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

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.

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

Blood ◽

10.1182/blood.v75.6.1247.bloodjournal7561247 ◽

1990 ◽

Vol 75 (6) ◽

pp. 1247-1251 ◽

Cited By ~ 3

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.

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Clonal assay of mouse mast cell colonies in methylcellulose culture

Blood ◽

10.1182/blood.v60.2.352.bloodjournal602352 ◽

1982 ◽

Vol 60 (2) ◽

pp. 352-361 ◽

Cited By ~ 5

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.

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Clonal assay of mouse mast cell colonies in methylcellulose culture

Blood ◽

10.1182/blood.v60.2.352.352 ◽

1982 ◽

Vol 60 (2) ◽

pp. 352-361 ◽

Cited By ~ 86

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.

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Mast Cell Involvement in Fibrosis in Chronic Graft-Versus-Host Disease

International Journal of Molecular Sciences ◽

10.3390/ijms22052385 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2385

Author(s):

Ethan Strattan ◽

Gerhard Carl Hildebrandt

Keyword(s):

Wound Healing ◽

Mast Cell ◽

Mast Cells ◽

Graft Versus Host Disease ◽

Chronic Gvhd ◽

Fibrotic Disease ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Host Disease ◽

Graft Versus Host

Allogeneic hematopoietic stem cell transplantation (HSCT) is most commonly a treatment for inborn defects of hematopoiesis or acute leukemias. Widespread use of HSCT, a potentially curative therapy, is hampered by onset of graft-versus-host disease (GVHD), classified as either acute or chronic GVHD. While the pathology of acute GVHD is better understood, factors driving GVHD at the cellular and molecular level are less clear. Mast cells are an arm of the immune system that are known for atopic disease. However, studies have demonstrated that they can play important roles in tissue homeostasis and wound healing, and mast cell dysregulation can lead to fibrotic disease. Interestingly, in chronic GVHD, aberrant wound healing mechanisms lead to pathological fibrosis, but the cellular etiology driving this is not well-understood, although some studies have implicated mast cells. Given this novel role, we here review the literature for studies of mast cell involvement in the context of chronic GVHD. While there are few publications on this topic, the papers excellently characterized a niche for mast cells in chronic GVHD. These findings may be extended to other fibrosing diseases in order to better target mast cells or their mediators for treatment of fibrotic disease.

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A liquid culture system for murine megakaryocyte progenitor cells

Blood ◽

10.1182/blood.v59.2.250.bloodjournal592250 ◽

1982 ◽

Vol 59 (2) ◽

pp. 250-257

Author(s):

T Nagasawa ◽

M Nakazawa ◽

T Abe

Keyword(s):

Progenitor Cells ◽

Conditioned Medium ◽

Culture System ◽

Liquid Culture ◽

Culture Medium ◽

Pokeweed Mitogen ◽

Plasma Clot ◽

Liquid Culture System ◽

Cell Conditioned Medium ◽

Number Of Cells

A liquid culture system is described for murine megakaryocyte progenitor cells (CFU-M) in the presence of pokeweed-mitogen-stimulated spleen-cell conditioned medium. There were dose-related responses between the number of CFU-M developed and the number of cells cultured and the dosage of conditioned medium in this liquid culture system. Murine CFU-M were abundantly cloned in this system an the plating efficiency was similar in comparison with that in a plasma clot system. The acetylcholinesterase-positive colonies (more than 4 acetylcholinesterase-positive cells) were clearly seen on day 3 of culture, and they reached a maximum (60.5 +/- 10.7/2 x 10(5) cells) on day 7 of culture. Ultrastructural analyses of megakaryocytic maturation in this system showed that a few megakaryocytes produced platelets that were released in the culture medium on day 5 of culture. This liquid culture system is suitable for the study of the dynamic process of the megakaryocyte-platelet system.

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Development of both human connective tissue-type and mucosal-type mast cells in mice from hematopoietic stem cells with identical distribution pattern to human body

Blood ◽

10.1182/blood-2003-04-1160 ◽

2004 ◽

Vol 103 (3) ◽

pp. 860-867 ◽

Cited By ~ 39

Author(s):

Naotomo Kambe ◽

Hidefumi Hiramatsu ◽

Mika Shimonaka ◽

Hisanori Fujino ◽

Ryuta Nishikomori ◽

...

Keyword(s):

Stem Cells ◽

Mast Cell ◽

Mast Cells ◽

Hematopoietic Stem Cells ◽

Cell Development ◽

Tissue Type ◽

Human Mast Cell ◽

Hematopoietic Stem ◽

Nog Mice

Abstract The transplantation of primitive human cells into sublethally irradiated immune-deficient mice is the well-established in vivo system for the investigation of human hematopoietic stem cell function. Although mast cells are the progeny of hematopoietic stem cells, human mast cell development in mice that underwent human hematopoietic stem cell transplantation has not been reported. Here we report on human mast cell development after xenotransplantation of human hematopoietic stem cells into nonobese diabetic severe combined immunodeficient \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{NOD{/}SCID}){/}{\gamma}_{\mathrm{c}}^{null}\) \end{document} (NOG) mice with severe combined immunodeficiency and interleukin 2 (IL-2) receptor γ-chain allelic mutation. Supported by the murine environment, human mast cell clusters developed in mouse dermis, but they required more time than other forms of human cell reconstitution. In lung and gastric tract, mucosal-type mast cells containing tryptase but lacking chymase located on gastric mucosa and in alveoli, whereas connective tissue-type mast cells containing both tryptase and chymase located on gastric submucosa and around major airways, as in the human body. Mast cell development was also observed in lymph nodes, spleen, and peritoneal cavity but not in the peripheral blood. Xenotransplantation of human hematopoietic stem cells into NOG mice can be expected to result in a highly effective model for the investigation of human mast cell development and function in vivo.

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Involvement of Bruton's Tyrosine Kinase in FcεRI-dependent Mast Cell Degranulation and Cytokine Production

Journal of Experimental Medicine ◽

10.1084/jem.187.8.1235 ◽

1998 ◽

Vol 187 (8) ◽

pp. 1235-1247 ◽

Cited By ~ 165

Author(s):

Daisuke Hata ◽

Yuko Kawakami ◽

Naoki Inagaki ◽

Chris S. Lantz ◽

Toshio Kitamura ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Tyrosine Kinase ◽

Bone Marrow Cells ◽

Late Phase ◽

Cell Development ◽

Mutant Mice ◽

Bruton’S Tyrosine Kinase ◽

Bruton's Tyrosine Kinase ◽

Retroviral Transfer

We investigated the role of Bruton's tyrosine kinase (Btk) in FcεRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcεRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcεRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcεRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed by the improvement in the ability of btk mutant mast cells to degranulate and to secrete cytokines after the retroviral transfer of wild-type btk cDNA, but not of vector or kinase-dead btk cDNA. Retroviral transfer of Emt (= Itk/Tsk), Btk's closest relative, also partially improved the ability of btk mutant mast cells to secrete mediators. Taken together, these results demonstrate an important role for Btk in the full expression of FcεRI signal transduction in mast cells.

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

Blood ◽

10.1182/blood.v108.11.1335.1335 ◽

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

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Failure of W/Wv mouse-derived cultured mast cells to enter S phase upon contact with NIH/3T3 fibroblasts

Blood ◽

10.1182/blood.v72.2.463.463 ◽

1988 ◽

Vol 72 (2) ◽

pp. 463-468 ◽

Cited By ~ 17

Author(s):

J Fujita ◽

H Nakayama ◽

H Onoue ◽

Y Ebi ◽

Y Kanakura ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

Cell Lines ◽

Bone Marrow Cells ◽

Fibroblast Cell ◽

S Phase ◽

Pokeweed Mitogen ◽

Congenic Mice ◽

Fibroblast Cell Lines ◽

Marrow Cells

Abstract Although W/Wv mutant mice are profoundly deficient in tissue mast cells, these mice do have cells with similar features of mast cells that develop from their bone marrow cells as efficiently as those from congenic +/+ mice in pokeweed mitogen-stimulated spleen cell- conditioned medium (PWM-SCM). With cultured mast cells (CMCs), we analyzed the mechanism of mast-cell deficiency in tissues of W/Wv mice. CMCs were established from bone marrow cells of W/Wv and congenic +/+ mice with PWM-SCM, and then co-cultured with various mouse fibroblast cell lines without PWM-SCM. All the examined mouse embryo-derived fibroblast cell lines maintained CMCs derived from +/+ mice, but not CMCs from W/Wv mice, for greater than 2 weeks. Mast cells in S phase were observed only in CMCs derived from +/+ mice under these conditions. The poor survival of W/Wv CMCs as compared with +/+ CMCs was not owing to a differential death rate but to the inability of W/Wv CMCs to continue active proliferation on fibroblasts without PWM-SCM. By synchronizing CMCs at the G1 phase of the cell cycle, the defect in W/Wv CMCs was further characterized as a failure to transit G1 and enter the S phase upon contact with fibroblasts. This finding indicates the indispensable function of the W gene product(s) for this response.

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