In vitro synthesis of immunoglobulins, secretory component, complement and lysozyme by human gastrointestinal tissues. I. Normal tissues.

An in vitro culture technique has been used to demonstrate synthesis of proteins by human gastrointestinal tissues cultured in vitro. Histologically normal tissues were obtained endoscopically and surgically. IgA and secretory component (SC) were produced in all sites, but the relative intensity of IgA synthesis and SC synthesis varied. In stomach and small intestine the intensity of IgA synthesis was greater than that of SC, but in large bowel mucosa, there appeared to be an excess of SC synthesis. Synthesis of IgG and IgM was also found in all sites. Complement proteins were produced by some of the intestinal biopsies, and by parotid gland. Lysozyme was synthesized by parotid gland and by gastric mucosa, and to a lesser extent in small intestine, and rarely in large intestine. The results suggest that in addition to the local mucosal IgA system the local production of other immunoglobulins, as well as non-immunoglobulin humoral defence factors, may be important host defences of the normal gastrointestinal tract.     

T lymphocytes of the human colonic mucosa: functional and phenotypic analysis.

Normal human colonic lymphocyte populations were isolated for both phenotypic analysis by double-label immunofluorescence and assessment for regulatory effects on Ig production by co-culture with responder cells from colonic mucosa and peripheral blood. Mean CD4:CD8 ratios for colonic intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) were comparable to values obtained from tissue sections. IEL alone did not produce Ig in vitro and were without effect on Ig production when co-cultured with LPL. However, T-enriched LPL had a marked helper effect for T-depleted LPL. Maximal help was for IgA production, increasing with numbers of T-enriched cells. In colonic LPL T-depleted and T-enriched co-cultures, pokeweed mitogen (PWM) had no significant effect. By contrast, in co-cultures of T-enriched and T-depleted peripheral blood mononuclear cells, Ig production was PWM-dependent. In all experiments with colonic mucosal responder cells, IgG production was low. The effects of unfractionated colonic biopsy lymphocytes on T-depleted peripheral blood mononuclear cells were additive for IgM production and synergistic for IgA synthesis, although almost no IgG was produced. Moreover, PWM had helper effects for IgM, but was suppressive for IgA production. These data suggest that colonic mucosal regulatory cells reside in the lamina propria, and predominantly provide help for IgA and IgM synthesis. The data further suggest the existence of a pre-stimulated IgA-specific T helper cell population.     

Polyclonal B cell activation in alcoholic patients with no evidence of liver dysfunction.

Unstimulated and pokeweed mitogen (PWM) stimulated immunoglobulin (Ig) synthesis by peripheral blood mononuclear cells (PBMC) in vitro and plasma Ig concentrations were measured in three groups of alcoholic patients: (i) with clinical or biochemical signs of liver disease, (ii) with evidence of an alcohol related disease but no overt signs of liver damage and (iii) with no evidence of any alcohol related disease. The concentrations of IgG and IgA were significantly raised in the supernatants of unstimulated cultures of PBMC from the patients, while the stimulation of Ig synthesis by PWM, measured as a stimulation index, was significantly reduced. The ratio of the concentration of IgG to IgA was reduced in the unstimulated cultures of PBMC from the alcoholics, indicating a greater relative increase in IgA synthesis compared to IgG synthesis. Comparing the alcoholics to the controls, it was found that the concentration of IgA in the plasma of the alcoholics was increased, but that the concentration of IgG was not altered. Comparing the different groups of patients, it was found that the concentration of IgG in the plasma was higher in the alcoholics with evidence of liver damage compared to alcoholics with alcohol related disease but no evidence of liver damage, and that the concentration of IgA in the plasma was higher in alcoholics with liver damage than those without. Otherwise there were no differences between the alcoholics with respect to the synthesis of IgG or IgA or the plasma Ig concentrations. These results indicate that IgG synthesis by PBMC in vitro, and serum Ig concentration in vivo, are abnormal in all alcoholics, not just those with overt clinical or biochemical signs of liver damage.     

Nerve growth factor inhibits immunoglobulin production by but not proliferation of human plasma cell lines

The effects of nerve growth factor (NGF) on human plasma cells were studied. NGF inhibited immunoglobulin (Ig) production but not thymidine uptake by human plasma cell lines IM-9 and AF-10 in a dose-dependent fashion. This NGF-induced inhibition of Ig production was specific, since inhibition was blocked by anti-NGF serum but not by control serum. Interleukin (IL)-6 did not affect Ig production by IM-9 and AF-10; however, IL-6 restored NGF-induced inhibition of Ig production. NGF also inhibited Ig production (IgG, IgM, and IgA) without affecting thymidine uptake by PCA-1+ plasma cells generated in vitro. This inhibition was also blocked by anti-NGF serum but not by control serum and was restored by IL-6. These results suggest that NGF may interact with IL-6 in control of Ig production by plasma cells.     

Acute autoimmune response in a case of 3yromellitic acid dianhydride-induced hemorrhagic alveolitis

A 17-year-old man was occupationally exposed to pyromellitic acid dianhydride dust during the production of epoxy resin in a chemical factory. He was clinically diagnosed as having acute hemorrhagic alveolitis associated with anemia. The serologic analysis revealed a high concentration of IgG antibodies against pyromellitic acid dianhydride-treated human serum albumin (PMDA-HSA). Immunoblotting with PMDA-treated human serum as antigen and the patient's serum as the first antibody showed that additional PMDA-modified serum proteins other than HSA were recognized by the patient's IgG antibodies in the higher mol. mass range (>67 kDa). No specific IgG could be detected against other anhydride conjugates (maleic acid, MA; phthalic acid, PA) with the exception of a reaction with the trimellitic acid anhydride-conjugated HSA (TMA-HSA). No specific IgE antibodies could be detected against any of the above mentioned antigens, but immunoblotting of the patient's serum indicated IgG 4-type autoantibodies against in vitro PMDA-treated Ig molecules of normal serum proteins.     

Regulation of ongoing IgE synthesis by human T-cell supernatants derived from atopic and nonatopic donors

Supernatants derived from T cells of donors with and without atopic disease were assessed to determine whether they could regulate ongoing IgE, IgG, or IgA synthesis of three different human B-cell lines. The results were compared with the ability of T-cell supernatants from the atopic donors to specifically induce IgE synthesis of nonatopic peripheral blood B lymphocytes (PBL-B cells). We found that, when tested on the B-cell lines, the T-cell supernatants from atopic donors doubled the IgE as well as the IgG and IgA synthesis. Although the T-cell supernatants from nonatopic donors had no effect on Ig synthesis of PBL-B cells or on the IgG of the IgG-secreting cell line, the supernatants doubled the IgE and moderately enhanced IgA synthesis of the IgE- and IgG-secreting cell lines. These results demonstrate that T-cell supernatants derived from atopic and nonatopic donors differ in their regulatory effects, not only on PBL-B cells, as had been shown previously by others, but also in their effects on ongoing Ig production.     

In vitro production of immunoglobulins of various classes and subclasses by cord blood B cells in African neonates: modeling and assessment of determination

Cord blood B cells obtained from neonates of healthy Senegalese mothers were assayed in vitro for their capacity to fully differentiate and secrete immunoglobulins (Ig) of various classes and subclasses. Stimulation of mononuclear cells with SAC particles or anti-micro antibodies in the presence of IL-4, or with IL-2 and IL-10 induced a strong production of IgG, provided that an additional CD40/CD40L signal was present, in contrast to adult cell cultures. Cord blood mononuclear cells differentially stimulated with various cytokines in order to lead to Ig heavy chain switching and production of the various classes/subclasses consistently produced IgG1, IgG3, IgG4, IgE and IgA. This system has been applied to immune cells from African neonates that have not been extensively studied previously. Estimation of Ig production as OD ratios could be applied to cultures where cord blood B cells are stimulated with defined antigens of human pathogens to which the fetus immune system was primed in utero.     

Suppressor Factors Generated from Human Mononuclear Cells by Means of Purified Myeloma Proteins

Peripheral blood mononuclear cells (PBMC) from normal human donors were cultured in Marbrook flasks in the presence of purified IgG or IgA myeloma proteins. The culture supernatants were tested for their ability to suppress pokeweed mitogen (PWM)- or Epstein-Barr virus (EBV)-driven Ig synthesis by normal PBMC. Two supernatants from PBMC cultured with IgG and one from PBMC cultured with IgA were tested and suppressed PWM-driven Ig synthesis as measured by a reverse haemolytic plaque assay and by quantitation of the Ig secreted into the culture medium of the PWM-driven cells. This suppression was not restricted to the Ig isotype of the 'inducing' myeloma protein, but was extended to IgG, IgA, and IgM. The suppressive effect could be absorbed out with human IgG.     

In Vitro Production of Monoclonal and Polyclonal Immunoglobulins by Peripheral Blood Mononuclear Cells in Human Plasma Cell Myeloma

The in vitro monoclonal and polyclonal immunoglobulin (Ig) production of peripheral blood mononuclcar cells was studied in human multiple myeloma (four IgG myelomas, one IgA myeloma) and in one patient with benign monoclonal gammopathy. Using an enzyme-linked immunosorbent assay with anti-class-specific antisera and antisera against idiotypic structures on the myeloma protein, it was possible to quantilate separately monoclonal and polyctonal Ig of the same class in cell culture supernatants. After stimulation with pokcweed mitogen (PWM) patients' cells produced lower amounts of polyclonal Ig than cells from healthy adults. In contrast, production of monoclonal Ig could not be enhanced by PWM. Moreover, the kinetics of monoclonal Ig production was different from that of polyclonal Ig. Myeloma cells contained large amounts of monoclonal Ig while their content of polyclonal Ig was low. A rapid release of preformed monoclonal Ig during the first day of culture was not inhibited by puromycin. A later phase of release was partly suppressed by puromycin and was probably caused by active protein synthesis.     

Decreased suppressor T cell activity in patients with hepatic cirrhosis (HC).

Hypergammaglobulinaemia (HGG) is frequently found in patients with hepatic cirrhosis (HC). Using an assay system of in vitro PWM-stimulated immunoglobulin (Ig) production, the amounts of IgG, IgA, and IgM produced by peripheral blood lymphocytes (PBL) from 15 HBs Ag-negative patients with HC and from 16 age-matched healthy subjects were quantitated by radioimmunoassay. We found that PBL from patients with HC produced significantly greater amounts of IgG (P less than 0.05) but not IgA or IgM than did those from control subjects. This increased IgG production by PBL from patients with HC was attributed to enhanced T helper activity and not to enhanced B cell function. We also searched for defects in naturally occurring suppressor T cell activity which is sensitive to irradiation. Irradiation-induced enhancement for IgG production was significantly lower in patients with HC compared with age-matched control subjects (P less than 0.01). Similarly, we examined the effect of Con A-induced suppressor T cells on the in vitro PWM-stimulated IgG production by allogeneic PBL and observed the decrease of Con A-induced suppressor T cell activity in patients with HC (P = 0.01). We conclude, therefore, that the increased serum levels of Ig, particularly IgG in patients with HC may result from in part on the basis of depressed ability of naturally occurring suppressor T cells or Con A-induced suppressor T cells to suppress Ig production.     

In vitro immunoglobulin secretion by normal human gastrointestinal mucosal tissues, and alterations in patients with inflammatory bowel disease.

Intestinal mucosal immunoglobulin secretion in vitro has been studied by culture of endoscopic biopsy tissues obtained from various sites along the gastrointestinal tract. IgA and IgM were the predominant immunoglobulins produced by intestinal tissues distal to the stomach and secretion of both reached a maximum in the small intestine of normal individuals. In patients with inflammatory bowel disease, characteristic alterations in immunoglobulin production were observed in cultures of large intestinal tissue. In ulcerative colitis (UC), significant reductions in IgA secretion (P less than 0.03) occurred in the rectum during remission and IgM in the colon in active disease (P less than 0.01). However, in active Crohn's disease (CD), rectal IgM secretion was enhanced (P less than 0.004) and IgA diminished (P less than 0.01). IgG secretion was increased throughout the colon in UC especially in distal sites, due largely to significant increases in pre-formed immunoglobulin (P less than 0.02). Similar total increases were observed in colonic tissues from patients with CD, although IgG synthesis in biopsies from rectal sites was normal. These findings suggest that specific abnormalities of intestinal mucosal immunoglobulin synthesis occur in patients with both UC and CD.     

Comparison of cervicovaginal humoral immunity in clinically asymptomatic (CDC Al and A2 category) patients with HIV-1 and HIV-2 infection

Paired sera and cervicovaginal secretions (CVS) from 11 HIV-1- and 11 HIV-2-infected women, all clinically asymptomatic (CDC A1 and A2 categories), were analyzed for total IgG, IgA, albumin (HSA), IgG, and IgA antibodies toenvencoded surface glycoproteins of HIV-1 (gp160) and of HIV-2 (gp105), by comparison to 15 age-matched healthy controls. Secretion rates of IgG and IgA into CVS were evaluated by calculation of their relative coefficients of excretion (RCE) by reference to HSA. Cervicovaginal production of anti-HIV antibodies was evaluated by comparison between specific antibody activities of IgG and of IgA to HIV in CVS and in sera. In HIV-1-infected women, total IgG and IgA in CVS were, respectively, 6- and 4-fold increased, whereas the secretion rate of total IgG was 2.1-fold increased and that of total IgA was 2.5-fold reduced. In contrast, total IgG and IgA as well as their secretion rates were normal in HIV-2-infected women. In HIV-1- but not in HIV-2-infected women, HSA levels in cervicovaginal washings were twofold increased, demonstrating alteration of the mucosal barrier in HIV-1 infection. In HIV-1-infected patients, IgG and IgA to gpl60 were detected in all sera and CVS. In HIV-2-infected patients, IgG to gp105 was detected in all sera and CVS, whereas IgA to gp105 could be detected in only half of sera and one-third of CVS. Cross-reactivity by IgG and/or IgA to HIV-1 or HIV-2 against the surface glycoprotein of the other HIV type was observed in sera as well as in CVS, and more frequently in HIV-2- than in HIV-1-infected women. Finally, the mean specific activities of IgG and of IgA to gp160 or gp105 were higher in CVS than in sera, evidencing a possible local synthesis of both isotypes in HIV-1 as well as in HIV-2 infections. As early as the asymptomatic stages, HIV-1 affects the cervicovaginal mucosa more than HIV-2 does, suggesting higher viral replication within the female genital tract in HIV-1 infection than in HIV-2 infection.     

Immunoglobulins of rnu/rnu ''nude'' rats with congenital aplasia of the thymus

The levels of immunoglobulins (Ig) in the serum and saliva of nude (rnu/rnu) congenitally athymic rats and control (rnu/+) thymic heterozygous rats have been studied. Serum IgM, and serum and salivary IgA levels were virtually identical between rnu/rnu and rnu/+ rats. Serum IgG was drastically reduced in nude rats during the period when endogenous IgG synthesis was predominant. The most consistent and pronounced differences in Ig levels between rnu/rnu and rnu/+ rats involved the IgG isotype.     

Murine Intestinal Humoral Responses in Chronic Schistosoma mansoni Infections

Specific and non-specific production of immunoglobulins (Ig) by the intestinal mucosa was examined in mice infected with the human blood fluke Schistosoma mansoni. Ileal and colonic mucosal tissue samples were cultured for 2 days, the medium replaced and the culture continued for a further 2 days. Ig concentrations and specific antibodies to soluble schistosome egg antigens in culture supernatants were estimated by isotype-specific ELISA. Cultured mucosae from control mice produced little IgG, but significant amounts of IgA and IgM on prolonged culture. IgG concentrations were increased in infected animals, mainly in the initial culture period, indicative of systemic, rather than local origins. By contrast, significantly increased local production of IgA and IgM occurred after the start of egg deposition in the intestinal mucosae. Although specific anti-egg antibodies of the IgG and IgM class were detected, none of the local IgA response was specific for schistosome eggs. We conclude that specific intestinal immune responses to schistosome eggs reflect systemic responses, whereas locally increased IgA production is largely non-specific. This pattern of response is likely to be related to the prior systemic exposure to schistosome eggs, which results in polyclonal local B-cell activation, but fails to trigger an antigen-specific IgA mucosal response.     

Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection

The immunoglobulin isotype-specific responses in serum and at the respiratory mucosa of pigs after a primary infection with influenza virus were studied. To do this, we developed an aerosol challenge model for influenza in specified pathogen-free (SPF) pigs and isotype-specific enzyme-linked immunosorbent assays (ELISAs). Ten-week-old pigs were inoculated without anesthesia in the nostrils with an aerosol of the field isolate influenza A/swine/Neth/St. Oedenrode/96 (H3N2). The infection caused acute respiratory disease that closely resembled the disease observed in some outbreaks of influenza among finishing pigs, which were not complicated by bacterial infections. Pigs showed clinical signs characterized by fever, dyspnea, and anorexia. At necropsy on postinfection days 1 and 2, an exudative endobronchitis was observed throughout the lung. Viral antigen was present in the epithelial cells of the bronchi and bronchioli and virus was isolated from bronchioalveolar and nasal lavage fluids and from pharyngeal swabs until 5 days after infection. With the isotype-specific ELISAs, viral nucleoprotein specific immunoglobulin (Ig) M, IgG1, and IgA antibody responses were measured in serum and bronchioalveolar and nasal lavage fluids. To determine whether the antibodies were produced and secreted at the respiratory mucosa or were serum-derived, the specific activity (ie, the ratio of antibody titer to Ig concentration) was calculated for each isotype. The IgA and interestingly also a substantial part of the IgG1 antibody response in pigs upon infection with influenza virus was shown to be a mucosal response. Local production of specific IgA in the nasal mucosa, and of specific IgA and IgG1 in the lung was demonstrated. These results indicate that protective efficacy of vaccination can be improved by an immunization procedure that preferentially stimulates a mucosal immune response. The aerosol challenge model in SPF pigs and the isotype-specific ELISAs that we developed can be useful for evaluating various strategies to improve efficacy of porcine influenza vaccines and to study the immune mechanisms underlying the observed protection.     

Topical application of human-derived Ig isotypes for the control of acute respiratory infection evaluated in a human CD89-expressing mouse model

Recurrent and persistent airway infections remain prevalent in patients with primary immunodeficiency (PID), despite restoration of serum immunoglobulin levels by intravenous or subcutaneous plasma-derived IgG. We investigated the effectiveness of different human Ig isotype preparations to protect mice against influenza when delivered directly to the respiratory mucosa. Four polyvalent Ig preparations from pooled plasma were compared: IgG, monomeric IgA (mIgA), polymeric IgA-containing IgM (IgAM) and IgAM associated with the secretory component (SIgAM). To evaluate these preparations, a transgenic mouse expressing human FcαRI/CD89 within the myeloid lineage was created. CD89 was expressed on all myeloid cells in the lung and blood except eosinophils, reflecting human CD89 expression. Intranasal administration of IgA-containing preparations was less effective than IgG in reducing pulmonary viral titres after infection of mice with A/California/7/09 (Cal7) or the antigenically distant A/Puerto Rico/8/34 (PR8) viruses. However, IgA reduced weight loss and inflammatory mediator expression. Both IgG and IgA protected mice from a lethal dose of PR8 virus and for mIgA, this effect was partially CD89 dependent. Our data support the beneficial effect of topically applied Ig purified from pooled human plasma for controlling circulating and non-circulating influenza virus infections. This may be important for reducing morbidity in PID patients.     

In vitro immunoglobulin production by mononuclear cells in rheumatoid arthritis

Since patients with rheumatoid arthritis (RA) exhibit serum hypergammaglobulinemia and autoantibody (rheumatoid factor) production, we compared elaboration and control of in vitro RA mononuclear cell (MNC), Ig assayed by enzyme-linked immunoassays or by hemolytic plaque formation, in 37 RA patients and 17 normal subjects. We found (1) RA spontaneous plaque-forming cells were significantly reduced (RA 344 vs normal 627 PFC/10(6) MNC, P less than 0.002); (2) RA spontaneous IgG and IgM (but not IgA) elaboration was significantly diminished (IgG RA 339, normal 776; IgM RA 255, normal 869 ng/ml, P less than 0.001; IgA RA 87, normal 124); (3) RA stimulated IgG and IgM production (but not IgA) was also decreased (IgG RA 2434, normal 3862, P less than 0.06; IgM RA, 1676, normal 3323, P less than 0.005; IgA RA 1859, normal 2315); (4) reduced RA Ig elaboration was not clearly due to altered numbers of T or non-T cells, age, medications, clinical features of disease, or response kinetics; (5) relative improvement of RA in vitro IgG, but not usually IgM, secretion followed removal of adherent cells, addition of indomethacin or addition of mitomycin C-treated T cells; (6) MNC from synovial fluids, but not bone marrows, exhibited spontaneous Ig production in excess of stimulated synovial fluid cellular or peripheral blood Ig elaboration. These observations indicate selective impairment of peripheral blood MNC IgG and, particularly, IgM secretion in RA. This defect appears to reflect accessory cell influences which differ from normal as well as the sequestration of primed or activated cells in the synovial fluid.     

Cleavage of immunoglobulin A1, A2 and G by proteases from clinical isolates of Pasteurella multocida.

Several Pasteurella multocida strains were examined for their ability to produce extracellular enzymes that cleave immunoglobulin A and G (Ig A and Ig G) molecules. Two strains isolated from human pulmonary and genital infections produced proteases that cleaved human IgA and IgG, colostral IgA and human myeloma IgA1 and IgA2. Human IgM was not degraded by these enzymes. Examination of cleavage digests showed two main fragments with different electrophoretic mobilities. The two P. multocida strains produced a protease that cleaved IgA and IgG heavy chains outside the hinge region, and differed in this respect from the hinge-cutting proteases of other bacteria. Protease production may be a virulence mechanism for P. multocida strains.     

T cell dependent differentiation of human B cells: direct switch from IgM to IgE, and sequential switch from IgM via IgG to IgA production.

Ig production by splenic human B cells that express different surface Ig isotypes were analysed in limiting dilution cultures. Therefore, FACS sorted IgM+, IgG+ and IgA1+ B cells were stimulated with PMA-activated EL4 thymoma cells as helper cells in the presence of IL-2 and IL-4. We found that at least every second B cell responded in vitro and secreted the antibody corresponding to its surface Ig isotype. IgE secreting cells developed from surface IgM+ D+ cells (1/31 to 1/167), but not from IgG+ or IgA1+ cells (much less than 1/5000). Negative signalling of the IgM+ B cells by addition of anti-IgM antibodies into the cultures reduced the number of single IgM producing cells by greater than 85%, and completely inhibited IgE switch. In contrast, anti-IgG and anti-IgA antibodies did not reduce the IgE response. The results indicate a direct switch from IgM to IgE secretion in vitro. In contrast to IgE, IgA secreting cells developed from IgM+D+ (1/30 to 1/51) and from IgG+ B cells (1/14 to 1/25). Negative signalling of the IgG+ B cell subset within total B cells by anti-IgG antibodies suppressed the development of IgG as well as IgA producing cells, but did not inhibit IgM and IgE responses. This indicates a sequential switch from IgM via IgG to IgA. Taken together, this study indicates that IgE secreting cells are derived directly from IgM+D+ B cells by non-sequential switching, whereas IgA producing cells preferentially develop by sequential switching via IgG+ B cells.