Tolerizing effect of DNP-ficoll on IgE antibody production.

A/J and DBA/1 mice were infected with 750 third-stage larvae of Nippostrongylus brasiliensis and immunized with 1 mug dinitrophenylated N. brasiliensis extract (DNP-Nb) with 1 mg Al(OH)3 to produce high titers of anti-hapten IgG1 and IgE antibody. Partial tolerance to the production of anti-hapten IgG1 and IgE antibody could be induced by DNP-Ficoll from 5 weeks before to 1 week after the DNP-Nb immunization. The tolerized state persisted through the duration of the experiments. However, no tolerizing effect could be demonstrated on secondary antihapten IgE antibody production induced by DNP-Nb. Moreover, DNP-Ficoll failed to evoke anti-hapten IgG1 or IgE antibody production.     

Cytotoxic T-cells specific for natural IgE peptides downregulate IgE production

Immunoglobulin E (IgE) plays a central role in IgE-mediated immediate type hypersensitivity. Since production of IgE depends on Th2, efforts to block IgE production and control allergic reactions include tolerization of Th2 or deviating development of Th2. We hypothesized that cytotoxic T lymphocytes targeting natural IgE peptides/MHC I complexes can eliminate IgE-producing cells and inhibit centrally IgE production. CTL to self-IgE peptides were elicited in mice immunized with nonameric p109-117, p113-121, and p103-141 (CHepsilon2 domain), which encompass both peptides with an OVA helper peptide (OVAp restricted for H-2d/b) in liposomes and presented by dendritic cells (DC). CTL from BALB/c lysed IgE peptide-pulsed P815 target as well as IgE-producing 26.82 hybridomas (H-2d). Natural tolerance to self-IgE peptides was tested in IgE sufficient (IgE +/+) as well as IgE-deficient (IgE -/-) 129/SvEv mice (H-2b). Comparable magnitude of CTL responses was observed in both strains immunized with p109-117 or p103-141 concomitantly with CD4 T-cell costimulation. CTL from 129/SvEv lysed not only IgE peptide-pulsed EL-4 but also IgE-producing B4 hybridomas (H-2b). This observation strongly suggests a correspondence of epitope of immunogenic peptide to that of physiologically processed IgE peptides presented on IgE-producing cells. Moreover, CTL were generated in 129/SvEv, immunized with the recombinant antigenized antibody in liposomes encompassing p107-123, p109-117, and p113-121 expressed in CDR3 of VH62/human gamma1. Polyclonal IgE production was inhibited by coincubation with MHC I-restricted CTL in vitro. Furthermore, antigen-specific IgE responses were inhibited in mice, immunized with p109-117 and p103-141 while IgG responses were not suppressed. Since IgE peptide sequences of CHepsilon2 are ubiquitous to all murine IgE heavy chain, peptides made as such can serve as a universal IgE vaccine to prevent allergy for a myriad of allergens in rodents. This observation suggests that similar human IgE peptides should be identified and employed to downregulate human IgE production.     

Regulation of IgE production requires oligomerization of CD23.

Here we describe the production of a rabbit polyclonal Ab (RAS1) raised against the stalk of murine CD23. RAS1 inhibits release of CD23 from the surface of both M12 and B cells resulting in an increase of CD23 on the cell surface. Despite this increase, these cells are unable to bind IgE as determined by FACS. CD23 has previously been shown to bind IgE with both a high (4-10 x 10(7) M(-1)) and low (4-10 x 10(6) M(-1)) affinity. Closer examination by direct binding of (125)I-IgE revealed that RAS1 blocks high affinity binding while having no effect on low affinity binding. These data support the model proposing that oligomers of CD23 mediate high affinity IgE binding. These experiments suggest that RAS1 binding to cell surface CD23 results in a shift from oligomers to monomers, which, according to the model, only bind IgE with low affinity. These experiments also suggest that high affinity binding of IgE is required for IgE regulation by CD23 and is demonstrated by the fact that treatment of Ag/Alum-immunized mice treated with RAS1 results in a significant increase in IgE production similar to the levels seen in CD23-deficient mice. These mice also had significantly decreased levels of serum soluble CD23 and Ag-specific IgG1. RAS1 had no effect on IgE or Ag-specific IgG1 production in CD23-deficient mice.     

Regulation of murine IgE production in SJA/9 and nude mice. Potentiation of IgE production by recombinant interleukin 4

Serum IgE levels were determined in different strains of mice with enzyme-linked immunosorbent assay by using rat monoclonal anti-murine IgE antibodies in normal and in Nippostrongylus brasiliensis-infected mice. After infection, serum IgE levels were high in BALB/c and CB-20, low in SJL/J and SJA/20 mice, and not detected at all in SJA/9 and nude mice. Surface IgE-positive cells were greatly increased in BALB/c and SJL/J mice after infection, but not in SJA/9 and nude mice. Most surface IgE-positive spleen cells were also surface IgM- and surface IgD-positive. When spleen cells from SJA/9 or nude mice were stimulated in vitro with lipopolysaccharide and recombinant interleukin 4 (formerly B cell-stimulating factor 1), IgE was produced and detected in the supernatants of these cultures. In addition, surface IgE-positive cells could be detected in these cultures. Most of the surface IgE-positive cells were surface IgM- and surface IgD-negative, unlike those seen in the spleens of Nippostrongylus-infected BALB/c and SJL/J mice. These observations show that SJA/9 and nude mice have IgE-producing precursor B cells, and after appropriate stimulation interleukin 4 can induce them to secrete IgE.     

Studies on IgE production in mice

IgE levels after Nippostrongylus brasiliensis infestation were high in all strains of mice examined except in the SJA/9 strain. However, surface IgE levels on B cells were not different from those observed in the SJL strain. Most surface IgE-bearing cells also had surface IgM molecules. We suggest that the surface IgE-bearing cells are precursors of IgE-producing cells. In adoptive transfer, hapten-specific IgE antibody was produced when hapten-primed cells from SJA/9 mice were transferred together with cells from SJL donors infested with N. brasiliensis but not when the cells were from SJA/9 donors. It is suggested that the lack of collaboration between hapten-primed B cells and cells from infested mice is a genetic trait of the SJA/9 strain.     

Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.     

Suppression of IgE antibody production in SJL mice. V. Effect of irradiation and adult thymectomy on the suppression of IgE antibody production in SJL mice

Anti-DNP IgE antibody production was low and transient in SJL mice which were immunized with 1 microgram DNP-Nb and 1 mg A1(OH)3. The immunized SJL mice were irradiated (60-540 R) 1 day after challenge. A dose higher than 180 R induced enhancement of anti-DNP IgE antibody production as compared to nonirradiated control mice, suggesting the existence of irradiation-sensitive suppressor cells. Anti-DNP IgE antibody production was suppressed when immunized and irradiated SJL mice were injected with spleen cells from adult-thymectomized SJL mice. The donors of the spleen cells were thymectomized 2 or 4 months previously, and this suggests that the suppressor cells from unprimed mice are long-living T cells.     

IgE peptide-specific CTL inhibit IgE production: a transient IgE suppression model in wild-type and HLA-A2.1 transgenic mice

Effect of IgE peptide-specific CTL on IgE antibody production was studied in mouse models. CTL elicited in B6.A2Kb tg mice against a human IgE peptide nonamer, pWV, lysed human IgE-secreting U266 myeloma cells and inhibit IgE production by these cells. U266 transfected with mouse A2Kb transgene (U266-A2Kb) were optimally lysed by these CTL, because the α3 domain of A2Kb interacts well with the CD8 co-receptors. The CTL generated were more effective in inhibiting IgE production by U266-A2Kb cells than lysing these cells. IgE production by and progression of U266 myeloma were suppressed in B6.A2Kb tg mice rendered tolerant to these cells and vaccinated with pWV along with CpG. We also studied the CTL response elicited in wild-type mice by a mouse nonameric IgE peptide, PI-1, along with CpG. This treatment caused a transient suppression of the IgE response in mice previously sensitized to an antigen. In mice treated with this regimen repeatedly, the IgE response was fully recovered 20 days after each treatment. Notably, while IgE peptide/CpG-treated mice remained unresponsive to antigen challenge in vivo, antigen-specific IgE production can be elicited by antigen in cultured splenocytes from these mice. Moreover, IgE peptide/CpG also inhibited an on-going IgE response, including IgE production by bone marrow cells. Taken together, these observations indicate that a CTL-based IgE peptide vaccine targeting IgE-secreting B/plasma cells may be safely employed as a therapeutic approach for suppressing IgE production.     

IL-4 can correct defective IgE production in SJA/9 mice.

SJA/9 mice are unable to produce significant serum IgE responses to either primary or secondary challenge with protein Ag or helminth parasites. Previous work has shown that this defect resides the T cell rather than with the B cells. These experiments test several possible explanations for the lack of IgE production in these mice. SJA/9 mice do not appear to overproduce IFN-gamma or any other antagonist of IL-4 function. Helminth parasite infection induces normal levels of IL-5-dependent eosinophilia, suggesting that there is not a complete absence of a Th2 response in these mice. The defect, however can be explained by a substantial reduction in IL-4 production in vivo and can be corrected by infusion with IL-4. The reason for this lack of IL-4 production is not yet clear and, paradoxically, SJA/9 T cells can be stimulated in vitro to produce IL-4 levels comparable to T cells from other strains.     

Capacity for IgE antibody production in ASK mice

The capacity for IgE anti body production in ASK mice, which are highly sensitivity to anaphylactic shock, was compared with that in C3H and AKR mice. Three strains of mice were immunized with DNP-Ascaris mixed with aluminum hydroxide gel. IgE antibody to DNP in the sera was titrated by the rat 48-hour passive cutaneous anaphylaxis test. Maximum IgE titers of each strain of mice were 1: 2560 in C3H, 1: 1280 in ASK and 1: 640 in AKR. IgE antibody was detected in the sera until 170 days in C3H, 290 days in ASK and for not less than 320 days in AKR. These results suggest that the ASK mouse is a high responder strain for IgE antibody.     

Inhibition of IgE production in B hybridomas by IgE class-specific suppressor factor from T hybridomas

The function of IgE class-specific suppressor factor (IgE-TsF) from T hybridomas was studied by employing IgE-producing B hybridomas. IgE-TsF was obtained from IgE class-specific T hybridomas, which had been established by the fusion of a phosphorylcholine-conjugated Mycobacterium-primed T cell population with the T lymphoma cell line BW5147. The absorption experiments showed that IgE-TsF from T hybridomas was composed of the binding site(s) for IgE and I region gene products as observed in conventional IgE-TsF. Incubation of IgE-producing B hybridomas with IgE-TsF for 1 hr at 37 degrees C resulted in the reduction of the number of IgE-secreting cells when assessed by a reverse plaque assay. The proportions of surface IgE-positive cells were concomitantly reduced. After 24 hr incubation with IgE-TsF, the number of cytoplasmic IgE-positive cells was reduced, showing that IgE synthesis was inhibited by IgE-TsF. Antigen-specific TsF from phosphorylcholine-specific T hybridomas did not show any inhibitory effect, and IgE-TsF did not block the antibody production of IgM-producing B hybridomas. Precapping of IgE receptors by anti-epsilon antibody or the simultaneous addition of soluble IgE with IgE-TsF abrogated the suppressive function, suggesting that IgE-TsF acted directly on B epsilon cells through binding with IgE receptors.     

Interleukin-13 and IgE production in rat experimental schistosomiasis

We have previously demonstrated in rat experimental schistosomiasis an upregulation of IL-4 expression at the mRNA and protein levels which could explain, at least in part, the increased IgE production observed during infection. Using this model, we have investigated the expression of IL-13 which is also involved in the induction of the IgE response. In the present study, we have shown a significant increase in IL-13 mRNA expression in spleen, liver and lungs following primary and secondary infection. IL-13 protein was detected by intracellular staining in spleen cells from infected rats, and in the supernatants of antigen-stimulated spleen cells. Furthermore, circulating levels of IL-13 were increased in sera from infected rats as compared to those from non-infected control animals. These findings show that, similarly to IL-4, IL-13 is upregulated and secreted during rat schistosomiasis, suggesting an involvement of both cytokines in IgE induction. In the in vivo experiments, only rats cotreated with neutralizing anti-IL-4 and anti-IL-13 antibodies showed significant decrease in the IgE levels. Moreover, administration of IL-13 enhanced total IgE levels. These results demonstrate the implication of IL-4 and IL-13 in vivo in IgE production, and provide a relevant animal model for a better understanding of the role of IL-4 and IL-13 in humans.     

Dual mechanisms of potentiation of murine antigen-specific IgE production by cyclosporin A in vitro.

Concomitant administration of cyclosporin A (CsA) with Ag has been shown to augment the production of Ag-specific IgE in vivo. We demonstrate that addition of CsA also markedly potentiated Ag-specific IgE in vitro. Low doses of CsA (3 and 10 ng/ml) added at the time of culture initiation selectively enhanced Ag-specific IgE but not IgA or IgG1 production, whereas higher doses (30 ng/ml) suppressed production of all the isotypes. Augmented IgE production was found to correlate with enhanced production of IL-4 and diminished production of IFN-gamma. Delayed addition (after 2 days) of low doses of CsA to Ag-stimulated cultures did not potentiate IgE production, even though CsA differentially affected levels of IL-4 and IFN-gamma. CsA enhanced Ag-mediated cognate T/B interaction was not affected by neutralizing doses of anti-IL-4, suggesting Ag-mediated lymphocytic "synapses" may be inaccessible to anti-IL-4. The effect of CsA on Ag presentation was determined by pulsing peritoneal exudate cells, spleen cells, or primed B cells with Ag and low doses of CsA before incubation with primed splenocytes. Enhanced Ag-specific IgE responses were detected with no effect on IL-4 or IFN-gamma levels. Thus, our study indicates that CsA potentiation of Ag-specific IgE response is due to cumulative action of CsA on two independent pathways: first, CsA differentially modulates IL-4 and IFN-gamma levels during the early phase of cognate Th2/B cell interaction; and second, CsA directly affects APC and IgE isotype-specific amplifying cellular components without apparently affecting the secretory levels of IL-4 and IFN-gamma. Dual mechanisms of CsA-potentiated IgE production are consistent with the hypothesis of two-tiered T cell regulation of Ag-specific IgE responses.     

Suppression by IL-2 of IgE production by B cells stimulated by IL-4.

IgE production was obtained from B cells of BALB/c or nude mice when these cells were cultured with IL-4 plus LPS. IL-2 added to these cultures at the start (day 0), 1 or 2 days later completely suppressed the production of IgE. The production of IgG1 was also inhibited, but only if IL-2 was added on day 0. The production of other isotypes (IgM, IgG2a, IgG2b) was only slightly decreased by addition of IL-2. No suppression of IgE or IgG1 production was observed if monoclonal anti-IL-2 was added, whereas anti-IFN-gamma had no effect on the suppression of the production of these isotypes. The expression of CD23 on the third day of culture on B cells stimulated with LPS and IL-4 was markedly decreased when IL-2 was added to the cultures on day 0. Addition of monoclonal anti-IL-2 suppressed all effects produced by IL-2, whereas addition of anti-IFN-gamma had no effect. These results show that the suppression by IL-2, at least for the first signaling processes, are different from the suppression produced by IFN-gamma.     

Mechanisms of IgE homeostasis. Sequestration of IgE by murine type II IgE Fc receptor-bearing B cell hybridomas.

Among all classes of Ig, IgE exhibits the highest rate of fractional catabolism of which the site and mechanisms is not understood. We construct a panel of murine B cell hybridomas to investigate the catabolism of IgE; one of these hybridomas, 17A11, constitutively expresses high levels of type II IgE FcR (Fc epsilon RII, CD23) (Kd:1.77 nM; B max: 1.65 x 10(5], and is capable of clearing receptor-bound IgE. Receptor-mediated endocytosis of IgE ligand ensues after binding monomeric and DNP-BSA:IgE immune complexes, and the binding is inhibited by treating 17A11 with anti-CD23. IgE ligands are sequestered and are not susceptible to acid stripping from the cell surface. The internalized IgE ligands redistributed into acid hydrolase containing high density lysosomal vesicles and were degraded; metabolic inhibitors such as chloroquine and monensin that elevate intracellular pH of 17A11 also prevent entry of IgE ligand into lysosomes. These observations raise the possibility that normal Fc epsilon RII-bearing mature B cells in the circulation and lymphoid tissues may function in sequestration and catabolic turnover of IgE molecules through IgE or IL-4 up-regulated Fc epsilon RII uptake; B cell Fc epsilon RII may perform an important role in determining the short biological half-life of IgE molecules, and contributes to IgE homeostasis.     

Regulation of antigen-specific versus by-stander IgE production after antigen sensitization

IgE is critical in the pathogenesis of allergic disorders. In this report, we investigated the differential regulation of antigen-specific and by-stander IgE. Ovalbumin (OVA) immunization did not increase IgE producing cells in the spleen, but significantly enhanced the intracellular IgE content of all IgE+ cells. In contrast, OVA induced a significant increase of IgE+ cells in the draining lymph nodes (LN). Furthermore, OVA-specific IgE was detected only in the ex vivo cultures of the draining LN but not the spleen cells, while total IgE was increased in both cultures. These results indicated that antigen-specific IgE was mainly produced in the draining LN, while the spleen was a major source for by-stander IgE. Anti-IL-4, but not anti-IL-13, antibody blocked the expansion of IgE producing cells in the draining LN as well as systemic OVA-specific and total IgE levels, indicating IL-4 was important in both antigen-specific IgE generation and total IgE upregulation.