Vitamin A is required for regulation of polymeric immunoglobulin receptor (pIgR) expression by interleukin-4 and interferon-gamma in a human intestinal epithelial cell line.

The secretory immunoglobulin A (IgA) antibody response to infections of mucosal surfaces requires transport of IgA from the basal to apical surface of mucosal epithelial cells by a specific transport protein, the polymeric immunoglobulin receptor (pIgR). We have tested the hypothesis that the vitamin A metabolite all-trans retinoic acid (RA) is required for the regulation of pIgR expression by the cytokines interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in HT-29 cells, a well-differentiated human epithelial cell line derived from a colonic carcinoma. pIgR expression is upregulated by IFN-gamma and IL-4 when HT-29 cells are grown in normal media, but this upregulation was significantly lower when cells were grown in vitamin A-depleted media. Treatment with RA at concentrations from 10(-9) to 10(-5) mol/L restored normal levels of pIgR expression. The percentages of cells expressing cell-surface pIgR after 24, 48 and 72 h of treatment with RA, IL-4 and IFN-gamma were 66 +/- 10, 90 +/- 5 and 92 +/- 1, respectively, significantly higher than the percentages seen without RA treatment, which were 32 +/- 2.3, 72 +/- 1.2 and 30 +/- 7, respectively. In addition, the intensity of fluorescence of pIgR-positive cells was significantly higher in the RA-treated cultures than in the cultures without RA treatment. Similarly, pIgR mRNA levels (adjusted for beta-actin mRNA levels) in RA-supplemented cultures were 404, 105 and 949% higher at 24, 48 and 72 h, respectively, than were pIgR mRNA levels in identical cultures grown in the absence of RA. These data indicate that RA strongly interacts with IL-4 and IFN-gamma to regulate pIgR expression in HT-29 cells, suggesting that vitamin A may be required for proper in vivo regulation of IgA transport in response to mucosal infections.

Antisperm antibodies in infertile women: subclass distribution of immunoglobulin (Ig) A antibodies and removal of IgA sperm-bound antibodies with a specific IgA1 protease.

OBJECTIVE: To determine the immunoglobulin (Ig) A subclass distribution of antibodies in the serum and cervical mucus (CM) of infertile women and to evaluate the effect of an IgA1 protease on the removal of sperm-bound antibodies. METHODS: Twenty infertile women with antisperm antibodies in serum (n = 10) or in CM (n = 10) were recruited for this study. Monoclonal antibodies to human IgA1 and IgA2 were conjugated to immunobeads and the IgA subclass distribution of antisperm antibodies was determined for positive serum and CM samples. The effect of an IgA1 protease (isolated from Neisseria meningitidis strain HF13) on sperm-bound antibodies was evaluated by immunobead binding. RESULTS: In serum, IgA1 subclass antisperm antibodies predominated (89%) when compared to IgA2 (11%). In CM IgA1 accounted for 62% and IgA2 accounted for 38% of the total IgA antisperm antibodies. Enzyme treatment was able to reduce dramatically the amount of serum IgA antibodies bound to sperm from 88% to 10%. Similarly, a significant reduction in CM antisperm antibodies was observed after enzymatic treatment with no loss in sperm motility. CONCLUSION: Cervical mucus antisperm antibodies have a higher proportion of IgA2 subclass suggesting a local production of IgA. Specific IgA1 protease treatment is capable of reducing the amount of immunobead-detectable IgA on sperm. Hamster sperm penetration assays are ongoing to determine if this treatment might improve sperm penetration rates with antibody positive sperm.

Antispermatozoal antibodies in human colostrum and milk.

Authors studied antisperm activity in human colostrum and milk by tray agglutination (TAT) and indirect mixed antiglobulin reaction (MAR) tests. Spermagglutination observed by TAT was found in 10 patients (24%) in colostrum, in 3 (5%) in milk. Character of spermantibodies by indirect MAR-test showed the presence of sIgA in 6 (14%), in IgG in 4 (9.5%) in colostrum, the presence of sIgA in 3 (4.8%), of sIgA and IgG together in 1 (1.7%) in milk. Authors discuss the origin of these antibodies.

Interactions of galactosyltransferase with serum and secretory immunoglobulins and their component chains.

Assay of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of beta-1,4-GT activity have shown that beta-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of beta-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in beta-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the beta-1,4-GT activity was detected in fractions that contained lactoferrin and alpha-lactalbumin. Interactions of beta-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and alpha-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the beta-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of beta-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the beta-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral beta-1,4-GT in the presence of Mn2+. Beta-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely alpha, mu and J chains, bound beta-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains.(ABSTRACT TRUNCATED AT 400 WORDS)

IgA subclasses of human colostral antibodies specific for microbial and food antigens.

The distribution of total and antigen-specific IgA1 and IgA2 antibodies in human colostrum was determined by ELISA using subclass-specific monoclonal reagents. In 18 samples of colostrum the mean ratio of total IgA1 to IgA2 was found to be 53:47, respectively, but significant individual variations were observed. In two samples we found unusually low levels of IgA1, while IgA2 was in the normal range. IgA1 and IgA2 antibody activities were determined against the following antigens: bovine gamma-globulin and beta-lactoglobulin, tetanus toxoid, protein antigen I/II of Streptococcus mutans, influenza virus vaccine, polysaccharides of pneumococcal, meningococcal and Haemophilus influenzae type b origin, and lipopolysaccharide (LPS) from Escherichia coli K235. The IgA antibody activity directed against the polysaccharides was almost equally distributed between the two subclasses. However, antibody activity specific for protein antigens was found predominantly in the IgA1 subclass while anti-LPS activity was mostly of the IgA2 subclass.

Natural killer cells in human colostrum.

The presence of natural killer cells in human colostrum was disclosed with the use of a fluorochrome-labeled monoclonal antibody HNK-1 (Leu-7) that recognizes cells with natural killer and killer activity. Approximately 0.5% of total colostral cells were stained with this reagent. These cells were separated by the fluorescence-activated cell sorter and examined for their morphology by electron microscopy and for their cytotoxic activity against 51Cr-labeled K562 target cells. Two morphological types of natural killer cells were observed in colostrum: the first was represented by large cells with numerous vacuoles but without dense cytoplasmic granules; the second type, which occurred with lower frequency, resembled the large granular lymphocytes associated with natural killer activity in peripheral blood. The HNK-1-positive cells from colostrum displayed low cytotoxic activity against K562 target cells. Incubation of HNK-1-positive cells from peripheral blood with cell-free colostrum resulted in a dose-dependent inhibition of the cytotoxic activity. The functional changes were accompanied by morphological alterations which included degranulation and the formation of numerous vacuoles. The variances in the cytotoxic activity of peripheral blood HNK-1-positive cells suspended in different dilutions of colostrum suggest that this fluid contains humoral factors which modify morphology and function depending on their concentrations.

Human colostral cells. II. Response to mitogens.

The ability of colostral lymphocytes to respond to pokeweed mitogen, phytohemagglutinin, or Epstein-Barr virus was examined. None of these mitogens induced colostral cells to differentiate into immunoglobulin-containing cells, either in the absence or in the presence of mitomycin C-treated mononuclear cells or T-cell-enriched populations from peripheral blood. Cocultivation of mononuclear cells from the peripheral blood of normal adults with mitomycin C-treated colostral cells resulted in a marked suppression of the generation of immunoglobulin-containing cells in response to pokeweed mitogen. The inhibitory effect was seen in peripheral blood mononuclear cell:colostral cell ratios of 1:1, 5:1, and 10:1. However, colostral cells had little effect on the ability of peripheral blood lymphocytes to proliferate in response to phytohemagglutinin or to allogeneic stimulation.

Localization of IgA and IgM in human colostral elements using immunoelectron microscopy.

In addition to the free form, IgA is associated with cellular and noncellular elements present in human colostrum. To resolve the existing controversy as to the cell type(s) containing IgA, we used immunoelectron microscopy with horseradish peroxidase-labeled F(ab')2 or Fab' fragments of anti-IgA or anti-IgM to determine the distribution of these immunoglobulins in colostral elements. IgA and IgM were localized in phagocytic vacuoles of polymorphonuclear leukocytes and macrophages in the vicinity of the cell membrane. In neutrophilic leukocytes, both immunoglobulins were occasionally found in phagocytic vacuoles distributed throughout the cytoplasm. Although the in vitro phagocytic activity of colostral cells was low, they retained the ability to ingest colloidal gold particles which were subsequently localized in phagocytic vacuoles that also contained IgA or IgM. IgA and IgM were not detected in lymphocytes, and plasma cells were not found in human colostrum. Numerous noncellular colostral globules of various shapes and sizes also contained IgA and IgM. These observations indicate that IgA and IgM were acquired by phagocytic cells and noncellular globules and were not actively synthesized by lymphoid cells present in human colostrum.

Human milk peroxidase is derived from milk leukocytes.

Peroxidase enzymes present in human colostrum, saliva, polymorphonuclear leukocytes, and bovine milk were compared with respect to their molecular exclusion chromatographic behavior and immunological cross-reactivity. Human milk peroxidase gave an elution profile similar to myeloperoxidase derived from blood polymorphonuclear leukocytes. Human salivary peroxidase reacted with an antibody directed against bovine lactoperoxidase, but with the same antibody preparation no reaction was detected either with human milk peroxidase or leukocyte myeloperoxidase. We conclude that the peroxidase enzyme in human milk is different from the human salivary and the bovine enzymes and is probably derived from milk leukocytes.

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.

Hemolytic plaque formation by cellular and noncellular elements of human colostrum.

The morphologic, histochemical, and immunohistochemical characteristics of colostral elements that form indirect IgA hemolytic plaques were determined. Microscopic examination of hemolytic plaques revealed noncellular, globular elements at the center of 82% of the plaques formed by colostral cell preparations. Similar globules in colostral cell preparations contained IgA, IgG, IgM, SC, lactoferrin, alpha-lactalbumin, and lipid. Lysates of colostral cell preparations contained large amounts (500 ng/10(6) cells) of polymeric, SC-associated IgA. These findings suggest that plaque formation by colostral cell preparations may be due to the release of passively acquired, preformed S-IgA from colostral elements, rather than active production of IgA by lymphoid cells.

Inhibition of the pokeweed mitogen-induced response of normal peripheral blood lymphocytes by humoral components of colostrum.

Colostral whey at dilutions up to 1 : 100 inhibited both the uptake of tritium-labelled thymidine and the differentiation of pokeweed mitogen-stimulated peripheral blood lymphocytes from normal adults into immunoglobulin-containing plasma cells. Upon gel filtration (Sephadex G-200), inhibitory activity was associated with high molecular weight fractions. Secretory component, but not monomeric, polymeric or colostral IgA, IgM, IgG, lactoferrin, casein or alpha-lactalbumin, inhibited the response to mitogen. Supernatants from cultures of colostral cells did not induce the differentiation of adult peripheral or cord blood lymphocytes into IgA-containing cells and did not stimulate the uptake of tritium-labelled thymidine.

Human colostral cells. I. Separation and characterization.

Analyses of the cells present in human colostrum obtained from fifty-four healthy donors during the first four days of lactation revealed that there were 3.3 x 10(6) (range 1.1 x 10(5)--1.2 x 10(7)) cells per ml of colostrum. Based on histochemical examinations, it was found that this population consisted of 30--47% macrophages, 40--60% polymorphonuclear leucocytes, 5.2--8.9% lymphocytes, and 1.3--2.8% colostral corpuscles; epithelial cells were rarely encountered. The identity of various cell types was confirmed by Wright's stain and by a series of histochemical techniques which disclosed the presence of non-specific esterase, peroxidase, and lipids. For further characterization, the different types of cells were separated by various methods, such as Ficoll-Hypaque density centrifugation, isokinetic centrifugation on a linear Ficoll gradient, adherence to glass or plastic, and phagocytosis of carbonyl iron. Immunohistochemical staining with FITC- and/or TRITC-labelled reagents to IgA, IgM, IgG, K- and lambda-chains, secretory component, lactoferrin, and alpha-lactalbumin were applied to unseparated as well as separated colostral cells. Polymorphonuclear leucocytes (staining for peroxidase) as well as macrophages and colostral corpuscles (staining for non-specific esterase) exhibited numerous intracellular vesicles that contained lipids as well as various combinations of milk proteins. Lymphoid cells did not stain with any of these reagents and plasma cells were not detected among the colostral cells. Individual phagocytic cells contained immunoglobulins of the IgA and IgM classes, both K and lambda light chains, secretory component, lactoferrin, and alpha-lactalbumin. The coincidental appearance of these proteins in single, phagocytic cells but not in lymphoid cells indicate that the cells acquired these proteins by ingestion from the environment. Markers commonly used for the identification of B lymphocytes (surface immunoglobulins) and T lymphocytes (receptors for sheep red blood cells) were unreliable for the analysis of colostral cells (unless accompanied by subsequent morphological characterization) because strong fluorescence was observed on the surface of many non-lymphoid cells and because numerous macrophages and colostral corpuscles formed rosettes with sheep red blood cells (SRBC). Lymphocytes, often found in association with colostral macrophages or corpuscles, were classified as T cells.