The polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces

Summary: Secretory antibodies of the immunoglobulin A (IgA) class form the first line of antigen‐specific immune protection against inhaled, ingested, and sexually transmitted pathogens and antigens at mucosal surfaces. Epithelial transcytosis of polymeric IgA (pIgA) is mediated by the polymeric immunoglobulin receptor (pIgR). At the apical surface, the extracellular ligand‐binding region of pIgR, known as secretory component (SC), is cleaved and released in free form or as a component of secretory IgA (SIgA). SC has innate anti‐microbial properties, and it protects SIgA from proteolytic degradation. Expression of pIgR is regulated by microbial products through Toll‐like receptor signaling and by host factors such as cytokines and hormones. Recent studies of the structure of the extracellular ligand‐binding domain of pIgR have revealed mechanisms by which it binds pIgA and other ligands. During transcytosis, pIgA has been shown to neutralize pathogens and antigens within intracellular vesicular compartments. The recent identification of disease‐associated polymorphisms in human pIgR near the cleavage site may help to unravel the mystery of how pIgR is cleaved to SC. The identification of novel functions for SC and SIgA has expanded our view of the immunobiology of pIgR, a key component of the mucosal immune system that bridges innate and adaptive immune defense.     

Increased immunoglobulin A levels in milk by over-expressing the murine polymeric immunoglobulin receptor gene in the mammary gland epithelial cells of transgenic mice

The polymeric immunoglobulin receptor (pIgR) transports dimeric immunoglobulin A (dIgA) across the epithelial cell layers into the secretions of various mucosal and glandular surfaces of mammals. At these mucosal sites, such as the gastrointestinal tract, respiratory tract, urogenital tract and the mammary glands, dIgA protects the body against pathogens. The pIgR binds dIgA at the basolateral side and transports it via the complex mechanism of transcytosis to the apical side of the epithelial cells lining the mucosa. Here, the extracellular part of the receptor is cleaved to form the secretory component (SC), which remains associated to dIgA, thereby protecting it from degradation in the secretions. One pIgR molecule transports only one dIgA molecule (1 : 1 ratio) and the pIgR is not recycled after each round of transport. This implies that the amount of available receptor could be a rate-limiting factor determining both the rate and amount of IgA transported per cell and therefore determining the total IgA output into the lumen or, in case of the mammary gland, into the milk. In order to test this hypothesis, we set up an in vivo model system. We generated transgenic mice over-expressing the murine pIgR gene under lactogenic control, by using a milk gene promoter, rather than under immunological control. Mice over-expressing the pIgR protein, in mammary gland epithelial cells, from 60- up to 270-fold above normal pIgR protein levels showed total IgA levels in the milk to be 1.5-2-fold higher, respectively, compared with the IgA levels in the milk of non-transgenic mice. This indicates that the amount of pIgR produced is indeed a limiting factor in the transport of dIgA into the milk under normal non-inflammatory circumstances.     

Polymeric IgA binding to the human pIgR elicits intracellular signalling, but fails to stimulate pIgR-transcytosis

The intracellular pathway of polymeric immunoglobulin receptor (pIgR) is governed by multiple signals that lead to constitutive transcytosis. In addition, in transfected polarized MDCK cells, polymeric immunoglobulin A (pIgA) binding stimulates rabbit pIgR-transcytosis, owing to phospholipase-C gamma 1 activation and increase of intracellular calcium. Transcytosis of rat pIgR across hepatocytes is similarly accelerated by pIgA injection. In contrast we show here that human Madrin-Darby Canine Kidney (pIgR)-transcytosis, in human Calu-3 and human pIgR-transfected MDCK cells, is not promoted by pIgA, as monitored by a continuous apical release of its secreted ectodomain. However, the incubation of cells expressing human or rabbit pIgR with pIgA induces a comparable IP3 production, and pIgR-transcytosis of either species is accelerated by the protein kinase C (PKC)-activator phorbol myristate acetate. Without pIgA, mimicking phospholipase-C activation by combining low concentrations of phorbol myristate acetate with ionomycin, or high concentrations of ionomycin alone, stimulates the rabbit, but not the human, pIgR transcytosis. These data suggest that the species difference in pIgA-induced pIgR-transcytosis does not stem from the defective production of second messengers, but from a different sensitivity of pIgR to intracellular calcium. Our results outline the danger of extrapolating to humans the abundant data obtained from mucosal vaccination of laboratory animals.     

Secretory Component Production by Polarized Epithelial Cells from the Human Female Reproductive Tract

At mucosal surfaces, the polymeric Ig receptor (pIgR) is responsible for transporting polymeric IgA across epithelial cells. The purpose of this study was to determine whether normal epithelial cells from the female reproductive tract form tight junctions and produce secretory component, the external domain of the pIgR. Uterine, cervical and vaginal tissues from women at different stages of the menstrual cycle and following menopause were used to prepare purified epithelial cell sheets, which were cultured in cell chambers. Transepithelial resistance was measured and the media from apical and basolateral compartments assayed for secretory component. Secretory component produced by uterine epithelial cells accumulated preferentially in apical compartment and correlated with increased transepithelial resistance. Seeding as epithelial sheets at 1×10⁶ cells/cm² of matrix coated cell chambers was required for growth. Epithelial cells from endo-cervix and ecto-cervix, but not the vagina, also showed preferential production and release of secretory component into the apical chamber. In conclusion, normal epithelial cells from the human female reproductive tract grow to confluence, become polarized and produce secretory component. Our results suggest that uterine and cervical epithelial cells play a key regulatory role in the control of IgA transcytosis from tissue into secretions.     

Immunoglobulin A antibodies against internal HIV-1 proteins neutralize HIV-1 replication inside epithelial cells

We show that intraepithelial cell neutralization of HIV by IgA antibodies to internal viral proteins can occur during antibody transcytosis from the basolateral to the apical surface. Polarized epithelial cells expressing the polymeric immunoglobulin receptor (pIgR) were transfected with HIV proviral DNA, and IgA was added to the basolateral side. Transcytosing IgA antibodies against Gag and RT significantly inhibited HIV replication as assessed by infection of HeLa-CD4-LTR/beta-Gal cells and direct p24 assay. Consistent with intracellular neutralization, colocalization of the internal virus proteins and their IgA antibodies was demonstrated by confocal microscopy. Thus, at least in the context of infections of polarized epithelia, antibody-mediated neutralization may not be restricted to viral surface antigens.     

In vivo stimulation of polymeric Ig receptor transcytosis by circulating polymeric IgA in rat liver

Binding of human polymeric IgA ligand to its epithelial cell polymeric Ig receptor, pIgR, has been shown to stimulate pIgR apical transcytosis in an in vitro system, based on polarized confluent MDCK cells expressing rabbit pIgR. The present study aimed at testing whether such a stimulation also occurs in vivo. Transcytosis of pIgR was monitored by rat liver output of total secretory component (SC) into bile, measured by radial immunodiffusion as the sum of free SC and pIgA-bound SC. Whereas in the perfused rat liver system addition of pIgA to the perfusate showed no effect, i.v. injection of human and rat pIgA, but not of monomeric IgA nor PBS, in living rats significantly increased total bile SC output for more than 1 h. Furthermore, depletion of the normal pIgA level circulating in the liver before injecting more pIgA was not required to show the stimulation. Our data thus strongly suggest that stimulation of liver pIgR transcytosis by pIgA ligand binding is physiologically relevant, helping to quickly adjust pIgA transport into bile to increase circulating pIgA levels, without need for increased SC/pIgR synthesis.      

In vitro transport of active alpha(1)-antitrypsin to the apical surface of epithelia by targeting the polymeric immunoglobulin receptor.

In cystic fibrosis (CF), the intense host inflammatory response to chronic infection largely accounts for the progressive pulmonary disease, and ultimately death. Neutrophils are the prominent inflammatory cells in the lungs of patients with CF, and large amounts of neutrophil elastase (NE) are released during phagocytosis. Besides having direct effects on structural elastin, NE stimulates the release of proinflammatory mediators from the respiratory epithelium and is a potent secretogogue. Therapeutic use of elastase inhibitors in CF has been complicated by difficulties in delivery to the critical site in the airway-the surface of the epithelium. We describe a unique strategy to protect the respiratory epithelial cell surface directly by capitalizing on the nondegradative transcytotic pathway of the polymeric immunoglobulin receptor (pIgR). A recombinant fusion protein was constructed consisting of an antihuman pIgR single-chain Fv (scFv) antibody linked to human alpha(1)-antitrypsin (A1AT), an inhibitor of NE. The recombinant scFv-A1AT fusion protein bound specifically to the pIgR on the basolateral surface of an epithelial cell monolayer, and was transported and released into the apical medium where the A1AT domain was capable of forming an inactivation complex with NE. Thus, A1AT linked to an antihuman pIgR scFv was delivered in receptor-specific fashion from the basolateral to apical surface and was released as an active antiprotease, indicating that it is feasible to deliver therapeutic proteins to the apical surface of epithelia by targeting the pIgR.     

Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis

There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining immunoglobulin A (IgA) transcytosis across Transwells. IgA transcytosis required induction of polymeric Ig receptor (pIgR) expression, which could be stimulated by a combination of lipopolysaccharide and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that tumor necrosis factor-α, interleukin (IL)-1β, IL-17, and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that among these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. Interferon-γ, however, did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology.     

Regulation of the polymeric immunoglobulin receptor and IgA transport: new advances in environmental factors that stimulate pIgR expression and its role in mucosal immunity

Secretory IgA (SIgA) antibodies represent the first line of antigen-specific immune defense protecting the mucosal surfaces against environmental pathogens and antigens, and maintaining homeostasis with the commensal microbiota. The polymeric immunoglobulin receptor (pIgR) has the dual role of transporting locally produced dimeric IgA across mucosal epithelia, and serving as the precursor of secretory component, a glycoprotein that enhances the immune functions of SIgA. The complex regulation of pIgR expression and transcytosis by host and microbial factors is finely tuned to optimize the role of SIgA in mucosal immunity. Disruption of this regulatory network in disease states similar to inflammatory bowel disease can result in profound consequences for mucosal homeostasis and systemic sequelae. Future research into the function and regulation of pIgR and SIgA may offer new insights into the prevention and treatment of infectious and inflammatory diseases that originate at mucosal surfaces.     

Monoclonal antibodies against avian paramyxovirus-3: antigenic mapping and functional analysis of the haemagglutinin-neuraminidase protein and the characterization of nonspecific monoclonal antibodies

Summary Sixteen monoclonal antibodies (Mabs) which immunoprecipitated the haemagglutinin neuraminidase (HN) of chorio-allantoic membrane-grown avian paramyxovirus-3 (PMV-3) of British turkeys were produced. Thirteen were PMV-3 specific. Three were nonspecific because they also bound to other viral proteins and to bovine kidney cells treated with neuraminidase enzyme or infected with influenza virus.The thirteen specific Mab defined four antigenic regions A-D by competition and variant selection assays. Region A was subdivided into five epitopes and region B into two epitopes. The thirteen Mab neutralized and were active in haemagglutination inhibition (HI) and twelve were active in haemolysis inhibition (HLI) tests. Neuraminidase inhibition (NI) was epitope-dependent.Mabs to five of the epitopes A₁, A₂, A₃, A₅, and C bound to the 1981 British turkey isolates but not the 1968 American turkey isolate. The Mab to epitope A₄ bound to both viruses. The Mabs to epitopes B₁, B₂, and D also bound to a parakeet isolate of PMV-3 which was the third PMV-3 tested. The Mab to B₂ gave identical titres to all three viruses and had HI, HLI, and NI activities. This made it a potential diagnostic reagent for avian PMV-3 viruses.One of the nonspecific Mabs bound to lactose-like moeities as reported on influenza virus and one to maltose-like moeities as on retroviruses. Immunoglobulin from all three nonspecific Mab had some HI activity.     

Interleukin-4 and interferon-gamma synergistically increase secretory component gene expression, but are additive in stimulating secretory immunoglobulin A release by Calu-3 airway epithelial cells

Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) synergize to express polymeric immunoglobulin receptor (pIgR) but their combined effect, and that of IL-4 alone, on secretory immunoglobulin A (sIgA) release is unknown. Recently, we have developed an airway epithelial cell model that allows assessment of the integrated effect of a stimulus on pIgR gene and protein expression and sIgA release. With this model we show here that IL-4 and IFN-gamma dose-dependently increased pIgR mRNA and protein expression, and sIgA release. IFN-gamma and IL-4 induced similar maximal expression of pIgR, but IFN-gamma enhanced sIgA release more than IL-4. When added together, IL-4 and IFN-gamma synergistically increased pIgR mRNA and protein expression, but sIgA release was stimulated in an additive manner. Thus, IL-4 and IFN-gamma may be implicated in the increase of sIgA levels as found in mucosal inflammatory diseases. In addition, our results indicate that transport and release of empty pIgR is subject to regulatory mechanisms different from those of pIgR with bound dimeric IgA.     

IgG transport across trophoblast-derived BeWo cells: a model system to study IgG transport in the placenta

In primates, prenatal transfer of IgG from mother to offspring occurs predominantly across the placenta. Although a number of Fcgamma-receptors and IgG binding proteins have been detected in human placental tissue, an involvement of any of these receptors in IgG transport across the syncytiotrophoblast remains to be demonstrated. Therefore, we investigated the mechanism of IgG transcytosis in trophoblast-derived BeWo cells. BeWo cells were not only found to express the MHC class I-related IgG Fc receptor, human FcRn, but also specifically bound fluorescein isothiocyanate (FITC)-labeled human IgG (FITC-hIgG) at the apical surface at mildly acidic pH. The cells preferentially transcytosed FITC-hIgG from the apical to the basolateral side when compared to the fluid-phase marker FITC-dextran and to FITC-hIgG transcytosis in the opposite direction. However, endocytosis of FITC-hIgG at the apical plasma membrane at physiological pH required the continuous presence of FITC-hIgG at concentrations similar to those present in the maternal circulation. These results suggest a mechanism by which IgG is internalized by BeWo cells via fluid-phase endocytosis. Tight binding of IgG to hFcRn may then occur in acidic endosomes, followed by selective sorting into the transcytotic pathway. Thus, the main function of this receptor is to prevent entry of IgG into the degradative pathway in lysosomes.     

Anti-inflammatory role for intracellular dimeric immunoglobulin a by neutralization of lipopolysaccharide in epithelial cells

Intestinal epithelial cells (IEC) play a central role in innate and acquired mucosal immunity. They ensure early signaling to trigger an inflammatory response against pathogens. Moreover, IEC mediate transcytosis of dimeric IgA (dIgA), through the polymeric-immunoglobulin receptor (pIgR), to provide secretory IgA, the major protective Ig in mucosal secretions. Using an in vitro model of polarized IEC, we describe an additional anti-inflammatory mechanism of dIgA-mediated protection against intracellular bacterial components involved in the proinflammatory activation of IEC. Specific dIgA colocalizes to lipopolysaccharide (LPS) in the apical recycling endosome compartment, preventing LPS-induced NF-kappaB translocation and subsequent proinflammatory response. Thus, intracellular neutralization by dIgA limits the acute local inflammation induced by proinflammatory pathogen-associated molecular patterns such as LPS.     

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.     

Antibody against the human J chain inhibits polymeric Ig receptor-mediated biliary and epithelial transport of human polymeric IgA

To emphasize the requirement for a J chain in native polymeric immunoglobulins for their selective transport into exocrine secretions, IgG, purified from two different antisera specific for the human J chain, was shown to: (i) bind in vitro to human polymeric IgA (pIgA) by density gradient ultracentrifugation; (ii) inhibit binding in vitro of rat secretory component to human pIgA; (iii) inhibit hepatic transport of human pIgA into rat bile in vivo; and (iv) inhibit apical transcytosis of pIgA in vitro by polarized human polymeric immunoglobulin receptor (pIgR)-expressing Madin-Darby canine kidney cells. Inhibition of biliary transport increased with the molar ratio of anti-J chain antibodies against pIgA and their incubation time. Anti-J chain F(ab ′ )₂ and Fab fragments also inhibited biliary transport, excluding a role for phagocytic clearance or excessive size of the immune complexes. Anti-human-Fcα Fab, bound to human pIgA in complexes of larger size than those with anti-J chain Fab, did not inhibit biliary transport of human pIgA. Propionic acid-denatured human pIgA, although containing J chains, was very poorly transported into rat bile. Altogether, our data strongly support, now also by in vivo experiments, the crucial role of the J chain of native pIgA in its selective pIgR-mediated transport into secretions, as suggested long ago by in vitro data only. Recent data on J chain-knockout mice, with low IgA levels in bile and feces, cannot explain the role of the J chain in contributing to the secretory component/pIgR-binding site of normal pIgA, but otherwise agree with our study.     

Multiple cleavage sites for polymeric immunoglobulin receptor

Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild-type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild-type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist.     

Domain deletions in the human polymeric Ig receptor disclose differences between its dimeric IgA and pentameric IgM interaction.

The human polymeric Ig receptor (pIgR), or transmembrane secretory component, is basolaterally expressed on secretory epithelial cells; its function is to transport externally J chain-containing dimeric IgA and pentameric IgM. The ligand-binding extracellular part of this receptor contains five disulfide-stabilized domains which show considerable homology with the variable domains of Ig chains. The N-terminal domain 1 (D1) mediates the initial noncovalent ligand interaction. In this study we made deletions of the human pIgR D2 and D3 (pIgRDelta2,3), or D4 and D5 (pIgRDelta4,5), to investigate the influence of these domains in receptor binding and transport of dimeric IgA and pentameric IgM across MDCK cells transfected with the truncated receptors. Both mutants were found to bind pentameric IgM, but only pIgRDelta4,5 bound dimeric IgA. These results showed that the two ligands interact differently with human pIgR; binding of pentameric IgM apparently depends fully on strong interactions with D1, while binding of dimeric IgA in addition depends on elements within D2 and / or D3 to support the initial noncovalent binding to D1. Moreover, our studies imply that dimeric human IgA binds differently to pIgR from various species. This observation cautions against interpretation of functional studies performed with non-homologous receptor-ligand pairs.