Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-beta-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia.

Mannose-binding lectin (MBL) is reported to bind to agalactosyl IgG, but not to normally galactosylated (native) IgG. It was recently reported that serum polymeric IgA in its native form reacts with MBL, whereas a more recent report has claimed that native IgD and IgE, and possibly IgM, do not. This led us to investigate whether IgA is truly reactive with MBL. To accomplish this, we collected purified human Igs, of various classes, subclasses, and allotypes, and tested their ability to bind to MBL using an ELISA method. Among these preparations, only one (monoclonal IgA2m(2):Kur) exhibited significant MBL binding. In particular, polymeric or monomeric forms of our normal serum IgA preparation lacked any ability to bind to MBL whatsoever. However, all the Ig preparations which had not bound to MBL became able to do so when they were degalactosylated with a galactosidase treatment, and the binding was further enhanced by acidic denaturation of the Igs. Among the degalactosylated and/or acid-denatured IgA, the IgA2 subclass exhibited a higher level of MBL binding than did IgA1. Our results suggest that MBL does not bind to native Igs (viewed in principle as “self” components), and that only Igs with abnormal glycosylation (degalactosylated forms) and/or denaturation would be MBL reactive.

Despite our knowledge on the role of IgA in mucosal homeostasis and host defense and clinical evidence suggesting deficient first-line defense mechanisms in chronic airway disorders, little is known regarding its role in asthma and chronic obstructive pulmonary disease (COPD). Studies suggest that the mucosal IgA response is impaired in COPD, and a deficient transport of IgA across the bronchial epithelium in COPD has been identified, possibly involving neutrophil proteinases, which may degrade the Ig receptor mediating this transepithelial routing. In contrast, the IgA response to allergens in patients with asthma may play a pathogenic role through eosinophil activation. Thus, secretory IgA can induce eosinophil degranulation in vitro, a feature in keeping with the correlations observed in vivo between airway IgA levels and eosinophil cationic protein during late asthmatic responses. Selective IgA deficiency is associated with an increased prevalence of atopy, and a protective role of IgA has been seen in murine models of asthma, delineating the complexity of the IgA system in the airway mucosa. Future studies will hopefully yield better knowledge of IgA biology and lung mucosal immunity and help to use more efficiently the mucosal route for immunotherapy or target specific genes in inflamed airways.

Patients with alcoholic liver cirrhosis (ALC) have high serum levels and spontaneous in vitro production of immunoglobulin (Ig) A. Deposits of IgA are also found in liver sinusoids. Increased interleukin 6 (IL-6) production is another feature of this disease. This study shows a linear correlation between increased lipopolysaccharide (LPS)-induced IL-6 production and increased spontaneous IgA and IgG secretion by peripheral blood mononuclear cells (PBMCs). PBMCs and purified monocytes isolated from healthy control subjects and patients with ALC contain elevated IL-6 messenger RNA levels and produce IL-6 in response to stimulation with soluble polymeric IgA (p-IgA) or attached monomeric IgA (m-IgA) but not with soluble m-IgA. The addition of monospecific antibody to human IL-6 inhibits spontaneous IgA production by PBMC. This inhibition is more pronounced in patients with ALC. These data provide evidence that IgA, possibly by attachment to cells possessing Fcα receptors and secreting IL-6, is involved in the production of this major mediator and the amplification of Ig secretion. Circulating IgA and IgA deposits could therefore initiate a process of autoamplification implicated in the development of hypergammaglobulinemia in ALC.

Oral administration to mice of ovalbumin (OVA), if given together with cholera toxin (CT) or its B subunit (CTB) prevented the hyporesponsiveness to OVA subsequently injected parenterally. Oral immunization with CT plus OVA or OVA plus CTB in fact primed the immune system, inducing a stronger response to a subsequent parenteral injection of OVA with complete Freund's adjuvant than in mice prefed only with OVA or with saline. Oral CT plus OVA also induced good serum IgG1 and IgA anti-OVA responses, with slightly (not significant) decreased IgG2a and IgG2b responses. Our in vivo findings agree well with earlier in vitro data from others, including CT inhibition of the T(h)1 CD4+ T cell subset and with CT effect on B cells (induction of LPS-stimulated IgM+ B cells to undergo increased switch differentiation to IgG1- and IgA-secreting cells).

Transcytosis is defined as the vesicular transfer of soluble or membranous constituents across polarized cells. Transcytosis can occur from basolateral-to-apical, or apical-to-basolateral in the same cell (Bomsel et al 1989). The three classical modes of endocytosis, namely fluid-phase, adsorptive and receptor-mediated, also apply to transcytosis. Receptor-mediated transcytosis makes the transcellular transfer of solutes both specific and efficient, by the selective uptake of ligands and their subsequent guidance along the complex transcellular route. This process is particularly important for the transfer of antibodies across epithelial barriers. Receptor-mediated transcytosis ensures the selective passage of IgG from the maternal blood across the placenta or, in some species, its intestinal absorption from mother’s milk, resulting in neonatal humoral immunity. Receptor-mediated transcytosis is also responsible for the transfer of J-chain containing polymeric immunoglobulins (pIg) A and M into secretions, generating mucosal immunity, conferring protection to suckling infants and allowing for the disposal of circulating pIg-immune complexes.

Another group of researchers has reported that seven intraperitoneal injections into mice of purified human serum IgA or normal human serum--but not IgA-deficient serum, mouse serum or saline--induced considerable growth of the extrahepatic bile duct epithelium. They stated that heterologous IgA was principally responsible for this effect. We have quantitated this growth by another method: enumeration of total and radiolabeled nuclei of the lumenal and glandular epithelium in transverse duct sections after tritiated thymidine injection and autoradiography. Our data show maximal incorporation between 18 and 24 hr after the last injection, with the highest labeling index in the lumenal epithelium after only three injections and before any duct enlargement. After four to seven injections, thymidine incorporation continued in juxtalumenal glands, together with massive glandular proliferation into the thickening wall and obvious inflammation. These changes were more pronounced in the liver-proximal part of the duct without affecting intrahepatic duct and gallbladder epithelia. Subcutaneous serum injections were less active. We confirm the inactivity of buffered saline, but, unlike previous authors, demonstrate strong activity in purified human milk secretory IgA and in IgA-deficient serum. We suggest that this different approach of quantitating epithelial proliferation will allow comparison of the bile duct growth effects of different, well-characterized human and animal IgA preparations.

Under endotoxin-free conditions, peripheral blood mononuclear cells and purified monocytes isolated from healthy control subjects and patients with alcoholic cirrhosis disclose elevated tumor necrosis factor alpha messenger RNA level and produce tumor necrosis factor alpha in response to stimulation by either soluble polymeric IgA or monomeric IgA bound to the surface of culture dishes but not by soluble monomeric IgA. Polymeric IgA induces tumor necrosis factor alpha secretion in a dose-dependent fashion. These results suggest that cross-linking of Fc-alpha receptors on human monocytes induces the messenger RNA accumulation and the secretion of the cytotoxic and immunoregulatory cytokine tumor necrosis factor alpha. Furthermore, it is shown that lipopolysaccharide-induced tumor necrosis factor alpha secretion by peripheral blood mononuclear cells is synergistically enhanced in the presence of solid phase monomeric IgA but not in the presence of either soluble monomeric or polymeric IgA. Although increased lipopolysaccharide-induced tumor necrosis factor alpha secretion is observed at baseline in alcoholic cirrhotic patients, this synergism is also expressed in this group of patients. These observations could be of pathophysiological relevance in alcoholic cirrhosis because monomeric IgA deposits along the liver sinusoids and increased serum levels of polymeric IgA are common even in the early stages of this disease.

Saccharomyces boulardii (S.b.) is largely used in Western European countries for the treatment of acute infectious enteritis and antibiotic-induced gastrointestinal disorders. To study the mechanisms of the protective effect of S.b. against enteral pathogen infection, we assessed the response of the intestinal secretion of secretory IgA (s-IgA) and of the secretory component of immunoglobulins (SC) to oral administration of high doses (0.5 mg/g body weight, three times per day) of S.b. cells in growing rats. S.b. cells (biological activity: 2.8× 109 viable cells/100 mg) were administered daily by gastric intubation to weanling rats from day 14 until day 22 postpartum. Control groups received either 0.9% saline or ovalbumin following the same schedule. Expressed per milligram of cell protein, SC content was significantly increased in crypt cells isolated from the jejunum (48.5% vs saline controls, P< 0.05) as it was in the duodenal fluid (62.8% vs saline controls, P<0.01) of rats treated with S.b. Oral treatment with S.b. had no effect on the secretion of SC by the liver. In the duodenal fluid of rats treated with S.b. cells, the mean concentration of s-IgA was increased by 56.9% (P<0.01) over the concentration of s-IgA measured in saline controls. Compared to control rats treated from day 14 until day 22 postpartum with an antigenic load of ovalbumin equivalent to the total protein load provided by Sb cells (0.05 mg protein/g body weight, three times per day), S.b.-treated rats also exhibited a significantly higher intestinal concentration of SC (69% in villus cells, P<0.025 and 80% in crypt cells, P<0.01 These changes in intestinal SC and s-IgA concentration appeared not to be due to an increase in enterocyte turnover rate, since the mucosal mass parameters and the incorporation rate of [3H]thymidine into DNA measured in the jejunum, ileum, and colon remained unchanged in S.b.- treated rats. Our findings suggest that one of the mechanisms by which S.b. exerts its immunoprotective effect in the gastrointestinal tract is a stimulation of the intestinal secretion of s-IgA and of the secretory component of immunoglobulins.

The serum IgA in young infants (6–240 days old) comprised a much larger proportion (\(\bar X\)=36%; range, 7.5–72%) of polymeric IgA than in normal adults (\(\bar X\)=13%; range, 4.7–22%), but with a normal subclass distribution. In contrast, in IgA-deficient adult serum, there was a higher proportion (\(\bar X\)=34%; range, 16.3–38%) of IgA2 than in normal adult serum (\(\bar X\)=21%; range, 5–36%), with, however, a normal proportion of polymers. The distribution of IgA subclasses in the separated monomeric and polymeric IgA fractions was similar to that in unfractionated sera. The proportions of IgA polymers and IgA2 in serum are unlinked parameters, suggesting that both do not necessarily represent mucosal contributions to blood IgA.

This chapter describes the different aspects of single radial immunodiffusion (SRID). SRID is an immunodiffusion technique, in which a single partner of the antigen–antibody reaction, usually the antigen (Ag), diffuses radially from a small well punched into a gel layer of constant thickness and the other partner, usually the antibody (Ab), has been uniformly incorporated into this gel layer and is, therefore, not considered as diffusing. The procedure of filling the Ag wells to the brim without measuring the volume is not recommended as it introduces comparatively large volumetric inaccuracies from well to well. The measurements of sharp precipitin rings may already be possible on the wet gel plates, either by using an ocular with a microcomparator scale or by enlarging the plate in a photographic or other optical enlarger. Some antigens may display the molecular size heterogeneity, usually consisting of a series of polymers, giving the reactions of immunological identity with the monomers. SRID may be used in a reversed version— that is, with diluted Ag incorporated into the gel layer and solutions of Ab or antiserum, diffusing from the wells.

Three classes of immunoglobulins have been identified in the pigeon. IgG and IgM were purified from pigeon serum whereas IgA was isolated from pigeon hepatic bile. Pigeon IgG and IgM had the same properties and immunohistological distribution as their chicken homologues. Pigeon IgA was identified on the following grounds: (1) it contains the same light chains as pigeon IgM and IgG; (2) it is relatively abundant in exocrine secretions such as bile, egg white, cropmilk and intestinal fluid, whereas it is present only in small amounts in serum; (3) it occurs in the cytoplasm of the majority of the immunocytes from the intestinal mucosa; (4) its electrophoretic mobility and molecular size are similar to those of chicken IgA. Surprisingly, no immunoglobulin-containing cells could be detected in sections of the wall of the cropmilk gland, despite the high IgA content of the cropmilk.

The inheritance and strain distribution of a genetic marker of the α-chains of rat IgA, which we propose to call Iα(1a), was tested by means of a precipitating anti-allotype antiserum obtained by immunizing August rats with a monoclonal IgA protein from a LOU/Wsl rat immunocytoma. The presence or absence of the Iα(1a) character appeared to be controlled by a single pair of codominant alleles, which appear to govern the biosynthesis of virtually all the rat serum IgA.

Antiserum from goats immunized with heavy polypeptide chains from a γA-type myeloma globulin was absorbed with serum from patients with selective absence of immunoglobulin A (γA). The resulting reagents could be used for the classification of 58 γA-myeloma proteins into two distinct antigenic types, respectively called subclasses He and Le. These differences were shown to be related to the heavy (alpha) polypeptide chains and independent of the integrity of interchain disulfide bridges. The γA-immunoglobulin from normal serum appears to consist, for the most part, of molecules with Le subclass specificity.