Search

In allergic inflammations of the skin, activation of CD4+ T cells was demonstrated to play an important role; however, a minor role for CD8+ T cells is implied. In the present study, we compared cutaneous lymphocyte-associated Ag (CLA)-expressing CD4+ and CD8+ subsets, which were isolated from peripheral blood and lesional skin biopsies in atopic dermatitis (AD) patients. We demonstrated that CD8+CLA+ T cells proliferate in response to superantigen and are as potent as CD4+CLA+ T cells in IgE induction and support of eosinophil survival. In atopic skin inflammation, the existence of high numbers of CD4+ and CD8+ T cells was demonstrated by immunohistochemistry and by culturing T cells from skin biopsies. In peripheral blood, both CD4+ and CD8+ subsets of CLA+CD45RO+ T cells were in an activated state in AD. The in vivo-activated CLA+ T cells of both subsets spontaneously released an IL-5- and IL-13-dominated Th2 type cytokine pattern. This was confirmed by intracytoplasmic cytokine staining immediately after isolation of the cells from peripheral blood. In consequence, both CD4+ and CD8+, CLA+ memory/effector T cells induced IgE production by B cells mainly by IL-13, and enhanced eosinophil survival in vitro by delaying eosinophil apoptosis, mainly by IL-5. These results indicate that in addition to the CD4+ subset, the CD8+CLA+ memory/effector T cells are capable of responding to superantigenic stimulation and play an important role in the pathogenesis of AD.

Background: In allergic inflammations of the skin, the pivotal role of CD45RO+ (memory/effector) T cells expressing the cutaneous lymphocyte-associated antigen (CLA) was demonstrated. In both atopic dermatitis (AD) and contact dermatitis (CD), T cells specific to skin-related allergens were confined to the CLA+ T cell population. Our research was aimed to further characterize these T cells in AD., Methods: CD4+ and CD8+ subsets of CLA+ CD45RO+ T cells were purified from peripheral blood of AD patients and healthy control individuals. We studied, in vivo activation patterns, cytokine profiles, immunoglobulin isotype regulation and the influence of these cells on eosinophil survival and apoptosis., Results: The CLA+ CD45RO+ T cells represent an in- vivo-activated memory/effector T cell subset as shown by surface expression of activation markers, spontaneous proliferation and a lower activation threshold via TCR/CD3 triggering. These cells contain and spontaneously release high amounts of preformed IL-5 and IL-13 but only very little IL-4 and IFN-gamma in their cytoplasm, as demonstrated by intracellular cytokine staining immediately after purification. Moreover, CLA+ memory/ effector T cells induce IgE production in B cells and enhance eosinophil survival by inhibiting eosinophil apoptosis in AD. In comparison, the CLA- population represents a resting memory T cell fraction, induces rather IgG4 in B cells and does not show any effect on eosinophil survival and apoptosis., Conclusion: Our results indicate that in-vivo-activated both CD4+ and CD8+ memory/effector T cells with skin-homing property play a specific and decisive role in the pathogenesis and exacerbation of AD. In contrast, resting memory T cells of atopic individuals retain normal, nonallergic immune functions.

Altered peptide ligands (APL) can modify T cell effector function by their diversity in binding to the TCR or MHC class II-presenting molecules. The capacity to inhibit Th2 cytokine production by allergen-specific T cells would contribute to combating allergic inflammation. The presence of APL generated by Ala-substitutions in a synthetic dodeca-peptide spanning an immunodominant epitope of bee venom phospholipase A2 (PLA) was investigated in human T cells. Four of five substituted peptides reduced proliferation, IL-4, and IFN-gamma production by cloned PLA-specific Th0 cells proportionately. However, one APL, PLA-F82A, inhibited IL-4 but had no effect on IFN-gamma production. This uncoupling of IL-4 from IFN-gamma production was also observed on immunogenic restimulation of the cloned T cells pre-exposed to the APL/APCs. It appeared to result from lower affinity of binding to MHC class II by the APL compared with the native peptide. The APL also inhibited IL-4 production by polyclonal T cells. In consequence of the change in cytokine secretion, the production of IgG4 in vitro increased by PLA-F82A stimulation, compared with the native peptide. Exposure of the cloned T cells to either the APL or the native peptide, in the absence of professional APC, induced anergy such that proliferation and production of IL-4, IL-5, and IL-13 was abrogated on immunogenic rechallenge. Defective T cell activation appeared to result from alterations in transmembrane signaling through the TCR, specifically to lack of tyrosine phosphorylation of the tyrosine kinase, ZAP-70.

Background: The soluble bee venom phospholipase A2 (PLA) represents the major allergen/antigen for allergic and hyperimmune individuals following bee sting. A number of studies implicate enzymes, and PLA in particular, as potent allergens. We have studied specific activation of T cells by enzymatically active and inactive mutants of PLA, and secretion of cytokines regulating IgE and IgG4 antibody formation., Methods: Recombinant (r) wild type PLA (rPLA-WT) and an enzymatically inactive rPLA (rPLA-H34Q) were produced in Escherichia coli. Eleven bee venom allergic patients and three hyperimmune, healthy individuals were included in the study. After specific stimulation of PBMC with the rPLA variants, proliferative response, IFNgamma, IL-2, IL-4, IL-5, and IL-13 production, as well as total and PLA-specific IgE and IgG4 production, were analysed., Results: Similar levels of specific B cell recognition, proliferative and cytokine responses were observed after stimulation with either enzymatically active or inactive rPLA. In addition, equal amounts of antigen-specific and total IgE and IgG4 antibodies were produced by stimulation with both forms of rPLA., Conclusions: The enzymatic activity of PLA does not influence the specific activation and cytokine production by T cells from bee venom-sensitized or hyperimmune individuals, or the IgE/IgG4 antibodies synthesis by B cells in vitro.

The induction of allergen-specific anergy in peripheral T cells represents a key step in specific immunotherapy (SIT). Here we demonstrate that the anergic state results from increased IL-10 production. In bee venom (BV)-SIT the specific proliferative and cytokine responses against the main allergen, the phospholipase A2 (PLA), and T cell epitope-containing PLA peptides were significantly suppressed after 7 d of treatment. Simultaneously, the production of IL-10 increased during BV-SIT. After 28 d of BV-SIT the anergic state was established. Intracytoplasmic cytokine staining of PBMC combined with surface marker detection revealed that IL-10 was produced initially by activated CD4(+)CD25(+), allergen-specific T cells, and followed by B cells and monocytes. Neutralization of IL-10 in PBMC fully reconstituted the specific proliferative and cytokine responses. A similar state of IL-10-associated T cell anergy, as induced in BV-SIT, was found in hyperimmune individuals who recently had received multiple bee stings. The addition of IL-10 to soluble CD40 ligand IL-4-stimulated PBMC or purified B cells inhibited the PLA-specific and total IgE and enhanced the IgG4 formation. Accordingly, increased IL-10 production by SIT causes specific anergy in peripheral T cells, and regulates specific IgE and IgG4 production toward normal IgG4-related immunity.

Background: Specific immunotherapy with honeybee venom (BV) is highly effective, but allergic side effects can occur during treatment. Immunotherapy with peptides containing major T-cell epitopes of the relevant allergen or allergens provides an alternative strategy without these problems., Objective: The study investigates the immunologic mechanisms and clinical effects of immunotherapy with T-cell epitope peptides of the major BV allergen, the phospholipase A2 (PLA)., Methods: Five patients with IgE-mediated systemic allergic reactions to bee stings were treated with a mixture of three T-cell epitope peptides of PLA. Ten patients allergic to BV receiving whole BV immunotherapy served as control subjects. Increasing doses of the peptide mixture, up to a maintenance dose of 100 microg, were administered subcutaneously within 2 months. The patients were then challenged with PLA and 1 week later with a bee sting. The cellular and humoral immune response was measured in vitro., Results: No allergic side effects were caused by the peptide immunotherapy, and all patients tolerated the challenge with PLA without systemic allergic symptoms. Two patients developed mild systemic allergic reactions after the bee sting challenge. After peptide immunotherapy, specific proliferative responses to PLA and the peptides in peripheral blood mononuclear cells were decreased in successfully treated patients. The production of TH2 and TH1 cytokines was inhibited, and B cells were not affected in their capacity to produce specific IgE and IgG4 antibodies. Their levels increased after allergen challenge in favor of IgG4., Conclusions: Immunotherapy of BV allergy with short T-cell peptides of PLA induces epitope-specific anergy in peripheral T cells and changes the specific isotype ratio in a fashion similar to that of conventional immunotherapy in successfully treated patients.

Bee venom phospholipase A2 (PLA) represents the major allergen and antigen in allergic and non-allergic individuals sensitized to bee sting. We have studied specific activation of peripheral T cells by different structural and conformational variants of PLA and secretion of cytokines regulating IgE and IgG4 antibody (Ab) formation. PLA molecules expressing the correctly folded tertiary structure, which show high affinity to membrane phospholipids and were recognized by Ab from bee sting allergic patients, induced high IL-4, IL-5 and IL-13 production in peripheral blood mononuclear cell cultures. In contrast, non-refolded recombinant PLA (rPLA) and reduced and alkylated native PLA (nPLA) induced more IFN-gamma and IL-2 and higher proliferative responses. Differences in proliferation and cytokine patterns among correctly folded and non-refolded PLA resulted from conformation-dependent involvement of different antigen-presenting cell (APC) types. Antigen (Ag)-presenting B cells recognized PLA only in its natural conformation, stimulated Th2 type cytokines and induced IgE Ab. Non-refolded PLA was recognized, processed and presented exclusively by monocytes and induced a Th1 dominant cytokine profile leading to IgG4 production by B cells. The possibility that production of particular cytokine patterns and Ig isotype was influenced by the enzymatic activity of PLA was excluded by using enzymatically inactive H34Q point-mutated, refolded rPLA. These findings demonstrate the decisive role of specific Ag recognition by different APC, depending on structural features, membrane phospholipid binding property and the existence of conformational B cell epitopes, in the differential regulation of memory IgE and IgG4 Ab. Furthermore, they show that a change from IgE-mediated allergy to normal immunity against a major allergen can be induced by rPLA variants that are not recognized by specific Ab and B cells but still carry the T cell epitopes. These features may enable new applications for safer immunotherapy.

Cutaneous lymphocyte-associated Ag (CLA) is a skin-homing receptor displayed by memory/effector T cells recognizing skin-related allergens. Here we demonstrate that peripheral blood CLA+ CD45RO+ T cells in patients with atopic dermatitis (AD) are in vivo activated. They spontaneously proliferate and release an IL-13-dominated Th2 cytokine profile and are capable of inducing IgE in autologous B cells without further activation. The spontaneous cytokine release occurred within the first hour of culture and was not inhibited by cycloheximide or by other immunosuppressive drugs, indicating that cytokine transcription and translation had been completed in vivo. In contrast, the CLA- CD45RO+ T cells from the same patients and from nonatopic controls represented a resting memory T cell subset, secreted borderline quantities of cytokines, and induced IgG4. Polyclonal activation by the anti-CD2/anti-CD3/anti-CD28 mAb mixture generated distinct cytokine patterns in the two memory/effector T cell subsets. CLA+ T cells secreted Th2 cytokines with high IL-13 levels, and the CLA- subset mainly produced IFN-gamma. There was no difference in in vitro activated cytokine pattern between AD patients and nonatopic subjects. These results indicate that the CLA+ memory/effector T cells of AD patients are activated in vivo and play a pivotal role in allergic inflammation by production of IL-13 and induction of IgE Abs. In contrast, the CLA- resting memory T cell population may exert immunoprotective properties toward allergen by high IFN-gamma secretion and induction of IgG4.

Although anti-inflammatory properties of glucocorticoids (GC) are well documented, their activity in allergic diseases is still controversial. Recently, it has been reported that GC can increase, both in vivo and in vitro, the polyclonal production of total IgE. In this study we investigated the effects of GC on the antigen (Ag)-specific IgE response in a human in vitro system with peripheral blood mononuclear cells or B cells of bee venom-sensitized individuals that allows the production of bee venom phospholipase A2 (PLA)-specific IgE and IgG4 antibodies (Ab). PLA-specific Ab were induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand (sCD40L) in the presence of interleukin (IL)-4. Indeed, dexamethasone and prednisolone enhanced the formation of total IgE and IgG4 in PBMC, while the production of PLA-specific IgE and IgG4 Ab was selectively inhibited in a dose-dependent manner. The suppressive effect of GC was mediated during Ag-specific stimulation and T cell-B cell interaction. This was due to GC suppressing specific T cell proliferation and cytokine production, whereas neither allergen-specific nor total IgE and IgG4 production by sCD40L/IL-4-stimulated pure B cells was affected. In contrast to GC, cyclosporine A inhibited both total and PLA-specific IgE and IgG4 secretion in peripheral blood mononuclear cells and B cell cultures. Further experiments showed that increase in nonspecific total isotype response resulted from inhibition of IL-4 uptake by cells other than B cells and sufficient availability of IL-4 to B cells for isotype switch and synthesis. Furthermore, demonstration of opposite regulatory effects of GC on specific and total isotype formation in vitro, including the inhibition of allergy-relevant Ag-specific IgE response, may contribute to a better understanding of apparently controversial observations, and explain why most allergic patients benefit from GC therapy.

Development of IgE-mediated allergic conditions is dependent on the secretion of a Th2 cytokine pattern, including IL-4, IL-5, and IL-13. The induction of anergy would be one mechanism to abrogate cytokine secretion by Th2 cells, which may be pivotal to the allergic response. We demonstrate here that incubation of cloned human CD4+ phospholipase A2 (PLA)-specific Th2 cells with antigenic peptide, in the absence of professional APC, results in a state of nonresponsiveness. The anergic T cells failed to proliferate or secrete IL-4 in response to optimal stimulation with PLA and autologous, professional APC. Secretion of IL-5 and IL-13, however, was only partially inhibited. The anergic state of the Th2 cells was not associated with CD3 or CD28 down-regulation. However, anergy did appear to be closely related to alterations in signaling pathways, mediated through the TCR, of the cells. In contrast to untreated Th2 cells, anergized Th2 cells failed to respond to anti-CD3 mAb with either increased tyrosine kinase activity or increased levels of tyrosine phosphorylation of p56(lck) or ZAP70. A strong and sustained intracellular calcium flux, observed in untreated Th2 cells in response to anti-CD3 mAb, was absent in anergic Th2 cells. Furthermore, the induction of anergy seems to represent an active process, associated with increased levels of basal tyrosine kinase activity, cytokine production, and CD25 up-regulation in anergic Th2 cells. Together, our results indicate that anergy in Th2 cells is associated with defective transmembrane signaling through the TCR.

Investigations on the mechanisms of IgE regulation in vitro have been conducted thus far in systems that allow the synthesis of total rather than specific IgE. To study the regulatory prerequisites of antigen-specific IgE antibody production, we have established a culture system that allows the generation of bee venom phospholipase A2-specific IgE and IgG4 antibodies. Allergen-specific IgE was induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand in the presence of IL-4. Additional stimulation of T cells through the CD2 activation pathway by two different anti-CD2 monoclonal antibodies enhanced both the allergen-specific and the total IgE and IgG4 responses. An optimal amount of allergen (0.1 ng/ml) resulted in the induction of both allergen-specific IgE and IgG4 antibodies. Higher antigen doses reduced allergen-specific antibodies and enhanced total isotype production. This differential regulation of allergen-specific and total isotypes reflects different allergen dose-dependent mechanisms in specific and polyclonal activation of T and B cells. Although both isotypes require IL-4 for initial induction, opposite regulatory effects by T cells were observed for IgE and IgG4 antibody expression. In peripheral blood mononuclear cell cultures stimulated with soluble CD40 ligand, IL-4, and phospholipase A2, stimulation of T cells with higher amounts of anti-CD2 enhanced IgG4 in parallel to increased IL-2 and interferon-gamma secretion but inhibited IgE synthesis. These results provide evidence for differential regulation of allergen-specific and total IgE and IgG4 by antigen concentration and demonstrate the pivotal role of T cells controlling the synthesis of the IgE and IgG4 antibody isotypes.

Bee venom phospholipase A2 (PLA) is the major allergen in bee sting allergy. It displays three peptide and a glycopeptide T cell epitopes, which are recognized by both allergic and non-allergic bee venom sensitized subjects. In this study PLA- and PLA epitope-specific T cell and cytokine responses in PBMC of bee sting allergic patients were investigated before and after 2 mo of rush immunotherapy with whole bee venom. After successful immunotherapy, PLA and T cell epitope peptide-specific T cell proliferation was suppressed. In addition the PLA- and peptide-induced secretion of type 2 (IL-4, IL-5, and IL-13), as well as type 1 (IL-2 and IFN-gamma) cytokines were abolished, whereas tetanus toxoid-induced cytokine production and proliferation remained unchanged. By culturing PBMC with Ag in the presence of IL-2 or IL-15 the specifically tolerized T cell response could be restored with respect to specific proliferation and secretion of the type 1 T cell cytokines, IL-2 and IFN-gamma. In contrast, IL-4, IL-5, and IL-13 remained suppressed. Treatment of tolerized T cells with IL-4 only partially restored proliferation and induced formation of distinct type 2 cytokine pattern. In spite of the allergen-specific tolerance in T cells, in vitro produced anti-PLA IgE and IgG4 Ab and their corresponding serum levels slightly increased during immunotherapy, while the PLA-specific IgE/IgG4 ratio changed in favor of IgG4. These findings indicate that bee venom immunotherapy induces a state of peripheral tolerance in allergen-specific T cells, but not in specific B cells. The state of T cell tolerance and cytokine pattern can be in vitro modulated by the cytokines IL-2, IL-4, and IL-15, suggesting the importance of microenvironmental cytokines leading to success or failure in immunotherapy.