SciCombinator

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Concept: Degranulation

114

Patients with autosomal dominant vibratory urticaria have localized hives and systemic manifestations in response to dermal vibration, with coincident degranulation of mast cells and increased histamine levels in serum. We identified a previously unknown missense substitution in ADGRE2 (also known as EMR2), which was predicted to result in the replacement of cysteine with tyrosine at amino acid position 492 (p.C492Y), as the only nonsynonymous variant cosegregating with vibratory urticaria in two large kindreds. The ADGRE2 receptor undergoes autocatalytic cleavage, producing an extracellular subunit that noncovalently binds a transmembrane subunit. We showed that the variant probably destabilizes an autoinhibitory subunit interaction, sensitizing mast cells to IgE-independent vibration-induced degranulation. (Funded by the National Institutes of Health.).

Concepts: Immune system, Protein, Amino acid, Cell biology, Mast cell, Histamine, Degranulation, Urticaria

28

Lipopolysaccharide (LPS) can exacerbate asthma; however, the mechanisms are not fully understood. This study investigated the effect of LPS on antigen-stimulated mast cell degranulation and the underlying mechanisms. We found that LPS enhanced degranulation in RBL-2H3 cells and mouse peritoneal mast cells upon FcεRI activation, in a dose- and time-dependent manner. Parallel to the alteration of degranulation, LPS increased FcεRI-activated Ca(2+) mobilization, as well as Ca(2+) entry through store-operated calcium channels (SOCs) evoked by thapsigargin. Blocking Ca(2+) entry through SOCs completely abolished LPS enhancement of mast cell degranulation. Consistent with functional alteration of SOCs, LPS increased mRNA and protein levels of Orai1 and STIM1, two major subunits of SOCs, in a time-dependent manner. In addition, LPS increased the mRNA level of Toll-like receptor 4 (TLR4) in a time-dependent manner. Blocking TLR4 with Cli-095 inhibited LPS, increasing transcription and expression of SOC subunits. Concomitantly, the effect of LPS enhancement of Ca(2+) mobilization and mast cell degranulation was largely reduced by Cli-095. Administration of LPS (1 μg) in vivo aggravated airway hyperreactivity and inflammatory reactions in allergic asthmatic mice. Histamine levels in serum and bronchoalveolar lavage fluid were increased by LPS treatment. In addition, Ca(2+) mobilization was enhanced in peritoneal mast cells isolated from LPS-treated asthmatic mice. Taken together, these results imply that LPS enhances mast cell degranulation, which potentially contributes to LPS exacerbating allergic asthma. Lipopolysaccharide increases Ca(2+) entry through SOCs by upregulating transcription and expression of SOC subunits, mainly through interacting with TLR4 in mast cells, resulting in enhancement of mast cell degranulation upon antigen stimulation.

Concepts: Immune system, Inflammation, Asthma, Immunoglobulin E, Allergy, Mast cell, Histamine, Degranulation

27

Store-Operated Calcium Entry (SOCE) represents a major calcium influx pathway in non-excitable cells and is central to many physiological processes such as T-cell activation and mast cell degranulation. SOCE is activated through intricate coordination between the Ca(2+) sensor on the ER membrane (STIM1) and the plasma membrane channel Orai1. When Ca(2+) stores are depleted, STIM1 oligomerizes and physically interacts with Orai1 through its SOAR/CAD domain resulting in Orai1 gating and Ca(2+) influx. Here we engineer novel inter- and intra-molecular FRET sensors in the context of the full-length membrane anchored STIM1, and show that STIM1 undergoes a conformational change in response to store depletion to adopt a stretched ‘open’ conformation that exposes SOAR/CAD allowing it to interact with Orai1. Mutational analyses reveal that electrostatic interactions between the predicted 1(st) and 3(rd) coiled-coil domains of STIM1 are not involved in maintaining the ‘closed’ inactive conformation. In addition, they argue that an amphipathic α-helix between residues 317-336 in the so-called inhibitory domain is important to maintain STIM1 in a closed conformation at rest. Indeed mutations that alter the amphipathic properties of this helix result in a STIM1 variant that is unable to respond to store depletion in terms of forming puncta, translocation to the cortical ER or activating Orai1.

Concepts: Immune system, Cell membrane, Mast cell, Degranulation, ORAI1, ER

24

Brain inflammation has a critical role in the pathophysiology of brain diseases. Microglia, the resident immune cells in the brain, play an important role in brain inflammation, while brain mast cells are the “first responder” in the injury rather than microglia. Functional aspects of mast cell-microglia interactions remain poorly understood. Our results demonstrated that site-directed injection of the “mast cell degranulator” compound 48/80 (C48/80) in the hypothalamus induced mast cell degranulation, microglial activation, and inflammatory factor production, which initiated the acute brain inflammatory response. “Mast cell stabilizer” disodium cromoglycate (cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced microglial activation, inhibition of MAPK and AKT pathways, and repression of protein expression of histamine receptor 1 (H1R), histamine receptor 4 (H4R), protease-activated receptor 2 (PAR2), and toll-like receptor 4 (TLR4) in microglia. We also demonstrated that C48/80 had no effect on microglial activation in mast cell-deficient Kit(W-sh/W-sh) mice. These results implicate that activated brain mast cells trigger microglial activation and stabilization of mast cell inhibits microglial activation-induced central nervous system (CNS) inflammation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.

Concepts: Immune system, Central nervous system, Nervous system, Brain, Asthma, Mast cell, Histamine, Degranulation

12

Rush desensitization (DS) is a widely used and effective clinical strategy for the rapid inhibition of IgE-mediated anaphylactic responses. However, the cellular targets and underlying mechanisms behind this process remain unclear. Recent studies have implicated mast cells (MCs) as the primary target cells for DS. Here, we developed a murine model of passive anaphylaxis with demonstrated MC involvement and an in vitro assay to evaluate the effect of DS on MCs. In contrast with previous reports, we determined that functional IgE remains on the cell surface of desensitized MCs following DS. Despite notable reductions in MC degranulation following DS, the high-affinity IgE receptor FcεRI was still capable of transducing signals in desensitized MCs. Additionally, we found that displacement of the actin cytoskeleton and its continued association with FcεRI impede the capacity of desensitized MCs to evoke the calcium response that is essential for MC degranulation. Together, these findings suggest that reduced degranulation responses in desensitized MCs arise from aberrant actin remodeling, providing insights that may lead to improvement of DS treatments for anaphylactic responses.

Concepts: Immune system, Protein, Cell biology, Allergy, Mast cell, Histamine, Anaphylaxis, Degranulation

8

Toxoplasma gondii is well-known to subvert normal immune responses, however, mechanisms are incompletely understood. In particular, its capacity to alter receptor-activated Ca(2+)-mediated signaling processes has not been well-characterized. In initial experiments, we found evidence that T. gondii infection inhibits Ca(2+) responses to fMetLeuPhe in murine macrophages. To further characterize the mechanism of inhibition of Ca(2+) mobilization by T. gondii, we used the well-studied RBL mast cell model to probe the capacity of T. gondii to modulate IgE receptor-activated signaling within the first hour of infection. Ca(2+) mobilization that occurs via IgE/FcεRI signaling leads to granule exocytosis in mast cells. We found that T. gondii inhibits antigen-stimulated degranulation in infected cells in a strain-independent manner. Under these conditions, we found that cytoplasmic Ca(2+) mobilization, particularly antigen-mediated Ca(2+) release from intracellular stores, is significantly reduced. Furthermore, stimulation-dependent activation of Syk kinase leading to tyrosine phosphorylation and activation of phospholipase Cγ is inhibited by infection. Therefore, we conclude that inhibitory effects of infection are likely due to parasite-mediated inhibition of the tyrosine kinase signaling cascade that results in reduced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Interestingly, inhibition of IgE/FcεRI signaling persists when tachyzoite invasion is arrested via cytochalasin D treatment, suggesting inhibition is mediated by a parasite-derived factor secreted into the cells during the invasion process. Our study provides direct evidence that immune subversion by T. gondii is initiated concurrently with invasion.

Concepts: Immune system, Signal transduction, Adenosine triphosphate, Cell biology, Apicomplexa, Mast cell, Phosphorylation, Degranulation

6

Atopic dermatitis is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca(2+)) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in Kit(W-sh/W-sh) mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

Concepts: Immune system, Bacteria, Asthma, Staphylococcus aureus, Staphylococcus, Allergy, Mast cell, Degranulation

4

Irritable bowel syndrome (IBS) is traditionally defined as a functional disorder since it lack of demonstrable pathological abnormalities. However, in recent years, low grade inflammatory infiltration, often rich in mast cells, in both the small and large bowel has been observed in some patients with IBS. The close association of mast cells with major intestinal functions, such as epithelial secretion and permeability, neuroimmune interactions, visceral sensation and peristalsis, makes researchers and gastroenterologists to focus attention on the key roles of mast cells in the pathogenesis of IBS. Numerous studies have been carried out to identify the mechanisms in the development, infiltration, activation and degranulation of intestinal mast cells, as well as the actions of mast cells in the processes of mucosal barrier disruption, mucosal immune dysregulation, visceral hypersensitivity, dysmotility, local and central stress in IBS. Moreover, therapies targeting mast cells, such as mast cell stabilizers (cromoglycate and ketotifen), antagonists of histamine and serotonin receptors, have been tried in IBS patients, and partly exhibited considerable efficacy. This review focuses on recent advances in the role of mast cells in IBS, with particular emphasis on bridging experimental data with clinical therapeutics for IBS patients.

Concepts: Immune system, Cell biology, Mast cell, Constipation, Gastroenterology, Histamine, Irritable bowel syndrome, Degranulation

3

Dengue virus (DENV) is the most significant human arboviral pathogen and causes ∼400 million infections in humans each year. In previous work, we observed that mast cells (MC) mediate vascular leakage during DENV infection in mice and that levels of MC activation are correlated with disease severity in human DENV patients (St John et al., 2013b). A major risk factor for developing severe dengue is secondary infection with a heterologous serotype. The dominant theory explaining increased severity during secondary DENV infection is that cross-reactive but non-neutralizing antibodies promote uptake of virus and allow enhanced replication. Here, we define another mechanism, dependent on FcγR-mediated enhanced degranulation responses by MCs. Antibody-dependent mast cell activation constitutes a novel mechanism to explain enhanced vascular leakage during secondary DENV infection.

Concepts: Immune system, Protein, Bacteria, Infection, Cell biology, Mast cell, Histamine, Degranulation

3

Development of specific inhibitors of allergy has had limited success, in part, owing to a lack of experimental models that reflect the complexity of allergen-IgE interactions. We designed a heterotetravalent allergen (HtTA) system, which reflects epitope heterogeneity, polyclonal response and number of immunodominant epitopes observed in natural allergens, thereby providing a physiologically relevant experimental model to study mast cell degranulation. The HtTA design revealed the importance of weak-affinity epitopes in allergy, particularly when presented with high-affinity epitopes. The effect of selective inhibition of weak-affinity epitope-IgE interactions was investigated with heterobivalent inhibitors (HBIs) designed to simultaneously target the antigen- and nucleotide-binding sites on the IgE Fab. HBI demonstrated enhanced avidity for the target IgE and was a potent inhibitor of degranulation in vitro and in vivo. These results demonstrate that partial inhibition of allergen-IgE interactions was sufficient to prevent mast cell degranulation, thus establishing the therapeutic potential of the HBI design.

Concepts: Immune system, Asthma, Immunology, Mast cell, Antigen, Epitope, Degranulation, Polyclonal B cell response