Pathology Of Allergic Diseases Research Articles

Pathophysiological Roles of Neuro-Immune Interactions between Enteric Neurons and Mucosal Mast Cells in the Gut of Food Allergy Mice.

Recently, the involvement of the nervous system in the pathology of allergic diseases has attracted increasing interest. However, the precise pathophysiological role of enteric neurons in food allergies has not been elucidated. We report the presence of functional high-affinity IgE receptors (FcεRIs) in enteric neurons. FcεRI immunoreactivities were observed in approximately 70% of cholinergic myenteric neurons from choline acetyltransferase-eGFP mice. Furthermore, stimulation by IgE-antigen elevated intracellular Ca2+ concentration in isolated myenteric neurons from normal mice, suggesting that FcεRIs are capable of activating myenteric neurons. Additionally, the morphological investigation revealed that the majority of mucosal mast cells were in close proximity to enteric nerve fibers in the colonic mucosa of food allergy mice. Next, using a newly developed coculture system of isolated myenteric neurons and mucosal-type bone-marrow-derived mast cells (mBMMCs) with a calcium imaging system, we demonstrated that the stimulation of isolated myenteric neurons by veratridine caused the activation of mBMMCs, which was suppressed by the adenosine A3 receptor antagonist MRE 3008F20. Moreover, the expression of the adenosine A3 receptor gene was detected in mBMMCs. Therefore, in conclusion, it is suggested that, through interaction with mucosal mast cells, IgE-antigen-activated myenteric neurons play a pathological role in further exacerbating the pathology of food allergy.

MicroRNAs in type 2 immunity

Type 2 immunity drives the pathology of allergic diseases and is necessary for expulsion of parasitic worms as well as having important implications in tumor progression. Over the last decade, a new research field has emerged describing a significant link between type 2 immunity and cancer development, called AllergoOncology. Thus, type 2 immune responses must be carefully regulated to mediate effective protection against damaging environmental factors, yet avoid excessive activation and immunopathology. Regulation of gene expression by microRNAs is required for normal behavior of most mammalian cells and has been studied extensively in the context of cancer. Although microRNA regulation of the immune system in cancer is well established and includes type 2 immune reactions in the tumor microenvironment, the involvement of microRNAs in these responses initiated by allergens, parasites or other environmental factors is just emerging. In this review, we focus on recent advances which increase the understanding of microRNA-mediated regulation of key mechanisms of type 2 immunity.

The Identification of Allergen-Derived T Cell Epitopes.

Type I allergy is a disease primarily mediated by immunoglobulin E (IgE) and T helper type 2 (Th2) cells. The role of Th2 and other T cell subsets in the pathology of allergic disease as well as induction of tolerance has become an area of intense research over the last decades. Studying allergen-specific T cells to gain a better understanding of their contribution to allergic pathology and how they are modulated by allergen-specific immunotherapy requires knowledge of the allergens targeted by these cells. Identification of T cell epitopes in allergy can be achieved by a variety of methods. In this chapter, we will focus on a technique named FluoroSpot, which relies on the detection of cytokines secreted by T cells in response to stimulation with an antigen (allergen), such as timothy grass (TG) extract or an allergen-derived peptide, for which the cell is specific. We will describe how to overcome the challenge of detecting rare, TG-specific, T cells that occur at low frequency in the blood by using an in vitro expansion culture and subsequent mapping of the precise T cell epitope using FluoroSpot.

The immunoglobulin superfamily member CD200R identifies cells involved in type 2 immune responses.

The pathology of allergic diseases involves type 2 immune cells, such as Th2, ILC2, and basophils exerting their effect by production of IL-4, IL-5, and IL-13. However, surface receptors that are specifically expressed on type 2 immune cells are less well documented. The aim of this investigation was to identify surface markers associated with type 2 inflammation. Naïve human CD4+ T cells were short-term activated in the presence or absence of IL-4 and analyzed for expression of >300 cell-surface proteins. Ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from peanut-allergic (PA) and nonallergic subjects were stimulated (14-16 h) with peanut extract to detect peanut-specific CD4+ CD154+ T cells. Biopsies were obtained for transcriptomic analysis from healthy controls and patients with extrinsic or intrinsic atopic dermatitis (AD) and psoriasis. Expression analysis of >300 surface proteins enabled identification of IL-4-upregulated surface proteins, such as CD90, CD108, CD109, and CD200R (CD200R1). Additional analysis of in vitro-differentiated Th0, Th1, and Th2 cultures identified CD200R as upregulated on Th2 cells. From ex vivo-isolated PBMCs, we found high expression of CD200R on Th2 and ILC2 cells and basophils. In PA subjects, the peanut-specific Th2 (CD154+ CRTh2+ ) cells expressed more CD200R than the non-allergen-specific Th2 (CD154- CRTh2+ ) cells. Moreover, costaining of CD161 and CD200R identified peanut-specific highly differentiated IL-4+ IL-5+ Th2 cells. Finally, transcriptomic analysis revealed upregulation of CD200R in lesional skin from subjects with an extrinsic AD phenotype compared to healthy skin. These results indicate that CD200R expression strongly correlates with Th2 pathology; though, the mechanism is as yet elusive.

Immunorehabilitation Children with Impaired Health Status

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Airway responses towards allergens - from the airway epithelium to T cells.

The prevalence of allergic diseases such as allergic rhinitis is increasing, affecting up to 30% of the human population worldwide. Allergic sensitization arises from complex interactions between environmental exposures and genetic susceptibility, resulting in inflammatory T helper 2 (Th2) cell-derived immune responses towards environmental allergens. Emerging evidence now suggests that an epithelial dysfunction, coupled with inherent properties of environmental allergens, can be responsible for the inflammatory responses towards allergens. Several epithelial-derived cytokines, such as thymic stromal lymphopoietin (TSLP), IL-25 and IL-33, influence tissue-resident dendritic cells (DCs) as well as Th2 effector cells. Exposure to environmental allergens does not elicit Th2 inflammatory responses or any clinical symptoms in nonatopic individuals, and recent findings suggest that a nondamaged, healthy epithelium lowers the DCs' ability to induce inflammatory T-cell responses towards allergens. The purpose of this review was to summarize the current knowledge on which signals from the airway epithelium, from first contact with inhaled allergens all the way to the ensuing Th2-cell responses, influence the pathology of allergic diseases.

Human mast cell and basophil/eosinophil progenitors.

Mast cell, basophil, and eosinophil lineages all derive from CD34(+) hemopoietic stem cells; however, mast cells are derived from a distinct, nonmyeloid progenitor, while eosinophils and basophils share a common myeloid progenitor. These progenitors likely evolved from an ancestral leukocyte population involved in innate immunity and currently play a central role in the pathology of allergic disease. Advances in isolation and analysis of mast cell and basophil/eosinophil progenitor populations have been critical to understanding lineage commitment, differentiation, function, and transcriptional regulation of these cells and have provided a way of monitoring the effect of novel investigational therapies on these cell populations in samples of blood, bone marrow, and airway secretions.

NKG2D+ IFN-γ+ CD8+ T Cells Are Responsible for Palladium Allergy

Nickel, cobalt, and chromium are well known to be causal agents of allergic contact dermatitis. Palladium (Pd) can also cause allergic disease and exposure results from wide use of this metal in dental restorations and jewelry. Metal allergy is categorized as a delayed-type hypersensitivity, and metal-responsive T cell clones have been isolated from allergic patients. However, compared to nickel, little is known about the pathology of allergic disease mediated by Pd, and pathogenic T cells are poorly understood. To identify the pathogenic T cells that are responsible for onset of Pd allergy, we enriched metal-responsive lymphocytes by sequential adoptive transfer of involved lymph node cells. Here we show that sequential adoptive transfer gradually increased the incidence and the intensity of Pd allergy, and CD8+ T cells are responsible for the disease as CD8+ T cell-depleted mice and β2-microglobulin-deficient mice did not develop Pd allergy. In addition, we found that draining lymph node cells skewed toward CD8+ T cells in response to Pd challenge in 8th adoptive transferred recipient mice. The CD8+ T cells expressed NKG2D, a costimulatory molecule involved in the production of IFN-γ. NKG2D ligand was also induced in Pd-injected tissues. Furthermore, both NKG2D ligand-transgenic mice, where NKG2D is downmodulated, and IFN-γ-deficient mice showed impaired Pd allergy. Taken together, these results indicate that IFN-γ-producing NKG2D+ CD8+ T cells are responsible for Pd allergy and suggest that NKG2D is a potential therapeutic target for treatment of metal allergy.

Suppressive effects of a novel CC chemokine receptor 4 antagonist on Th2 cell trafficking in ligand- and antigen-induced mouse models

CC chemokine receptor 4 (CCR4) has been implicated as a preferential marker for T helper type 2 (Th2) cells, and is believed to be involved in the pathology of allergic diseases by controlling Th2 cell trafficking into inflamed tissues. The objective of the study was to characterize the pharmacological properties of E0001-163, a novel CCR4 antagonist. E0001-163 was tested in both in vitro chemotaxis assays as well as in vivo mouse models of CCR4 ligand-induced air pouch and antigen-induced airway inflammation by utilizing in vitro-polarized Th2 cells. In vitro, E0001-163 inhibited migratory response of human Th2-polarized cells to CCL22, a CCR4 ligand, with an IC50 value of 11.9nM. E0001-163 significantly suppressed CCL22-induced Th2 cell trafficking into mouse air pouch in a dose-dependent manner at doses of 3 and 10mg/kg, suggesting that E0001-163 has an inhibitory effect on CCR4-mediated T cell trafficking in vivo. In addition, E0001-163 partially decreased Th2 cell trafficking and the level of IL-4 in the lungs in Th2-tansferred and ovalbumin (OVA)-challenged mice. T cell trafficking involves multiple chemokine receptors both in acute and chronic phases, and our findings suggest that CCR4, together with other chemokine receptors, may be involved in Th2 cell trafficking under disease conditions.

Spatial and temporal regulation of interleukin 4 and interleukin 13 expression identified by dual reporter mice. (P1129)

Abstract Type II immune responses are associated with protection against helminth infections, as well as the pathology of allergic diseases that are initiated against innocuous antigens. Two integral cytokines associated with type II immunity are interleukin (IL-)4 and IL-13. The 4C13R transgenic dual reporter mouse has been created to allow identification of IL-4 and IL-13 producing cells by the production of two intracellular fluorescent molecules, AmCyan and DS-Red respectively. The expression of the distinct reporter proteins are under the normal transcriptional control of the Il4 or Il13 genes. This technology allows for the analysis of in situ IL-4 and/or IL-13 production by the relevant differentiated immune cell types without any effect on the endogenous cytokine genes or their effector activities in the mouse. Using this reporter system we have identified that in vitro generated CD4+ Th2 cells have divergent expression of IL-4 and IL-13, suggesting cytokine specific Th2 subsets. Additionally, IL-13 expression is delayed compared with IL-4 in this system. Reporter expression in vivo has also highlighted that IL-4/IL-13 double producers are only a subpopulation of CD4+ T cells, and cytokine expression profiles differ significantly between lymph node and effector tissues during type II responses. Studying the expression and regulation of these cytokines will allow us to understand their contribution in both disease and protection.

Characterization of IgE+ B cells during primary and secondary immune responses (P6035)

Abstract IgE antibodies may have a protective role in parasite immunity, but they are best known for contributing to the pathology of allergic diseases. Exaggerated IgE responses can have dangerous consequences such as systemic anaphylaxis. However, direct studies of the B cells that express IgE have been hindered by technical challenges. We have generated fluorescent IgE reporter mice and also developed an improved flow cytometry procedure, enabling specific detection of IgE+ B cells in vivo. With these tools, we have analyzed various types of immune responses and determined general features of the genesis and fate of IgE+ B cells. The frequency of IgE+ B cells was typically very low compared with other isotypes such as IgG1. The majority of IgE+ B cells differentiated into short-lived plasma cells. IgE+ B cells transiently participated in germinal centers, and showed limited affinity maturation. Consistent with these findings, IgE+ B cells expressing memory cell markers were virtually undetectable, and no persistent population of IgE+ B cells could be identified several weeks after immunization. However, secondary immunizations generated faster IgE+ plasma cell responses compared with primary immunizations, suggesting that other memory precursors likely give rise to IgE plasma cells in recall responses.

TH2 heterogeneity: Does function follow form?

T(H)2 immune responses are required for the 2 fundamental pathological processes characteristic of allergic disease: IgE-mediated hypersensitivity and eosinophilic inflammation. The 3 established T(H)2 cytokines, IL-4, IL-5, and IL-13, each play a nonredundant role in allergic disease pathology. The recent explosion of T(H) subpopulations combined with the wide availability of polychromatic cytokine staining has facilitated the discovery of T(H)2 lineage heterogeneity. In this article we review T(H)2 heterogeneity and ask the following question: At what point do these subpopulations graduate from in vitro curiosities to immunologically robust therapeutic targets? We propose criteria to establish a T-cell subset as a biologically relevant entity and address the evidence to support these T(H)2 subpopulations having a unique function or specific contribution to allergic pathology or host defense.

In vivo regulation of the allergic response by the IL-4 receptor α chain immunoreceptor tyrosine-based inhibitory motif

Signaling by IL-4 and IL-13 through the IL-4 receptor alpha chain (IL-4Ralpha) plays a critical role in the pathology of allergic diseases. The IL-4Ralpha is endowed with an immunoreceptor tyrosine-based inhibitory motif (ITIM) centered on tyrosine 709 (Y709) in the cytoplasmic domain that binds a number of regulatory phosphatases. The function of the ITIM in the in vivo regulation of IL-4 receptor signaling remains unknown. We sought to determine the in vivo function of the IL-4Ralpha ITIM by using mice in which the ITIM was inactivated by mutagenesis of the tyrosine Y709 residue into phenylalanine (F709). F709 ITIM mutant mice were derived by means of knock-in mutagenesis. Activation of intracellular signaling cascades by IL-4 and IL-13 was assessed by means of intracellular staining of phosphorylated signaling intermediates and gene expression analysis. In vivo responses to allergic sensitization were assessed by using models of allergic airway inflammation. The F709 mutation increased signal transducer and activator of transcription 6 phosphorylation by IL-4 and, disproportionately, by IL-13. This was associated with exaggerated T(H)2 polarization, enhanced alternative macrophage activation by IL-13, augmented basal and antigen-induced IgE responses, and intensified allergen-induced eosinophilic airway inflammation and hyperreactivity. These results point to a physiologic negative regulatory role for the Y709 ITIM in signaling through IL-4Ralpha, especially by IL-13.

Pimecrolimus Reduces Eosinophil Activation Associated with Calcium Mobilization

Background: Pimecrolimus is a calcineurin inhibitor that inhibits T cell and mast cell activation and effectively treats atopic dermatitis. However, its effects on eosinophils, a cell type implicated in allergic disease pathology, are unknown. Therefore, we examined the effects of pimecrolimus on eosinophil superoxide anion production, degranulation and survival. Methods: Purified eosinophils from normal or atopic donors were incubated with serial dilutions of pimecrolimus (μM to nM) and then stimulated with platelet activating factor (PAF), interleukin 5 (IL5), secretory immunoglobulin A (sIgA) or Alternaria alternata (Alt) fungus extract. Eosinophil activation was monitored by cytochrome c reduction resulting from superoxide anion production and by a 2-site immunoassay for eosinophil-derived neurotoxin (EDN) in cellular supernatants, as a marker of degranulation. Eosinophil survival was measured by propidium iodide exclusion using flow cytometry after 4 days in culture. Results: Normal and atopic eosinophil superoxide anion production induced by PAF, and associated with increased intracellular calcium, was inhibited up to 37% with 1 μM pimecrolimus. However, superoxide anion production induced by IL5 and sIgA was not consistently inhibited. EDN release, which ultimately depends on calcium, was inhibited about 30% with PAF, IL5 and sIgA stimulation for normal and atopic donor eosinophils. Furthermore, calcium-dependent Alt-induced EDN release was inhibited up to 49% with nanomolar pimecrolimus. Finally, increased eosinophil survival promoted by IL5 and sIgA was not influenced by pimecrolimus. Conclusion: Pimecrolimus moderately inhibits eosinophil superoxide anion production and EDN release associated with calcium mobilization, which may contribute to its efficacy in treating atopic dermatitis.

IgE-receptor activation induces survival and Bfl-1 expression in human mast cells but not basophils

The contribution of mast cells to the pathology of allergic diseases are facilitated by their long life span in tissue and ability to regranulate. Bcl-2 genes are one of the main regulators of cell death and survival. The aim of this study was to elucidate the mechanisms responsible for mast cell survival in allergy. Bcl-2 family gene expression in human mast cells and basophils was analyzed by ribonuclease protection assay and by reverse-transcriptase polymerase chain reaction. Cell survival was measured by mixing cells with the vital dye, trypan blue, and the number of living cells was enumerated. Apoptotic cells were measured by a Cell Death Detection ELISA. We found that cross-linking of FcepsilonRI on human cord blood cultured mast cells (CBCMCs) promoted cell survival and induced expression of the pro-survival gene Bfl-1. CBCMCs were found to express both Bfl-1 and Bfl-1S, two splicing variants of Bfl-1. Bfl-1 induction was mediated through Syk, PI3-kinase and intracellular calcium mobilization, since piceatannol, wortmannin and EDTA, respectively, significantly reduced Bfl-1 expression levels. In contrast to CBCMCs, no evidence was found for Bfl-1 expression and survival promotion in human basophils. Immunoglobulin E (IgE)-dependent activation-induced mast cell survival was correlated with Bfl-1 gene upregulation, providing a possible explanation for mast cell longevity in allergic reactions.

Animal models of type I allergy using recombinant allergens

Various animal models including guinea pigs, monkeys, dogs, rats, and mice have been established in an attempt to provide insights into the complex immunological and pathophysiological mechanisms of human type I allergic diseases. The detailed knowledge of the murine genome, the various components of the murine immune system, and the generation of engineered mice has made the murine system the most attractive among all animal models. The availability of multitude technologies and reagents to characterize and manipulate immunological pathways and mediators adds to the outstanding opportunities to assess the pathology of allergic diseases and to develop novel therapeutic strategies in mice. Numerous sensitization protocols with food and aero-allergens are used to establish an allergic/asthma-like phenotype in mice. Requirements for an appropriate murine model include a close resemblance to the pathology of the disease in humans, the objective measurement of the physiologic parameters, as well as reliability and reproducibility of the experimental data. With respect to reproducible experimental conditions, it has been recognized that extract preparations from natural allergen sources can vary in their allergen-content and -composition. This might influence the degree of sensitization or the outcome of treatment strategies in dependence of the applied extract preparation. The use of recombinant allergens in experimental in vivo and in vitro systems can overcome these problems. Another aspect, that has become obvious from the experimental studies, is that allergens can differ in their immunogenicity as well as in their capacity to act as tolerogens. Therefore, it seems important that the efficacy of the different allergen-molecules to act as therapeutic agents is individually examined. In this review, examples of animal models are described, in which recombinant allergens have been used for sensitization and/or treatment of allergic responses and how they have been used to enhance our understanding of the pathology of allergic diseases.

Molecular pathology of allergic disease: II: Upper airway disease

Allergic upper airway diseases such as allergic rhinitis and chronic sinusitis are an increasing problem. Although the pathogenesis remains elusive, an individual’s genetic predisposition as well as exposure to the allergen are currently considered factors in their development. Clinical symptoms of sneezing, rhinorrhea, and congestion are primarily a consequence of granulocyte release of chemical mediators such as histamine, prostanoids, and leukotrienes as well as the infiltration of inflammatory cells. Observations subsequent to allergen provocation are comparable to natural exposure and as such much of our understanding of allergic responses is derived from this model. A prominence of CD4+ T cells and eosinophils, synthesis and release of TH2 cytokines, and the coordinate expression of chemokines and adhesion molecules are all characteristic of the allergic response observed in rhinitis and sinusitis. Corticosteroids and immunotherapy target these inflammatory processes and have been observed to successfully reduce and shift the predominantly TH2 environment toward TH1 cytokine expression. As our understanding of the pathophysiologic features of allergic upper airway disease improves, as well as the relationship between their development and that of lower airway disease, new strategies of diagnosis and treatment will allow for more effective modulation of the allergic process and associated morbidity. (J Allergy Clin Immunol 2000;105:211-23.)

Molecular pathology of allergic diseases.

Molecular pathology of allergic diseases.

Molecular pathology of allergic disease: I: Lower airway disease

Asthma is a complex disorder associated with eosinophil infiltration and the activation of T lymphocytes within the airways. Recent advances in the pathophysiologic mechanisms of asthma point to the importance of eosinophil-basophil progenitor cells and a family of transcription factors that underlie the development of TH2-type responses. Further research is needed to address the development of chronic inflammatory changes, the role of profibrotic cytokines, and especially their reliance on eosinophils in the lungs. (J Allergy Clin Immunol 2000;105:20-36.)

Immunogenetics of human IgE

Immunoglobulin E plays a central role in mediating the pathology of allergic disease. Conversely, it is involved in the normal protective immune responses against parasite infection. Both these biological processes depend on interaction between the variable regions (VH and VL) of IgE antibodies and target antigen. It is now feasible to investigate the molecular nature of VH regions used to encode IgE at the genetic level. Using this technology to analyze IgE in patients with asthma has revealed features which may have relevance for allergic disease. First, preferential choice of VH genes, with dominance of the small VH5 family, particularly the VH32 gene, has been found. This may implicate a B cell superantigen (superallergen) selectively driving the use of these genes. Second, VH5 genes in IgE are somatically mutated with clear hot spots of mutational activity. Mutational hotspots, which are a feature of the VH5 gene, are supplemented in IgE by ongoing mutations which may be involved in affinity maturation. Third, a single B cell can switch to either IgE or IgG4, with both variants coexisting in blood. These findings may provide clues to the mechanism by which IgE is generated, and suggest options for therapeutic intervention.