Cell-derived Mast Cells Research Articles

Inhibition of mast cell degranulation by novel small molecule MRGPRX2 antagonists

BackgroundMas-related G-protein coupled receptor X2 (MRGPRX2) is a promiscuous receptor on mast cells that mediates IgE-independent degranulation and has been implicated in multiple mast cell-mediated disorders, including chronic urticaria, atopic dermatitis, and pain disorders. Although it is a promising therapeutic target, few potent, selective, small molecule antagonists have been identified, and functional effects of human MRGPRX2 inhibition have not been evaluated in vivo. ObjectiveWe identified and characterized novel, potent, and selective orally active small molecule MRGPRX2 antagonists for potential treatment of mast cell-mediated disease. MethodsAntagonists were identified using multiple functional assays in cell lines overexpressing human MRGPRX2, LAD2 mast cells, human peripheral stem cell-derived mast cells, and isolated skin mast cells. Skin mast cell degranulation was evaluated in Mrgprb2em(-/-) knockout (KO) and Mrgprb2em(MRGPRX2) transgenic human MRGPRX2 knock-in (KI) mice by assessment of agonist-induced skin vascular permeability. Ex vivo skin mast cell degranulation and associated histamine release was evaluated by microdialysis of human skin tissue samples. ResultsMRGPRX2 antagonists potently inhibited agonist-induced MRGPRX2 activation and mast cell degranulation in all mast cell types tested, in an IgE-independent manner. Orally administered MRGPRX2 antagonists also inhibited agonist-induced degranulation and resulting vascular permeability in MRGPRX2 KI mice. In addition, antagonist treatment dose dependently inhibited agonist-induced degranulation in ex vivo human skin. ConclusionMRGPRX2 small molecule antagonists potently inhibited agonist-induced mast cell degranulation in vitro and in vivo as well as ex vivo in human skin, supporting potential therapeutic utility as a novel treatment for multiple human diseases involving clinically relevant mast cell activation.

The Uptake of Heparanase into Mast Cells Is Regulated by Its Enzymatic Activity to Degrade Heparan Sulfate.

Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.

Cytotoxicity assessment of exfoliated MoS2 using primary human mast cells and the progenitor cell-derived mast cell line LAD2.

Molybdenum disulfide is an emerging 2D material with several potential applications in medicine. Therefore, it is crucial to ascertain its biocompatibility. Mast cells are immune cells that are found in many organs and tissues in contact with the extracellular environment, and can be cultured from progenitor cells present in the bone marrow. Given the long period required for differentiation and proliferation of primary mast cells, human mast cell lines have emerged as a tractable model for biological and toxicological studies. Here, we compare two types of industrial MoS2 using CD34+-derived primary human mast cells and the LAD2 cell line. Minimal effects were observed on early-stage activation endpoints such as β-hexosaminidase release and expression of surface markers of mast cell activation. Transmission electron microscopy revealed limited uptake of the tested materials. Overall, MoS2 was found to be biocompatible, and the LAD2 cell line was validated as a useful in vitro model of mast cells.

Human mast cells induce osteoclastogenesis through cell surface RANKL.

We employed the co-culture of CD34+ stem cell-derived human mast cells (HMC) and human monocyte-derived osteoclast precursors to evaluate if mast cells contribute to the pathogenesis of osteoporosis through regulation of osteoclast proliferation and activation. Mature HMC and osteoclast precursors were cultured from monocytes isolated from human buffy coat. The osteoclast precursors were incubated with HMC or receptor activator of nuclear factor kappa-B ligand (RANKL) for a week prior to determination of osteoclast maturation through characterization by their morphology and tartrate resistant acid phosphatase (TRAP) expression. The bone absorption activity was determined by pit formation on osteo-assay plate. Mature osteoclasts were identified following co-culture of osteoclast precursors with HMC for one week in the absence of RANKL and they were capable of bone resorption. These actions of HMC on osteoclasts were not affected by mast cell activators such anti-IgE or substance P but could be reversed by osteoprotegerin (OPG) in the co-culture system suggesting the involvement of RANKL. The expression of RANKL on the cell surface of HMC was confirmed by flow cytometry and the density was not affected by activation of HMC. Our study provided direct evidence confirming the initiation of osteoclast proliferation and activation by mast cells through cell surface RANKL suggesting that mast cells may contribute to bone destruction in pathological conditions such as osteoporosis.

Morphological and Functional Characterization of CD34+cell-Derived Mast Cells from Sickle Cell Anemia Patients

Introduction: Mast cells (MC) activation has been described as a key factor on several disorders, but their role on sickle cell anemia (SCA) remains unclear. SCA is characterized by chronic inflammatory events orchestrated by activated leukocytes, which contribute to vaso-occlusive crisis. Physical interplay, release or production of mediators upon MC activation could affect the response of other cells. Stable MC and eosinophils (Eos) interplay and bidirectional modulation has been described on allergic and inflammatory diseases. We aimed to determine whether MC obtained from CD34+ stem cells of SCA patients, compared to healthy volunteers (HV), show differences in phenotype, function, or ability to interact with Eos.Methods: CD34+cellswere isolated from peripheral blood (HV: 60 ml; SCA: 30 ml) and cultured in StemPro34® medium with glutamine (2mM), LDL (10 mg/ml), SCF, IL-6 and IL-3 (100 ng/ml each). Culture was weekly expanded (medium without IL-3); differentiation was confirmed by toluidine blue staining and FACS (CD117+, FceRI+). After 4 weeks, surface marker expression of MC differentiation/activation, adhesion and mast cell/eosinophil related of HV and SCA was compared by FACS. Imaging flow cytometry (IFC) was used to define nuclear morphology, to identify differences in other marker expression and to characterize in vitro physical contact between MC and Eos isolated from peripheral blood from HV and SCA individuals. Cells were separately marked with fluorescent, lipophilic stains, and Feature Finder tool was used to pinpoint frequency and remarkable features for heterotypic conjugates formation. Standardized conditions were a 1:3 MC-Eos ratio, incubation for 5 minutes at 37°C and plotting Shape Ratio and Circularity features to quantify the occurrence of MC-Eos conjugates. In addition, cells were stained with fluorescent antibodies after 2 hours of interaction to evaluate the changes in marker expression during this process.Results: There was not difference in CD34+ cell count between HV and SCA individuals (p=0.848); however, a higher number of MC was obtained from SCA samples (4th week) (HV: 1.67x105±0.40; SCA: 4.38x105±1.22; p=0.027). Mature SCA from HV (n=10) and SCA (n=10) were obtained after 5 weeks (CD117+, FceRI+>50%). FACS showed higher percentage of CD117+ cells in SCA cultures (HV 18.35±5.83%; SCA 65.25±12.28%; p<0.01). Regarding CD244 and Siglec-8, SCA samples showed higher positivity, although not statistically significant because of marked variation among individuals (CD244: HV 3.32±0.84%, SCA 16.30±10.89%; Siglec-8: HV 5.94±2.48%, SCA 8.8±5.32%, p>0.05). No difference in adhesion molecules expression was detected (data not shown). IFC analysis revealed no difference in nuclear morphology, p=0.352). Differences in CD117 and Siglec-8 expression allowed a clear separation of HV- and SCD-MC (Bkgd Mean CD117: RD 2.78; Bkgd Mean Siglec8 RD: 1.84). Heterotypical (MC-Eos) conjugates formation was a rare event (less than 5% of images); there was no difference in the percentage of conjugates (HV-MC/HV-Eos: 1.61±0.37%; HV-MC/SCA-Eos: 1.92±0.37%; SCA-MC/HVEos: 1.12±0.31%; SCA-MC/SCA-Eos: 1.26±0.27%; p>0.05). Analysis of marker expression after interaction showed that HV-MC changed CD48, CD63 and CD117 expression patterns in response to SCA-Eos challenge (Bkgd Mean RD: CD48 1.09; CD63 1.16; CD117, 1.31); similarly, SCA-MC co-cultures with SCA-Eos not only modified CD48 and CD63 (Bkgd Mean RD values: BV421-CD48 2.32; PB-CD63 2.27), but also altered CD193, in a lesser extent (Bkgd Mean CD193: RD 0.88).Conclusion: We report for the first time the morphological and functional characteristics of CD34+-derived MC obtained from samples of SCA in comparison with HV. Our data suggest that MC from SCA patients may have a different expression profile of markers related to activation even after being withdrawn from exposure to chronic inflammation and vaso-occlusion in vivo . Surface markers corresponding to CD117, CD48, CD63 and CD193 in SCA animal models could be helpful to assess the activation state of MC and their response to experimental treatments targeting these cells. DisclosuresFertrin:Alexion Pharmaceuticals: Consultancy.

Mast Cell-Mediated Reactions In Vivo.

Mast cells are involved in many physiological reactions in which their functions can be very diverse. Models of allergic skin inflammation and systemic anaphylactic reactions in mice are validated methods in which the role of mast cells is well established. In this chapter, we will therefore present protocols for passive cutaneous anaphylaxis and contact hypersensitivity, i.e., models which can be used to identify and characterize the role of mast cells as well as mast cell mediators and receptors in allergic IgE-dependent and IgE-independent skin inflammation, and for passive systemic anaphylaxis, a model ideally suited to characterize the systemic effects of mast cell-derived mediators and mast cell receptors.

Generation and Culture of Peripheral CD34+ Stem Cell-Derived Mast Cells (PSCMCs).

Mast cells (MCs) are long-living tissue-resident cells that play an important role in inflammatory and allergic reactions. In vitro models of mast cell functions have allowed better understanding of the function of mast cells and mast cell-mediated disorders. In this unit, we describe a protocol used for the generation and culture of peripheral CD34+ stem cell-derived mast cells (PSCMCs). It provides a useful tool for the investigation of the biology of human MCs in vitro.

Imiquimod enhances DNFB mediated contact hypersensitivity in mice

Imiquimod (Imiq) is a synthetic imizoquinoline compound which can act on Toll-like receptor (TLR)7 and transduce signals involved in cell activation. We investigated the role of Imiq on contact hypersensitivity (CHS) and explored the potential mechanisms of mast cells involved in the process. Topical application of Imiq cream augmented DNFB mediated CHS in C57BL/6 mice. Imiq application induced skin inflammation and increased the number of dendritic cells (DCs) in the draining lymph nodes (DLNs). The splenic cell proliferation to DNBS in DNFB and Imiq treated mice was greater than that in mice of DNFB treatment alone. Peritoneal cell-derived mast cells (PCMCs) expressed TLR7 mRNA. The results from toluidine blue staining for mast cells and histamine detection indicated that Imiq alone did not induce mast cell degranulation while Imiq plus DNFB significantly induced mast cell degranulation. Cromolyn, pyrilamine and cimetidine attenuated CHS reaction induced by Imiq. Our findings suggest that Imiq could augment the intensity of CHS reaction. The mechanisms underlying the effect may relate to histamine release by mast cells and induction of DC homing to DLNs. Blocking histamine action in early time of allergen contact is beneficial to the alleviation of CHS.

The Inhibitory Effects on Cytotoxicity and Nitric Oxide (NO) of the Nano-Encapsulated Extraction of Lipid-Soluble Green Tea Leaves

The purpose of this study was to investigate the bioactive substances and extraction conditions of lipid-soluble green tee extract using supercritical CO extraction from green tea leaves growing in Boseong-gun, Jeollanam-do and to demonstrate cell safety and effects on inflammatory response of the nano-encapsulated lipid-soluble green tee extract. The polyphenol content of green tea extract using supercritical CO₂ extraction was analyzed. The highest polyphenol was extracted in the condition of 400 bar/50 °C. A total of polyphenol content was 2.47 mg/g. The lipid-soluble vitamin content of supercritical extracts of lipid-soluble green tea was analyzed. Beta-carotene, a vitamin A precursor (4.131 mg/100 g) and vitamin E (537.9 mg/100 g) were detected. ECG content was the highest of catechin of lipid-soluble green tea extract. For cell safety and NO inhibition, human skin cell-derived HaCaT cells and RBL-2H3 mast cells did not have cytotoxicity in 1% of the nano-encapsulated lipid-soluble green tea leaf extract. RAW 264.7 cells did not show cytotoxicity in 5%. NO production was significantly decreased in RAW 264.7 cells in a concentration-dependent manner and within the concentration range. Based on the findings, this study suggested the extraction standards and ingredients of polyphenol according to temperature and pressure in extracting green tea leaf ingredients using supercritical CO₂ extraction. This study also objectively demonstrated the safety and anti-inflammatory effect of the nano-encapsulated lipid-soluble green tea leaf extract which is used as functional materials in the cosmetic industry.

Human induced pluripotent stem cell-derived mast cells useful for in vitro mast cell activation assay exhibiting phenotypes and morphological characteristics of human mast cells.

Mast cells are key players in the inflammatory response with an important role in allergic reactions and are therefore useful for assessing the risk of anaphylaxis. However, they are difficult to isolate due to their low abundance and wide distribution. To overcome this, we generated and characterized mast cell-like cells derived from human induced pluripotent stem (hiPS) cells. These hiPS cell-derived mast cells (hiPS-MCs) were generated using recombinant human bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor 165 (VEGF), stem cell factor (SCF), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-9 (IL-9) in a StemPro-34 medium. The hiPS-MCs exhibited the morphological characteristics of human mast cells, expressing high affinity-IgE receptor (FcεRI) and mast cell markers such as tryptase, chymase, and CD117. In addition, FcεRI stimulation with agonistic anti-IgE functionally increased the expression of activation markers CD63 and CD203c, as well as the amount of released histamine. We think the hiPS-MCs generated in this study will be useful for assessing the pharmacology and toxicity of anti-allergy medicines.

Incorporation, intracellular trafficking and processing of extracellular heparanase by mast cells: Involvement of syndecan-4-dependent pathway

We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway.

Mechanistic Insights of an Immunological Adverse Event Induced by an Anti-KIT Antibody Drug Conjugate and Mitigation Strategies.

Purpose: Hypersensitivity reactions (HSRs) were observed in three patients dosed in a phase I clinical trial treated with LOP628, a KIT targeted antibody drug conjugate. Mast cell degranulation was implicated as the root cause for the HSR. Underlying mechanism of this reported HSR was investigated with an aim to identifying potential mitigation strategies.Experimental Design: Biomarkers for mast cell degranulation were evaluated in patient samples and in human peripheral blood cell-derived mast cell (PBC-MC) cultures treated with LOP628. Mitigation strategies interrogated include pretreatment of mast cells with small molecule inhibitors that target KIT or signaling pathways downstream of FcεR1, FcγR, and treatment with Fc silencing antibody formats.Results: Transient elevation of serum tryptase was observed in patients 1-hour posttreatment of LOP628. In agreement with the clinical observation, LOP628 and its parental antibody LMJ729 induced degranulation of human PBC-MCs. Unexpectedly, KIT small molecule inhibitors did not abrogate mast cell degranulation. By contrast, small molecule inhibitors that targeted pathways downstream of Fc receptors blunted degranulation. Furthermore, interference of the KIT antibody to engage Fc receptors by pre-incubation with IgG or using engineered Fc silencing mutations reduced or prevented degranulation. Characterization of Fcγ receptors revealed human PBC-MCs expressed both FcγRII and low levels of FcγRI. Interestingly, increasing the level of FcγRI upon addition of IFNγ, significantly enhanced LOP628-mediated mast cell degranulation.Conclusions: Our data suggest LOP628-mediated mast cell degranulation is the likely cause of HSR observed in the clinic due to co-engagement of the FcγR and KIT, resulting in mast cell activation. Clin Cancer Res; 24(14); 3465-74. ©2018 AACR.

Mast cell chymase decreases the severity of group B Streptococcus infections

Group B Streptococcus (GBS) or Streptococcus agalactiae are β-hemolytic gram-positive bacteria that colonize the lower genital tracts of women and are frequently associated with infections during pregnancy. Innate immune defenses are critical for controlling GBS dissemination and systemic infection. Mast cells are resident sentinel cells that come into contact with pathogens early during colonization and infection. We aimed to investigate the contribution of chymase to systemic GBS infection and rates of preterm birth. Pharmacologic and genetic approaches using mice deficient in mast cell protease (MCPT) 4, the mouse functional homologue of human chymase, were used. Our studies show that mast cells release a protease with chymotrypsin-like cleavage specificity in response to GBS. Additionally, increased GBS systemic infection and preterm births were observed in MCPT4-deficient mice versus MCPT4-sufficient mice. Furthermore, we observed that proteolytic cleavage of the host extracellular matrix protein fibronectin by peritoneal cell-derived mast cell lysates diminished GBS adherence. Consistent with this observation, the increase in GBS dissemination and preterm births observed in MCPT4-deficient mice was abolished when GBS was deficient in expression of the fibronectin-binding protein SfbA. Taken together, our results suggest that the protective effect of MCPT4 against GBS dissemination and preterm labor can be attributed in part to MCPT4-mediated proteolysis of fibronectin. Our studies reveal a novel role of mast cells in defense against bacterial infections.

Long-term survival of the mouse ES cell-derived mast cell, MEDMC-BRC6, in mast cell-deficient KitW-sh/W-sh mice.

Mast cells (MCs) play pivotal roles in allergic reactions and the host defense against microbial infection through the IgE-dependent and IgE-independent signaling pathways. MC lines that can be analyzed both in vitro and in vivo would be useful for the study of MC-dependent immune responses. Here, we investigated the functional characteristics of a mouse embryonic stem cell-derived MC-like cell line, MEDMC-BRC6. The cell line expressed FcεRI and c-Kit and showed degranulation and production of inflammatory cytokines and chemokines, including TNF-α, IL-6 and MCP-1, upon cross-linking FcεRI with IgE. These cytokines and chemokines were also produced by the cell line by stimulation of TLR2 and TLR4. MEDMC-BRC6 survived in the peritoneal cavity and the ear skin for at least 6 months after the transfer into genetically compatible MC-deficient KitW-sh/W-sh mice, in which systemic anaphylaxis was successfully induced. Thus, MEDMC-BRC6 cells represent a potent tool for investigating the functions of MCs in vitro and in vivo.

Phospholipid scramblase 1 amplifies anaphylactic reactions in vivo.

Mast cells are critical actors of hypersensitivity type I (allergic) reactions by the release of vasoactive and proinflammatory mediators following their activation by aggregation of the high-affinity receptor for immunoglobulin E (FcεRI). We have previously identified Phospholipid Scramblase 1 (PLSCR1) as a new molecular intermediate of FcεRI signaling that amplifies degranulation of the rat mast cell line RBL-2H3. Here we characterized primary mast cells from Plscr1-/- mice. The absence of PLSCR1 expression did not impact mast cell differentiation as evidenced by unaltered FcεRI expression, general morphology, amount of histamine stored and expression of FcεRI signal effector molecules. No detectable mast cell deficiency was observed in Plscr1-/- adult mice. In dose-response and time-course experiments, primary cultures of mast cells (bone marrow-derived mast cells and peritoneal cell-derived mast cells) generated from Plscr1-/- mice exhibited a reduced release of β-hexosaminidase upon FcεRI engagement as compared to their wild-type counterparts. In vivo, Plscr1-/- mice were protected in a model of passive systemic anaphylaxis when compared to wild-type mice, which was consistent with an observed decrease in the amounts of histamine released in the serum of Plscr1-/- mice during the reaction. Therefore, PLSCR1 aggravates anaphylactic reactions by increasing FcεRI-dependent mast cell degranulation. PLSCR1 could be a new therapeutic target in allergy.

Live Staphylococcus aureus Induces Expression and Release of Vascular Endothelial Growth Factor in Terminally Differentiated Mouse Mast Cells.

Mast cells have been shown to express vascular endothelial growth factor (VEGF), thereby implicating mast cells in pro-angiogenic processes. However, the mechanism of VEGF induction in mast cells and the possible expression of VEGF in fully mature mast cells have not been extensively studied. Here, we report that terminally differentiated peritoneal cell-derived mast cells can be induced to express VEGF in response to challenge with Staphylococcus aureus, thus identifying a mast cell–bacteria axis as a novel mechanism leading to VEGF release. Whereas live bacteria produced a robust upregulation of VEGF in mast cells, heat-inactivated bacteria failed to do so, and bacteria-conditioned media did not induce VEGF expression. The induction of VEGF was not critically dependent on direct cell–cell contact between bacteria and mast cells. Hence, these findings suggest that VEGF can be induced by soluble factors released during the co-culture conditions. Neither of a panel of bacterial cell-wall products known to activate toll-like receptor (TLR) signaling promoted VEGF expression in mast cells. In agreement with the latter, VEGF induction occurred independently of Myd88, an adaptor molecule that mediates the downstream events following TLR engagement. The VEGF induction was insensitive to nuclear factor of activated T-cells inhibition but was partly dependent on the nuclear factor kappa light-chain enhancer of activated B cells signaling pathway. Together, these findings identify bacterial challenge as a novel mechanism by which VEGF is induced in mast cells.

Proteome analysis of mast cell releasates reveals a role for chymase in the regulation of coagulation factor XIIIA levels via proteolytic degradation

Mast cells are significantly involved in IgE-mediated allergic reactions; however, their roles in health and disease are incompletely understood. We aimed to define the proteome contained in mast cell releasates on activation to better understand the factors secreted by mast cells that are relevant to the contribution of mast cells in diseases. Bone marrow-derived cultured mast cells (BMCMCs) and peritoneal cell-derived mast cells were used as "surrogates" for mucosal and connective tissue mast cells, respectively, and their releasate proteomes were analyzed by mass spectrometry. Our studies showed that BMCMCs and peritoneal cell-derived mast cells produced substantially different releasates following IgE-mediated activation. Moreover, we observed that the transglutaminase coagulation factor XIIIA (FXIIIA) was one of the most abundant proteins contained in the BMCMC releasates. Mast cell-deficient mice exhibited increased FXIIIA plasma and activity levels as well as reduced bleeding times, indicating that mast cells are more efficient in their ability to downregulate FXIIIA than in contributing to its amounts and functions in homeostatic conditions. We found that human chymase and mouse mast cell protease-4 (the mouse homologue of human chymase) had the ability to reduce FXIIIA levels and function via proteolytic degradation. Moreover, we found that chymase deficiency led to increased FXIIIA amounts and activity, as well as reduced bleeding times in homeostatic conditions and during sepsis. Our study indicates that the mast cell protease content can shape its releasate proteome. Moreover, we found that chymase plays an important role in the regulation of FXIIIA via proteolytic degradation.

Peritoneal Mast Cell Stabilization Potential of Ziziphus Xylopyrus (Retz) Wild Extract in Rat Mesenteric Model

Objective: To investigate the peritoneal mast cell stabilization activity of Ziziphus xylopyrus extracts. Materials and Methods: Ziziphus Xylopyrus L. (family- Rhamnaceae) stem part was successively extracted with ethanol and aqueous to prepare extract of the plant. The extracts of Ziziphus Xylopyrus (Z. xylopyrus) were evaluated for stabilization of mast cell in rat for the evaluation of Hypersensitivity type I allergic reaction in animal models. The extract of Z. xylopyrus ethanolic and aqueous (1, 10 and 100 μg/ml) was evaluated for peritoneal mast cell stabilization activity in rat mesenteric preparation induced by C 48/80. Result: Preliminary phytochemical analysis revealed the presence of carbohydrates, fixed oil, proteins, alkaloids, glycosides, flavonoids and phenolic compounds. The ethanolic and aqueous extracts of Z. xylopyrus showed dose dependent increase in the number of intact cells when compare with C 48/80 at the concentration of 10 and 100 μg/ml. It virtues further work towards the isolation of phyto constituents from this plant. Conclusion: This finding provides evidence that the Z. xylopyrus inhibits mast cell-derived immediate-type allergic reactions and mast cell degranulation. Z. xylopyrus has a potential as allergic anti-asthmatic agent.

Tie2 Signaling Enhances Mast Cell Progenitor Adhesion to Vascular Cell Adhesion Molecule-1 (VCAM-1) through α4β1 Integrin.

Mast cell (MC) activation contributes considerably to immune responses, such as host protection and allergy. Cell surface immunoreceptors expressed on MCs play an important role in MC activation. Although various immunoreceptors on MCs have been identified, the regulatory mechanism of MC activation is not fully understood. To understand the regulatory mechanisms of MC activation, we used gene expression analyses of human and mouse MCs to identify a novel immunoreceptor expressed on MCs. We found that Tek, which encodes Tie2, was preferentially expressed in the MCs of both humans and mice. However, Tie2 was not detected on the cell surface of the mouse MCs of the peritoneal cavity, ear skin, or colon lamina propria. In contrast, it was expressed on mouse bone marrow–derived MCs and bone marrow MC progenitors (BM-MCps). Stimulation of Tie2 by its ligand angiopoietin-1 induced tyrosine phosphorylation of Tie2 in MEDMC-BRC6, a mouse embryonic stem cell-derived mast cell line, and enhanced MEDMC-BRC6 and mouse BM-MCp adhesion to vascular cell adhesion molecule-1 (VCAM-1) through α4β1 integrin. These results suggest that Tie2 signaling induces α4β1 integrin activation on BM-MCps for adhesion to VCAM-1.

A novel method to generate and culture human mast cells: Peripheral CD34 + stem cell-derived mast cells (PSCMCs)

The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 108 differentiated human MCs per batch after only three weeks of culture starting with 106 total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4–20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1β or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs.