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As central effectors of the adaptive immune response, immunoglobulins, or antibodies, provide essential protection from pathogens through their ability to recognize foreign antigens, aid in neutralization, and facilitate elimination from the host. Mammalian immunoglobulins can be classified into five isotypes-IgA, IgD, IgE, IgG, and IgM-each with distinct roles in mediating various aspects of the immune response. Of these isotypes, IgA and IgM are the only ones capable of multimerization, arming them with unique biological functions. Increased valency of polymeric IgA and IgM provides high avidity for binding low-affinity antigens, and their ability to be transported across the mucosal epithelium into secretions by the polymeric immunoglobulin receptor allows them to play critical roles in mucosal immunity. Here we discuss the molecular assembly, structure, and function of these multimeric antibodies.

Immunoglobulin G (IgG) is currently the most studied immunoglobin class and is frequently used in antibody therapeutics in which its beneficial effector functions are exploited. IgG is composed of two heavy chains and two light chains, forming the basic antibody monomeric unit. In contrast, immunoglobulin A (IgA) and immunoglobulin M (IgM) are usually assembled into dimers or pentamers with the contribution of joining (J)-chains, which bind to the secretory component (SC) of the polymeric Ig receptor (pIgR) and are transported to the mucosal surface. IgA and IgM play a pivotal role in various immune responses, especially in mucosal immunity. Due to their structural complexity, 3D structural study of these molecules at atomic scale has been slow. With the emergence of cryo-EM and X-ray crystallographic techniques and the growing interest in the structure-function relationships of IgA and IgM, atomic-scale structural information on IgA-Fc and IgM-Fc has been accumulating. Here, we examine the 3D structures of IgA and IgM, including the J-chain and SC. Disulfide bridging and N -glycosylation on these molecules are also summarized. With the increasing information of structure-function relationships, IgA- and IgM-based monoclonal antibodies will be an effective option in the therapeutic field.

Secretory (S) Immunoglobulin (Ig) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here, we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a β-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen-binding fragments (Fabs) and preserves steric accessibility to receptor-binding sites, likely influencing antigen binding and effector functions., Competing Interests: SK, BP, AM, NH, KH, ET, BS No competing interests declared, (© 2020, Kumar Bharathkar et al.)

The N -glycosylation of immunoglobulin (Ig) G, the major antibody in the circulation of human adults, is well known for its influence on antibody effector functions and its alterations with various diseases. In contrast, knowledge on the role of glycans attached to IgA, which is a key immune defense agent in secretions, is very scarce. In this study we aimed to characterize the glycosylation of salivary (secretory) IgA, including the IgA joining chain (JC), and secretory component (SC) and to compare IgA and IgG glycosylation between human plasma and saliva samples to gain a first insight into oral cavity-specific antibody glycosylation. Plasma and whole saliva were collected from 19 healthy volunteers within a 2-h time window. IgG and IgA were affinity-purified from the two biofluids, followed by tryptic digestion and nanoLC-ESI-QTOF-MS(/MS) analysis. Saliva-derived IgG exhibited a slightly lower galactosylation and sialylation as compared to plasma-derived IgG. Glycosylation of IgA1, IgA2, and the JC showed substantial differences between the biofluids, with salivary proteins exhibiting a higher bisection, and lower galactosylation and sialylation as compared to plasma-derived IgA and JC. Additionally, all seven N -glycosylation sites, characterized on the SC of secretory IgA in saliva, carried highly fucosylated and fully galactosylated diantennary N -glycans. This study lays the basis for future research into the functional role of salivary Ig glycosylation as well as its biomarker potential.

Objective: To conduct a proof-of-concept study on preferential binding of polymeric IgA (pIgA) using a novel recombinant rabbit/human chimeric secretory component (cSC) and preliminary assessment of the diagnostic potential of virus-specific pIgA in discriminating acute hepatitis A, E, and C (HAV, HEV, HCV) patients and uninfected controls using an indirect enzyme-linked immunoassay., Results: cSC binds > 0.06 μg/ml of purified human and mouse pIgA with negligible cross-reactivity against IgM and IgA. Virus-specific pIgA was significantly higher in serum of acute HAV (n = 6) and HEV (n = 12) patients than uninfected samples (HEV: p < 0.001; HAV: p = 0.001), and had low correlation with virus-specific IgM (HEV r: - 0.25, 95% CI - 0.88 to 0.71, p = 0.636; HAV r: 0.05, 95% CI - 0.54 to 0.60, p: 0.885). Anti-HCV pIgA peaked early in HCV seroconversion panels (n = 14), and was undetectable after 4 weeks post-primary bleed, even in ongoing infections, while serum anti-HCV IgA, IgG and IgM persisted. Patients with early acute HCV infection had significantly higher levels of anti-HCV pIgA compared to those with chronic infections (p < 0.01). The use of novel cSC demonstrates the presence of virus-specific pIgA in sera of patients with acute HAV, HEV, and HCV infection, and posits its potential utility as a diagnostic biomarker that warrants further validation on larger sample populations.

Background & Aims: Anti-neutrophil cytoplasmic antibodies (ANCA) are a non-uniform family of antibodies recognizing diverse components of neutrophil granulocytes. ANCA formation might be induced by protracted bacterial infections or probably reflect an abnormal immune response to commensal microorganisms. Bacterial infections are common complications in cirrhosis with high incidence of episodes caused by enteric organisms, therefore, we sought to study the presence and clinical importance of ANCA in cirrhosis., Methods: Sera of 385 patients with cirrhosis of different etiologies were assayed for ANCA of IgG, IgA, IgA1, IgA2, and secretory IgA subtypes by indirect immunofluorescence and ELISAs. The control group comprised 202 patients with chronic liver diseases without cirrhosis and 100 healthy subjects. In cirrhosis, a 2-year follow-up, observational study was conducted to assess a possible association between the presence of ANCA and clinically significant bacterial infections., Results: Prevalence of ANCA IgA was significantly higher in cirrhosis (52.2%) compared to chronic liver diseases (18.6%) or healthy controls (0%, p<0.001 for both). ANCA IgA subtyping assays revealed marked increase in the proportion of IgA2 subtype (46% of total ANCA IgA) and presence of the secretory component concurrently. Presence of ANCA IgA was associated with disease-specific clinical characteristics (Child-Pugh stage and presence of ascites, p<0.001). During a 2-year follow-up period, risk of infections was higher among patients with ANCA IgA compared to those without (41.8% vs. 23.4%, p<0.001). ANCA IgA positivity was associated with a shorter time to the first infectious complication (pLogRank <0.001) in Kaplan-Meier analysis and was identified as an independent predictor in multivariate Cox-regression analysis (HR:1.74, 95% CI: 1.18-2.56, p=0.006)., Conclusions: Presence of IgA type ANCA is common in cirrhosis. Involvement of gut mucosal immune system is in center of their formation and probably reflects sustained exposure to bacterial constituents., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

This paper describes the separation and characterization of several IgA fractions from the same human monoclonal source, based on their ability to bind secretory component (SC). The study was undertaken to elucidate features of the immunoglobulin-binding site for SC, and to examine the dependence of mucosal transport on IgA-SC interaction. Enrichment or depletion of SC-binding activity was accomplished on an affinity adsorbant made with SC from human colostral whey. The affinity-purified human IgA fractions contained IgA polymers and were 77% active in rebinding to the adsorbant; this activity was diminished significantly by direct radioiodination. The non-adherent IgA fractions contained both polymer and monomer, and were only 8% active in rebinding to the adsorbant. When the polymer and monomer components were separated from each other, the non-adherent polymer was found to resemble the affinity-purified fraction by all criteria examined including J-chain content, except that the SC-binding capacity was greater than five-fold lower. These findings have two implications for the SC-binding site on human IgA: first, the presence of J-chain is insufficient to bestow IgA with SC-binding activity; second, a critical tyrosine participates in maintaining the SC-binding region, possibly on the IgA heavy chain. The relationship between SC binding and mucosal transport was tested in the rat hepatobiliary model. All radiolabeled human IgA fractions were captured rapidly from blood by the rat liver, but only the SC-binding fractions underwent substantial intact transport to bile (greater than 70% of the injected dose). Even though a nominal proportion of the SC-non-adherent IgA appeared in bile (4-15% of the dose), most IgA in these fractions was rapidly degraded within the liver. Thus, only a small amount of monomeric and polymeric IgA can use alternative receptors to get to bile by diversion from the degradative pathway. Polymeric IgA can undergo efficient transport across the cell, strictly conditional on a high binding capacity for SC. This demonstrates that membrane SC is the receptor conferring specificity on the mucosal-transport pathway.

Rat hepatic bile contains three proteins as major constituents: secretory IgA (SIgA), free secretory component (FSC) and albumin. Traces of alpha-macroglobulin, transferrin, IgG and IgM are also detectable. The bile duct daily pours between 5-12 mg each of SIgA and FSC into the rat duodenum. The origin and function of these proteins in bile may represent important clues in the understanding of the SIgA system.

The net stimulation of dextransucrase EC 2.4.1.5) activity from Streptococcus mutans HS6 by dextran, secretory immunoglobulin A, or secretory component was investigated. Approximately equal stimulation resulted from treatment with these three components.

Clinical signs of hyperviscosity syndrome in a 6-year-old dog included listlessness, polydipsia, anorexia, vomiting, and recurrent bleeding from the gums. Fundoscopy showed the typical retinal changes associated with this syndrome. A polymeric IgA cryoglobulin characterized in the dog's serum was later isolated and structurally studied. It was found to contain J chain but no secretory component. The heavy and light chains were covalently bound.

The arrangement of disulfide bonds joining secretory component (SC) to the alpha chains in secretory IgA was studied by determining the molecular size of the principal fragments resulting from CNBr digestion of secretory dimeric Fc fragments from IgA (Fc)2alpha fragments). In vitro complexes formed by incubating 125I-free SC and myeloma 131I-(Fc)2alpha fragments were isolated by gel filtration and subsequently digested with cyanogen bromide. The CNBr digests of SC-(Fc)2alpha fragments were analyzed by gel filtration in 5 M guanidine. Two principal fragments were obtained, one containing a monomeric Fc fragment from IgA (Fcalpha) associated with SC (m.w. congruent to 110,000) and a second containing the second Fcalpha monomer (m.w. congruent to 50,000) from the dimeric SC-(Fc)2alpha. Similar results were obtained when secretory (Fc)2alpha fragments isolated from native secretory IgA dimer were subjected to CNBr digestion. The data indicate that SC is disulfide bonded to a single monomer subunit in secretory IgA dimer.

The secretory component (SC) isolated from human milk was labeled with 2 mol of the fluorescent thiol reagent N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide (DACM) per mol of SC through the reactive disulfide bond of SC. The binding of the labeled SC to polymeric immunoglobulins was examined by gel filtration by measuring the fluorescence of DACM at 478 nm. The labeled SC was bound to immunoglobulin M (IgM) and its (Fc)5 mu fragment and to dimeric immunoglobulin A (IgA). When the labeled SC was bound to IgM or the (Fc)5 mu fragment, the fluorescence of DACM increased about 30%. By use of this fluorescence change, quantitative studies were made on the equilibrium and kinetics of the reversible interactions of the labeled SC with two IgM proteins and their (Fc)5 mu fragments at pH 7.0 and 25 degrees C. All the IgM proteins and their (Fc)5 mu fragments had one binding site per mole of polymers. The affinity constant (6 X 10(8) M-1), the association rate constant (7 X 10(7) M-1 min-1), and the dissociation rate constant (0.1 min-1) of one IgM were different from those of the other IgM (1.7 X 10(9) M-1, 1.0 X 10(8) M-1 min-1, and 0.06 min-1, respectively). However, the values for the (Fc)5 mu fragments of the two proteins were the same (1.9 X 10(9) M-1, 1.1 X 10(8) M-1 min-1, and 0.06 min-1, respectively) and were very similar to those of the IgM with the higher affinity constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymeric IgA antibody mediates the transport of corresponding antigens, in the form of IgA immune complexes (IC), from the circulation into the bile, whereas monomeric IgA, IgG, and IgM are ineffective. Transport has been shown with dinitrophenyl albumin and bacterial polysaccharides such as pneumococcal SIII and C-substance. The process does not result in breakdown of the antigen, which can be detected in the bile in intact free and IgA-bound forms. Although IgG promotes clearance of antigen from the circulation mainly to the liver, only low levels of breakdown fragments are detectable in bile. In mice, the mechanism of IgA IC transport does not appear to involve Kupffer cells, the complement system, or the glycoprotein receptors on liver cells because attempts to block these systems failed to affect transport. The mechanism appears to be analogous to that for free IgA. Transport could be inhibited by antigen-nonspecific IgA, but not IgG or IgM. IgA IC were found in the bile but not in the saliva, milk, urine, or bronchial or intestinal washings. A similar pattern of appearance was seen when IgA alone was injected. Thus hepatobiliary transport appears to be the major pathway for the clearance of both IgA IC and free IgA from the circulation.

We studied the translocation of dimeric IgA across epithelium, using neoplastic human colon cells in culture as a source of epithelial cells, and immunoelectronmicroscopy with peroxidase-labeled antigens and antibodies. The cells had some of the ultrastructural characteristics of normal, mature epithelial cells, i.e., polarity, desmosomal junctions, and secretory component on their basal and lateral plasma membranes. Horseradish peroxidase-labeled dimeric IgA, exposed to the cells at 0 degrees C, bound selectively to secretory component on the cell surfaces. At 37 degrees C, the bound dimeric IgA was taken into the cells by endocytosis and transported apically through the cytoplasm in vesicles. After 30 min, IgA was discharged across the apical surface. Neither colchicine (10(-4) M) nor cytochalasin B (10(-5) M) interfered with binding or endocytosis of dimeric IgA, but colchicine inhibited intracellular transport of the IgA-containing vesicles. These experiments demonstrated that dimeric IgA can be transported through living intestinal epithelial cells in vitro. The transport includes 1) specific binding of IgA dimers to secretory component on plasma membranes, 2) endocytosis of IgA in vesicles, 3) transcytoplasmic transport of the IgA-containing vesicles by a process involving microtubules, and 4) discharge of IgA at the apical surfaces.

A method was developed for the preparation of a proteolytic fragment of rabbit secretory immunoglobulin A (sIgA) which contains the variable region of the alpha-chain; this fragment is suitable for primary-sequence studies. The serologically defined subclasses of sIgA are shown to correlate partially with the nature of the binding of a constituent chain of sIgA, called secretory piece. Data are also presented on the relative resistance of sIgA to enzymic and reductive cleavage, compared with immunoglobulin G.

Plasma-derived dimeric immunoglobulin A is transported through liver parenchymal cells into bile, in association with its glycoprotein receptor secretory component, by a vesicular transport system. This study was designed to determine the effects of colchicine, a microtubule-disrupting agent, and thus the role of microtubules on the uptake, intracellular transport, and subsequent biliary secretion of dimeric immunoglobulin A. In vivo studies in rats showed that colchicine treatment reduced the amount of intraportally injected 125I-dimeric immunoglobulin A that appeared in the bile. It was also found that although the livers in colchicine-treated animals could sequester and internalize immunoglobulin A, it was not readily secreted into bile. In vitro studies using peroxidase-labeled antisecretory component and 125I-dimeric immunoglobulin A autoradiography were both used to determine the site of this block in immunoglobulin A secretion. These studies demonstrate that colchicine disruption of microtubules (a) has little initial effect on the binding and internalization of dimeric immunoglobulin A; (b) has a major effect on the translocation of immunoglobulin A-containing vesicles within the hepatocyte, and (c) most likely prevents the translocation of newly synthesized secretory component to the plasma membrane.

Proteins in hepatic bile from cannulated rabbits were analysed by gel filtration, electrophoresis, density gradient ultracentrifugation, and immunochemical methods. Bile-to-serum concentration ratios (no. = 8) demonstrated that IgA and free secretory component (SC) were major constituents of rabbit bile. Relative concentration ratios, obtained by dividing the bile to serum level ratio for each protein by the corresponding ratio for albumin, were 310, 1.6, 1.0, 0.82, and 0.54 for IgA, IgM, albumin, transferrin, and IgG respectively, suggesting that both IgA and IgM are selectively secreted into bile. Ligation of the bile duct (no. = 5) led to a selective increase of the serum levels of IgA and SC, the latter as secretory IgA. After intravenous injection (no. = 4) of human polymeric IgA, 37-69% of the injected dose was recovered in bile after 3 h, in contrast to 4-5% for human monomeric IgA. The active transfer of both rabbit and human polymeric IgA into rabbit bile occurs via the same hepatic SC-mediated transport process as that described for the rat.

Human or rat purified secretory IgA (sIgA) injected intravenously (i.v.) into rats is transferred to bile much less (seven to twenty-four times) than human or rat polymeric IgA (pIgA) devoid of secretory component (SC). A polymeric Fc alpha (pFc alpha) fragment of a human IgAl myeloma protein, obtained by IgA-protease digestion, bound in vitro to rat SC and was actively transferred in vivo into bile, in contrast to the corresponding Fab alpha. The IgA recovered in bile was not degraded, as judged by sedimentation in density gradients. Purified rabbit IgG anti-rat SC antibody was also efficiently transported in vivo into bile, about forty times more than normal rabbit IgG. The biliary transport of anti-Sc antibody could be reduced and retarded by the simultaneous i.v. injection of purified rat SC or human pIgA. The transfer of rat 125I-pIgA into bile was also significantly reduced and retarded by the concomitant i.v. injection of purified rat or human SC. Moreover, i.v. injection of purified rat or human SC induced a marked and prolonged decrease of the sIgA level in the bile. Rat SC was more effective than human SC in this respect. All these in vivo experiments confirm the in vitro findings of Orlans, Peppard, Fry, Hinton : Mullock, (1979) and Socken, Jeejeebhoy, Bazin & Underdown, (1979) showing that Sc is the IgA-receptor on the hepatocyte membrane for the transfer of pIgA from rat plasma into bile.

The specificity of rabbit receptors for IgA (RFc alpha) was investigated on lymphocytes isolated from the Peyer's patches and spleen. Specifically was determined by the inhibition of IgA rosette formation with intact and proteolytic fragments of rabbit and human secretory IgA (SIgA), human myeloma proteins, and secretory component. RFc alpha bound human SIgA as efficiently as rabbit SIgA. Of the human IgA tested, RFc alpha preferentially bound to the IgA2 subclass and most avidity to a multimeric IgA molecule. In addition., the site of the RFc alpha-IgA interactions was localized to the C alpha 2 domain in the Fc portion of IgA. RFc alpha on spleen lymphocytes showed the same pattern of binding specificity toward the human IgA subclasses as the RFc alpha on lymphocytes from Peyer's patches. However, differences between the spleen and Peyer's patches were observed in the interaction of RFc alpha and the rabbit IgA subclasses. RFc alpha on lymphocytes from the Peyer's patches bound the rabbit IgA-g subclass more efficiently than the rabbit IgA-f subclass, whereas the converse was true for the spleen RFc alpha. Human secretory component inhibited IgA but not IgG or IgM rosette formation as a result of its interaction with lymphocytes. These studies suggest: 1) that there may be two types of RFc alpha expressed by lymphocytes in certain tissues, and 2) that there are two closely associated receptors on the lymphocyte--one specific for secretory component and the other for structures within the C alpha 2 domain of IgA.

Rat hepatocytes in short-term monolayer cultures bound radiolabeled polymeric rat IgA but not IgG. The binding of 125I-IgA was inhibited equally well by unlabeled polymeric IgA and by antiserum to rat secretory component (SC). The antibody to SC, after specific purification and radiolabeling, was bound to hepatocytes as effectively as the IgA. These results indicate that SC acts as the receptor for polymeric IgA on rat hepatocytes as it does on human gut epithelia, and that the transport of IgA from blood to bile in rats across the liver is analogous to that of IgA across human enterocytes.

Specific IgA and sIgA antibodies were studied in the sera of patients suffering from various intestinal diseases (dysentery, salmonellosis, typhoid fever, chronic typhoid carrier state) and in the sera of healthy persons immunized by parenteral route with typhoid alcohol vaccine. The nature of antibodies was identified in Coombs' test, using monospecific antisera to alpha-chain and to the secretory component. IgA and sIgA antibodies were revealed most frequently in the sera of dysentery patients and of chronic typhoid carriers. No sIgA antibodies were found in the sera of subcutaneously immunized persons. The presence of specific sIgA antibodies in the serum reflects the participation of local immune mechanisms in the formation of systemic immunity in the intestinal infections.

Viable suspensions of columnar cells were obtained from the surface and outer crypt epithelium of human colon mucosa by a combined treatment with citrate and EDTA solutions and mechanical disruption, but without the use of enzymes. A substantial fraction of the isolated cells contained free SC and secretory IgA in a cytoplasmic distribution that corresponded to previously reported immunohistochemical and immunoelectron-microscopical results obtained with tissue sections. On their surface the same cells were shown to bear SC complexed with J-chain-containing polymeric IgA, whereas only occasional traces of free SC were found. IgM was detected in the surface patches which contained the largest concentrations of SC and IgA. These findings demonstrate that in SC-producing epithelial cells SC molecules become exposed on the plasma membrane, thereby being able to bind specifically J-chain-containing IgA and IgM present in the tissue fluid. Such adsorptive complexing is probably a prerequisite for the selective pinocytotic transport of these two Ig classes through glandular epithelium in man.

The ability of the liver to transport antigen-antibody complexes containing polymeric IgA was tested in a model system using the isolated perfused rat liver and soluble complexes formed between trinitrophenylated (TNP) antigens and MOPC 315, a polymeric mouse IgA protein with anti-TNP activity. A double-label strategy (125I and 131I) was used to separately follow antigen and antibody during isolation and transport by the isolated perfused liver. Complexes formed in antigen excess and isolated by gel filtration were added to the perfusate. The quantity of antigen or antibody transported was determined by counting the radioactivity in collected bile fractions. TNP-human albumin (TNP-HSA) complexed to polymeric IgA antibody was transported from blood to bile while the same antigen complexed to IgG antibody was not. The transport of IgA-(TNP-HSA) complexes was inhibited by preincubation with human secretory component (SC), which indicated that transport of such complexes proceeds though an SC-dependent mechanism previously described for uncomplexed polymeric IgA antibody. Complexes (m.w. congruent to 970,000) of trinitrophenylated bovine thyroglobulin (TNP-TG) and polymeric IgA were transported less well than IgA-(TNP-HSA) complexes (m.w. less than or equal to 460,000), even though both types of complexes bound SC. The possibility that the poor transport of IgA-(TNP-TG) complexes reflected a size restriction on hepatic transport from blood to bile is discussed.

The functional importance of the intestinal immune system will be shown in the following report. The secretory immune system, the pool of T-lymphocytes, the K-(killer-) and NK-(natural killer-) cells, the accessory cell-system (macrophages, mast cells, granulocytes), the so-called homing and the intestinal antigenabsorption will be described and discussed.

IgA nephropathy, as Berger defined it, is characterized by mesangial deposits of IgA, which are easily visualized by immunofluorescence on kidney biopsies. The structure (mono- or dimeric) of these IgA has not been clearly defined so far. Fifteen renal biopsies were studied to find out whether these IgA are serum monomers, or are polymers from a different origin. This was done by tissue fixation in vitro of free secretory component, which was then visualized by immunofluorescence (IF). In all 15 cases, the IgA deposits were shown to lack bound secretory component, but were able to bind, specifically, with the free secretory component. The presence of J chain in these deposits was also evidenced by indirect IF. These findings favour the hypothesis that these immunoglobulins are polymeric.

The relative contribution of secretory IgA, monomeric and polymeric IgA and IgA/secretory component-containing immune complexes was investigated in sera of diabetic patients. Secretory IgA and immune complexes containing IgA and secretory component seem to participate in the hyper-IgA of patients with Type 2 (non-insulin-dependent) diabetes only, suggesting an altered hepatic clearance via secretory component receptors on hepatocytes. In Type 1 (insulin-dependent) diabetes, the high serum IgA levels might be explained by an increase in IgA production in response to antigenic stimuli. Evidence is also accumulated that immune complexes containing IgA of mucosal origin may be involved in microangiopathy production in Type 2 diabetes.

A disulfide-interchange enzyme from rat liver microsomes was found to promote binding in vitro of human free secretory component (SC) to dimeric serum-type IgA containing J chain, as assessed by immune precipitation and gel filtration. This effect was greater withe native than with partially reduced SC. Most of the bound SC was covalently linked, as determined by electrophoresis in polyacrylamide gels in detergent. The enzyme did not promote binding of native or partially reduce SC to IgG, IgA monomer, IgA dimer without J chain, or IgM. In the case of IgM, the enzyme did, however, promote covalent bonding of previously non-covalently linked SC. The results overall suggest that a disulfide-interchange enzyme could play a role in vivo in the cell-associated assembly of secretory IgA by promoting the covalent attachment of SC to a dimer of serum-type IgA and that the J chain in the IgA dimer contributes to the enzyme effect.

Dimeric IgA-forming cells were studied by a secretory component (SC) affinity test on 20 palatine and 7 pharyngeal tonils from children. This study also included an investigation on the immunoflurescent localization of IgA immunocytes and IgA and SC deposits. The results showed that IgA immunocytes capable of binding SC in tissue sections are present in both palatine and pharyngeal tonsils. However, the number of cells positive for the SC affinity test was significantly lower than that of IgA immunocytes not binding SC. IgA immunocytes were located mainly in the subepithelial area, medullary portion, and occasionally the intraepithelial layer. SC determinants were detected only in some epithelial cells of the pharyngeal tonsils. The findings of the present study suggest that the pharyngeal tonsils share in the local immunological mucosal resistance regulated by secretory IgA, although its activity might be limited.

A sensitive solid phase radioimmunoassay method was established for the specific quantitative determination of secretory IgA (sIgA) by taking advantage of the dual antigenicities of sIgA, one specific for alpha-chain and the other for secretory component (SC). The sIgA and IgA in the sample were first bound by anti-IgA antibodies coated on the polystyrene tube, then the amount of bound sIgA was quantified by the use of 125I-labeled anti-SC antibodies. This method is quite sensitive and allows us to distinguish sIgA from IgA and free SC which usually coexist in exocrine secretions. Linear relationship was observed between the bound radioactivity of radioiodinated anti-SC and the amount of sIgA in the range of 5 to 60 ng of sIgA. With this method, the urinary sIgA levels in normal children and adults were measured. Urinary sIgA was detected in half of infants within 7 days after birth, while it could be demonstrated in almost all of the infants by 14th day. Then, it gradually increased and reached about a half of the adult level by the age of puberty. This method will be useful for the sensitive and specific measurement of sIgA in various exocrine secretions.

The number of antigenic determinants of human SC and sIgA was determined with specific anti-SC antibodies isolated from a pool of two hyperimmunized sheep. The Fab' antibody fragments were prepared and [125I] labelled, while pure SC and pure sIgA, isolated from colostrum, were labelled with 131I. The [125I] Fab' antibodies were added, in very large molar excess, to the [131I] antigens at various ratios. The Fab'-Ag complexes were separated from the antibody excess by gel-filtration. The highest Fab'/Ag molar ratios of the complexes, which correspond to the maximal number of accessible antigenic determinants, were calculated. We found at least 16 sites (16.6 +/- 1.36) on free SC while only 12 (12.27 +/- 0.51) were located on sIgA. These results confirm the existence of a significant number of hidden determinants of the SC subunit of sIgA and establish that about 1/4 of the SC molecule is implied in this binding.

The secretory component (SC) has recently been found to be associated with IgM in external secretions, although in a less stable complex than secretory IgA. Moreover, SC combines spontaneously in vitro with both IgA and IgM. A prerequisite is that the immunoglobulins contain the J chain, which is present only in dimers and polymers. This polypeptide is essential for the formation of an SC-binding site which appears already at the cytoplasmic level in IgA- and IgM-producing immunocytes. Locally formed J-chain-containing immunoglobulins are therefore readily available for complexing with SC present in the membranes of columnar secretory epithelial cells of glandular sites. This complexing initiates pinocytosis and external transport. Immunohistochemically the gland cells are shown to contain SC, IgA and IgM in identical locations, except that SC alone appears in the Golgi zone. Locally formed IgA and IgM antibodies are thus efficiently transferred to the mucosal surface where they exert an immunological exclusion of antigens. Conversely, IgG antibodies, which are not actively drained away from the lamina propria, may rather become engaged in complement activation and cell-mediated cytotoxicity with potentially deleterious effects on the tissue. Secondary to severe inflammatory reactions, secretory epithelium may show decreased production of SC; the selective external transport of SC-stabilized secretory IgA and IgM is thus jeopardized, and a vicious circle may be set up in the mucosa.

Secretory IgA is the prevailung immunoglobulin on the mucous membranes of different tissues. It is a polymer immunoglobulin and in comparison to the serum IgA the secretory IgA has a additional polypeptide chain, the secretory component. The secretory IgA is synthesized locally in the mucosa. Secretory IgA is a very important factor in the immune defence of the mucous membranes. Secretory antibodies are regulated independently from serum antibodies. They show a virus neutralizing effect and activities against bacteria and lifeless noxa. The mechanism is not yet clear. It is possible, that the reaction of the secretory IgA with the antigen prevents settlement of microorganisms on the mucous membranes. The clinical importance of secretory IgA is undoubted especially the deference of the mucosa in cases of a virus infection.

Hepatic bile IgA from 6 patients was measured by radioimmunoassay and characterized immunochemically. The concentration of IgA was 0.14 to 0.88 mg/ml. IgA associated with secretory component (SC) as well as unassociated with SC was demonstrated in all 6 samples; The proportion associated with SC (secretory IgA) was 72% to 95%. IgA and SC were localized immunocytochemically in liver and bile duct tissues by the peroxidase-labeled antibody method. Evidence favoring endocytic, SC-mediated transfer of IgA by intrahepatic and extrahepatic biliary epithelium, but not by hepatocytes, was found.

The translocation of dimeric IgA across epithelial cells was studied by immunoelectron microscopy in an in vitro system with cultured neoplastic human colon cells (HT-29). Ultrastructurally, the cells were found to be well-polarized epithelial cells connected by intercellular junctions. Secretory component (SC) was localized to the basolateral plasma membranes. Dimeric human IgA, when reacted with the cells at 0 C, bound selectively to SC. When incubated at 37 C, the bound dimeric IgA was internalized by pinocytosis and transported apically through the cytoplasm in vesicles. The vesicles were opened to the lumen at the apical surfaces or discharged into the lumen. We conclude that the translocation of dimeric IgA across intestinal epithelial cells has been defined at the ultrastructural level in cultured neoplastic colon cells in vitro.

Secretory IgA (sIgA) and monomeric IgA (mIgA) were purified from normal rat bile, and their role in the gastrointestinal tract was investigated. Biliary sIgA has a molecular weight of approximately 400,000 daltons and a sedimentation constant of 11.7S. Thus, sIgA obtained from rat bile had physicochemical properties similar to those reported for sIgA in other secretions, and probably consists of L-chains, alpha-chains and secretory component. After in vitro incubation with trypsin or intestinal fluid, sIgA remained intact, whereas mIgA was hydrolyzed. Rats challenged repeatedly with dinitrophenylated bovine serum albumin (DNP-BSA) had specific IgA antibody against DNP in the bile and feces as measured by a radioimmunoassay using [3H]-DNP-lysine. Our results demonstrate a possibility that biliary sIgA, not mIgA, has an important role which inhibits the absorption of foreign antigen from intestine.

Immunoglobulin A from human milk binds to neonatal buccal cells. Pretreatment of the cells with anti-secretory component antiserum blocks this binding, suggesting that secretory component (SC) contributes to the adhesive process. The concentrations of SC in saliva of preterm neonates is lower than that of full-term infants. This may lower the effectiveness of mucosal immunity passively transferred from the mother.