Search

Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with specific immune and inflammatory mechanisms. Atopy is among the major features of the diagnosis criteria for AD but is not an essential feature. Thus, patients diagnosed with AD can be atopic or non-atopic. This review focuses on the role of IgE, mast cells, and eosinophils in the pathogenesis of AD. The known functions of IgE in allergic inflammation suggest that IgE and IgE-mediated mast cell and eosinophil activation contribute to AD, but direct evidence supporting this is scarce. The level of IgE (thus the degree of allergic sensitization) is associated with severity of AD and contributed by abnormality of skin barrier, a key feature of AD. The function of IgE in development of AD is supported by the beneficial effect of anti-IgE therapy in a number of clinical studies. The role of mast cells in AD is suggested by the increase in the mast cell number and mast cell activation in AD lesions and the association between mast cell activation and AD. It is further suggested by their role in mouse models of AD as well as by the effect of therapeutic agents for AD that can affect mast cells. The role of eosinophils in AD is suggested by the presence of eosinophilia in AD patients and eosinophil infiltrates in AD lesions. It is further supported by information that links AD to cytokines and chemokines associated with production, recruitment, and activation of eosinophils.

Nanostructures containing 2,4-dinitrophenyl (DNP) as antigen were designed and produced to investigate antibody-mediated activation of mast cells. The design consists of nanogrids of DNP termini inlaid in alkanethiol self-assembled monolayers (SAMs). Using scanning probe-based nanografting, nanometer precision was attained for designed geometry, size, and periodicity. Rat basophilic leukemia (RBL) cells exhibited high sensitivity to the geometry and local environment of DNP presented on these nanostructures. The impact included cellular adherence, spreading, membrane morphology, cytoskeleton structure, and activation. The highest level of spreading and activation was induced by nanogrids of 17 nm line width and 40 nm periodicity, with DNP haptens 1.4 nm above the surroundings. The high efficacy is attributed to two main factors. First, DNP sites in the nanostructure are highly accessible by anti-DNP IgE during recognition. Second, the arrangement or geometry of DNP termini in nanostructures promotes clustering of FcεRI receptors that are prelinked to IgE. The clustering effectively initiates Lyn-mediated signaling cascades, ultimately leading to the degranulation of RBL cells. This work demonstrates an important concept: that nanostructures of ligands provide new and effective cues for directing cellular signaling processes., (© 2011 American Chemical Society)

Background: At present, it is highly controversial whether pure mast cells can serve as antigen presenting cells, and it is not known whether the capacity of antigen presenting function is temporally restricted to a particular subset of differentiated mast cells. Evidence is presented for a novel surface FcepsilonRIhi , MHC II +, and c-kit + pure mast cell subset, temporally restricted as antigen-presenting cells in the immune axis of T-cell activation., Results: Bone marrow-derived mast cells (BMMC) cultured in the presence of IL-3 for three weeks are pure mast cells based on surface expression of lineage-specific marker, c-kit and FcepsilonRI. Herein we present the first demonstration that approximately 98.7% c-kit + and FcepsilonRI expressing BMMC, further depleted of any contaminated professional antigen-presenting cells, are still fully capable of presenting antigens, i.e., OVA protein, OVA peptide, and IgE-TNP-OVA, to OVA peptide-specific T-cell hybridomas. Notably, IgE-dependent antigen presentation is more efficient compared to that resulting from direct antigen uptake. Importantly, we present the novel finding that only surface FcepsilonRIhi mast cells, also expressing surface MHC II exhibited antigen-presenting function. In contrast, surface FcepsilonRIlo mast cells without expressing surface MHC II were not capable of antigen presentation. Interestingly, the antigen-presenting function of BMMC was irrevocably lost during the third and fourth week in IL-3 or SCF containing cultures., Conclusions: This is the first observation to attribute a spatiotemporally restricted antigen-presenting function to a subset of three-week old pure BMMC expressing both high levels of surface FcepsilonRI and surface MHC II. We propose that mast cells play an important role in immune deviating and/or sustaining the activation of infiltrating CD4 T-cells, and modulating T-cell mediated allergic inflammation via its flexibility to present antigens and antigen-IgE complexes.

This article addresses the question of what information and new insights atomic force microscopy (AFM) provides that are of importance and relevance to cellular biophysical chemistry research. Three enabling aspects of AFM are discussed: (a) visualization of membrane structural features with nanometer resolution, such as microvilli, ridges, porosomes, lamellapodia, and filopodia; (b) revealing structural evolution associated with cellular signaling pathways by time-dependent and high-resolution imaging of the cellular membrane in correlation with intracellular components from simultaneous optical microscopy; and (c) qualitative and quantitative measurements of single cell mechanics by acquisition of force-deformation profiles and extraction of Young's moduli for the membrane as well as cytoskeleton. A future prospective of AFM is also presented.

Background: Allergic conjunctivitis is characterized by allergen-specific IgE in the serum and infiltration of eosinophils into the conjunctiva. The role of IgE and mast cells in allergic conjunctivitis is largely unknown, however., Objectives: We investigated the importance of conjunctival mast cells in a murine model of IgE-mediated allergic conjunctivitis., Methods: IgE-mediated allergic conjunctivitis was initiated in C57BL/6-Kit(+/+) wild-type mice, mast cell-deficient Kit(W-sh/W-sh) mice, and Kit(W-sh/W-sh) mice that had been subconjunctivally or systemically engrafted with bone marrow-derived, cultured mast cells (BMCMCs) from Kit(+/+) wild-type mice, and clinical symptoms and infiltration of eosinophil of the eyes were evaluated. Total numbers of mast cells in the conjunctiva were counted, and the phenotypes of these cells were characterized by means of immunostaining and PCR analysis of murine mast cell proteases., Results: No mast cells were detected in the conjunctiva or eyelid dermis of adult Kit(W-sh/W-sh) mice. Subconjunctival injection of BMCMCs resulted in local mast cell reconstitution, with the numbers of reconstituted mast cells in the conjunctiva and eyelid dermis comparable with those observed in wild-type Kit(+/+) littermates. Reconstituted and naive conjunctival mast cells expressed proteases ascribed to connective tissue-type mast cells but not mucosal mast cells. Passive transfer of ragweed-specific IgE followed by antigen challenge resulted in both early-phase clinical symptoms and late-phase eosinophilic inflammation in Kit(+/+) mice. These responses, which were significantly decreased in Kit(W-sh/W-sh) mice, were restored on reconstitution of the conjunctival mast cell population., Conclusions: These results suggest a direct contribution of IgE-activated mast cells to both the early-phase reaction and late-phase inflammation during ocular allergy.

Degranulation of bone marrow-derived mast cells (BMMCs) triggered by antigens (e.g., 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) and secretagogues (e.g., poly-L-lysine) was investigated by combined atomic force microscopy (AFM) and laser scanning confocal microscopy (LSCM). This combination enables the simultaneous visualization and correlation of membrane morphology with cytoskeletal actin arrangement and intracellular granules. Two degranulation mechanisms and detailed membrane structures that directly corresponded to the two stimuli were revealed. In DNP-BSA triggered activation, characteristic membrane ridges formed in accordance with the rearrangement of underlying F-actin networks. Individual granules were visualized after they released their contents, indicating a "kiss-and-run" pathway. In BMMCs stimulated by poly-L-lysine, lamellopodia and filopodia were observed in association with the F-actin assemblies at and near the cell periphery, whereas craters were observed on the central membrane lacking F-actin. These craters represent a new membrane feature resulting from the "kiss-and-merge" granule fusion. This work provides what we believe is important new insight into the local membrane structures in correlation with the cytoskeleton arrangement and detailed degranulation processes.

The immune response is regulated, in part, by effector cells whose activation requires multiple signals. For example, T cells require signals emanating from the T cell antigen receptor and co-stimulatory molecules for full activation. Here, we present evidence indicating that IgE-mediated hypersensitivity reactions in vivo also require cognate signals to activate mast cells. Immediate hypersensitivity reactions in the conjunctiva are ablated in mice deficient in eotaxin-1, despite normal numbers of tissue mast cells and levels of IgE. To further define the co-stimulatory signals mediated by chemokine receptor 3 (CCR3), an eotaxin-1 receptor, effects of CCR3 blockade were tested with an allergic conjunctivitis model and in ex vivo isolated connective tissue-type mast cells. Our results show that CCR3 blockade significantly suppresses allergen-mediated hypersensitivity reactions as well as IgE-mediated mast cell degranulation. We propose that a co-stimulatory axis by CCR3, mainly stimulated by eotaxin-1, is pivotal in mast cell-mediated hypersensitivity reactions.

Mast cells play an important role in both adaptive and innate immunity, and a large body of literature demonstrates their functions in skin immunity. This article reviews the literature on the role of this cell type in the pathogenesis of a number of immunological skin diseases, including contact dermatitis, atopic dermatitis, immunobullous disease, scleroderma, and chronic graft-vs.-host disease. In all these diseases, mast cells are noted to increase in number and undergo degranulation in the affected skin, and in some cases, their specific mediators are detected. Elucidation of the contribution of mast cells to the pathogenesis of these diseases has been aided significantly by the use of animal models, especially mouse models. The studies of mast cell-deficient mice in conjunction with normal congenic mice have been particularly fruitful, although in some cases, such as contact dermatitis, a definitive conclusion has not been achieved despite extensive efforts. The role of mast cells in atopic dermatitis has also been suggested by studies of gene polymorphism, which have linked some of the mast cell-related genes to the disease. In the case of scleroderma and chronic graft-vs.-host disease, the function of mast cells in fibrosis is further supported by the ability of these cells and their mediators to induce activation and proliferation of fibroblasts. Therapies targeting mast cells may prove beneficial for treatment of these inflammatory and autoimmune diseases.

Galectin-3 is a member of the beta-galactoside-binding animal lectin family expressed in various cell types, including mast cells. To determine the role of galectin-3 in the function of mast cells, we studied bone marrow-derived mast cells (BMMC) from wild-type (gal3(+/+)) and galectin-3-deficient (gal3(-/-)) mice. Cells from the two genotypes showed comparable expression of IgE receptor and c-Kit. However, upon activation by FcepsilonRI cross-linkage, gal3(-/-) BMMC secreted a significantly lower amount of histamine as well as the cytokine IL-4, compared with gal3(+/+) BMMC. In addition, we found significantly reduced passive cutaneous anaphylaxis reactions in gal3(-/-) mice compared with gal3(+/+) mice. These results indicate that there is a defect in the response of mast cells in gal3(-/-) mice. Unexpectedly, we found that gal3(-/-) BMMC contained a dramatically lower basal level of JNK1 protein compared with gal3(+/+) BMMC, which is probably responsible for the lower IL-4 production. The decreased JNK1 level in gal3(-/-) BMMC is accompanied by a lower JNK1 mRNA level, suggesting that galectin-3 regulates the transcription of the JNK gene or processing of its RNA. All together, these results point to an important role of galectin-3 in mast cell biology.

We demonstrate that binding of different IgE molecules (IgEs) to their receptor, FcepsilonRI, induces a spectrum of activation events in the absence of a specific antigen and provide evidence that such activation reflects aggregation of FcepsilonRI. Highly cytokinergic IgEs can efficiently induce production of cytokines and render mast cells resistant to apoptosis in an autocrine fashion, whereas poorly cytokinergic IgEs induce these effects inefficiently. Highly cytokinergic IgEs seem to induce more extensive FcepsilonRI aggregation than do poorly cytokinergic IgEs, which leads to stronger mast cell activation and survival effects. These effects of both types of IgEs require Syk tyrosine kinase and can be inhibited by FcepsilonRI disaggregation with monovalent hapten. In hybridoma-transplanted mice, mucosal mast cell numbers correlate with serum IgE levels. Therefore, survival effects of IgE could contribute to the pathogenesis of allergic disease.

We have studied murine models of asthma using FcepsilonRIalpha-chain-deficient (FcepsilonRIalpha(-/-)) mice to investigate the role of IgE-dependent mast cell activation in these models. When mice were either 1) immunized once with OVA in alum i.p. and then challenged with OVA intranasally, or 2) repeatedly immunized with OVA in the absence of adjuvant and subsequently challenged with nebulized OVA, FcepsilonRalpha(-/-) mice had significantly fewer eosinophils and lower IL-4 levels in their bronchoalveolar lavage fluid compared with wild-type mice. When mice were given anti-IL-5 antibody before OVA challenge in protocol 1, eosinophilic infiltration into the airways was significantly suppressed in both genotypes, but only FcepsilonRIalpha(-/-) mice showed significantly reduced airway hyperresponsiveness (AHR). In addition, when mice immunized and challenged with OVA also received a late OVA provocation at a higher concentration and were then exposed to methacholine, only wild-type mice developed a substantial increase in AHR. Since FcepsilonRI is expressed mainly on mast cells in mouse airways, we conclude that IgE-dependent activation of this cell type plays an important role in the development of allergic airway inflammation and AHR in mice. The models used may be of value for testing inhibitors of IgE or mast cells for development of therapeutic agents for human asthma.

A recent finding reports that co-stimulation of the high-affinity immunoglobulin E (IgE) receptor (FcεRI) and the chemokine receptor 1 (CCR1) triggered formation of membrane nanotubes among bone-marrow-derived mast cells. The co-stimulation was attained using corresponding ligands: IgE binding antigen and macrophage inflammatory protein 1α (MIP1 α), respectively. However, this approach failed to trigger formation of nanotubes among rat basophilic leukemia (RBL) cells due to the lack of CCR1 on the cell surface (Int. Immunol. 2010, 22 (2), 113-128). RBL cells are frequently used as a model for mast cells and are best known for antibody-mediated activation via FcεRI. This work reports the successful formation of membrane nanotubes among RBLs using only one stimulus, a hapten of 2,4-dinitrophenyl (DNP) molecules, which are presented as nanostructures with our designed spatial arrangements. This observation underlines the significance of the local presentation of ligands in the context of impacting the cellular signaling cascades. In the case of RBL, certain DNP nanostructures suppress antigen-induced degranulation and facilitate the rearrangement of the cytoskeleton to form nanotubes. These results demonstrate an important scientific concept; engineered nanostructures enable cellular signaling cascades, where current technologies encounter great difficulties. More importantly, nanotechnology offers a new platform to selectively activate and/or inhibit desired cellular signaling cascades.