IgE hidden in immune complexes with anti-IgE autoantibodies in children with asthma

Serum levels of IgE, anti-IgE autoantibodies (Abs), and IgE/IgG anti-IgE immune complexes (ICs) were measured in 110 children with asthma and 90 healthy control children. Significantly enhanced levels of IgE/anti-IgE IC were detected in children with asthma. However, only a weak correlation was found between anti-IgE auto-Ab serum levels and the degree of lung function abnormalities in children with asthma. However, children with asthma with low serum IgE levels had elevated IC serum levels of IgE/anti-IgE auto-Abs, suggesting that IgE might be hidden within these ICs and is therefore not measurable in vitro. The significant elevation of IgE/anti-IgE IC serum levels raises the question whether IgE within ICs is neutralized or might still be involved in immunologic mechanisms responsible for clinical symptoms of bronchial asthma.     

Rat monoclonal anti-murine IgE antibody removes IgE molecules already bound to mast cells or basophilic leukemia cells,resulting in the inhibition of systemic anaphylaxis and passive cutaneous anaphylaxis

IgE plays a central role in allergic reactions. Some anti-IgE antibodies (HMK-12, 6HD5) inhibit the binding of IgE to the FcepsilonRI of mast cells/basophilic leukemia cells (PT-18, RBL/2H3), but less inhibition is seen with the anti-allotypic JKS-6 and the anti-idiotypic Eb-1. Anti-IgE HMK-12 can detach bound IgE molecules from the FcepsilonRI. When mast cells or basophils were incubated with monoclonal anti-DNP-IgE SPE-7, washed and treated with anti-IgE HMK-12, anti-IgE/IgE complexes were found in the supernatant. Similar results were obtained with the Fab fragment of HMK-12. Mice injected with anti-DNP-IgE SPE-7 and later with DNP-BSA had the typical systemic anaphylactic shock. However, if they were injected with the anti-IgE antibody (HMK-12) before the challenge, they did not get an anaphylactic shock. In the sera of mice injected with monoclonal IgE SPE-7 and anti-IgE antibody (HMK-12), IgE/anti-IgE complexes were detected. No passive cutaneous anaphylaxis occurred if the rats were injected with anti-IgE antibodies before the challenge. In summary, anti-IgE antibodies can remove IgE antibodies from the FcepsilonRI; anti-IgE/IgE complexes can be detected in vitro and in vivo, and anti-IgE antibodies can inhibit IgE-mediated systemic or local anaphylactic reactions.     

Monoclonal anti-human IgE: histamine release capacity and effect onin vitro sensitization of human basophils

IgE-anti-IgE complexes were formed by preincubation of a human myeloma IgE with a monoclonal anti-human IgE antibody at different concentrations. Binding of IgE onto the anti-IgE inhibited the histamine release capacity of anti-IgE from basophils. The IgE cell-binding capacity was altered by the IgE/anti-IgE ratio in the preincubation buffer. Heating of the complexes gave a partial recovery of the anti-IgE degranulating capacity, suggesting that the monoclonal anti-IgE is specific for an epitope present on the D2 domain.     

Natural anti-IgE auto-antibodies interfere with diagnostic IgE determination

A simple method is described which enables measurement of anti-IgE antibodies in free form as well as in immune complexes of IgE and anti-IgE. Anti-IgE antibodies were purified from serum of one selected blood donor with highly elevated levels of such autoantibodies. These purified anti-IgE auto-antibodies inhibited the measurement of myeloma IgE. Purification also revealed that 98% of the subject's serum IgE was masked by anti-IgE auto-antibodies. Our data suggest that IgE determinations in sera containing anti-IgE antibodies might be underestimated.     

IgE autoantibodies in atopic dermatitis -occurrence of different antibodies against the CH3 and the CH4 epitopes of IgE

Levels of "free" anti-IgE autoantibodies and IgE/anti-IgE immune complexes were measured in the sera of patients with atopic dermatitis before and after treatment, psoriasis patients, and nonatopic controls. In this measurement, we used two monoclonal antibodies with distinct in vitro functions (LE 27, BSW 17), directed against the epsilon CH3 and CH4 domains of the IgE Fc-fragment, in a novel immunobinding assay. In patients with atopic dermatitis, elevated levels of "free" anti-IgE antibodies and IgE/anti-IgE immune complexes were detected in comparison to psoriasis patients and controls. In addition, there was a positive correlation between total IgE and the amount of IgE/anti-IgE complexes detected by LE 27 ( r =0.7; P < 0.001) or BSW 17 ( r = 0.64; P < 0.001) in patients with atopic dermatitis. In contrast, an inverse correlation was observed between total IgE and "free" anti-IgE antibodies ( r =−0.34; P < 0.05) in atopic dermatitis. However, serum levels of anti-IgE autoantibodies before and after therapy in patients with atopic dermatitis did not differ, and levels of anti-IgE antibodies did not correlate with clinical severity, as evaluated by an established clinical scoring system. Our data clearly indicate that significantly elevated amounts of anti-IgE antibodies could be observed in patients with atopic dermatitis, which are directed against different epitopes on the IgE molecule. It is tempting to speculate that these autoantibodies exert different effects on IgE-receptor-bearing effector cells and may play an important role in IgE regulation.     

IgG autoantibody to IgE in atopic patients

An IgG type of antibody directed against IgE has been studied in serum from healthy and allergic individuals. The technique used is based on a solid phase paper radioimmunoassay in which the discs were sensitized with purified IgE myeloma. After incubation with patients' serum, human IgG labeled with iodine 125 was added. The anti-IgE antibodies were partially blocked by endogenous IgE in the serum and heating the serum samples at 56 degrees C disrupted the immune complexes (ie, IgG-aIgE:IgE), thereby increasing the detectable levels of IgG anti-IgE. The specificity of anti-IgE autoantibody was confirmed by both competitive inhibition and absorption experiments, using IgG, IgM, IgA, IgE, and rabbit anti-human IgG. Significantly raised levels of anti-IgE autoantibody were found in patients suffering from atopic disorders in comparison to the controls. These observations may suggest that the anti-IgE autoantibody could play a certain role in the modulation of IgE-mediated immune system and the pathogenesis of atopic diseases.     

Molecular and cellular targets of anti-IgE antibodies

Immunoglobulin E (IgE) was the last of the immunoglobulins discovered. It is present in very low amounts (nano- to micro-gram per ml range) in the serum of normal healthy individuals and normal laboratory mouse strains and has a very short half-life. This contrasts with the other immunoglobulin classes, which are present in much higher concentrations (micro- to milligram per ml range) and form a substantial component of serum proteins. Immunoglobulins play a role in homeostatic mechanisms and they represent the humoral arm of defence against pathogenic organisms. Since IgE antibodies play a key role in allergic disorders, a number of approaches to inhibit IgE antibody production are currently being explored. In the recent past the use of nonanaphylactic, humanized anti-IgE antibodies became a new therapeutic strategy for allergic diseases. The therapeutic rational beyond the idea derives from the ability of the anti-IgE antibodies to bind to the same domains on the IgE molecule that interact with the high-affinity IgE receptor, thereby interfering with the binding of IgE to this receptor without cross-linking the IgE on the receptor (nonanaphylactic anti-IgE antibodies). Treatment with anti-IgE antibodies leads primarily to a decrease in serum IgE levels. As a consequence thereof, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells reducing the release of inflammatory mediator such as histamine, prostaglandins and leukotrienes. Experimental studies in mice indicate that injection of some monoclonal anti-IgE antibodies also inhibited IgE production in vivo. The biological mechanism behind this reduction remains speculative. A possible explanation may be that these antibodies can also interact with membrane bound IgE on B cells, which could interfere the IgE production.     

Effect of a serum factor on IgE-mediated histamine release from whole blood

IgE-mediated histamine release from whole blood was analyzed in 44 patients with bronchial asthma by observing maximum present release and dose-response curves of histamine release induced by anti-IgE and house dust extract. The maximum histamine release from whole blood induced by anti-IgE correlated with total serum IgE levels. There was a close correlation between allergen-induced release from whole blood and the serum levels of specific IgE antibodies. In the maximum histamine release from whole blood induced by both anti-IgE and allergen, the interaction with a serum factor was not clearly recognized. Effect of a serum factor was shown in the dose-response curves of anti-IgE-induced histamine release, but not in those of allergen-induced histamine release. The dose-response curves caused by anti-IgE showed that basophils from cases with a high serum IgE level require much more anti-IgE to produce maximum histamine release than basophils from cases with a low serum IgE level. The results showed that IgE molecules contained in the serum participate in anti-IgE-induced histamine release from whole blood.     

Mimotope and anti-idiotypic vaccines to induce an anti-IgE response

We have defined epitopes on human IgE by screening different phage display random peptide libraries with a monoclonal anti-IgE antibody termed BSW17. The selected mimotopes and epitopes within the Cepsilon3 and Cepsilon4 region of IgE induced antibodies that were nonanaphylactogenic and had biological activity similar to BSW17. The chemically synthesized and KLH-coupled IgE epitopes or mimotopes were used to induce an anti-IgE response in rhesus monkeys. The immunized rhesus monkeys were subsequently protected in a PCA test when sensitized with human IgE and triggered with the corresponding allergen. Furthermore, using the same monoclonal anti-IgE antibody, we also generated an anti-idiotypic antibody that showed sequence homology with the IgE epitope in the Cepsilon3 domain. This anti-idiotypic antibody as well as the mimotopes were then used in a mouse model to induce orally an anti-IgE immune response. For this purpose mice were fed by intragastric gavages with bacteriophages displaying the small IgE-homologous structures. Orally immunized mice produced serum anti-IgE antibodies that were inhibited by BSW17 suggesting that it may be possible to induce a systemic anti-IgE response orally.     

Mouse and rat IgE Cross-sensitization of mast cells and antigenic relationships.

Mouse and rat IgE fix firmly to the peritoneal mast cells from the other species, sensitizing them for anaphylactic reaction. Sensitization with IgE can be demonstrated by inducing degranulation either with specific antigens or with corresponding anti-IgE. Sensitization of rat mast cells by mouse IgE antibodies is more easily obtained than that of mouse mast cells by rat IgE antibodies. In this case, anti-IgE-induced degranulation is higher than antigen-induced degranulation. Heterologous sensitization by IgE is time requiring and temperature-dependent. Its kinetics depend upon IgE concentration. Cross-reactions between IgE from one species and anti-IgE from another species have been observed: anti-IgE for one species is able to neutralize PCA reaginic activity of sera from the other species; anti-rat IgE induces degranulation of mouse actively sensitized mast cells. The results suggest strongly that there exists a structural and functional similarity between the IgE molecules from the two species.     

Are allergen-specific IgG mainly IgG anti-IgE autoantibodies?

A large proportion of specific IgE occurred in immune complexes with anti-IgE autoantibodies in sera from nonhyposensitized allergic patients. These autoantibodies were misinterpreted as 'specific IgG' in different immunoassays such as dot immunoassays and the radioallergosorbent test (RAST), leading to overestimation of specific IgG. Purified immune complexes contained even more IgG than IgE antibodies. Heating of the complexes liberated specific IgE, producing an upwards RAST class shift. Thus anti-IgE autoantibodies are hiding the specific IgE, which is thereby underestimated. It is not known whether the hiding anti-IgE autoantibodies are also effectively neutralizing circulating or cell-bound IgE and might represent the actual blocking antibody.     

Differences in response to anti-IgE and to anti-IgG in basophils from patients with bronchial asthma

Peripheral blood basophils of thirty-eight patients with bronchial asthma were examined for their reactivity to anti-IgE and anti-IgG antisera. Basophils of patients with serum IgE levels of more than 1001 i.u./ml reacted strongly to anti-IgE. Basophils of patients with serum IgE levels of less than 100 i.u./ml had a tendency to react more strongly to anti-IgG. An index (basophil ratio) was devised to compare the patient basophil reactivity to anti-IgE and anti-IgG. This basophil ratio was lower (IgE dominant) in the atopic cases which usually exhibited a high serum IgE level. Most cases with low serum IgE exhibited a high basophil ratio (IgG dominant). The basophils of seven intractable patients reacted more strongly to anti-IgG than to anti-IgE regardless of the serum IgE level.     

Effect of anti-IgE antibodies on IgE binding to CD23

Human sera contain anti-IgE autoantibodies. Using a human B lymphoblastoid cell line (Wil-2WT cells) and monoclonal murine anti-IgE antibodies (BSW17 and Le27) we investigated a possible role of such anti-IgE antibodies. A 100-fold excess of monoclonal anti-IgE antibodies inhibited binding of 125I labeled IgE to Fc epsilon RII on Wil-2WT cells. Further, both monoclonal anti-IgE antibodies dissociated surface bound IgE from Fc epsilon RII on Wil-2WT cells. However, BSW17 which does not trigger histamine release from human leucocytes, was much more effective in dissociating Fc epsilon RII bound IgE than Le27 which triggers histamine release. These results may suggest that naturally occurring IgG anti-IgE antibodies are able to inhibit binding of IgE to its receptor.     

Circulating IgG autoantibodies to IgE in atopic syndromes

Sera from nonatopic healthy donors and patients with hyper-IgE syndrome, allergic respiratory disease, i.e., allergic rhinitis and asthma, and atopic dermatitis were assayed for the presence of IgG and IgM antibodies to IgE. The assay used was based on an ELISA method that measured the binding of IgG or IgM in test sera to myeloma IgE (PS)-coated microtiter wells. The levels of IgG anti-IgE but not of IgM anti-IgE were elevated in patient sera of all three categories tested. The same sera failed to demonstrate increased levels of IgG anti-IgM or IgG anti-IgA. Significant IgG anti-IgE activity remained after absorption of patient sera over pooled human IgG F(ab')2 Sepharose. The IgG anti-IgE activity appeared to be directed toward the Fc portion of IgE because absorption of positive sera over IgE (ADZ) Sepharose but not over myeloma IgG Sepharose completely removed their reactivity with IgE (PS) and because sera from atopic individuals but not from normal subjects contained IgG anti-IgE activity against the protein backbone of the Fc portion of IgE synthesized from a fragment of the cloned gene of human myeloma IgE (ND) heavy chain. Regression analysis demonstrated a weak but significant correlation (r = 0.31; p less than 0.05) between serum IgE levels and IgG anti-IgE activity. Fractionation of sera from the three patient categories by gel filtration over Sepharose 6B revealed that IgG anti-IgE activity was present both as monomeric IgG and in IgE containing immune complexes (IC). Intermediate molecular size IC (between 7S and 19S) were present in all three patient groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Administration of an anti-IgE antibody inhibits CD23 expression and IgE production in vivo.

High IgE responder BDF1 mice were immunized intraperitoneally (i.p.) with dinitrophenol4 (DNP4)-ovalbumin (OVA) in alum concomitant with intravenous (i.v.) administration of an anti-IgE monoclonal antibody (mAb). IgE levels were undetectable in mice treated with the anti-IgE antibody, whereas mice treated with isotype-matched irrelevant mAb had IgE levels comparable to that of untreated, immunized mice. Subsequent antigen challenges with DNP4-OVA, either at weekly or monthly intervals, failed to evoke an IgE response for greater than 2 months in mice treated with anti-IgE during the primary sensitization, even though the terminal half-life of the anti-IgE antibody was 7 days. This inhibition was specific for DNP4-OVA since the DNP4-OVA-suppressed mice were able to respond to keyhole limpet haemocyanin (KLH). To investigate the effects of antibody treatment at the cellular level, passive transfer experiments were performed. The primary DNP-specific IgE response of adoptive transfer recipient mice was the same whether the donor cells were from mice treated with IgG or anti-IgE. Transfer of enriched T- or B-cell populations indicated that T-cell help was not compromised by administration of the anti-IgE mAb. However, splenocytes from the anti-IgE-treated mice failed to synthesize IgE in vitro, and flow cytometric analysis of B cells from anti-IgE-treated mice showed a dose-dependent decrease in CD23+ cells following antibody treatment, which correlated with decreased serum IgE levels. Taken together, the results of these studies suggest that anti-IgE treatment suppresses IgE responses via effects on B cells rather than T cells, possibly through effects on CD23-dependent pathways.     

Rabbit F(ab')2 antihuman IgE is a universal skin test reagent in the evaluation of skin mast cell degranulation in vivo

Antihuman IgE is often used to study basophil- and mast cell-mediator release in vitro but is infrequently used in vivo. To evaluate in vivo skin reactivity to anti-IgE, an affinity-purified rabbit F(ab')2 fragment of antihuman IgE was injected intradermally in 22 nonallergic and 27 allergic subjects. All 49 subjects (including a subject with less than 1 ng/ml of total serum IgE) had positive immediate cutaneous reactions to anti-IgE. Although total serum IgE level was weakly correlated (r = -0.51; p less than 0.005) with in vivo skin reactivity to anti-IgE for the entire population, allergic subjects did not have significantly increased skin reactivity compared to nonallergic subjects (p = 0.18), despite having higher total serum IgE levels (p less than 0.002). A late-phase cutaneous response (LPR) to anti-IgE occurred in 60% of the allergic and in 50% of the nonallergic subjects. Subjects with an LPR required approximately tenfold higher concentrations of anti-IgE to produce an immediate wheal of 10 mm compared to subjects who did not develop an LPR (p = 0.02), suggesting that the concentration of the stimulus injected is more important for the development of a LPR than the size of the immediate cutaneous response. Skin reactivity to codeine phosphate (a non-IgE-dependent secretagogue) was correlated with skin reactivity to anti-IgE (r = 0.47; p less than 0.05), suggesting that in vivo skin mast cell degranulation is partially a function of mast cell releasability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Do IgE-IgG complexes occur in the circulation?

IgG anti-IgE autoantibodies in human allergic sera have been investigated using an enzyme immunoassay on microplates coated with anti-human IgE MoAb. After incubation with serum, the plates were developed with peroxidase-labelled anti-human IgE MoAb for the determination of total IgE levels, or with anti-IgG MoAb for evaluating IgG anti-IgE autoantibodies. Using this methodology, no correlation was found between total IgE and IgG anti-IgE levels in groups of sera of allergic individuals. Although the results obtained with enzyme-labelled anti-IgG are often interpreted as indicative of IgE-IgG complexes captured from the serum, molecular sieving on gel columns as well as direct ultrafiltration experiments through 300-kD membranes demonstrate that such complexes do not occur preformed in the circulation, but arise de novo on the anti-IgE-coated solid phase during in vitro incubation with human serum. It is suggested that IgG anti-IgE autoantibodies react with IgE only after the latter has undergone a conformational change, either by colloidal manipulation or after reaction with allergen.     

Dot-immunobinding assay with monoclonal anti-IgE antibodies for the detection and quantitation of human IgE

This paper describes a dot immunobinding assay for determining total human IgE with a tandem of monoclonal anti-IgE antibodies. Minute quantities of monoclonal anti-IgE antibodies were adsorbed on nitrocellulose discs. IgE bound to this solid phase monoclonal anti-IgE antibody was detected by a second monoclonal antibody conjugated with horseradish peroxidase. Using 4-chloro-1-naphthol as a chromogen results in a stable colour reaction that can be semiquantitatively analysed by the naked eye. The colour intensities of the reaction were also analysed by densitometry, yielding a very reproducible quantitation of human serum IgE. Using a serum dilution of 1:50, IgE could be detected in the range of 12.5-2500 U/ml. Using non-diluted serum samples IgE levels between 0.05-50 U/ml were reproducibly measured. Total serum IgE as determined by this dot assay correlated very well with IgE determinations performed by the commercial PRIST assay.     

IFN-gamma potentiates the release of TNF-alpha and MIP-1alpha by alveolar macrophages during allergic reactions

Viral infections play an important role in the exacerbation of asthma. The production of interferons (IFNs) is well known to limit viral spread, but IFN-gamma can also prime alveolar macrophages to release more inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha). Given the importance of these cytokines, we have investigated the effect of IFN-gamma on their release by alveolar macrophages during stimulation by immunoglobulin (Ig)E/anti-IgE. Alveolar macrophages from normal or Nippostrongylus brasiliensis-infected rats, the latter having increased numbers of low-affinity receptors for IgE (Fcepsilon RII) on their alveolar macrophages, were treated with IgE for 2 h and stimulated with anti-IgE for 18 h. The increase of TNF-alpha release (153 +/- 48 pg/10(6) cells) by IgE/anti-IgE occurred only with alveolar macrophages from infected rats. The messenger RNA level for TNF-alpha in rat alveolar macrophages was also increased by stimulation with IgE/anti-IgE. Treatment with IFN-gamma prior to stimulation with IgE/anti-IgE showed a time- and concentration-dependent increase of TNF-alpha release. Interestingly, IgE/anti-IgE treatment did not stimulate the release of MIP-1alpha (15 +/- 5 pg/10(6) cells), but IFN-gamma treatment alone and with IgE /anti-IgE significantly increased and potentiated MIP-1alpha release (98 +/- 40 pg/10(6) cells) by alveolar macrophages, respectively. These results suggest that IFN-gamma produced at times such as during viral infections primes alveolar macrophages for enhanced release of inflammatory mediators during allergic reactions, thereby contributing to the inflammatory process.     

Serum and cell bound IgE in chronic urticaria

Basophil leucocyte-bound IgE has been investigated in patients with chronic urticaria by the method of reversed anaphylaxis. In this reaction basophil-bound IgE behaves as an antigen, the amount present being inversely proportional to the concentration of anti-IgE producing maximum histamine release. Dose-response curves in which histamine release was plotted against the concentration of anti-IgE failed to reveal any substantial difference in the optimum concentration of anti-IgE for maximum release from basophils of urticarial subjects compared with control subjects, thus suggesting there is no quantitative abnormality of basophil bound IgE in chronic urticaria. However, the magnitude of maximum histamine release by anti-IgE from basophils of urticarial subjects was reduced compared with controls and this was not related to serum IgE concentrations since the mean serum IgE concentration was slightly higher in the urticaria group. Studies of spontaneous and compound 48/80-evoked histamine release in the two groups did not reveal any differences in stability of histamine stores or in the biochemical histamine release mechanism over a wide range of concentrations of 48/80. These results raise the possibility of a qualitative abnormality of basophil bound IgE in chronic urticaria.