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.     

Interaction of specific and innate factors of immunity: IgA enhances the antimicrobial effect of the lactoperoxidase system against Streptococcus mutans

The antimicrobial effect of the lactoperoxidase (LPO) system (enzyme with the thiocyanate ion and hydrogen peroxide) on Streptococcus mutans NCTC 10449 (serotype c) was significantly enhanced when the system was combined with secretory IgA. Similar enhancement was observed with LPO-myeloma IgA1 or IgA2 combinations. This enhancement of the antimicrobial efficiency was not dependent on the presence of specific antibodies to S. mutans in the IgA preparation, but seemed to require binding between LPO and immunoglobulin. However, neither human polyclonal nor myeloma IgG or IgM nor rabbit IgG enhanced the antibacterial activity of the LPO system. None of the immunoglobulins, when added alone, produced antimicrobial effects. LPO was shown to bind to colostral secretory IgA, myeloma IgA1, IgA2, and to a lesser degree to monoclonal and polyclonal IgG and monoclonal IgM. This binding had a stabilizing effect on the enzyme activity. Our results suggest that IgA significantly enhances the antibacterial efficiency of one of the innate immune factors--the LPO system.     

Analysis of paraprotein transport into the saliva by using anti-idiotype antibodies

To determine the extent of clonal involvement of the secretory immune system and the origin of salivary immunoglobulins (Ig) in monoclonal gammopathy patients, saliva and serum samples were collected from five affected individuals (two IgA myelomas, one IgG myeloma, one IgG benign monoclonal gammopathy, and one IgM lymphoma) and were assayed for the presence of monoclonal Ig. Purified polyclonal or monoclonal anti-idiotype (Id) antibodies were prepared against each of the isolated serum paraproteins. In all five individuals, the patient saliva samples inhibited the binding of 125I-labeled homologous Ig to the corresponding anti-Id antibodies, but normal saliva did not. The concentration of Id in patients' saliva varied from 1 to 400 micrograms/ml; i.e., 0.004 to 1.0% of the corresponding serum values. Saliva of a lymphoma patient whose IgM kappa protein exhibited rheumatoid factor (RF) activity also contained RF. The salivary Id-bearing molecules were found to have the same Ig isotype as the serum paraproteins. The myeloma IgA represented a minor component (0.4 and 3.9%) of the total salivary IgA. The salivary IgA myeloma proteins were associated at least in part with secretory component, but the salivary IgG paraproteins were not. In an IgA myeloma patient, a minority (17%) of the IgA+ plasma cells found in the lacrymal gland biopsy specimen were Id+, whereas the great majority (98%) of bone marrow IgA plasma cells were Id+. The results suggest active transport rather than passive transudation of myeloma IgA into the patients' saliva, and the integrity of the secretory immune system was not compromised by the neoplastic process.     

The specificity of the IgA receptor purified from human neutrophils.

A receptor for IgA was purified from human polymorphonuclear neutrophils (PMN) by affinity chromatography on human serum IgA-Sepharose. The receptor appeared on SDS/polyacrylamide gels as a diffuse band with an apparent molecular mass of 50-70 kDa, whether reduced or non-reduced. During purification, the protein showed remarkable stability to proteolytic digestion by endogenous PMN proteinases. Purified radioiodinated receptor re-bound to IgA-Sepharose, but not to IgG-Sepharose or BSA-Sepharose. The binding of the receptor to IgA-Sepharose was inhibited in a dose-dependent manner by human serum IgA1 or IgA2 or secretory IgA1 or IgA2, but not by IgG or IgM. Binding of receptor to IgA-Sepharose was also inhibited by the Fc fragment of IgA, but not by the Fab fragment. An IgA fragment produced by digestion with pepsin which lacks the CH3 domain also inhibited binding, but to a more limited extent than did the whole IgA molecule.     

Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient.     

Human IgM molecules that bind staphylococcal protein A contain VHIII H chains

Staphylococcal protein A (SPA) is a bacterial membrane protein which has distinct binding sites for Fc gamma and for the Fab region of some IgM, IgG, IgA, and IgE molecules. This study establishes a structure-function correlation responsible for the binding of Ig Fab regions to SPA. Binding of 24 isolated human monoclonal IgM proteins to SPA was measured in a solid phase RIA. VH and V kappa subgroups of each IgM were determined by SDS-PAGE, transfer blotting, and detection with antisera prepared against specific first framework region peptides. Binding to SPA was seen with 10 of 11 VHIII IgM, but none of the 7 VHI or 6 VHII. Similarly, polyclonal IgM fractionated on a SPA-Sepharose CL4B column showed nearly complete partition of VHIII molecules into the SPA-binding fraction, and VHI and VHII subgroup proteins into the fall-through. We conclude that SPA binding is a functional marker for VHIII H chains in human IgM molecules.     

Immunoglobulins in uterine secretions of mares with differing resistance to endometritis

The immunoglobulins IgA, IgG, IgG(T) and IgM were measured in uterine secretions from mares with normal uterine defense capability against bacterial contamination, and in mares with lowered resistance. Samples were collected for analysis at two stages of estrus and two stages of diestrus. All mares were then challenged with a pathogenic culture of Streptococci inoculated into the uterus. The immunoglobulins were quantitated on a similar schedule post-inoculation. Generally higher amounts of IgA, IgG and IgG(T) were found in the uterine secretions of mares which had an imparied resistance to endometritis than in mares with an efficient defense mechanisms. IgM was not detected in enough samples to suggest any differences.     

Secretory IgA and secretory component in women affected by recidivant vaginal candidiasis

Local immunity was evaluated in 47 patients affected by recidivant vaginal candidiasis and 33 control women. IgG, IgA, IgM and secretory component (SC) were determined by single radial immunodiffusion in samples of cervicovaginal secretion. IgG in dosable levels was detected in 17/47 samples (36.2%) and IgA in 15/47 patients (31.9%) whereas in the controls, the incidence was 31/33 (93.9%) for IgG and 24/33 (72.7%) for IgA. The difference was significative (P< 0.001) for both immunoglobulins. Significant differences were not obtained for IgM. The SC was detected in 4/47 cervicovaginal secretions of patients affected by candidiasis (8.5%) whereas in the control samples the incidence was 21/33 (63.6%) (P<0.001). In only 2/15 patients with dosable levels of IgA (13%) the secretory nature of this immunoglobulin could be shown by its reaction with anti-SC serum. In the control group, secretory IgA was detected in 19/24 cases (79%) (P< 0.001). Serum immunoglobulins levels were normal. The lack of secretory IgA and SC in the secretion could be related to the adherence capacity of the Candida albicans to epithelial cells.     

IgG antibodies to secretory component in normal human serum

While isolating free secretory component (FSC) by monoclonal antibody affinity chromatography, we demonstrated FSC-IgG complexes in human milk. We hypothesized that IgG antibody to secretory component (SC) might be transported into the milk from the serum. We therefore examined sera from 10 normal adults and 10 infants for IgG capable of binding to FSC in an enzyme-linked immunosorbent assay. Eight of 10 normal adult sera and nine of 10 infant sera demonstrated IgG binding to FSC with titers ranging from 1:54 to 1:4096. Quantitation of the IgG bound to FSC was hampered in adult sera by the binding of IgM and polymeric IgA to the FSC. Quantitation in five infant sera ranged from 0.5 to 6.4 micrograms/ml. A pepsin digest of an IgG fraction of serum demonstrated binding of the F(ab')2 fragments to the FSC. The specificity of the antibodies in human serum was evaluated by examining the binding to secretory IgA (sIgA) and FSC isolated from pooled human milk and polymeric IgA isolated from the ascitic fluid of a patient with an IgA myeloma. Eight of the 10 adults had antibody specific for FSC. Three of the eight, all female, also had antibody specific for sIgA. Two of the eight had antibody either to FSC and sIgA or to FSC plus an antibody that could bind to an epitope shared by sIgA and FSC. Competition experiments with monoclonal antibodies to human secretory component and sIgA were used to confirm and further define these specificities. The results of this study indicate that antibody to SC is common in normal adult and infant sera. The majority of antibodies seem to be directed against epitopes present on FSC but not on sIgA, which suggests sensitization to circulating or membrane-bound SC. The significance of these antibodies in normal human sera remains to be elucidated.     

Immunohistochemical localization of immunoglobulins A, G and M in the mouse female genital tract

The localization of immunoglobulins A, G and M (IgA, IgG, IgM) in the mouse genital tract was studied by immunoperoxidase techniques at oestrus, on the day of mating and at the time of implantation. In the horns and body of the uterus, IgA and IgG were located in plasma cells in the endometrium surrounding uterine glands and in the gland lumina. The numbers of these plasma cells increased markedly between Day 1 and Days 4 and 5 of pregnancy and the ratio of plasma cells containing IgA and IgG was about 3 or 4 to 1 at all stages. Area measurements indicated that the increased number of plasmacytes was not due to an increase in the amount of endometrial, myometrial or glandular tissues. Plasma cells were not detected in the cervix and vaginal fornix at oestrus and Day 1, but a few were present on Day 5. In the oviduct, plasma cells containing IgA and IgG were present only in the preampulla and both immunoglobulins were present in the extracellular space of the lamina propria only in this region. No IgM was detected in any part of the reproductive tract at any of the times studied. Uteri on Day 1 of pregnancy contained bacteria of several kinds, some of which were aggregated and coated with IgA. This suggests that the uterine lumen at this time may contain specific anti-bacterial IgA antibodies. Our observations indicate that the horns and body of the uterus and the preampulla of the oviduct are major sites of a local immune system in the female mouse genital tract.     

The Interactions of Porcine and Ovine,Serum and Colostral Immunoglobulins with Staphylococcal Protein A

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produced by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG₁, IgG₂, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG₁ from most ewes possessed a low affinity for Protein A whereas ovine IgG₂ generally possessed a high affinity; 100% of the IgG₂ in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.     

Human intestinal epithelial cells express a novel receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.     

Heterogeneity of binding of human IgA subclasses to protein A.

The ability of human IgA myeloma immunoglobulins to interact with protein A-containing Staphylococcus aureus was examined. Some IgA1 and IgA2 immunoglobulins bound to S. aureus although others of both subclasses failed to do so. These results were obtained by using both direct binding of radiolabeled immunoglobulins to S. aureus and with inhibition-type assays. Binding was dependent on the Fc fragment of IgA since there was no binding to S. aureus by an F(ab')2 fragment of IgA1. Nonprotein A-containing bacteria did not bind these immunoglobulins and isolated protein A interacted with radiolabeled immunoglobulins. This strongly suggested that protein A was responsible for the observed binding to S. aureus. These data indicate, in contrast to previous reports, that there is no simple relationship between IgA subclass and the capacity to bind to protein A.     

The localization of the antibody response in milk or bile depends on the nature of the antigen

Immunization in the Peyer's patches of rats with horse spleen ferritin or Escherichia coli 06 carrying type 1 pili resulted in an IgA antibody response detected in milk and bile and an IgG and IgM antibody response in serum, milk, and bile. The IgA antibody response to type 1 pili was as a mean 5.0-fold higher in milk than in bile. In contrast IgA antibody activity to 06 LPS was as a mean 6.3-fold higher in bile than in milk. The IgA antibodies to ferritin were randomly distributed between milk and bile. The IgG and IgM antibody activity to all three antigens studied were higher in the milk than in the bile. The secretory antibody response could be transferred from immunized rats to unimmunized rats with mesenteric lymph node cells (MLN) taken from donor rats 4 days after immunization in the Peyer's patches. IgA antibodies to pili and ferritin appeared solely in the milk of the recipients, whereas IgA antibodies to the 06 LPS only appeared in the bile. The ratios serum:milk and serum:bile for the IgG and IgM antibodies indicated an antigen-specific direction of homing with local production of these two isotypes primarily in the mammary gland. Antibody-forming cells of the IgA class could not be detected in the MLN on the day the cells were transferred. It is concluded that the difference seen in antibody distribution between milk and bile is not due to dissemination of antigen, but instead a result of different homing or expansion at the mucosal-glandular site dependent on the antigen specificity of the migrating cells.     

In vitro refolding of recombinant human free secretory component using equilibrium gradient dialysis

Human secretory component (SC) is associated with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived from the polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. In this present study, we describe the first cloning, expression, in vitro refolding and purification of a free form of human secretory component (rSC) containing the five functional ligand binding domains using Escherichia coli BL21 (DE3). Free rSC was refolded from inclusion bodies by equilibrium dialysis after purification by nickel affinity chromatography under denaturing conditions. Refolded rSC was purified by gel filtration chromatography. Surface plasmon resonance and dot blot association analysis have shown that purified rSC binds IgM with a physiological equilibrium dissociation constant (KD) of 4.6x10(-8) M and shares structural similarity to native SC. This provides an important step in the elucidation of the structure of this immunologically important receptor.     

Differences in fragmentation between bound and unbound bovine secretory component suggest a model for its interaction with polymeric immunoglobulin.

Unbound bovine secretory component was cleaved into two-domain and one-domain fragments by trypsin within 1 h. Bovine secretory component covalently bound to bovine IgA dimer, as in secretory IgA, was much more resistant to fragmentation, which did not proceed beyond the three-domain stage even after 5 h. Bovine secretory component non-covalently bound to bovine IgM or to human IgM or IgA polymer was also relatively resistant to fragmentation, which again was largely arrested at the three-domain stage. A model for the binding of secretory component to polymeric immunoglobulin is proposed.     

The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Calpha3 and Cmu4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Calpha3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component.     

Shifts in immunoglobulin (IgG,IgM and IgA) levels in the milk of southern elephant seals,at Potter Peninsula,King George Island,Antarctica

We quantified immunoglobulins (Ig) in mammary secretions of 12 female southern elephant seals ( Mirounga leonina) at King George Island, Antarctica, using single radial immunodiffusion on agarose plates. Seals were chemically immobilized for milk sample collection at four time points during the 3- to 4-week suckling period. All three major mammalian immunoglobulins (IgG, IgM, IgA) were detected in southern-elephant seal milk. Total immunoglobulin levels and the ratio of Ig subclasses varied throughout the suckling period. Total immunoglobulin levels were highest on the 1st day of lactation, when they represented over 57% of the mean total protein concentration of the milk, and declined steadily throughout lactation, representing approximately 40% of the mean total protein concentration at the end of the suckling period. IgG was the most abundant immunoglobulin class (85.93-93.78% of total milk immunoglobulins), followed by IgM (6.06-13.93%), and IgA (0.14-0.23%). There was no significant difference in IgA levels throughout the suckling period. IgG levels were significantly lower during the second stage (3-6 days post-parturition) than during the first, third or fourth stages (1, 13-15, and 19-25 days post-parturition, respectively). IgM levels were highest during the first stage of lactation; these values were significantly higher than levels measured during the second, third and fourth stages of lactation. Transfer of passive immunity from female to offspring in other mammalian species is correlated with the subclass of immunoglobulin secreted in the milk; species acquiring passive immunity in utero, via the placenta, secrete a preponderance of IgA, whereas species acquiring immunity post-partum, via lacteal secretions and gut resorption, secrete a preponderance of IgG. The Ig patterns and concentrations observed in our study of southern elephant seals are consistent with an important role of post-partum transmission of passive immunity during the pinniped lactation period.     

Receptor for IgA in group A streptococci: cloning of the gene and characterization of the protein expressed in Escherichia coli

The gene for an IgA-binding protein from a group A streptococcal strain was cloned and expressed in Escherichia coli. The IgA-binding protein, called protein Arp, was purified on IgA-Sepharose, allowing complete purification in a single step. Analysis of protein Arp by Western immunoblotting demonstrated a major IgA-binding band, with an apparent molecular weight of 42 kD. The purified protein was shown to bind serum IgA and secretory IgA, as well as monoclonal IgA of both subclasses. There was no binding to IgM, IgD or IgE, but a weak binding to IgG. Inhibition experiments with whole bacteria indicated that IgA and IgG bind at separate sites. Experiments with immunoglobulin fragments showed that protein Arp binds to the Fc region of both IgA and IgG. The equilibrium constant of the reaction between protein Arp and polyclonal human IgA was determined to be 5.6 x 10(8) M-1. Amino acid sequencing of protein Arp demonstrated a direct repeat of 7 amino acids in the NH2-terminal region, a feature previously found in several streptococcal M proteins. This suggests that protein Arp, like M proteins, may be a streptococcal virulence factor.     

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.