In vitro production of IgE by human peripheral blood mononuclear cells. IV. Modulation by allergen of the spontaneous IgE antibody biosynthesis.

Peripheral blood lymphocytes (PBL) from a proportion of grass-sensitive patients, studied during or immediately after the grass pollination period, showed spontaneous production in vitro of grass-specific IgE antibody, whereas PBL from atopic patients sensitive to allergens other than grass pollens or non-atopic individuals did not. Pre-incubation of IgE antibody producing PBL from grass-sensitive patients with minute amounts of a mixed grass pollen (MGP) extract or Rye grass antigen Group I (Rye I) usually resulted in a reduction of the spontaneous production in vitro of IgE protein and in a marked inhibition of the spontaneous production in vitro of grass-specific IgE antibody. This antigen-specific inhibition was not mediated by T lymphocytes, but it was apparently due to a signal directly delivered by antigen to the spontaneously IgE antibody producing cells. The results support the concept that the activity of cells responsible for the persistent IgE antibody formation in vitro in atopic patients can be modulated by antigen.     

Effect of in vitro irradiation and cell cycle-inhibitory drugs on the spontaneous human IgE synthesis in vitro

The in vitro effects of radiation, diterpine forskolin (FK), and hydrocortisone (HC) on the in vitro spontaneous IgE synthesis by peripheral blood B-lymphocytes from atopic patients were investigated. Without affecting cell viability, in vitro irradiation inhibited in a dose-dependent fashion de novo IgE synthesis in vitro by B cells from all patients examined with a mean 40% reduction of in vitro IgE product after treatment with 100 rads. In contrast, the in vitro IgE production by the U266 myeloma cell line was unaffected, even by irradiation with 1600 rads. The addition to B cell cultures from atopic patients of FK consistently resulted in a dose-dependent inhibition of the spontaneous IgE production in vitro. The addition to cultures of 10(-5) and 10(-6) molar concentrations of HC was also usually inhibitory, whereas lower HC concentrations were uneffective or even enhanced the spontaneous in vitro IgE synthesis. When 10(-6) molar concentrations of both HC and FK were combined in culture, a summation inhibitory effect on the spontaneous IgE synthesis was observed. In contrast, neither FK nor HC had inhibitory effect on the in vitro spontaneous IgE synthesis by the U266 myeloma cell line. The spontaneous in vitro IgE synthesis by B cells from patients with Hodgkin's disease, demonstrating high levels of serum IgE, was strongly reduced or virtually abolished after patients underwent total nodal irradiation to prevent the spread of the disease. In addition, the in vitro spontaneous IgE synthesis by B cells from atopic patients was markedly decreased or abolished by in vivo administration of betamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-stimulation of T cells via CD28 inhibits human IgE production; reversal by pertussis toxin.

In lymphocyte cultures, IgE production was achieved by stimulating T cells with anti-CD2 and IL-2. Here we show that anti-CD28, in the presence or absence of IL-2, reduces this IgE production approximately 10-fold. This inhibition of IgE production was almost completely reversed by Pertussis toxin (PT). PT had no effect on IgE production when the cells were stimulated in the absence of anti-CD28. No major effects of PT were found on IgM production. PT had no effect on purified B cells, stimulated with IL-4 and anti-CD40. In the presence of saturating amounts of rIL-4 similar results were obtained, albeit the absolute amounts of IgE produced were higher in all situations. Furthermore, PT-induced IgE production was still dependent on IL-4, as was evident from experiments in which anti-IL-4 was added to the culture. The IgE enhancing effect was dependent on the adenosine diphosphate (ADP)-ribosyltransferase activity of PT, because a mutant molecule lacking this activity was not able to restore anti-CD28-induced inhibition of IgE synthesis. Thus, we show that co-stimulation with anti-CD28 causes an inhibition of T cell-dependent IgE production by B cells, which inhibition can be specifically overcome by PT. An analysis of the molecular pathways underlying this phenomenon may contribute to our understanding of the regulation of IgE synthesis in (patho)physiological conditions.     

In vitro production of IgE by human peripheral blood mononuclear cells. II. Cells involved in the spontaneous IgE production in atopic patients

Spontaneous IgE production in vitro was investigated in 7-day cultures of unfractionated mononuclear cells (MNC) and MNC subpopulations from atopic patients. Depletion of either phagocytic or adherent cells decreased the amount of IgE detectable in 7-day culture supernatants, but this decrease was due, at least in part, to a loss of cytophilic IgE. Depletion of immunoglobulin-bearing cells (SIg⁺) reduced significantly but did not abolish the spontaneous IgE production in vitro. On the other hand, depletion of IgM-bearing lymphocytes (SIgM⁺), which virtually abolished the production of immunoglobulins of the IgM class, did not change significantly the spontaneous production of IgE. Similarly, no change in the spontaneous production of IgE was found when lymphocyte suspensions were depleted of complement receptor-bearing cells (CR⁺). In contrast, spontaneous IgE production was significantly increased by depletion of T lymphocytes and this increase did not simply reflect the enrichment for IgE-producing cells caused by the fractionation procedure. No significant change in the spontaneous IgE production was found when small numbers of autologous T lymphocytes were added to B cell fractions, whereas the addition of higher concentrations of autologous T cells induced a marked inhibition of the spontaneous IgE production. On the other hand, the addition in culture of pokeweed mitogen (PWM) resulted in a marked reduction of the spontaneous IgE production by B cells, also in the presence of small concentrations of autologous T lymphocytes. Normal T cells were consistently effective in inducing a partial inhibition of the spontaneous IgE production by B cells from atopic patients, whereas T cells from a noticeable proportion of atopic patients were not. These data suggest that MNC responsible for the spontaneous IgE production in atopic subjects are SIgM- and CR-deficient well-differentiated lymphocytes which probably represent the result of an activation which has occurred in vivo. However, this spontaneous IgE production can still be influenced by in vitro manipulation, such as variations in T–B cell ratios or addition of PWM. The results here reported also indicate that normal T cells are generally more effective than T cells from atopic patients in regulating the activity of spontaneous IgE-producing cells present in the blood of atopic subjects.     

Modulation of cell-bound and soluble CD23, spontaneous and ongoing IgE synthesis of human peripheral blood mononuclear cells by soluble IL-4 receptors and the partial antagonistic IL-4 mutant protein IL-4 (Y124D).

We studied the influence of human recombinant soluble interleukin-4 receptors (sIL-4R) and a partial antagonistic mutant IL-4 protein, IL-4(Y124D), on the in vitro CD23 expression, soluble (s)CD23 release and IgE synthesis of human peripheral blood mononuclear cells (PBMC). The data show that sIL-4R suppressed the IL-4-induced IgE synthesis of PBMC. sIL-4R fusion protein stabilized with human Fc gamma fragments showed a more pronounced effect than unconjugated sIL-4R. IL-4(Y124D) also suppressed the IL-4-induced IgE synthesis. The IL-4-induced antigen CD23 and its soluble fragments were suppressed by sIL-4R and IL-4(Y124D). PBMC of atopic donors with a spontaneous in vitro IgE synthesis showed a partial suppression of the IgE production after sIL-4R or IL-4(Y124D) application. The IgE synthesis and sCD23 release of normal donor PBMC were suppressed when the substances were applied 0-4 days after IL-4 treatment. After 4 days of IL-4 stimulation, sIL-4R and IL-4(Y124D) enhanced the IgE synthesis. These data demonstrate that sIL-4R and IL-4(Y124D) are suppressive for the primary IgE synthesis induced by IL-4. In contrast, the ongoing IgE synthesis was only partially modulated by sIL-4R and IL-4(Y124D), and in some conditions even an enhancement of the IgE production was observed. These data suggest a differential function for IL-4 in the early and late phase of PBMC IgE production.     

Role of Serine Proteases of Schistosoma mansoni in the Regulation of IgE Synthesis

The regulation of the IgE response by schistosomula-released products (SRP) was studied either in vitro with rat and human cell cultures or in vivo by injection into rats of SRP with an unrelated allergen at primary or secondary immunization. The results obtained in vitro showed that non-dialysable factors present in SRP potentiate the IgE synthesis by rat and human cells. This enhancing effect was supported by molecules with serine protease activities. On the other hand, the inhibition or depletion of SRP in serine proteases induced a weak synthesis of IgM by rat cells in vitro. The injection of SRP into rats on day 0 with an unrelated allergen led to a potentiation of total IgE production, but an inhibition of specific IgE response. In contrast, a marked elevation of specific IgE response was obtained when SRP was injected upon secondary immunization. Serine proteases of SRP were partly responsible for this potentiative effect.     

IL-4-producing murine T helper cell line provides help for in vitro production of IgE.

Studies of the regulation of the IgE synthesis have been hampered by the difficulties in making lymphocytes produce IgE in vitro. Synthesis of IgE, IgG1 and IgM were investigated in a murine coculture system in which a conalbumin-specific T helper cell line of Th2 type was cultured together with nonadherent splenocytes from normal MHC-matched mice. Help provided by the antigen-stimulated T cell line induced significant IgE production (20 ng/ml), along with IgG1 (5 micrograms/ml) and IgM (250 micrograms/ml). Immunoglobulin synthesis in cultures was detectable at day 3-4 and culminated at day 7-8. IL-4 production was observed within the first 2 days of culture. The kinetics of the synthesis of the three isotypes were parallel. Stimulation of the Th2 cell line with Con A produced dose-response curves resembling those after antigen stimulation, whereas supernatant from these cells was unable to induce synthesis of IgE. The presence of interferon-gamma in the cultures, inhibited synthesis of IgE, IgM and IgG1, but this inhibition was not due to interference with the IL-4 production, which increased in the presence of high doses of interferon-gamma.     

Disodium cromoglycate (DSCG) selectively inhibits IgE production and enhances IgG4 production by human B cell in vitro.

The effect of DSCG on human IgE production in vitro was studied. DSCG selectively inhibited interleukin-4 (IL-4) induced IgE production by mononuclear cells (MNC) from normal donors without affecting IgM, IgA, IgG1, IgG2 or IgG3 production. In contrast, DSCG enhanced IgG4 production. To achieve this effect, DSCG must be added to the culture at the initiation and be present throughout the entire culture period. Interferon-gamma (IFN-gamma) also inhibited IL-4-induced IgE production, but IgG4 production was not affected by IFN-gamma. Monoclonal anti-IFN-gamma antibody blocked the inhibition of IgE production by IFN-gamma, but did not block the inhibition of IgE production by DSCG. DSCG also selectively inhibited spontaneous IgE production and enhanced IgG4 production by B cells from atopic patients in the presence of T cells and monocytes. These results indicate that there is a mechanism of IgE production inhibition which is not mediated by IFN-gamma. We also found that DSCG is an excellent reagent for the study of IgE and IgG4 regulation in vitro.     

Differential effect of phosphonoacetic acid on early antigen synthesis in two Epstein-Barr virus producer cell lines.

The effect of phosphonoacetic acid (PAA) on the expression of Epstein-Barr virus (EBV)-determined antigens in producer (P3HR-1 and B95-8) lymphoblastoid cell lines (LCLs) and iododeoxyuridine (IUDR)-treated nonproducer cell lines (Raji and F137) was investigated. PAA effectively inhibited spontaneous early antigen (EA) synthesis in the P3HR-1 LCL only, while it had no effect on EA production in the B95-8 cell line nor on the IUDR-induced EA in nonproducer LCLs. The inhibition of spontaneous EA in P3HR-1 cell was dose and time dependant, and removal of PAA from cultures and subsequent propagation of these cells in PAA-free medium did not restore spontaneous EA production. The data reported show that EA is synthesized by at least two mechanisms in P3HR-1 cells, one of which is sensitive to PAA, thus suggesting that viral DNA synthesis is required for spontaneous EA synthesis only in the P3HR-1 cells; however, in these same cells, viral DNA synthesis would not be required for the IUDR-induced EA production.     

In vitro synthesis of IgE by human lymphocytes. II. Enhancement of the spontaneous IgE synthesis by IgE-binding factors secreted by RPMI 8866 lymphoblastoid B cells.

RPMI 8866 lymphoblastoid cells, known to express surface Fc epsilon R, were tested for their ability to regulate the in vitro synthesis of human IgE. Cell-free supernatants (CFS) of RPMI 8866 cells enhanced in a dose-dependent fashion the spontaneous IgE synthesis by B cells of allergic individuals. For maximum activity the CFS had to be added during the first 3 days of culture. CFS did not significantly alter the spontaneous synthesis of IgM or IgG, but they suppressed IgA synthesis both in B cell cultures and in pokeweed mitogen-stimulated peripheral blood mononuclear cells cultures. Cyclosporin A did not suppress either the spontaneous Ig production by B cells nor the IgE-potentiating activity of CFS. The enhancing activity of CFS was related to its content in IgE binding factors (IgE-BFs); these factors were detected by their ability to inhibit the rosetting of RPMI 8866 cells with IgE-coated erythrocytes (E-IgE). Both the IgE-BFs and the IgE-potentiating activity of the supernatants of RPMI 8866 cell cultures could be removed by absorption with IgE-Sepharose, from which they could subsequently be eluted with glycine-HCl buffer. IgE-BFs were identified as glycoproteins on the basis of their sensitivity to trypsin and to neuraminidase. By filtration of the RPMI 8866 cell supernatants through a Sephadex G75 column, IgE-binding activity was found to be associated with two fractions with molecular sizes in the range of 10,000-15,000 and 30,000-40,000. The IgA-suppressing activity of the RPMI 8866 culture filtrates could be absorbed with sIgA-Sepharose from which it was subsequently recovered by elution with glycine-HCl buffer. Most unexpectedly, sIgA-Sepharose also removed IgE-BFs and IgE-potentiating activity from the RPMI 8866 supernatants; both could be recovered by subsequent elution from sIgA-Sepharose with gycline-HCl buffer. These data are provisionally interpreted as indicating that the IgE-BFs secreted by RPMI 8866 cells had affinity for both IgE and sIgA and that they exerted a reciprocal effect on the in vitro synthesis of IgE and IgA.     

The lack of competition between two unrelated IgE antibodies in in vivo sensitization of mouse mast cells

The relation between IgE antibodies of different specificity in in vivo sensitization of mouse mast cells were studied. In the system of active immunization with EA and KLH mixed with Al(OH)3 no inhibition of an IgE and IgGl response to one antigen by simultaneous administration of other antigen was observed. The lack of inhibition was reflected in serum anti-EA and anti-KLH antibody levels as well as in mast cell sensitization expressed in antigen-induced histamine release. Furthermore, we were unable to detect an inhibitory effect of active immunization of mice with EA on in vivo passive sensitization of peritoneal mast cells. These observations indicated that in such a system immunization actively produced IgE antibodies do not saturate mast cell IgE receptors and do not inhibit subsequent sensitization of these cells with IgE antibodies of another specificity.     

Natural killer cell interaction with IgE in the control of ongoing human IgE synthesis

Fresh natural killer (NK) cells from normal donors inhibited IgE synthesis from U266/AF-10 cells via a direct cytolytic effect. This inhibition was 'reversed' by incubation of NK cells with human IgE-anti-IgE immune complexes (IgE-IC) for 16 h without a decrease in NK-mediated cytotoxicity. Upon incubation with IgE-IC, Fc epsilon receptors (FcER) were induced on 3-9% of NK cells. These IgE-IC induced FcER+ NK cells from normal donors secreted (an) IgE-specific factor(s) which enhanced U266/AF-10 IgE production without increasing DNA synthesis. Production of this IgE differentiation factor(s) explains the apparent reversal of NK cell inhibition of IgE production.     

Effect of cyclic AMP-elevating agents on human spontaneous IgE synthesis in vitro

The cyclic AMP (cAMP)-elevating substances dibutyryl-cAMP (dbcAMP), isoproterenol and theophylline were found to suppress the spontaneous in vitro IgE synthesis of peripheral blood mononuclear cells (MNC) from patients with atopic dermatitis, when added at high concentrations (10(-3) -10(-4) M) to the IgE-producing cell cultures. In contrast, low concentrations of the substances (10(-8) -10(-12) M) significantly enhanced IgE production. This enhancement was probably due to effects of cAMP on T cells since pretreatment of allogeneic MNC or T cells with dbcAMP abrogated their suppressive effect or resulted in enhancement of IgE synthesis in coculture experiments. Likewise, pretreated T cells from atopics stimulated the IgE production of autologous B cells more than did untreated T cells. These findings may possibly have bearing on the pathogenesis of the atopic diseases, which are associated with abnormalities of the cyclic nucleotide metabolism.     

Effects of interleukin-4 and interferon-γ on the secretion of IgG4 from human peripheral blood mononuclear cells

Whereas IgE antibodies are linked with allergy, IgG4 antibodies may reflect the state of immunity and protection against a particular antigen. It has been shown that interleukin (IL)-4 is required for induction of IgE synthesis. In order to elucidate the role of IL-4 in the production of IgG4 and to compare IgG4 and IgE regulatory processes, we quantified these immunoglobulin isotypes after in vitro culture of peripheral blood mononuclear cells (PBMC) in the presence of IL-4. The production of IgG4 was increased by IL-4 under the same conditions which are optimal for IgE production but not among PBMC from all donors, depending on the magnitude of spontaneous IgG4 secretion: IL-4 was effective only when the spontaneous secretion of IgG4 was < 7% of the total IgG secretion; it had no effect when spontaneous IgG4 production was >7% of total IgG. The IL-4-induced IgE response was consistently obtained when IgG4 was < 7% of total IgG but was markedly diminished or absent when IgG4 was > 7% of total IgG. If Staphylococcus aureus strain Cowan 1 (SAC) was present during the 48-h preincubation step, spontaneous IgG4 production was increased, but the stimulatory effect of this mitogen on immunoglobulin production, including IgG4, was markedly blocked by the addition of IL-4. In contrast, IL-4-induced IgE synthesis was strongly blocked by the presence of SAC. Finally, secretion of IgG4 (spontaneous and IL-4-induced) was suppressed among cells from most donors by interferon-γ (IFN-γ). These results suggest that IL-4 has opposite effects on in vitro IgG4 production and that the in vitro synthesis of both IgG4 and IgE appears to be regulated similarly by IL-4 and IFN-γ, whereas additional signals promote the production of one isotype in preference to the other. It is possible that activated B cells respond to IL-4 less well than do nonactivated cells.     

Modulation of human IgE synthesis by transforming growth factor-beta.

Exogenous transforming growth factor-beta 2 (TGF-beta 2) markedly inhibits the interleukin 4 (IL4)-stimulated synthesis of human IgE in three models where the B cell co-stimulation signals are contact dependent. This concerns T cell-dependent IgE production by (i) unfractionated peripheral blood mononuclear cells (PMBC) cultured with IL4 and (ii) highly purified B cells cocultured with irradiated EL4 thymoma cells in the presence of IL4 and phorbol myristate acetate, as well as monocyte-dependent IgE production by rigorously T cell-depleted PBMC cultured with IL4 and hydrocortisone. The suppression is not isotype specific. TGF-beta exerts its effect by inhibiting the proliferation of B cells and perhaps also the differentiation of proliferating B cells. However, at a later stage of differentiation, IgE B cells are refractory to the inhibitory effect of TGF-beta, as shown by the slight but significant increase of the spontaneous secretion of IgE by PBMC of atopic patients. This enhancement is due to the suppression of endogenous interferon-gamma production. Most interestingly the synthesis of IgE by highly purified B cells costimulated with IL4 and Epstein-Barr virus is unaffected by TGF-beta. It is concluded that TGF-beta mainly acts by inhibiting IL4-supported B cell proliferation; however, its effects depend upon the B cell costimulation signals that are required together with IL4 for the induction of IgE synthesis.     

Effect of antigens from Nippostrongylus brasiliensis and cytokines on the ongoing IgE synthesis in vitro.

The modulation of an ongoing IgE-mediated immune response induced by the infection of mice with the nematode Nippostrongylus brasiliensis (N.b.) was investigated in vitro. In previous experiments antigens derived from the homogenate of adult worms (WH) and third stage larvae (LH) were characterized by immunoblotting. Our results demonstrate that only antigens of the WH were recognized by IgE antibodies. The effect of worm antigens and cytokines (IL-2, IL-4, IL-5, IFN gamma) on the IgE and IgG synthesis in different culture systems was studied. The IgG synthesis of B cells was stimulated by WH or WH/cytokines. The IgE production of B cells was enhanced only by WH/cytokines or when T cells were present. Individual antigen fractions (WH 5, 8, 9, 10, 13) increased the IgE production, which was enhanced in the presence of IL-4 2- to 3.5-fold) but had no significant effects on the IgG production.     

In vitro IgE production by interleukin 4-stimulated human peripheral blood mononuclear cells is suppressed by rapamycin

Rapamycin (RAPA) is a new immunosuppressant which is 50-fold to 100-fold more potent than cyclosporin A (CyA) in inhibiting cellular immune responses and allograft rejection in animal models of organ transplantation. The drug's effect on in vitro IgE synthesis by interleukin (IL)4-stimulated human peripheral blood mononuclear cells was examined and compared with CyA's effect in this study. RAPA was found to be about 100-fold more potent than CyA in inhibiting IgE synthesis. Its inhibitory effect on IgE production was significant if it was added to the culture before Day 6 of a 14-day culture. The suppression was accompanied by the inhibitory effect on cell proliferation and on IgE-binding factor (IgE-BF) production. IL2 was able to partially reverse CyA- but not RAPA-induced inhibition of IgE production. Commercial B cell growth factor (cBCGF) was not able to reverse either RAPA- or CyA-induced suppression of IgE synthesis. The strong inhibitory effect of RAPA in IgE synthesis may be useful in certain clinical applications where overproduction of pathogenic IgE is a key issue. RAPA can also be used as a tool to dissect the regulation of IgE production.     

Antigenic competition in IgE antibody production. III. Suppressive effect on Th and B cells

An adoptive cell transfer system was utilized to evaluate the site of action of the suppressive mechanism involved in antigenic competition in IgE antibody production. Carrier-primed (OA) and hapten-primed (DNP-KLH) spleen cells were transferred to syngeneic irradiated recipients that were challenged with a heterologous conjugate (DNP-OA). To study the effect of antigenic competition on T and B cells, donor mice of one or the other cell type received in addition the competitor antigen (Asc) at immunization. The adoptive secondary IgE anti-DNP antibody response was suppressed in both situations. This effect could not be attributed to transfer of Asc-primed cells. Irradiation of donor mice before immunization with the two antigens abrogated the suppressive effect. These results indicate that both Th and B cells primed to the test antigen were affected by antigenic competition.     

CD40 antibodies defining distinct epitopes display qualitative differences in their induction of B-cell differentiation.

IgE production can be obtained in vitro by stimulating B lymphocytes with CD40 antibodies and interleukin-4 (IL-4). This stimulation also results in homotypic aggregation and cell proliferation. We have shown previously that IgE synthesis may be dependent on additional signals provided by the close cellular contact. Thus inhibition of the aggregation by lymphocyte function-associated antigen-1 (LFA-1) antibodies leads to a decrease in IgE production. In the present study we show that the inhibitory effect of LFA-1 antibodies is critically dependent on the CD40 antibody used for stimulation. Thus, while previously using the monoclonal antibody (mAb) S2C6, IgE production induced by the CD40 antibody mAb89 was generally higher and could be enhanced more than fivefold in the presence of LFA-1 antibodies. Similarly, the addition of the CD23 mAb MHM6, which blocked aggregation to a similar degree as the LFA-1 antibodies, inhibited S2C6-induced IgE production but enhanced that induced by mAb89. In contrast to these opposing effects on IgE synthesis, proliferation induced by the two CD40 antibodies was affected similarly by the blocking antibodies. As the interaction between CD23 and CD21 has been suggested to involve recognition of carbohydrate structures on CD21 by the lectin-like domain on CD23, we also tested the effect of some different sugars on IgE synthesis and proliferation. Addition of fucose-1-phosphate to anti-CD40 and IL-4-stimulated B cells completely inhibited IgE synthesis and proliferation. Inhibition was also seen with mannose-6-phosphate but not with glucose-1-phosphate. In contrast to the MHM6 antibody, the effect of the sugars was similar irrespective of the CD40 antibody used for stimulation. The study shows that different antibodies to CD40 may give rise to qualitatively distinct signals depending on the epitope recognized.