Mature Mast Cells Research Articles

Abstract P131: A Selective Chymase Inhibitor, TEI-f00806, Eliminates the Ang II Production From Rat Ang-(1-12) in Bone Marrow Tissue

Background: Rat bone marrow (BM)-chimeric experiments reveal a critical role of locally formed angiotensin II (Ang II) in mediating the oxidant-rich inflammatory milieu associated with cardiovascular disease pathogenesis. Extending our first demonstration of renin angiotensin system (RAS) components in the BM, we recently reported a primary role of chymase rather than angiotensin converting enzyme (ACE) in BM generation of Ang II from the novel substrate angiotensin-(1-12) [Ang-(1-12)]. To verify chymase contribution over other proteases such as cathepsins, Ang-(1-12) metabolism studies in rat BM were carried out in the absence and in the presence of a selective orally active chymase inhibitor (TEI-F00806). Methods: Plasma membrane (PM) of BM and left ventricle (LV) tissues were obtained from 12 week-old Sprague Dawley rats following systemic perfusion with phosphate buffered saline with or without the secondary addition of Zamboni’s fixative. PMs were incubated with 125 I-Ang-(1-12) and the metabolic products during incubation were determined by high-performance liquid chromatography. All data were analyzed using GraphPad PRISM 7.0 with P<0.05 to determine statistical significance. Results: Assessment of 125 I-Ang-(1-12) hydrolysis in the absence of the chymase inhibitor revealed that chymase activity in BM membranes was 280 fold higher than that in LV (4,531 ± 137 fmol/mg/min and 16.3 ± 0.3 fmol/mg/min, respectively, n = 6, P < 0.001). In the presence of TEI-F00806, 96% of the chymase-mediated Ang II production was inhibited in BM. Confocal microscopy showed chymase-positive but tryptase-negative cells as the most dominant in BM, indicating that stem/progenitor cells rather than mature mast cells might be the source of chymase in BM. Conclusion: The current findings demonstrate that BM has tremendously higher chymase-mediated Ang II producing activity from Ang-(1-12) compared to heart tissue. The fact that TEI-00806 inhibited the Ang II producing activity of chymase underscores a tissue-selective enzymatic mechanism for Ang II formation from Ang-(1-12) contributing to the chronic inflammatory processes accompanying the pathogenesis of cardiovascular disease.

Deletion of ΔdblGata motif leads to increased predisposition and severity of IgE-mediated food-induced anaphylaxis response.

BackgroundPrevious studies have revealed an important role for the transcription factor GATA-1 in mast cell maturation and degranulation. However, there have been conflicting reports with respect to the requirement of GATA-1 function in mast cell dependent inflammatory processes. Herein, we examine the requirement of GATA-1 signaling in mast cell effector function and IgE-mast cell-dependent anaphylaxis.ObjectiveTo study the requirement of GATA-1 dependent signaling in the development and severity of IgE-mast cell-dependent anaphylaxis in mice.MethodsWild type (Balb/c) and mutant ΔdblGata (Balb/c) mice were employed to study the role of GATA-1 signaling in in vitro IgE-mediated activation of bone marrow derived mast cells (BMMCs). Murine models of passive IgE-mediated and oral antigen-induced IgE-mediated anaphylaxis were employed in mice. Frequency of steady state mast cells in various tissues (duodenum, ear, and tongue), peritoneal cavity, and clinical symptoms (diarrhea, shock, and mast cell activation) and intestinal Type 2 immune cell analysis including CD4+ Th2 cells, type 2 innate lymphoid cells (ILC2), and IL-9 secreting mucosal mast cells (MMC9) were assessedResultsIn vitro analysis revealed that ΔdblGata BMMCs exhibit a reduced maturation rate, decreased expression of FcεRIα, and degranulation capacity when compared to their wildtype (WT) counterparts. These in vitro differences did not impact tissue resident mast cell numbers, total IgE, and susceptibility to or severity of IgE-mediated passive anaphylaxis. Surprisingly, ΔdblGata mice were more susceptible to IgE-mast cell-mediated oral antigen induced anaphylaxis. The increased allergic response was associated with increased Type 2 immunity (antigen-specific IgE, and CD4+ TH2 cells), MMC9 cells and small intestine (SI) mast cell load.ConclusionDiminished GATA-1 activity results in reduced in vitro mast cell FcεRIα expression, proliferation, and degranulation activity. However, in vivo, diminished GATA-1 activity results in normal homeostatic tissue mast cell levels and increased antigen-induced CD4+ Th2 and iMMC9 cell levels and heightened IgE-mast cell mediated reactions.

Regulation of Cardiac Mast Cell Maturation and Function by the Neurokinin-1 Receptor in the Fibrotic Heart

Cardiac fibrosis is an underlying cause of diastolic dysfunction, contributing to heart failure. Substance P (SP) activation of the neurokinin-1 receptor (NK-1R) contributes to cardiac fibrosis in hypertension. However, based on in vitro experiments, this does not appear to be via direct activation of cardiac fibroblasts. While numerous cells could mediate the fibrotic effects of SP, herein, we investigate mast cells (MC) as a mechanism mediating the fibrotic actions of SP, since MCs are known to play a role in cardiac fibrosis and respond to SP. Spontaneously hypertensive rats (SHR) were treated with the NK-1R antagonist L732138 (5 mg/kg/d) from 8 to 12 weeks of age. L732138 prevented increased MC maturation of resident immature MCs. NK-1R blockade also prevented increased cardiac MC maturation in angiotensin II-infused mice. MC-deficient mice were used to test the importance of MC NK-1Rs to MC activation. MC-deficient mice administered angiotensin II did not develop fibrosis; MC-deficient mice reconstituted with MCs did develop fibrosis. MC-deficient mice reconstituted with MCs lacking the NK-1R also developed fibrosis, indicating that NK-1Rs are not required for MC activation in this setting. In conclusion, the NK-1R causes MC maturation, however, other stimuli are required to activate MCs to cause fibrosis.

Adipocytokines leptin and adiponectin function as mast cell activity modulators.

A growing body of data indicates that adipocytokines, including leptin and adiponectin, are critical components not only of metabolic regulation but also of the immune system, mainly by influencing the activity of cells participating in immunological and inflammatory processes. As mast cells (MCs) are the key players in the course of those mechanisms, this study aimed to evaluate the impact of leptin and adiponectin on some aspects of MC activity. We documented that in vivo differentiated mature tissue MCs from the rat peritoneal cavity express a receptor for leptin (OB-R), as well as receptors for adiponectin (AdipoR1 and AdipoR2). We established that leptin, but not adiponectin, stimulates MCs to release of histamine as well as to generation of cysteinyl leukotrienes (cysLTs) and chemokine CCL2. We also found that both adipocytokines affect mRNA expression of various cytokines/chemokines. Leptin and adiponectin also activate MCs to produce reactive oxygen species. Moreover, we documented that leptin significantly augments the surface expression of receptors for cysLTs, i.e. CYSLTR1, CYSLTR2, and GPR17 on MCs, while adiponectin increases only GPR17 expression, and decreases CYSLTR2. Finally, we showed that both adipocytokines serve as potent chemoattractants for MCs. In intracellular signaling in MCs activated by leptin Janus-activated kinase 2, phospholipase C, phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK1/2), and p38 molecules play a part whereas the adiponectin-induced activity of MCs is mediated through PI3K, p38, and ERK1/2 pathways. Our observations that leptin and adiponectin regulate MC activity might indicate that adipocytokines modulate the different processes in which MCs are involved.

Expression of suppressor of cytokine signaling 3 (SOCS3) and interleukin-6 (-174-G/C) polymorphism in atopic conditions.

Hypersensitivity of the immune system is caused by elevated immunoglobulin E (IgE) levels in the serum, in response to a discrete allergen leading to allergic reactions. IgE-mediated inflammation is regulated by the cascade of defense related signaling molecules including interleukin-6 (IL-6) that plays pivotal role in the survival and maturation of mast cells during an allergic reaction. IL-6 mediated defense responses are tightly regulated by Suppressor of Cytokine Signaling 3 (SOCS3), an inhibitory molecules of Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling, in a negative feedback mechanism. The given study focuses on the assessment of crosstalk between SOCS3 and IL-6 to unravel the molecular significance of SOCS3 and IL-6 in the diagnosis and prognosis of allergy. The expression study of SOCS3 through real-time PCR analysis revealed, a 5.9 mean fold increase in SOCS3 expression in atopic cases in comparison to control cases. Moreover, IL-6 has, also, been found significantly enhanced in the serum level of atopic cases (26.4 pg/ml) as compared to control cases (3.686 pg/ml). Female population was found to be at a higher risk to develop atopic condition than male population as females exhibited higher expression of both SOCS3 and IL-6 than males. Furthermore, the polymorphic study of IL-6 promoter region (IL-6 174-G/C) in atopic population has reasserted the importance of SOCS3 and IL-6 in the diagnosis and prognosis of allergy. Expression of SOCS3 and IL-6 serum levels were found to be highly correlated. Therefore establishing the role of IL-6 (-174-G/C) polymorphism on the expression of SOCS3 and IL-6 in atopic cases. Notably, the study established SOCS3 and IL-6 as potential targets for the diagnosis/prognosis of allergy and for the development of reliable therapeutic strategies to control atopic conditions in the near future.

Skin microbiome regulates SCF level in Keratinocytes and defines mast cell maturation

Abstract Background Mast cell (MC) progenitors enter the skin and become mature in the surrounding microenvironment during embryogenesis and they fully mature 8 to 15 days after birth (in mouse). We published that skin commensal bacteria work together with skin resident cells (especially keratinocytes), in order to retain and differentiate mast cells into the skin. In the present study, we aim to demonstrate that this effect is due to TLR2 sensing of microbiome. Methods we used germ free mice (GF), hairless (hr/hr) mice and hr/hr/Tlr2−/− mice. Analyses were performed by immunohistochemistry, qPCR and FACS. IgE cross-linking and 48/80 have been used to test in vivo MCs degranulation. Results we found that GF mice present with less mature skin MCs than pathogen free mice (PFM). MCs immaturity correlated with a low level of stem cell factor (SCF) in keratinocytes, a critical MC differentiation and growth factor. Furthermore, compound 48/80, a known degranulation agent, induced less edema in the paws of GF mice than in those of conventional mice. A smaller population of c-Kit positive MCs in the GF mice normalized after being co-housed with PFM mice for 5 weeks to reconstitute their microbiome. On the other hand, hr/hr mice, have a skin increased number of mature and proliferating MCs that correlates with a more gram positive microbiome than their littermates. When hr/hr were backcrossed into Tlr2−/− (so they cannot sense the gram positive commensals) the number of MCs in skin dramatically decreased. Conclusions While baseline keratinocyte SCF expression is essential to recruit MCs into the skin, TLR2 stimulation by gram positive skin microbiome, promotes SCF keratinocyte production, which then enhances the maturation and proliferation of skin MCs.

Single-cell analysis reveals the KIT D816V mutation in haematopoietic stem and progenitor cells in systemic mastocytosis.

BackgroundSystemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ haematopoietic progenitors can carry the mutation; however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells.MethodsFluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34+ bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential.FindingsWe found that in SM 60–99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time.InterpretationThe KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

Characterization of mast cell-derived rRNA-containing microvesicles and their inflammatory impact on endothelial cells.

Tissue-resident mast cells (MCs) are well known for their role in inflammatory responses and allergic and anaphylactic reactions, but they also contribute to processes of arterial remodeling. Although ribosomes and cytosolic RNAs are located around secretory granules in mature MCs, their functional role in MC responses remains unexplored. Previous studies by our group characterized extracellular RNA (eRNA) as an inflammatory and pathogenetic factor in vitro and in vivo. In the present study, RNA-containing MCs and eRNA were located in close proximity to growing collateral arteries in vivo. In vitro, various agonists were found to induce the degranulation of MCs and the concomitant release of eRNA in association with microvesicles (MVs). The liberation of eRNA from MCs was abolished by MC stabilizers or by preventing the increase of intracellular Ca2+ in MCs. eRNA was found to be mainly contained inside MVs, as demonstrated by electron microscopy and immunocytochemistry. The exposure to and the uptake of MC-released MVs by cultured endothelial cells increased their expression of cytokines, such as monocyte chemoattractant protein or IL-6, in a dose- and time-dependent manner. These results indicate that RNA-containing MC-derived MVs are likely to be involved in inflammatory responses, relevant, for example, to processes of vascular remodeling.-Elsemüller, A.-K., Tomalla, V., Gärtner, U., Troidl, K., Jeratsch, S., Graumann, J., Baal, N., Hackstein, H., Lasch, M., Deindl, E., Preissner, K. T., Fischer, S. Characterization of mast cell-derived rRNA-containing microvesicles and their inflammatory impact on endothelial cells.

Temporal and Spatially Regulated Oncogenic KIT Expression and Loss of Dnmt3a Cooperate to Drive MPN Development: Role of PI3Kinase in Dnmt3a Loss Induced Hyperproliferation in Myeloid Cells

Recent studies, including studies from our group have shown that somatic mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with systemic mastocytosis (SM), myelodysplastic syndrome and AML. Importantly, the presence of this mutation is associated with poor prognosis and overall patient survival. We have shown that approximately 12% of patients with SM possess DNMT3A mutations, which are commonly co-expressed with an activating mutation of KIT (KITD816V in humans and KITD814V in mice). However, it is unclear how DNMT3A mutations contribute to myeloid lineage derived mast cell growth and differentiation either alone or in conjunction with KITD816V. Further, the mechanisms by which loss of DNMT3A impairs various mast cell functions either alone or in cooperation with KITD816V are poorly understood. Utilizing mice conditionally deficient in Dnmt3a in the hematopoietic compartment, we show that loss of Dnmt3a in vivo results in increased numbers of mast cells relative to controls. In vitro, loss of Dnmt3a results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors. Previous studies have shown that the level of expression of the transcription factor, Microthalamia (MITF) (high MITF expression drives mast cell commitment) and CEBPα (high C/EBPα drives basophil commitment) in Basophil-Mast cell common progenitor (BMPs) drives the fate of these cells towards either basophilic lineage or mast cell lineage. To assess why loss of Dnmt3a results in increased mast cell lineage commitment, we performed RNAseq analysis and found elevated expression of MITF and a profound repression in the expression of C/EBPα in BM precursors. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with the expression of MITF and C/EBPα. To our surprise, no appreciable differences in the expression of Gata-1, Gata-2 or PU.1 were noted in the absence of Dnmt3a relative to controls. These results demonstrate that early loss of C/EBPα in Dnmt3a null cells contributes to enhanced maturation of mast cells from their precursors. We next assessed the growth and survival potential of Dnmt3a mast cells and found a significant increase in SCF-mediated growth of these cells compared to controls. Biochemical analysis revealed greater PI3Kinase activation in Dnmt3a null cells, and these findings were supported by genome wide transcriptome analysis involving Ingenuity pathway analysis (IPA) and Gene set enrichment analysis (GSEA). Importantly, mast cells derived from Dnmt3a null BM lacking the expression of p85α regulatory subunit of PI3Kinase (Dnmt3a-/-:p85α-/-) or pharmacologic inhibition of PI3Kinase completely corrected SCF induced hyper proliferation in these cells to near normal levels. The increase in PI3Kinase activity in Dnmt3a null cells was associated with the loss of PTEN expression. To understand how loss of Dnmt3a and expression of an oncogenic form of KIT cooperate to drive MPN, we generated mice in which the expression of oncogenic KIT was temporally and spatially regulated in a cell type and tissue specific manner using the Mx-Cre system along with the expression of Dnmt3a. Consistent with earlier studies, we observed that KitD814V/+ mutation alone only resulted in the development of lymphoid neoplasms. In contrast, Dnmt3a-/- :KitD814V/+ mice manifested robust signs of MPN development including splenomegaly with increased numbers of neutrophils and monocytes in the peripheral blood associated with a significant reduction in lymphoid cells including B cells. Importantly, we noted a 5-fold increase in the fraction of LSKs and GMPs in the BM of Dnmt3a-/- :KitD814V/+ mice compared to controls and consequently an increase in the presence of mature Gr-1 and Mac-1 positive myeloid cells in the BM, spleen and in the PB of these mice relative to controls. The observed MPN was more aggressive in Dnmt3a-/- :KitD814V/+ mice compared to Dnmt3a+/- :KitD814V/+ mice and of stem cell origin as transplantation of BM cells from these mice recapitulated the development of MPN in recipient mice, similar to that observed in the original animals. The impact of PI3Kinase inhibition on mitigating oncogenic KIT and loss of Dnmt3a induced MPN and overall survival will be discussed. DisclosuresNo relevant conflicts of interest to declare.

The Mast Cell-Aryl Hydrocarbon Receptor Interplay at the Host-Microbe Interface.

Mast cells are increasingly being recognized as crucial cells in the response of the organism to environmental agents. Interestingly, the ability of mast cells to sense and respond to external cues is modulated by the microenvironment that surrounds mast cells and influences their differentiation. The scenario that is emerging unveils a delicate equilibrium that balances the effector functions of mast cells to guarantee host protection without compromising tissue homeostasis. Among the environmental components able to mold mast cells and fine-tune their effector functions, the microorganisms that colonize the human body, collectively known as microbiome, certainly play a key role. Indeed, microorganisms can regulate not only the survival, recruitment, and maturation of mast cells but also their activity by setting the threshold required for the exploitation of their different effector functions. Herein, we summarize the current knowledge about the mechanisms underlying the ability of the microorganisms to regulate mast cell physiology and discuss potential deviations that result in pathological consequences. We will discuss the pivotal role of the aryl hydrocarbon receptor in sensing the environment and shaping mast cell adaptation at the host-microbe interface.

Leptin receptor is expressed by tissue mast cells

Leptin, the adipose tissue-derived product of the obese (ob) gene, is known to function as the hormone of energy expenditure. It has also been established that leptin regulates immune and inflammatory processes. All leptin-induced biological activities depend on binding to the membrane-spanning leptin receptor (Ob-R), belonging to the class I cytokine receptor family. The available data relating to the Ob-R on mature mast cells (MCs), and consequently leptin significance in the modulation of MC activity within the tissue, are limited. Immunohistochemistry was used to establish Ob-R expression by MCs in the mesenteric adipose tissue. Flow cytometry and confocal microscopy were used to evaluate both constitutive and leptin-induced expression of Ob-R on freshly isolated peritoneal MCs. MCs in the mesenteric adipose tissue and native peritoneal MCs express Ob-R constitutively. Additionally, leptin influences its receptor expression on these cells. Leptin at lower concentrations caused Ob-R expression increase both at the cell surface and in the cell interior. MC stimulation with higher concentrations of leptin results in a decline of Ob-R from the cell surface and significant enhancement of this receptor not only in the nuclear region but also in the endoplasmic reticulum. In conclusion, one can be assumed that leptin regulates MC activity within tissues. These findings might provide an additional link among the leptin, innate immune function, and inflammatory processes and diseases.

Rapid Mast Cell Generation from Gata2 Reporter Pluripotent Stem Cells.

SummaryMast cells are tissue-resident immune cells. Their overgrowth/overactivation results in a range of common distressing, sometimes life-threatening disorders, including asthma, psoriasis, anaphylaxis, and mastocytosis. Currently, drug discovery is hampered by use of cancer-derived mast cell lines or primary cells. Cell lines provide low numbers of mature mast cells and are not representative of in vivo mast cells. Mast cell generation from blood/bone marrow gives poor reproducibility, requiring 8–12 weeks of culture. Here we report a method for the rapid/robust production of mast cells from pluripotent stem cells (PSCs). An advantageous Gata2Venus reporter enriches mast cells and progenitors as they differentiate from PSCs. Highly proliferative mouse mast cells and progenitors emerge after 2 weeks. This method is applicable for rapid human mast cell generation, and could enable the production of sufficient numbers of physiologically relevant human mast cells from patient induced PSCs for the study of mast cell-associated disorders and drug discovery.

Cultivation and identification of mouse peritoneal and bone marrow-derived mast cells

Objective To explore the in vitro culture methods for oriented differentiation of peritoneal cells and bone marrow cells into high-purity mast cells, and to identify the function of these mast cells. Methods Peritoneal cells and bone marrow cells were isolated from the peritoneal cavity lavages and femur of C57BL/6 mice, and cultured with both interleukin-3 (IL-3) and stem cell factor for 2 and 4 weeks respectively. Light microscopy was performed to observe the morphology of these cells, toluidine blue staining to identify the degree of maturity of these mast cells, and flow cytometry to measure the expression of cell surface markers CD117 and FceRⅠα. After the stimulation with compound 48/80 at different concentrations, the degranulation rate of mast cells was counted under the microscope, and β-hexosaminidase release rate was measured by spectrophotometry. Results After 2- or 4-week culture, the mouse peritoneal and bone marrow cells all manifested as refractive suspension cells of uniform size. Toluidine blue staining showed violaceous metachromatic granules in the cytoplasm of the two kinds of cells. The proportions of CD117 or FceR Ⅰ α single-positive peritoneal and bone marrow-derived mast cells were all more than 95%, and the proportions of CD117/FceRⅠα double-positive peritoneal and bone marrow-derived mast cells were 97.68% ± 0.80% and 96.12% ± 0.76% respectively. The degranulation rates of mast cells in the 100- and 1 000-mg/L compound 48/80 groups significantly differed from those in the blank control group (all P < 0.01) . Compared with the blank control group, the β-hexosaminidase release rates significantly increased in bone marrow-derived mast cells in the 100-mg/L compound 48/80 group and peritoneal mast cells in the 10- and 100-mg/L compound 48/80 groups (P < 0.01 or 0.05) . Conclusion IL-3 and stem cell factor can co-induce the directed differentiation and proliferation of mouse bone marrow stem cells and peritoneal cells, so as to harvest high-purity mature degranulated mast cells, and lay a foundation for subsequent cell biology research. Key words: Mast cells; Myeloid progenitor cells; Induced pluripotent stem cells; Mice; Cells, cultured; Interleukin-3; Stem cell factor

Degradation of Monocyte Chemoattractant Protein-1 by Tryptase Co-Released in Immunoglobulin E-Dependent Activation of Primary Human Cultured Mast Cells

Background: Mast cells are key immune effector cells which release chemokines, proteases, and other inflammatory mediators upon activation by immunological stimuli. The aim of this study was to investigate the effects of co-releasing proteases on the kinetics of release of the chemokine monocyte chemoattractant protein-1 (MCP-1) in immunoglobulin E (IgE)-mediated activation of human mast cells. Methods: Homogenous populations of mature and functional primary human mast cells were generated from CD34⁺ progenitors originated from buffy coats of healthy adult donors. The releases of MCP-1 from human mast cells in basal conditions and in response to FcεRI cross-linking were assessed at different time points. The effects of different types of protease inhibitors on MCP-1 release from these mast cells under stimulated or unstimulated conditions were also investigated. Results: Cultured human mast cells released MCP-1 in basal conditions and its levels increased in a time-dependent manner. When stimulated by FcεRI cross-linking, the levels of MCP-1 detected in the medium gradually decreased over time after the initial peak induction. Such a decline in MCP-1 levels after IgE-dependent activation was completely prevented by pretreatment with a cocktail of protease inhibitors or the specific tryptase inhibitor APC366. Conclusions: Direct regulation of MCP-1 expression by co-release of tryptase in cultured human mast cells upon IgE-dependent activation demonstrates a role of the serglycin:serine protease axis in modulation of inflammatory reactions through proteolytic degradation of mediators such as chemokines.

Leptin stimulates tissue rat mast cell pro-inflammatory activity and migratory response

ObjectiveThe aim of this study was to determine whether leptin, a member of the adipocytokines involved in immune and inflammatory response regulation, may influence some aspects of mast cell biology.Materials and methodsExperiments were done in vitro on fully mature tissue rat mast cells isolated from the peritoneal cavity, and leptin was used at concentrations 0.001–100 ng/ml. The effect of leptin on mast cell degranulation (histamine release assay), intracellular Ca2+ level (fluorimetry), pro-inflammatory mediator release (ELISA technique), surface receptor expression (flow cytometry and confocal microscopy), and migration (Boyden microchamber assay) was estimated.ResultsLeptin was found to stimulate mast cells to degranulation and histamine release. It induced the intracellular Ca2+ increase, as well. In response to leptin stimulation, mast cells generated and released cysLTs and chemokine CCL3. Leptin-induced upregulation of CYSLTR1 and CYSLTR2 surface expression was observed. Moreover, this adipocytokine stimulated mast cells to migratory response, even in the absence of extracellular matrix (ECM) proteins.ConclusionsOur observations clearly documented that leptin promotes the pro-inflammatory activity of mast cells, and it thereby engages these cells in the inflammatory processes.

Deep-Sea-Derived Butyrolactone I Suppresses Ovalbumin-Induced Anaphylaxis by Regulating Mast Cell Function in a Murine Model.

Deep-sea-derived butyrolactone I (BTL-I), which was identified as a type of butanolide, was isolated from Aspergillus sp. Ovalbumin (OVA)-induced BALB/c anaphylaxis was established to explore the antifood allergic activity of BTL-I. As a result, BTL-I was able to alleviate OVA-induced allergy symptoms, reduce the levels of histamine and mouse mast cell proteinases, inhibit OVA-specific IgE, and decrease the population of mast cells in the spleen and mesenteric lymph nodes. BTL-I also significantly suppressed mast-dependent passive cutaneous anaphylaxis. Additionally, the maturation of bone marrow-derived mast cells (BMMCs) declined as BTL-I caused down-regulation of c-KIT receptors. Furthermore, molecular docking analyses revealed that BTL-I interacted with the inhibitory receptor, FcγRIIB. In conclusion, the reduction of mast cell function by deep-sea-derived BTL-I as well as its interactions with the inhibitory receptor, FcγRIIB, may contribute to BTL-I-related protection against food anaphylaxis.

IL-6 potentiates FcɛRI-induced PGD2 biosynthesis from human skin mast cells by a STAT3-dependent mechanism.

Abstract Mast cells are tissue resident immune cells that cause allergies. The agents responsible for allergic reactions are pre-formed mediators like histamine and inflammatory lipids such as Prostaglandin D2 (PGD2) that are rapidly biosynthesized following crosslinking of FcɛRI with allergen. In humans, increased IL-6 levels are consistently correlated with allergic diseases like asthma and urticaria in which mast cells play a vital role. However, the exact role of IL-6 in allergic disease is not known. In this study, we show that human in situ-matured skin mast cells (huSMCs) express functional membrane-bound receptors for IL-6, and demonstrate our novel finding that IL-6 potentiates FcɛRI-induced PGD2 biosynthesis. Treatment of huSMCs with IL-6 alone induced the phosphorylation of STAT3 at the activating tyrosine 705, and the expression of SOCS3 and VEGF mRNA. IL-6 alone did not induce PGD2 secretion, but significantly and dose-dependently enhanced PGD2 secretion from FcɛRI-activated huSMCs. Accordingly, IL-6 enhanced the FcɛRI-induced expression of COX-2, which is directly involved in PGD2 biosynthesis. In addition, phosphorylation of STAT3 at both tyrosine 705 and serine 727, which is required for maximal transcriptional activity, was increased in huSMCs treated with IL-6 + FcɛRI crosslinking. Moreover, the IL-6-mediated increase in FcɛRI-induced COX-2 expression was inhibited with C188-9, the small molecule inhibitor of STAT3 tyrosine phosphorylation. Together, these data demonstrate that IL-6 potentiates FcɛRI-induced PGD2 biosynthesis from huSMCs by a STAT3-dependent mechanism. Thus, we hypothesize that IL-6 contributes to allergic disease by enhancing the production of inflammatory PGD2 from mature mast cells.

Munc18-2, but not Munc18-1 or Munc18-3, controls compound and single-vesicle–regulated exocytosis in mast cells

Mast cells (MCs) play pivotal roles in many inflammatory conditions including infections, anaphylaxis, and asthma. MCs store immunoregulatory compounds in their large cytoplasmic granules and, upon stimulation, secrete them via regulated exocytosis. Exocytosis in many cells requires the participation of Munc18 proteins (also known as syntaxin-binding proteins), and we found that mature MCs express all three mammalian isoforms: Munc18-1, -2, and -3. To study their functions in MC effector responses and test the role of MC degranulation in anaphylaxis, we used conditional knockout (cKO) mice in which each Munc18 protein was deleted exclusively in MCs. Using recordings of plasma membrane capacitance for high-resolution analysis of exocytosis in individual MCs, we observed an almost complete absence of exocytosis in Munc18-2-deficient MCs but intact exocytosis in MCs lacking Munc18-1 or Munc18-3. Stereological analysis of EM images of stimulated MCs revealed that the deletion of Munc18-2 also abolishes the homotypic membrane fusion required for compound exocytosis. We confirmed the severe defect in regulated exocytosis in the absence of Munc18-2 by measuring the secretion of mediators stored in MC granules. Munc18-2 cKO mice had normal morphology, development, and distribution of their MCs, indicating that Munc18-2 is not essential for the migration, retention, and maturation of MC-committed progenitors. Despite that, we found that Munc18-2 cKO mice were significantly protected from anaphylaxis. In conclusion, MC-regulated exocytosis is required for the anaphylactic response, and Munc18-2 is the sole Munc18 isoform that mediates membrane fusion during MC degranulation.

Loss of epigenetic regulator TET2 and oncogenic KIT regulate myeloid cell transformation via PI3K pathway.

Mutations in KIT and TET2 are associated with myeloid malignancies. We show that loss of TET2-induced PI3K activation and -increased proliferation is rescued by targeting the p110α/δ subunits of PI3K. RNA-Seq revealed a hyperactive c-Myc signature in Tet2-/- cells, which is normalized by inhibiting PI3K signaling. Loss of TET2 impairs the maturation of myeloid lineage-derived mast cells by dysregulating the expression of Mitf and Cebpa, which is restored by low-dose ascorbic acid and 5-azacytidine. Utilizing a mouse model in which the loss of TET2 precedes the expression of oncogenic Kit, similar to the human disease, results in the development of a non-mast cell lineage neoplasm (AHNMD), which is responsive to PI3K inhibition. Thus, therapeutic approaches involving hypomethylating agents, ascorbic acid, and isoform-specific PI3K inhibitors are likely to be useful for treating patients with TET2 and KIT mutations.

Interleukin-6 potentiates FcεRI-induced PGD2 biosynthesis and induces VEGF from human in situ-matured skin mast cells

BackgroundInterleukin-6 is a gp130 utilizing cytokine that is consistently associated with allergic diseases like asthma and urticaria in humans where mast cells are known to play a critical role. However, the role of IL-6 in allergic disease in not known. IL-6 was reported to enhance degranulation of in vitro-derived mast cells, but the effect of IL-6 on mediator release from human in situ-matured tissue-isolated mast cells had not been reported. MethodsHuman mature mast cells were isolated and purified from normal skin tissue from different donors. The expression of surface-expressed IL-6 receptors was demonstrated by flow cytometry. The effect of IL-6 on FcεRI-induced degranulation, PGD2 biosynthesis, and cytokine production was determined with β‑hexosaminidase release assay, Western blotting, quantitative real-time PCR, and ELISA. The small molecule inhibitor of STAT-3, C188-9, was used to demonstrate STAT3 dependency.Results: IL-6 significantly potentiated FcεRI-induced PGD2 biosynthesis, but had no effect on degranulation. IL-6 also induced VEGF gene expression and protein secretion, and enhanced FcεRI-induced IL-8 production. Mechanistically, IL-6 enhanced FcεRI-induced COX‑2 expression, PGD2 biosynthesis, and VEGF production in a STAT3 dependent manner. ConclusionHere, we demonstrate that IL-6 is a potentiator of FcεRI-induced PGD2 biosynthesis, and can induce or enhance production of pro-angiogenesis factors VEGF and IL-8 from human in situ-matured skin mast cells. General significanceThese findings from this study indicate that IL-6 contributes to human allergic disease by enhancing the production of inflammatory PGD2 from tissue-resident mast cells. Moreover, the data suggest a novel role for IL-6 in mast cell-mediated angiogenesis.