KLF4 is required for suppression of histamine synthesis by polyamines during bone marrow-derived mast cell differentiation.

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Constitutive Association of Lyn A and B Kinases with the High Affinity IgE Receptor (FceRI) Positively Regulates Mast Cell Secretion

Serotonin and histamine storage in mast cell secretory granules is dependent on serglycin proteoglycan

Mast cells synthesize and store histamine, a key immunomodulatory mediator. Polyamines are essential for every living cell. Previously, we detected an antagonistic relationship between the metabolisms of these amines in established mast cell and basophilic cell lines. Here, we used the IL-3-driven mouse bone marrow-derived mast cell (BMMC) culture system to further investigate this antagonism in a mast cell model of deeper physiological significance. Polyamines and histamine levels followed opposite profiles along the bone marrow cell cultures leading to BMMCs. alpha-Difluoromethylornithine (DFMO)-induced polyamine depletion resulted in an upregulation of histidine decarboxylase (HDC, the histamine-synthesizing enzyme) expression and activity, accompanied by increased histamine levels, specifically during early stages of these cell cultures, where an active histamine synthesis process occurs. In contrast, DFMO did not induce any effect in either HDC activity or histamine levels of differentiated BMMCs or C57.1 mast cells, that exhibit a nearly inactive histamine synthesis rate. Sequence-specific DNA methylation analysis revealed that the DFMO-induced HDC mRNA upregulation observed in early bone marrow cell cultures is not attributable to a demethylation of the gene promoter caused by the pharmacological polyamine depletion. Taken together, the results support an inverse relationship between histamine and polyamine metabolisms during the bone marrow cell cultures leading to BMMCs and, moreover, suggest that the regulation of the histamine synthesis occurring during the early stages of these cultures depends on the concentrations of polyamines.

Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow-derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor-alpha (TNF-alpha) storage and secretion, and TNF-alpha-dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two-dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC-associated enzyme, rat mast cell proteinase-2 (RMCP-2), but not RMCP-1, mast cell proteinase-5 (MCP-5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the periotoneal MC (PMC). BMMC demonstrated levels of TNF-alpha-dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF-alpha, in comparison with PMC, but both MC types generated substantial amounts of TNF-alpha 6 hr following IgE-mediated activation. Pretreatment of PMC with recombinant rat interferon-gamma (IFN-gamma) for 20 hr inhibited anti-immunoglobulin E (anti-IgE)-mediated release of the granule-associated enzyme, beta-hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC.

Bone marrow-derived mast cells as well as dendritic cells, macrophages and B lymphocytes express major histocompatibility complex (MHC) class II molecules. In mast cells, the majority of MHC class II molecules reside in intracellular cell type-specific compartments, secretory granules. To understand the molecular basis for the localisation of MHC class II molecules in secretory granules, MHC class II molecules were expressed, together with the invariant chain, in the mast cell line, RBL-2H3. Using electron and confocal microscopy, we observed that in RBL-2H3 cells, mature and immature class II molecules accumulate in secretory granules. Two particular features of class II transport accounted for this intracellular localization: first, a large fraction of newly synthesized MHC class II molecules remained associated with invariant chain fragments. This defect, resulting in a slower rate of MHC class II maturation, was ascribed to a low cathepsin S activity. Second, although a small fraction of class II dimers matured (i.e. became free of invariant chain), allowing their association with antigenic peptides, they were retained in secretory granules. As a consequence of this intracellular localization, cell surface expression of class II molecules was strongly increased by cell activation stimuli which induced the release of the contents of secretory granules. Our results suggest that antigen presentation, and thereby antigen specific T cell stimulation, are regulated in mast cells by stimuli which induce mast cell activation.

SHP‐1 is a cytoplasmic tyrosine phosphatase and, in mast cells, has been shown to negatively regulate the initial signaling triggered by high affinity receptors for IgE (FcεRI). To clarify the molecular mechanisms of SHP‐1 in mast cells, we generated bone marrow‐derived mast cells (BMMCs) from SHP‐1‐deficient motheaten (me) and control (wt) mice and examined downstream signaling events and final outputs following FcεRI crosslinking. FcεRI‐induced tyrosine phosphorylation of SLP‐76 and LAT was increased in me‐derived BMMCs. The activity of Syk that is responsible for the phosphorylation of SLP‐76 and LAT was comparable in both BMMCs, implying that SLP‐76 and LAT might be substrates of SHP‐1 in mast cells. Lack of SHP‐1 expression resulted in enhanced activation of mitogen activated protein kinases, Erk, JNK, and p38, and transcription of cytokine genes such as TNFα and IL‐6, in good accordance with the previous implication of SHP‐1 as a negative regulator. In contrast, FcεRI‐induced calcium mobilization was severely impaired in me‐derived BMMCs. Whereas wt‐BMMCs showed sprolonged increase in calcium flux upon high intensity stimulation, BMMCs derived from me mice demonstrated decreased and transient responses. In addition, me‐BMMCs exhibited diminished calcium flux at low intensity stimulation. Thapsigargin induced similar level of calcium flux in both BMMCs, indicating that BMMCs lacking SHP‐1 expression are still capable of mobilizing calcium. These studies revealed that SHP‐1 not only regulates FcεRI‐signaling negatively but can act as a positive regulator in mast cells. The experiments to clarify possible molecular mechanisms for the positive regulation of calcium signaling by SHP‐1 are currently under study.

Role of common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of murine mast cells

Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A1-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A1 and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.

Ceramide kinase is not essential but might act as an Ca 2+-sensor for mast cell activation

Introduction: Our laboratory previously demonstrated that miR-9 promotes the invasive properties of normal and malignant mast cells and that overexpression of miR-9 is associated with spontaneous metastasis in canine mast cell tumors (MCTs), a well-established large animal model of malignant mast cell disease. The purpose of this study was to further investigate the biologic effect and mechanisms by which mir-9 enhances normal mast cell invasion. Methods: Bone marrow-derived mast cells (BMMCs) were generated from Cpa3-Cre; miR-9Δfl/Δfl, and Cpa3-Cre; miR-9Δfl/Δfl mice and Taqman miRNA assays were used to quantify mature miR-9 expression. BMMCs were evaluated for differences in proliferative capacity, the ability to migrate through Matrigel, sensitivity to degranulation, and the expression and release of growth factor expression and release following chemical stimulation. RNA sequencing was performed by the OSUCCC Genomics Resource and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. Results and Conclusions: We generated a transgenic mouse carrying a floxed STOP-miR-9 transgene (miR-9Δfl/Δfl) in which expression of miR-9 occurs only after Cre-mediated recombination and crossed these mice with Cpa3-Cre mice which express Cre recombinase under the control of the carboxypeptidase A3 (Cpa3) promoter, restricting expression of miR-9 to mast cells and basophils. Bone marrow-derived mast cells (BMMCs) derived from Cpa3-Cre;miR-9Δfl/Δfl mice significantly enhanced the invasive phenotype of mast cells. Moreover, the enforced expression of miR-9 in BMMCs markedly changed the production and release of cytokine and growth factors in response to chemical stimulation, including substantial up-regulation of IL-4, IL-13, and GM-CSF transcript and enhanced TGF-β1 release. RNA sequencing revealed a unique transcriptional profile associated with miR-9 over-expression in the Cpa3-Cre;miR-9Δfl/Δfl derived BMMCs and identified increased expression of several mast cell-restricted proteases involved in extracellular matrix and tissue remodeling including CMA1 and MCP-6. Furthermore, transduction of Cpa3-Cre;miR-9Δfl/Δfl derived BMMCs with CMA1 shRNA abrogated miR-9-dependent mast cell invasion. Studies are underway to investigate the regulatory pathway mediated by miR-9 that contributes to CMA1 up-regulation and promotes the invasive phenotype. In summary, these data provide evidence supporting a critical role for miR-9 in mast cell invasion and suggest that dysregulation of miR-9 may contribute to pathologic conditions involving mast cell-mediated tissue remodeling. Citation Format: Joelle M. Fenger, Feng Xu, Xaioli Zhang, Peter Y. Yu, Misty D. Bear, Shanice L. Reynolds, Michael L. Pennell, Vincenzo Coppola, William C. Kisseberth, Stephen J. Galli, Cheryl A. London. A novel Cpa3-Cre; miR-9fl/fl mouse reveals a functional role for miR-9 in promoting mast cell invasion via up-regulation of CMA1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3043. doi:10.1158/1538-7445.AM2017-3043

Identification of Biological and Pharmaceutical Mast Cell‐ and Basophil‐Related Targets

The expression of neutral glycosphingolipids by mouse T cell-dependent, bone marrow-derived mast cells (BMMC) obtained in vitro was determined by chromatographic and immunochemical criteria. Neutral glycosphingolipids were isolated from BMMC by extraction of 3 to 5 X 10(8) cells in chloroform/methanol (1/1, v/v) and chromatography on DEAE-Sephadex, and were analyzed by thin layer chromatography with orcinol staining. The predominant neutral glycosphingolipids of BMMC were glucosylceramide (CMH), lactosylceramide (CDH), globotriosylceramide (CTH), globotetraosylceramide (globoside), and a molecule migrating slightly faster than gangliotetraosylceramide (asialo GM1) and slower than globopentaosylceramide (Forssman glycolipid). The profiles on thin layer chromatograms of the neutral glycosphingolipids were the same for BMMC derived from BALB/c, C57BL/6, WBBF1-W/Wv, and WBBF1-+/+ mice, and for cells differentiated in either WEHI-3 conditioned medium or concanavalin A-splenocyte conditioned medium. High performance liquid chromatography of benzoylated neutral glycosphingolipids of BMMC on a Zipax column confirmed the identity of the four neutral glycosphingolipids identified by thin layer chromatography. The fifth major glycosphingolipid had an elution time greater than that of globotetraosylceramide and did not co-elute with any of the standards tested. Direct biochemical analyses of the neutral glycosphingolipids of mouse serosal mast cells (SMC) were not feasible because only 2 X 10(6) SMC could be isolated per 100 mice. However, mouse SMC bound a rat monoclonal anti-globopentaosylceramide antibody (M1/87.27.7) and rat monoclonal B1.1 antibody, as assessed by indirect immunofluorescence and flow cytometry, whereas mouse BMMC did not. The binding of B1.1 antibody to SMC could be blocked by the anti-globopentaosylceramide antibody, and the specificity of B1.1 antibody for globopentaosylceramide was confirmed immunochemically with the use of a solid phase radioimmunoassay. As estimated immunochemically, the amount of globopentaosylceramide in mouse SMC was 62 ng/10(6) cells, whereas BMMC contained less than 8 ng/10(6) cells. Thus, the expression of globopentaosylceramide is a characteristic of the mouse SMC that is lacking in the T cell-dependent BMMC.

Lactic acid (LA) is present in tumors, asthma, and wound healing, environments that also include mast cells and IL-33. The goal of this work is to evaluate how LA modifies IL-33 activation in mast cells. Bone marrow derived mast cells (BMMC) were cultured in the presence of LA and activated with IL-33. BMMC cultured in LA had reduced IL-33-mediated cytokine production. However, LA increased IL-33-mediated VEGF production. Using an MCT1 inhibitor or replacing LA with sodium lactate revealed that LA effects are MCT1 and pH-dependent. Examining IL-33 signaling demonstrated that LA decreased TAK1, JNK, and NFkB phosphorylation, yet increased Akt phosphorylation. An Akt inhibitor blocked VEGF production, but surprisingly enhanced IL-33-mediated cytokine production. Because miR-155 has recently been reported to suppress Akt activation in mast cells, we examined LA effects on miR-155. LA decreased miR-155-5p expression in BMMC. Moreover, BMMC transfected with miR-155-5p were resistant to LA-mediated suppression. Our data demonstrate that lactic acid, present in inflammatory and malignant microenvironments, can alter mast cell behavior to suppress inflammation while increasing angiogenesis.

Mast cells (MCs) play a central role in allergic and inflammatory disorders by rapid degranulation and release of inflammatory mediators upon antigen-driven engagement of the FcεRI. Receptor-mediated MC responses are controlled by the activation of different isoforms of phosphoinositide-3-kinase (PI3K) and the downstream signaling processes. Recent evidence suggests that miRNAs are important molecular players regulating the PI3K/Akt pathway. The role of miR-155 in the regulation of MC functions in vivo was studied in the passive cutaneous anaphylaxis (PCA) MC-dependent model. WT and miR-155(-/-) mice were injected intradermally with anti-DNP-IgE and intravenously with the antigen DNP-HSA. Ear swelling was assessed to evaluate the anaphylactic response. All investigations, to characterize miR-155 specific activities in MCs, were conducted comparing WT and miR-155(-/-) bone marrow-derived MCs (BMMCs). We report that miR-155(-/-) mice display enhanced anaphylaxis reactions. Although miR-155(-/-) BMMCs show normal development, proliferation, and survival, miR-155 deficiency enhances FcεRI-mediated degranulation and release of TNF-α, IL-13, and IL-6. Interestingly, the level of Akt phosphorylation on both of its regulatory residues Thr308 and Ser473 was increased in miR-155(-/-) compared to WT BMMCs. Gene expression profiling showed that miR-155(-/-) BMMCs exhibited significantly increased expression of the adapter PI3Kγ subunits Pik3r5 (p101) and Pik3r6 (p84, p87(PIKAP) ). Furthermore, selective blockade of the PI3Kγ pathway inhibited degranulation in miR-155(-/-) BMMCs. Thus, we suggest that miR-155 plays a critical role in FcεRI-mediated MC responses by modulating components of the PI3Kγ pathway. This newly identified mechanism of miRNA-controlled MC activation may affect the initiation and maintenance of allergic disorders.