Developmental changes in humoral suppressor activity released from concanavalin A-stimulated human lymphocytes on B cell differentiation

Cell-free culture supernatants (Con A-activated supernatants) were obtained by incubating peripheral blood lymphocytes (PBL) from cord blood, healthy children of various ages, and healthy adults with mitogenic doses of concanavalin A (Con A) for 48 hr. It is well known that human T lymphocytes are activated by Con A to manifest suppressor function in vitro. One mechanism whereby these suppressor cells act has been shown to be by the secretion of a soluble suppressor factor. The present study has investigated the Con A-inducible suppressor cell function in cord blood, children of various ages, and adults by comparing the ability of each Con A-activated supernatant to inhibit the generation of immunoglobulin-producing cells (Ig-PC) in pokeweed mitogen- (PWM) stimulated cultures of adult PBL. Con A-activated supernatants from adults could markedly suppress the generation of Ig-PC by allogeneic as well as autologous PBL in response to PWM. Such suppression appeared to be equally effective on the generation of IG-PC of 3 major classes, IgG, IgM, and IgA. On the contrary, Con A-activated supernatants from cord blood and newborn infants showed only a negligible suppression on PWM-induced adult B cell differentiation. But the suppressor activity found in Con A-activated supernatants gradually increased with advancing age, and reached approximately to the adult level at 4 yr of age or later. The results suggest that human T lymphocytes may be relatively deficient in their Con A-induced suppressor cell function in the early period of life.     

Preparation and properties of cloning inhibitory factor. I. Inhibition of HeLa cell cloning by stimulated lymphocytes and their culture supernatants

Lymphocytes stimulated by either phytohemagglutinin or tuberculin inhibit the cloning of HeLa cells. Cell-free supernatants prepared from such cultures of activated lymphocytes produced similar inhibition, whereas supernatants from unstimulated lymphocytes did not. The cloning inhibitory factor (CIF) produced by activated lymphocytes was nondialyzable and inactivated at 56 °C for 30 minutes. CIF did not produce cytolysis of cloned HeLa cells; time-lapse cinematography instead disclosed an additional, normally timed mitosis. Subsequent divisions, however, were markedly delayed. CIF did not depend for its activity on depletion of an essential nutrient from the HeLa medium.     

Neuroendocrine regulation of salivary IgA synthesis and secretion: implications for oral health

Secretory immunoglobulin A (S-IgA) represents the main adaptive immune mechanism in the oral cavity. The regulation of secretion and synthesis of S-IgA is not only dependent on prior antigenic stimulation, but is also under strong neuroendocrine control. Thus, alterations in neuroendocrine functioning (such as induced by stress, exercise, pregnancy, menstrual cycle, and pharmacological interventions) may affect salivary IgA levels. This review deals with the neuroendocrine regulation of synthesis and secretion of salivary IgA and its potential role in the maintenance of oral health.     

IgA and IgA diphtheria antitoxin responses from human tonsil lymphocytes.

Human tonsil lymphocytes were stimulated with diphtheria toxoid and then cultured in a Marbrook culture system so that antibodies could be measured in the culture supernatant. Specific antibodies were measured with excess radiolabeled antigen and antisera specific for each immunoglobulin class. Good IgG and IgA diphtheria antitoxin responses have been obtained and responding culture supernatants were shown to neutralize toxin. The relationship between antitoxin response in vitro and immunization of donors with toxoid was investigated. It was found that at least two immunizations after the age of 6 months were necessary to prime the tonsils for an in vitro antibody response. The IgG and IgA in culture supernatants were demonstrated by immunodiffusion and were measured by radioimmunoassay. By sucrose density gradient ultracentrifugation, it was shown that 40% of the IgA produced in the cultures was greater than 7S. Evidence was obtained that neither the IgA nor the specific IgA antitoxin bears secretory piece. It appears that human lymphocytes from tonsils produce polymer IgA in vitro without secretory piece.     

Role of mesenteric lymph nodes and aging in secretory IgA production in mice

Although it is known that Peyer's patches are the major inductive site for S-IgA production and B1 cells contribute to half of the IgA plasma cells detected in the gut lamina propria, the type of contribution of mesenteric lymph nodes to the process is still unclear. Cytokines such as TGF-beta, IL-10, IL-4, IL-5, and IL-6, are required to promote IgA class switching and IgA synthesis. Aging-related alterations in T and B cells and in cytokine production are already known. Some reports have also proposed that S-IgA production might be altered in aged animals. Herein, we investigated the role of MLN and aging in S-IgA production. Two- to 18-month-old BALB/c mice were used to evaluate aging-related alterations and MLN were removed to study its role in S-IgA production. We found that MLN are important, although not essential for S-IgA production. In addition, we showed that production of IgA-related cytokines are well preserved in MLN but not in PP of aged mice and that S-IgA levels are not affected by aging. Our results suggest that MLN may play a complementary role in S-IgA production mostly in aged animals.     

The amount of secreted IgA may not determine the secretory IgA coating ratio of gastrointestinal bacteria

It is reported that some, but not all, bacteria in human faeces are coated with secretory immunoglobulin A (S-IgA). We evaluated the proportion of S-IgA-coated bacteria to total intestinal bacteria (S-IgA coating ratio) in the gastrointestinal tract of two different strains of mice supplied by two different suppliers. The S-IgA coating ratio was significantly different in each gastrointestinal segment and between mouse suppliers. The amount of non-bacteria-bound IgA (free IgA) in each gastrointestinal segment indicated that this difference in the S-IgA coating ratio might not be due to the amount of secreted IgA. Furthermore, immunoblotting analysis revealed that only a small amount of IgA (<5% to free-IgA) was used for the coating. This indicates that, although sufficient S-IgA was secreted to coat the entire intestinal population of bacteria, only some part of the bacteria were coated with S-IgA. This study suggests that the amount of luminal S-IgA may not determine the S-IgA coating ratio, and that the amount of IgA coating intestinal commensal bacteria is very small.     

Cellular origins of human polymeric and monomeric IgA: intracellular and secreted forms of IgA

Intracellular and secreted IgA from pokeweed mitogen (PWM)-stimulated normal peripheral blood lymphocytes, from 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated peripheral blood lymphocytes of a patient with chronic lymphocytic leukemia (CLL), or from an IgA-producing human Epstein Barr virus (EBV)-transformed lymphoblastoid cell line were analyzed by molecular-sieve chromatography, electrophoresis in sodium dodecyl sulfate, and sucrose density ultracentrifugation. Fluorochrome-labeled anti-human IgA and secretory component (SC) were used as probes for the detection of polymeric IgA in individual cells. These methods demonstrated that the majority of intracellular IgA occurred in monomeric form, even when the predominant form of secreted IgA was polymeric. Sequential analyses of the IgA secreted by PWM-stimulated normal peripheral blood lymphocytes revealed that the proportion of polymeric IgA increased with the time of culture and that polymers represented the prevalent form of secreted IgA from the fifth day of culture. Although approximately one-half of TPA-stimulated CLL cells bound fluorochrome-labeled SC, only trace amounts of extracellular and intracellular polymeric IgA were detected in both culture supernatants and lysates. Culture supernatants of an IgA-secreting EBV-transformed cell line contained predominantly polymeric IgA. However, intracellular IgA was largely represented by monomers. The predominance of intracellular monomers in polymeric IgA-secreting cells suggested that the pathway of the assembly of human IgA molecules is analogous to that described for mouse IgA synthesis.     

Lymphotoxin production by human lymphocytes. I. Study of the supernatants of phytohemagglutinin stimulated human lymphocyte cultures]

Lymphotoxin was found to be present in supernatants from 22 human lymphocytes cultures stimulated with phytohemagglutinin in a dose of 5 and 10 microgram/ml. The lymphocytes were obtained from the peripheral blood of 6 apparently healthy persons. Lymphotoxin activity was determined by simple and objective method, i.e. by staining the target cells (mouse L-cells) monolayer with crystal violet, with the following determination of optic densities of the L-cells lysates at 570 nm in the spectrophotometer. As revealed, 1 : 5 dilutions of the supernatants from the lymphocyte cultures incubated for 48 hours inhibited the L-cells growth by from 40 to 60%. With further incubation of the cultures (up to 72 and 96 hours) the cytotoxicity of their supernatants for the target cells showed no increase, whereas the blasttransformation index reaches the maximal value by 72nd incubation hour. Supernatants from unstimulated lymphocyte cultures failed to produce any cytotoxic effect on L-cells.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. II. Responsible factors for nonspecific destruction

Cell-free supernatants of thoracic duct lymphocyte cultures which were stimulated in vitro by horse serum on syngeneic fibroblast monolayers are demonstrated to be cytotoxic on syngeneic embryonic fibroblasts by means of a direct cell count using microtest plates. Experimental supernatants showed up to 100% suppression of fibroblast growth at $\text{1}\text{3}$ dilution and up to 96% suppression at $\text{1}\text{4}$ dilution as compared to the control supernatants. Evidence is presented indicating that lymphocytes cultured on mosaic monolayers, which were comprised of syngeneic and xenogeneic fibroblasts, were reacting both to xenogeneic cells and horse serum in the medium at the cellular level. A hapten-to-carrier type relationship is suggested between xenogeneic antigen and horse serum. Absence of horse serum in the test cultures using these lymphocytes resulted in the abrogation of nonspecific toxic activity of lymphocytes while the specific activity, though diminished, remained. This again indicates the difference in the mechanisms underlying the specific and nonspecific target cell destruction by T cells.     

Role of beta 2-adrenoceptors in the biological effect of autoimmune orchitis spleen cells

Autoimmune orchitis induced an increment in beta 2-adrenoceptor populations in intact mouse spleen lymphocytes. Normal and autoimmune lymphocytes incubated with soterenol increased the mechanical response of isolated atria. Autoimmune cells were more effective than normal cells in inducing this response. Soterenol or spleen cells alone did not modify the contractility at the concentration used. Inhibitors of beta 2-adrenoceptors of spleen cells completely blunted the reaction between soterenol and lymphocytes, while when atria were exposed to butoxamine, mechanical activity induced by soterenol plus lymphocytes was not affected. Cell-free supernatants of lymphocytes exposed to soterenol elicited the reaction in the same way as soterenol-treated lymphocytes. Direct contact of cells with the assay organ was not necessary. Inhibitors of cyclooxygenase on lymphocytes blocked the reaction of soterenol-treated lymphocytes on atria, while inhibitors of lipoxygenase(s) completely blocked the reaction of atria exposed to soterenol-stimulated lymphocytes or supernatants. These results suggest that soterenol reacts with beta 2-adrenoceptors of normal and autoimmune cells. From this reaction, soluble factors are released that in turn trigger stimulation of the atrial contractility as a consequence of the release of oxidative products of the lipoxygenase(s) pathway of arachidonic acid from atria. The high activity of atria in the presence of autoimmune spleen cells is probably related to the increment in number of beta 2-adrenoceptors of these cells.     

Anti-tumor antibody produced by human tumor-infiltrating and peripheral blood B lymphocytes

Cell suspensions from 69 human tumor biopsies and malignant effusions depleted of infiltrating T cells were incubated for 10–14 days with mitomycin-C-treated cells of the transformed T cell line MOT as feeder cells. B lymphocytes proliferated and differentiated as indicated by immunoglobulin (Ig) seerction in the culture supernatants (B cell expansion). Ig was present in culture supernatants of tumor cell suspensions incubated without MOT feeder cells (non-expanded cells), but the addition of MOT feeder cells to these cultures invariably resulted in a significant increase in Ig concentration. While IgG, IgA. and IgM isotypes were all detected in supernatants of both expanded- and nonexpanded tumor cell suspensions, the increase in total Ig induced by MOT feeder cells was mainly due to an increase in IgG. Peripheral blood B lymphocytes (PBBL) from 15 cancer patients and 4 healthy individuals were also successfully expanded by the same method. In these it was shown that IgA was the predominant Ig isotype. Using a modified enzyme-linked immunosorbent assay, IgG of 25/36 expansions from tumor cell suspensions showed reactivity with autologous tumor targets, and that from 10/13 expansions reacted with allogeneic tumor targets of the same histological diagnosis. No reactivity was found against tumor targets of different histology. IgG of 4/10 expansions of PBBL from cancer patients showed reactivity with allogeneic tumor targets of the same histology, while no reactivity was demonstrated against tumor targets of different histology. IgG of expanded PBBL from healthy individuals showed no reactivity against tumor targets. This method allows detailed study of the specific humoral antitumor immune response of intratumoral and peripheral blood B lymphocytes in cancer.Work supported by grants from the Share and Concern Foundations and grant CA MOPP from the National Institutes of Health, C.J.A.P. is a visiting scientist from the University of Nijmegen, Department of Medical Oncology, Nijmegen, The Netherlands, and is supported by a Fulbright Senior Research Grant and grants from the Dutch Cancer Society and the Regional Cancer Center of the East Netherlands (IKO). J.A.M.B. is a visiting scientist from the University of Sao Paolo, Department of Immunology, Brazil, and is supported by grant 90/1844-4 from the FAPESP     

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.     

Membrane-mediated killing of Saccharomyces cerevisiae by glycoproteins from Torulopsis glabrata.

Cell-free supernatants from cultures of Torulopsis glabrata contained glycoprotein toxins that killed sensitive and killer strains of Saccharomyces cerevisiae with single-hit kinetics. Growing S. cerevisiae treated with the toxins showed a leakage of cellular potassium, partial dissipation of the adenosine triphosphate pool, and a coordinate shutdown of macromolecular synthesis. These pool efflux-stimulating toxins have been partially purified and at least three toxic glycoproteins have been separated. Pool efflux-stimulating toxin activity was stable from pH 3 through 7, though killing was maximal close to pH 4.     

Transforming growth factor-beta inhibits the production of IgG, IgM, and IgA in human lymphocyte cultures.

Transforming growth factor beta (TGF beta) has potent immunoregulatory effects acting on both T and B cells. It strongly inhibits secretion of IgG and IgM in human and murine B cell cultures, but has been shown to have an enhancing effect on IgA production in the mouse. We have studied the effect of TGF beta on the production of IgA in human lymphocyte cultures. The addition of TGF beta to pokeweed-stimulated peripheral blood lymphocytes resulted in a suppression of IgA production of both subclasses, similar in magnitude to the suppression of IgG and IgM production. Membrane IgA expression was not increased by culturing tonsillar lymphocytes with TGF beta. In conclusion, we find no evidence for a selective enhancing effect of TGF beta on IgA synthesis in humans, in contrast to the findings reported in mice.     

Functional and biochemical disposition of 7,12-dimethylbenz[a]anthracene in murine lymphoid cells

The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.     

Enteric immunization reveals a T cell network for IgA responses and suggests that humans possess a common mucosal immune system

The local IgA response is a result of two related events, the induction of sensitized T and B cells in gut- or b ronchial-associated lymphoreticular tissues (GALT or BALT) and the final differentiation of IgA plasma cells in mucosal tissues where IgA is produced and transported to become secretory IgA (S-IgA) antibodies into external secretions. Oral administration of various types of antigens/vaccines may result in two types of response, i.e., S-IgA antibodies at mucosa and systemic unresponsiveness to antigen, a state termed oral tolerance. Regulatory T cells in GALT help account for both S-IgA responses and oral tolerance and thus serve to fine tune responses to orally encountered antigens. Studies in animal models and humans have shown that oral administration of antigens sensitize lymphoid cells in GALT which subsequently home to mucosa and result in S-IgA responses in several external secretions. The significant promise of oral vaccines for prevention of microbial diseases including neisserial diseases is discussed.     

Inhibition of interleukin 2 production by factors released from tumor cells

In previous studies, cultured melanoma cells were shown to have a suppressive influence on the induction of cytotoxic T cells. Our investigation of the mechanism of these effects revealed that supernatants from certain cultures of melanoma cells contained inhibitory activity against the production of interleukin 2 (IL 2) from phytohemagglutinin (PHA)-stimulated cultures of lymphocytes. These supernatants did not inhibit interleukin 1 production, and also did not inhibit the mitogenic activity of performed IL 2 on IL 2-dependent target cells. Production of the inhibitory activity could be reduced by inhibitors of protein synthesis, but this activity was not inhibited by digestion with the proteolytic enzymes trypsin or pronase. Gel filtration analysis of tumor supernatants revealed that the majority of the inhibitory activity was detected in fractions of approximately 44 and 7 Kd. The addition of supernatants with inhibitory activity to PHA-stimulated cultures of lymphocytes was associated with reduced transition of cells from G1 to S phase of cell division, which could be reversed by the addition of IL 2. Preliminary studies suggest that the release of the factor(s) from melanoma cells may be related to rapid progression of tumor growth in patients, and therefore may be of prognostic significance in tumor host relationships.     

The development of an immunoenzyme method for determining human secretory IgA

The results of the work on the development of an enzyme immunoassay (EIA) system for the determination of secretory IgA (S-IgA) are presented. A first, S-IgA was isolated from human colostrum and used as the basis for obtaining biologically active immunosorbent; then antibodies to S-IgA were isolated and the specific conjugate was obtained. The determination of S-IgA was carried out by the method of sandwich EIA. The newly developed EIA system permitted the determination of S-IgA only, giving no positive reactions with serum immunoglobulins. The data thus obtained make it possible to regard this assay system as specific, sensitive and suitable for further trials.     

Relationship between canine mucosal and serum immunoglobulin A (IgA) concentrations: serum IgA does not assess duodenal secretory IgA

A double-sandwich enzyme immunoassay method was developed for determination of serum immunoglobulin A (S-IgA) and mucosal secretory immunoglobulin A (sIgA) in duodenal brush samples obtained via endoscopy and the relationship between enteric mucosal sIgA, salivary sIgA and S-IgA in dogs was examined. Twenty healthy dogs underwent routine endoscopy. A brush sample from the duodenal mucosa was obtained and washed in PBS, with a serum sample being taken concurrently. A saliva sample was collected from twelve of these dogs. S-IgA and sIgA with total protein concentrations in the duodenal washings and saliva samples were determined. A significant negative correlation (r = -0.64, P = 0.0059) was found between duodenal sIgA/protein ratios and S-IgA concentrations. Saliva sIgA/protein ratios did not correlate with sIgA/protein ratios of duodenal samples. The method described here allows for direct assessment of duodenal IgA; therefore indirect measures based on serum IgA or salivary IgA can be avoided. In addition, these indirect measures appear to be poor indicators of duodenal sIgA competence in dogs.