Ongoing IgE synthesis by atopic B cells is enhanced by interleukin-3 and suppressed directly by interferon-gamma in vitro

Human recombinant interleukin-3 (rIL-3; 10 U/ml) consistently augmented spontaneous IgE synthesis by isolated atopic B cells in vitro, whereas rIL-4 (1-1,000 U/ml) failed to induce IgE synthesis by these cells. Recombinant interferon-gamma (rIFN-gamma) suppressed ongoing IgE production by atopic B cells in a dose-dependent manner. IFN-gamma also inhibited IgE synthesis by a human myeloma cell line (U-266), demonstrating the direct effect of IFN-gamma on the terminal differentiation of IgE-secreting plasma cells. IL-3 and IFN-gamma from different sources displayed the same effects on IgE synthesis. Neutralizing antibodies toward IL-3 or IFN-gamma abolished their activities toward IgE synthesis, supporting the specificity of the effect of these cytokines. The quantity of endogenous IFN-gamma produced by stimulated T cells was significantly decreased in atopic patients compared to nonatopic controls, which might be responsible for the propensity of a high blood IgE level in atopic patients.     

IgE synthesis in vitro induced by T cell factors from patients with elevated serum IgE levels

The effect of unstimulated T cell culture supernatants (TCS) from patients with atopic dermatitis and high serum IgE levels on the IgE production in vitro by B cell rich suspensions from normal individuals or grass pollen sensitive patients with mild atopy was evaluated. TCS from patients with raised IgE enabled B cell suspensions from normal individuals to produce detectable amounts of IgE and potentiated the spontaneous IgE synthesis in vitro by B cell suspensions from grass sensitive patients. In contrast, the addition of TCS from normal subjects with low serum IgE levels did not increase or even reduced IgE synthesis by B cell cultures. When the same B cell cultures were analysed for their ability to produce IgG or IgM protein, no significant differences were observed. These findings indicate that T lymphocytes from patients with high serum IgE levels can release soluble factor(s) possessing isotype (IgE) specific potentiating activity.     

In vitro synthesis of human IgE: reappraisal of a 5-year study

In the last 5 years some models of human IgE production in vitro have been investigated in our laboratory. Spontaneous IgE synthesis was found in cultures of B cells from most patients with atopic dermatitis or atopic patients with multiple sensitivities and from some patients with pollenosis, but only during the pollination period. A small and variable increase of the spontaneous IgE synthesis was induced by soluble factor(s) produced by T cells from patients with severe atopy. Selected helper T cell clones were also able to induce IgE synthesis in vitro by both atopic and normal B cells.     

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.     

Hydrocortisone enhances allergen-specific IgE production by peripheral blood mononuclear cells from atopic patients with high serum allergen-specific IgE levels.

BACKGROUND: Although there is convincing evidence that human B cells can be induced to produce IgE by a combination of interleukin 4 (IL-4) and hydrocortisone (HC) in atopic subjects, it is still uncertain if this performs the same functions in allergen-specific IgE synthesis. OBJECTIVE: This study was designed to investigate the differences of IgE regulation between atopics and nonatopics, interactions of HC with IL-4, and the correlation between in vitro total IgE, allergen-specific IgE synthesis and serum IgE levels. METHODS: Peripheral blood mononuclear cells (PBMCs) from 16 atopic asthma patients sensitive to Dermatophagoides farinae and seven nonatopic controls were cultured with IL-4 and/or HC. Total IgE and D. farinae-specific IgE in culture supernatant were measured by ELISA and FAST. RESULTS: IL-4 increased total IgE synthesis in PBMCs from both atopics and nonatopics, whereas, HC had this effect only in some atopics who showed spontaneous IgE production in vitro. HC acted synergistically with IL-4 in total IgE synthesis. Their effects were more remarkable in cases with lower total serum IgE levels. PBMCs from eight of 16 atopics produced D. farinae-specific IgE in vitro either spontaneously or by IL-4 and/or HC. HC had more profound effects than IL-4 in these patients. They also showed higher total IgE synthesis by HC, and higher specific serum IgE levels than the others. IL-4 and/or HC did not induce any D. farinae-specific IgE synthesis by PBMCs from nonatopics. CONCLUSION: HC had a more profound effect than IL-4 on the induction of D. farinae-specific IgE synthesis in atopic patients with high serum allergen specific IgE levels. Further studies to determine the causes of these effects, such as the presence of long lived allergen specific B cells as the result of the priming effect of IL-4 in vivo, may be needed.     

Comparison of IgE expression at the mRNA and protein levels in vitro.

The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay.     

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.     

Enhancement of IgE synthesis and histamine release by T cell factors derived from atopic patients with bronchial asthma

Culture supernatants of unstimulated T cells (TCS) derived from normal donors or from atopic patients with bronchial asthma were tested for their ability to regulate the spontaneous IgE synthesis by B cells of normal and atopic subjects. The same TCS were also tested for their influence on the histamine release from leukocytes of house dust mites-sensitive patients. Addition of TCS to B cell cultures from allergic donors induced a dose-dependent increase of the spontaneous IgE production without affecting the synthesis of IgG, IgM, and IgA. The potentiating activity of TCS was observed only in B cell cultures spontaneously producing IgE; TCS were still active on irradiated B cells. The maximal IgE-enhancing activity was observed when TCS were added at the onset of B cell cultures. The supernatants of T cells lysed at day 0 did not contain IgE-potentiating factors. The antigen-induced but not the spontaneous histamine release from leukocytes of house dust mite-sensitive patients was enhanced by pretreatment with TCS from allergic donors. The enhancing activities of TCS on IgE synthesis and on histamine release could be removed by absorption with IgE-Sepharose and subsequently recovered by elution with glycine buffer. The results indicate that T cells of patients with asthma spontaneously release IgE-binding factors capable of increasing both the spontaneous IgE synthesis by B cells and the antigen-induced histamine release.     

Detection and characterization of IgE-producing cells in patients with clonorchiasis

The ability of peripheral B cells of patients with Clonorchis sinensis infections to secrete IgE spontaneously was investigated in vitro. The de novo synthesis of IgE was observed in unstimulated B-cell cultures of patients. There was a significant relationship between the serum IgE level and the amount of IgE spontaneously secreted by B cells. Pretreatment of patients' B cells with 10 micrograms/ml of rabbit anti-human IgE resulted in the clear suppression of spontaneous IgE synthesis without affecting the IgG synthesis. Their B cells capable of spontaneously secreting IgE were partially sensitive to irradiation with 1,000 rad. The results obtained suggest that such IgE-forming cells may be responsible for at least part of the persistent IgE formation in patients with helminthic infections as well as in those with atopic disease.     

Human IgE Synthesis in vitro

Using monoclonal anti-human IgE antibodies recognizing the D epsilon 1 or D epsilon 2 epitope, we have developed a sandwich radioimmunoassay (RIA) to determine IgE in human cell cultures. With the help of this assay, various methods of measuring an actual de novo IgE synthesis were compared. It was necessary to subtract performed IgE from released IgE in the culture supernatant and the IgE associated to the cultured cells, in order to determine a net IgE synthesis which would reflect de novo synthesized IgE. Using this differential in vitro IgE assessment, no net IgE synthesis could be demonstrated in culture conditions which lead to strong IgG synthesis. Actual net in vitro IgE antibody production was only found in approximately 30% of cell cultures from atopic donors. This spontaneous IgE synthesis did not correlate to the serum IgE levels of the patients. However, correlations were found between serum IgE levels and amount of performed, released or cell-associated IgE of the cultures.     

In vitro synthesis of IgE by human lymphocytes. III. IgE-potentiating activity of culture supernatants from Epstein-Barr virus (EBV) transformed B cells.

Seven Epstein--Barr virus (EBV)-transformed B cell lines were derived from circulating lymphocytes of two atopic and two non-atopic individuals, two preparations of cord blood lymphocytes and one tonsillar lymphocyte preparation. All the cell lines contained a significant proportion of cells expressing Fc epsilon R as detected by rosette formation with IgE-coated bovine erythrocytes (E-IgE) and by flow cytometry using IgE-linked to fluorescent microspheres. None of the cell lines displayed FcR for IgA, IgM or IgG. The cell-free supernatants (CFS) of EBV-transformed cells contained IgE-binding factors (IgE-BFs) detected by their ability to inhibit the binding to RPMI 8866 cells of either E-IgE or IgE-linked to microspheres. Whereas these CFS enhanced the synthesis of IgE and suppressed the synthesis of IgG by purified B lymphocytes isolated from the blood of allergic donors and cultured in the absence of stimulant, their effect on the synthesis of IgA or IgM was not predictable. CFS significantly enhanced the secretion of IgE by the U266 myeloma cell line without interfering with secretion of IgM, IgG or IgA by EBV-transformed cells. These data are in accord with similar properties of RPMI 8866 cells and suggest that B lymphocytes might play a regulating role in the IgE synthesis.     

Mononuclear cells from patients with the hyper-IgE syndrome produce little IgE when they are stimulated with recombinant human interleukin-4.

To investigate whether B cells from patients with the hyper-IgE syndrome are more sensitive to the effects of interleukin-4 in vitro than B cells of normal or atopic individuals, we stimulated blood mononuclear cells (MNC) with varying doses of recombinant human interleukin 4 (rhIL-4) and measured supernatant IgE concentrations after 18 days of culture. Geometric mean spontaneous IgE synthesis after 18 days of culture without rhIL-4 was low (less than 3 ng/ml) and similar for MNCs from nine patients with the hyper-IgE syndrome, nine atopic and nine normal subjects. As found in our previous studies, MNCs from the nine atopic and the nine normal donors produced significant and similar quantities of IgE (geometric mean maximum IgE, 25.2 and 18.7 ng/ml, respectively) when MNCs were stimulated with rhIL-4. MNCs from both donor groups had similar sensitivity to the concentration of IL-4 eliciting the IgE response. In striking contrast, MNCs from the nine patients with the hyper-IgE syndrome failed to produce significant IgE over that produced spontaneously when MNCs were stimulated by a wide range of rhIL-4 concentrations. Coculture of B cell-enriched subpopulations from patients with the hyper-IgE syndrome with T cell-enriched subpopulations from nonatopic and atopic donors failed to restore responsiveness to rhIL-4. The addition of anti-CD40 monoclonal antibody to MNC cultures did result in enhancement of rhIL-4 IgE synthesis by MNCs from patients with the hyper-IgE syndrome, but the concentration of anti-CD40 required to elicit this enhancement was tenfold higher than for control MNCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory effects of human IgE-binding factors in the IgE synthesis by human and rat lymphocytes

We have previously established human T cell hybridomas which produce IgE-binding factors. Incubation of one of the T cell hybridomas, 166A2, with human IgE dimer in the presence of 1 microgram/ml bradykinin resulted in the formation of IgE-binding factors having affinity for lentil lectin. The factors selectively enhanced both IgE-forming cell responses of rat mesenteric lymph node (MLN) cells and spontaneous IgE synthesis by human peripheral blood B cells of atopic patients, without affecting the IgG response. The same factors that enhanced IgE synthesis of B cells from atopic patients also enhanced IgE synthesis induced under bystander conditions by activated alloreactive T cells. Fractionation of the affinity-purified IgE-binding factors by gel filtration revealed three molecular mass species, i.e., 60 kDa, 30 kDa and 15 kDa. The 60-kDa and 15-kDa IgE-binding factors selectively enhanced both the spontaneous IgE synthesis by B cells of atopic patients and IgE response of rat MLN cells. In contrast, the 30-kDa IgE-binding factors had only marginal enhancing effects on the IgE synthesis by both human B cells and rat MLN cells. When the 166A2 hybridoma cells were incubated with IgE dimer in the presence of glycosylation-inhibiting factor (GIF), essentially all IgE-binding factors formed by the cells had affinity for peanut agglutinin (PNA) but for neither lentil lectin nor concanavalin A. All of the 60-kDa, 30-kDa and 15-kDa species, having affinity for PNA, selectively suppressed the potentiating factor-enhanced IgE response of rat MLN cells. The factors also suppressed the IgE synthesis of human B cells from atopic patients when the synthesis was enhanced by IgE-potentiating factor. The results indicate that human IgE-binding factors regulate IgE synthesis by both human and rat lymphocytes.     

Effect of recombinant human interleukin 4 on spontaneous in vitro human IgE synthesis

Recombinant human interleukin 4 (IL 4) alone enhanced the spontaneous IgE synthesis in cultures of peripheral blood leukocytes (PBL) from atopic patients as well as from nonatopic individuals, suggesting the existence of preactivated PBL sensitive for IL 4. Preactivated cells were also obtained by stimulation with Staphylococcus aureus strain Cowan I (SAC). However, co-stimulation of PBL by IL 4 with SAC or anti-IgM antibody and pokeweed mitogen did not result in an enhanced IgE synthesis. Optimal IL 4 concentrations for the induction of IgE synthesis coincided with optimal proliferative responses in PBL. The effect of IL 4 was not isotype specific, and in terms of protein even more IgG and IgM antibodies were formed. The effect of IL 4 on IgE synthesis was counteracted by very low concentrations of interferon-gamma (IFN-gamma), suggesting that both IL 4 and IFN-gamma might be decisive cytokines for the human in vitro IgE synthesis.     

Abnormal regulation of in vitro IgE synthesis by T cells obtained from patients with atopic dermatitis

The regulatory influence of atopic eczema and non-atopic T cells on spontaneous IgE synthesis by eczema B cells was examined. Eczema B cells were cocultured with either autologous or allogeneic T cells in RPMI 1640 with 10% fetal calf serum at 0.75 X 10(6) cells/ml (B/T = 0.5) and supernatant IgE was measured by a modified PRIST assay. Net IgE synthesis was obtained by subtracting preformed IgE (+ cycloheximide at Day 0) from total IgE in 7-day supernatants. T cells were either untreated, heat-killed, exposed to 2000 rad, or depleted of helper/suppressor T cells by "panning" with monoclonal antibodies (Leu 3a and Leu 2a). Atopic eczema B cells spontaneously synthesized IgE when cultured alone. No significant suppression of net IgE synthesis occurred when atopic eczema T cells were cocultured with autologous B cells. In allogeneic recombinations, non-atopic T cells significantly suppressed net IgE synthesis by atopic eczema B cells (mean suppression = 59%; P less than 0.05). This suppression was abrogated if allogeneic control T cells were heat-killed, irradiated, or depleted of Leu 2a+ suppressor cells. In order to exclude an "allogeneic effect" as the sole mechanism to explain the suppression of IgE synthesis observed by coculturing non-atopic T cells with eczema B cells, the latter were recombined with either T cells from HLA-DR and mixed lymphocyte culture-matched sibling or autologous T cells. Greater suppression of net IgE synthesis was seen in the presence of histoidentical non-atopic T cells than in the presence of autologous eczema T cells, indicating that the latter have a partial defect in their suppressor function. This apparent "defect" in immunoregulatory function may be overcome by in vitro activation of atopic eczema T cells by concanavalin A.     

Effect of dexamethasone on de novo IgE synthesis by human blood lymphocytes

The effect of dexamethasone (DM) on de novo in vitro total IgE synthesis by blood mononuclear cells (MNC) was studied in atopic patients with eczema and in nonatopic control subjects. Unfractionated blood MNC were cultured at 1 X 10(6) cells per milliliter for 7 days in RPMI 1640 with 10% fetal calf serum with or without decreasing concentrations of DM (10(-7) to 10(-11). Net IgE synthesis was calculated by subtracting preformed (+ cycloheximide at day 0) from total IgE in 7-day supernatants. Supernatant IgE was measured by use of a modified PRIST assay. A significant increase in net IgE synthesis occurred in the presence of DM in 11 of 11 atopic patients with eczema (mean percent increase = 68%; p less than 0.05) and five of five atopic patients without eczema (mean percent increase = 53%; p 0.05) but not in seven of seven nonatopic controls. This increase in de novo IgE synthesis could not be explained by a significant change in cell viability. In five of five experiments, a mean increase of 78% was still noted when DM was added to atopic blood MNCs depleted of T cells by sheep red blood cell rosetting. The addition of 10(-9)M of DM to eczema B+ T cell recombinations enriched for suppressor cells (depleted of Leu 3a+ helper T cells) resulted in a loss of suppressor-T cell activity and maximal augmentation of IgE synthesis. Enhancement of IgE synthesis was also noted when DM was added to eczema B+ T cell recombinations enriched for helper T cells (depleted of suppressor Leu 2a+ T cells).(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of IgE synthesis in vitro by allogeneic T cells from atopic and non-atopic subjects.

The role of T cells in the regulation of IgE synthesis by human PBMC was studied. PBMC or separated and recombined populations of T and B cells from both normal and atopic donors were cultured for 10 days with and without cycloheximide. IgE and IgG synthesis were determined by specific RIA. IgE synthesis was detected in 0/30 non-atopic, 6/34 mildly atopic and 25/31 severely atopic subjects. Autologous T cells from 10/26 atopic donors, whose B cells synthesised IgE, significantly suppressed this IgE synthesis. The addition of allogeneic T cells from atopic or non-atopic subjects to atopic B cells resulted in greater suppression of IgE synthesis than the addition of autologous T cells. These data support the notion that atopic subjects have naturally occurring IgE isotype-specific suppressor T cells as well as suppressor T cells which can be activated during incubation with alloantigen.     

Hydrocortisone enhances total IgE levels--but not the synthesis of allergen-specific IgE--in a monocyte-dependent manner.

Recently, hydrocortisone (HC), when combined with human IL-4, has been reported to increase IgE levels in supernatants (SN) of in vitro cultured leucocytes. In this study we investigated the influence of HC on allergen-specific IgE synthesis. Moreover, we examined the relevance of different cell types in this respect. Peripheral blood mononuclear cells (PBMC), T-cell depleted PBMC, CD14-depleted PBMC and highly purified B cells from 10 allergic (birch pollen and/or grass pollen) patients and five non-allergic individuals were investigated. The cells were incubated with HC and/or recombinant human IL-4 (rIL-4) for 8 days. A considerable increase of total IgE was observed in HC/rIL-4-stimulated cultures compared with rIL-4 alone, HC alone or non-stimulated cultures. We demonstrate that this effect depends on the presence of monocytes in in vitro cultures. These results were seen in every experiment, irrespective of healthy or atopic state of the blood donor. The increase of IgE could not be attributed to a rise of birch pollen-and/or grass pollen-specific IgE in patients allergic to these allergens, as shown by IgE-immunoblot. Radio-allergosorbent test (RAST) investigations of HC/rIL-4-stimulated cells cultures from allergic and non-allergic patients confirmed that HC/rIL-4-induced elevated IgE production was also not due to increased production of IgE, specific for important aero-allergens (pollens, house dust mite or animal dander). Therefore we conclude that newly synthesized IgE is not specific for allergens, but that sequential isotype switching in human B cells leads to increased polyclonal IgE production.     

Characterization of human T cell-derived IgE-potentiating factor

We have previously shown that Fc epsilon receptor-positive (Fc epsilon R+) T cell lines from patients with the hyper IgE syndrome secrete IgE-binding factors which selectively enhance IgE but not IgG synthesis in cultures of B cells obtained from patients with allergic rhinitis but not from nonatopic subject. In the present study we have tested the effect of supernatants from Fc epsilon R+ T cell lines on a large panel of B cells from atopic patients (n = 20). We found that IgE synthesis was selectively enhanced only in B cell cultures in which there was ongoing spontaneous synthesis of IgE. The target of IgE-potentiating factor(s) was a large low-density B cell present in the circulation of responding atopic donors. In addition, we further characterized IgE-potentiating factors derived from Fc epsilon R+ T cell lines. The factor(s) fractionated into 2 peaks on Sephadex G-75 with approximate molecular masses of 15,000 and 60,000 kDa, and had affinity for lentil lectin but not for peanut agglutinin. Release of IgE-potentiating factor(s) was enhanced by the addition of exogenous human IgE to Fc epsilon R+ T cell cultures and was inhibited by tunicamycin, an inhibitor of N-glycosylation. These studies suggest a close homology between the physicochemical characteristics of human and rodent IgE-potentiating factors and the immune signals which modulate production of these IgE regulatory factors.