Structure of the human secretory immunoglobulin M core.

Immunoglobulins (Ig) A and M are the only human antibodies that form oligomers and undergo transcytosis to mucosal secretions via the polymeric Ig receptor (pIgR). When complexed with the J-chain (JC) and the secretory component (SC) of pIgR, secretory IgA and IgM (sIgA and sIgM) play critical roles in host-pathogen defense. Recently, we determined the structure of sIgA-Fc which elucidated the mechanism of polymeric IgA assembly and revealed an extensive binding interface between IgA-Fc, JC, and SC. Despite low sequence identity shared with IgA-Fc, IgM-Fc also undergoes JC-mediated assembly and binds pIgR. Here, we report the structure of sIgM-Fc and carryout a systematic comparison to sIgA-Fc. Our structural analysis reveals a remarkably conserved mechanism of JC-templated oligomerization and SC recognition of both IgM and IgA through a highly conserved network of interactions. These studies reveal the structurally conserved features of sIgM and sIgA required for function in mucosal immunity.

Structure of the secretory immunoglobulin A core.

Secretory immunoglobulin A (sIgA) represents the immune system's first line of defense against mucosal pathogens. IgAs are transported across the epithelium, as dimers and higher-order polymers, by the polymeric immunoglobulin receptor (pIgR). Upon reaching the luminal side, sIgAs mediate host protection and pathogen neutralization. In recent years, an increasing amount of attention has been given to IgA as a novel therapeutic antibody. However, despite extensive studies, sIgA structures have remained elusive. Here, we determine the atomic resolution structures of dimeric, tetrameric, and pentameric IgA-Fc linked by the joining chain (JC) and in complex with the secretory component of the pIgR. We suggest a mechanism in which the JC templates IgA oligomerization and imparts asymmetry for pIgR binding and transcytosis. This framework will inform the design of future IgA-based therapeutics.

Free and complexed-secretory immunoglobulin A triggers distinct intestinal epithelial cell responses.

Secretory immunoglobulin A (SIgA) antibodies play an important role in protecting the mucosal surfaces against pathogens and maintaining homeostasis with the commensal microbiota. Because a substantial portion of the gut microbiota is coated with SIgA, we hypothesized that microbiota-SIgA complexes are important for the maintenance of gut homeostasis. Here we investigated the relationship between microbiota-SIgA complexes and inflammatory epithelial cell responses. We used a multi-cellular three-dimensional (3D) organotypical model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes/monocytes, endothelial cells and fibroblasts. We also used human SIgA from human colostrum, and a prominent bacterial member of the first colonizers, Escherichia coli, as a surrogate commensal. We found that free and microbiota-complexed SIgA triggered different epithelial responses. While free SIgA up-regulated mucus production, expression of polymeric immunoglobulin receptor (pIgR) and secretion of interleukin-8 and tumoir necrosis factor-α, microbiota-complexed SIgA mitigated these responses. These results suggest that free and complexed SIgA have different functions as immunoregulatory agents in the gut and that an imbalance between the two may affect gut homeostasis.

Secretory component mediates Candida albicans binding to epithelial cells.

OBJECTIVES: Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. MATERIALS AND METHODS: Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. RESULTS: Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. CONCLUSION: These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

Acute effects of black currant consumption on salivary flow rate and secretion rate of salivary immunoglobulin a in healthy smokers.

The role of saliva in maintaining oral health and homeostasis is based on its physicochemical properties and biological activities of its components, including salivary immunoglobulin A (IgA). Both salivary rates and immunological status of saliva are found to be compromised in smokers. The aim of this study was to investigate the acute time-dependent effect of smoking and black currant consumption on the salivary flow rate (SFR) and salivary IgA secretion rate (sIgA SR) in healthy smokers. SFR, sIgA levels in saliva, and sIgA SRs were determined in healthy smokers (n=8) at eight times of assessment within three consecutive interventions: at the baseline; 5, 30, and 60 min after smoking; 5, 30, and 60 min after black currant consumption (100 g), followed by smoking; and 5 min after black currant consumption. Smoking induced a significant delayed effect on SFR measured 60 min after smoking (P=.03), while black currant consumption preceding smoking prevented that effect. Salivary IgA concentrations and sIgA flow rates were not acutely influenced by smoking. Black currant consumption preceding smoking induced a significant decrease in sIgA concentrations 5 min after the intervention compared with the baseline (P=.046), with a further increasing trend, statistically significant, 60 min after the intervention (P=.025). Although smoking cessation is the most important strategy in the prevention of chronic diseases, the obtained results suggest that the influence of black currant consumption on negative effects of tobacco smoke on salivary flow and immunological status of saliva could partly reduce the smoking-associated risk on oral health.

Leite de mulheres brasileiras apresenta anticorpos IgA secretores (SIgA) que neutralizam o rotavírus G9P[5] / Milk from Brazilian women presents secretory IgA antibodies and neutralizes rotavirus G9P[5]

OBJETIVO: Verificar a presença de SIgA anti-rotavírus sorotipo G9P[5] e a capacidade de neutralização do vírus de amostras de leite de mulheres brasileiras. MÉTODOS: Foram determinados os níveis de anticorpos SIgA reativos contra rotavírus G9 em 30 amostras de leite materno por ELISA usando suspensões purificadas do vírus. A capacidade das amostras de neutralizarem o rotavírus G9P[5] foi analisada em ensaio de de Neutralização utilizando células MA-104. RESULTADOS: Foram observadas grandes variações individuais referentes aos níveis de SIgA e títulos de neutralização, mas todas as amostras mostraram certa capacidade de neutralizar o G9P[5]. Verificamos uma correlação positiva altamente significativa entre os níveis de anticorpos e os títulos de neutralização. CONCLUSÕES: A alta correlação entre níveis de anticorpos anti-rotavírus e a capacidade neutralizante das amostras de leite sugere um possível papel protetor desses anticorpos contra a infecção. Esses resultados também apoiam o incentivo à prática do aleitamento materno.

OBJECTIVE: To verify the presence of anti-rotavirus serotype G9P[5] SIgA and the virus neutralization capacity of milk samples from Brazilian women. METHODS: SIgA antibody levels reactive to rotavirus G9 were determined in 30 maternal milk samples by enzyme-linked immunosorbent assay (ELISA) using purified virus suspensions. The samples' capacity to neutralize rotavirus G9P[5] was analyzed using the MA-104 cells neutralization assay. RESULTS: Great individual variations were observed regarding the SIgA levels and neutralization titers, but all samples showed some G9P[5] neutralizing ability. A highly significant positive correlation was observed between antibody levels and neutralization titers. CONCLUSIONS: The high correlation between anti-rotavirus antibody levels and neutralizing capacity of the milk samples suggests a possible protective role of these antibodies against infection. These results also support the encouragement of the breast-feeding practice.

Human milk sIgA molecules contain various combinations of different antigen-binding sites resulting in a multiple binding specificity of antibodies and enzymatic activities of abzymes.

In the classic paradigm, immunoglobulins are monospecific molecules that have stable structures and two or more identical antigen-binding sites. However, we show here for the first time that the sIgA pool of human milk contains, depending on the donor, only 35±5% λ-sIgAs, 48±7% κ-sIgAs, and 17±4% of chimeric λ-κ-sIgAs. sIgA preparations contained no traces of canonical enzymes. However, all sIgA fractions eluted from several specific affinity sorbents under the conditions destroying even strong immune complexes demonstrated high catalytic activities in hydrolysis of ATP, DNA, and oligosaccharides, and phosphorylation of proteins, lipids, and oligosaccharides. Sequential re-chromatographies of the sIgA fractions with high affinity to one affinity sorbents on the second, third and then fourth affinity sorbents bearing other immobilized antigens led to the distribution of Abs and all catalytic activities all over the profiles of these chromatographies; in all cases some fractions eluted from affinity sorbents only under the conditions destroying strong immune complexes. In vitro, only an addition of reduced glutathione and milk plasma containing no Abs to two sIgA fractions with different affinity for DNA-cellulose led to a transition of up to 11-20% of Ab from one fraction to the other. Our data are indicative of the possibility of half-molecule exchange between different IgA and sIgA molecules. In addition, it cannot be excluded that during the penetration of IgAs through the specific milk barrier, the secretory component (S) and the join chain (J) can combine molecules of dimeric H(2)L(2) λ-IgAs and κ-IgAs against different antigens forming many different variants of H(4)L(4)SJ sIgA molecules. Therefore, some chimeric molecules of sIgA can contain from two to four HL-fragments to various antigens interacting with high affinity with different sorbents and catalyzing various chemical reactions. Our data essentially expand the ideas concerning explanation of the phenomenon of polyspecificity and cross-reactivity of Abs.

N-Glycans on secretory component: mediators of the interaction between secretory IgA and gram-positive commensals sustaining intestinal homeostasis.

Human beings live in symbiosis with billions of microorganisms colonizing mucosal surfaces. The understanding of the mechanisms underlying this fine-tuned intestinal balance has made significant processes during the last decades. We have recently demonstrated that the interaction of SIgA with Gram-positive bacteria is essentially based on Fab-independent, glycan-mediated recognition. Results obtained using mouse hybridoma- and colostrum-derived secretory IgA (SIgA) consistently show that N-glycans present on secretory component (SC) play a crucial role in the process. Natural coating may involve specific Gram-positive cell wall components, which may explain selective recognition at the molecular level. More widely, the existence of these complexes is involved in the modulation of intestinal epithelial cell (IEC) responses in vitro and the formation of intestinal biofilms. Thus, SIgA may act as one of the pillars in homeostatic maintenance of the microbiota in the gut, adding yet another facet to its multiple roles in the mucosal environment.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Variant size- and glycoforms of the scavenger receptor cysteine-rich protein gp-340 with differential bacterial aggregation.

Glycoprotein gp-340 aggregates bacteria in saliva as part of innate defence at mucosal surfaces. We have detected size- and glycoforms of gp-340 between human saliva samples (n = 7) and lung gp-340 from a proteinosis patient using antibodies and lectins in Western blots and ELISA measurements. Western blots of saliva samples, and of gp-340 purified, from the seven donors using a gp-340 specific antibody distinguished four gp-340 size variants, designated I to IV (n = 2,2,2 and 1). While saliva gp-340 variants I to III had single bands of increasing sizes, variant IV and lung gp-340 had double bands. Purified I to IV proteins all revealed a N-terminal sequence TGGWIP upon Edman degradation. Moreover, purified gp-340 from the seven donors and lung gp-340 shared N-glycans, sialylated Galbeta1-3GalNAc and (poly)lactosamine structures. However, the larger size gp-340 grouping II/III (n = 4) and smaller size grouping I/IV correlated with a secretor, Se(+), and a non secretor, Se(-), dependent glycoform of gp-340, respectively (p = 0.03). The Se(+) glycoforms contained ABH, Le(b), Le(y) and polylactosamine structures, while the Se(-) glycoforms lacked ABH antigens but expressed Le(a), Le(x) and lactosamine structures. By contrast, lung gp-340 completely lacked ABH, Le(a/b), Le(x/y) or sLe(x) structures. Gp-340 and secretor typing of saliva from additional donors (n = 29) showed gp-340 glycoforms I to IV for 6, 16, 4 and 0 donors, respectively, and 3 non-typeable donors, and verified that gp-340 glycoforms I and II/III correlate with Se(-) and Se(+) phenotypes, respectively (p < 0.0001). The glycoforms of saliva and lung gp-340 mediated differential aggregation of Le(b)- (Helicobacter pylori), sialylpolylactosamine- (Streptococcus suis) or sialic acid- (Streptococcus mutans) binding bacteria. In conclusion, variant size- and glycoforms of gp-340 are expressed by different individuals and may modulate the biological properties of gp-340 pertinent to health and disease.

Pathogenic IgA in IgA nephropathy: still the blind men and the elephant?

IgA nephropathy (IgAN), the most common glomerulonephritis worldwide, remains an important cause of end-stage renal failure. The pathology is characterized by mesangial deposition of IgA. The disease is now recognized as arising from anomalies of the IgA molecule and the kidneys are innocent bystanders. The immunochemical nature of the IgA molecule and its mesangial uptake command a pivotal role in the pathogenesis of IgAN.

Human colostrum: identification of minor proteins in the aqueous phase by proteomics.

Human colostrum is an important source of protective, nutritional and developmental factors for the newborn. We have investigated the low abundance proteins in the aqueous phase of human colostrum, after depletion of the major proteins secretory IgA, lactoferrin, alpha-lactalbumin and HSA by immunoabsorption, using 2-D LC and gel-based proteomic methods. One hundred and fifty-one proteins were identified, 83 of which have not been previously reported in human colostrum, or milk. This is the first comprehensive proteomic analysis of human colostrum produced during the first 48 h of lactation.

Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems.

Secretory IgA (SIgA) is a multi-polypeptide complex consisting of a secretory component (SC) covalently attached to dimeric IgA containing one joining (J) chain. We present the analysis of both the N- and O-glycans on the individual peptides from this complex. Based on these data, we have constructed a molecular model of SIgA1 with all its glycans, in which the Fab arms form a T shape and the SC is wrapped around the heavy chains. The O-glycan regions on the heavy (H) chains and the SC N-glycans have adhesin-binding glycan epitopes including galactose-linked beta1-4 and beta1-3 to GlcNAc, fucose-linked alpha1-3 and alpha1-4 to GlcNAc and alpha1-2 to galactose, and alpha2-3 and alpha2-6-linked sialic acids. These glycan epitopes provide SIgA with further bacteria-binding sites in addition to the four Fab-binding sites, thus enabling SIgA to participate in both innate and adaptive immunity. We also show that the N-glycans on the H chains of both SIgA1 and SIgA2 present terminal GlcNAc and mannose residues that are normally masked by SC, but that can be unmasked and recognized by mannose-binding lectin, by disrupting the SC-H chain noncovalent interactions.

Recombinant secretory immunoglobulin A in passive immunotherapy: linking immunology and biotechnology.

The use of monoclonal antibodies has become routine in the research and diagnostic laboratories, but the potential of antibody molecules in public health and medical applications is still far from its maximum. Most infections begin at mucosal surfaces, and this is certainly not only a stroke of good fortune if mother's milk serves as a natural delivery vehicle for antibodies protecting the gastrointestinal tract of nursing infants. Mammary gland or other mucous secretions containing numerous antibody specificities provide an efficient mean to immediately protect a mucosal surface against pathogens, which have never been encountered by the host. From a public health perspective, topical passive immunization of mucosal surfaces with monoclonal antibodies can block entry and transmission of bacteria, viruses, fungi and parasites that infect humans, and thus defeat some key immune evasion strategies designed by many pathogens. The chief antibody on most mucosal surfaces is secretory immunoglobulin A (SIgA), a polypeptide complex comprising dimeric IgA, the connecting J chain, and the secretory component. The molecular stability, tetravalency, and strong anti-inflammatory properties make SIgA particularly well suited to fulfill the function of passive protective immunity when applied exogenously to mucosal surfaces. The review will give an overview of the basic concepts underlying mucosal immunity, present the molecular mechanisms whereby SIgA prevents mucosal infections, cover the last advances in the topic of recombinant SIgA production, and examine how structure-function relationship in SIgA will help designing molecules with novel properties for passive immunotherapy.

Recombinant immunoglobulin A: powerful tools for fundamental and applied research.

The use of monoclonal antibodies has become routine in research and diagnostic laboratories but the potential level of antibodies in use in public health and medical applications is still far from its maximum. From a clinical perspective, topical immunotherapy of mucosal surfaces with monoclonal antibodies can block entry and transmission of bacteria, viruses, fungi and parasites that infect humans, and defeat some key strategies, evolved by many pathogens, to evade the host immune system. The chief antibody at mucosal surfaces is secretory immunoglobulin A (SIgA), a multi-polypeptide complex originating from two cell types. The recent design of heterologous expression systems, coupled with modern biotechnology processes, should form a sound basis for studying the functional properties of SIgAs and evaluate their value as biotherapeutics. Here, we discuss the principles underlying mucosal immunity and review the application of recombinant SIgA to the dissection of mechanisms in passive and active protection at mucosal surfaces.

[Properties of mucosal antibodies]. / Propriétés des anticorps des muqueuses.

Mucosal antibodies consist of a variety of molecules, including secretory IgA and local IgG, involved in the first immune barrier of defence against pathogens. They account for the majority of daily synthesized immunoglobulins in the body and mostly depend on the secretory immune system which is independent from its systemic counterpart. Acting by immune exclusion and immune elimination, these immunoglobulins correspond to preimmune poly-reactive natural antibodies and to antigen-induced antibodies. Recent progress in this field have suggested new approaches of mucosal vaccines preventing the entry of pathogens in the body.

Tracking membrane and secretory immunoglobulin alpha heavy chain mRNA variation during B-cell differentiation by real-time quantitative polymerase chain reaction.

Primary transcripts for all Ig heavy chain isotypes are alternatively processed to encode either secreted or membrane forms of the same antibody and, in plasma cells, a shift towards the secreted form occurs. In principle, measuring the relative quantities of secreted and membrane forms for a particular isotype could monitor B-cell plasmacytoid differentiation. Ratios of alpha heavy chain mRNA secreted (alphas) to membrane (alpham) form were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR; TaqMan) using an IgA plasma cell line (NCI-H929), a surface IgA+ line (Dakiki) and human tonsillar B cells. While NCI-H929 cells showed the highest alphas: alpham ratio as expected, alphas mRNA predominated for all unstimulated B cells and Dakiki cells. Treatment of B cells and Dakiki cells with IL-2 and IL-10 resulted in a further progression towards the alphas form, correlating with increased human plasma cell antigen-1 (HPC1) mRNA levels. However, alpha mRNA processing and HPC1 expression were independently regulated, as IFN-gamma treatment suppressed HPC1 levels while increasing alphas: alpham ratios. Cytokine-mediated increases in the alphas: alpham ratio resulted from strongly enhanced levels of alphas with relatively constant alpham values. Differentiation-related changes in mRNA processing can thus be tracked by automated quantitative PCR.

[The role of airway submucosal glands in the airway mucosal defense system].

Despite constant inhalation of air-borne particles including a variety of microbes and antigens, the normal lungs hardly ever develop infection or airway injury. This is because the normal lung is equipped with sophisticated defense mechanisms against foreign substances. It has been reported that the airway mucosa, especially the submucosal glands, play important roles not only in nonspecific defense using airway secretions but also in specific defense in cooperation with immune cells. In contrast to the nasopharyngeal or intestinal mucosa, which is always exposed to many foreign antigens, the mucosal surface of the lower respiratory tract in known to be kept in a germ-free condition. This fact indicates that immunological information derived from the antigen-rich mucosa, such as the intestine, might be transmitted to the airway mucosa, thus resulting in efficient removal of unwanted substances. This immunological elimination requires specific antibodies (Abs) against harmful antigens, and the major population of Abs in the airway is dimeric IgA. Airway submucosal glands synthesize a secretory component (SC), a transporter of secretory IgA, and immunoglobulin-containing plasma cells have been identified preferentially around the glandular tissue. Overall, the submucosal glands play a key role in the integrity of airway mucosal immunity.

Polymeric IgA are sulfated proteins.

The main sulfated proteins secreted by rabbit mammary gland tissue had M(r) of approximately 67 000, 63 000 and 23 000, and one component which most likely corresponded to proteoglycans had a diffuse electrophoretic mobility (M(r)200 000). The sulfate groups in the 67-63 kDa proteins were mostly linked to carbohydrates. These proteins and the 23 kDa protein were co-purified and identified to heavy chains of immunoglobulin A (IgA) and J chain, respectively. Sulfation of alpha-chains also occurred in rat mammary and rabbit lacrimal glands. We conclude that polymeric IgA which are produced by plasma cells and released in secretion fluids after transcytosis through epithelia are sulfated.