[Show abstract][Hide abstract] ABSTRACT: 1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25[OH](2)D(3)) exerts its effects on the immune system, particularly through suppression of T helper/cytotoxic cell 1 (T(H)/T(C)1)-mediated reactions, although direct actions of 1 alpha,25(OH)(2)D(3) on human T lymphocytes have not yet been studied in detail.
We evaluated the effect of 1 alpha,25(OH)(2)D(3) on basal and cytokine-driven T-cell functions at the single-cell level.
We used 4-color flow cytometry for simultaneous detection of intracellular cytokines in CD4(+) and CD8(+) human PBMCs that had been cultured in the presence of 1 alpha,25(OH)(2)D(3) singly or in combination with either IL-12 or IL-4. According to the exploratory nature of these investigations, the Bonferroni correction was not applied for data analysis and presentation.
1 alpha,25(OH)(2)D(3) had little effect on T(H)/T(C)1 cytokines but significantly inhibited IL-12-induced IFN-gamma production. Constitutive synthesis of T(H)/T(C)2-related cytokines was also only modestly affected by 1 alpha,25(OH)(2)D(3) alone. When T(H)/T(C)2 differentiation was induced by IL-4, 1 alpha,25(OH)(2)D(3) significantly expanded the percentages of IL-4(+) and IL-13(+) cells. However, the predominant effect of 1 alpha,25(OH)(2)D(3) on T lymphocytes, particularly in the presence of IL-4, was the induction of separate CD4(+) and CD8(+) subpopulations with almost exclusive expression of IL-6. This might be an important facet of the immunomodulatory action of 1 alpha,25(OH)(2)D(3) because IL-6 might act in parallel with 1 alpha,25(OH)(2)D(3) in modulation of T(H)/T(C) effector cell functions.
Our data imply that the specific actions of 1 alpha,25(OH)(2)D(3) on cytokine-stimulated T-cell functions could play a role in the prevention of T(H)/T(C)1-related autoimmune diseases but also predispose toward T(H)/T(C)2-mediated allergic reactions.
Journal of Allergy and Clinical Immunology 10/2005; 116(3):683-9. DOI:10.1016/j.jaci.2005.05.013 · 11.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coli in tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 microg rLTB pentamers per gram fresh plant material and was capable of binding G(M)1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 microg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.
[Show abstract][Hide abstract] ABSTRACT: The prevalence of type I allergy to Hevea brasiliensis latex is particularly high among individuals with frequent exposure to latex products, such as health-care workers (HCW) and patients with spina bifida (SB). Treatment of latex allergy seems problematic as preventive measures, such as allergen avoidance, are not always possible and conventional immunotherapy with standardized latex extracts is not performed routinely. Thus, the aim of the present study was to establish a mouse model of latex allergy using two major latex allergens for HCWs and SB patients, Hev b 1 and Hev b 3, for sensitization. Prophylactic measures on the basis of mucosal tolerance induction with the recombinant allergens were tested in this model. Female BALB/c mice immunized intraperitoneally with recombinant (r)Hev b 1 or rHev b 3 displayed strong immune responses in vivo and in vitro. Intranasal treatment with rHev b 1 and rHev b 3 prior to sensitization led to reduced allergen-specific IgG1/IgE levels and significantly suppressed allergen-induced basophil degranulation. Moreover, lymphocyte proliferation and cytokine production (IL-4, IL-5, IFN-gamma) in vitro were significantly suppressed after pretreatment with both allergens. Suppressive cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-beta, remained unchanged after the intranasal pretreatment, indicating mechanism of anergy rather than active immunosuppression. Taken together, these results suggest that mucosal tolerance induction with recombinant allergens could present a promising prevention strategy against latex allergy.
[Show abstract][Hide abstract] ABSTRACT: The protozoan parasite Entamoeba histolytica, which is responsible for intestinal amebiasis and amebic liver abscess, is causing significant morbidity and mortality worldwide. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) are major surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against the PPGs protected severe combined immune-deficient mice from amebic liver abscess. The structure of the PPGs is very complex and only known in part. To find peptide mimics of E. histolytica PPG antigens, we had screened phage-displayed random peptide libraries with the antibody EH5. We identified various peptide mimics of E. histolytica PPGs, all sharing a consensus sequence Gly-Thr-His-Pro-X-Leu. Several of the phage clones induced a significant, specific IgG response against membrane antigens of E. histolytica after immunization of mice with whole phage particles. In the present work, in order to avoid the use of phage particles for immunization, we coupled two selected chemically synthesized peptides to keyhole limpet hemocyanin (KLH). The two KLH-conjugated peptides were immunogenic in mice and induced the production of high titers of anti-peptide antibodies, and one of the two peptides was also able to induce significant titers of antibodies against E. histolytica PPGs. Our results demonstrate that the KLH-conjugated peptides are able to mimic the EH5 epitope without the M13 phage sequences flanking the peptide inserts and independent of the structural framework of the phage.
[Show abstract][Hide abstract] ABSTRACT: In previous studies we have generated mimotopes of Bet v 1, the major birch pollen allergen, by biopannings of phage-display random peptide libraries. In the present study, we analysed the humoral and cellular immune response to Bet v 1-mimotopes.
The mimotope CFPYCYPSESA, designated Bet mim 1, was used for intraperitoneal immunizations of BALB/c mice in phage-displayed form. For examination of the humoral immune response, enzyme-linked immunosorbent assay (ELISA) experiments were applied. Stimulation capacities were investigated in cultured mouse splenocytes and in humoral Bet v 1-specific T cell clones.
We demonstrated that the Bet mim 1-induced murine antibody response against Bet v 1 was predominated by the IgG1 isotype. In these mice only the phage-displayed mimotopes, but neither the allergen nor the synthetic Bet mim 1-mimotopes were able to stimulate proliferation of cultured splenocytes. Using Bet v 1-specific T cell clones of allergic patients, phage-displayed and synthetic mimotopes were unable to stimulate T cell proliferation. Moreover, tolerance induction to Bet v 1 in mice by intranasal administration of Bet mim 1-phages or Bet mim 1-peptide failed.
Taking these results together, our data indicate that Bet mim 1 mimics a Bet v 1-epitope on the B cell but not on the T cell level. We suggest that the phage itself is responsible for the recruitment of T cells providing bystander help in the formation of a mimotope-specific humoral response.
[Show abstract][Hide abstract] ABSTRACT: Several studies have demonstrated that mucosal administration of soluble antigens can prevent the onset or reduce the severity of certain autoimmune diseases or allergies. Few studies exist showing the efficacy of mucosal tolerance for therapy of such diseases.
The aim of the present study was to modulate an allergic immune response by intranasal antigen administration in an already sensitized organism.
A murine model of allergic asthma to birch pollen (BP) and its major allergen Bet v 1 was utilized. Sensitized mice were intranasally treated with recombinant (r)Bet v 1 in different concentrations and at different intervals. On the day the mice were killed, blood and bronchoalveolar lavage fluids were taken and immediate type I skin tests were performed. T cell proliferation and cytokine production (interleukin (IL)-5, interferon (IFN)-gamma) were measured in spleen and lung cell cultures.
Mucosal treatment with rBet v 1 (3 x 50 microg in 4 day intervals) led to a reduction of type I skin reactions, suppressed immunoglobulin (Ig)G1/IgE antibody levels and markedly decreased IL-5 and IFN-gamma production in vitro in spleen and lung cell cultures. Moreover, lung inflammation (i.e. eosinophilia and IL-5 levels in bronchoalveolar lavage fluids) was significantly suppressed by the treatment.
Our results demonstrate that intranasal treatment with rBet v 1 reduced systemic allergic immune responses as well as airway inflammation in BP-sensitized mice. We therefore suggest that mucosal tolerance induction with recombinant allergens could be a promising concept for the therapy of allergic diseases.
[Show abstract][Hide abstract] ABSTRACT: Regional differences in immune responsiveness have been studied by comparing the frequency of cytokine producing T cells in healthy African children and adults and their age-matched European counterparts. By use of flow cytometry for the intracellular detection of cytokines an overall expansion of CD4+ and CD8+ T cells producing the Type 1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma was observed in adults when compared with children, giving credit to the cumulative effect of contacts with environmental antigens. The CD4+ cells expressing the Type 2 cytokines IL-4 and IL-13, however, increased only in Africans, probably reflecting continuously present challenges with antigens that preferentially drive Type 2 responses. A striking increased frequency of both Type 1 and Type 2 cytokines producing T cells was found in African adults when compared with their European counterparts. The quantitative and qualitative regional differences in immune reactivity are likely to be of significance for all immune intervention strategies, especially for the design of vaccines.
The American journal of tropical medicine and hygiene 12/2001; 65(5):504-9. · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The major birch pollen allergen Bet v 1 represents one of the most prevalent environmental allergens responsible for allergic airway inflammation.
In the present study we sought to compare the complete recombinant Bet v 1 allergen molecule with genetically produced hypoallergenic fragments of Bet v 1 regarding mucosal tolerance induction in a mouse model of allergic asthma.
BALB/c mice were intranasally treated with recombinant Bet v 1 or with two recombinant Bet v 1 fragments (F I: aa 1-74; F II: aa 75-160) prior to aerosol sensitization with birch pollen and Bet v 1.
Intranasal application of F II, containing the major T cell epitope, led to significant reduction of IgE/IgG1 antibody responses, in vitro cytokine production (IL-5, IFN-gamma, IL-10) and negative immediate cutaneous hypersensitivity reactions comparable to the pretreatment with the complete rBet v 1 allergen. Moreover, airway inflammation (eosinophilia, IL-5) was inhibited by the pretreatment with either the complete Bet v 1 or F II. However, for prevention of airway hyperresponsiveness the complete molecule was required. The mechanisms leading to immunosuppression seemed to differ in their dependence on the conformation of the molecules, since tolerance induced with the complete Bet v 1, but not with F II, was transferable with spleen cells and associated with increased TGF-beta mRNA levels.
We conclude that mucosal tolerance induction with recombinant allergens and genetically engineered hypoallergenic derivatives thereof could provide a convenient and safe intervention strategy against type I allergy.
International Archives of Allergy and Immunology 10/2001; 126(1):68-77. DOI:10.1159/000049496 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied the possible regulatory effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] on cytokine production and differentiation of subsets of CD4+ [T helper 1 (Th1) and Th2] and CD8+ [T cytotoxic 1 (Tc1) and Tc2] lymphocytes at the single cell level. PBMC from healthy donors were cultured with or without 1alpha,25-(OH)2D3 for up to 21 days. On days 0, 7, 14, and 21, the percentage of cytokine-producing T lymphocytes was analyzed by intracellular cytokine detection with mAb and flow cytometry. Simultaneous staining for cell surface markers allowed discrimination of CD4+ and CD8+ T cell subsets. After culture with 1alpha,25-(OH)2D3 (10(-8) mol/L), no significant effects on the proportion of interferon-gamma (IFNgamma)- or interleukin-4 (IL-4)-producing cells were detected, whereas reduced frequencies of IL-2-producing cells in the CD4+ as well as in the CD8+ population were found. An increase in the low percentage of CD4+ and CD8+ T cells producing the Th2 cytokine IL-13 was noticed. Most interestingly, IL-6-producing CD4+ and CD8+ T cells could only be detected in cultures with 1alpha,25-(OH)2D3, reaching a plateau after 14 days. The percentage of IL-6-producing T cells induced by 1alpha,25-(OH)2D3 after a given time period remained stable for at least 7 weeks. Studies of cytokine coexpression revealed that about 70% of IL-6-producing CD4+ and CD8+ cells were also positive for IL-2, but more than 90% were negative for IFNgamma, IL-4, or IL-13, respectively. This suggests that the IL-6-producing population does not match the Th1/Tc1-like (IFNgamma+) or Th2/Tc2-like (IL-4+ or IL-13+) subset. The influence of 1alpha,25-(OH)2D3 on cytokine production by lymphocytes is probably an important point of intersection between the endocrine and the immune system.
[Show abstract][Hide abstract] ABSTRACT: The frequency of cytokine-producing CD4-/CD8- mononuclear cells was assessed in patients of different age groups (29 infants, aged 1-5 years; 30 schoolchildren, aged 6-14 years, 26 adults, aged > 15 years) with acute Plasmodium falciparum malaria, from Gabon. Fifteen patients were followed up before antimalarial treatment (day 0), during parasite clearance (day 3) and after resolution of parasitemia (day 10). By using flow cytometry for intracellular detection of cytokines, a striking expansion of CD4-/CD8- cells producing the type 1 cytokines interleukin (IL)-2-/interferon (IFN)-gamma+, IL-2+/IFN-gamma+ and IL-2+/IFN-gamma- was observed in adults as compared with children. Type 2 cytokine expression (IL-4+/IFN-gamma-, IL-13+/IFN-gamma-) and type 0 cells (IL-4+/IFN-gamma+, IL-13+/IFN-gamma+) were not significantly different between the three age groups. Patients with severe malaria had a significantly increased frequency of type 2 cytokine-producing CD4-/CD8- cells. Drug-induced clearance of parasitemia was characterized by a decrease of IL-2+/IFN-gamma- and type 2 cytokine expressing CD4-/CD8- cells and by a gradual increase of IL-10+/IFN-gamma- expression. The type 1/type 2 dichotomy observed within the CD4-/CD8- cell population is likely to be of significance in the host response against P. falciparum malaria.
European cytokine network 07/1999; 10(2):155-60. · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the past few years, the role of polymorphonuclear neutrophils (PMN) in specific immune responses has gained significance due to their ability to express a variety of immunoregulatory molecules. However, controversial results concerning the potential of neutrophils for cytokine production have been obtained by sensitive molecular biological techniques. This problem might be related to contaminating leukocytes in conventionally isolated neutrophil suspensions as outlined by our study. We have established a novel method yielding highly purified neutrophils by combining a discontinuous Percoll gradient with fluorescence activated cell sorting of CD16bright cells. The latter step exploits the exceptionally high expression of Fc gammaRIIIB on PMN. Neutrophils could be enriched to homogeneity (> 99.9%) with a viability exceeding 90%. Contamination with NK cells or other lymphocytes, monocytes and eosinophils could be excluded as evaluated by reverse transcription-polymerase chain reaction (RT-PCR) with primers for HLA-DR, c-fms and CD52. The transcriptional potential of such purified neutrophils was confirmed by their ability to express MHC class II molecules after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Our method should permit studies of PMN at the mRNA level and future investigations concerning the specificity of immunoregulatory molecule synthesis by neutrophils.
[Show abstract][Hide abstract] ABSTRACT: The frequency of cytokine-producing peripheral blood mononuclear cells was assessed in 28 subjects with microfilaremic loiasis and in 14 amicrofilaremic individuals. In addition, a subgroup of seven microfilaremic individuals coinfected with Plasmodium malariae was evaluated. By using flow cytometry for the intracellular detection of cytokines, a more pronounced T helper (Th)2 cell-type response with the expansion of interleukin (IL)-4, IL-10, and IL-13 expressing CD4+ cells in the microfilaremic compared with the amicrofilaremic group was noted. Expression of IL-5 was equivalent in both groups as was the frequency of Th2-type cytokines expressing CD8+ cells and of Th1-type cytokines (interferon [IFN]-gamma, IL-2, IFN-gamma/IL-2) producing CD4+ and CD8+ cells. Th0-type cytokine-expressing cells, represented by IL-4/IFN-gamma, IL-10/IFN-gamma, and IL-13/IFN-gamma, were equally distributed within groups. Coinfection of P. malariae did not significantly alter the cytokine expression compared with microfilaremic individuals without P. malariae infections. By identifying a large panel of cytokine-producing T cell subpopulations, a Th2-driven immune response in microfilaremic Loa loa patients was noted.
The American journal of tropical medicine and hygiene 05/1999; 60(4):680-6. · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The frequency of cytokine-producing T cells was assessed in patients of different age groups (29 infants, aged 1-5 years; 30 schoolchildren, aged 6-14 years; 26 adults, aged >15 years) with acute Plasmodium falciparum malaria from Gabon. By using flow cytometry for the intracellular detection of cytokines, a striking expansion was seen, in adults compared with children, of CD4+ and CD8+ T cells with the following profiles of type 1 cytokine production: interleukin (IL)-2-/interferon (IFN)-gamma+, IL-2+/IFN-gamma+, and IL-2+/IFN-gamma-. Patients with hyperparasitemia had a significantly lower frequency of IL-2-/IFN-gamma+ CD4+ cells. Type 2 cytokine expression (IL-4+/IFN-gamma-, IL-13+/IFN-gamma-) and type 0 cytokine expression (IL-4+/IFN-gamma+, IL-13+/IFN-gamma+) were also increased in adults within the CD4+ subset. Frequencies of IL-5+/IL-4+, IL-10+/IFN-gamma-, and IL-10+/IFN-gamma+ cells were similar in all groups. The increased frequency of both type 1 and type 2 cytokine-producing T cells in adults is likely to be of significance in the protection against P. falciparum malaria.
The Journal of Infectious Diseases 02/1999; 179(1):209-16. DOI:10.1086/314571 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flow cytometry for the intracellular detection of T-cell cytokines was performed for 15 Gabonese patients during acute uncomplicated Plasmodium falciparum malaria. A striking expansion of CD4(+) and CD8(+) T cells producing gamma interferon (IFN-gamma) was found during drug-induced clearance of parasitemia, paralleled by a decrease of interleukin-2 (IL-2) production. The frequency of IL-4- and IL-13-producing CD4(+) cells gradually decreased, whereas the frequency of T cells producing IL-2(+)-IFN-gamma+, IL-4(-)-IL-5(+), and IL-4(+)-IL-5(+) cytokines as well as IL-4(+)-IFN-gamma+ and IL-13(+)-IFN-gamma+ cytokines was not significantly altered. The capacity for IL-10 production within the CD4(+) subset increased due to an expansion of both IL-10(+)-IFN-gamma- and IL-10(+)-IFN-gamma+ cytokine-expressing cells. Thus, a more pronounced Th2-driven immune response during acute untreated P. falciparum infection with a shift towards Th1 responsiveness induced by parasite clearance is suggested.
Infection and Immunity 01/1999; 66(12):6040-4. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the possibility of using surface molecules as markers for human T helper cell subsets, we studied the expression of surface molecules on T cell clones (TCCs) specific for the major birch pollen allergen, Bet v 1. No difference in the expression of the respective receptors for interferon-gamma (IFN-gamma), IL-4, IL-6, or IL-10 could be detected on T(H) subsets, nor did CD25 expression (IL-2R alpha-chain) differ significantly. However, high expression of CD26 antigen (dipeptidyl peptidase IV) correlated with a T(H1)/T(H0)-like phenotype, whereas T(H2)-like clones displayed a lower expression of CD26 antigen. Comparing cytokine production and CD26 expression simultaneously, we found a correlation between the IL-4/IFN-gamma ratio and the density of CD26 per cell. We could show that the amount of IL-4 secretion, and not of IFN-gamma secretion, was responsible for this correlation. To evaluate whether CD26 antigen expression is regulated by stimuli inducing a T(H1)- or T(H2)-like phenotype, peripheral blood mononuclear cells (PBMC) were cultured in the presence of IL-4, IFN-gamma, and IL-12, respectively. Incubation with IL-4 led to T cells with a T(H2)-like cytokine pattern and a significantly lower expression of CD26; IFN-gamma and IL-12 led to a T(H1) shift associated with an increased expression of CD26 on CD4+ T cells. By means of intracellular cytokine detection we analyzed expression of CD26 on CD4+ PBMC stimulated to produce IFN-gamma or IL-4 on a single cell level. All activated, cytokine-producing CD4+ T cells expressed CD26, but the increase in CD26 expression was higher in cells producing IFN-gamma. These data suggest that regulation of CD26 cell surface expression correlates with the production of T(H1)-like cytokines.
Journal of Allergy and Clinical Immunology 10/1997; 100(3):348-55. DOI:10.1016/S0091-6749(97)70248-3 · 11.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, hydrocortisone (HC), when combined with human IL-4, has been reported to increase IgE levels in supernatants (SN) of in vitro cultured leucocytes. In this study we investigated the influence of HC on allergen-specific IgE synthesis. Moreover, we examined the relevance of different cell types in this respect. Peripheral blood mononuclear cells (PBMC), T-cell depleted PBMC, CD14-depleted PBMC and highly purified B cells from 10 allergic (birch pollen and/or grass pollen) patients and five non-allergic individuals were investigated. The cells were incubated with HC and/or recombinant human IL-4 (rIL-4) for 8 days. A considerable increase of total IgE was observed in HC/rIL-4-stimulated cultures compared with rIL-4 alone, HC alone or non-stimulated cultures. We demonstrate that this effect depends on the presence of monocytes in in vitro cultures. These results were seen in every experiment, irrespective of healthy or atopic state of the blood donor. The increase of IgE could not be attributed to a rise of birch pollen-and/or grass pollen-specific IgE in patients allergic to these allergens, as shown by IgE-immunoblot. Radio-allergosorbent test (RAST) investigations of HC/rIL-4-stimulated cells cultures from allergic and non-allergic patients confirmed that HC/rIL-4-induced elevated IgE production was also not due to increased production of IgE, specific for important aero-allergens (pollens, house dust mite or animal dander). Therefore we conclude that newly synthesized IgE is not specific for allergens, but that sequential isotype switching in human B cells leads to increased polyclonal IgE production.