Polymeric immunoglobulin receptor traffics through two distinct apically targeted pathways in primary lacrimal gland acinar cells.

The polymeric immunoglobulin receptor (pIgR) mediates transcytosis of dimeric immunoglobulin A (dIgA) and its release into mucosal secretions. The present study reveals the complexity of the trafficking of pIgR to the apical plasma membrane in epithelial cells with exocrine secretory functions; in rabbit lacrimal gland acinar cells (LGACs), trafficking of pIgR involves both the transcytotic pathway and one arm of the regulated secretory pathway. By specifically tracking pIgR endocytosed from the basolateral membrane, we show here that the Rab11a-regulated transcytotic pathway mediates the basal-to-apical transport of pIgR, and that pIgR sorted into the transcytotic pathway does not access the regulated secretory pathway. However, previous work in LGACs expanded in the present study has shown that some pIgR is localized to Rab3D-enriched mature secretory vesicles (SVs). Myosin Vb and myosin Vc motors modulate release of proteins from the Rab11a-regulated transcytotic pathway and the Rab3D-enriched secretory pathway in LGACs, respectively. Confocal fluorescence microscopy and biochemical assays showed that inhibition of myosin Vb and myosin Vc activity by overexpression of their dominant-negative mutants each significantly but differentially impaired aspects of apically targeted pIgR trafficking and secretory component release, suggesting that these motors function to regulate pIgR trafficking in both the transcytotic and exocytotic pathways. Intriguingly, a second mature SV population enriched in Rab27b was devoid of pIgR cargo, suggesting the specialization of Rab3D-enriched mature SVs to carry a particular subset of cargo proteins from the trans-Golgi network to the apical plasma membrane.

Functional and structural characterisation of human colostrum free secretory component.

Secretory component (SC) in association with polymeric IgA (pIgA) forms secretory IgA (SIgA), the major antibody active at mucosal surfaces. SC also exists in a free form in secretions, with innate neutralizing properties against important pathogens. IgA-bound SC and free secretory component (FSC) are both produced by proteolytic cleavage of the polymeric Ig receptor whose function is to transport IgA and IgM across mucosal epithelia. Although the proteases have not been characterised and the site(s) of cleavage of the polymeric Ig receptor has been debated, it has been assumed that bound and free SC are produced by cleavage at the same site. Here we show by SDS-PAGE analyses that FSC is slightly smaller than SIgA1- or SIgA2-bound SC when purified simultaneously. The FSC preparation was functionally active, shown by binding to dimeric and polymeric IgA, and by its ability to trigger a respiratory burst by binding to 'SC receptors' on eosinophils. We also show that FSC from different human secretions have different molecular sizes. The solution structure of FSC from colostrum was studied by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient of 4.25S is close to that for recombinant FSC. The X-ray scattering curve showed that FSC adopts a compact structure in solution which corresponds well to the J-shaped domain arrangement determined previously for recombinant FSC which terminates at residue Arg585. The smaller sizes of the FSC forms are attributable to variable cleavages of the C-terminal linker region, and may result from the absence of dimeric IgA. The FSC modelling accounts for the lack of effect of the C-terminal linker on the known functions of FSC.

Solution structure of human secretory component and implications for biological function.

Secretory component (SC) in association with polymeric IgA (pIgA) forms secretory IgA, the major antibody active at mucosal surfaces. SC also exists in the free form, with innate-like neutralizing properties against pathogens. Free SC consists of five glycosylated variable (V)-type Ig domains (D1-D5), whose structure was determined by x-ray and neutron scattering, ultracentrifugation, and modeling. With a radius of gyration of 3.53-3.63 nm, a length of 12.5 nm, and a sedimentation coefficient of 4.0 S, SC possesses an unexpected compact structure. Constrained scattering modeling based on up to 13,000 trial models shows that SC adopts a J-shaped structure in which D4 and D5 are folded back against D2 and D3. The seven glycosylation sites are located on one side of SC, leaving known IgA-binding motifs free to interact with pIgA. This work represents the first analysis of the three-dimensional structure of full-length free SC and paves the way to a better understanding of the association between SC and its potential ligands, i.e. pIgA and pathogenic-associated motifs.

Human milk glycoproteins inhibit the adherence of Salmonella typhimurium to HeLa cells.

The ability of human milk, as well as its protein fractions, to inhibit the adhesion and invasion of Salmonella typhimurium to HeLa cells was investigated. The results revealed that milk secretory immunoglobulin A (sIgA) inhibited neither the adherence nor the bacterial invasion; however, free secretory component and lactoferrin inhibited the bacterial adhesion and interacted with several bacterial proteins. Our data indicated that glycoproteins such as free secretory component and lactoferrin could act as protective compounds against infant enteric diseases, possibly binding to bacterial surface and blocking adhesion, the primordial step of S. typhimurium infection.

Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo.

Secretory immunoglobulin (Ig) A (SIgA) is essential in protecting mucosal surfaces. It is composed of at least two monomeric IgA molecules, covalently linked through the J chain, and secretory component (SC). We show here that a dimeric/polymeric IgA (IgA(d/p)) is more efficient when bound to SC in protecting mice against bacterial infection of the respiratory tract. We demonstrate that SC ensures, through its carbohydrate residues, the appropriate tissue localization of SIgA by anchoring the antibody to mucus lining the epithelial surface. This in turn impacts the localization and the subsequent clearance of bacteria. Thus, SC is directly involved in the SIgA function in vivo. Therefore, binding of IgA(d/p) to SC during the course of SIgA-mediated mucosal response constitutes a crucial step in achieving efficient protection of the epithelial barrier by immune exclusion.

Role of J chain in secretory immunoglobulin formation.

The joining (J) chain is a small polypeptide, expressed by mucosal and glandular plasma cells, which regulates polymer formation of immunoglobulin (Ig)A and IgM. J-chain incorporation into polymeric IgA (pIgA, mainly dimers) and pentameric IgM endows these antibodies with several salient features. First, a high valency of antigen-binding sites, which makes them suitable for agglutinating bacteria and viruses; little or no complement-activating potential, which allows them to operate in a noninflammatory fashion; and, most importantly, only J-chain-containing polymers show high affinity for the polymeric Ig receptor (pIgR), also known as transmembrane secretory component (SC). This epithelial glycoprotein mediates active external transfer of pIgA and pentameric IgM to exocrine secretions. Thus, secretory IgA (SIgA) and SIgM, as well as free SC, are generated by endoproteolytic cleavage of the pIgR extracellular domain. The secretory antibodies form the 'first line' of defence against pathogens and noxious substances that favour the mucosae as their portal of entry. The J chain is involved in creating the binding site for pIgR/SC in the Ig polymers, not only by determining the polymeric quaternary structure but apparently also by interacting directly with the receptor protein. Therefore, both the J chain and the pIgR/SC are key proteins in secretory immunity.

Secretory component: a potential regulator of endometrial-decidual prostaglandin production in early human pregnancy.

OBJECTIVE: We sought to investigate the production of secretory component, an inhibitor of phospholipase A(2), and prostaglandins by human endometrium-decidua. STUDY DESIGN: The production of secretory component and prostaglandins by explants and dispersed glandular and stromal cells of secretory endometrium and first-trimester and term decidua were measured by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. RESULTS: Explants of first-trimester decidua produced significantly more secretory component and less prostaglandins than secretory endometrium. Immunohistochemical studies localized secretory component to epithelial glandular cells. At term, when fewer glandular cells are present, both secretory component and prostaglandin production were low. Exposure of first-trimester decidua to progesterone significantly increased secretory component production. CONCLUSION: Secretory component and prostaglandins localize primarily to epithelial glandular cells in endometrium-decidua, and their production appears to be inversely correlated. The increase in secretory component by first-trimester decidua after progesterone stimulation may account for the down-regulation of endometrial prostaglandin synthesis after implantation, a process thought to be necessary for pregnancy success.

The relationship of smoking, preeclampsia, and secretory component.

OBJECTIVE: We sought to determine whether total secretory component in serum is increased in women in whom preeclampsia subsequently develops. STUDY DESIGN: Serum samples were collected serially throughout pregnancy and post partum from nulliparous women (N = 1496). Serum concentrations of total secretory component were measured by an enzyme-linked immunosorbent assay in all women in whom preeclampsia developed (n = 71) and a randomly selected group of normotensive women (n = 83). RESULTS: Secretory component increased with smoking (P =.0003) and with gestation (P =.0001). In the whole group secretory component was not different in women with preeclampsia (P =.10), but there was a significant interaction of smoking, gravidity, and preeclampsia (P =.04). Among the women who smoked, secretory component was lower in women in whom preeclampsia subsequently developed compared with those who remained normotensive (P =.02). This difference was significant from 15 to 19 weeks' gestation. CONCLUSION: Very high serum concentrations of secretory component in smokers may protect against the development of preeclampsia and may indicate the involvement of mucosal tolerance.

Role of secretory IgA, secretory component, and eosinophils in mucosal inflammation.

Eosinophils and their products are important in the pathophysiology of allergic inflammation in mucosal tissues. Secretory component (SC) bound to IgA mediates transepithelial transport of IgA. As another biological activity of SC, we have reported that secretory IgA (sIgA) and SC preferentially activate human eosinophils. When eosinophils were stimulated with immobilized sIgA, degranulation and superoxide production were greater than when stimulated with serum IgA. In contrast, neutrophils responded similarly to sIgA and serum IgA. Superoxide production by eosinophils stimulated with cytokines was enhanced synergistically by immobilized SC, while SC showed no effect on neutrophil activation. Eosinophil superoxide production stimulated with sIgA was abolished by anti-CD18 mAb, suggesting that beta2 integrins might be crucial for this reaction. There are several reports that SC and sIgA may play important roles in regulating eosinophil functions in vivo in diseases associated with mucosal eosinophilia and in various allergic diseases. It is speculated that eosinophils in the mucosa are activated by SC or sIgA, and that subsequent degranulation and superoxide production are induced.

Polymerization of IgA and IgM: roles of Cys309/Cys414 and the secretory tailpiece.

We have investigated how the secretory tailpiece (tp), Cys414 and the amino acids flanking Cys414 or Cys309 are involved in regulating the different polymerization of IgM and IgA to pentamers and dimers/monomers, respectively. Whereas changing the tp of IgM to that of IgA has little effect on IgM polymerization, introducing the mu tp to IgA leads to the formation of larger than wild-type IgA polymers, including pentamers and hexamer. This shows that the secretory tp can differentially regulate polymerization depending on the heavy chain context. Cys414, which is engaged in intermonomeric disulfide bonds in IgM, is not crucial for the difference in IgM and IgA polymerization; IgM with a C414S mutation forms more large polymers than IgA. Also, IgA with IgM-like mutations in the five amino acids flanking Cys309, which is homologous to Cys414, oligomerize similarly as IgA wild type. Thus, IgA appears to have an inherent tendency to form monomers and dimers that is partially regulated by the tp, while the Cys309 region has only a minor effect. We also show that complement activation by IgM is sensitive to alterations in the polymeric structure, while IgA is inactive in classical complement activation even for polymers such as pentamers and hexamers.

Regulation of the formation and external transport of secretory immunoglobulins.

Secretory IgA (SIgA) is the best defined effector component of the mucosal immune system. Generation of SIgA and secretory IgM (SIgM) in exocrine glands and mucous membranes depends on a fascinating cooperation between local plasma cells that produce polymeric IgA (pIgA, mainly dimers and some larger polymers) and pentameric IgM, and secretory epithelial cells that express the polymeric Ig receptor (pIgR)--also known as transmembrane secretory component. After release from the local plasma cells and diffusion through the stroma, pIgA and pentameric IgM become readily bound to pIgR, and are then actively transported across secretory epithelial cells for extrusion into external secretions after cleavage of pIgR. Much knowledge has recently been obtained at the molecular level about the regulation of pIgR-mediated transport of antibodies. This mechanism is of considerable biological interest because SIgA and SIgM form the first line of specific immunological defense against infectious agents and other harmful substances that may enter the body through the mucosae.

Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense.

Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

Secretory IgA induces degranulation of IL-3-primed basophils.

We examined whether secretory IgA (sIgA), known to mediate eosinophil stimulation, has an effect on basophil functions. An immobilized preparation of sIgA, but not of monomeric IgA, induced histamine release (approximately 15% of total histamine contents) from human basophils in vitro. sIgA-induced basophil histamine release was totally dependent on pretreatment with IL-3. IL-5 and granulocyte-macrophage CSF also primed basophils for sIgA-mediated release. Exogenous divalent ions, i.e., Ca2+ and Mg2+, were essential for sIgA-mediated basophil degranulation, and the degranulation was completed within 45 min. A newly synthesized lipid mediator, leukotriene C4, was also liberated from IL-3-primed, sIgA-stimulated basophils. Enzyme digestion experiments revealed that the (Fc)2 x secretory component portion of sIgA is important for sIgA-mediated basophil activation, but the functional binding sites of sIgA on basophils were surmised to be different from FcalphaR. These observations reveal the novel finding that sIgA is able to stimulate basophils as well as eosinophils. Since sIgA is the most abundant Ig isotype in the secretions from mucosal tissues, and basophils are active participants in allergic late-phase reactions, sIgA-mediated basophil mediator release is potentially involved in exacerbation of the inflammation associated with allergic disorders.

Mucosal immunity--a major adaptive defence mechanism.

The epithelial glycoprotein called secretory component (SC) is quantitatively the most important receptor of the immune system because it is responsible for external transport of locally produced polymeric IgA (pIgA) to generate remarkably large amounts of secretory IgA. Antibodies of this type constitute the major mediators of specific humoral immunity. Transmembrane SC belongs to the Ig supergene family and functions as a common pIg receptor, also translocating pentameric IgM externally to form secretory IgM. The B cells responsible for mucosal pIg production are initially stimulated in organized mucosa-associated lymphoepithelial structures, particularly the Peyer's patches in the distal small intestine; from these inductive site they migrate as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory effector site. There is currently great interest in exploiting this integrated or "common" mucosal immune system for oral vaccination against pathogenic infectious agents and also to induce tolerance in T cell-mediated autoimmune diseases. However, much remains to be learned about mechanisms for antigen uptake and processing necessary to elicit stimulatory or suppressive mucosal immune responses. Moreover, evidence is emerging for the existence of considerable regionalization with regard to functional links between inductive sites and effecter sites of mucosal immunity.

[Molecular aspects of secretory IgA (S-IgA) in gut-associated lymphoid tissues].

Secretory IgA, which plays an important role in the defense of the exocrine tissue, is composed of a polymeric IgA, joining (J) chain and secretory component (SC). Polymeric IgA and J chain are produced by plasma cells and SC by glandular epithelial cells. We here described the molecular aspects of J chain and SC. Study of the J chain has been confined to vertebrates which produce immunoglobulin (Ig) because the function of J chain is considered to be a polymerization of Ig. Recent molecular studies indicate that the role of J chain has been questioned. The J chain is expressed in invertebrates, as well as, representative species of vertebrates and that J chain is a primitive polypeptide that arose before the evolution of Ig molecules. SC is a 80 kDa glycoprotein functioning as a receptor for J chain-containing polymeric Ig. The expression of SC is regulated by various inflammatory cytokines such as, IL-1, IL-4, IL-6, IL-8, IFN-gamma and TNF-alpha, suggesting SC upregulation in vivo in inflammatory conditions. The human SC cDNA analysis reveals that it consisted of 11 exons with no functional TATA-box or CCAAT-box in the putative promoter region. Further upstream, there are several interesting motifs such as NF-kB and IFN-gamma response element, suggesting possible regulation of SC by cytokines through cellular signal transduction pathways.

Dimeric and tetrameric IgA are transcytosed equally by the polymeric Ig receptor.

Polymeric IgA (pIgA) is transcytosed across epithelial cells and into external secretions by the polymeric Ig receptor (pIgR). Binding of dimeric IgA (dIgA) to the pIgR stimulates transcytosis of the pIgR. The pIgA in secretions is found as dimers (dIgA) and higher polymers, such as tetramers (tIgA), but little is known of the functional significance of the different sizes. Here we compared the ability of dIgA and tIgA to perform three functions that are essential to their transport into mucosal secretions. 1) Equilibrium binding studies showed that there were twice as many binding sites for tIgA as dIgA at the basolateral cell surface, but that the affinity of these sites for tIgA was one-half of that for dIgA. 2) Both dIgA and tIgA were rapidly transcytosed by the pIgR, although transcytosis of tIgA was slower. 3) Both dIgA and tIgA could stimulate transcytosis of the pIgR, although tIgA was less effective. The possible implications of these findings for the relative biologic roles of dIgA and tIgA are discussed.

Human eosinophils express a receptor for secretory component. Role in secretory IgA-dependent activation.

The existence of a functional receptor for secretory component (SC) on the eosinophil membrane might explain the preferential degranulation induced by secretory IgA (sIgA) when compared to serum IgA. Indeed, flow cytometry analysis revealed that purified human SC could bind to a subpopulation (4-59%) of blood eosinophils purified from 19 patients with eosinophilia. Binding of radiolabeled human SC could be competitively inhibited using unlabeled SC or secretory IgA but not with serum IgA or IgG. Immunoprecipitation and immunosorbent chromatography using human SC revealed the presence of a major component at 15 kDa in eosinophil extracts as well as in culture supernatants but not in neutrophils. The 15-kDa protein eluted from the human SC immunosorbent was able to bind to SC or to sIgA but not to serum IgA. Eosinophils preincubated with human SC or sIgA released eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) after addition of anti-SC or anti-IgA monoclonal antibody as respective cross-linking reagents. These results indicated that binding of free or complexed SC to human eosinophils could induce eosinophil degranulation. Furthermore, the dose-dependent inhibition by SC of mediator release induced by sIgA but not by serum IgA, suggested that the receptor for SC could be involved in the preferential degranulation mediated by sIgA. These results indicate a novel pathway of eosinophil activation and its potential involvement in mucosal immunity, particularly in inflammatory diseases associated with infiltration of eosinophils and the enhanced production of sIgA.

Secretory component (polymeric immunoglobulin receptor) as an intrinsic inhibitor of biological functions of interferon gamma in keratinocytes.

Secretory component (SC) acts as a transmembrane polymeric immunoglobulin receptor of epithelial cells and is known to bind to polymeric IgA and to contribute to the secretion of secretory IgA (sIgA). We describe a new biological function for free SC (FSC) by which the expression of intercellular adhesion molecule-1 (ICAM-1) and HLA-DR induced by interferon gamma (IFN gamma) is inhibited in human keratinocytes. This activity coincided with suppression of adenosine cyclic 3,5-monophosphate (cyclic AMP) production in keratinocytes. Keratinocytes produced SC after stimulation with IFN gamma and this production was suppressed by the addition of H-7 or propranolol. The addition of propranolol resulted in prolongation of ICAM-1 expression on keratinocytes induced by IFN gamma. These results suggest that endogenously produced SC, as well as exogenously added FSC, acts as an inhibitor of IFN gamma. Therefore, our results suggest that SC plays an antiinflammatory role in the pathogenesis of inflammatory skin diseases via inhibition in keratinocytes of IFN gamma induced expression of ICAM-1 and HLA-DR.