The Regulatory Effect of Macrophages on the in Vitro Synthesis of IgE

The effects of indomethacin or macrophage-depletion on the in vitro unstimulated and pokeweed mitogen (PWM)-induced synthesis of IgG and IgE were compared. The effect on the unstimulated synthesis of IgG and IgE was similar, namely, no effect due to indomethacin and enhancement due to macrophage depletion. In contrast, the effect on PWM-induced synthesis was dissimilar. Macrophage depletion enhanced IgG but inhibited IgE production. The effect of indomethacin paralleled that of macrophage depletion suggesting the involvement of prostaglandins. It seems that the regulatory effect of macrophages is different in the IgE system compared with the IgG system.     

Basophil Histamine Release in Cord Blood Regulatory Role of IgE

Thirty-two cord blood samples were studied for histamine releasing capability by using a sensitive glass microfibre-based histamine analysis. Histamine was obtained after challenge with anti-IgE in 24 of the 32 samples. However, the net release in cord blood was only 25% of that in adult blood and no relationship was found between histamine release response, total IgE in cord plasma, and a family history of atopic diseases. The low histamine release in cord blood seemed to be associated with the immunological IgE receptor complex activation and not with an immature basic cell function, since the calcium ionophore A23187 which bypasses the receptor complex induced identical histamine release curves in cord and adult blood. Furthermore, when comparing the results of passive sensitization of basophils from new-born and adult persons, the new-born basophils possessed a significant fraction of free IgE receptors, whereas in adults most of the receptors were occupied by IgE.     

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.     

Affinity of IgE antibody to antigen influences allergen-induced histamine release.

BACKGROUND: Although affinity of an antibody for an antigen is recognized to be an important factor in determining its biological effects, little is known about the relevance of such affinity of IgE antibodies to the functional response. OBJECTIVES: To investigate the effect of IgE antibody affinity to Der p 2 on Der p 2-induced histamine release from human basophils. METHODS: The most probable value of the dissociation constant (Kd) of IgE antibody to Der p 2 was calculated and histamine release by Der p 2-challenged leucocytes was used to evaluate the biological efficacy of the IgE antibody. RESULTS: The most probable Kd value of IgE antibody to Der p 2 ranged from 5.6 to 177.8 pM in 14 asthmatic patients sensitive to Der p 2. A significant correlation was observed in Der p 2-induced histamine release between the sensitivity and the Kd value for Der p 2-specific IgE antibody (rs = 0.797, P = 0.0040), suggesting that the higher the affinity, the lower the amount of allergen required for the release of a specific level of histamine. CONCLUSION: Apart from the changes associated with the reactivity, the sensitivity of histamine release is closely related to the affinity of IgE antibody for its antigen.     

IgE-mediated basophil releasability is influenced by intrinsic factors and by IgE on the cell surface

Experiments were done to clarify the mechanisms associated with releasability of histamine. First, washed leukocytes from 23 asthmatic patients sensitive to mite allergen were challenged with Der p 1, a major allergen isolated from Dermatophagoides pteronyssinus , or anti-IgE. A significant correlation was observed between the ratio of Der p 1-specific IgE liter to total IgE level (S/T) in the patient's plasma and either the reactivity (maximal percentage of histamine release; r_s= 0.514, P = 0.016, n = 23) or the sensitivity (the minimum allergen concentration required to achieve 25% histamine release; r_s= -0.790, P = 0.0002) to Der p 1. Additionally, the reactivity to Der p 1 was significantly correlated with that to anti-IgE (r_s= 0.690, P = 0.0012), indicating that an intrinsic cellular property may be one of the contributing factors in immunologic histamine release. In a second series of experiments, sinus mast cells were passively sensitized with immunoglobulins prepared from the patient's plasma. A statistically significant correlation was found between either the reactivity or the sensitivity to Der p 1 and S/T, thus indicating that S/T is an indicator of the releasability of histamine. When basophils or mast cells were passively sensitized with mouse IgE and subsequently stimulated with antimouse IgE, the reactivity to antihuman IgE was significantly correlated with that to antimouse IgE ( r _s= 0.966, P = 0.0023, n = 11). These observations suggest that an intrinsic cellular property regulates reactivity in immunologic histamine release. Taken together, our results suggest that an intrinsic cellular property, as well as specific IgE antibody levels on the cell surface, is an important factor in determining histamine release in response to IgE-dependent activation.     

IgE Synthesis in vitro: Attempt to Isolate Stimulating Factors

Studies made in our laboratory have demonstrated the presence in atopic serum of enhancing factors(s) for in vitro IgE synthesis by peripheral blood lymphocytes (BPL). Here we show that these enhancing factors can be recovered from human serum by means of immunoabsorbents. This factor is able to stimulate in vitro IgE synthesis by PBL independent of presence of IgE.     

IL-4 和sCD40 L 协同增强体外IgE 的生成

应用 Ｅ Ｌ Ｉ Ｓ Ａ 和 Ｃ Ａ Ｐ 变应原系统, 检测２４ 例对屋尘螨过敏的Ⅰ型超敏反应患者血浆中总 Ｉｇ Ｅ 和特异性 Ｉｇ Ｅ 的水平,以及外周血单个核细胞（ Ｐ Ｂ Ｍ Ｃ） 在 Ｉ Ｌ ４ 、可溶性 Ｃ Ｄ４０ 配体（ｓ Ｃ Ｄ４０ Ｌ） 作用下, 其培养上清液中总 Ｉｇ Ｅ 和特异性 Ｉｇ Ｅ 的水平。结果表明： （１ ） Ⅰ型超敏反应患者血浆中 Ｉｇ Ｅ 水平比正常组高（ Ｐ＜ ００１ ） ; （２ ） 在 Ｉ Ｌ ４ 和ｓ Ｃ Ｄ４０ Ｌ 共同作用下, Ｐ Ｂ Ｍ Ｃ 培养上清液中总 Ｉｇ Ｅ 和特异性 Ｉｇ Ｅ 的水平比 Ｉ Ｌ ４ 或ｓ Ｃ Ｄ４０ Ｌ单独作用下明显升高（ Ｐ＜ ００１ ） 。提示 Ｉｇ Ｅ 的生成不仅需要 Ｉ Ｌ ４ 的作用, 还需要 Ｔ、 Ｂ 细胞接触介导的信号传导或 Ｔ 细胞释放的细胞因子作用, 而ｓ Ｃ Ｄ４０ Ｌ 就是其中的重要因子之一。     

Effect of Cimetidine on Exogenous Histamine Inhibition of Histamine Release in Vivo

We previously reported that exogenous histamine inhibits in vivo histamine release and eosinophil accumulation in ragweed-challenged skin sites of sensitive human subjects. The mechanism(s) involved were unclear. In this study, we repeated similar approaches in four of the same subjects pretreated for 3 days with cimetidine, an H2 receptor antagonist. The pattern of exogenous histamine effects was now different in that local exogenous histamine (50 ng/ml) did not significantly alter ragweed-induced mast cell alteration, histamine release, or the degree of eosinophil accumulation in skin challenge sites. These findings suggest that the observed exogenous histamine inhibitory effects may be mediated through the H2 receptor.     

Reactivity to IgE-dependent histamine-releasing factor is due to monomeric IgE

BACKGROUND: IgE-dependent histamine-releasing factor (HRF) can distinguish between IgE+ and IgE-. In contrast to IgE-, IgE+ sensitizes basophils to release histamine in response to HRF. But we do not know what particular feature distinguishes IgE+ from IgE-. The objective was to investigate the hypothesis that IgE+ is polymeric IgE. METHODS: IgE+ plasma was separated by size-exclusion chromatography. The basophil-sensitizing capacity of the fractions was analyzed in response to HRF produced by mononuclear cells. RESULTS: We showed that monomeric IgE sensitized basophils to release histamine in response to HRF and to house-dust mite, whereas no enhanced reactivity was found in the fractions containing polymeric IgE. CONCLUSIONS: HRF reacts with monomeric IgE, and not (exclusively) with polymeric IgE.     

Human auto-anti-idiotypic antibodies to mite-specific IgE can degranulate human basophils in vitro

Background Anti-idiotypic antibodies (anti-Ids) to specific IgE antibodies are formed spontaneously during an anti-allergen immune response and can be induced by immunotherapy. Although anti-Ids can down-regulate the production of IgF. antibodies, at least in experimental models, their possible role in the modulation of target cell reactivity remains ill-defined. Objective The capacity of human anti-Ids to modulate the release of histamine was examined in an in vitro system of human basophil degranillation. Anti-Ids were prepared from the serum of six Dermatophagoides pteranyssinus (DP)-hypersensitive patients suffering from atopic dermatitis and who had never been desensitized. Basophils were obtained from the blood of atopic donors. The extent of histaminc release was determined using a fluorometric assay. Results We show that: anti-Ids trigger the release of histamine in an allergen-specific, dose- and IgE-dependent manner; the release is not due to the presence of allergen and/ or anti-IgE antibodies: and that the degranulating activity can be removed by absorption with affinity-purified anti-Dp antibodies of the corresponding patient. Conclusion These results indicate that spontaneously produced human anti-Ids can modulate the reactivity of human basophils.     

The Glucocorticosteroid, Budesonide, Partially Blocks Histamine Release from Human Lung Tissue in Vitro

IgE-receptor dependent, but not A23187-induced, histamine release from passively sensitized chopped human lung tissue, or from a dispersed lung cell population obtained from the tissue after enzymatic digestion, is reduced after incubation overnight of cells/tissue with the glucocorticosteroid budesonide (10(-7) M). Since the inhibitory effect of budesonide is reduced in the continuous presence of diluted reagin-rich serum used for passive sensitization, but not in the presence of heat-inactivated serum, it is suggested that the glucocorticosteroid acts by reducing the binding of IgE-antibody to the target cell(s).     

Effects of a Beta-Receptor Blocking Agent (Propranolol) on Synthesis of IgE in vitro by Peripheral Blood Lymphocytes from Atopic Patients

Peripheral blood lymphocytes from patients with atopy were studied for IgE production in vitro. Addition of a beta-receptor blocking agent (propranolol) to lymphocytes from 9 of 10 patients with low spontaneous IgE production in vitro caused increased IgE production. In 10 tests with lymphocytes from patients, who were undergoing hyposensitization treatment for cat epithelium and/or birch pollen allergy, no spontaneous in vitro production of the relevant antigen-specific IgE antibodies was detected. However, when propranolol was added to the lymphocyte cultures in vitro, production of antigen-specific IgE antibodies was found. No such production was found when lymphocytes from patients who were not allergic to either of these antigens were studied. Szentivanyi's theory of a partial blockade of beta-adrenergic receptors in atopy and a possible linkage between this theory and the hypotheses of disturbed regulatory functions in the immune system of patients with atopy is discussed.     

IgE及其介导的Ⅰ型变态反应对免疫复合物在肾脏沉积的影响

本文采用豚鼠血清过敏症的动物模型,以热凝聚IgG(HAG)代替免疫复合物(IC),用直接免疫荧光技术,测检了IC在豚鼠肾脏的沉积情况。结果表明,在血清IgE水平增高的豚鼠和发生血清过敏症的豚鼠的肾脏中,IC的沉积量明显高于对照组。这说明,血清IgG水平增高及其介导的Ⅰ型变态反应均可促进IC在肾脏的沉积。从而提示,IgE及其介导的Ⅰ型变态反应可能参与某些免疫复合物性疾病的发病过程。     

Influence of Neuraminidase and N-Acetylneuraminic Acid on Basophil Histamine Release in Vitro

Since N-acetylneuraminic acid (NANA) in cell membrane glucocalyx mediates or modulates a variety of actions, such as mediator release, we examined a possible modulating role of this amino sugar in histamine release from human basophil leukocytes. Removal of NANA from the cell membrane by the enzyme neuraminidase caused a dose-dependent histamine release. Removal of smaller amounts of NANA enhanced histamine release induced by anti-IgE, Concanavalin A and the calcium ionophore A23187, and reduced the interval between addition of antigen and initiation of histamine release. Pretreatment with free NANA had the opposite effects, i.e. a diminished and delayed maximal histamine release. The hypothesis that NANA in the cell membrane modulates the cellular response to stimulation was further substantiated by demonstrating that the altered response was reflected by a change in the sensitivity of the cell to extracellular calcium. NANA in the cell membrane glucocalyx thus seems to modulate the basophil response to stimulation by modulating transmembraneous calcium transport.     

过敏性鼻炎患者总IgE、嗜酸性粒细胞和特异性IgE结果分析

目的 探讨血清总IgE(immunoglobulin E,IgE)水平及外周血嗜酸性粒细胞(eosinophil,EOS)计数在过敏性鼻炎(allergic rhinitis,AR)诊断中的作用,以及两者与血清特异性IgE(specific immunoglobulin E,sIgE)水平之间的关系。方法选择对蒿属花粉过敏的过敏性鼻炎患者60例作为鼻炎组,60例健康人作为对照组。将鼻炎组分为男性组与女性组,青年组(18~40岁)与中年组(41~65岁),将鼻炎组患者根据sIgE结果分为:中低水平(0.35 IU/mL≤sIgE<3.5IU/mL)、高水平(3.5 IU/mL≤sIgE<17.5 IU/mL)与极高水平(sIgE≥17.5 IU/mL)。结果 鼻炎组总IgE水平和EOS计数均高于对照组,差异有统计学意义( P <0.05);鼻炎组、男性组、女性组、青年组和中年组总IgE与sIgE均成正相关( r s =0.541,0.490,0.599,0.566,0.462,均 P <0.05);鼻炎组患者总IgE与sIgE等级成正相关( rs =0.449, P <0.05)。结论 血清总IgE可作为AR诊断辅助指标,联合检测血清总IgE及sIgE可为AR的诊断提供更充分的依据。     

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.     

Tomato profilin Lyc e 1: IgE cross-reactivity and allergenic potency

BACKGROUND: To date, very little data are available about the nature of tomato allergens. Immunoglobulin E (IgE) cross-reactive profilins have been suggested to account for allergic symptoms in patients suffering from tomato allergy. METHODS: The cDNA of tomato profilin was amplified by reversely transcribed polymerase chain reaction (RT-PCR) from total RNA extracted from ripe tomato fruit. The gene was cloned into the pET101D expression plasmid and the protein was produced in Escherichia coli BL21. Purification was performed via poly-l-proline (PLP) affinity chromatography. IgE reactivity of recombinant tomato profilin was investigated by immunoblot and enzyme-linked immunosorbent assay. IgE-inhibition studies were performed to analyse cross-reactivity with other profilins. To determine the allergenic activity of the recombinant protein, basophil histamine release assays using sera of patients with adverse reactions to tomato were performed. RESULTS: Profilin was identified as a new minor allergen in tomato fruits. The recombinant tomato profilin comprises 131 amino acids and high sequence identity to other allergenic food and pollen profilins. It was shown to be IgE-reactive with a prevalence of 22% (11/50) in tomato-allergic patients. In patients with tomato allergy and multiple sensitization to other foods and birch pollen, IgE directed against tomato profilin showed a strong cross-reactivity with profilins from plant food sources and birch pollen. The tomato profilin was able to induce mediator release from human basophils. CONCLUSION: The tomato profilin is a minor allergen in tomato fruit. Thus, it shows biological activity, as confirmed by in vitro histamine release assays with human basophils and thereby has the potential to account for clinical symptoms in tomato-allergic patients.     

Effect of recombinant human erythropoietin on human IgE production in vitro.

Recombinant human erythropoietin enhanced spontaneous IgE production (200-300% enhancement) in cultures of peripheral blood mononuclear cells (MNC) from atopic patients. In contrast, IgG and IgA production were only slightly enhanced (30-50% enhancement), and IgM production was not affected by erythropoietin. The enhancement of IgE production by erythropoietin was indirect since it required T cells and monocytes. However, erythropoietin effect was specific since enhancement was blocked by anti-erythropoietin antibody but not by control antibody. Interleukin-4 (IL-4) also enhanced spontaneous IgE production from atopic MNC. However, the enhancing effect by erythropoietin is different from that by IL-4, since the erythropoietin effect was not blocked by anti-IL-4 antibody, and conversely IL-4 effect was not blocked by anti-erythropoietin antibody. In contrast to the enhancing effect on atopic MNC, erythropoietin failed to induce IgE production in cultures of MNC from normal donors while IL-4 induced IgE production from normal MNC. However, when normal MNC were pre-incubated with IL-4, erythropoietin enhanced IgE production from IL-4-pre-incubated MNC. Moreover, B cells separated from IL-4-pre-incubated MNC produced IgE which was enhanced by erythropoietin. However, this effect required T cells and monocytes. These results indicate that erythropoietin could regulate ongoing IgE production in vitro by T cell- and monocyte-dependent mechanisms.