多聚免疫球蛋白受体（pIgR）在粘膜免疫中的重要功能

多聚免疫球蛋白受体（pIgR）属于Ⅰ型跨膜糖蛋白,可与多聚免疫球蛋白A和多聚免疫球蛋白M特异性结合,通过穿胞转运,将它们从上皮细胞基底侧膜转运到顶膜,并最终分泌到外分泌液中去. 在此过程中,多聚免疫球蛋白受体的细胞外段被水解,释放出与多聚免疫球蛋白A或多聚免疫球蛋白M相结合的细胞外段（又称为分泌成分）. 分泌成分是sIgA分子的重要组成部分,直接参与sIgA的粘膜防御功能,而且在被动粘膜免疫中也有重要作用. 多聚免疫球蛋白受体通过介导细胞内多聚免疫球蛋白的转运,可以在粘膜的腔面阻止病原体粘附,在上皮细胞内中和病毒,也可以将固有层内的抗原分泌出去. 因此,多聚免疫球蛋白受体的有效分泌是多聚免疫球蛋白发挥粘膜防御功能的必要条件. 但在某些情况下,该受体也可以介导微生物对上皮屏障的入侵. 多聚免疫球蛋白受体是高度 N -糖基化的,其分子中独特的糖链结构,可能与受体的穿胞转运、sIgA在粘膜的正确定位,以及抗原对上皮细胞的粘附有关. 多聚免疫球蛋白受体和分泌成分参与的多重分子机制,使它们在粘膜免疫中起着举足轻重的作用.     

The IgA/IgM receptor expressed on a murine B cell lymphoma is poly-Ig receptor

T560, a mouse B lymphoma that originated in gut-associated lymphoid tissue, expresses receptors that bind dimeric IgA and IgM in a mutually inhibitory manner but have little affinity for monomeric IgA. Evidence presented in this paper indicates that the receptor is poly-Ig receptor (pIgR) known in humans and domestic cattle to bind both IgA and IgM. The evidence includes the demonstration that binding of IgM is J chain dependent, and that pIg-precipitated receptor has an appropriate Mr of 116-120 kDa and can be detected on immunoblots with specific rabbit anti-mouse pIgR. Overlapping RT-PCR performed using template mRNA from T560 cells and oligonucleotide primer pairs designed from the published sequence of mouse liver pIgR indicate that T560 cells express mRNA virtually identical with that of the epithelial cell pIgR throughout its external, transmembrane, and intracytoplasmic coding regions. Studies using mutant IgAs suggest that the Calpha2 domain of dimeric IgA is not involved in high-affinity binding to the T560 pIgR. Inasmuch as this mouse B cell pIgR binds IgM better than IgA, it is similar to human pIgR and differs from rat, mouse, and rabbit epithelial cell pIgRs that bind IgA but not IgM. Possible explanations for this difference are discussed. All clones of T560 contain some cells that spontaneously secrete both IgG2a and IgA, but all of the IgA recoverable from the medium and from cell lysates is monomeric; it cannot be converted to secretory IgA by T560 cells.     

Synergistic effect of IL-4 and IFN-gamma on the expression of polymeric Ig receptor (secretory component) and IgA binding by human epithelial cells

The expression of secretory component (SC), the epithelial receptor for polymeric Ig, was enhanced by the addition of human rIFN-gamma or rIL-4, as revealed by the binding of radiolabeled polymeric, J chain-containing IgA or anti-SC antisera to the human colonic adenocarcinoma epithelial cell line HT-29. In combination, these cytokines exhibited a synergistic effect, and the potentiating effect of IL-4 was inhibitable by polyclonal anti-IL-4 antisera. Because the binding of radiolabeled polymeric IgA (pIgA) to HT-29 cells was inhibited by unlabeled pIgA or a polyclonal anti-SC reagent, but not by IgG, monomeric IgA, or Fab alpha fragments, we conclude that the receptor involved in the increased binding of pIgA is indeed SC. These data suggest that the expression of SC on human epithelial cells and the subsequent binding of pIgA (produced in mucosal tissues and glands by subepithelial plasma cells) is regulated by lymphokines such as IL-4 and IFN-gamma that are presumably derived from T cells found in abundant numbers in these tissues. These findings demonstrate a novel pathway of interaction between T cell products and epithelial cells that may result in enhanced translocation of large amounts of locally produced pIgA through epithelial cells into external secretions.     

Fine specificity of ligand-binding domain 1 in the polymeric Ig receptor: importance of the CDR2-containing region for IgM interaction

The human polymeric Ig receptor (pIgR), also called transmembrane secretory component, is expressed basolaterally on exocrine epithelia, and mediates specific external transport of dimeric IgA and pentameric IgM. The extracellular part of pIgR consists of five Ig-like domains (D1-D5), and a highly conserved D1 region appears to mediate the initial noncovalent ligand interaction. While the human pIgR binds both dimeric IgA and pentameric IgM with high affinity, the rabbit counterpart has virtually no binding capacity for pentameric IgM. This remarkable disparity constitutes evidence that the binding site of the two ligands differs with regard to essential receptor contact elements. Therefore, we expressed human/rabbit chimeric pIgRs in Madin-Darby canine kidney cells and found that human pIgR D1 is crucial for the interaction with pentameric IgM when placed in the context of a full-length receptor regardless of its backbone species. D1 contains three complementarity-determining region-like loops (CDR1-3), and to further map human D1 regions involved in pentameric IgM binding, we transfected Madin-Darby canine kidney cells with human/rabbit chimeric receptors in which the regions containing the CDR-like loops had been interchanged. Our results showed that the region containing the CDR2-like loop is the most essential for pentameric IgM binding. The region containing the CDR1-like loop also contributed substantially to this interaction, whereas only little contribution was provided by the region containing the CDR3-like loop, although it appeared to be necessary for maximal pentameric IgM binding.     

Identification of a polymeric Ig receptor binding phage-displayed peptide that exploits epithelial transcytosis without dimeric IgA competition

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity.     

Generation of polymeric immunoglobulin receptor-deficient mouse with marked reduction of secretory IgA.

We generated mouse lacking exon 2 of polymeric Ig receptor (pIgR) gene by a gene-targeting strategy (pIgR-deficient mouse; pIgR-/- mouse) to define the physiological role of pIgR in the transcytosis of Igs. pIgR-/- mice were born at the expected ratio from a cross between pIgR+/- mice, indicating that disruption of the pIgR gene in mice is not lethal. pIgR and secretory component proteins were not detected in pIgR-/- mice by Western blot analysis. Moreover, immunohistochemical analysis showed that pIgR protein is not expressed in jejunal and colonic epithelial cells of pIgR-/- mice, whereas IgA+ cells are present in the intestinal mucosa of pIgR-/- mice as well as wild-type littermates. Disruption of the pIgR gene caused a remarkable increase in serum IgA concentration and a slight increment of serum IgG and IgE levels, leaving serum IgM level unaltered. In contrast, IgA was much reduced but not negligible in the bile, feces, and intestinal contents of pIgR-/- mice. Additionally, IgA with a molecular mass of 280 kDa preferentially accumulated in the serum of pIgR-/- mice, suggesting that transepithelial transport of dIgA is severely blocked in pIgR-/- mice. These results demonstrate that dIgA is mainly transported by pIgR on the epithelial cells of intestine and hepatocytes, but a small quantity of IgA may be secreted via other pathways.     

Dimerization of the Polymeric Immunoglobulin Receptor Controls Its Transcytotic Trafficking

Binding of dimeric immunoglobulin (Ig)A to the polymeric Ig receptor (pIgR) stimulates transcytosis of pIgR across epithelial cells. Through the generation of a series of pIgR chimeric constructs, we have tested the ability of ligand to promote receptor dimerization and the subsequent role of receptor dimerization on its intracellular trafficking. Using the cytoplasmic domain of the T cell receptor-ζ chain as a sensitive indicator of receptor oligomerization, we show that a pIgR:ζ chimeric receptor expressed in Jurkat cells initiates a ζ-specific signal transduction cascade when exposed to dimeric or tetrameric IgA, but not when exposed to monomeric IgA. In addition, we replaced the pIgR’s transmembrane domain with that of glycophorin A to force dimerization or with a mutant glycophorin transmembrane domain to prevent dimerization. Forcing dimerization stimulated transcytosis of the chimera, whereas preventing dimerization abolished ligand-stimulated transcytosis. We conclude that binding of dimeric IgA to the pIgR induces its dimerization and that this dimerization is necessary and sufficient to stimulate pIgR transcytosis.     

Tumor necrosis factor-alpha up-regulates expression of secretory component, the epithelial receptor for polymeric Ig

Secretory component (SC) is the receptor that facilitates transcytosis of polymeric IgA and polymeric IgM through secretory epithelial cells and into exocrine fluids. The present study showed that rTNF-alpha enhanced the cellular pool, membrane expression, and secretion of functionally SC in a human colonic carcinoma cell line (HT-29m2) which is known to express and process SC like normal glandular cells. TNF-alpha also up-regulated membrane expression of the constitutive HLA class I molecules, whereas the cells remained HLA class II-negative.     

The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Calpha3 and Cmu4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Calpha3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component.     

Secretory antibody formation: conserved binding interactions between J chain and polymeric Ig receptor from humans and amphibians

Abs of the secretory Ig (SIg) system reinforce numerous innate defense mechanisms to protect the mucosal surfaces against microbial penetration. SIgs are generated by a unique cooperation between two distinct cell types: plasma cells that produce polymers of IgA or IgM (collectively called pIgs) and polymeric Ig receptor (pIgR)-expressing secretory epithelial cells that mediate export of the pIgs to the lumen. Apical delivery of SIgs occurs by cleavage of the pIgR to release its extracellular part as a pIg-bound secretory component, whereas free secretory components are derived from an unoccupied receptor. The joining chain (J chain) is crucial in pIg/SIg formation because it serves to polymerize Igs and endows them with a binding site for the pIgR. In this study, we show that the J chain from divergent tetrapods including mammals, birds, and amphibians efficiently induced polymerization of human IgA, whereas the J chain from nurse shark (a lower vertebrate) did not. Correctly assembled polymers showed high affinity to human pIgR. Sequence analysis of the J chain identified two regions, conserved only in tetrapods, which by mutational analysis were found essential for pIgA-pIgR complexing. Furthermore, we isolated and characterized pIgR from the amphibian Xenopus laevis and demonstrated that its pIg binding domain showed high affinity to human pIgA. These results showed that the functional site of interaction between pIgR, J chain and Ig H chains is conserved in these species and suggests that SIgs originated in an ancestor common to tetrapods.     

Alternative endocytic pathway for immunoglobulin A Fc receptors (CD89) depends on the lack of FcRgamma association and protects against degradation of bound ligand

IgA is the most abundant immunoglobulin in mucosal areas but is only the second most common antibody isotype in serum because it is catabolized faster than IgG. IgA exists in monomeric and polymeric forms that function through receptors expressed on effector cells. Here, we show that IgA Fc receptor(s) (FcalphaR) are expressed with or without the gamma chain on monocytes and neutrophils. gamma-less FcalphaR represent a significant fraction of surface FcalphaR molecules even on cells overexpressing the gamma chain. The FcalphaR-gamma2 association is up-regulated by phorbol esters and interferon-gamma. To characterize gamma-less FcalphaR functionally, we generated mast cell transfectants expressing wild-type human FcalphaR or a receptor with a point mutation (Arg --> Leu at position 209) which was unable to associate with the gamma chain. Mutant gamma-less FcalphaR bound monomeric and polymeric human IgA1 or IgA2 but failed to induce exocytosis after receptor clustering. The two types of transfectant showed similar kinetics of FcalphaR-mediated endocytosis; however, the endocytosis pathways of the two types of receptor differed. Whereas mutant FcalphaR were localized mainly in early endosomes, those containing FcalphaR-gamma2 were found in endo-lysosomal compartments. Mutant gamma-less FcalphaR recycled the internalized IgA toward the cell surface and protected against IgA degradation. Cells expressing the two forms of FcalphaR, associated or unassociated with gamma chains, may thus have differential functions either by degrading IgA antibody complexes or by recycling serum IgA.     

In vitro refolding of recombinant human free secretory component using equilibrium gradient dialysis

Human secretory component (SC) is associated with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived from the polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. In this present study, we describe the first cloning, expression, in vitro refolding and purification of a free form of human secretory component (rSC) containing the five functional ligand binding domains using Escherichia coli BL21 (DE3). Free rSC was refolded from inclusion bodies by equilibrium dialysis after purification by nickel affinity chromatography under denaturing conditions. Refolded rSC was purified by gel filtration chromatography. Surface plasmon resonance and dot blot association analysis have shown that purified rSC binds IgM with a physiological equilibrium dissociation constant (KD) of 4.6x10(-8) M and shares structural similarity to native SC. This provides an important step in the elucidation of the structure of this immunologically important receptor.     

Interferon-gamma enhances expression of secretory component, the epithelial receptor for polymeric immunoglobulins

Recombinant interferon-gamma (IFN-gamma) increased in a dose-dependent manner the intracellular pool, the membrane expression, and the shedding of secretory component (SC) in human colonic adenocarcinoma cell line (HT-29). A similar dose-response relationship was observed when we examined the binding of polymeric IgA to HT-29 cells treated with IFN-gamma, thus reflecting expression of functional SC. Because IFN-gamma is produced by T cells during immune responses, activated T cells may be able to promote the external transport of dimeric IgA and pentameric IgM and thereby enhance the efferent limb of the secretory immune system. This is, therefore, the first observation indicating how the secretory transport capacity may be adjusted to increased local immunoglobulin production.     

Excretion of human immunodeficiency virus type 1 through polarized epithelium by immunoglobulin A

Human immunodeficiency virus (HIV) is transmitted primarily sexually across mucosal surfaces. After infection, HIV propagates initially in the lamina propria below the polarized epithelium and causes extensive destruction of mucosal T cells. Immunoglobulin A (IgA) antibodies, produced in the lamina propria and then transcytosed across the mucosal epithelium into the lumen, can be the first line of immune defense against HIV. Here, we used IgA monoclonal antibodies against HIV envelope proteins to investigate the abilities of polarized primate and human epithelial cells to excrete HIV virions from the basolateral to the apical surface via polymeric Ig receptor (pIgR)-mediated binding and the internalization of HIV-IgA immune complexes. African green monkey kidney cells expressing pIgR demonstrated HIV excretion that was dependent on the IgA concentration and the exposure time. Matched IgG antibodies with the same variable regions as the IgA antibodies and IgA antibodies to non-HIV antigens had no HIV excretory function. A mixture of two IgA anti-bodies against gp120 and gp41 showed a synergistic increase in the level of HIV excreted. The capacity for HIV excretion correlated with the ability of IgA antibodies to bind HIV and of the resulting immune complexes to bind pIgR. Consistent with the epithelial transcytosis of HIV-IgA immune complexes, the colocalization of HIV proteins and HIV-specific IgA was detected intracellularly by confocal microscopy. Our results suggest the potential of IgA antibodies to excrete HIV from mucosal lamina propria, thereby decreasing the viral burden, access to susceptible cells, and the chronic activation of the immune system.