Peculiar secretory IgA system identified in chickens. II. Identification and distribution of free secretory component and immunoglobulins of IgA, IgM, and IgG in chicken external secretions.

A homologue of a free secretory component (SC) was identified in chicken intestinal secretion by criteria based on its antigenic relationship with intestinal secretory IgA (SIgA), molecular size, sugar content, and electrophoretic mobility, as well as its elution characteristic from ion-exchange chromatography. SC was obtained in a form free from IgA from the intestinal secretion by salting out and DEAE chromatography, followed by density ultracentrifuguation or Sephadex G-200 gel-filtration. However, the free SC revealed some antigenic deficiency when compared to bound SC of intestinal SIgA and showed a failure of binding to serum-type-polymeric IgA of biliary IgA in vitro. Several kinds of chicken external secretions were examined for detection of SC and immunoglobulin classes of IgG, IgA, and IgM. In spite of the wide distribution of immunoglobulins in the external secretions, SC antigen could be detected only in intestinal secretion. Most IgA in the secretions had a molecular structure of a tetramer of serum-type IgA, lacking in SC and having 17S to 18.5S and 600,000 to 700,000 daltons. On the other hand, IgA in the intestinal secretion showed close similarity to the mammalian SIgA, associated with SC and having 11.2S and 350,000 daltons. Presence of antibody activity in the intestinal IgA to avian reovirus was confirmed by plaque reduction tests.     

Quantification of human urinary free secretory component, secretory and nonsecretory IgA by ELISA

The specific quantification of human urinary free secretory component (FSC), secretory IgA (SIgA) and total IgA using ELISA has been hampered by mutual interferences of these three molecules. Using affinity chromatographically purified antisera an attempt was therefore made to reduce these interferences without necessitating further assay steps. FSC and total IgA were measured in unprocessed urine by means of anti-FSC and anti-IgA as well as alkaline phosphatase-coupled anti-FSC or anti-IgA antisera. SIgA was determined using anti-IgA as well as alkaline phosphatase-coupled anti-FSC. Nonsecretory urinary IgA was calculated from the measured SIgA and total IgA. The mutual interferences of FSC, SIgA or nonsecretory IgA in the three assay systems were low and not relevant for normal samples. Normal urinary concentrations were: FSC 344 +/- (SD) 208 ng/ml (n = 120), SIgA 1,874 +/- 1,133 ng/ml (n = 123) and nonsecretory IgA, depending on the way of standardization, 712 +/- 699 (n = 56) or 878 +/- 732 ng/ml (n = 51). SIgA excretion increased with age. Lower urinary SIgA as well as total and nonsecretory IgA levels were observed in males as compared to females. No correlation evolved between the hormonal status of women and the excretion of FSC, SIgA or IgA. In IgA-deficient patients virtually no nonsecretory IgA or SIgA was detected in the urine while the FSC concentration was in the normal range.     

Recognition of gram-positive intestinal bacteria by hybridoma- and colostrum-derived secretory immunoglobulin A is mediated by carbohydrates

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of gram-positive bacteria by control IgG was observed. The interaction with gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.     

Mannose-containing oligosaccharides of non-specific human secretory immunoglobulin A mediate inhibition of Vibrio cholerae biofilm formation

The role of antigen-specific secretory IgA (SIgA) has been studied extensively, whereas there is a limited body of evidence regarding the contribution of non-specific SIgA to innate immune defenses against invading pathogens. In this study, we evaluated the effects of non-specific SIgA against infection with Vibrio cholerae O139 strain MO10 and biofilm formation. Seven day old infant mice deficient in IgA (IgA(-/-) mice) displayed significantly greater intestinal MO10 burden at 24 hr post-challenge when compared to IgA(+/+) pups. Importantly, cross-fostering of IgA(-/-) pups with IgA(+/+) nursing dams reversed the greater susceptibility to MO10 infection, suggesting a role for non-specific SIgA in protection against the infection. Since biofilm formation is associated with virulence of MO10, we further examined the role of human non-specific SIgA on this virulence phenotype of the pathogen. Human non-specific SIgA, in a dose-dependent fashion, significantly reduced the biofilm formation by MO10 without affecting the viability of the bacterium. Such an inhibitory effect was not induced by human serum IgA, IgG, or IgM, suggesting a role for the oligosaccharide-rich secretory component (SC) of SIgA. This was supported by the demonstration that SIgA treated with endoglycosidase H, to cleave the high-mannose containing terminal chitobiose residues, did not induce a reduction in biofilm formation by MO10. Furthermore, the addition of free mannose per se, across a wide dose range, induced significant reduction in MO10 biofilm formation. Collectively, these results suggest that mannose containing oligosaccharides within human non-specific secretory IgA can alter important virulence phenotypes of Vibrio cholerae such as biofilm formation, without affecting viability of the microorganism. Such effects may contribute significantly to innate immune defenses against invading pathogens in vivo in the gastrointestinal tract.     

SpsA, a novel pneumococcal surface protein with specific binding to secretory Immunoglobulin A and secretory component

The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 × 10⁻⁹ M. Free secretory component (SC) also binds to S . pneumoniae , whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA- or SC-binding domain is located in the N-terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA-binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S . pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S . pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.     

Simplified procedure to recover recombinant antigenized secretory IgA to be used as a vaccine vector

Induced protection mechanisms at mucosal surfaces involve secretory IgA (SIgA), a complex structure made of polymeric-dimeric IgA (IgA(p/d)) antibody associated with secretory component (SC). SIgA can adhere to M cells of the intestinal and nasal epithelia, are transported across these latter, and are thus available to the immune cells underlying the epithelia. This property makes SIgA suitable as potential mucosal vaccine delivery vector. It remains that production and purification of SIgA is a complex task since IgA(p/d) and SC are naturally synthesized by two different cell types. Furthermore, only IgA(p/d) are capable to associate with SC. Thus, we sought to separate IgA(p/d) and monomeric IgA (IgA(m)) antibodies secreted by hybridoma cells in CELLine bioreactors. To this aim, we connected together two 1-m long columns filled with Sephacryl S-300 beads and placed them under the control of a automatized chromatographic system. In parallel, we produced recombinant antigenized human SC (ra-hSC) in Chinese hamster ovary (CHO) cells adapted to suspension culture in CELLine bioreactors. To avoid intermediate purification of ra-hSC, culture supernatants (SN) containing this latter were combined with purified IgA(p/d), and the recombinant antigenized SIgA (raSIgA) complex was resolved on a 1-m long column filled with Superdex 200 beads. Biochemical characterization based on SDS-PAGE, silver staining, immunodetection and enzyme-linked immunosorbent assay (ELISA) indicates that highly purified raSIgA can be recovered using this simple two-step procedure. Such preparations are currently used to immunize mice to induce mucosal and systemic responses.     

The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA.

Local production of secretory (S)IgA provides adaptive immunologic protection of mucosal surfaces, but SIgA is also protective when administered passively, such as in breast milk. Therefore, SIgA is a potential candidate for therapeutic administration, but its complex structure with four different polypeptide chains produced by two distinct cell types complicates recombinant production. The J chain is critical in the structure of SIgA because it is required for efficient polymerization of IgA and for the affinity of such polymers to the secretory component (SC)/polymeric (p)IgR. To better understand the role of the J chain in SIgA production, we have generated various mutant forms of the human J chain and analyzed the function of these mutants when coexpressed with IgA. We found that the C terminus of the J chain was not required for the formation of IgA polymers, but was essential for the binding of pIgA to SC. Likewise, we found that two of the intrachain disulfide bridges (Cys(13):Cys(101) and Cys(109):Cys(134)) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys(72):Cys(92)) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys(15) or Cys(69) was sufficient to support polymerization of IgA, but that these polymers were mostly noncovalently stabilized. Nevertheless, these polymers bound free SC with nearly the same affinity as pIgA containing wild-type J chain, but were transcytosed by pIgR-expressing polarized epithelial cells at a reduced efficiency.     

Characterization of epitopes of human secretory component on free secretory component, secretory IgA, and membrane-associated secretory component

We have compared the epitopes present in various forms of human secretory component by using a panel of hybridoma-derived antibodies elicited by immunizing mice with free secretory component (FSC) or secretory IgA (sIgA). Enzyme-linked immunosorbent binding assays (ELISA) were used to assess antibody binding to FSC- and SC-containing antigens, including sIgA isolated from milk, reduced and alkylated sIgA, and sIgA assembled in vitro by incubating dimeric IgA with FSC. Immunofluorescence assays were also used to assess binding to a human epithelial tumor cell line (HT29) that expresses secretory component as an integral protein of the plasma membrane. The results can be summarized as follows. 1) Most antibodies from fusions in which sIgA was the immunizing antigen bound preferentially to sIgA. 2) Most antibodies from fusions in which FSC was the immunizing antigen bound preferentially to FSC. 3) Antibodies that bound preferentially to sIgA invariably bound sIgA assembled in vitro; antibodies that bound preferentially to FSC invariably did not. 4) Antibodies that bound readily to both sIgA and FSC were rare in all fusions. 5) The monoclonal antibodies defined at least six classes of epitopes on SC, including epitopes that were a) FSC specific and reduction sensitive, b) FSC specific and reduction insensitive, c) sIgA specific and reduction-sensitive, d) sIgA specific and reduction insensitive, e) shared by FSC and sIgA and reduction-sensitive, and f) shared by FSC and sIgA and reduction-insensitive. 6) Antibodies that mediated intense immunofluorescent staining of secretory component on HT29 cell membranes were rare and constituted a distinct subset of those which recognized epitopes shared by FSC, reduced and alkylated sIgA, and some preparations of native sIgA. Results of these antibody-binding studies indicate that most SC epitopes are not shared by FSC and sIgA. Most SC-related epitopes on sIgA appear to be generated by the physical interaction of SC with dimeric IgA, whereas most epitopes on FSC are masked or altered by this interaction. Finally, epitopes that are shared by membrane SC and FSC and/or sIgA represent a minor and immunochemically distinct subset of epitopes on SC. The high proportion of unique epitopes on the different physical forms of SC suggest that the epitopes of this molecule are highly sensitive to its molecular environment. The monoclonal reagents described here will be useful in studying the structure and function of SC; quantitating FSC, sIgA, and membrane SC; purifying various molecular forms of SC by immunoaffinity chromatography; and localizing SC in human tissues and cultured cells by immunocytochemical techniques.     

抗禽流感病毒H5N1 分泌型IgA 在中国仓鼠卵巢细胞中的表达

分泌型IgA (SIgA) 在机体的粘膜免疫中具有重要作用,在外分泌道中比单体IgA和IgG抗体具有更好的抗感染活性。为了表达抗禽流感病毒H5N1人-鼠嵌合分泌型IgA抗体,首先以本室先前构建的稳定表达IgA的中国仓鼠卵巢细胞 (CHO) 细胞系为基础,共转染分泌片和J链表达质粒,然后用抗生素Zeocin选择阳性克隆细胞,利用倍比稀释的方法筛选分泌SIgA的单克隆细胞,通过Western blotting分析培养上清中SIgA的表达情况。结果表明,在CHO细胞中成功表达了SIgA抗体,上述研究为研制分泌型     

Chromatographic Separation and Purification of Secretory IgA from Human Milk

Defatted and decaseinated human milk was concentrated and was fractionated on a preparative DEAE cellulose column. Elution with various concentrations of sodium chloride in Tris-HCl buffer (pH 8.0, 0.01 M) resulted in fractions that were rich in either secretory immunoglobulin A (SIgA) (0.1 M Nad) or free secretory component (SC) (0.05 M NaCl). The fractions, which were eluted with 0.10 M NaCl from the preparative column, were further fractionated on a G-200 Sephadex column. Repeated fractionation on this column resulted in a single purified fraction, which contained very high SIgA activity and showed immunological cross-reaction with both SC and serum IgA. Additional studies indicated that this fraction was homogeneous as shown by immunoprecipitin and disc gel electrophoresis. Injection of this purified SIgA into rabbits resulted in the production of monospecific antiscil.     

Antisera to the secretory component recognize the murine Fc receptor for IgA

Studies were undertaken to determine a possible structural relationship between the secretory component (SC) and the receptor for IgA (Fc alpha R). An IgA-mediated rosetting technique was used to assess the presence of Fc alpha R+ cells in various lymphoid tissues from normal BALB/c mice and mice bearing an IgA plasmacytoma (MOPC 315). Tissues from the MOPC 315-bearing BALB/c mice were found to have a significantly higher percentage of Fc alpha R+ cells; thus, nonadherent spleen cells from MOPC 315-bearing mice were used as a source of Fc alpha R+ cells in these studies. The cells were preincubated with anti-SC and then assayed for the ability of IgA to bind to the Fc alpha R. Antisera to SC from various species inhibited the formation of IgA-mediated rosettes, although preincubation of the Fc alpha R+ cells with antisera directed against other cell surface molecules (e.g., Thy1.2, Lyt1, Lyt2, Fc gamma R, MHC class I and II) or preimmune sera had no significant effect on IgA-mediated rosette formation. Preabsorption of the anti-SC with secretory IgA or with free SC removed the inhibitory effect; preabsorption with myeloma IgA had no effect. These data suggest that SC and Fc alpha R are related serologically and may be structurally related, possible in the IgA-binding region.     

SIgA分泌片分离、纯化研究

为研究SIgA分泌片（Secretory component,SC)分离,纯化及鉴定方法,以SC存在游离和结合两种形式,本研究应用凝胶过滤和盐析法直接从初乳中分离纯化游离SC,并进行鉴定。分离纯化获得蛋白溶液12.4ml,蛋白含量0.85mg/ml,免疫双扩仅与抗SC多克隆抗体（PcAb)反应,分子量约75kD,免疫印迹实验与抗SC单克隆抗体（McAb)和PcAb特异反应,表明纯化蛋白为SC。该SC的分离纯化和鉴定处于不断完善之中,其方法的改进有利于获得更纯的SC,值得粘膜免疫研究者借鉴。     

分泌型免疫球蛋白A的研究进展

大部分感染都起源于黏膜表面,而黏膜免疫的主要抗体是分泌型免疫球蛋白A（SIgA）,它能有效地阻断病原体的感染和侵入。SIgA是由1个IgA二聚体、1条J链和1个分泌片（SC）共价结合构成的异源十聚体。IgA和J链由活化B细胞产生,SC则由黏膜上皮细胞合成。SIgA分子具有极高的稳定性和极强的抗微生物活性。我们就SIgA合成的相关机制、IgA单体和SIgA的结构与功能,以及重组SIgA的研究进展简要综述。     

Some Properties of Secretory IgA Purified from Bovine Colostrum

The major component of a purified sample of secretory IgA (SIgA) in colostrum was revealed as a single peak on gel filtration with Sepharose 6B, having an estimated molecular weight of 540,000. The existence of a higher molecular weight component was suggested by a small shoulder on the ascending limb of the peak, but another component of IgA reported as IgA lacking the secretory component (SC) could not be found. When the purified SIgA was concentrated by dialysis against polyethylene glycol, its molecular size was apparently significantly decreased.

Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) showed that all SC in SIgA binds covalently. The band corresponding to the J chain was easily detected when a reduced and alkylated sample was analysed. Estimation of the molecular weight by SDS-PAGE gave the following values for each of the constituent polypeptide chains of bovine colostral SIgA: SC, 76,000; H chain, 62,000; L chain, 23,000; and J chain, 18,000. The molecular weight of the whole molecule was calculated to be 434,000.

Analysis of carbohydrates by gas-liquid chromatography showed 6.8% neutral and amino hexoses, consisting of 0.4% fucose, 1.8% mannose, 1.1% galactose and 3.5% glucosamine. Galactosamine, which has been found in bovine free secretory component from milk, could not be detected.     

Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin in plants

Secretory immunoglobulin (Ig) A is a decameric Ig composed of four alpha-heavy chains, four light chains, a joining (J) chain, and a secretory component (SC). The heavy and light chains form two tetrameric Ig molecules that are joined by the J chain and associate with the SC. Expression of a secretory monoclonal antibody in tobacco (Nicotiana tabacum) has been described: this molecule (secretory IgA/G [SIgA/G]) was modified by having a hybrid heavy chain sequence consisting of IgG gamma-chain domains linked to constant region domains of an IgA alpha-chain. In tobacco, about 70% of the protein assembles to its final, decameric structure. We show here that SIgA/G assembly and secretion are slow, with only approximately 10% of the newly synthesized molecules being secreted after 24 h and the bulk probably remaining in the endoplasmic reticulum. In addition, a proportion of SIgA/G is delivered to the vacuole as at least partially assembled molecules by a process that is blocked by the membrane traffic inhibitor brefeldin A. Neither the SC nor the J chain are responsible for vacuolar delivery, because IgA/G tetramers have the same fate. The parent IgG tetrameric molecule, containing wild-type gamma-heavy chains, is instead secreted rapidly and efficiently. This strongly suggests that intracellular retention and vacuolar delivery of IgA/G is due to the alpha-domains present in the hybrid alpha/gamma-heavy chains and indicates that the plant secretory system may partially deliver to the vacuole recombinant proteins expected to be secreted.     

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

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

The amino-terminal domain of rabbit secretory component is responsible for noncovalent binding to immunoglobulin A dimers

Rabbit secretory components (SC) constitute a family of markedly heterogeneous glycoproteins which are released in the secretions as free SC or as SC bound to polymeric immunoglobulins. The aim of this work was to determine the region of the SC polypeptides which is involved in IgA binding. The high and the low Mr forms of free SC (or IgA-dissociated bound SC) and the native secretory IgA complex were subjected to limited tryptic digestion. Chemically characterized peptides ranging in apparent size from 15 to 20 kDa, depending upon the allotype, were shown to be necessary and sufficient for efficient noncovalent binding to IgA dimers (subclass g). These fragments encompass the amino-terminal first domain of SC, i.e. residues 1-126, when aligned with the predicted amino acid sequence from a cDNA clone encoding the rabbit polymeric Ig receptor (Mostov, K.E., Friedlander, M., and Blobel, G. (1984) Nature 308, 37-43). The high and the low Mr forms of SC exhibited the same relative affinity for IgA dimers, suggesting that the postulated internal deletion in the smaller polypeptide (Kühn, L. C., Kocher, H.-P., Hanly, W.C., Cook, L., Jaton, J.-C., and Kraehenbuhl, J.-P. (1983) J. Biol. Chem. 258, 6653-6659) does not impair the IgA dimer recognition function.     

Variations in free secretory component levels in mucosal secretions of the rat

The present studies were conducted to compare the levels of free secretory component (SC) in a number of rat mucosal secretions and to determine whether SC content varies significantly during the four stages of the estrous cycle. Levels of SC, as measured by radioimmunoassay, were markedly different in various external secretions. Bile contained the highest amount, irrespective of whether SC was normalized to volume or protein. Concentrations of SC in saliva or uterine fluid from intact rats were approximately 20- to 30-fold less than measured in bile. When SC levels were normalized to protein, the SC to protein ratios in uterine, vaginal, and respiratory secretions were six to 18 times greater than values calculated in salivary and small intestinal fluids. Analysis of SC levels in mucosal secretions during the estrous cycle indicated significant variations occur in uterine and vaginal samples, but not in saliva or small intestinal secretions. In the uterine lumen, SC levels were highest at proestrus, partially elevated at estrus, and lowest at both days of diestrus. In contrast, vaginal SC levels were maximal at estrus and reduced at all other stages of the cycle. Immunoglobulin A content was also measured in uterine and vaginal secretions during the estrous cycle. Significant changes in IgA levels were found and these coincided with the changing pattern of SC. These results suggest hormones may modulate SC levels in reproductive tissues. In addition, our findings indicate variations in SC during the estrous cycle may direct the movement of IgA from tissue to lumen.     

Glycans on secretory component participate in innate protection against mucosal pathogens

In mucosal secretions, secretory component (SC) is found either free or bound to polymeric IgA within the secretory IgA complex. SC displays numerous and various glycans, which are potential ligands for bacterial compounds. We first established that human SC (hSC) purified from colostrum (hSCcol) or produced in Chinese hamster ovary cells (hSCrec) exhibits the same lectin reactivity. Both forms bind to Clostridium difficile toxin A and functionally protect polarized Caco-2 cell monolayers from the cytopathic effect of the toxin. The interaction is mediated by glycans present on hSC and involves galactose and sialic acid residues. hSCcol and hSCrec were also shown to bind enteropathogenic Escherichia coli adhesin intimin and to inhibit its infectivity on HEp-2 cells in a glycan-dependent manner as well. SC remained operative in the context of the whole secretory IgA molecule and can therefore enhance its Fab-mediated neutralizing properties. On the contrary, hSC did not interact with three different strains of rotavirus (RF, RRV, and SA11). Accordingly, infection of target MA104 cells with these rotavirus strains was not reduced in the presence of either form of hSC tested. Although not a universal mechanism, these findings identify hSC as a microbial scavenger contributing to the antipathogenic arsenal that protects the body epithelial surfaces.