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.     

Soluble fragments of the low-affinity IgE receptor (CD23) inhibit the spontaneous migration of U937 monocytic cells: neutralization of MIF-activity by a CD23 antibody.

U937 monocytic cells were found to respond by diminished spontaneous migration when confronted with affinity-purified soluble fragments of the low-affinity receptor for IgE (FcER2/CD23). Unlike B lymphoma cells, U937 cells could not be activated to respond with enhanced DNA synthesis through their membrane-bound CD23 antigen by MHM6, a monoclonal antibody within the CD23 cluster. MHM6 did, however, effectively neutralize the U937-directed MIF (migration inhibition factor) activity contained within the soluble CD23 preparations. The findings not only suggest a role for soluble CD23 as a novel cytokine at sites of inflammation but also indicate different functions for the membrane-bound forms expressed on B cells and monocytes.     

T cell clones providing helper function for IgE synthesis release soluble factor(s) that induce IgE production in human B cells: possible role for interleukin 4 (IL-4).

We have previously demonstrated that a proportion of human T cell clones (TCC) derived from tonsil or peripheral blood (PB) of non-allergic donors, upon triggering with phytohaemagglutinin (PHA) or anti-CD3 monoclonal antibody (MoAb), were able to provide help for IgE synthesis in B cells from both allergic and non-allergic individuals. In this study we show that, upon PHA stimulation, culture supernatants from 10 selected TCC active on IgE synthesis also provided helper activity for IgE, whereas supernatants from unstimulated cultures of the same TCC were ineffective. In contrast, culture supernatants derived from five PHA-stimulated TCC, unable to provide helper function for IgE synthesis, consistently failed to elicit production of IgE. While the induction of IgE synthesis by TCC occurred in B cells from virtually all allergic and non-allergic donors, their soluble factor(s) were found to be able to provide substantial help for IgE production only in B cells from a proportion of donors tested. In addition, B cells from non-atopic donors usually appeared to be less responsive than atopic B cells to the activity of such factor(s). In contrast, synthesis of both IgG and IgM was induced in every B cell donor by both TCC and their supernatants. Partial characterization of the factor(s) providing helper function for IgE synthesis in B cells showed that it apparently had a mol. wt between 10 and 50 kD and did not bind to immobilized IgE. Such an activity appeared to be associated with the presence of interleukin 4 (IL-4) in supernatants and it was inhibited by adding both gamma-interferon and anti-human IL-4 antibody in culture.     

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)     

Regulation of the Human IgE Antibody Response

The frequent association of elevated serum IgE in patients with T cell immunodeficiencies suggest a role for T cells in the regulation of the human IgE antibody response. Unlike the situation with other isotypes the polyclonal B cells activators, pokeweed mitogen and Epstein Barr virus, do not routinely induce IgE synthesis in normal B cells. However, B cells from normal donors will synthesize immunoglobulins of all isotypes (including IgE) when cultured with T cell clones that recognize determinants expressed on the B cells (cognate stimulation). T cells with Fc receptors for IgE can be isolated from patients with hyper IgE syndrome and maintained as long term continuous T cell lines. These cells secrete IgE binding factors which enhance IgE synthesis but not IgG synthesis by preactivated IgE bearing B cells from allergic subjects but not resting B cells from normal donors. IgE binding factors isolated from sera of normal donors selectively suppress IgE synthesis. In contrast, IgE binding factors isolated from sera of patients with hyper IgE syndrome contain IgE potentiating activity as well as IgE suppressor activity. These results suggest that IgE synthesis in man is activated by T cells and isotype specific secretion of this immunoglobulin is modulated by IgE binding factors.     

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.     

Human T cell clones can induce in vitro IgE synthesis in normal B cells regardless of alloantigen recognition or specificity for peculiar antigens

A total number of 119 (98 CD 4+ and 21 CD 8+) T cell clones were established from tonsil and peripheral blood of three nonallergic individuals and examined for their ability to induce in vitro IgE synthesis in normal B cells. Following preactivation for 24 h with phytohemagglutinin, 34 clones (33 CD 4+ and 1 CD 8+) induced normal B cells to synthesize remarkable amounts of IgE in vitro. In contrast, equal numbers of T blasts of phytohemagglutinin-induced T cell lines obtained from unfractionated T lymphocyte suspensions of the same donors did not show such an effect. The in vitro IgE synthesis evoked by T cell clones was detectable between day 6 and 9 and peaked on day 12. Most clones maintained their ability to stimulate in vitro IgE synthesis in repeated assays over a 3-month period. The induction of IgE synthesis by cloned T cells did not reflect alloantigen recognition on target B cells, since T cell clones induced IgE synthesis in B cells from all randomly selected donors tested, including autologous B cells. Preincubation for 24 h with optimal stimulatory concentrations of anti-CD 3 (OKT 3) monoclonal antibody or its addition through the entire culture period also enabled T cell clones to stimulate de novo IgE synthesis in vitro in normal B cells. Virtually all the T cell clones active on IgE synthesis induced the in vitro production of remarkable amounts of IgM and IgG as well. These data indicate that several human T cell clones can induce normal B cells to synthesize immunoglobulin of different classes, including IgE, regardless of alloantigen recognition on target B cells or specificity for peculiar antigens. The activity of these clones was apparently mediated by triggering of the monomorphic molecular complex CD 3, immediately before or during the incubation of T cell clones with the target B cells.     

Regulation of the human IgE antibody response

The frequent association of elevated serum IgE in patients with T cell immunodeficiencies suggest a role for T cells in the regulation of the human IgE antibody response. Unlike the situation with other isotypes the polyclonal B cells activators, pokeweed mitogen and Epstein Barr virus, do not routinely induce IgE synthesis in normal B cells. However, B cells from normal donors will synthesize immunoglobulins of all isotypes (including IgE) when cultured with T cell clones that recognize determinants expressed on the B cells (cognate stimulation). T cells with Fc receptors for IgE can be isolated from patients with hyper IgE syndrome and maintained as long term continuous T cell lines. These cells secrete IgE binding factors which enhance IgE synthesis but not IgG synthesis by preactivated IgE bearing B cells from allergic subjects but not resting B cells from normal donors. IgE binding factors isolated from sera of normal donors selectively suppress IgE synthesis. In contrast, IgE binding factors isolated from sera of patients with hyper IgE syndrome contain IgE potentiating activity as well as IgE suppressor activity. These results suggest that IgE synthesis in man is activated by T cells and isotype specific secretion of this immunoglobulin is modulated by IgE binding factors.     

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.     

Expression of Fc epsilon receptors and surface and cytoplasmic IgE on human fetal and adult lymphopoietic tissue

The appearance during ontogeny of IgE-positive and Fc epsilon receptor (FcER)-bearing cells was studied. Monoclonal antibody to the constant region (Fc) of IgE (CIA-E-7.12) was used to detect cytoplasmic and surface IgE. A monoclonal antibody to the low-affinity Fc epsilon receptor (FcER-II = CD23) and immune complexes composed of human IgE and mouse monoclonal anti-human IgE Fc were used to detect FcER. Cryostat sections of human fetal tissues (liver, lung, spleen, and thymus) from 11 to 22 weeks gestation as well as adult tonsil tissues were examined for IgE, FcER, and other lymphoid markers by immunoperoxidase staining. Although both IgE- and FcER-positive cells were present in adult tissues, we found that, in contrast to an earlier report, such cells were not present in the fetal tissues examined. The in situ location of FcER on cells in human lymphoid tissues revealed that the FcER-bearing cells were localized predominantly in the germinal centers (mature B cell and macrophage areas) of the tonsil follicles with some staining in the mantle (resting and less mature B cell areas).     

Human mast cell progenitors in peripheral blood from atopic subjects with high IgE levels.

BACKGROUND: It remains unclear whether the number of circulating mast cell progenitors is increased in patients with atopic diseases. Distinct genotypes are reported to affect mast cell/basophil activation. OBJECTIVE: We compared the number and function of mast cell progenitors present in the peripheral blood from donors with normal IgE (IgE < 400 U/mL) and those with atopic dermatitis accompanied by high serum IgE (IgE > 5000 U/mL). METHODS: Purified peripheral blood cells were cultured in serum-free methylcellulose containing stem cell factor (SCF), IL-6 plus IL-3. Fresh methylcellulose containing the cytokines was layered over every 2 weeks. The cultured mast cells were retrieved from the methylcellulose and were functionally analysed. RESULTS: Mast cell colonies were distinguished at 6 weeks of culture as other colony types had been degenerated. The number of mast cell colony-forming cells varied depending on donors and was not significantly increased in peripheral blood from the hyper-IgE atopic patients. A significant inversed correlation was found between the number of mast cells per one colony and the ages of donors. The cultured mast cells derived from atopic patients and those from normal IgE donors equally expressed Fc epsilon RI and released histamine through Fc epsilon RI, although IL-4 priming in vitro markedly enhanced the function of mast cells regardless of donors. CONCLUSIONS: These results indicate that the number of circulating mast cell progenitors may be regulated by unknown individual factors unrelated to IgE levels. Mast cell function may be regulated largely by environmental factors, such as IL-4, but not determined by their progenitors' genotypes.     

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.     

Characterization of specific IgE response in vitro against protein and drug allergens using atopic and normal donors

BACKGROUND: As the incidence of allergy to different compounds increases in society, the need to understand and characterize specific IgE responses becomes obvious. Different cell culture systems have been evaluated for their ability to support such IgE secretion. METHODS: One system employed human peripheral lymphocytes (PBL) from normal donors stimulated with anti-CD3 activated T cells with or without the presence of allergens like benzylpenicillin (BP) and Phlenum pratense (PP). Secretion of IgE was analyzed in ELISA and compared to the IgG response to the nonallergenic antigen tetanus toxoid (TT). Another system employed stimulation of T and B cells with a heterotope, consisting of a T helper cell epitope derived from TT, and a B cell allergen epitope derived from BP. The specific IgE secretion was compared, using lymphocytes from normal as well as BP-allergic donors. RESULTS: Anti-CD3 stimulated T cells supported BP-specific IgE secretion in six of 11 normal donors. This response was inhibited in four donors and enhanced in two donors by the addition of the BP-allergen to the culture. In contrast, addition of the protein allergen (PP) or antigen (TT) to the same culture system inhibited both IgE and IgG synthesis in all experiments. Cells from the majority (10/16) of the BP-allergic donors failed to produce BP-specific IgE in vitro, when cultured in the presence of allergen. CONCLUSIONS: An allergen specific immune response is readily generated in vitro. The differential response against benzylpenicillin between different donor categories most probably reflects the level of pre-exposure to this allergen in vivo.     

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.     

Biomolecular regulation of the IgE immune response

Several cell types, including mast cells, basophils, macrophages/monocytes, lymphocytes, platelets and eosinophils, may bind or contain IgE. To investigate the source of cell-associated IgE and its relation to spontaneous IgE synthesis, peripheral blood mononuclear cells from allergic and non-allergic donors were examined. Using a combination of different cell fractionation techniques and varying methods for extraction of cell-associated IgE, data were obtained indicating that monocytes constitute a major source of cell-associated IgE in human blood. The amount of cell-associated IgE in peripheral blood mononuclear cells from allergic and non-allergic donors varied more than 100-fold but correlated closely to the level of serum IgE (r = 0.84, p < 0.001, n = 38). Spontaneous and mitogen-induced in vitro syntheses of IgA, IgE and IgG were compared for allergic and non-allergic donors. Only one donor with very high serum IgE demonstrated spontaneous or mitogen-induced in vitro IgE synthesis despite synthesis of IgA and IgG.