scholarly journals Clonal assay of mouse mast cell colonies in methylcellulose culture

Blood ◽  
1982 ◽  
Vol 60 (2) ◽  
pp. 352-361 ◽  
Author(s):  
T Nakahata ◽  
SS Spicer ◽  
JR Cantey ◽  
M Ogawa
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Time Course ◽  
Membrane Receptors ◽  
Pokeweed Mitogen ◽  
Spleen Cells ◽  
Transmission Electron ◽  
Blast Cells ◽  
Mouse Mast Cell

Abstract When mouse marrow and spleen cells were cultured for over 12 days in methylcellulose containing media conditioned by pokeweed-mitogen- stimulated spleen cells, colonies containing mast cells and blast cells were observed. The characteristic morphology of the colonies and the time course of their development allowed in situ identification of the mast cell colonies. Identification of the mast cells was confirmed by metachromatic staining with toluidine blue and alcian blue, transmission electron microscopy, and by demonstration of the membrane receptors for IgE. Coculture studies with male and female marrow cells strongly indicated the single cell origin of individual colonies. Detailed cytologic analyses of mixed hemopoietic colonies and replating experiments of individual mixed hemopoietic and mast cell colonies clearly established the hemopoietic origin of mast cells. In replating experiments of individual mast cell colonies, those without blast cells did not yield secondary mast cell colonies. This result strongly indicated that morphologically recognizable mast cells have lost their self-renewing capabilities. The quantitative nature of the mast cell colony assay was supported by linearity studies and provides a method for studies of the progenitors of mouse mast cells.

scholarly journals Clonal assay of mouse mast cell colonies in methylcellulose culture

Blood ◽  
1982 ◽  
Vol 60 (2) ◽  
pp. 352-361 ◽  
Author(s):  
T Nakahata ◽  
SS Spicer ◽  
JR Cantey ◽  
M Ogawa
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Time Course ◽  
Membrane Receptors ◽  
Pokeweed Mitogen ◽  
Spleen Cells ◽  
Transmission Electron ◽  
Blast Cells ◽  
Mouse Mast Cell

When mouse marrow and spleen cells were cultured for over 12 days in methylcellulose containing media conditioned by pokeweed-mitogen- stimulated spleen cells, colonies containing mast cells and blast cells were observed. The characteristic morphology of the colonies and the time course of their development allowed in situ identification of the mast cell colonies. Identification of the mast cells was confirmed by metachromatic staining with toluidine blue and alcian blue, transmission electron microscopy, and by demonstration of the membrane receptors for IgE. Coculture studies with male and female marrow cells strongly indicated the single cell origin of individual colonies. Detailed cytologic analyses of mixed hemopoietic colonies and replating experiments of individual mixed hemopoietic and mast cell colonies clearly established the hemopoietic origin of mast cells. In replating experiments of individual mast cell colonies, those without blast cells did not yield secondary mast cell colonies. This result strongly indicated that morphologically recognizable mast cells have lost their self-renewing capabilities. The quantitative nature of the mast cell colony assay was supported by linearity studies and provides a method for studies of the progenitors of mouse mast cells.

scholarly journals Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1247-1251 ◽  
Author(s):  
Y Ebi ◽  
T Kasugai ◽  
Y Seino ◽  
H Onoue ◽  
T Kanemoto ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Culture Medium ◽  
Fibroblast Cell ◽  
Mutant Mice ◽  
Pokeweed Mitogen ◽  
Spleen Cells ◽  
Hematopoietic Stem ◽  
Nih 3T3 ◽  
Cell Conditioned Medium

Abstract Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.

scholarly journals Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1247-1251 ◽  
Author(s):  
Y Ebi ◽  
T Kasugai ◽  
Y Seino ◽  
H Onoue ◽  
T Kanemoto ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Culture Medium ◽  
Fibroblast Cell ◽  
Mutant Mice ◽  
Pokeweed Mitogen ◽  
Spleen Cells ◽  
Hematopoietic Stem ◽  
Nih 3T3 ◽  
Cell Conditioned Medium

Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.

Magnesium and sulfur in mast cell and basophil granules of lower vertebrates in relation with histamine

1992 ◽  
Vol 50 (2) ◽  
pp. 1596-1597
Author(s):  
Kenichi Takaya
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Mast Cell ◽  
Mast Cells ◽  
Tree Frog ◽  
X Ray ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Fresh Frozen ◽  
Ultrathin Sections

Mast cell and basophil granules of the vertebrate contain heparin or related sulfated proteoglycans. Histamine is also present in mammalian mast cells and basophils. However, no histamine is detected in mast cell granules of the amphibian or fish, while it is shown in those of reptiles and birds A quantitative x-ray microanalysis of mast cell granules of fresh frozen dried ultrathin sections of the tongue of Wistar rats and tree frogs disclosed high concentrations of sulfur in rat mast cell granules and those of sulfur and magnesium in the tree frog granules. Their concentrations in tree frog mast cell granules were closely correlated (r=0.94).Fresh frozen dried ultrathin sections and fresh air-dried prints of the tree frog tongue and spleen and young red-eared turtle (ca. 6 g) spleen and heart blood were examined by a quantitative energy-dispersive x-ray microanalysis (X-650, Kevex-7000) for the element constituents of the granules of mast cells and basophils. The specimens were observed by transmission electron microscopy (TEM) (80-200 kV) and followed by scanning transmission electron microscopy (STEM) under an analytical electron microscope (X-650) at an acceleration voltage of 40 kV and a specimen current of 0.2 nA. A spot analysis was performed in a STEM mode for 100 s at a specimen current of 2 nA on the mast cell and basophil granules and other areas of the cells. Histamine was examined by the o-phthalaldehyde method.

scholarly journals Biochemical and immunological characterization of multiple glycoforms of mouse mast cell protease 1: comparison with an isolated murine serosal mast cell protease (MMCP-4)

1993 ◽  
Vol 294 (1) ◽  
pp. 127-135 ◽  
Author(s):  
G F J Newlands ◽  
D P Knox ◽  
S R Pirie-Shepherd ◽  
H R P Miller
Keyword(s):  
Mast Cell ◽  
Amino Acid ◽  
Mast Cells ◽  
Western Blotting ◽  
Trichinella Spiralis ◽  
Double Diffusion ◽  
Terminal Amino Acid ◽  
Mast Cell Protease ◽  
Terminal Amino ◽  
Mouse Mast Cell

Five highly soluble, chymotrypsin-like, neutral serine proteases, with molecular masses in the range 30-33 kDa, were isolated from Trichinella spiralis-infected mouse small intestine. These enzymes were closely related antigenically on Western blotting and by Ouchterlony double diffusion using a polyclonal, cross-absorbed, sheep antibody raised against mouse mast cell protease-1 (MMCP-1) and on the basis of N-terminal amino acid sequence analysis, were identified as variant forms of MMCP-1. Substrate and inhibitor analysis confirmed that the five variants (MMCP-1 A-E) had similar characteristics, although highly significant (P = 0.025 to P < 0.0001) variations in Km and kcat, were detected. Against human alpha 1-proteinase inhibitor the Ki for MMCP-1C (45 pM) was significantly (P < 0.0001) greater than those for the other proteases (0.76-2.2 pM). The differences in electrophoretic mobility are probably a result of variable glycosylation, since removal of N-linked carbohydrate produced a polypeptide of approx. 28 kDa in each case which was, like the native enzyme, immunoreactive on Western blotting. A much less soluble 28 kDa enzyme was isolated from serosal mast cells and identified as MMCP-4 by N-terminal amino acid sequencing. Like MMCP-1 it has chymotrypsin-like substrate specificities with activity at neutral pH. However, it was antigenically distinct from MMCP-1 and, using sheep anti-MMCP-1, was not detected on Western blotting or by Ouchterlony double diffusion, e.l.i.s.a. or immunohistochemistry. This last technique established that the MMCP-1 variants were uniquely present in enteric mast cells, thereby providing a highly selective means of distinguishing the mucosal and connective tissue mast cell subsets in the mouse.

scholarly journals Low c-kit expression of cultured mast cells of mi/mi genotype may be involved in their defective responses to fibroblasts that express the ligand for c-kit

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1454-1462 ◽  
Author(s):  
Y Ebi ◽  
Y Kanakura ◽  
T Jippo-Kanemoto ◽  
T Tsujimura ◽  
T Furitsu ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tyrosine Phosphorylation ◽  
Messenger Rna ◽  
Tissue Type ◽  
Phenotypic Change ◽  
3T3 Fibroblasts ◽  
Mouse Mast Cell ◽  
And Function

Abstract Mutant mice of mi/mi genotype are osteopetrotic and deficient in tissue mast cells due to a defect in osteoclasts and mast cells. In an effort to further understand the mechanisms behind why mi/mi mouse-derived cultured mast cells (mi/mi-CMC) responded to interleukin-3 (IL-3), but not to the proliferative stimuli presented by fibroblasts, mi/mi-CMC and congenic normal (+/+) mouse-derived CMC (+/+-CMC), both of which expressed the phenotypic characteristics of immature mast cells, were cocultured with Swiss albino/3T3 fibroblasts in a medium containing IL- 3. In the in vitro CMC/fibroblast coculture, mi/mi-CMC did not acquire the phenotypes of connective tissue-type mast cells (CTMC), while +/+- CMC did. In addition, attachment of mi/mi-CMC to the fibroblasts was found to be significantly lower than that of +/+-CMC. Because the interaction of c-kit product with its ligand (stem cell factor [SCF]) is known to play an important role not only in proliferation and differentiation of mast cells but also in attachment of CMC to fibroblasts, the expression and function of c-kit were investigated in mi/mi-CMC and +/+-CMC. Recombinant rat SCF (rrSCF164) induced a dose- dependent proliferation of +/+-CMC. Also, rrSCF164 induced +/+-CMC to acquire the phenotypes of CTMC in the medium containing IL-3. By contrast, rrSCF164 did not stimulate the proliferation of mi/mi-CMC nor induce a phenotypic change of the cells from immature mast cells to mature, CTMC-like mast cells. Immunoblotting with antiphosphotyrosine antibody showed that rrSCF164 induced considerable tyrosine phosphorylation of 145- to 165-Kd protein, the product of c-kit, in +/+- CMC, whereas tyrosine phosphorylation of the protein was barely detectable in mi/mi-CMC. Northern blot and flow cytometry analyses showed that mi/mi-CMC expressed much less c-kit at both protein and message levels than +/+-CMC. Further, mi/mi-CMC were found to differ from +/+-CMC in the expression of mouse mast cell protease-6 (MMCP-6) and MMCP-2 messenger RNA transcripts. These results suggest that the gene product of the mi locus may be important in regulating the expression of gene products such as c-kit, and that mast cell deficiency of mi/mi mice appears to be due, at least in part, to impaired signaling through the c-kit receptor because of the low c-kit expression.

scholarly journals IgE Enhances Mouse Mast Cell FcεRI Expression In Vitro and In Vivo: Evidence for a Novel Amplification Mechanism in IgE-dependent Reactions

1997 ◽  
Vol 185 (4) ◽  
pp. 663-672 ◽  
Author(s):  
Masao Yamaguchi ◽  
Chris S. Lantz ◽  
Hans C. Oettgen ◽  
Ildy M. Katona ◽  
Tony Fleming ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Activation ◽  
Immunoglobulin E ◽  
Specific Antigen ◽  
Mast Cell Activation ◽  
Proinflammatory Mediators ◽  
Mouse Mast Cell

The binding of immunoglobulin E (IgE) to high affinity IgE receptors (FcεRI) expressed on the surface of mast cells primes these cells to secrete, upon subsequent exposure to specific antigen, a panel of proinflammatory mediators, which includes cytokines that can also have immunoregulatory activities. This IgE- and antigen-specific mast cell activation and mediator production is thought to be critical to the pathogenesis of allergic disorders, such as anaphylaxis and asthma, and also contributes to host defense against parasites. We now report that exposure to IgE results in a striking (up to 32-fold) upregulation of surface expression of FcεRI on mouse mast cells in vitro or in vivo. Moreover, baseline levels of FcεRI expression on peritoneal mast cells from genetically IgE-deficient (IgE −/−) mice are dramatically reduced (by ∼83%) compared with those on cells from the corresponding normal mice. In vitro studies indicate that the IgE-dependent upregulation of mouse mast cell FcεRI expression has two components: an early cycloheximide-insensitive phase, followed by a later and more sustained component that is highly sensitive to inhibition by cycloheximide. In turn, IgE-dependent upregulation of FcεRI expression significantly enhances the ability of mouse mast cells to release serotonin, interleukin-6 (IL-6), and IL-4 in response to challenge with IgE and specific antigen. The demonstration that IgE-dependent enhancement of mast cell FcεRI expression permits mast cells to respond to antigen challenge with increased production of proinflammatory and immunoregulatory mediators provides new insights into both the pathogenesis of allergic diseases and the regulation of protective host responses to parasites.

scholarly journals Fate of two mast cell tryptases in V3 mastocytosis and normal BALB/c mice undergoing passive systemic anaphylaxis: prolonged retention of exocytosed mMCP-6 in connective tissues, and rapid accumulation of enzymatically active mMCP-7 in the blood.

1996 ◽  
Vol 184 (3) ◽  
pp. 1061-1073 ◽  
Author(s):  
N Ghildyal ◽  
D S Friend ◽  
R L Stevens ◽  
K F Austen ◽  
C Huang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Mast Cell ◽  
Mast Cells ◽  
Chromosome 17 ◽  
Macromolecular Complex ◽  
Connective Tissues ◽  
Systemic Anaphylaxis ◽  
Rich Domain ◽  
Passive Systemic Anaphylaxis ◽  
Mouse Mast Cell

The mouse mast cell protease granule tryptases designated mMCP-6 and mMCP-7 are encoded by highly homologous genes that reside on chromosome 17. Because these proteases are released when mast cells are activated, we sought a basis for distinctive functions by examining their fates in mice undergoing passive systemic anaphylaxis. 10 min-1 h after antigen (Ag) was administered to immunoglobulin (Ig)E-sensitized mice, numerous protease/proteoglycan macromolecular complexes appeared in the extracellular matrix adjacent to most tongue and heart mast cells of normal BALB/c mice and most spleen and liver mast cells of V3 mastocytosis mice. These complexes could be intensively stained by anti-mMCP-6 Ig but not by anti-mMCP-7 Ig. Shortly after Ag challenge of V3 mastocytosis mice, large amounts of properly folded, enzymatically active mMCP-7 were detected in the plasma. This plasma-localized tryptase was approximately 150 kD in its multimeric state and approximately 32 kD in its monomeric state, possessed an NH2 terminus identical to that of mature mMCP-7, and was not covalently bound to any protease inhibitor. Comparative protein modeling and electrostatic calculations disclosed that mMCP-6 contains a prominent Lys/Arg-rich domain on its surface, distant from the active site. The absence of this domain in mMCP-7 provides an explanation for its selective dissociation from the exocytosed macromolecular complex. The retention of exocytosed mMCP-6 in the extracellular matrix around activated tissue mast cells suggests a local action. In contrast, the rapid dissipation of mMCP-7 from granule cores and its inability to be inactivated by circulating protease inhibitors suggests that this tryptase cleaves proteins located at more distal sites.

scholarly journals Mast cells protect from post-traumatic spinal cord damage in mice by degrading inflammation-associated cytokines via mouse mast cell protease 4

2014 ◽  
Vol 62 ◽  
pp. 260-272 ◽  
Author(s):  
Sofie Nelissen ◽  
Tim Vangansewinkel ◽  
Nathalie Geurts ◽  
Lies Geboes ◽  
Evi Lemmens ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Mast Cell ◽  
Mast Cells ◽  
Mast Cell Protease ◽  
Spinal Cord Damage ◽  
Post Traumatic ◽  
Mouse Mast Cell

scholarly journals Abundant expression of transcription factor GATA-2 in proliferating but not in differentiated mast cells in tissues of mice: demonstration by in situ hybridization

Blood ◽  
1996 ◽  
Vol 87 (3) ◽  
pp. 993-998 ◽  
Author(s):  
T Jippo ◽  
H Mizuno ◽  
Z Xu ◽  
S Nomura ◽  
M Yamamoto ◽  
...  
Keyword(s):  
Mast Cell ◽  
In Situ Hybridization ◽  
Mast Cells ◽  
Differentiation Markers ◽  
Two Parameters ◽  
High Affinity Ige Receptor ◽  
Relationship Of ◽  
The Relationship ◽  
Receptor Beta

Although GATA-binding transcription factors (GATA-1 and GATA-2) are strongly expressed in cultured mast cells (CMCs), their expression in mast cells within tissues has not been reported. We examined the expression of GATA-1 and GATA-2 in skin tissues of mice using Northern blot analysis and in situ hybridization. mRNA for GATA-2 but not for GATA-1 was expressed in skin mast cells of WB-+/+ embryos between days 15 and 17 postcoitum (pc). The expression was downregulated on and after day 18 pc. Skin mast cells did not express GATA-2 after birth either. When the number of skin mast cells was compared with the number of GATA-2 mRNA-expressing cells, GATA-2 mRNA appeared to be expressed by mast cells only when the number was increasing. When the mRNA expression of high-affinity IgE receptor beta-subunit and mast cell carboxypeptidase A was used as differentiation markers, the expression of these mRNAs continued even after the downregulation of GATA-2 expression. To clarify the relationship of the proliferation and GATA-2 expression, proliferating CMCs derived from WBB6F1-+/+ mice were transplanted into the peritoneal cavity of mast cell-deficient WBB6F1- W/Wv mice. The CMCs stopped both the proliferation and GATA-2 expression after the transplantation, suggesting the association of these two parameters in mast cells within tissues of mice.

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