact that there are both membrane and soluble forms of SC, four other variables have to be taken into account in discussing the biochemical structure of SC: (a) genetic heterogeneity; (b) differences in SC between different species and even between different tissues of the same species; (c) the susceptibility of SC to proteolytic degradation; and (d) the extraordinary carbohydrate heterogeneity. The genetic heterogeneity of SC has best been worked out in the rabbit. Originally, Knight et al. (68) demonstrated the presence of two allotypes of rabbit SC, designated T6l and T62. Rabbits homozygous for the T6l allotype had four molecular forms of SC: an upper doublet of -83 and 80 kilodaltons and a lower doublet of approximately 58 and 55 kilodaltons. Rabbits homozygous for the T62 allotype also had four molecular forms of SC, but these forms were slightly smaller than the T6l allotype (69). Heterozygous rabbits had all eight molecular forms of SC. This genetic heterogeneity of rabbit SC is not due to posttranslational events, in that primary translation products of mRNA purified from mammary glands of the two strains also displayed the same differences in size of the SC molecule (70). Thus, different strains of the same species may not necessarily have identical forms of SC. There also are major differences in molecular forms of SC among species. In contrast to the four molecular forms of SC in the milk of homozygous rabbits, SC in rat bile consists of a protein doublet of

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molecular weight of -82,000 and 78,000(71),and SC in human milk consists of only a single 80kilodalton molecular form (18,72). It is not yet known if there are any functional differences between the multiple molecular forms of SC found in the rabbit or the rat and the single form found in humans. It has been reported recently that there may even be differences in SC structure between different tissues of the same species. Singleton et al. (73) reported that soluble SC isolated from rat small intestinal secretions is -15 kilodaltons smaller than soluble SC in bile. This difference in molecular size is not due to proteolysis of SC in the intestinal lumen by pancreatic proteases, as the smaller soluble SC was found also in small intestinal loops that had been isolated from pancreatic secretions for up to 4 wk. More recent studies from our laboratory have suggested that soluble SC in rat colonic secretions and milk are the same size as SC in bile, and that the small intestinal form is smaller because of proteolytic cleavage by an intestinal brush border protease (74). Secretory component is very susceptible to proteolytic degradation (71): SC in bile is rapidly cleaved to smaller molecular forms unless protease inhibitors (phenylmethylsulfonyl fluoride and trypsin inhibitor) are added. Proteolytic degradation of SC undoubtedly has contributed to some of the confusion regarding the molecular size of SC, at least in humans. Early reports suggested that SC in human milk was -50-70 kilodaltons (75,76), whereas subsequent studies (18,32,77-80) have demonstrated that the milk SC, as well as human biliary SC, have a molecular weight of -80,000. The fourth variable in the molecular forms of SC is the marked heterogeneity in its carbohydrate chains. Soluble SC in humans has been reported to contain from 9% to 23% carbohydrate by weight (72,81-83). The carbohydrate structure of SC has been most extensively studied in soluble SC isolated from human milk. Mizoguchi et al. (83) demonstrated that the SC glycoprotein contained 18.6% carbohydrate by weight and contained only four asparagine-linked side chains, but 19 different oligosaccharide structures were identified. These 19 oligosaccharide chains contained a common core carbohydrate, with the heterogeneity arising from variation in the terminal sugars. It is conceivable that purification of selected populations of the heterogeneous SC molecules accounts for the differences between the reported carbohydrate composition of SC. The amino acid composition of soluble SC has been studied by several laboratories. Although there are some differences in the reported composition, the common finding is that soluble SC does not

w membranespannmg

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Soluble SC +

Membrane SC Membrane Anchoring Peptide

Figure

3.

Proteolytic processing of secretory component (SC). Secretory component is synthesized as an integral membrane protein [membrane SC) with a glycosylated ectoplasmic domain, a membrane spanning segment, and a cytosolic tail. Before its secretion into external fluids, a proteolytic step cleaves the ectoplasmic domain from membrane SC to produce a soluble protein (soluble SC). Presumably, the membrane spanning segment and cytosolic tail (membrane anchoring peptide) remain behind in the lipid bilayer. This proteolytic step occurs whether or not polymeric immunoglobulin binds to the membrane SC.

contain any methionine residues (72,84-86). Recently, the cDNA for rabbit mSC has been cloned and sequenced; thus the sequence of the mSC mRNA and the deduced amino acid sequence of mSC have been predicted (20). The entire protein consists of 773 amino acids. At the N-terminal end the molecule contains an 18-amino acid hydrophobic signal sequence. The ectoplasmic region of the molecule consists of approximately the next 630 amino acids. Two potential sites for N-linked glycosylation are available on this region. The amino acid sequence predicts a membrane spanning segment of -23 uncharged, predominantly hydrophobic residues and a cytoplasmic tail consisting of -100 amino acids extending to the C-terminus of the molecule. The relationship of the membrane form of SC as predicted from the amino acid sequence to that of the secreted form of SC is diagrammed in Figure 3. It has been shown in humans (7O), rabbits (19,20,70,85), and rats (71,87) that soluble SC in secretions is a proteolytic product of an integral membrane protein. In the case of the liver, mSC is initially inserted into the sinusoidal membrane (87-89); in the case of the enterocyte, it is present in the laterobasal plasma membrane (21,73). Polymeric immunoglobulin A, if present, may bind to mSC available on the cell surface. Regardless of whether pIgA binds to SC or not, the mSC molecule is subsequently internalized and transported across the cell and its ectoplasmic portion is proteolytically cleaved to form the soluble SC before its secretion (Figure 3). The SC biosynthetic pathway has been character-

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ized extensively in the rabbit and to a lesser extent in humans and rats. In the rabbit, both cell-free translation and pulse-chase experiments have contributed complementary information to the understanding of SC biosynthesis. As noted previously, four molecular forms of soluble SC have been identified in milk from homozygous rabbits. These four forms consist of two protein doublets (upper doublet -80 kilodaltons, lower doublet -60 kilodaltons). Cellfree translation experiments in the absence of microsomes (70) have shown that these four proteins result from four different primary mRNA translation products that are about 10 kilodaltons larger than the soluble SC in milk. All four of these primary translation products contain an IgA binding site as evidenced by their ability to bind to IgA-Sepharose. Cell-free translation in the presence of microsomes results in an increase in the size of all four SC bands (upper doublet -100 kilodaltons, lower doublet -80 kilodaltons) and their acquisition of concanavalin-A binding capacity. Thus, in the rabbit, four SC proteins are synthesized on membrane-bound ribosomes and cotransiationally core-glycosylated. (85) characterized the Solari and Kraehenbuhl kinetics of SC biosynthesis in ex vivo preparations of the rabbit mammary gland. They found that the earliest molecular forms of SC detected consisted of two doublets (upper doublet -100 kilodaltons, lower doublet -80 kilodaltons) that corresponded in size to the core-glycosylated cell-free translation products. Within -30 min after the time of synthesis, these doublets were converted to two sets of doublets with slightly higher molecular weight. This step-up in molecular weight coincided with the acquisition of resistance to endoglycosidase H, which suggested that it resulted from the addition of terminal carbohydrates to the core-glycosylated molecules. It is presumably this complex glycosylated protein that is inserted initially into the plasma membrane. Beginning about 30-60 min after synthesis, the complex glycosylated form was converted to the smaller soluble SC found in secretions (upper doublet -80 kilodaltons, lower doublet -60 kilodaltons). Soluble SC was found in the culture medium by 2 h after the pulse. Experiments in the HT-29 human colonic cancer cell line (18) have suggested a similar SC biosynthetic pathway. Cell-free translation experiments demonstrated an 80-kilodalton SC protein that was converted to a 95kilodalton glycoprotein in the presence of microsomes (presumably because of core glycosylation). In pulse-chase experiments, the coreglycosylated 95kilodalton protein was converted after -30-90 min to an endoglycosidase H-resistant lOO-kilodalton protein. The 100-kilodalton protein was subsequently cleaved to an 80-kilodalton solu-

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ble form of SC which was secreted into the medium. The available evidence in the rat is also consistent with this biosynthetic pathway for SC. Membrane forms of SC in the Golgi and plasma membranes of rat liver have been reported to be 15-20 kilodaltons larger than soluble SC in bile (71,87). Similarly, mSC identified in jejunal laterobasal coionic or mammary membranes is 20 kilodaltons larger than SC in their respective secretions (74). Thus, SC biosynthesis initially follows the pattern of many plasma membrane glycoproteins: synthesis and cotranslational core glycosylation in the endoplasmic reticulum; transport to the Golgi, where terminal glycosyiation occurs; and transport and insertion into the plasma membrane. Later, however, the biosynthesis of SC differs from that of most other plasma membrane proteins in that SC is internalized from the plasma membrane, transported across the cell, and proteolytically cleaved to a soluble protein that is secreted into external fluids. The importance of this pathway in the transcellular transport of pIgA is described later. The mechanisms responsible for the regulation of SC biosynthesis and secretion are only beginning to be understood. It is clear, though, that the rate of synthesis of SC is not regulated by the presence of pIgA. Histochemical data have shown that SC is present in normal or increased amounts in intestinal tissue of patients who are IgA deficient (6,28,90). Similarly, soluble SC has been detected in external secretions of immunoglobulin-deficient subjects (78,79,91,92). Furthermore, SC has been demonstrated in neoplastic epithelial cells maintained in long-term culture in the absence of IgA (26,93) as well as in fetal epithelium before the appearance of immunoglobulin-forming cells in the mucosa (94). Finally, studies in the isolated perfused rat liver (95) have demonstrated that SC biosynthesis continues at approximately an equal rate in the presence or absence of IgA. A substantial amount of data suggest that SC synthesis is hormonally responsive in the uterus and the lacrimal glands. Secretory component is present in rat uterine secretions during proestrus but not at diestrus (96,97), and in studies of oophorectomized rats, estradiol, but not progesterone, cortisol, or testosterone, was capable of increasing the rate of secretion of soluble SC into uterine fluids (98). The estradiol effect is likely due to stimulation of SC synthesis rather than increased secretion of stored SC as it can be blocked by cyclohexamide treatment (99). Secretory component concentrations in human uterine secretions have also been found to be highest during the secretory phase, significantly reduced during the proliferative phase, and lowest during menstruation (100). Moreover, the total amount of

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SC in uterine tissue extracts was about two times greater during the secretory phase than during the proliferative phase. Concentrations of SC in lacrimal fluids are approximately five times greater in male than in female rats and do not vary during the estrous cycle. Castration of males resulted in a timedependent decrease in the SC content of the tears, which could be reversed by the administration of testosterone (101).It is not yet known if steroid hormones are capable of regulating SC synthesis or secretion in other tissues, including the digestive tract.

Secretory Component Expression During Normal Development and in Disease States Distinctly different patterns of appearance of SC during normal development occur in the rat and human. In the rat (102), SC appears in the cytoplasm of enterocytes sometime between day 10 and 15 after birth, coincident with the time of appearance of IgA plasma cells in the lamina propria. In contrast, SC appears in human epithelial tissues between 8 and 22 wk of fetal development, although neither IgA nor IgM plasma cells are present in any of the epithelial tissues through week 22 of fetal development (103). The reason for the differences between species in the time of appearance of SC in tissues remains unclear. The tissue localization of SC suggests that its expression may be a marker of epithelial cell differentiation. In the small intestine, SC is expressed predominantly by the immature cells within the crypts, with diminished expression by cells on the villi (Figure 2a). Similarly, in the colon, immature cells in the lower two-thirds of the crypt express more SC than do mature luminal cells (104). These patterns of expression of SC suggest that SC is a marker of immature cells during normal intestinal cell differentiation. The histochemical data of SC and IgA localization (Figures 2a and 2b) suggest also that crypt cells are primarily responsible for IgA secretion into the intestine. In addition to its selective expression during cellular differentiation, SC is selectively expressed on only a single domain of the plasma membrane of epithelial cells or hepatocytes. For example, in both small intestinal (21) and colonic (104) epithelial cells, SC is present on the basolateral but not on the imapical plasmalemma (Figure 4). Similarly, munoelectron microscopic studies have demonstrated the selective expression of SC on the basolateral plasmalemma of glandular and ciliated bronchial epithelial cells (105) as well as on the sinusoidal plasma membrane of hepatocytes (49). In adult human tissue, SC has been localized by

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immunofluorescence and immunoperoxidase methods to numerous secretory epithelia. The initial survey by Tourville et al. (12) demonstrated the presence of SC in bronchial epithelial cells, the salivary glands, gallbladder, renal tubules, pancreatic glandular and ductular epithelial cells, as well as the ductular epithelium of sweat glands. Tourville et al. (12) suggested that SC was present within goblet cells of the small intestine and colon, but later studies demonstrated that SC is synthesized and expressed instead by columnar absorptive cells (FigOther studies ures 2a and 4) (21,23,104,106,107). have demonstrated SC in the lacrimal gland (108), mammary gland, tonsils (log), uterus (loo), gallbladder (42),and biliary epithelia (110). In the human liver, SC has been reported by several labo(110-112), ratories to be absent from hepatocytes although one laboratory has reported finding SC on these cells (113). The explanation for these differences is as yet unknown. However, absence of SC from hepatocytes accords much better with the limited hepatic clearance of plasma IgA that has been observed in humans (58). The tissue localization of SC in carcinomas of several glandular epithelia has been studied, and it has been suggested that SC expression may be useful as a marker of metastatic epithelial cancers. Harris et al. (114) demonstrated that SC was present in all of 45 epithelial carcinomas examined, including nine metastatic sites. The authors suggested that the presence of SC on a metastatic tumor of unknown primary might be a useful indicator of a glandular epithelial origin of the malignantly transformed cell. Other studies (104,107,115,116), however, have found SC to be present in only about 50% of colonic carcinomas, and SC expression seemed to correlate with the degree of morphologic differentiation of the carcinomas. Thus, the presence of SC on a metastatic adenocarcinoma might well indicate a glandular epithelial origin, but the absence of SC on the tumor does not exclude that possibility. There have been four reports of the expression of SC in premalignant mucosa. Poger et al. (107)demonstrated that in adenomatous polyps of the colon, SC was generally expressed in a pattern similar to that in normal colonic mucosa, but focal areas of atypia were marked by the absence of SC expression. This suggested that the loss of SC expression is a marker of early malignant transformation. Rognum et al. (117) examined SC expression in colonic biopsy specimens of patients with ulcerative colitis that had dysplastic foci. They demonstrated that SC expression was diminished in the dysplastic biopsy specimens and that the loss of SC correlated with the degree of dysplasia, i.e., biopsy specimens with mild dysplasia tended to have more SC immunofluores-

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4. Immunoelectron microscopic localization of secretory component (SC) in the human jejunal crypt cell. Black reaction product indicating the sites of SC is seen in the perinuclear space (PS), the rough endoplasmic reticulum (RER), the Golgi (G), and along the lateral plasma membrane to the level of the tight junctions (TJ). No SC is seen on the microvilli at the luminal (L) surface of the cell. Reprinted with permission from Brown et al., Gastroenterology 1976;71:985-95.

cence than biopsy specimens with moderate or severe dysplasia. However, a similar decrease in intensity of SC staining was demonstrated in the presence of inflammation alone with no associated dysplasia. Thus, it appears that the loss of SC expression in ulcerative colitis is not a specific marker of the premalignant mucosa. Two groups have reported enhanced expression of SC in intestinal metaplasia of the stomach (118-120). Nagura et al. (118) demonstrated that SC was not present in normal gastric mucosa, but in intestinal metaplasia the protein was expressed on the basolateral surface of the gastric epithelial cells. Secretory component was present also in gastric carcinomas, but was expressed over the entire surface of the cancer cells. Thus, there was a progression from lack of SC in normal gastric tissue to polar surface expression of SC in preneoplastic tissue and, finally, to an alteration of the surface distribution of the SC in cancer. The studies on SC in neoplasia indicate that SC expression is altered to variable degrees in malignant and premalignant mucosa but fail to provide a unifying concept. Thus, in the colon, SC expression seems to be lost during transformation from normal through premalignant to poorly differentiated malig-

nant cells, whereas SC expression is enhanced premalignant and malignant gastric mucosa.

in

Secretory Component-Dependent Binding, Transport, and Exocytosis of Polymeric Immunoglobulin The molecular ratio of SC to IgA in human exocrine IgA predicted that one molecule of SC is associated with either two (75,77) or three (76) molecules of IgA. Later, studies with rat bile directly demonstrated the association of SC with various multimers of IgA (121,122). Subsequently, the specificity of SC binding for polymeric forms of immunoglobulins was formally established: both soluble and membrane SC molecules prefer association with dimeric and polymeric immunoglobulins-most notably dimers and polymers of IgA and pentameric IgM; monomeric forms of IgA and IgM display lOOfold less affinity for binding SC, and IgG fails to bind SC (22,30,32,123). The biochemical nature of the association between SC and IgA was at first controversial, in part because of variation in SC-IgA bonds among species. In studies of rabbit milk sIgA, Cebra and Small (124)

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reported that the bulk of SC could be released by treatment with 5 M guanidine, which suggested that the sIgA complex was held together by noncovalent interactions. By contrast, only 10%20% of SC can be released from human colostral sIgA upon treatment with denaturing agents (75,76,78); the remainder of the SC is covalently linked to the IgA molecule via disulfide bonds and can be released only by treatment with reducing agents. In the rat, Altamirano et al. (121) found that all of the SC associated with pIgA in bile was attached covalently and could not be released with 6 M guanidine. It has been observed, though, that only part of the pIgA in rat bile is linked with SC (46,125); this finding may be interpreted as the free pIgA in bile being molecules that were associated with SC by noncovalent bonds at the time of secretion but that spontaneously dissociated in the bile. Lindh and Bjork (31) examined the chemical and physical association between human free SC (purified from milk) and human myeloma IgA. The authors reported that the purified pIgA molecule contained 0.4 free thiol groups per mole of protein on both the heavy and light chains, whereas no free thiols were present on free SC or the J chain. Their studies suggested that the initial, noncovalent interaction between SC and IgA is followed by disulfide interchange, in which the free sulfhydryl groups on IgA initiate the reaction by reducing a reactive disulfide bond on the SC molecule. Interestingly, the reaction does not progress to completion and results in an equilibrium in which a proportion of the SC molecules are not attached covalently. The site of binding of SC to pig molecules is in the Fc region of the Ig molecule (Figure l), which is also the same portion of the pig molecule that binds J chain (30). The J chain of pigs may facilitate or provide the correct conformation for binding to SC but probably does not play a role in the disulfide interchange (28,126). The binding of antigen to the Fab region of pIgA appears not to affect the SC combining site, as IgA-antigen complexes can be removed from the circulation by an SC-mediated event (50,127). The binding affinity of human colostral SC with isolated human myeloma proteins has been calculated from Scatchard analysis to be 10” M-’ (30). Similar values were obtained for polymeric myeloma proteins of the IgA and IgM classes as well as for the IgAl and IgA2 subclasses. Nearly identical affinity constants (K = 1.9 x 10’ M-l) were observed for soluble SC and pIgA isolated from rabbit milk (128). In the rabbit studies, association and dissociation constants also were calculated (2.4 X lo5 M-l . min-l and 1.8 x low3 min-‘, respectively). In membrane binding studies, however, the affinity constants of dimeric IgA binding to liver and mam-

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mary membrane SC molecules was greater (K = 10’ M-l) than binding to soluble SC; these data suggest that the membrane form of SC contains a more favorable conformation for the binding of IgA. Studies using isolated soluble SC have also addressed the species specificity of the SC-immunoglobulin reaction. Mach (32) observed that purified human SC could bind to pIgA and pIgM molecules from many different mammalian species. Socken and Underdown (129) similarly reported that SC molecules from different species would displace lz51-human SC that was complexed with human pIgM or pIgA; however, the degree of displacement was dissimilar for each species, probably because of different affinities of the various SC molecules for human immunoglobulins. It is noteworthy that when SC from a human or rabbit source was bound to rabbit pIgA, the interaction was noncovalent; in contrast, when human or rabbit SC was bound to human pIgA, the interaction between SC and pIgA was covalent (129). Thus, the immunoglobulin, n& the SC molecule, may play the determining role in the formation of covalent bonds. The finding of both SC and IgA on the plasma membrane of epithelial cells (Figure 2) was the first piece of evidence suggesting that the binding of IgA to SC occurs at the cell surface (21,130-132). Subsequently, Brown et al. (21) documented by means of immunohistochemical techniques that dimeric IgA and IgM could bind to SC in intestinal epithelium, whereas monomeric IgA, IgG, or dimeric IgA preincubated with SC did not bind to the epithehum. Corroborative experiments in a human colonic carcinoma cell line and human fetal intestine further documented that SC was the cell surface receptor for pigs that mediates the transport of pig across glandular epithelia (25,26). It is now well established that the transport of serum pIgA across rat and rabbit hepatocytes into bile is an SC-dependent process similar to that occurring in glandular epithelia. It was initially demonstrated in the rat that serum pIgA was rapidly cleared from the circulation and actively transported to the bile (43,45). Electron-microscopic autoradiographic analysis showed that intravenously administered pIgA initially binds to the hepatocyte sinusoidal membrane, then is transported through the hepatocyte to the canalicular area (133). Socken et al. (47) and Orlans et al. (48) demonstrated that the binding of radiolabeled pIgA to isolated rat hepatocytes was mediated by SC. Although IgA has been detected on human hepatocytes (113) and sIgA is present in human bile, SC was not observed on human hepatocytes, but on the basolateral surface of bile duct epithelial cells (110). Thus, the biliary secretion of IgA in the human

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appears to be facilitated by ductal epithelial cells and not by hepatocytes. Irrespective of the cell type responsible for the transport of pIgA into bile, the secretion is mediated by an SC-associated process, and this process is initiated by pIgA binding to cell surface-associated SC. The SC-mediated transport of IgA across epithelial cells or hepatocytes occurs via intracellular transport vesicles (26,49,134). Electron-microscopic immunocytochemical analysis of the transport of IgA in the rat showed that pIgA injected into the perfused rat liver was present initially on the sinusoidal plasma membrane of the hepatocyte and in endocytic vesicles. The endocytic vesicles containing IgA then migrated across the cell, avoiding interaction with lysosomes, and fused with the bile canalicular membrane. At that point, the SC-IgA complex was released into the biliary lumen. The transport of pIgA from blood to bile requires about 20-40 min (45,133,135). The biliary secretion of pIgA is considerably less efficient than the clearance of pIgA from serum. Whereas virtually 100% of intravenously injected pIgA is cleared from the serum within 10 h, only lo%-70% (depending on species) is secreted into bile (125,136). Some of this observed inefficiency in hepatic translocation of IgA may be artifactual, resulting from the use of exogenous purified IgA proteins. [Radioiodinated, isolated proteins or myeloma proteins from heterologous species may behave differently from endogenous proteins (137)]. Indeed, the processing of human IgA by the rat hepatocyte is less efficient than the processing and translocation of rat IgA (138). Additionally, the finding that human IgA molecules of the IgAl subclass may interact with the receptor for asialoglycoproteins suggests that not all IgA molecules enter the hepatocyte by the SCmediated pathway (139). The presence of Fc receptors for IgA on many cells throughout the body may also contribute to the inefficient biliary secretion of labeled IgA. Thus, the processing of injected IgA may follow several different routes. It is generally agreed, however, that IgA that enters the cell via SC is directly transported and secreted into bile. Relatively little is known about the intracellular events that occur after endocytosis of the SC-pig complex. The transcellular transport of the complex appears to require microtubules, as colchicine treatment blocks biliary secretion of pIgA or transport of pIgA-SC-containing vesicles by cultured epithelial cells (26,88,89,140). After colchicine treatment, vesicles containing IgA accumulate near the area of initial binding, suggesting that the colchicine block is not at the endocytic step but at the level of transcellular vesicular transport. The release of SC-Ig complexes from the secreting cells is preceded by a

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proteolytic processing step in which the mSC is converted to the soluble form. The exact morphologic location of the proteolytic processing (within the transport vesicle or after fusion with the luminal or canalicular membrane) has not been elucidated. The SC endocytic transcellular pathway is in some respects similar to other receptor-mediated endocytic events, but in other ways is unique. Whereas most receptor-mediated endocytic processes involve internalization and degradation of the ligand, the SC-pig pathway is one of the few that results in the transcellular movement of macromolecules from one environment across the cell into a different environment. The transcellular transport of IgG across the neonatal intestine (141) or the placenta (142,143) is the transport system most analogous to the IgA transport; the IgG transport also involves a specific cell membrane receptor for the Fc region of the immunoglobulin molecule, and it delivers the ligand intact across the cell. Unlike the SC-pig pathway, however, the IgG Fc receptor dissociates from its ligand before release of the ligand from the cell. In contrast with the few known examples of receptor-mediated transcellular transport of macromolecules, many kinds of ligands are internalized by receptor-mediated endocytosis but not transported across the cell. Studies with ligands such as low density lipoprotein, o-2-macroglobulin, insulin, asialoglycoproteins, epidermal growth factor, lysosomal enzymes, and transferrin have shown that many similarities exist among these receptormediated endocytic pathways. Generally, internalization of these ligands is initiated by binding of the ligand to receptors that are diffusely distributed on the cell surface (144-149). This initial interaction is followed by clustering of ligand-receptor complexes in clathrin-coated pits (149-151) and internalization in clathrin-coated endocytic vesicles. Soon after endocytosis, the clathrin coat is lost; the smooth, ligand-containing, endocytic vesicle, now termed an endosome or receptosome (152), is acidified through the action of an adenosine triphosphatase-dependent proton pump (153-155). In most cases, the acidic environment promotes dissociation of the ligand from the receptor (156,157). At this point, the ligand is transported to a lysosome for degradation, and the receptor is recycled to the cell surface for reutilization. The dissociation of ligand from receptor appears to occur in a prelysosomal vesicle or compartment termed CURL (compartment for uncoupling receptor from ligand) (156,158). The acidification of the prelysosomal vesicle appears to be necessary for the successful routing of endocytosed receptor-ligand complexes, as the addition of weak bases such as chloroquine or ammonium chloride prevent the dissociation of the receptor-ligand com-

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plex as well as the subsequent degradation of the ligand and recycling of the receptor to the cell such as monensurface (156,159-161).Ionophores, sin, that disrupt proton gradients, also block the endocytic pathways by preventing vesicle acidification (157). The SC-pig pathway is clearly distinct from most of the above-mentioned receptor-mediated endocytic pathways in several important ways: (a) the ligand (pig) is not ultimately degraded by lysosomes; (b) the receptor (mSC) is not recycled to the cell surface for reutilization; (c) the receptor-ligand complex (mSC-pIgA) is not dissociated after internalization; and (d) the ligand (pIgA) is released from the cell in complex with its receptor (as sIgA). At what point the SC-pig pathway diverges from other receptor-mediated endocytic pathways is unclear. Schiff et al. (137) examined the hepatic transport and fate of asialoglycoproteins and pIgA and suggested that sorting or discrimination between the two ligands occurs at the cell surface. Geuze et al. (162),however, used antiligand and antireceptor antibodies in combination with different-sized gold-labeled protein A complexes to demonstrate the colocalization of IgA and asialoorosomucoid as well as colocalization of SC and the asialoglycoprotein receptor in the same endocytic vesicle. This report suggested that sorting of the two ligands begins after endocytosis and occurs in the acidified prelysosomal compartment, designated CURL. Whether the SC-pIgcontaining vesicle actually undergoes acidification as described for other endocytic events is unknown. Acidification, however, appears not to be a prerequisite event for SC-pig transport, as chloroquine does not inhibit the transport and biliary secretion of pIgA (163). Similarly, monensin treatment does not block the biliary secretion of SC (95). In summary, the existing evidence suggests that the SC-pig pathway diverges from other endocytic pathways after internalization and before lysosomal fusion.

Summary The primary function of the SC-pig system is to secrete pigs into various external secretions. The cellular mechanism responsible for this transport is schematically depicted in Figure 5. Polymeric immunoglobulin A, which is synthesized by plasma cells that are part of the mucosa-associated lymphoid tissue, gains access to the SC on the albuminal surface of epithelial cells by diffusion from sites of synthesis in mucosae or enters the blood circulation and is cleared, largely by hepatic transport, into bile. The pIgA binds to SC on the abluminal surface of the epithelial cells (and probably hepatocytes) initially

Figure

677

5. Summary of secretory component-polymeric immunoglobulin A (SC-pIgA) transport system. Secretory component is synthesized and core-glycosylated in the (1). In the Golgi apparatus, the endoplasmic reticulum terminal sugars are added to the SC glycoprotein, and SC is packaged into vesicles for transport to the plasma membrane (2). After insertion into the abluminal domain of the plasma membrane (3). SC is available for binding of pIgA (4). Receptor-mediated endocytosis of the SC-pIgA complex (51, followed by vesicular transcellular transport (6) and fusion of the transport vesicle with the luminal plasma membrane (7). allows the release of sIgA into the lumen. The site of proteolytic conversion of membrane secretory component to soluble SC is not known, but it must occur after endocytosis (5) and before secretion of sIgA. Reprinted with permission from Kuhn and Kraehenbuhl, Trends Biochem Sci 1982:7:299.

by noncovalent interactions that are saturable, reversible, and specific for pIgA and IgM. Subsequently, covalent interaction between SC and its ligand occurs to a variable degree in different species. The SC-IgA complex is endocytosed by the epithelial cell or hepatocyte and is transported across the cell into the external secretions by a microtubule-dependent vesicular transport mechanism. At some point during the transport, the complex is rendered soluble by proteolytic cleavage of the membrane-associated SC molecule to release the soluble sIgA into the gland lumen or the canaliculus. In the intestinal lumen, SC helps protect the sIgA molecule from proteolytic degradation. The sIgA may play a major role in the mucosal defense against pathogenic organisms or harmful antigens. The SC-pig system differs from many of the other known receptor-ligand interactions in several important ways. First, the synthesis or expression of the receptor (SC), or both, are not regulated by the concentration of the ligand. Second, SC probably is

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not dissociated from its ligand or recycled to the cell surface as it is secreted in complex with its ligand (pIg) into the external secretions. Third, the interaction of pigs with their receptor does not function to regulate an intracellular process, but results in transcellular transport of the ligand, which acts in the external environment. Fourth, after initial noncovalent, reversible binding between the receptor and its ligand, the interaction becomes covalent by the formation of disulfide linkages between SC and the pig. Finally, SC is initially inserted into the abluminal domain of epithelial cells as an integral membrane protein and subsequently is proteolytitally cleaved to a soluble molecule which is secreted by the cell. Thus, in contrast to many cell-surface receptor-ligand interactions in which the ligand is ultimately degraded and the receptor is conserved, the SC-pIgA interaction results in partial proteolytic degradation of the receptor and conservation of the ligand. Despite the wealth of information that has accumulated about this receptor-ligand system, a number of very important questions remain to be answered: How is the synthesis and expression of SC in tissues such as the intestine and the liver regulated? What determines how SC is expressed during normal differentiation? What are the cellular mechanisms responsible for the sorting of membrane SC to the abluminal domain of the plasma membrane and then rerouting of SC to and through the luminal plasma membrane? Where and how does the SC-pig transcellular transport pathway diverge from other receptor-mediated endocytic pathways? What are the specific functions of the sIgA or secretory IgM after secretion into the extracellular environment? From the standpoint of pathophysiology of the liver and digestive organs, it is likely that full understanding of SC and SC-pig interactions will greatly contribute to understanding of many fundamental aspects of the cellular biology of hepatocytes and intestinal epithelial cells. Moreover, important clues concerning the pathogenesis of major digestive tract diseases, e.g., malignancies and chronic inflammatory diseases, may lie in a better appreciation of SC and the secretory immune system. Thus, study of the SC-pig system will likely continue to be a fruitful and exciting area of investigation for years to come.

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