IgE-producing hybridomas established after B-cell culture in the CD40 system.

While total IgE synthesis can be easily induced in human PBL or B cells by different stimuli, no systems are known for the induction of allergen-specific IgE in vitro. In this study we investigated whether a specific Ig response could be induced using the CD40 culture system with the final intention to generate B-cell hybridomas secreting IgE of defined specificity. B cells derived from immunized donors normally give rise to many specific hybridomas after cell fusion. However, if cultured in the CD40 system and then immortalized and screened for anti-tetanus specificity, no tetanus-specific clones were found but a large number of IgE-secreting hybridomas had been generated. Also allergen-specific B cells could not be expanded in the CD40 system but long-term cultures yielded again B cells that were efficiently immortalized by cell fusion resulting in stable IgE-secreting hybridomas but of undefined specificity. One of these IgE-producing clones was further characterized and had an IgE production rate of 4.5 micrograms/10(6) cells/24 h. This paper provides two findings. (1) Our cell lines represent a valuable new source of human IgE. (2) Most importantly, our data indicate that the CD40 system is not suitable to expand specific B cells, suggesting that other systems have to be developed for the induction of a significant antigen-specific Ig response.

Inhibition of human IgE synthesis by anti-IgE antibodies requires divalent recognition.

We used a selection of well-characterized murine monoclonal anti-IgE antibodies to investigate their effect on human in vitro IgE synthesis. We found anti-IgE antibodies that either inhibited or enhanced interleukin-4 plus anti-CD40-induced in vitro IgE synthesis in peripheral blood mononuclear cells (PBMC). This differential activity was isotype specific as neither IgM nor IgG synthesis were affected. Interestingly, only coding IgE mRNA was down-regulated, whereas germ-line epsilon RNA expression was not influenced by anti-IgE monoclonal antibody (mAb). On purified B cells all anti-IgE mAb inhibited interleukin-4 plus anti-CD40-induced IgE synthesis, implying a role of non-B cells for the enhancing activity observed in PBMC. Using Fab and F(ab')2 of an inhibitory anti-IgE mAb we could show that divalent recognition was required for inhibition of IgE synthesis.

Rapid detection of autoantibodies to dsDNA with the particle gel immunoassay (ID-PaGIA).

OBJECTIVE: To describe a new particle agglutination test for the detection of autoantibodies to double stranded DNA (dsDNA). PATIENTS AND METHODS: Serum samples were collected from 40 unselected healthy blood donors and 200 patients with systemic lupus erythematosus (SLE) or a positive antinuclear antibody screen, or both. The samples were tested in the presence of red high density polystyrene particles coated with purified human dsDNA using the gel technique (Micro Typing System, ID-PaGIA, particle gel immunoassay). The results were compared with those obtained by the two standard anti-dsDNA antibody detection methods, Crithidia luciliae immunofluorescence test (CLIF) and enzyme linked immunosorbent assay (ELISA). RESULTS: The three anti-dsDNA assays exhibited an overall agreement of 87% and significant correlation with each other (p<0.0001). In the SLE group (n=71), 45 patients (63%) were found to be positive by ID-PaGIA compared with only 11/129 (9%) patients in the non-SLE group. Thus the ID-PaGIA had a sensitivity of 63%, and a specificity of 92% for SLE. In comparison, the standard detection methods showed sensitivities of 62% (CLIF) and 70% (ELISA) and specificities of 99% (CLIF) and 84% (ELISA) for SLE. Anti-dsDNA reactivity in the agglutination assay correlated closely with the quantities of antibody obtained by CLIF (r=0.81, p<0.0001) and ELISA (r=0.73, p<0.0001). CONCLUSIONS: The new particle gel agglutination test is a sensitive and specific immunoassay. It is a simple test procedure that might be well suited as a rapid screening method.

Neuropeptides accentuate interleukin-4 induced human immunoglobuline E synthesis in vitro.

Corticotropin releasing factor, adrenocorticotropic hormone (ACTH) and alpha-melanocyte stimulating hormone either inhibit or enhance in a dose-dependent fashion an interleukin-4 (IL-4) driven human IgE synthesis in vitro. Here, we show that culture conditions strongly influence the earlier observed dose- and donor-dependent effects of adrenocorticotropic hormone. The effect of ACTH on IgE synthesis became only apparent late during culture periods, suggesting an indirect effect via the cellular microenvironment rather than by acting directly at the level of B-cell isotype switching. Thus, we studied other proopiomelanocortin (POMC) derived peptides and neuropeptides known to influence the cellular microenvironment. Indeed, similar modulatory effects on IgE synthesis were also observed by the addition of other proopiomelanocortin-derived peptides such as alpha-, beta-, and gamma-endorphins as well as by the opioid binding pentapeptide Leu-enkephalin. Furthermore the neuropeptide substance P accentuated an IL-4 or an IL-4 and anti-CD40 antibody driven class switch to IgE. In contrast to ACTH, substance P interfered not only with IgE synthesis but also with the synthesis of the other immunoglobulin isotypes. Thus, systemically acting neuroendocrine peptides such as ACTH and locally acting neuropeptides such as the enkephalins and substance P can modulate the magnitude of an IL-4 induced IgE response.

IgG anti-IgE autoantibodies in immunoregulation.

Human sera contain anti-IgE autoantibodies with diverse biological functions in vitro. Opposite functions can be shown for triggering of IgE-mediated histamine release from human basophils in terms of anaphylactogenic or nonanaphylactogenic autoantibodies. Furthermore, autoantibodies are either capable of removing IgE from the surface of CD23-positive cells of binding more IgE to such cells. A similar dichotomy seems also to exist for the effect of autoantibodies on human IgE mRNA synthesis as well as IL-4-induced proliferation of human mononuclear cells. Thus, anti-IgE autoantibodies may represent a key factor in the manifestation and the development of allergic disease.

Antigen interaction and heat inactivation expose new epitopes on human IgE.

It is well established that heat-denatured IgE is no longer capable of binding to FcepsilonRI. We have found an antibody that interacts with heat-denatured IgE. Interestingly, this antibody can also be used to detect some serum IgE, but not IgE synthesized de novo in vitro. However, native IgE can be transformed into an IgE that is recognized by this antibody, if antigen is added. Our data indicate that physiological mechanisms exist that biologically inactivate IgE which might still be mistaken for 'functional' IgE by assays based on polyclonal antibodies.

Rapid detection of antibodies to immunoglobulin A molecules by using the particle gel immunoassay.

BACKGROUND AND OBJECTIVES: Antibodies to immunoglobulin A (IgA) molecules are thought to be frequently responsible for anaphylactic reactions in transfusion medicine, but practical tests for the detection of antibodies to IgA are not yet available. MATERIAL AND METHODS: Red, high-density polystyrene beads were coated with purified IgA molecules and then used to test serum samples collected from unselected healthy blood donors (n = 105) and patients with common variable immunodeficiency and/or IgA deficiency (n = 44). For testing, the standard gel-agglutination technique (ID-Micro Typing System) was employed. RESULTS: None of the normal serum samples were reactive with IgA-coated beads and samples from only 10 patients were positive (titre range 1 : 2 to 1 : 256). Only one out of all patients studied had a history of an anaphylactic reaction and this was related to the administration of Rh(D) prophylaxis (anti-D immunoglobulin). The beads did not show non-specific agglutination and could be used repeatedly for longer than 6 months. The results were reproducible in all patients tested. CONCLUSION: The new test allows a specific and rapid detection of antibodies to IgA molecules. In order to evaluate the clinical relevance of the test, analysis is required of a wider range of antibodies that produce anaphylactic reactions.

A specific feedback by anti-IgE autoantibodies on the cytokine network in allergy.

During recent years we have shown that anti-IgE antibodies can have different biological functions. Depending on their epitope specificity they can be anaphylactogenic or not, they interfere with IgE binding to its receptor or not, and they enhance or inhibit IgE synthesis. Therefore we propose a theoretical model implying that anti-IgE autoantibodies are specific feed back molecules that neutralize IgE induced by the cytokine network. In the normal individual this system would be beneficial, where as the atopic individual, due to differences in its B cell repertoire, will produce the wrong type of anti-IgE antibody. The wrong type of anti-IgE antibody may even aggravate the disease as some of these autoantibodies may induce IgE synthesis or trigger effector cells that in turn generate a Th2 like cytokine pattern.

Neuropeptides are potent modulators of human in vitro immunoglobulin E synthesis.

We determined the effect of adrenocorticotropin hormone (ACTH) on the regulation of IgE synthesis. Depending on the concentration, ACTH enhanced or inhibited IgE synthesis in a culture system where IgE synthesis was induced with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody in peripheral blood mononuclear cells. Similar effects on IgE synthesis were observed by adding ACTH-related peptides, e.g. corticotropin-releasing factor (CRF), the inducer of ACTH, or alpha-melanocyte stimulating hormone (alpha-MSH), a cleavage product of ACTH. However, ACTH had no effect on IgG or IgM synthesis in this culture system. ACTH did not act directly on either B or T cells as there was no influence on IgE synthesis in a system using purified B cells alone or co-cultured with T cells. The effect of ACTH on IgE synthesis was mediated by accessory cells. This was shown by priming purified CD14-positive monocytes with ACTH and reconstitution experiments. Therefore, these findings suggest that ACTH and the related peptides CRF and alpha-MSH can influence the microenvironment modulating an IL-4 and anti-CD40 monoclonal antibody driven class switching to IgE via accessory cells.

Restricted TCR Valpha gene rearrangements in T cells recognizing an immunodominant peptide of myelin basic protein in DR2 patients with multiple sclerosis.

T cell responses to myelin basic protein (MBP) are thought to play an important role in the pathogenesis of multiple sclerosis (MS). The response to the 83-99 region of MBP represents a dominant response to MBP in patients with MS and is associated with HLA-DR2 that is linked with susceptibility to MS. Although T cell clones reactive to various regions of MBP have been found to exhibit heterogeneous TCR Vbeta gene usage in patients with MS, it is unclear whether T cell clones uniformly recognizing the 83-99 peptide of MBP in the context of the same DR molecule would have restricted TCR V gene rearrangements and recognition motifs. In this study, a panel of DR2- or DR4-restricted T cell clones specific for the MBP83-99 peptide were derived from 11 patients with MS and examined for TCR V gene usage by PCR and the recognition motifs using analog peptides. Our study revealed that despite a few T cell clone pairs having similar recognition motifs and shared sequence homology in the CDR3, the overall recognition motifs of MBP83-99-specific T cells were considerably diverse. Interestingly, the DR2-restricted T cell clones displayed a biased V gene usage for Valpha3 and Valpha8, while Vbeta gene rearrangements were highly heterogeneous. This study provided experimental evidence suggesting a limited heterogeneity in TCR Valpha gene rearrangements of MBP-reactive T cells in DR2 patients with MS.

T cell recognition motifs of an immunodominant peptide of myelin basic protein in patients with multiple sclerosis: structural requirements and clinical implications.

Myelin basic protein (MBP)-reactive T cells may play an important role in the pathogenesis of multiple sclerosis (MS). The T cell response to the 83-99 region of MBP represents a dominant autoreactive response to MBP in MS patients of DR2 haplotype. In this study, a large panel of DR2- and DR4-restricted T cell clones specific for the MBP83-99 peptide were examined for the recognition motifs and structural requirements for antigen recognition using alanine-substituted peptides. Our study revealed that although the recognition motifs of the T cell clones were diverse, the TCR contact residues within the 83-99 region of MBP were highly conserved. Two central residues (Phe90 and Lys91) served as the critical TCR contact points for both DR2- and DR4-restricted T cell clones. Single alanine substitution at residue 90 or residue 91 abolished the responses of 81-95 % of the T cell clones while a double alanine substitution rendered all T cell clones unresponsive. It was also demonstrated in this study that the substituted peptides altered the cytokine profile of some, but not all, T cell clones. Some MBP83-99-specific T cell clones were able to sustain alanine substitutions and were susceptible to activation by microbial antigens. The study has an important implication in designing a peptide-based therapy for MS.