Recent advances in the use of nanoparticles for allergen-specific immunotherapy.

The number of patients suffering from allergic asthma and rhinoconjunctivitis has increased dramatically within the last decades. Allergen-specific immunotherapy (AIT) is the only available cause-oriented therapy so far. AIT reduces symptoms, but has also a disease-modifying effect. Disadvantages are a long-lasting procedure, and in a few cases potential systemic adverse reactions. Encapsulation of allergens or DNA vaccines into nanostructures may provide advantages compared to the conventional AIT with noncapsulated allergen extracts: The protein/DNA molecule can be protected from degradation, higher local concentrations and targeted delivery to the site of action appear possible, and most importantly, recognition of encapsulated allergen by the immune system, especially by IgE antibodies, is prevented. AIT with nanoparticles (NPs) may offer a safer and potentially more efficient way of treatment for allergic diseases. In this review, we summarize the use of biodegradable NPs consisting of synthetic or natural polymers, liposomes, and virus-like particles as well as nonbiodegradable NPs like dendrimers, and carbon- or metal-based NPs for AIT. More or less successful applications of these NPs in prophylactic as well as therapeutic vaccination approaches in rodents or other animals as well as first human clinical trials are discussed in detail.

Adjuvant effects of aluminium hydroxide-adsorbed allergens and allergoids - differences in vivo and in vitro.

Allergen-specific immunotherapy (SIT) is a clinically effective therapy for immunoglobulin (Ig)E-mediated allergic diseases. To reduce the risk of IgE-mediated side effects, chemically modified allergoids have been introduced. Furthermore, adsorbance of allergens to aluminium hydroxide (alum) is widely used to enhance the immune response. The mechanisms behind the adjuvant effect of alum are still not completely understood. In the present study we analysed the effects of alum-adsorbed allergens and allergoids on their immunogenicity in vitro and in vivo and their ability to activate basophils of allergic donors. Human monocyte derived dendritic cells (DC) were incubated with native Phleum pratense or Betula verrucosa allergen extract or formaldehyde- or glutaraldehyde-modified allergoids, adsorbed or unadsorbed to alum. After maturation, DC were co-cultivated with autologous CD4(+) T cells. Allergenicity was tested by leukotriene and histamine release of human basophils. Finally, in-vivo immunogenicity was analysed by IgG production of immunized mice. T cell proliferation as well as interleukin (IL)-4, IL-13, IL-10 and interferon (IFN)-γ production were strongly decreased using glutaraldehyde-modified allergoids, but did not differ between alum-adsorbed allergens or allergoids and the corresponding unadsorbed preparations. Glutaraldehyde modification also led to a decreased leukotriene and histamine release compared to native allergens, being further decreased by adsorption to alum. In vivo, immunogenicity was reduced for allergoids which could be partly restored by adsorption to alum. Our results suggest that adsorption of native allergens or modified allergoids to alum had no consistent adjuvant effect but led to a reduced allergenicity in vitro, while we observed an adjuvant effect regarding IgG production in vivo.

An alternative allergen:adjuvant formulation potentiates the immunogenicity and reduces allergenicity of a novel subcutaneous immunotherapy product for treatment of grass-pollen allergy.

BACKGROUND: Subcutaneous specific immunotherapy (SCIT) has proven sustained clinical efficacy against allergy. The recommended regimen for SCIT is a gradual updosing over a period of weeks. Commonly, in commercial products for SCIT, the specific allergen is formulated with an adjuvant, most often in the form of aluminium hydroxide (AlOH). It has been shown that allergen-specific IgG antibodies are induced as a result of successful SIT. OBJECTIVE: To investigate the possibility of optimizing the formulation of AlOH-based grass-pollen allergy vaccines for SCIT in a way that allows for shorter updosing regimens while maintaining the immunogenicity of the vaccine. METHODS: Mice were immunized with various concentrations of Phleum pratense (Phl p) allergen extract and AlOH or a fixed dilution of the maintenance doses of one conventional and one alternatively formulated vaccine. The kinetics of Phl p-specific IgG antibody responses in serum and spleen T cell responses were determined. Allergenicity, measured as the ability of the formulations to activate human basophils, was also determined. In addition, human T cell responses and the expression of dendritic cell surface markers after vaccine challenge in vitro were analysed. RESULTS: Specific IgG antibody responses were shown to depend on the AlOH concentration, but not on the allergen concentrations. The immunogenicity of the conventional formulation and the alternative formulation was shown to be similar with regard to the in vivo-induced IgG and T cell responses. In contrast, the allergenicity of the alternative formulation was significantly reduced compared with the conventional formulation. CONCLUSION: The optimization of the formulation allows for administration of a lower dose of allergen while maintaining the immunogenicity of the product and at the same time reducing allergenicity. CLINICAL RELEVANCE: This study indicates that the optimization of the allergen and the adjuvant formulation could benefit the safety/efficacy profile and allow for shorter updosing.

Gram-positive bacteria on grass pollen exhibit adjuvant activity inducing inflammatory T cell responses.

BACKGROUND: Recently, it has been established that pollen grains contain Th2-enhancing activities besides allergens. OBJECTIVE: The aim of this study was to analyse whether pollen carry additional adjuvant factors like microbes and what immunological effects they may exert. METHODS: Timothy pollen grains were collected and disseminated on agar plates, and the growing microorganisms were cultivated and defined. Furthermore, the immunologic effects of microbial products on DC and T cell responses were analysed. RESULTS: A complex mixture of bacteria and moulds was detected on grass pollen. Besides Gram-negative bacteria that are known to favour Th1-directed immune responses, moulds were identified as being sources of allergens themselves. Herein, we focused on Gram-positive bacteria that were found in high numbers, e.g. Bacillus cereus and Bacillus subtilis. Contact of immature dendritic cells (DC) from grass pollen allergic donors with supernatants of homogenized Gram-positive bacteria induced maturation of DC as measured by up-regulation of CD80, CD83 and CD86 and by enhanced production of IL-6, IL-12p40 and TNF-α, which was less pronounced compared with effects induced by lipopolysaccharide (LPS). Consequently, stimulation of autologous CD4(+) T cells with supernatants of homogenized Gram-positive bacteria plus grass pollen allergen-pulsed DC led to an enhanced proliferation and production of IL-4, IL-13, IL-10, IL-17, IL-22 and IFN-γ production compared with T cells that were stimulated with allergen-pulsed immature DC alone, whereas production of the transcription factor for regulatory T cells FoxP3 was not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that grass pollen is colonized by several microorganisms that influence the immune response differently. Similar to LPS, supernatants of homogenized Gram-positive bacteria may serve as adjuvants by augmenting DC maturation and inflammatory Th1, Th2 and Th17 responses helping to initiate allergic immune responses.

Allergological implication of the quaternary hexameric structure of the cockroach allergen Per a 3.

BACKGROUND: Cockroach allergens play a very important role in allergic diseases, especially asthma. The major allergen of the American cockroach (Periplaneta americana), Per a 3, naturally occurs as isoforms of hexamers. OBJECTIVE: The aim of this study was to investigate whether the hexameric structures of Per a 3 influence their allergenicity and immunogenicity. METHODS: Therefore, we compared the different effects of native hexamers and dissociated monomers of cockroach haemolymph (HL), containing almost only Per a 3 proteins (HL-Per a 3), on proliferation and T-helper type 1 (Th1)/Th2 cytokine production of human CD4(+) T cells in co-culture with allergen-pulsed monocyte-derived autologous dendritic cells (DC) as well as the leukotriene release of basophils. RESULTS: In P. americana-sensitized and non-sensitized donors the HL-Per a 3 monomers were internalized faster by immature DC and induced higher proliferation and IFN-gamma production than the hexamers. While in non-sensitized donors IL-4 and IL-5 as well as IL-10 production were also increased after stimulation with monomeric HL-Per a 3-pulsed DC, Th2 cytokine and IL-10 production were only enhanced in P. americana-sensitized donors using hexameric HL-Per a 3-pulsed DC. Furthermore, in the leukotriene release assay the monomers were less effective than the hexamers. CONCLUSION: Our data indicate that the quaternary structure can influence both allergenicity and immunogenicity, also depending on the sensitization status. The monomeric variant of Per a 3 allergens could be a possible candidate for a specific immunotherapy because the IgE-mediated allergic reaction and the Th2-inducing capacity are diminished while the Th1-inducing capacity is retained.

Inhibition of human allergic T-helper type 2 immune responses by induced regulatory T cells requires the combination of interleukin-10-treated dendritic cells and transforming growth factor-beta for their induction.

BACKGROUND: In grass pollen-allergic individuals, T cell anergy can be induced by IL-10-treated dendritic cells (IL-10-DC) resulting in the suppression of T helper type 1 (Th1) as well as Th2 cells. This study was performed to analyse whether such IL-10-DC-treated T cells are able to act as regulatory T cells (Treg) suppressing the function of other T cells in the periphery. As transforming growth factor (TGF)-beta is also a potential inducer of Treg, we additionally analysed the inhibitory capacity of TGF-beta-treated T cells in this system. MATERIALS AND METHODS: Freshly isolated CD4+ or CD4+ CD25- T cells from grass pollen-allergic donors were stimulated with autologous mature monocyte-derived allergen-pulsed DC in the presence or absence of T cells previously cultured with IL-10-DC- and/or TGF-beta. RESULTS: Anergic T cells induced by allergen-pulsed IL-10-treated DC or allergen-pulsed DC and TGF-beta enhanced IL-10 production and strongly inhibited IFN-gamma production of freshly prepared peripheral CD4+ or CD4+ CD25- T cells while proliferation and Th2 cytokine production were only slightly reduced. The combination of allergen-pulsed IL-10-treated DC and TGF-beta had an additional effect leading to a significant suppression also of Th2 cytokine production and proliferation. Suppression was not antigen-specific and was mainly mediated by cell-to-cell contact and by the molecule-programmed death-1 and only partially by CTLA-4, TGF-beta and IL-10. CONCLUSION: These data demonstrate that regulatory T cells that also suppress Th2 cytokine production are induced by two signals: TGF-beta and IL-10-DC. This is of importance for the regulation of allergic immune responses and might be exploited for future therapeutic strategies for allergic diseases.

[Mechanisms of specific immunotherapy]. / Wirkmechanismen der spezifischen Immuntherapie.

Specific immunotherapy (SIT) is a well-established and clinically effective treatment for allergic rhinitis and bronchial asthma. The immunological mechanisms of the subcutaneously applied immunotherapy have been partially elucidated over the past several years but are not completely understood. This report summarizes the immunological and immunoregulatory changes during SIT of wasp venom allergic patients. The induction of IL-10-producing regulatory T cells is described.

The role of interleukin 10 in the regulation of allergic immune responses.

Several clinical studies and animal models have shown that Th2 lymphocytes play a key role in the pathophysiology of IgE-mediated allergic immune responses like allergic rhinitis and asthma or venom anaphylaxis. Classical specific immunotherapy (SIT) that has been proven to be clinically effective can serve as a role model for immunological changes that are associated with amelioration of allergic diseases. During SIT, the Th2-dominated immune response is modified towards a Th1 response leading to a decline in allergen-specific IgE and an increase in allergen-specific IgG production. Most importantly, however, production of the immunosuppressive/-regulatory cytokine interleukin 10 (IL-10) is also induced leading to T cell tolerance and prevention of tissue inflammation. In this article the role of IL-10-producing T cells in the regulation of allergic immune responses will be discussed.

Inhibition of human allergic T-cell responses by IL-10-treated dendritic cells: differences from hydrocortisone-treated dendritic cells.

BACKGROUND: Dendritic cells (DCs) are able to induce human allergic T(H)1 responses as well as T(H)2 responses. OBJECTIVE: In this study, we examined the effect of antiinflammatory agents such as IL-10 and hydrocortisone (HC) on the accessory function of DCs and the resulting T-cell response, especially that of T(H)2 cells. METHODS: Naive and memory CD4(+) T cells from atopic donors were stimulated with autologous allergen-pulsed DCs generated from CD14(+) monocytes by culture with GM-CSF/IL-4 and fully matured with IL-1 beta, TNF-alpha, and PGE(2) in the presence or absence of IL-10 or HC. RESULTS: IL-10-treated DCs and, to a lesser extent, HC-treated DCs showed a decreased expression of MHC II molecules, the costimulatory molecule CD86, and the DC-specific marker CD83, as well as a strongly reduced IL-12 secretion. Consequently, T-cell proliferation was reduced after stimulation with IL-10- or HC-treated DCs alike. However, pretreatment of DCs with IL-10 inhibited the production of T(H)1 and T(H)2 cytokines by T cells, whereas HC-treated DCs inhibited production of IFN-gamma but induced an increased release of IL-4 and no change in IL-5. Both effects were long-lasting; cytokine production remained low (which was due not to enhanced apoptosis but to functional hyporesponsiveness) or even increased after restimulation with fully matured DCs. CONCLUSION: These data indicate that IL-10- or HC-treated DCs differ in their ability to influence human allergic T-cell responses. This has major implications for therapeutic strategies aiming at the downregulation of proallergic T(H)2 responses.

Comparison of allergen-stimulated dendritic cells from atopic and nonatopic donors dissecting their effect on autologous naive and memory T helper cells of such donors.

BACKGROUND: Because of their production of IL-12, mature dendritic cells (DC) are potent inducers of T(H)1 responses. However, recent reports have demonstrated that DCs can also induce T(H)2 differentiation. OBJECTIVE: In the current study we investigated which immune response is induced by DCs in naive CD45RA(+) or memory CD45R0(+) CD4(+) T cells from atopic individuals (patients with grass pollen, birch pollen, or house dust mite allergy) compared with nonatopic control subjects. METHODS: Immature DCs, generated from peripheral blood monocytes from atopic and nonatopic donors, were pulsed with the respective allergen and fully matured. Then the mature DCs were cocultured in vitro with autologous naive (CD45RA(+)) and memory (CD45R0(+)) CD4(+) T cells and cytokine and IgE production were measured by ELISA. RESULTS: After the second restimulation with allergen-pulsed DCs, naive as well as memory autologous CD4(+) T cells from atopic but not from nonatopic donors showed an enhanced production of the T(H)2-type cytokines IL-4, IL-5, and IL-10, resulting in an increased IgE production, whereas IFN-gamma production and proliferation were not different. IL-12 production and surface marker expression of DCs derived from atopic and nonatopic donors did not differ and addition of neutralizing anti-IL-12 mAbs did not increase IL-4 but diminished IFN-gamma production. CONCLUSION: These data indicate that mature DCs are able to induce naive and activate allergen-specific T helper cells to produce T(H)2 cytokines if the T cells are derived from atopic donors. This phenomenon is not due to diminished IL-12 production by DCs of atopic donors.

Allergen-specific immune deviation from a TH2 to a TH1 response induced by dendritic cells and collagen type I.

BACKGROUND: Atopy and IgE production are associated with enhanced allergen-specific T(H)2 responses. Therefore a causative treatment may result from the deviation of this T(H)2-dominated immune response toward a T(H)1 response. OBJECTIVE: This study was carried out to analyze whether dendritic cells, the most potent antigen-presenting cells that are also known to induce antigen-specific T(H)1 responses, are suitable for therapy of atopic diseases by shifting the allergen-specific T(H)2 response toward a T(H)1 response. METHODS: Monocyte-derived dendritic cells were used to present allergens in vitro to autologous CD4(+) T cells of allergic persons. Because collagen type I activates dendritic cells and enhances the secretion of IL-12, we performed allergen presentation assays also in the presence of collagen type I. RESULTS: After stimulation with allergen-pulsed dendritic cells the production of IFN-gamma as well as that of IL-4 and IL-5 by CD4(+) T cells was enhanced. In the presence of collagen type I, however, a significant shift toward a T(H)1 response with increased production of IFN-gamma and a decreased production of IL-5 could be observed. When T cells were stimulated directly with anti-CD3 and anti-CD28 in the absence of antigen-presenting cells, it was demonstrated that collagen type I also exerted a direct effect on T cells, increasing their IFN-gamma production. CONCLUSION: These data indicate that collagen type I influences dendritic cells as well as T cells in a way that a shift in cytokine production results in a T(H)1 response even in already-sensitized atopic individuals.

Signals involved in the early TH1/TH2 polarization of an immune response depending on the type of antigen.

BACKGROUND: The early production of distinct cytokines by epidermal cells (ECs) in response to antigen exposure may govern the development of TH1 -like immune responses, such as contact sensitivity, or TH2 -like immune responses, such as IgE-dependent allergies of the immediate type, depending on the type of antigen. OBJECTIVE: The aim of this study was to compare the signals induced by protein allergens with those induced by haptens in ECs and subsequently in local draining lymph node cells (LNCs) or splenocytes. METHODS: BALB/c mice were primed in vivo with the protein allergens ovalbumin or birch pollen or the haptens 2, 4-dinitrofluorobenzene or trinitrochlorbenzene, respectively, and cytokine and immunoglobulin secretions of responding splenocytes were measured by ELISA after in vitro coculture with ECs. Induction of cytokine mRNA expression in ECs and LNCs was analyzed by reverse transcriptase-PCR. RESULTS: In the presence of protein allergens, ECs enhance the induction of a TH2 immune response (IL-4 and IgE production of splenocytes), whereas a TH1 immune response (IFN-gamma and IgG2a production) was only induced in the context of haptens. Heat inactivation of ovalbumin did not diminish the development of a TH2 immune response. One direct effect of antigen on ECs was the earlier expression of IL-10 mRNA after stimulation with protein allergens (30 minutes) than with haptens (2 hours) in vitro. By using an in vivo approach, sensitization of the skin with trinitrochlorbenzene, but not with ovalbumin, resulted in an early induction of IL-1beta, IL-12p40, and IFN-gamma mRNA in LNCs, whereas IL-18 was induced by both. CONCLUSION: These data indicate that the type of antigen strongly influences the type of immune response by eliciting distinct signals already in the epithelium.

Decreased release of histamine and sulfidoleukotrienes by human peripheral blood leukocytes after wasp venom immunotherapy is partially due to induction of IL-10 and IFN-gamma production of T cells.

BACKGROUND: Recent studies provide evidence that venom immunotherapy (VIT) alters the pattern of cytokine production by inducing an allergen-specific T-cell shift in cytokine expression from TH2 (IL-4, IL-5) to TH1 (IFN-gamma) cytokines and also inducing the production of IL-10. OBJECTIVE: This study was carried out to analyze whether these changes in cytokine production of T cells already observed 1 week after the initiation of VIT in subjects with wasp venom allergy also influence the reactivity of effector cells, such as mast cells and basophils. METHODS: All subjects included in this study had a history of severe systemic allergic reactions to wasp stings and positive skin test responses with venom and venom-specific IgE in the sera. Peripheral blood leukocytes were isolated before and after the initiation of VIT (rush therapy reaching a maintenance dose of 100 microg venom injected subcutaneously within 1 week) and preincubated with or without addition of IL-10, IFN-gamma, IL-10 + IFN-gamma, anti-IL-10, or anti-IFN-gamma. After stimulation with wasp venom, histamine and sulfidoleukotriene release were assessed by ELISA and compared with spontaneous release and total histamine content. RESULTS: After the induction of VIT, venom-induced absolute and relative histamine and sulfidoleukotriene release were reduced. This was at least partially due to the induction of IFN-gamma and IL-10 production, because (1) neutralization of IL-10 and IFN-gamma by mAbs partially restored the release after the initiation of VIT and (2) the addition of exogenous IFN-gamma and IL-10 caused a statistically significant diminution of the venom-induced histamine and sulfidoleukotriene release before VIT. Depletion of CD2(+) T cells also restored the releasability after VIT. CONCLUSION: These data indicate that T cells (producing IL-10 and IFN-gamma after VIT) play a key role for the inhibition of histamine and sulfidoleukotriene release of effector cells.

Active suppression induced by cutaneous exposure to bacterial superantigen is prevented by interleukin-12 treatment in vivo.

Exposure to the bacterial superantigen staphylococcal enterotoxin B (SEB) leads to inhibition of several immune responses and the induction of regulatory cells. The aim of this study was to characterize these regulatory cells further and to investigate the effect of interleukin-12 (IL-12) on superantigen-induced suppression. For this purpose BALB/c mice were injected subcutaneously with low doses of SEB that did not deplete the SEB-reactive V beta T cells. Intravenous transfer of unseparated local-draining lymph node cells from these SEB-treated animals suppressed the proliferative response of mononuclear spleen cells of naive syngeneic recipients for at least 3 weeks. The regulatory cells did not produce the type 2 cytokines, interleukin-4 (IL-4) or interleukin-10 (IL-10), or increased amounts of transforming growth factor-beta (TGF-beta). Depletion of CD8+ or SEB-reactive V beta 7+ and V beta 8+ T cells, prior to transfer, abrogated the suppressive effect. Intraperitoneal injections of IL-12 into donors, prior to SEB treatment, prevented the induction of functional regulatory cells, and treatment of recipients with IL-12, prior to receipt of cells from SEB-treated donors, prevented the suppressive effect of regulatory cells that were already induced. The data indicate that exposure to minute amounts of superantigens directly induces superantigen-reactive and CD8+ regulatory T cells and that superantigen-induced suppression can be prevented and reversed by IL-12 treatment in vivo.

Influence of extracellular matrix proteins on the development of cultured human dendritic cells.

The development of dendritic cells (DC) is still only partly understood. Recently established culture systems using CD34+ cells or monocytes as precursor cells for the generation of DC indicate the necessity of pro-inflammatory cytokines for their development. In vivo the contact to other cells or to the proteins of the extracellular matrix might also be essential for their development. In our experiments we used granulocyte-macrophage colony-stimulating factor- and IL-4-treated human monocytes as precursor cells to investigate the interaction of DC at different maturation stages with the matrix proteins fibronectin, collagen type I and collagen type IV. We demonstrate a strong beta1-integrin-mediated adherence of immature DC to fibronectin that is lost completely during maturation. The binding to collagen type I was less strong but induced a maturation of the precursor cells. After 3 days of culture on this protein, the cells showed all features of fully matured DC such as expression of CD83 and an excellent allostimulatory capacity. The reason for this effect was shown to be the induction of TNF-alpha production by the DC themselves. In contrast to the adhesion to fibronectin, the maturation and the cytokine production of DC induced by collagen type I could not be inhibited by blocking of beta1-integrins. These results indicate that proteins of the extracellular matrix play an important role in the development and function of human DC.

Insect venom immunotherapy induces interleukin-10 production and a Th2-to-Th1 shift, and changes surface marker expression in venom-allergic subjects.

The current study was carried out to elucidate the immunoregulatory changes induced by venom immunotherapy (VIT) in bee or wasp allergic subjects. All subjects included in this study had a history of severe systemic allergic reactions to stings of the respective insect as well as positive skin tests with the respective venom or venom-specific IgE in the sera. Parameters assessed in peripheral blood mononuclear cells (PBMC) before and after initiation of VIT (rush therapy reaching a maintenance dose of 100 micrograms venom injected subcutaneously within 1 week) were expression of CD3, CD4, CD8, CD45RA, CD45RO, interleukin (IL)-2 receptor (R) alpha, IL-4R, IL-12R, Fc epsilon RII, CD40, and CD40 ligand (CD40L), cells producing interferon (IFN)-gamma and IL-10 after stimulation with phorbol 12-myristate 13-acetate + ionomycin in the presence of monensin measured by flow cytometry; secretion of IFN-gamma, IL-4, and IL-10 measured by ELISA (IFN-gamma and IL-10 were additionally measured by PCR), and proliferation after stimulation with the respective venom. Significant decreases were observed after VIT for proliferative response to venom and venom + IL-4, IL-4 secretion, Fc epsilon RII, CD40, and CD40L expression. Significant increases were observed after VIT for IFN-gamma concerning the amount secreted and the number of producing cells, and IL-10, IL-10 was mainly produced by CD4+ cells that were negative for IFN-gamma, but some double-positive (IL-10 and IFN-gamma) cells were always detected. Addition of blocking anti-IL-10 antibodies, but not isotype control antibodies, prevented down-regulation of proliferation (but not IL-4 secretion) and further enhanced IFN-gamma secretion after VIT. These data indicate that in insect venom allergic subjects, VIT not only induces a rapid shift in cytokine expression from Th2 to Th1 cytokines, but also leads to induction of the immunosuppressive cytokine IL-10, which may be important for the limitation of potentially harmful allergen-specific Th1 responses. The described changes in cytokine expression may be responsible for subsequent increases in allergen-specific IgG and decreases in IgE production, as well as suppressive activity observed in earlier studies.

Epidermal cells enhance interleukin 4 and immunoglobulin E production after stimulation with protein allergen.

Exposure to certain allergens via epithelial tissues is the primary route for the induction of immunoglobulin E-dependent allergies of the immediate type associated with atopic diseases. In order to address the question whether and how epithelial cells might contribute to the induction or increase of TH2-dependent IgE production, we performed co-culture experiments of syngeneic epidermal cells and cells from the associated lymphoid tissue or spleen (responder cells) of BALB/c mice primed with ovalbumin in vivo. In the presence of ovalbumin in vitro, immunoglobulin E but not immunoglobulin G2a production was significantly enhanced by the addition of epidermal cells, and separation of epidermal cells from responder cells by a membrane that prevented cellular contacts or addition of antibodies against intercellular adhesion molecule-1 reduced the enhancement of immunoglobulin E production induced by epidermal cells. Depletion of major histocompatibility complex class II+ antigen presenting Langerhans cells from the epidermal cells prior to co-culture also reduced the enhancement of immunoglobulin E production induced by epidermal cells. The enhanced immunoglobulin E production was dependent on the induction of TH2 cell-derived interleukin-4 detected in co-cultures because it was completely inhibited after addition of anti-interleukin-4 antibodies that also lead to increased immunoglobulin G2a production. Whereas interleukin-4 was not produced by epidermal cells, interleukin-10 seemed to be one important mediator contributed by epidermal cells. Interleukin-10 skewed the response toward a TH2-mediated IgE response because antibodies against interleukin-10 inhibited interleukin-4 and immunoglobulin E production, whereas they enhanced interferon-gamma and immunoglobulin G2a production.

Prevention and reversal of superantigen-induced anergy by contact allergen exposure.

The superantigen Staphylococcal enterotoxin B (SEB) and the contact allergen 2,4-dinitrofluorbenzene (DNFB) both react with V beta 8+ T-cells delivering distinct signals. Pre-treatment with DNFB painted onto the same skin site where SEB was to be injected, prevented the induction of anergy in V beta + T-cells that was otherwise induced after SEB had been injected intradermally over a period of 2 weeks. Application of the irritant sodium dodecyl sulfate (SDS) instead of DNFB did not exert this effect. Application of DNFB at a site distant from the site where SEB was injected resulted in a much weaker inhibitory influence on the induction of anergy by SEB. Established anergy of V beta 8+ T-cells (proliferative non-responsiveness to SEB in vitro that could be overcome by addition of exogenous interleukin 2 (IL-2)) could be largely reversed by repeated cutaneous exposure to DNFB painted to the site where SEB had been injected before. The moderate decrease of V beta 8+ T-cells normally induced by SEB-treatment was also partially prevented by DNFB pre-treatment. The data indicate the importance of the sequence of signals delivered to T cells and the plasticity of the responsiveness of this cell type.

Modulation of contact sensitivity responses by bacterial superantigen.

Superantigens are potent modulators of the immune system, especially T cells. Therefore, we determined the influence of superantigens on the T-cell-mediated immune response, contact sensitivity. We chose the combination of staphylococcal enterotoxin B (SEB) as superantigen and 2,4-dinitrofluorbenzene (DNFB) as the contact sensitizer, because in BALB/c mice SEB reacts almost exclusively with V beta 8+ T cells, and these cells are capable of transferring contact sensitivity to DNFB from sensitized donors to naive syngeneic recipients. Pretreatment with a single intradermal injection of 50 ng SEB 24 h before DNFB exposure at the same site on the lower abdomen enhanced the induction of contact sensitivity: its intradermal injection permitted sensitization with non-sensitizing concentrations of DNFB as assessed by ear swelling responses after challenge with DNFB. In contrast, pretreatment with repeated intradermal injections of 50 ng SEB every other day over at least 1 week inhibited the induction of contact sensitivity following sensitization. The enhancing effect of SEB may be explained by the creation of a proinflammatory milieu in the skin after a single intradermal injection of the bacterial toxin, whereas the inhibitory effect may be due to tolerization of V beta 8+ T cells. The data indicate that products of skin-colonizing bacteria that can serve as superantigens are able to augment or inhibit the development of contact sensitivity.