Identification of secretory component as an IgA receptor on rat hepatocytes.

Secretory component (SC) was found to be synthesized by isolated rat hepatocytes. SC was detected by radioimmunoassay and cultured hepatocytes were found to synthesize 0.078 microgram SC/10(6) hepatocytes in a 48-h period. SC was also present on the surface of hepatocytes as detected by the specific binding of radiolabeled anti-SC antibodies as well as by the detection of specific membrane staining in indirect immunofluorescence tests using specifically purified anti-SC antibodies. Rat SC was detected on hepatocytes and intestinal epithelial cells but not on peripheral blood lymphocytes, unfractionated spleen cells, or erythrocytes. Specific binding of radiolabeled rat dimeric IgA to rat hepatocytes was also observed and evidence was obtained to indicate that such binding was mediated by SC. Thus, prior incubation of hepatocytes with anti-SC prevented binding of radiolabeled IgA. Moreover, prior incubation of radiolabeled IgA with rat SC prevented binding of the IgA to isolated hepatocytes. Cells treated with 0.25% trypsin lost their ability to bind to radiolabeled dimeric IgA.

Receptor-mediated biliary transport of immunoglobulin A and asialoglycoprotein: sorting and missorting of ligands revealed by two radiolabeling methods.

In the rat, all receptor-bindable immunoglobulin A (IgA), and 1-4% of injected asialoglycoprotein (ASG), are transported from blood to bile intact. The major fraction of the ASG is degraded in hepatic lysosomes. The study described here was designed to elucidate the sorting that occurs in hepatocytes subsequent to receptor binding of ligands not sharing the same fate. We show that conjugation of protein with the Bolton and Hunter reagent can be used as a probe for the lysosomal pathway, since 50% of the reagent is released into bile after lysosomal degradation of internalized protein. Radiolabeling by iodine monochloride was alternatively used to follow the direct pathways that deliver intact IgA and ASG to bile. After intravenous injection of labeled proteins, first intact ASG and IgA, and then radioactive catabolites from degraded protein, were released into bile. No proteolytic intermediates were detected, and the transport of IgA or ASG directly to bile was not affected by the lysosomal protease inhibitor leupeptin. These observations indicate that divergence of the direct biliary transport pathways from the degradation pathway occurs at a stage preceding delivery to lysosomes, possibly at the cell surface. Competition studies showed that all three pathways (including the biliary transport of intact ASG) are receptor mediated, but even at supersaturating doses the uptake and processing of IgA and ASG occur independently. We propose that IgA and ASG receptors are not frequently in juxtaposition on the plasma membrane, but that ASG, after binding to its receptor, is occasionally missorted into the biliary transport pool.

Human IgA as a heterovalent ligand: switching from the asialoglycoprotein receptor to secretory component during transport across the rat hepatocyte.

Asialoglycoproteins are taken up by the rat liver for degradation; rat polymeric IgA is taken up via a separate receptor, secretory component (SC), for quantitative delivery to bile. There is negligible uptake of these ligands by the converse receptor, and only a low level of missorting of ligands to opposite destinations. The two pathways are not cross-inhibitable and operate independently (Schiff, J.M., M. M. Fisher, and B. J. Underdown, 1984, J. Cell Biol., 98:79-89). We report here that when human IgA is presented as a ligand in the rat, it is processed using elements of both pathways. To study this in detail, different IgA fractions were prepared using two radiolabeling methods that provide separate probes for degradation or re-secretion. Behavior of intravenously injected human polymeric IgA in the rat depended on its binding properties. If deprived of SC binding activity by affinity adsorption or by reduction and alkylation, greater than 80% of human IgA was degraded in hepatic lysosomes; radioactive catabolites were released into bile by a leupeptin-inhibitable process. If prevented from binding to the asialoglycoprotein receptor by competition or by treatment with galactose oxidase, human IgA was cleared and transported to bile directly via SC, but its uptake was about fivefold slower than rat IgA. Untreated human IgA was taken up rapidly by the asialoglycoprotein receptor, but depended on SC binding to get to bile: the proportion secreted correlated 1:1 with SC binding activity determined in vitro, and the IgA was released into bile with SC still attached. These results demonstrate that human IgA is normally heterovalent: it is first captured from blood by the asialoglycoprotein receptor, but escapes the usual fate of asialoglycoproteins by switching to SC during transport. Since the biliary transit times of native human and rat IgA are the same, it is probable that the receptor switching event occurs en route. This implies that the two receptors briefly share a common intracellular compartment.

Evidence for a vesicular transport mechanism in hepatocytes for biliary secretion of immunoglobulin A.

Quantitative electron microscopic autoradiography and diaminobenzidine cytochemistry provide evidence for an uptake and vesicular transport mechanism for iodine-125-labeled immunoglobulin A from plasma to bile by hepatocytes in vivo. The data confirm the existence of a hepatobiliary pathway for secretion of immunoglobulin A into the intestine and are consistent with a vesicular transport mechanism for biliary proteins within liver parenchymal cells.

Studies on the specificity of the IgA-binding lectin, jacalin.

The interactions of IgA with the jackfruit lectin, jacalin, were investigated with regard to the specificity of jacalin for species and subclasses of IgA. It was found that jacalin selectively bound to human IgA1, but not to human IgA2, mouse IgA or rat IgA. Binding studies with human IgA1 fragments produced by different IgA1 proteases revealed that jacalin bound to galactose-terminal oligosaccharides in the hinge region of human IgA1. Affinity chromatography employing jacalin-Sepharose provided a means to separate the subclasses of IgA in human whey.

Secretory component as the mucosal transport receptor: separation of physicochemically analogous human IgA fractions with different receptor-binding capacities.

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.

Binding of secretory component to protein 511, a pIgA mouse protein lacking 36 amino acid residues of the C alpha 3 domain.

Protein 511, a murine IgA protein described previously by Robinson and Appella [Proc. natn. Acad. Sci. U.S.A. 77, 4909-4913 (1980)] which lacks 36 amino acids in the C alpha 3 domain, was tested for its ability to bind to radiolabelled secretory component (125I-rat SC) and to be transported from blood to bile in the rat, a function described previously to be mediated by the poly Ig receptor (pIg R). When compared to other mouse pIgA proteins, the naturally occurring mutant protein 511 bound 125I-rat SC and was transported from blood to bile in a manner indistinguishable from wild-type pIgA protein. We conclude that the region of Fc alpha which is missing in protein 511, is not involved in mediating the binding of pIgA to the pIg R.

Low level transport of IgA to bile via the asialoglycoprotein receptor.

The rat and rabbit transport IgA from blood to bile by a highly efficient transcellular pathway mediated by secretory component (SC). Other mammals do not express SC on liver hepatocytes, but they do transport a small amount of IgA to bile. In the first part of this study, human polymeric IgA was radiolabeled and depleted of SC binding activity by successive affinity adsorption. Transport of this preparation intact to rat bile was 4%, but was reduced to 2% when 50 mg unlabeled asialoglycoprotein was preadministered. The 2% decline corresponds to the percent of asialo-orosomucoid diverted to bile from the lysosomal pathway. In guinea-pigs, missorting of asialo-orosomucoid intact to bile was 10% of the injected dose. Transport of normal human IgA to bile was 1-2%, even though guinea-pigs do not express SC in the liver. Excess unlabeled asialofetuin reduced the transport of asialo-orosomucoid by 10-fold and IgA by 6-fold. This demonstrates that the asialoglycoprotein receptor can mediate transport of IgA to bile in small amounts, but that this transport may be only a biological artifact resulting from limited fidelity of intracellular protein sorting.

A comparison of secretory component - immunoglobulin interactions amongst different species.

1) SC from many species may be isolated by affinity chromatography to human IgA-Sepharose. 2) In some species SC may exist in two molecular forms. 3) The SC-binding sites on polymeric IgA and IgM are not identical. 4) In some species SC binds to IgM with higher affinity than to polymeric IgA while in other species SC binds best to polymeric IgA. This difference may influence the relative concentrations of these two immunoglobulin classes in the secretions of different species. 5) The SC-binding site is present on high molecular weight immunoglobulin in species as primitive as the nurse shark.

Acquired resistance to Giardia muris in X-linked immunodeficient mice.

A previous study from this laboratory (D. P. Snider, D. Skea, and B. J. Underdown, Infect. Immun. 56:2838-2842, 1988) indicated that immunodeficient mice expressing the xid gene develop prolonged infections with Giardia muris, unlike immunocompetent mice, which eliminate the intestinal protozoan parasite in 8 to 10 weeks. In this study, CBA/N (xid) and CBA/Ca mice were infected with G. muris cysts and at various times following this primary infection were cured by treatment with metronidazole. In contrast to the marked differences in the ability of xid and normal mice to eliminate a primary infection, mice of both strains were resistant to a secondary challenge of G. muris cysts. These data imply that the mechanism(s) responsible for elimination of a primary infection is not identical to those required to resist a secondary challenge infection. Splenocytes from immunocompetent CBA/Ca mice (but not immunodeficient CBA/N mice) could transfer the ability to eliminate a primary G. muris infection to irradiated mice of either strain. In contrast, splenocytes from previously infected CBA/Ca mice could not transfer resistance to a challenge infection, further supporting the hypothesis that there are differences between mechanisms required to eliminate a primary infection and those necessary to resist a second challenge infection.

Quantitative and temporal analyses of murine antibody response in serum and gut secretions to infection with Giardia muris.

We analyzed the appearance and level of Giardia muris-specific antibody of immunoglobulin A (IgA), IgG, and IgM isotypes, at weekly intervals, over the course of a 7-week infection in BALB/c and C57BL/6 mice. Using sensitive immunoradiometric assays, we observed that IgA antibody was the only detectable anti-G. muris antibody in intestinal secretions throughout the course of infection. No secreted IgG or IgM anti-G. muris antibody was detected even in concentrated intestinal secretions. The expulsion of G. muris by the mice was associated closely with the appearance and increasing levels of secreted anti-G. muris IgA antibody. Both IgG and IgA serum antibody to G. muris were detected, but no serum IgM antibody was detected. Serum IgA and IgG anti-G. muris antibody remained at high levels up to 10 weeks following clearance of the parasite. An interesting observation indicated that serum IgA antibody to G. muris developed more slowly in response to infection than secreted IgA antibody. An analysis of the molecular weight distribution of total serum IgA in infected mice determined that infection produced a transient but significant shift in serum IgA to high-molecular-weight (greater than or equal to dimeric IgA) forms. The results indicate that a substantial IgA antibody response occurs in sera and in gut secretions of G. muris-resistant mice and that IgA antibody is the dominant and possibly the only effector antibody active in intestinal secretions during G. muris infection in mice.

A study of the association of human secretory component with IgA and IgM proteins.

Human secretory component (SC) was isolated from colostral whey, and the binding of 125I-SC to purified IgA and IgM monoclonal proteins was studied using two methods to separate free from immunoglobulin-bound 125I-SC: a) gel filtration on Sephadex G-200, and b) precipitation of 125I-SC-Ig complexes with anti-Ig antibody. Both IgA dimeric proteins and IgM pentamers bound 125I-SC with approximately one SC-binding site per mole of polymer and similar affinity. Assuming a reversible equilibrium, an apparent association constant congruent to 10-8 M-1 was calculated to govern the binding of 125I-SC to immunoglobulin polymers. The assignment of a single association constant may be an oversimplication, particularly for the case of IgA polymers, since evidence was obtained that disulfide bonds were formed in the 125I-SC-IgA complex. Despite the complexity of the reaction, binding of 125I-SC to both IgA and IgM polymers could be analyzed by standard methods of saturation analysis, and both were shown to have a similar affinity for 125I-SC. No differences were noted in the affinity of 125I-SC binding to the IgA1 and IgA2 subclasses. Binding of monomeric IgA and IgM proteins could not be measured and was at least 100-fold lower than that found for IgA and IgM polymers. Complexes of 125I-SC with IgA dimers were presumed to involve covalent bond formation, since these complexes did not dissociate in guanidine-HCl. One IgA2 trimer did not form a covalent bond since it was completely dissociated in guanidine. In contrast, 125I-SC-IgM complexes were dissociated in denaturing solvent, indicating that such complexes were held together primarily by non-covalent bonds. Experiments with (Fc)5 mu isolated by high temperature tryptic digestion of IgM showed that binding of 125I-SC was to the Fc region of IgM proteins. It was suggested that the binding of SC with similar affinity to both IgA and IgM polymers may be important in the biologic function of both these immunoglobulin classes.

Early expression in mice of genetic resistance to intestinal amebiasis.

Some strains of mice are known to be relatively resistant to hepatic or intestinal amebic infections. In order to know if the intestinal resistance is expressed few hours after infection, we inoculated axenic amebae in three inbred strains of mice either by direct intracecal injection or by infection of a washed-closed cecal loop. We found that amebae do not survive in conventional animals but they colonize longer in animals with the cecal loop. However, the survival was low after 24 hours postinfection. Balb/c mice were more susceptible and CBA mice more resistant. Our results suggest that genetic resistance to intestinal amebiasis is expressed in mice in the early phases of infection.

Lymphocyte proliferation to antigen E: demonstration of the restriction of antigen E-specific T cells to ragweed-allergic donors.

Allergic sensitivity to ragweed is common among atopic individuals in North America and can be associated with symptoms of seasonal hay fever and increased airway reactivity in asthma. This sensitivity is mediated by IgE antibody to ragweed antigens that in turn is presumed to be the product of B-lymphocytes regulated by various T cell subsets. Proliferation in vitro by lymphocytes obtained from individuals allergic to ragweed and cultured in the presence of ragweed antigen E (AgE) has been repeatedly described, but a comprehensive study of this proliferation has questioned the specificity of this response. We have examined this question and found that in the first week of culture, the specific lymphocyte proliferation to AgE may be obscured by high background and mitogen-like proliferation. However, by carrying the cells for a longer period of time in culture and providing a second in vitro boost with AgE, specific proliferation could be clearly documented. Lymphocytes from atopic ragweed-allergic donors proliferated at levels 20 to 50 times beyond background in the presence of AgE. Cells from nonragweed-allergic donors (either nonatopic or atopic) did not do so. The AgE-responsive cells could be expanded in culture and demonstrated to be T cells. Moreover, AgE-responsive T cells could only be cloned from AgE-allergic donors and, after expansion and subcloning, demonstrated to respond to AgE but not partially purified dust mite antigen. In contrast, a clone of T cells from a dust mite-sensitive individual proliferated in response to the dust mite antigen but not AgE.

Chronic giardiasis in B-cell-deficient mice expressing the xid gene.

The role of antibody in immunity to Giardia muris infection was investigated by studying B-cell-deficient CBA/N mice expressing the xid gene. After gastric administration of infective G. muris cysts, CBA/N male and female mice developed prolonged G. muris infection, whereas BALB/c mice eliminated their infection in 6 to 8 weeks. Male F1 progeny obtained from matings between female CBA/N mice and male BALB/c mice expressed the xid gene and developed prolonged infections. In contrast, all other F1 progeny of CBA/N and BALB/c matings, which did express the xid gene, eliminated G. muris. The link between the xid gene and prolonged infection was confirmed by studies of C57BL/6 mice congenic for the xid gene. When compared with BALB/c or F1 mice, CBA/N mice produced large quantities of immunoglobulin A (IgA) anti-G. muris antibody in serum and gut secretions during prolonged infection. Serum IgG anti-G. muris antibody levels were reduced in CBA/N and F1 male mice that expressed the xid gene. The inability of xid mice to eliminate G. muris is consistent with the importance of antibody in the development of immunity to G. muris. We hypothesize that mice bearing the xid gene fail to produce IgA antibody of appropriate specificity to an antigen or antigens whose recognition by antibody is critical for successful elimination of the parasite.

Intranasal antigen targeting to MHC class II molecules primes local IgA and serum IgG antibody responses in mice.

Covalent conjugates of hen egg lysozyme (HEL) and anti-major histocompatibility complex (MHC) class II monoclonal antibodies (mAb) were used to immunize mice intranasally. Three weeks after intranasal (IN) priming, mice responded rapidly to IN challenge with a mixture of HEL and cholera toxin (CT), by producing large titres of anti-HEL IgA and IgG1 antibody in serum, and IgA antibody in nasal secretions. No secondary response to HEL plus CT occurred in mice that received no priming or mice primed with HEL alone. The secondary serum IgG antibody response was dominated by the IgG1 subclass. HEL combined with CT adjuvant worked much better than HEL alone in producing the secondary response. Control conjugates, containing an IgG isotype-matched mAb without specificity for mouse tissues, provided poor priming. Mice expressing MHC class II molecules, to which the anti-MHC II mAb could not bind, produced a weak antibody response compared with those that expressed the appropriate. MHC class II molecule. Our results demonstrate that immunotargeting to MHC class II molecules via the IN route allows priming of the local IgA and circulating IgG antibody, and indicate that this technique is a feasible approach for delivery of subunit vaccines in the upper respiratory tract.

Steroid hormone regulation of free secretory component in the rat uterus.

The present studies analysed the uterine free secretory component (SC) response to steroid hormones, and correlated effects on SC with those on IgA. Administration of oestradiol for 3 days to ovariectomized rats significantly increased the levels of SC in uterine secretions, when compared to those in saline-injected controls. This response was dose-dependent and specific for oestrogens, since progesterone, testosterone and glucocorticoids had no effect. The oestradiol-induced elevation in SC levels occurred in parallel with that of IgA. Time course studies of SC and IgA in uterine secretions indicated that both proteins accumulated in nearly identical patterns following oestradiol administration. The oestradiol-stimulated accumulation of SC, however, appears to be independent of IgA since dexamethasone treatment with oestradiol decreased IgA but not SC levels in uterine secretions. In contrast to dexamethasone, progesterone antagonized the oestradiol effect on both uterine IgA and SC. The uterine SC response to oestradiol was also observed in vitro. Incubation of uterine tissue following oestradiol exposure in vivo resulted in a significant accumulation of SC in the culture medium, when compared to levels from control uteri. Addition of either cycloheximide or colchicine to uterine incubation media significantly decreased the effect of oestradiol on SC accumulation. These results suggest that oestrogen regulation of uterine SC may involve stimulation of its synthesis. In addition, our findings indicate that oestradiol control of SC in uterine secretions may be responsible for the movement of IgA from uterine tissues to lumen.