Colostral IgA Research Articles

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.

Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor

Characterization of the human placental membrane receptor for human 125I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10 7 mole −1, and 2.0 ± 0.16 × 10 15 IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 m M), and periodate (4 m M) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 m M or 20 m M) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of 125I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.

The differential localization of IgA, IgM and IgG in the gut of suckled neonatal piglets

The localization of immunoglobulins IgG, IgM and IgA in tissue sections prepared from the ileum of neonatal and adult swine were compared. Eighty percent of the immunoglobulin-containing lymphoid cells in the lamina propria of conventional adult German Landrasse swine were IgA-positive with lower numbers of IgM cells and occasionally an IgG cell. Anti-μ and α-chain reagents also stained the cytoplasm of the crypt epithelial cells. By comparison to these adult control tissues, the ileum of unsuckled neonates contained no immunoglobulins although after the ingestion of colostrum, the entire cytoplasm of the villus epithelial cells stained intensely when tested for IgG with only faint staining for IgM and IgA. On the other hand, IgA and IgM were readily localized on what appears to be only the apical border of the crypt epithelial cells but in contrast to the adult, the cytoplasm of these cells was unlabelled. IgG was absent from the crypt region. We interprete these findings to indicate an important, selective role for the villus epithelium in the absorption into the neonatal circulation of colostral IgG and probably IgA and IgM, and a specialized role for the crypt epithelium in adsorbing colostral IgA and IgM; possibly by complexing with mucin-bound secretory component.

Interactions of pig Fab mu and Fab alpha fragments with protein A from Staphylococcus aureus.

Forty % of serum IgM and 47% of dimeric colostral IgA were bound to protein A-Sepharose. Fab mu and Fab alpha fragments showed a reactivity similar to that of the whole immunoglobulins. Complete elutions of IgM and IgA from the protein A-Sepharose were obtained in lower cocentrations of MgCl2 than the complete elution of IgG. However, IgM and IgA were completely eluted at higher concentrations of MgCl2 in the presence of IgG than in its absence.

Restricted dietary protein in pregnant beef cows: II. Effect on the immune response

Cows fed 0.37 Kg crude protein per day for four months before calving showed no decrease in colostral IgG, IgA, or IgM from cows fed a normal ration. Similarly, no difference in serum immunoglobulin concentration was detected in their calves. Cows in the low protein group developed significantly lower (P < 0.01) titers to Salmonella pullorum antigen than did the normal group. The data show significant positive linear regressions of calf agglutination titer to S. pullorum on the dam's colostral titers. Serum levels of IgG measured at 24 hours were significantly lower in calves from second-calf heifers than from these of other age groups. The IgA levels of serums from calves from the six-year-old cows were significantly higher than the serum levels of calves from younger dams (P < 0.01).

Neutralizing Activity against E. coli Enterotoxin in Colostral IgA

Neutralizing Activity against E. coli Enterotoxin in Colostral IgA

Kinetic Assessment of Alternative Complement Pathway Activity in A Hemolytic System

Abstract By using a kinetic assay, we have examined the role of antibody in the lysis of rabbit erythrocytes (RaRBC) through the alternative complement (C) pathway. Sera from some hypogammaglobulinemic (Hγ) humans and all agammaglobulinemic chickens tested had subnormal activity in the assay. Heated normal human or chicken sera, but not heated Hγ sera, restored activity to deficient Hγ serum and initiated hemolysis in the presence of rabbit serum as C source. Absorption of heated normal human serum with RaRBC, but not with sheep erythrocytes or zymosan, removed its ability to reconstitute deficient Hγ serum. Normal hemolytic activity could be restored to Hγ serum with human IgM, IgG, or colostral IgA, with goat anti-RaRBC IgG, or with an eluate from serum-sensitized RaRBC, but not with myeloma IgA. Restoration of hemolytic activity to Hγ serum could be achieved in a dose-dependent fashion with the F(ab′)2 fragment of IgG. These results suggest that antibody exerts a significant rate-limiting effect on alternative pathway activity in the RaRBC lytic system. This raises the possibility that antibody may be required for efficient alternative pathway activity in vivo and that the pyogenic infections that occur in Hγ individuals are due to inefficient activation and fixation of C3 through either the classical or alternative pathway.

Influence of Antibody on the Rate of Alternative Pathway Activation

Abstract Using a kinetic assay, we have examined the role of antibody in the lysis of rabbit erythrocytes (RaRBC) through the alternative complement pathway. Sera from 7 of 11 hypogammaglobulinemic (Hγ) humans and all agammaglobulinemic chickens tested had subnormal activity in the assay. γ-Globulin injections restored normal activity to the two Hγ patients studied. Heated normal human or chicken sera, but not heated Hγ sera, normalized activity in deficient Hγ serum and initiated hemolysis in the presence of rabbit serum as complement source. Absorption of heated normal human serum with RaRBC, but not with sheep erythrocytes or zymosan, removed its ability to reconstitute deficient Hγ serum. Normal hemolytic activity could be restored to Hγ serum with human IgM, IgG, or colostral IgA, with small amounts of goat anti-RaRBC IgG, or with an eluate from serum-sensitized RaRBC, but not with myeloma IgA. Restoration of hemolytic activity to Hγ serum could be achieved in a dose-dependent fashion with the F(ab′)2 fragment of IgG.

Studies on Translocation of Immunoglobulins Across Intestinal Epithelium: IV. Evidence for binding of IgA and IgM to secretory component in intestinal epithelium

We conducted studies concerning the issue of whether secretory component (SC) is a specific receptor on intestinal epithelial cells for IgA and IgM. Initially, frozen sections of human intestinal mucosa were incubated with dimeric monoclonal human IgA, conjugated to horseradish peroxidase; adjacent sections were reacted with peroxidase-conjugated antibodies to SC. The conjugated IgA and anti-SC bound to similar sites in the epithelium, that is to basolateral margins and supranuclear cytoplasm of columnar epithelial cells, principally in gland crypts. In subsequent tests of binding specificity, binding of the dimeric IgA conjugate was inhibited by pretreating the tissues with unconjugated dimeric IgA or 19S IgM, pretreating the tissues with unconjugated antibodies to SC, or preincubating the dimeric IgA conjugate with free SC. Binding was not inhibited or only partially inhibited by pretreating the tissues with monomeric IgA or IgG, pretreating the tissues with antibodies to human or heterologous immunoglobulins, or preincubating the dimeric IgA conjugate with 11S secretory colostral IgA. The findings indicate that dimeric IgA and 19S IgM are capable of binding in vitro to specific sites on intestinal epithelial cells, most likely to SC. This supports the hypothesis that transport of these immunoglobulins into intestinal fluids involves their combination with SC in the epithelium.

Comparison of immunoglobulin determinations in pathological sera by radial immunodiffusion and laser nephelometry.

Immunoglobulins G, A, and M were measured by radial immunodiffusion and by laser nephelometry in sera from 33 patients and in a preparation of purified colostral IgA. In general the results agreed for the two methods, with some exceptions attributed to the physicochemical characteristics of the immunoglobulins assayed: low-molecular-weight (monomeric) IgM gives falsely high values and IgA proteins (consisting predominantly of polymeric molecules) give falsely low values when assayed by radial immunodiffusion. Results from laser nephelometry apparently are not affected by the molecular-size characteristics of the proteins assayed.

Determination du rapport IgA 7 S/IgA 11 S dans la secretion bronchique humaine par une technique associant la gel-filtration en couche mince a une immunoelectrophorese bidimensionnelle

After extraction by treatment with Ecteola-cellulose, IgA 11 S and IgA 7 S from human bronchial secretion were first separated by thin-layer gel filtration in Ultrogel AcA 3–4, and then were analyzed by crossed immunoelectrophoresis with an immune serum against α chain. Precipitation areas were measured separately. A correction factor was necessary to express IgA 7 S in terms of IgA 11S: it was calculated with standard curves made with serum and colostral IgA.

Human secretory component—V: Lack of association with γ-glutamyl transpeptidase in colostrum

Pooled human colostrum contained high levels of macromolecular γ-glutamyl transpeptidase (γ-GT), which appeared in the secretory IgA fraction after gel filtration. Only a trace of enzyme activity was detected in retarded fractions with a molecular size slightly smaller than that of native free secretory component (SC). No decrease in the level of macromolecular γ-GT was observed when bound SC was separated from colostral IgA under reducing conditions. Moreover, it was possible to distinguish between secretory IgA and macromolecular γ-GT by immunological precipitation. Finally, native free SC did not acquire detectable enzyme activity after complexing with dimeric serum IgA in vitro. These results do not support the previously suggested function of SC as a γ-GT enzyme in the glandular transport of IgA.

Suppression of leukocyte chemotaxis by human IgA myeloma components.

Sera from patients with IgA myeloma suppressed polymorphonuclear leukocyte (PMN) chemotaxis, while generally little or no suppression was observed with sera from patients with IgG myeloma or Waldenstrom's macroglobulinemia. Chemotactic inhibitory activity was not limited to a single chemotactic factor and was equivalent when C5a, bacterial factor, casein, or normal serum were used as chemotactic attractants. No association was noted between the degree of inhibitory activity and the IgA subclass or light chain type. Chemotactic inhibitory activity was found to be directly associated with isolated IgA M components, and similar chemotactic suppression was noted with purified preparations of normal human colostral IgA. By comparison, IgA preparations were most effective in suppressing PMN chemotaxis and had a much lesser effect on monocyte chemotaxis. The mode of IgA chemotactic inhibition was cellular and at least partially reversible after a 37degreesC incubation in the absence of IgA. Some inhibition of PMN random mobility was noted with certain IgA preparations, although such effects did not parallel the degree of chemotactic inhibition. Fractionation of IgA myeloma sera and IgA M components by sucrose density gradient centrifugation showed multiple peaks of inhibitory activity in 10 to 13S fractions. The majority of IgA inhibitory activity was lost after pepsin digestion or sulfhydryl reduction and alkylation of isolated M components. When isolated IgA M components were fractionated on Sephadex G-200, inhibitory activity was associated with the exclusion volume and was abolished by reduction and alkylation procedures which resulted in a conversion of polymeric IgA to monomeric IgA.

Synchronous Regulation of VH and CH Allotypes among Ig Molecules in Multi-Heterozygous Rabbits Suppressed with Anti-VH Allotype Antibody: Emergence of IgA from Suppression Prior to IgG and IgM

Heterozygous rabbits of genotype a1n81f73g74/a2n82f71g75 were suppressed at birth for the VH region a1 allotype. At 8 weeks of age, quantitative analysis of serum IgG, IgM, and IgA molecules showed that the VHa1 specificity was effectively suppressed in the three classes of Ig and that the suppression was extended to the CH region n81 specificity on mu-chains as well as to the CH region f73 and g74 specificities on alphaf and alphag chains. At 26 weeks of age, analysis of serum IgG and IgM molecules showed that a1 was still suppressed to approximately the same extent in both Ig classes and the suppression was still extended to the CH region n81 specificity. However, at 26 weeks, the percentage of molecules with a1 specificity had doubled among serum and colostral IgA molecules and this increase was extended to the CH region f73 and g74 specificities. Thus, the suppressed allotypes reappeared first among IgA molecules. Our data are consistent with a regulatory mechanism which controls and synchronizes the expression of the VHa and the CH allotypes expressed on the same heavy chain. The order of the re-expression of the suppressed allotypes with respect to Ig class may allow further definition of selective regulatory mechanisms for the synthesis of Ig classes.

Influence of maternal gut flora and colostral and cord serum antibodies on presence of Escherichia coli in faeces of the newborn infant.

From 29 healthy newborn infants and their mothers faecal, serum and milk specimens were obtained on several occasions from one to nine weeks after delivery. Predominant faecal E. coli were serotyped with regard to the O antigen and milk and serum were analysed for their content of E. coli O antibodies by the enzyme-linked immunosorbent assay. In five cases the babies acquired the same O serotype as was found in the stools of their mothers but in 12 out of 29 cases infant and mother never had any dominating faecal E. coli O type in common. There was no apparent correlation between the patterns of feeding and interchange of bacteria. Klebsiella/Enterobacter was the dominating facultative organism on at least one occasion in half the infants. The newborns received colostral IgA and transplacental circulating IgG antibodies against a great number of E. coli O serotypes. These antibodies did not prevent intestinal colonization, as judged from cultures of faeces.

Human colostral IgA interacting with staphylococcal protein A.

Human colostral IgA from three apparently healthy mothers were all divided into protein A reactive and protein A non-reactive fractions on a Sepharose-protein A column. The reactive fractions, giving no direct precipitation but co-precipitations, were found to interact with protein A through the Fc-region.

The respiratory tract immune system in the pig. I. Distribution of immunoglobulin-containing cells in the respiratory tract mucosa.

The number of cells containing immunoglobulins A, G and M in the respiratory tract mucosa of pigs from birth to maturity was assessed using the immunoperoxidase technique. Immunoglobulin-containing cells first appeared at 6-7 days of age and rose rapidly to reach levels at 3-4 weeks similar to those in the adult. IgA-containing cells predominated at all sites in all age groups, although there were significant proportions of cells containing IgM and IgG. Our findings suggest that IgA is transported into secretions via the mixed mucoserous glands of the nasal and tracheobronchial mucosa, and that this route is also operative for colostral IgA absorbed from the gut in the baby pig.

Immunochemical studies on free and bound J chain of human IgA and IgM.

J chains purified from colostral IgA, polymeric (mainly dimeric) polyclonal IgA from serum, and monoclonal IgM appeared to be immunochemically identical in tests with antisera produced against the two latter antigens. None of the antisera precipitated with the native immunoglobulins, but J-chain antibodies became bound to all three types of Ig polymer. On a molar basis polymeric IgA was almost twice as efficient as IgM and 17 times more efficient than colostral IgA in antibody inhibition tests. The J-chain antibodies were able to link two or more IgA dimers and two or more IgM pentamers. After incubation in large antibody excess, the sedimentation properties of most IgM molecules were altered, whereas about 40% of the IgA dimers were unaffected by the treatment. THus, although the J chain on the whole was slightly more exposed in polymeric IgA than in IgM, it was more homogeneously available in the latter polymer. After denaturation in acid urea, the J chain became markedly more exposed in all three types of Ig polymer, and they were all precipitated by the most potent antiserum. Immunochemical quantitation of J chain released after reduction with 20mM dithiothreitol indicated that on the average there are about 2 mol of J chain per mol of polymeric IgA, and 3 to 4 mol per mol of IgM.

Purification of J chain after mild reduction of human immunoglobulins.

Two methods are described for the purification of J chain from polymeric IgA after mild reduction without the use of alkylating or dissociating reagents. The released peptide was separated from other protein components by immunoadsorption combined with gel filtration or anionic-exchange chromatography, or both. J chain was thus obtained in a yield of about 30% of the total release. Most of it consisted of dimers (molecular weight, approximately 25,000 to 30,000) or larger polymers, but re-reduction and alkylation produced a quite homogeneous fraction that sedimented slightly more slowly than egg-white lysozyme. The purity was high enough for successful immunization. When J chain coupled to bovine serum albumin was used as an antigen, all of five rabbits showed a good immune response. Although the same principle could be used for the purification of J chain from IgM and colostral IgA, high purity was more difficult to achieve and the yield was much lower. These preparations contained an unidentified slow-moving component, and the J chain was more prone to become rapidly degraded to smaller fragments.

Comparative studies on human and rabbit exocrine IgA antibodies to an albumin

To study some mechanisms involved in mucoantibody production and to compare immunological activities of exocrine IgA antibodies and IgG antibodies, we isolated albumin from the blood serum of Alligator mississippiensis, alligator serum albumin (ASA), and used it to immunize rabbits and one human. The human subject responded to intranasal administration of ASA with the delayed production of IgG and IgA antibodies in his nasal fluids and serum. Most of the nasal IgA, and some of the nasal IgG antibodies were produced and secreted locally in the nose, as indicated by the results of measuring primary antigen-binding IgA and IgG separately. The pattern of responses to subsequent administrations of intranasal ASA moreover suggested the operation of immunological memory in the production of human exocrine IgA antibodies. The human nasal exocrine IgA and serum IgG antibodies were compared with rabbit colostral exocrine IgA and serum IgG antibodies produced in response to intramammary or parenteral immunization with ASA. Antibody concentrations were measured in terms of antigen-binding capacity by ammonium sulfate coprecipitation. Relative avidities of the antibody preparations, as reflected in the effect of antigen concentration on antigen-binding capacities, were similar for the various preparations. Nevertheless, exocrine IgA antibody was more efficient in agglutinating passively sensitized red blood cells than the IgG antibodies. Human and rabbit IgG antibodies precipitated ASA from solution, unlike either human or rabbit exocrine IgA antibodies. Low concentration of polyethylene glycol however induced precipitation of immune complexes composed of human, but not rabbit exocrine IgA antibodies. A probable explanation for this difference was found in experiments on the ability of antibodies to cross-link ASA molecules, using an insoluble immunoadsorbent containing ASA. Human exocrine IgA antibody that was combined with insoluble antigen was at least effective as IgG antibody in binding soluble ASA molecules, whereas rabbit exocrine IgA antibody was far less able to cross-link. Although these experiments do not allow a decision as to whether monogamous polyvalency, steric hindrance, or some other mechanism can explain the inability of the rabbit exocrine IgA antibody to cross-link ASA molecules, these findings suggest fundamental differences between the exocrine IgA antibodies from different species with respect to their reactions with polyvalent antigens.