Kurt Blaser

An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils

A simple method is described for the isolation of human peripheral blood eosinophils by an immunomagnetic procedure. Superparamagnetic particles were coupled to a monoclonal antibody against CD16, a molecule present on neutrophils but not on eosinophils. A peripheral blood granulocyte preparation, containing neutrophils and eosinophils, was incubated with these anti-CD16 particles. In the magnetic field of a permanent magnet, magnetically labelled neutrophils were then retained on columns with a ferromagnetic matrix. By this negative selection procedure, eosinophils of 99.5% purity were obtained from normal individuals and 99.6% purity from patients with eczema. A comparison was made between the immunomagnetic method and eosinophil isolation after N-formyl-methionyl-leucyl-phenyl-alanine treatment. Even in the case of individuals with very low eosinophil counts, the immunomagnetic method permits the efficient isolation of highly purified and functionally active eosinophils.

Immune Responses in Healthy and Allergic Individuals Are Characterized by a Fine Balance between Allergen-specific T Regulatory 1 and T Helper 2 Cells

The mechanisms by which immune responses to nonpathogenic environmental antigens lead to either allergy or nonharmful immunity are unknown. Single allergen-specific T cells constitute a very small fraction of the whole CD4+ T cell repertoire and can be isolated from the peripheral blood of humans according to their cytokine profile. Freshly purified interferon-γ–, interleukin (IL)-4–, and IL-10–producing allergen-specific CD4+ T cells display characteristics of T helper cell (Th)1-, Th2-, and T regulatory (Tr)1–like cells, respectively. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals; in contrast, there is a high frequency of allergen-specific IL-4–secreting T cells in allergic individuals. Tr1 cells use multiple suppressive mechanisms, IL-10 and TGF-β as secreted cytokines, and cytotoxic T lymphocyte antigen 4 and programmed death 1 as surface molecules. Healthy and allergic individuals exhibit all three allergen-specific subsets in different proportions, indicating that a change in the dominant subset may lead to allergy development or recovery. Accordingly, blocking the suppressor activity of Tr1 cells or increasing Th2 cell frequency enhances allergen-specific Th2 cell activation ex vivo. These results indicate that the balance between allergen-specific Tr1 cells and Th2 cells may be decisive in the development of allergy.

Role of interleukin 10 in specific immunotherapy.

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.

IL‐10 and TGF‐β cooperate in the regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy

The regulation of normal and allergic immune responses to airborne allergens in the mucosa is still poorly understood, and the mechanism of specific immunotherapy (SIT) in normalizing the allergic response to such allergens is currently not clear. Accordingly, we have investigated the immunoregulatory mechanism of both normal and allergic responses to the major house‐dust mite (HDM) and birch pollen allergens — Dermatophagoides pteroynyssinus (Der p)1 and Bet v 1, respectively — as well as the immunologic basis of SIT to HDM in rhinitis and asthma patients. In normal immunity to HDM and birch pollen, an allergen‐specific peripheral T cell suppression to Der p 1 and Bet v 1 was observed. The deviated immune response was characterized by suppressed proliferative T celland Th1 (IFN‐γ) and Th2 (IL‐5, IL‐13) cytokine responses, and increased IL‐10 and TGF‐β secretion by allergen‐specific T cells. Neutralization of cytokine activity showed that T cell suppression was induced by IL‐10 and TGF‐β during SIT and in normal immunity to the mucosal allergens. In addition, SIT induced an antigen‐specific suppressive activity in CD4+ CD25+ T cells of allergic individuals. Together, these results demonstrate a deviation towards a regulatory/suppressor T cell response during SIT and in normal immunity as a key event for the healthy immune response to mucosal antigens.

Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells (TH1 and TH2) at the site of inflammation. The diversity of TH1 and TH2 function is not predetermined but depends on signals that drive the cells towards either subset. Histamine, released from effector cells (mast cells and basophils) during inflammatory reactions can influence immune response. Here we report that histamine enhances TH1-type responses by triggering the histamine receptor type 1 (H1R), whereas both TH1- and TH2-type responses are negatively regulated by H2R through the activation of different biochemical intracellular signals. In mice, deletion of H1R results in suppression of interferon (IFN)-γ and dominant secretion of TH2 cytokines (interleukin (IL)-4 and IL-13). Mutant mice lacking H2R showed upregulation of both TH1 and TH2 cytokines. Relevant to T-cell cytokine profiles, mice lacking H1R displayed increased specific antibody response with increased immunoglobulin-ε (IgE) and IgG1, IgG2b and IgG3 compared with mice lacking H2R. These findings account for an important regulatory mechanism in the control of inflammatory functions through effector-cell-derived histamine.

Activated T cells and eosinophilia in bronchoalveolar lavages from subjects with asthma correlated with disease severity

Activated T-lymphocytes may regulate the eosinophilic inflammation of bronchial asthma. In the present study, we investigated T cell activation and eosinophilia in blood and bronchoalveolar lavage (BAL) in 17 patients with asthma not receiving steroid treatment. Compared to normal individuals, BAL from patients with asthma contained significantly increased numbers of both lymphocytes and eosinophils (EOSs). The lymphocytosis consisted of increased numbers of both CD4+ and CD8+ T cells, and these T cell populations expressed elevated levels of T cell activation markers (interleukin-2 receptor [CD25], HLA-DR, and very late activation antigen 1). Close correlation was found between numbers of BAL CD4+ IL-2R+ T cells and numbers of EOSs. Moreover, the numbers of activated T cells and EOSs were related to the severity of asthma as measured by impairment of FEV1 and increased methacholine bronchial responsiveness. We demonstrate in both blood and BAL a close correlation between T cell activation, eosinophilia, and severity of asthma, suggesting that recruitment and activation of lymphocytes and EOSs are fundamental to the pathogenesis of bronchial asthma.

Mechanisms of immune suppression by interleukin-10 and transforming growth factor-β: the role of T regulatory cells

Specific immune suppression and induction of tolerance are essential processes in the regulation and circumvention of immune defence. The balance between allergen‐specific type 1 regulatory (Tr1) cells and T helper (Th) 2 cells appears to be decisive in the development of allergy. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals. In contrast, there is a high frequency of allergen‐specific interleukin‐4 (IL‐4)‐secreting T cells in allergic individuals. Allergen‐specific immunotherapy can induce specific Tr1 cells that abolish allergen‐induced proliferation of Th1 and Th2 cells, as well as their cytokine production. Tr1 cells utilize multiple suppressor mechanisms, such as IL‐10 and transforming growth factor‐β (TGF‐β) as secreted cytokines and various surface molecules, such as cytotoxic T‐lymphocyte antigen 4 and programmed death‐1. IL‐10 only inhibits T cells stimulated by low numbers of triggered T‐cell receptors, which depend on CD28 costimulation. IL‐10 inhibits CD28 tyrosine phosphorylation, preventing the binding of phosphatidylinositol 3‐kinase p85 and consequently inhibiting the CD28 signalling pathway. In addition, IL‐10 and TGF‐β secreted by Tr1 cells skew the antibody production from immunoglobulin E (IgE) towards the non‐inflammatory isotypes IgG4 and IgA, respectively. Induction of antigen‐specific Tr1 cells can thus re‐direct an inappropriate immune response against allergens or auto‐antigens using a broad range of suppressor mechanisms.

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment

Type I allergy is an immunoglobulin E (IgE)‐mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen‐containing sources but cannot identify the disease‐eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients’ IgE reactivity profiles to large numbers of disease‐causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

Molecular Mechanisms Underlying FOXP3 Induction in Human T Cells

FOXP3 is playing an essential role for T regulatory cells and is involved in the molecular mechanisms controlling immune tolerance. Although the biological relevance of this transcription factor is well documented, the pathways responsible for its induction are still unclear. The current study reveals structure and function of the human FOXP3 promoter, revealing essential molecular mechanisms of its induction. The FOXP3 promoter was defined by RACE, cloned, and functionally analyzed using reporter-gene constructs in primary human T cells. The analysis revealed the basal, T cell-specific promoter with a TATA and CAAT box 6000 bp upstream the translation start site. The basal promoter contains six NF-AT and AP-1 binding sites, which are positively regulating the trans activation of the FOXP3 promoter after triggering of the TCR. The chromatin region containing the FOXP3 promoter was bound by NF-ATc2 under these conditions. Furthermore, FOXP3 expression was observed following TCR engagement. Promoter activity, mRNA, and protein expression of T cells were suppressed by addition of cyclosporin A. Taken together, this study reveals the structure of the human FOXP3 promoter and provides new insights in mechanisms of addressing T regulatory cell-inducing signals useful for promoting immune tolerance. Furthermore, the study identifies essential, positive regulators of the FOXP3 gene and highlights cyclosporin A as an inhibitor of FOXP3 expression contrasting other immunosuppressants such as steroids or rapamycin.

T cell–mediated Fas-induced keratinocyte apoptosis plays a key pathogenetic role in eczematous dermatitis

Clinical and histologic similarities between various eczematous disorders point to a common efferent pathway. We demonstrate here that activated T cells infiltrating the skin in atopic dermatitis (AD) and allergic contact dermatitis (ACD) induce keratinocyte (KC) apoptosis. KCs normally express low levels of Fas receptor (FasR) that can be substantially enhanced by the presence of IFN-gamma. KCs are rendered susceptible to apoptosis by IFN-gamma when FasR numbers reach a threshold of approximately 40,000 per KC. Subsequently, KCs undergo apoptosis induced by anti-FasR mAbs, soluble Fas ligand, supernatants from activated T cells, or direct contact between T cells and KCs. Apoptotic KCs show typical DNA fragmentation and membrane phosphatidylserine expression. KC apoptosis was demonstrated in situ in lesional skin affected by AD, ACD, and patch tests. Using numerous cytokines and anti-cytokine neutralizing mAbs, we found no evidence that cytokines other than IFN-gamma participate in this process. In addition, apoptosis-inducing pathways other than FasR triggering were ruled out by blocking T cell-induced KC apoptosis by caspase inhibitors and soluble Fas-Fc protein. Responses of normal human skin and cultured skin equivalents to activated T cells demonstrated that KC apoptosis caused by skin-infiltrating T cells is a key event in the pathogenesis of eczematous dermatitis.