IgE responses to exogenous and endogenous allergens in atopic dermatitis patients under long‐term systemic cyclosporine A treatment

Atopic dermatitis (AD) patients mount IgE antibody responses to a variety of environmental allergens and also to autoantigens. We analyzed serum samples from four AD patients who had received oral cyclosporine A (CyA) treatment for up to 17 months regarding IgE autoreactivity to nitrocellulose‐blotted human epithelial cell extracts and IgE levels to environmental allergens by quantitative ImmunoCap measurements. Skin inflammation was assessed by SCORAD. During full‐dose treatment, a strong reduction in T‐cell‐mediated skin symptoms was observed which reappeared when CyA treatment was reduced or stopped. The intensity of IgE autoreactivity seemed to follow skin inflammation as it was reduced during full‐dose treatment and increased upon inflammation. Interestingly, IgE levels to exogenous allergens were boosted by allergen exposure, declined thereafter, and seemed to be unaffected by CyA. Our data thus indicate that allergen‐specific IgE production is boosted by allergen contact and cannot be reduced by CyA‐mediated T‐cell suppression.     

Antigenic competition in IgE antibody production. I. Establishment of parameters involved in primary and secondary responses.

Antigenic competition was demonstrated in IgE antibody response in mice immunized with ovalbumin or DNP-ovalbumin associated with several non-related proteins: DNP-Ascaris, DNP-keyhole limpet haemocyanin or Ascaris. Simultaneous injection of two antigens caused a suppression of IgE antibody production to the test antigen, IgG1 antibody formation being only diminished under certain conditions. Competition was dose-dependent and effective only in the primary response. However, the secondary response could be also partially suppressed if the competitor antigen was given in both first and second antigenic stimulation. Competition was abolished by irradiation prior to immunization.     

In vitro IgE production by interleukin 4-stimulated human peripheral blood mononuclear cells is suppressed by rapamycin

Rapamycin (RAPA) is a new immunosuppressant which is 50-fold to 100-fold more potent than cyclosporin A (CyA) in inhibiting cellular immune responses and allograft rejection in animal models of organ transplantation. The drug's effect on in vitro IgE synthesis by interleukin (IL)4-stimulated human peripheral blood mononuclear cells was examined and compared with CyA's effect in this study. RAPA was found to be about 100-fold more potent than CyA in inhibiting IgE synthesis. Its inhibitory effect on IgE production was significant if it was added to the culture before Day 6 of a 14-day culture. The suppression was accompanied by the inhibitory effect on cell proliferation and on IgE-binding factor (IgE-BF) production. IL2 was able to partially reverse CyA- but not RAPA-induced inhibition of IgE production. Commercial B cell growth factor (cBCGF) was not able to reverse either RAPA- or CyA-induced suppression of IgE synthesis. The strong inhibitory effect of RAPA in IgE synthesis may be useful in certain clinical applications where overproduction of pathogenic IgE is a key issue. RAPA can also be used as a tool to dissect the regulation of IgE production.     

Effect of Cyclosporin A on the in Situ Inflammatory Response of Rat Renal Allograft Rejection

The impact of cyclosporin A (CyA) on a normal kidney parenchyma and on the in situ inflammatory response of rejection was investigated in normal DA rats and after transplantation of DA renal allografts to Lewis recipients. In a normal, non-transplanted DA kidney more than 80 mg/kg/day of CyA induced light-microscopic changes in the distal tubular cells of the renal cortex and outer medulla. These changes were not accompanied by any visible inflammation and were directly proportional to the dose of the drug and to the duration of drug administration. Treatment of a transplant recipient with 40 mg/kg/day of CyA abolished or at least efficiently reduced the in situ inflammatory response of rejection both as analysed from tissue sections and as quantified from the recovery of inflammatory cells after enzymatic digestion. It also reduced efficiently not only the number of T and B blast cells of the inflammatory infiltrate but also the number of other inflammatory cells, such as in situ lymphocytes, monocytes, and macrophages, and abolished or at least reduced the generation of (T) killer cells in situ and in the recipient spleen. These effects were inversely proportional to the time elapsed between grafting and initiation of treatment: although a complete suppression of all three features was obtained if the drug treatment was initiated already on the day of transplantation, a significant reduction of these functions was still found if the treatment was initiated later when the blastogenic response was already underway.     

Antigen dose-dependent regulation of B epsilon-memory cell expression

The data presented in this study document that the phospholipase A2 (PLA2)-specific IgE antibody response in high responder CBA/J mice is solely dependent on the antigen dose used for immunization. Repeated injections of minute doses (MD) of antigen (0.1 microgram/mouse) induce a persisting high level of PLA2-specific IgE antibody titer, whereas large doses (LD) (10 micrograms/mouse) induce a persisting low level of IgE. The IgG antibody titers are the same under both conditions. The low level IgE immune status induced by repeated LD is irreversible and cannot be boosted by MD. In contrast a single LD primes for a secondary IgE response which can be recalled by MD. A high level of PLA2-specific IgE antibodies induced by MD can be downregulated by a single intervening LD of antigen. A low level IgE immune status can be transferred with spleen cells of mice immunized with LD into naive syngeneic recipients, which then fail to mount a high level IgE response upon injection of MD of antigen. The experiments reveal two countercurrent processes, induction of B epsilon-memory cells after a single LD and additional activation of a persisting IgE-specific cellular suppression mechanism after repeated LD of antigen. These properties make the system suitable for the analysis of cellular interactions and of potential desensitization protocols.     

Direct action of cyclosporin A on human B-lymphocytes with regard to immunoglobulin production

Highly purified preparations of human B-lymphocytes were cultured with or without cyclosporin A (CyA; 1 microgram/ml) for 8 days with pokeweed mitogen (PWM) in the presence of helper factors or with Epstein-Barr virus (EBV). The amounts of IgG, IgM, IgA and IgE produced in cultures were measured by radioimmunoassays or by reverse hemolytic plaque-forming cell assays. The results demonstrate that whereas CyA had a strong suppressive effect on the production of immunoglobulins (Ig) by PWM-activated B-cells, it had an enhancing effect on EBV-activated B-cells. It is concluded that CyA has a direct effect on human B-lymphocytes and that it may suppress or enhance their activation depending on the stimulant employed to trigger the cells.     

The effect of cyclosporin A on the interstitial mononuclear cell infiltration and the induction of Heymann''s nephritis.

Heymann's nephritis was induced with brush-border (BB) antigen. Interstitial mononuclear cell infiltration was studied with cytological examinations of fine-needle aspiration biopsies (FNAB), and with immunoperoxidase stains of frozen sections with monoclonal antisera. The effect of cyclosporin A (CyA), 20 mg/kg when administered intraperitoneally for 8 days in association with both initial immunization, and with the booster 4 weeks later, on the interstitial leukocyte infiltration and on the development of membranous glomerulonephritis (MGN) and proteinuria were investigated. Another group of rats was immunized, but not given CyA. Experimental animals were killed in groups 3, 6 and 20 weeks after initial immunization. CyA inhibited significantly the initial interstitial lymphocyte and blast cell response at 3 weeks (FNAB), but did not inhibit the secondary response after the booster. The anti-BB titre reacted in a similar fashion. Immunoperoxidase stains indicated a clearly suppressed T suppressor/cytolytic (T s/c) cell response. Glomerular basement membrane (GBM) deposits of IgG developed more slowly and were more scarce in the CyA-treated rats, when compared with the untreated group. Only one out of 15 CyA treated rats developed C3 deposits in the GBM during the course of the study, and none developed proteinuria, when most untreated rats (10/17) had C3 deposits and were nephrotic at 20 weeks. Thus, CyA depressed the initial interstitial cellular response after immunization with BB antigen, and also inhibited the development of antibody response, C3 deposits and proteinuria of Heymann nephritis. These effects of CyA may be contributed to an inhibited amplification of the autoimmune response associated with interstitial damage and continuous release of autoantigen.     

IgE-isotype-specific suppressor cells in the mouse: characterization using tetraparental chimera mice of high (DBA/2) and low (SJL) IgE responder embryos

Tetraparental chimera mice were developed by aggregation of IgE high responder (DBA/2) and IgE low responder (SJL) embryos. Anti-dinitrophenyl (DNP) IgE antibody response in such mice (SJL----DBA/2) upon challenge with DNP-keyhole-limpet hemocyanin (KLH) in alum was clearly suppressed, while anti-DNP IgG antibody response was not. High-titer anti-DNP IgE and IgG antibody response developed in F1 hybrid mice of SJL and DBA/2 (SDF1) mice. The experimental results suggest that high IgE antibody production is the dominant trait, and the IgE-specific suppressor gene in SJL mice is autosomal recessive. IgE-specific suppressor T cells in SJL mice actively suppressed IgE antibody formation by DBA/2 immuno-competent cells across the histocompatibility barrier. Hapten-specific B cells and carrier-specific T cells were prepared in SJL----DBA/2 and SDF1 mice by immunization with DNP-KLH or ovalbumin (OA) in alum and transferred to irradiated SDF1 mice followed by challenge with DNP-OA. Hapten-specific B cells and carrier-specific helper T cells clearly developed in SDF1 mice. Recipient mice transferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells showed high-titer anti-DNP IgE and IgG antibody responses. OA-primed SJL----DBA/2 spleen cells cotransferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells completely abolished secondary anti-DNP IgE antibody response. The data suggest that carrier-specific helper T cells for IgE and IgG antibody responses are distinct. The regulatory role of IgE-isotype-specific suppressor cells were considered to be the interference of cooperative cellular interaction between IgE B cells and carrier-specific, IgE-specific helper T cells.     

Inhibition of primary and secondary IgE-response by a schistosome-derived inhibitory factor

Schistosome-derived inhibitory factor (SDIF) previously shown to inhibit lymphocyte proliferation, markedly decreased the primary IgE response of rats immunized with dinitrophenylated ovalbumin (DNP-OVA) when injected either simultaneously or shortly after antigen administration. No effect however was observed when SDIF was injected before the immunization. An inhibition of non-IgE anti-DNP antibodies was also found in SDIF-treated rats although the decrease was lower than with IgE antibody. IgE responses of both low and high IgE responder rats were reduced but a lower dose of SDIF was required in the case of high IgE responder Brown Norway rats. When SDIF was only given at the time of priming, the secondary IgE response was no longer modified. However, the administration of SDIF together with the second injection of the antigen induced marked decrease in the secondary IgE response. The effects of SDIF on primary and secondary IgE responses could be attributed to the inhibitory activity of the parasite-derived factor on lymphocyte proliferation. The observed inhibition of secondary IgE antibody responses confers to SDIF a pharmacological interest in allergic diseases.     

Early Expansion of Secondary B Cells after Primary Immunization with Antigen Complexed with IgE

IgE antibodies have potent immunoregulatory effects in vivo , and mice immunized with IgE–antigen (IgE/Ag) complexes exhibit a several hundred-fold higher humoral Ag-specific response than mice immunized with non-complexed Ag. In vitro studies indicate that this is a result of efficient endocytosis of the IgE/Ag complexes via the low-affinity receptor for IgE (CD23) on B cells, leading to efficient antigen presentation to T cells. Previous studies of IgE-induced Ab responses in vivo have only measured serum responses. The authors have now studied the up-regulated response as the number of IgG-, IgA-, IgE- and IgM-secreting single B cells in spleen, lymph nodes and bone marrow of mice immunized with IgE-anti-TNP + BSA-TNP (2,4,6-trinitrophenylated bovine serum albumin). IgE and Ag induced a greater than 500-fold increase of specific IgG-secreting spleen cells with the peak of the response 6 days after primary immunization. The response of other Ab isotypes and the response in other lymphoid organs was marginal. The rapid increase in the number of IgG-secreting cells in the spleen suggests that IgE/Ag complexes induce a secondary type of antibody response without requirement for conventional priming.     

New immunomodulator lobenzarit disodium (CCA): activation of IgE class-specific suppressor T lymphocytes and regulation of IgE antibody response enhanced by sublethal X-irradiation in SJL/J mice

The effect of the new immunomodulator lobenzarit disodium (CCA, disodium-2,2'-iminodibenzoate) on IgE antibody response was studied in X-irradiated SJL/J mice. IgE antibody response to dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) was enhanced by sublethal X-irradiation (400 R), and this enhanced IgE production was suppressed by unprimed normal spleen cell transfer. Oral administration of CCA, every day from 1 day after immunization with DNP-KLH to 1 day before bleeding out, reduced IgE antibody response in sublethally X-irradiated SJL/J mice. The suppressive effect of CCA in the mice was exerted at an early stage of the IgE antibody response. Moreover, CCA showed IgE class-specific suppression and did not suppress IgG class antibody production. CCA-induced reduction of IgE antibody response in SJL/J mice seems to be mediated by suppressor T cells, since depletion of T cells by treatment with antithymocyte serum and complement abolished the CCA-induced reduction and splenic T cells from CCA-treated mice were able to transfer the suppressive effect.     

Role of Serine Proteases of Schistosoma mansoni in the Regulation of IgE Synthesis

The regulation of the IgE response by schistosomula-released products (SRP) was studied either in vitro with rat and human cell cultures or in vivo by injection into rats of SRP with an unrelated allergen at primary or secondary immunization. The results obtained in vitro showed that non-dialysable factors present in SRP potentiate the IgE synthesis by rat and human cells. This enhancing effect was supported by molecules with serine protease activities. On the other hand, the inhibition or depletion of SRP in serine proteases induced a weak synthesis of IgM by rat cells in vitro. The injection of SRP into rats on day 0 with an unrelated allergen led to a potentiation of total IgE production, but an inhibition of specific IgE response. In contrast, a marked elevation of specific IgE response was obtained when SRP was injected upon secondary immunization. Serine proteases of SRP were partly responsible for this potentiative effect.     

Serological markers during dengue 3 primary and secondary infections.

BACKGROUND: The detection of the IgM antibody for the dengue virus in serum by ELISA has become one of the most important and useful methods for diagnosis of dengue using a single acute-phase serum sample. Currently, this system is an invaluable tool for the surveillance of dengue fever (DF) and dengue hemorrhagic fever (DHF). The usefulness of other serological markers such as IgA and IgE have been less studied. OBJECTIVE: To study the IgM, IgA and IgE specific antibody response in dengue 3 infected patients with different clinical picture and type of infection. STUDY DESIGN: One hundred and twenty-seven serum samples collected on days 5-7 at the onset of fever from clinically and serologically confirmed dengue cases were studied. Forty-two were classified as primary dengue fever cases, 48 as secondary dengue fever cases and 37 as secondary dengue hemorrhagic fever cases. All samples were tested by capture ELISA in order to detect dengue IgM, IgA and IgE antibodies. RESULTS AND CONCLUSIONS: In this study, significant differences were observed in the IgM, IgA and IgE response between the study groups. High IgA and IgE OD ratios in secondary dengue cases were found. The usefulness of serotype specific IgM antibody detection is also analyzed and discussed. A priority for future dengue research in terms of protection, recovery of infection and immunopathogenesis is to elucidate the role of these immunoglobulins. The cross reactivity response to IgM between dengue virus serotypes in primary and secondary cases should also be more studied.     

The production of IgE and IgGa antibodies in normal rats and rats infected with Nippostrongylus brasiliensis.

The time courses of production of IgE and IgGa homocytotropic antibodies were measured in Wistar rats during a primary and secondary response to egg albumin with pertussis or Freund's adjuvants. An anamnestic IgE antibody response occurred in animals previously sensitized to antigen with killed Bordetella pertussis as adjuvant. IgGa antibodies were formed in the primary response with Freund's complete adjuvant only, but were found during the secondary response with all adjuvants used. The time courses of formation of IgE and IgGa antibodies were very different during the secondary response. The production of both classes of antibody to egg albumin was studed in Wistar and Hooded Lister rats infected with Nippostrongylus brasiliensis. IgGa antibody formation was not potentiated by the infection. However, increased levels of IgE antibody, formed during a secondary response to antigen in infected animals, were consistently higher in both strains than during a primary response.     

Soluble CD23 displays T-cell growth enhancing activity.

Soluble CD23 (sCD23) enhances, in a dose-dependent manner, the number of secondary T-cell colonies generated by peripheral blood-derived agar T-colony cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2). This effect is not affected by IL-1 or IL-4 but is abolished by an anti-CD23 monoclonal antibody (mAb) or by IgE. No colonies were observed when sCD23 was added to PHA- or IL-2-free cultures. sCD23 also enhanced the cloning frequency of primary T-colony cells in a limiting dilution assay. These data provide the first direct evidence that sCD23 recruits T-cell clones in peripheral blood-born T cells and may be involved indirectly in the regulation of IgE response.     

IgE suppressor factor induced by phytohemagglutinin

Suppression of IgE response induced by phytohemagglutinin (PHA) inoculation near the time of immunization is studied. Donor spleen cells injected with PHA on day -1 before transfer were either depleted from Lyt 1+ or Lyt 2+ T cells and inoculated to isogenic recipients. Animals were immunized with ovalbumin in aluminum hydroxide gel 1 h later. IgE response was determined by passive cutaneous anaphylactic (PCA) reaction and ELISA. Results show that suppression of the IgE response caused by PHA only affects PCA reaction. In contrast, IgE response measured by ELISA is not modified. Depletion of Lyt 1+ T cell abolished the PHA effect. Thus, as a provocative notion, we propose the generation of an IgE suppressor factor which inhibited the PCA reaction. It was present in the sera of treated animals. IgM and/or IgG production was not affected.     

The effect of hapten-specific suppression of IgE on antigen-induced histamine release from mouse peritoneal mast cells.

Subcutaneous injections of a mixture of dinitrophenylated ovalbumin (DNP3-OA) and dextran sulfate into Swiss-Webster mice elicited short-lived primary and long-lasting secondary IgE antibody responses to both DNP and OA. Histamine was released on in vitro challenge with antigen (OA or DNP22-BSA) of washed peritoneal mast cells (PMC) obtained from mice during a primary or a secondary IgE response. Administration of an intravenous injection of a tolerogenic conjugate of DNP8-mouse gamma-globulin, either prior to immunizationor during an ongoing IgE response, resulted in almost complete disappearance of circulating anti-DNP IgE antibody and in a very marked decrease in histamine release from PMC on challenge with DNP22-BSA. However, the IgE response to OA of these mice and the histamine release from their PMC on challenge with OA were not affected. Moreover, the PMC of mice, which had been tolerized to DNP, could be passively sensitized with serum containing DNP-specific IgE antibody for the release of histamine on DNP22-BSA challenge. The most significant finding of this study is the observation that the time course for the loss of reactivity of PMC to DNP22-BSA, after administration of the tolerogen during an ongoing secondary response, paralleled the decrease in circulating anti-GNP IgE antibody.     

IgE and IgG1 antibody production by a soluble product of Ascaris suum in the guinea-pig.

Third-stage larvae of Ascaris suum cultured to the fourth stage in a chemically defined culture medium produced a substance, the 'ACF antigen', which was allergenic in the guinea-pig. When three different concentrations (3.1, 31 and 62 micrograms) of the ACF antigen were given intraperitoneally, only the highest concentration induced a primary IgE specific antibody response (1:100 titre) as determined with the passive cutaneous anaphylaxis reaction. Upon secondary exposure all concentrations demonstrated a strong IgE response (1:50,000 peak titre) with very little IgG1 activity (1:100). The secondary IgE responses began to rise on the fourth day, peaked on the sixth day and returned to relatively low levels by the fourteenth day (1:100). The intramuscular administration of the ACF antigen did not induce the extremely high titres of IgE as found with the intraperitoneal injection, but rather a low level response (1:500 peak) which did not differ greatly from the IgG1 response.     

Effect of cyclosporin A on autoimmune tubulointerstitial nephritis in the brown Norway rat.

The effect of cyclosporin A (CyA) on the development of tubulointerstitial nephritis (TIN) in the brown Norway (BN) rat was assessed. All manifestations of TIN were prevented in rats by subcutaneously injected CyA (20 mg/kg/day). A short 7-day course of CyA beginning the day before immunization suppressed the primary and ongoing antibody response. In addition, delayed CyA treatment (starting on day 10 after immunization), when antibody response was established, drastically reduced the levels of serum anti-TBM IgG, and abrogated the interstitial inflammatory cell response, in spite of persistent kidney-bound TBM antibodies. These results indicate that CyA has a therapeutic effect on the BN rat model of TIN.     

In vitro antigen-specific IgE response is refractory to suppression by interferon-gamma.

Although interferon (IFN)-gamma has been shown to be involved in the down-regulation of polyclonal IgE response in murine B cells that were activated by lipopolysaccharide (LPS) and interleukin 4 (IL4), effects of IFN-gamma on antigen-specific IgE responses have not been fully investigated. We have developed the following culture systems for inducing antigen-specific IgE responses in murine lymphocytes, and examined the effects of IFN-gamma on the following responses in vitro. (1) Anti-trinitrophenyl (TNP) IgE response induced by the stimulation with TNP-keyhole limpet hemocyanin (KLH) of BALB/c spleen cells that had been primed in vivo with the same antigen. (2) Anti-TNP IgE response induced by the coculture of unprimed C3H B cells with conalbumin (CA)-specific helper T cell clone, D10.G4.1, in the presence of TNP-CA. The former anti-TNP IgE response was not suppressed, and the latter suppressed only partially (less than 30%) by the addition of 100-200 U/ml IFN-gamma. In contrast, polyclonal IgE response in murine B cells that were stimulated by LPS and IL4 was abolished by 10 U/ml IFN-gamma. These results indicate that IgE production from antigen-stimulated B cells, in contrast to those activated polyclonally, are refractory to direct suppression by IFN-gamma.