Release Of Preformed Mediators Research Articles

Acute activation of SERCA with CDN1163 attenuates IgE-mediated mast cell activation through selective impairment of ROS and p38 signaling.

Mast cells are granulocytic immune sentinels present in vascularized tissues that drive chronic inflammatory mechanisms characteristic of allergic pathologies. IgE-mediated mast cell activation leads to a rapid mobilization of Ca2+ from intracellular stores, which is essential for the release of preformed mediators via degranulation and de novo synthesized proinflammatory cytokines and chemokines. Given its potent signaling capacity, the dynamics of Ca2+ localization are highly regulated by various pumps and channels controlling cytosolic Ca2+ concentrations. Among these is sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), which functions to maintain low cytosolic Ca2+ concentrations by actively transporting cytosolic Ca2+ ions into the endoplasmic reticulum. In this study, we characterized the role of SERCA in allergen-activated mast cells using IgE-sensitized bone marrow-derived mast cells (BMMCs) treated with the SERCA activating compound, CDN1163, and simultaneously stimulated with allergen through FcεRI under stem cell factor (SCF) potentiation. Acute treatment with CDN1163 was found to attenuate early phase mast cell degranulation along with reactive oxygen species (ROS) production. Additionally, treatment with CDN1163 significantly reduced secretion of IL-6, IL-13, and CCL3, suggesting a role for SERCA in the late phase mast cell response. The protective effects of SERCA activation via CDN1163 treatment on the early and late phase mast cell response may be driven by the selective suppression of p38 MAPK signaling. Together, these findings implicate SERCA as an important regulator of the mast cell response to allergen and suggest SERCA activity may offer therapeutic potential targeting allergic pathologies, warranting further investigation.

IL-25 participates in keratinocyte-driven dermal matrix turnover and is reduced in systemic sclerosis epidermis.

Evidence shows that dysfunctional SSc keratinocytes contribute to fibrosis by altering dermal homeostasis. Whether IL-25, an IL-17 family member regulating many epidermal functions, takes part in skin fibrosis is unknown. Here we address the role of IL-25 in skin fibrosis. The expression of IL-25 was evaluated by immunofluorescence and in situ hybridization in 10 SSc and seven healthy donor (HD) skin biopsies. Epidermal equivalents (EE) reconstituted by primary HD keratinocytes were used as a model to study transcriptomic changes induced by IL-25 in the epidermis. RNA expression profile in EEs was characterized by RNAseq. The conditioned medium (CM) from primary SSc and HD keratinocytes primed with IL-25 was used to stimulate fibroblasts. IL-6, IL-8, MMP-1, type-I collagen (Col-I), and fibronectin production by fibroblasts was assessed by ELISA. SSc epidermis expressed lower levels of IL-25 compared with HDs. In EEs, IL-25 regulated several molecular pathways related to wound healing and extracellular matrix remodelling. Compared with control CM, the CM from IL-25-primed keratinocytes enhanced the fibroblast production of MMP-1, IL-6 and IL-8, but not of Col-I nor fibronectin. However, IL-25 significantly reduced the production of Col-I when applied directly to fibroblasts. The activation of keratinocytes by IL-25 was receptor-dependent and evident after a very short incubation time (10 min), largely mediated by IL-1, suggesting enhanced and specific release of preformed mediators. These results show that IL-25 participates in skin homeostasis, and its decreased expression in SSc may contribute to skin fibrosis by favouring extracellular matrix deposition over degradation.

Calcineurin Aα Contributes to IgE-Dependent Mast-Cell Mediator Secretion in Allergic Inflammation

Mast cells (MCs) are key mediators of allergic inflammation through the activation of cross-linked immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcϵRI) on the cell surface, leading to the release of biologically potent mediators, either from preformed granules or newly synthesized. Pharmacological inhibitors have been developed to target a key signaling protein phosphatase in this pathway, calcineurin, yet there is a lack of genetic and definitive evidence for the various isoforms of calcineurin subunits in FcϵRI-mediated responses. In this study, we hypothesized that deficiency in the calcineurin Aα isoform will result in a decreased allergic immune response by the MCs. In a model of passive cutaneous anaphylaxis, there was a reduction in vascular permeability in MC-deficient mouse tissues reconstituted with calcineurin subunit A (CnAα) gene-knockout (CnAα^−/−) MCs, and in vitro experiments identified a significant reduction in release of preformed mediators from granules. Furthermore, released levels of de novo synthesized cytokines were reduced upon FcϵRI activation of CnAα^−/− MCs in vitro. Characterizing the mechanisms associated with this deficit response, we found a significant impairment of nuclear factor of kappa light polypeptide gene enhancer in B cell phosphorylation and impaired nuclear factor kappa-light-chain-enhancer of activated B-cell inhibitor alpha (NF-κB) activation. Thus, we concluded that CnAα contributes to the release of preformed mediators and newly synthesized mediators from FcϵRI-mediated activation of MCs, and this regulation includes NF-κB signaling.

A MAST KNOWN CAUSE OF SHOCK

TOPIC: Critical Care TYPE: Fellow Case Reports INTRODUCTION: The main types of shock include cardiogenic, hypovolemic, distributive, and neurogenic shock. Management of shock requires identifying and correcting the underlying cause, and supporting end-organ perfusion with fluid resuscitation and vasoactive medications. Here we present a rare cause of distributive shock. CASE PRESENTATION: A 65-year-old man presented with 2-month history of progressive weakness. Laboratory studies reviewed new thrombocytopenia to 25 x10(9)/L and acute renal failure with creatinine of 3.24 mg/dL. His bone marrow biopsy was concerning for acute mast cell leukemia, and he was started on treatment with 1 gram of methylprednisolone. About 1 hour after receiving this infusion, he developed chest pain, tachycardia and tachypnea. He was urgently transferred to the ICU. In the ICU, his condition progressed into ventricular fibrillation requiring CPR, defibrillation, and intubation. He was in refractory circulatory shock requiring high doses of norepinephrine, epinephrine, vasopressin, and stress dose steroids. Given his underlying mast cell leukemia, empirical treatment for mast cell activation syndrome was started. He received scheduled diphenhydramine, famotidine, montelukast and cromolyn. Patient was quickly weaned off vasoactive medications in the next 24 hours. Tryptase obtained at time of his hypotensive event was markedly elevated at greater than 2000 ng/mL. He had worsening renal failure requiring renal replacement therapy, and given his underlying diagnosis of mast cell leukemia, a rare hematological malignancy with limited treatment options and high mortality, the focus of his care was transitioned to concentrate solely on his comfort. DISCUSSION: Mast cell disorders include cutaneous and systemic mastocytosis, allergic diseases, and idiopathic disorders. Acute mast cell leukemia is a rare form of systemic mastocytosis characterized by at least 20% immature mast cells in bone marrow. Sudden mast cell activation leads to degranulation of mast cell and release of pre-formed mediators including histamine. It also stimulates de novo synthesis of other cytokines and chemokines. Typical symptoms include flushing, lightheadedness and abdominal cramping, but can progress to syncope and distributive shock, as seen in our patient. Treatment includes identification and avoidance of triggers, and supportive care with antihistamines (both H1 and H2 blockers) and cromolyn (mast cell stabilizer). CONCLUSIONS: It is essential for critical care clinicians to recognize mast cell activation syndrome as a potential cause of refractory shock, especially in at risk patients with systemic mastocytosis. Prompt treatment with antihistamines and cromolyn can facilitate resolution of shock. REFERENCE #1: Akin C, Valent P, Metcalfe DD. Mast cell activation syndrome: Proposed diagnostic criteria. J Allergy Clin Immunol. 2010;126(6):1099-104.e4. doi:10.1016/j.jaci.2010.08.035 REFERENCE #2: Molderings GJ, Haenisch B, Brettner S, et al. Pharmacological treatment options for mast cell activation disease. Naunyn Schmiedebergs Arch Pharmacol. 2016;389(7):671-694. doi:10.1007/s00210-016-1247-1 DISCLOSURES: No relevant relationships by Alice Gallo De Moraes, source=Web Response No relevant relationships by Zhenmei Zhang, source=Web Response

Mechanisms Governing Anaphylaxis: Inflammatory Cells, Mediators, Endothelial Gap Junctions and Beyond.

Anaphylaxis is a severe, acute, life-threatening multisystem allergic reaction resulting from the release of a plethora of mediators from mast cells culminating in serious respiratory, cardiovascular and mucocutaneous manifestations that can be fatal. Medications, foods, latex, exercise, hormones (progesterone), and clonal mast cell disorders may be responsible. More recently, novel syndromes such as delayed reactions to red meat and hereditary alpha tryptasemia have been described. Anaphylaxis manifests as sudden onset urticaria, pruritus, flushing, erythema, angioedema (lips, tongue, airways, periphery), myocardial dysfunction (hypovolemia, distributive or mixed shock and arrhythmias), rhinitis, wheezing and stridor. Vomiting, diarrhea, scrotal edema, uterine cramps, vaginal bleeding, urinary incontinence, dizziness, seizures, confusion, and syncope may occur. The traditional (or classical) pathway is mediated via T cells, Th2 cytokines (such as IL-4 and 5), B cell production of IgE and subsequent crosslinking of the high affinity IgE receptor (FcεRI) on mast cells and basophils by IgE-antigen complexes, culminating in mast cell and basophil degranulation. Degranulation results in the release of preformed mediators (histamine, heparin, tryptase, chymase, carboxypeptidase, cathepsin G and tumor necrosis factor alpha (TNF-α), and of de novo synthesized ones such as lipid mediators (cysteinyl leukotrienes), platelet activating factor (PAF), cytokines and growth factors such as vascular endothelial growth factor (VEGF). Of these, histamine, tryptase, cathepsin G, TNF-α, LTC4, PAF and VEGF can increase vascular permeability. Recent data suggest that mast cell-derived histamine and PAF can activate nitric oxide production from endothelium and set into motion a signaling cascade that leads to dilatation of blood vessels and dysfunction of the endothelial barrier. The latter, characterized by the opening of adherens junctions, leads to increased capillary permeability and fluid extravasation. These changes contribute to airway edema, hypovolemia, and distributive shock, with potentially fatal consequences. In this review, besides mechanisms (endotypes) underlying IgE-mediated anaphylaxis, we also provide a brief overview of IgG-, complement-, contact system-, cytokine- and mast cell-mediated reactions that can result in phenotypes resembling IgE-mediated anaphylaxis. Such classifications can lead the way to precision medicine approaches to the management of this complex disease.

Differential Use of the Signaling Molecules BTK and PLC-γ in Mast Cells Stimulated Via the Receptors FcεRI and KIT

Mast cells belong to the myeloid lineage of the hemopoietic system, are localized to the tissue-environment interphase, and execute regulatory and protective roles in both innate and adaptive immunity. However, mast cells also are involved in pathophysiological processes ranging from allergic diseases to autoimmune diseases and tumors. Two receptors expressed on the surface of mast cells and prominently involved in physiology and pathophysiology of mast cell biology are the high-affinity receptor for IgE (FcεRI) and the receptor tyrosine kinase KIT (a.k.a. CD117), the receptor for stem cell factor (SCF). Whereas antigen (Ag)-triggered activation of the IgE-bound FcεRI results in immediate release of preformed mediators (degranulation) and production of arachidonic acid metabolites and cytokines/chemokines, SCF-induced mast cell activation is only causing the latter. On the other side, SCF is inducing mast cell proliferation and chemotaxis, two responses that are not caused by FcεRI activation. Intriguingly, the signal transduction downstream of these receptors involves activation of the same signaling pathways and engagement of comparable sets of signaling proteins, raising the question, how differential mast cell effector functions are controlled by these receptors. Therefore, it is essential to learn more about the molecular pathways leading to FcεRI and KIT-mediated activation of mast cells. In the presented work, primary murine mast cells were stimulated either with Ag via the IgE-bound FcεRI or with SCF via its receptor KIT and differential signal transduction was analyzed. We put our focus on two central signaling elements in mast cells, the cytosolic tyrosine kinase BTK and its target, phospholipase C-γ, and also addressed differences between using suboptimal and optimal ligand concentrations. Cell responses like degranulation, proinflammatory cytokine production, activating protein phosphorylation, and intracellular calcium mobilization were analyzed making use of mast cells from wild-type and transgenic (knock-out) mice as well as selective pharmacological inhibitors. Interestingly, marked differences were found with respect to pathway activation, importance of particular signaling proteins, and induction of effector responses. Ag-triggered degranulation is thought to be dependent on BTK/PLC-γ-mediated calcium mobilization and thus it was unexpected to observe only a weak effect of PLC-γ inhibition. On the other side, SCF-induced calcium mobilization was strictly dependent on PLC-γ, however, only a minor involvement of BTK could be observed. In combination with the additional results obtained in our study, future analyses of differential pathway organization by multiprotein assemblies as well as kinetic control mechanisms appear to be important. Detailed knowledge of these mechanisms will help to precisely promote beneficial and strictly control detrimental mast cell responses. DisclosuresNo relevant conflicts of interest to declare.

Hypersensitivity Reactions and Anaphylaxis

Allergic reactions vary in intensity from mild rash or allergic rhinitis to devastating anaphylactic shock. Anaphylaxis, often underrecognized and undertreated, can be a life-threatening syndrome leading to multiorgan dysfunction. This review covers the etiology, pathophysiology, and treatment of severe allergic reactions and anaphylaxis. It is precipitated by exposure to particular allergens—commonly food, medications, insect stings, and environmental exposures—in a previously sensitized individual. Symptoms develop from an IgE-mediated immune response leading to degranulation of mast cells and basophils and the release of preformed mediators, lipid-derived metabolites, and inflammatory cytokines. First-line treatment for anaphylaxis involves epinephrine. Secondary treatments are antihistamines and corticosteroids. Further treatments for patients refractory to standard therapies involve vasopressor agents, nebulized albuterol, and glucagon. Frequency and duration of biphasic reactions are variable, limiting the development of consensus guidelines for monitoring of anaphylactic reactions. Figures show the immune activity and inflammatory pathways in allergic responses, mast cell degranulation, and a depiction of common organs involved and corresponding clinical manifestations. Tables list the criteria for diagnosis of anaphylaxis, classification of hypersensitivity reactions, common clinical manifestations, and etiology and mediators of anaphylaxis. This review contains 4 highly rendered figures, 11 tables, and 43 references. Key words: allergy, anaphylaxis, antihistamine, corticosteroid, epinephrine, mast cells

HIV gp120 Induces the Release of Proinflammatory, Angiogenic, and Lymphangiogenic Factors from Human Lung Mast Cells.

Human lung mast cells (HLMCs) express the high-affinity receptor FcεRI for IgE and are involved in chronic pulmonary diseases occurring at high frequency among HIV-infected individuals. Immunoglobulin superantigens bind to the variable regions of either the heavy or light chain of immunoglobulins (Igs). Glycoprotein 120 (gp120) of HIV-1 is a typical immunoglobulin superantigen interacting with the heavy chain, variable 3 (VH3) region of human Igs. The present study investigated whether immunoglobulin superantigen gp120 caused the release of different classes of proinflammatory and immunoregulatory mediators from HLMCs. The results show that gp120 from different clades induced the rapid (30 min) release of preformed mediators (histamine and tryptase) from HLMCs. gp120 also caused the de novo synthesis of cysteinyl leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) from HLMCs. Incubation (6 h) of HLMC with gp120 induced the release of angiogenic (VEGF-A) and lymphangiogenic (VEGF-C) factors from HLMCs. The activating property of gp120 was mediated through the interaction with IgE VH3+ bound to FcεRI. Our data indicate that HIV gp120 is a viral superantigen, which induces the release of different proinflammatory, angiogenic, and lymphangiogenic factors from HLMCs. These observations could contribute to understanding, at least in part, the pathophysiology of chronic pulmonary diseases in HIV-infected individuals.

The Role of Mast Cells in Stroke.

Mast cells (MCs) are densely granulated perivascular resident cells of hematopoietic origin. Through the release of preformed mediators stored in their granules and newly synthesized molecules, they are able to initiate, modulate, and prolong the immune response upon activation. Their presence in the central nervous system (CNS) has been documented for more than a century. Over the years, MCs have been associated with various neuroinflammatory conditions of CNS, including stroke. They can exacerbate CNS damage in models of ischemic and hemorrhagic stroke by amplifying the inflammatory responses and promoting brain–blood barrier disruption, brain edema, extravasation, and hemorrhage. Here, we review the role of these peculiar cells in the pathophysiology of stroke, in both immature and adult brain. Further, we discuss the role of MCs as potential targets for the treatment of stroke and the compounds potentially active as MCs modulators.

Clinical implications of mast cell involvement in allergic conjunctivitis.

The conjunctiva is a common site for the allergic inflammatory response due to it being highly vascularized, having constant exposure to environmental pollutants and allergenic pollens and having a unique conjunctival associated lymphoid tissue. The primary morbidity of anterior surface conjunctival disorders that include allergic conjunctivitis and tear film disorders is associated with its high frequency of involvement rather than its severity, although the more chronic forms can involve the cornea and lead to sight-threatening conditions. Ocular allergy is associated with IgE-mediated mast cell activation in conjunctival tissue leading to the release of preformed mediators including histamine and proteases and subsequent de novo formation of lipid-derived mediators and cytokines that trigger a cascade of cellular and molecular events leading to extensive migration and infiltration of inflammatory cells to the ocular surface. The trafficking of neutrophils, eosinophils, and lymphocytes to the ocular surface is due to establishing various chemokine gradients (mainly CCL11, CCL24, CCL5, MCP-3, and MCP-4), cell surface expression of adhesion molecules (such as VCAM-1 the ligand for VLA-4), and leukocyte adhesion to vascular endothelium. The release of preformed mediators underlies the acute ocular surface response while the secondary influx of inflammatory cells leading to the recruitment and activation of eosinophils and the subsequent activation of Th2 and Th1 lymphocytes at the level of the conjunctiva reflects the late-phase reaction.

Anti-allergic activity of 2,4,6-trihydroxy-3-geranylacetophenone (tHGA) via attenuation of IgE-mediated mast cell activation and inhibition of passive systemic anaphylaxis

tHGA, a geranyl acetophenone compound originally isolated from a local shrub called Melicope ptelefolia, has been previously reported to prevent ovalbumin-induced allergic airway inflammation in a murine model of allergic asthma by targeting cysteinyl leukotriene synthesis. Mast cells are immune effector cells involved in the pathogenesis of allergic diseases including asthma by releasing cysteinyl leukotrienes. The anti-asthmatic properties of tHGA could be attributed to its inhibitory effect on mast cell degranulation. As mast cell degranulation is an important event in allergic responses, this study aimed to investigate the anti-allergic effects of tHGA in cellular and animal models of IgE-mediated mast cell degranulation. For in vitro model of IgE-mediated mast cell degranulation, DNP-IgE-sensitized RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA to induce degranulation. For IgE-mediated passive systemic anaphylaxis, Sprague Dawley rats were sensitized by intraperitoneal injection of DNP-IgE before challenged with DNP-BSA. Both in vitro and in vivo models showed that tHGA significantly inhibited the release of preformed mediators (β-hexosaminidase and histamine) as well as de novo mediators (interleukin-4, tumour necrosis factor-α, prostaglandin D2 and leukotriene C4). Pre-treatment of tHGA also prevented IgE-challenged RBL-2H3 cells and peritoneal mast cells from undergoing morphological changes associated with mast cell degranulation. These findings indicate that tHGA possesses potent anti-allergic activity via attenuation of IgE-mediated mast cell degranulation and inhibition of IgE-mediated passive systemic anaphylaxis. Thus, tHGA may have the potential to be developed as a mast cell stabilizer for the treatment of allergic diseases in the future.

Mast Cell Degranulation Is Accompanied by the Release of a Selective Subset of Extracellular Vesicles That Contain Mast Cell-Specific Proteases.

Mast cells (MC) are well known for their effector role in allergic disorders; moreover, they are associated with diverse modulatory effects in innate and adaptive immunity. It is largely unclear how MC exert these modulating functions. In this article, we show that IgE-mediated MC degranulation leads to a rapid release of high quantities of extracellular vesicles (EV), comparable to the release of preformed mediators. EV are submicron structures composed of lipid bilayers, proteins, and nucleic acids that are released by cells in a regulated fashion and are involved in intercellular communication. Primary murine mucosal-type MC and connective tissue-type MC released phenotypically different EV populations depending on the stimulus they received. Although unstimulated MC constitutively released CD9+ EV, degranulation was accompanied by the release of CD63+ EV, which correlated with release of the soluble mediator β-hexosaminidase. This CD63+ EV subset was smaller and exhibited a higher buoyant density and distinct phospholipid composition compared with CD9+ EV. Marked differences were observed for phosphatidylinositol, phosphatidic acid, and bis(monoacylglycero)phosphate species. Strikingly, proteomic analysis of CD63+ EV from connective tissue-type MC unveiled an abundance of MC-specific proteases. With regard to carboxypeptidase A3, it was confirmed that the enzyme was EV associated and biologically active. Our data demonstrate that, depending on their activation status, MC release distinct EV subsets that differ in composition and protease activity and are indicative of differential immunological functions. Concerning the strategic tissue distribution of MC and the presence of degranulated MC in various (allergic) disorders, MC-derived EV should be considered potentially important immune regulators.

SLAP negatively regulates FcɛRI receptor-mediated mast cell response in mice

Abstract Mast cells (MCs) are key mediators of innate immune response to pathogens and allergens, etiological factors for drugs, dust and peanut-induced anaphylaxis. Degranulation of MCs in response to binding of high affinity FcɛRI receptor (IgE receptor) to allergen-induced IgE results in the release of preformed mediators including histamine, and de novo synthesis of cytokines causing hypersensitivity symptoms including anaphylaxis. Src-like adapter protein (SLAP), has been shown to regulate signaling downstream of T cell receptor (TCR), B cell receptor (BCR) and receptor tyrosine kinases (RTK) including c-Kit, FLT3 and CSF-1 via recruitment of the Cbl ubiquitin ligase. Here, we show that bone marrow-derived mast cells (BMMCs) from SLAP knockout (SLAP KO) mice displayed significantly enhanced degranulation and de novo synthesis of IL-6 but not TNFα compared to wild type (WT) BMMCs. SLAP KO BMMCs also displayed significantly reduced F-actin polymerization upon stimulation of IgE receptor in comparison to WT BMMCs. In addition, SLAP KO BMMCs maintained enhanced cell surface expression of FcɛRI receptor post stimulation suggesting reduced endocytosis/degradation. While WT and SLAP KO mice have equal numbers of MCs in ear skin, SLAP KO mice showed enhanced passive cutaneous anaphylaxis response. Thus, these results suggest that SLAP negatively regulates MC-induced anaphylaxis reaction via IgE-DNP pathway.

Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines.

Mast cells are immunoregulatory cells that participate in inflammatory processes. Cross-linking mast cell specific GD1b derived gangliosides by mAbAA4 results in partial activation of mast cells without the release of preformed mediators. The present study examines the release of newly formed and newly synthesized mediators following ganglioside cross-linking. Cross-linking the gangliosides with mAbAA4 released the newly formed lipid mediators, prostaglandins D2 and E2, without release of leukotrienes B4 and C4. The effect of cross-linking these gangliosides on the activation of enzymes in the arachidonate cascade was then investigated. Ganglioside cross-linking resulted in phosphorylation of cytosolic phospholipase A2 and increased expression of cyclooxygenase-2. Translocation of 5-lipoxygenase from the cytosol to the nucleus was not induced by ganglioside cross-linking. Cross-linking of GD1b derived gangliosides also resulted in the release of the newly synthesized mediators, interleukin-4, interleukin-6, and TNF-α. The effect of cross-linking the gangliosides on the MAP kinase pathway was then investigated. Cross-linking the gangliosides induced the phosphorylation of ERK1/2, JNK1/2, and p38 as well as activating both NFκB and NFAT in a Syk-dependent manner. Therefore, cross-linking the mast cell specific GD1b derived gangliosides results in the activation of signaling pathways that culminate with the release of newly formed and newly synthesized mediators.

Intestinal and peritoneal mast cells differ in kinetics of quantal release

5-hydroxytriptamine (5-HT, serotonin) storage and release in mast cell (MC) secretory granules (SG) are dependent on serglycin proteoglycans (PG). This notion is based on the studies of MC of the connective tissue subtype that predominantly contain PG of the heparin type, whereas intestinal mucosal MC, which contain predominantly chondroitin sulfate, have been poorly explored. In the present study, we addressed the possibility that PG contents may differently affect the storage and release of preformed mediators in these two MC subclasses and explain in part their different functional properties. Rat peritoneal (PMC) and intestinal mast cells (IMC) were isolated and purified using a percoll gradient, and the efflux of 5-HT from each SG was measured by amperometric detection. IMC exhibited a ∼34% reduction in the release of 5-HT compared with PMC because of a lower number of exocytotic events, rather than a lower secretion per single exocytotic event. Amperometric spikes from IMC exhibited a slower decay phase and increased half-width but a similar ascending phase and foot parameters, indicating that the fusion pore kinetics are comparable in both MC subclasses. We conclude that both PG subtypes are equally efficient systems, directly involved in serotonin accumulation, and play a crucial role in regulating the kinetics of exocytosis from SG, providing specific secretory properties for the two cellular subtypes.

Mast Cell Serotonin Immunoregulatory Effects Impacting on Neuronal Function: Implications for Neurodegenerative and Psychiatric Disorders.

Mast cells (MCs) are derived from hemopoietic precursor cells, undergo their maturation in peripheral tissues, and play a significant role in both the innate and adaptive immune response. Cross-linking of the FcεRI on MCs initiates activation of several cytoplasmic protein tyrosine kinases which rapidly lead to phosphorylation and recruitment of adaptor molecules. These effects trigger the release of preformed mediators stored in the cytoplasmic granules, including histamine, serotonin and tryptase, as well as newly synthesized mediators, such as cytokines/chemokines, prostaglandins, leukotrienes, and growth factors. Serotonin (5-HT) is a bioactive monoamine, which has seven specific cell surface membrane bound receptors which are coupled to G-proteins, plays an important role in the central and peripheral nervous system, and is one of the key mediators in signaling between nervous and immune systems. Serotonin is not stored in all MC types but is implicated in MC adhesion, chemotaxis, tumorigenesis, and tissue regeneration through smooth muscle differentiation of stromal cells. Recent evidence indicates that serotonin has immunoregulatory actions that may be important in neuropsychiatric conditions. Chemokines, RANTES/CCL5, MCP-1/CCL2, and related molecules, constitute the C-C class of chemokine supergene family, play a role in regulating T helper-cell cytokine production and MC trafficking, and are involved in histamine and serotonin generation and MC functions. Pro-inflammatory cytokines such as interleukin-1-β and tumor necrosis factor which mediate MC response, are capable of activating p38 MAPK, and might increase serotonin generation through p38 MAPK activation. Here, we review the relationship between MCs and serotonin and its role in inflammatory diseases and neuroimmune interactions.

Influence of physicochemical properties of silver nanoparticles on mast cell activation and degranulation

Silver nanoparticles (AgNPs) are increasingly being incorporated into products for their antimicrobial properties. This has resulted in increased human exposures and the possibility of adverse health effects. Mast cells orchestrate allergic immune responses through degranulation and release of pre-formed mediators. Little data exists on understanding interactions of AgNPs with mast cells and the properties that influence activation and degranulation. Using bone marrow-derived mast cells and AgNPs of varying physicochemical properties we tested the hypothesis that AgNP physicochemical properties influence mast cell degranulation and osteopontin production. AgNPs evaluated included spherical 20nm and 110nm suspended in either polyvinylpyrrolidone (PVP) or citrate, Ag plates suspended in PVP of diameters between 40–60nm or 100–130nm, and Ag nanowires suspended in PVP with thicknesses <100nm and length up to 2μm. Mast cell responses were found to be dependent on the physicochemical properties of the AgNP. Further, we determined a role for scavenger receptor B1 in AgNP-induced mast cell responses. Mast cell degranulation was not dependent on AgNP dissolution but was prevented by tyrosine kinase inhibitor pretreatment. This study suggests that exposure to AgNPs may elicit adverse mast cell responses that could contribute to the initiation or exacerbation of allergic disease.

Granule protein processing and regulated secretion in neutrophils.

Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines, and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First, we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking, and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection.

Rac1 and Rac2 control distinct events during antigen-stimulated mast cell exocytosis

The release of preformed mediators from immune cells is through a process described as exocytosis. In mast cells, exocytosis is regulated by several coordinated intracellular signaling pathways. Here, we investigated the role of the hematopoietic-specific Rho GTPase, Rac2, and the ubiquitously expressed Rac1, in controlling mast cell exocytosis. These two isoforms showed equivalent levels of expression in mouse BMMCs. Although Rac1 and Rac2 share 92% sequence identity, they were not functionally redundant, as Rac2-/- BMMCs were defective in exocytosis, even though Rac1 levels were unaffected. Antigen-stimulated WT mast cells underwent a series of morphological transitions: initial flattening, followed by actin-mediated peripheral membrane ruffling and calcium influx, which preceded exocytosis. Whereas membrane ruffling was unaffected in Rac2-/- BMMCs, calcium influx was decreased significantly. Calcium influx was studied further by examining SOCE. In Rac2-/- BMMCs, the activation of PLCγ1 and calcium release from intracellular stores occurred normally; however, activation of plasma membrane calcium channels was defective, shown by the lack of extracellular calcium influx and a reduction of YFP-STIM1 puncta at the plasma membrane. Additionally, we used the small molecule Rac inhibitor, EHT 1864, to target Rac signaling acutely in WT BMMCs. EHT 1864 blocked exocytosis and membrane ruffling completely in conjunction with exocytosis. Our findings suggest that antigen-stimulated membrane ruffling in mast cells is a Rac1-mediated process, as this persisted in the absence of Rac2. Therefore, we define distinct modes of Rac-regulated mast cell exocytosis: Rac2-mediated calcium influx and Rac1-mediated membrane ruffling.

Regulation of FcεRI signaling by lipid phosphatases.

Mast cells (MCs) are tissue-resident sentinels of hematopoietic origin that play a prominent role in allergic diseases. They express the high-affinity receptor for IgE (FcεRI), which when cross-linked by multivalent antigens triggers the release of preformed mediators, generation of arachidonic acid metabolites, and the synthesis of cytokines and chemokines. Stimulation of the FcεRI with increasing antigen concentrations follows a characteristic bell-shaped dose-responses curve. At high antigen concentrations, the so-called supra-optimal conditions, repression of FcεRI-induced responses is facilitated by activation and incorporation of negative signaling regulators. In this context, the SH2-containing inositol-5'-phosphatase, SHIP1, has been demonstrated to be of particular importance. SHIP1 with its catalytic and multiple protein interaction sites provides several layers of control for FcεRI signaling. Regulation of SHIP1 function occurs on various levels, e.g., protein expression, receptor and membrane recruitment, competition for protein-protein interaction sites, and activating modifications enhancing the phosphatase function. Apart from FcεRI-mediated signaling, SHIP1 can be activated by diverse unrelated receptor systems indicating its involvement in the regulation of antigen-dependent cellular responses by autocrine feedback mechanisms or tissue-specific and/or (patho-) physiologically determined factors. Thus, pharmacologic engagement of SHIP1 may represent a beneficial strategy for patients suffering from acute or chronic inflammation or allergies.