scholarly journals Dynamics of Plasma and Granule Membrane in Murine Bone Marrow-Derived Mast Cells after Re-stimulation

2015 ◽  
Vol 8 (1) ◽  
pp. 14-22
Author(s):  
Masahiro Kaneko ◽  
Arisa Yamada
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Plasma Membrane ◽  
Mast Cells ◽  
Morphological Changes ◽  
Membrane Dynamics ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Cell Functions ◽  
Granule Membrane

Mast cells are derived from hematopoietic stem cells and play important roles in allergic responses. Mast cells are long-lived compared with other granular cell types. Since the response of the individual mast cell after FcεRI-induced degranulation is unclear, the aim of this study was to analyze morphological changes in individual mast cells after restimulation. To observe plasma and granule membrane dynamics, AcGFP-actb (β-actin) and DsRed-monomer (DRM)- CD63 fusion constructs were introduced into bone marrow-derived mast cells (BMMCs). Furthermore, AcGFP-CD63 and DRM-Cma1 (mMCP-5) were introduced into BMMCs. Re-stimulation resulted in increased β-hexosaminidase release and cytokine mRNA expression similar to those observed during initial stimulation. Moreover, expression of FcεRI on BMMCs 24 h after initial stimulation was similar to that measured before initial stimulation. Changes in morphology of the plasma membrane and colocalization of granules and plasma membrane were observed after initial stimulation. BMMCs returned to normal 120 min after the initial stimulation. These phenomena were also observed in BMMCs after re-stimulation. BMMC chymase content decreased 20 min after stimulation but returned to near normal 24 h after stimulation. These findings suggest that mast cell functions can be maintained and that these cells can be repeatedly degranulated after FcεRI-mediated stimulation.

scholarly journals Strain difference of murine bone marrow-derived mast cell functions

2005 ◽  
Vol 78 (3) ◽  
pp. 605-611 ◽  
Author(s):  
Junko Noguchi ◽  
Etsushi Kuroda ◽  
Uki Yamashita
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Strain Difference ◽  
Murine Bone Marrow ◽  
Cell Functions

Murine Mast Cells Express Mpl, the Thrombopoietin Receptor, and Thrombopoietin Is a Potent Regulator of Mast Cell Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1335-1335
Author(s):  
Fabrizio Martelli ◽  
Giovanni Amabile ◽  
Barbara Ghinassi ◽  
Rodolfo Lorenzini ◽  
Alessandro M. Vannucchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Mast Cells ◽  
Progenitor Cells ◽  
Peritoneal Cavity ◽  
Surface Protein ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Progenitor Cell Proliferation

Abstract Mast cells are hematopoietic cells localized in extramedullary sites where they engage themselves in the process of allergic response and in the immune reaction against parasites. Mast cells derive from multilineage c-KitlowCD34lowSca-1pos progenitor cells present in the marrow. These cells give rise to Linnegc-KitposSca-1neg T1/ST2pos mast cell restricted progenitor cells (MCP) whose futher maturation in the marrow remains limited under steady state conditions. MCP migrate through the blood in extramedullary sites were they mature into tissue-retricted c-KitposFceRIpos mast cells characterized by a specific mast cell protease (MMCP) profiling (dermal, mucosal and serosal mast cells in skin, gut and peritoneal cavity, respectively). The molecular mechanism that, in normal mice, restricts the mastocytopoietic potential of progenitor cells to the extramedullary sites, as well as the factors that guide the tissue-restricted differentiation of these cells, are unknown. Thrombopoietin (TPO)-Mpl interactions play an important role in the regulation of hematopoietic stem/progenitor cell proliferation and differentiation in the marrow. Here we report that mast cells, and their precursors, express Mpl (both as mRNA and cell surface protein) (see Table). Furthermore, targeted deletion of this gene (Mplnull mutation) decrease the number of MCP (by 1-log) and increases that of mast cells in dermis (by 3-fold), peritoneal cavity (by 3-fold), bone marrow (2-log) and spleen (2-log). Furthermore, because of their higher (by 2-log) MMCP-7 expression, serosal Mplnull mast cells resemble more wild-type dermal rather than serosal mast cells. On the other hand, either treatment of mice with TPO or addition of TPO to bone marrow-derived mast cell cultures induces mast cell apoptosis (by Tunel and Annexin staining) and severely hampers mast cell differentiation (by expression profiling). These data are consistent with a regulatory mechanism for murine mastocytopoiesis according to which TPO favours the transition from multilineage progenitors to CMP but blocks differentiation of MCP to mature mast cells. We propose TPO as the growth factor that restrict mast cell differentiation to extramedullaty sites and that control the switch between serosal vs dermal mast cell differentiation. Mpl expression mRNA 2-ΔCt Protein (AFU) Cy7-A Protein (AFU) Cy7-AMM2 AFU= arbitrary fluorescence intensity. p< 0.01 with respect to Cy7-A (irrilevant antibody) Wild type Marrow B cells (B220pos) b.d. 120±4 205±4 Wild type Marrow Megakaryocytes (CD61pos/CD41pos) 5.0±0.1 × 10-2 178±3 978±74* Wild type Marrow MCP (cKitpos/T1ST2pos) 1.3±0.01 × 10-2 139±16 1658±73* Wild-type Marrow Mast Cells (cKitpos/Fcε RIpos) 1.9±0.1 × 10-2 110±1 868±71* Serosal Mast Cells (cKitpos/FcεRIpos) 7.2±2.1 × 10-4 393±1 1374±25* Mplnull Marrow Megakaryocytes (CD61pos/CD41pos) b.d. 365±28 469±50 Mplnull Marrow Mast Cells (cKitpos/FcεRIpos) b.d 107±1 109±3

Differential Role of Class 1A PI 3-Kinase Regulatory Subunits in Mast Cell Growth, Maturation and Survival.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 77-77
Author(s):  
Raghuveer Mali ◽  
Subha Krishnan ◽  
Ramdas Baskar ◽  
Veerendra Munugalavadla ◽  
Emily Sims ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Bone Marrow Cells ◽  
Full Length ◽  
Receptor Internalization ◽  
Regulatory Subunits ◽  
Growth And Survival ◽  
Cell Functions ◽  
Kit Receptor

Abstract Abstract 77 Stem cell factor (SCF) mediated c-Kit receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from c-Kit are poorly understood. Mast cells express multiple regulatory subunits of class 1A PI 3-kinase including p85α, p85β, p50α, and p55α. While it is known that PI 3-kinase plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including maturation, growth, and survival are not known. Using mice deficient in the expression of p85α or p85β or combination of both p85α/p55α/p50α as well as all four subunits we have examined the role of these subunits in mast cell functions. We show that loss of p85α subunit alone results in impaired bone marrow derived mast cell (BMMC) maturation, growth, and survival compared to wild-type (WT) controls, in spite of the continuous expression of p85β, p55α, and p50α subunits in these cells. Restoring the expression of p85α in p85α deficient mast cells restores the maturation and growth defects. To assess the contribution of p50α and p55α subunits, we generated mice using the Cre lox system that were deficient in the expression of all three subunits (i.e. p85α/p55α/p50α). Deficiency of p85α/p55α/p50α subunits in bone marrow cells completely blocked mast cell maturation and growth, suggesting an essential role for the smaller subunits p50 and p55 in addition to the full length form of p85. Curiously, over-expression of p50α in p85α deficient BMMCs only marginally rescued mast cell maturation and growth, suggesting that the full length form of p85α functions with specificity in regulating mast cell functions. Since the major difference between the shorter isoforms and the full length form of p85α is the absence of the amino terminal SH3 and BH domains, we generated two mutants of p85α lacking either the SH3 or the BH domain and expressed them in p85α−/− BMMCs. While both these mutants completely restored the maturation defect associated with p85α deficiency and showed normal binding to the c-Kit receptor upon SCF stimulation as well as to the p110 catalytic subunits; none of these mutants completely rescued SCF induced proliferation (50% and 70% respectively, n=3, p<0.004). Biochemically, lack of SCF induced growth rescue in p85α−/− BMMCs expressing p85αΔSH3 and p85αΔBH mutants was associated with a lack of rescue in the activation of Akt and Erk, but complete rescue in the activation of JNK (n=3). Consistently, while transplantation of p85α deficient bone marrow cells transduced with p85α into mast cell deficient Wsh mice resulted in complete restoration of gastrointestinal mast cells as well as mast cells in the stomach and spleen, p85αΔSH3 and p85αΔBH mutants restored mast cells only partially. These results indicate that other domains (SH3 and BH) of p85α are required for mast cell growth. In contrast to p85α, deficiency of p85β alone resulted in increased BMMC maturation, growth and survival compared to controls (1.2 fold, n=3, p<0.003). Consistently, over-expression of p85β in WT bone marrow cells resulted in a profound reduction in the maturation of mast cells as well as proliferation. We studied whether reduced maturation and proliferation due to the loss or over-expression of p85β was a result of altered c-Kit receptor internalization and degradation. Our results revealed significantly more c-Kit receptor internalization and degradation in p85β expressing cells compared to p85α expressing cells (2 fold, n=5, p<0.001). Since Cbl family of ubiquitin ligases are involved in the down-regulation of tyrosine kinase receptors, we analyzed whether c-Cbl is involved in p85β mediated c-Kit receptor internalization and degradation. Phosphorylation of c-Cbl and ubiquitination of c-Kit receptor was more in p85β expressing cells compared to p85 expressing cells (n=3). In conclusion, while the current dogma in the field of PI3Kinase signaling suggests that all regulatory subunits of PI3Kinase function in a similar manner; we provide genetic and biochemical evidence to suggest that p85 regulatory subunits differentially regulate growth and maturation of mast cells. Disclosures: Munugalavadla: Genentech: Employment, Patents & Royalties.

scholarly journals Busulfan-induced chronic bone marrow failure: changes in cortical bone, marrow stromal cells, and adherent cell colonies

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1036-1041
Author(s):  
PM McManus ◽  
L Weiss
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Cortical Bone ◽  
Stromal Cells ◽  
Morphological Changes ◽  
Bone Marrow Failure ◽  
Marrow Stromal Cells ◽  
Adherent Cell ◽  
Vitro Assay ◽  
Hematopoietic Stem

This study represented an effort to determine if there were quantitative or morphological changes in marrow stromal cells in busulfan-induced marrow failure and to relate these changes to other disturbances in blood and bone marrow. Mice received four busulfan (BU) injections at two-week intervals and were killed at various time points up to 40 weeks after the first injection. Evaluation techniques included complete blood counts, in vitro assay of short-term adherent cell colonies per femur (STACC per femur) and colony-forming unit- culture (CFU-C) per femur, light microscopy of sternebral marrow and spleen, and electron microscopy (EM) of sternebral marrow taken at 40 weeks. STACC per femur were acutely reduced to 25% of control, but recovered to 76% by 40 weeks. CFU-C per femur dropped to below 10% of control and never recovered. Histologically, we found that hypoplasia of acutely affected marrow was associated with heightened endosteum and cortical bone thickening. In the chronic phase of BU injury, bones became osteoporotic, and the frequency of adipocytes and mast cells rose. BU-affected spleens generally had enhanced erythropoiesis. No stromal cell changes in 40-week marrow were discernible by EM. We concluded that there were morphological changes in BU marrow stroma specifically involving endosteum, bone, adipocytes, and mast cells. Also, there was quantitative depression in stromal cells measured by the STACC assay, but this improved substantially with time, unlike damage to hematopoietic stem cells measured by the CFU-C.

scholarly journals Wnt-β-Catenin Signaling Promotes the Maturation of Mast Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Tomoko Yamaguchi ◽  
Misae Nishijima ◽  
Katsuhisa Tashiro ◽  
Kenji Kawabata
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Wnt Signaling ◽  
Molecular Mechanisms ◽  
Histidine Decarboxylase ◽  
Wnt Signaling Pathway ◽  
Hematopoietic Stem ◽  
Peripheral Tissues ◽  
Wnt Proteins

Mast cells play an important role in the pathogenesis of allergic diseases. Immature mast cells migrate into peripheral tissues from the bone marrow and undergo complete maturation. Interestingly, mast cells have characteristics similar to hematopoietic stem cells (HSCs), such as self-renewal and c-kit expression. In HSCs, Wnt signaling is involved in their maintenance and differentiation. On the other hand, the relation between Wnt signaling and mast cell differentiation is poorly understood. To study whether Wnt signals play a role in the maturation of mast cells, we studied the effect of Wnt proteins on mast cell maturation of bone marrow-derived mast cells (BMMCs). The expression levels of CD81 protein and histidine decarboxylase mRNA and activity of mast cell-specific protease were all elevated in BMMCs treated with Wnt5a. In addition, Wnt5a induced the expression of Axin2 and TCF mRNA in BMMCs. These results showed that Wnt5a could promote the maturation of mast cells via the canonical Wnt signaling pathway and provide important insights into the molecular mechanisms underlying the differentiation of mast cells.

scholarly journals Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors

2005 ◽  
Vol 201 (12) ◽  
pp. 1961-1971 ◽  
Author(s):  
Charlotte L. Weller ◽  
Sarah J. Collington ◽  
Jeremy K. Brown ◽  
Hugh R.P. Miller ◽  
Adam Al-Kashi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
The Body ◽  
Active Principle ◽  
Human Cord Blood ◽  
Hematopoietic Stem

Mast cells are tissue-resident cells with important functions in allergy and inflammation. Pluripotential hematopoietic stem cells in the bone marrow give rise to committed mast cell progenitors that transit via the blood to tissues throughout the body, where they mature. Knowledge is limited about the factors that release mast cell progenitors from the bone marrow or recruit them to remote tissues. Mouse femoral bone marrow cells were cultured with IL-3 for 2 wk and a range of chemotactic agents were tested on the c-kit+ population. Cells were remarkably refractory and no chemotaxis was induced by any chemokines tested. However, supernatants from activated mature mast cells induced pronounced chemotaxis, with the active principle identified as leukotriene (LT) B4. Other activation products were inactive. LTB4 was highly chemotactic for 2-wk-old cells, but not mature cells, correlating with a loss of mRNA for the LTB4 receptor, BLT1. Immature cells also accumulated in vivo in response to intradermally injected LTB4. Furthermore, LTB4 was highly potent in attracting mast cell progenitors from freshly isolated bone marrow cell suspensions. Finally, LTB4 was a potent chemoattractant for human cord blood–derived immature, but not mature, mast cells. These results suggest an autocrine role for LTB4 in regulating tissue mast cell numbers.

scholarly journals Plasma membrane folds on the mast cell surface and their relationship to secretory activity.

1977 ◽  
Vol 74 (3) ◽  
pp. 690-697 ◽  
Author(s):  
S J Burwen ◽  
B H Satir
Keyword(s):  
Mast Cell ◽  
Plasma Membrane ◽  
Mast Cells ◽  
Cell Surface ◽  
Normal Population ◽  
Secretory Activity ◽  
Cell Type ◽  
Distinct Cell Type ◽  
Granule Membrane ◽  
Fold Formation

Changes in the surface morphology of secreting mast cells have been followed by scanning electron microscopy. Mast cells isolated from the rat peritoneal cavity have folds of plasma membrane that form snake-like ridges on their surfaces. Fold length varies considerably from cell to cell, whereas fold width and depth appear to remain relatively constant. To assess the possible relationship between secretory activity and surface folding, a seimquantitative method was used for measuring fold length in control and secreting populations. A positive correlation is found between secretion of histamine and the extent of membrane folds on the mast cell surface. The source of the membrane required for fold formation is probably secretory granule membrane incorporated into the plasma membranene as a result of exocytosis. Furthermore, a distinct cell type devoid of surface folds, designated as a raspberry-type cell, is found to occur as an integral part of a normal population of mast cells. This cell type is resistant to stimulation by polymyxin.

scholarly journals Loss of Dnmt3a Dysregulates Hematopoietic Homeostasis and Myeloid Skewing Via the PI3Kinase Pathway: PI3Kα/β Inhibition Corrects Extramedullary Hematopoiesis and Myeloid Skewing Due to Loss of Dnmt3a

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 723-723
Author(s):  
Lakshmi Reddy Palam ◽  
Baskar Ramdas ◽  
Katelyn Pickerell ◽  
Reuben Kapur
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Transcriptome Analysis ◽  
Extramedullary Hematopoiesis ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Cell Functions ◽  
Genome Wide ◽  
Erythroid Development ◽  
Inhibitor Treatment

Mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with myeloid malignancies including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN) including systemic mastocytosis (SM). Presence of this mutation is associated with poor prognosis and reduced overall patient survival. Earlier studies have shown that hematopoietic specific loss of Dnmt3a in mice results in lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by extramedullary hematopoiesis, peripheral cytopenias, splenomegaly and hepatomegaly. However, it's unclear how DNMT3A mutations contribute to myeloid skewing, and induce myeloid malignancies. Further, the mechanism(s) by which loss of DNMT3A impairs various mast cell functions leading to mastocytosis are also not defined. We show that loss of Dnmt3a in vitro results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors and in vivo results in increased number of mast cells. Genome wide transcriptome analysis revealed elevated expression of the transcription factor MITF and a profound repression in the expression of C/EBPα in BM precursors derived from Dnmt3a-/- mice. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with correction in levels of MITF and C/EBPα, suggesting that early loss of C/EBPα in Dnmt3a-/- cells contributes to enhanced maturation of mast cells from its precursors. Furthermore, biochemical analysis revealed greater PI3Kinase activation in Dnmt3a-/- cells, and these findings were supported by genome wide transcriptome analysis. Importantly, mast cells derived from Dnmt3a-/- mice lacking the expression of p85α regulatory subunit of PI3Kinase or pharmacologic inhibition of PI3Kinase completely corrected cytokine induced hyperproliferation in these cells to near normal levels. Insights from mast cell studies led us to hypothesize that the aberrant hematopoietic regulation in Dnmt3a-/- mice described above may be due to hyperactivation of the PI3Kinase signaling in hematopoietic stem and progenitors (HSCPs). We investigated the role of PI3Kinase signaling in Dnmt3a loss induced myeloid malignancy using pharmacological inhibitor, GDC0941 (PI3Kα/δ inhibitor) and BAY1082439 (PI3Kα/β inhibitor). PI3Kα/β inhibitor treatment of Dnmt3a-/- mice markedly reduced monocytosis, neutrophilia, enhanced WBC counts, and improved RBCs, hematocrits and platelet numbers compared to control mice. In contrast, PI3Kα/δ inhibition moderately corrected the monocytosis and WBC aberrancy, however correction in erythroid dysregulation was not observed. Inhibiting PI3Kα/β signaling dramatically reduced splenomegaly and hepatomegaly in Dnmt3a-/- mice. In contrast, PI3Kα/δ inhibition moderately reduced the spleen size, however the correction in hepatomegaly was insignificant. Importantly, clonal hematopoiesis due to loss of Dnmt3a in a competitive transplant setting was rescued by inhibiting the PI3Kinase pathway. In the BM, PI3Kα/β inhibition moderately decreased LSK cells, however a marked decrease in GMP, and significant increase in MEP, and CLP was observed compared to controls. Flow cytometry analysis revealed that PI3Kα/β inhibition reduced Dnmt3a-/- BM cell migration to liver, and completely corrected the extramedullary hematopoiesis in the spleen compared to controls. Further, Dnmt3a loss induced alteration in erythroid development in the BM, spleen and PB was corrected upon PI3Kα/β blockade compared to controls. Genome wide transcriptome analysis revealed that PI3Kα/β inhibition markedly reduced the expression of GMP related genes, fetal liver hematopoiesis transcription program, and expression levels of inflammatory cytokines, growth factors, and chemokines in Dnmt3a depleted malignant mice. However PI3Kα/β inhibition increased the expression of genes involved in erythroid development in Dnmt3a-/- mice compared to controls. These results demonstrate that Dnmt3a ablation induces liver specific expansion of hematopoietic cells, and extramedullary hematopoiesis in spleen via aberrant activation of PI3Kinase signaling in HSCPs. Consistent with this notion, PI3K inhibitor treatment of malignant Dnmt3a-/- bearing mice showed significantly improved overall survival compared to controls. Disclosures No relevant conflicts of interest to declare.

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