Sylvia S. Crago

Concept of the Local and Common Mucosal Immune Response

The available information concerning the origin of IgA-producing plasma cells and the spectrum of IgA-associated antibodies found in external secretions provide arguments that support two pathways of stimulation for a secretory humoral immune response. In addition to an explicitly local immune response induced by a topical antigen application, a second mechanism of induction operating through the sensitization of GALT, and possibly BALT, emerges. The latter pathway of stimulation leads to the appearance of specific IgA-associated antibodies in secretions of mammary, salivary, and lacrymal glands (and perhaps other sites), all of which suggests the existence of a common mucosal secretory system. Not yet explained are the mechanisms involved in the homing to secretory glands of cells sensitized in the remote lymphoid tissues (GALT and BALT), the differentiation patterns of these cells, and the regulation of selective IgA expression.

Inhibition of natural killer cell activity by IgA

The in vitro effect of IgA on natural killer (NK) activities of human peripheral blood mononuclear cells was investigated. Purified myeloma polymeric IgA2 (pIgA2) and secretory IgA (S-IgA) from human colostrum inhibited NK activity, while myeloma polymeric IgA1 (pIgA1), monomeric IgA1 (mIgA1), IgG, and IgM were ineffective. Inhibition was proportional to the concentration of pIgA2 (0.125-1 mg/ml) and was observed after as little as 1 hr of incubation at various effector to K562 target cell ratios. pIgA2 and S-IgA also inhibited NK activity of NK cell-enriched lymphoid cells and gamma-interferon-treated effector cells, but did not interfere with effector-target cell binding. The inhibitory effect was slightly diminished after 24 hr culture in pIgA2-free medium. Inhibition of cytotoxicity was not due to direct toxicity on lymphoid cells by IgA because PBL treated with pokeweed mitogen in the presence of pIgA2 or S-IgA differentiated into immunoglobulin-producing cells. Viability after 24 hr of preculture with pIgA2 and S-IgA was comparable to that of untreated control cells. Morphological examination of effector cells cultured with pIgA2 or S-IgA showed a decrease in the number of granules, and the formation of cytoplasmic vacuoles. These morphological changes appeared to coincide with the depressed cytotoxicity of NK cells. The results demonstrate that purified pIgA2 and S-IgA have significant immunomodulatory effects on human NK activity.

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 thein 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.

IgA Antibodies to Oral and Ocular Bacteria in Human External Secretions

Abstract Human saliva contains secretory IgA (sIgA) antibodies to Streptococcus mutans ; however, lower antibody levels to serotype c than other serotypes of S. mutans are found. Oral challenge with S. mutans c and d resulted in implantation of c but not d , suggesting that naturally occurring salivary IgA antibodies regulate S. mutans colonization. Saliva, tears and milk contain naturally occurring sIgA antibodies to S. mutans and ocular bacteria, implying that these bacteria stimulate gut-associated lymphoreticular tissue (GALT) leading to sIgA responses in distant mucosal sites. Oral administration of S. mutans c to humans induced elevated levels of sIgA antibodies in external secretions and lowered numbers of S.mutans in dental plaque. Peripheral blood lymphocyte cultures stimulated with pokeweed mitogen and S. mutans gave antigen-specific IgA responses. These results show that ingested bacterial antigens stimulate GALT, and precursor-IgA B cells from this tissue migrate via the circulation to distant mucosal tissues; supporting the concept of a common mucosal IgA immune system in humans.

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.

Evidence for a Common Mucosal Immune System in Humans

The finding that ingestion of antigens results in the selection induction of IgA antibodies in external secretions suggests that antigen sensitizes Peyers patch lymphoid cells, which migrate to mucosal sites and generate local secretory IgA (S-IgA) antibody responses. Evidence for a common mucosal immune system in humans has been scanty because of the difficulty in demonstrating migratory behavior of Peyers patch cells. In the present study, peripheral blood mononuclear cells (PBMC) from human volunteers who had ingested capsules containing killed Streptococcus mutans were assayed for spontaneous antibody-producing cells. Four of five volunteers exhibited circulating IgA-producing cells within 7 days and reached maximum responses by days 10-12. One IgA-deficient subject exhibited IgM responses with identical kinetics. Pokeweed-mitogen-stimulated PBMC produced anti-S. mutans antibodies predominantly of the IgA isotype. Significant S-IgA anti-S. mutans antibodies were detected in saliva and tears by day 14, and the antibodies reached maximum titers by 3 weeks. No changes in serum anti-S. mutans antibodies were noted. The IgA-deficient subject produced salivary secretory IgM antibodies. These results suggest that, after antigen ingestion, peripheral blood contains antigen-specific precursors of IgA plasma cells and that their presence precedes the appearance of S-IgA antibodies in external secretions. Therefore, these experiments provide further support for the existence of a common mucosal immune system in humans.

Molecular-cellular interactions in the secretory IgA system.

1) SC receptors were not detected on the surface of human PBL before or after PWM stimulation or on the surface of established lymphoblastoid cell lines. 2) SC binding was detected in the cytoplasm of differentiated lymphoid cells. The majority of the SC-binding cells contained intracellular IgA. 3) The binding of polymeric IgA to the surface of human epithelial cells (colonic carcinoma HT-29) was dependent on the presence of SC. 4) These findings indicate that SC is a receptor and possible transport protein for polymeric immunoglobulins, but that it is not directly involved in the homing of the IgA precursor cells to secretory tissues.

Presence of Antibodies to Food Antigens in Human Colostral Cells

Abstract Cellular and non-cellular components of human colostrum were examined by solid-phase radioimmunoassay for the presence of IgA-, IgM- or IgG- associated antibodies to common food antigens, including ovalbumin, bovine casein, bovine gammaglobulin, soy protein and wheat gliadin. Significant levels of IgA- and IgM-associated antibodies were detected against all antigens examined in both cell-free colostral whey and in colostral cell lysates. Food antigen-specific antibodies in the IgG class were not present in whey or cell lysates in levels detectable by this assay.

Immunological Properties and Differentiation Potential of Human Colostral Lymphocytes of B Cell Lineage

Human colostrum and milk contain approximately 1–5×106 cells/ml, identified by histochemical and immunohistochemical staining as macrophages (~40% of total cells), polymorphonuclear leukocytes (PMN), (~50%), lymphocytes (7–25%), and epithelial cells (~1%)1–4. Phenotypic analyses of lymphocytes revealed the presence of both B and T cells; the latter population is represented by CD4- and CD8-positive cells as well as γ/δ cells and NK cells2,5–7. Studies of colostral and milk lymphocytes of B cell lineage yielded controversial results with respect to their morphological properties, and their ability to respond in vitro to various stimuli and secrete immunoglobulins (Ig)1,2,4,8. The purpose of our studies was to re-evaluate the characteristics of colostral Ig-containing cells and to determine the transformability of B cells by the Epstein-Barr virus (EBV) which has been used in many studies of the differentiation potential of human B cells from various sources9.

Inhibition of Natural Killer (NK) Activity by Human Colostral and Serum IgA

Natural killer (NK) cells are defined as effector cells with spontaneous cytotoxicity against tumor, virus-infected and undifferentiated normal cells. NK activity is closely associated with a subpopulation of lymphocytes, large granular lymphocytes (LGL), that contain azurophilic cytoplasmic granules. The NK cells show a characteristic organ distribution: they are numerous in peripheral blood and spleen, but relatively low numbers are found in lymph nodes, peritoneal cavity and bone marrow (1,2). Previous investigations have demonstrated that NK activity is influenced by various factors such as interferon, interleukin-2, hormones, suppressor cells, environmental factors and age. Recently, mucosal LGL have been isolated from the small intestines of humans, mice and rats (3–6). In the mouse and rat, mucosal LGL showed moderate levels of NK activity, while human mucosal LGL demonstrated low NK activity.