Evaluation of Mast Cells in Myeloproliferative Disorders and Myelodysplastic Syndromes

Abstract Context.—Mast cells may be increased as a reactive mastocytosis in various hematologic disorders and malignant neoplasms, as well as in systemic mast cell disease (SMCD). There are no statistical differences in mast cell numbers in reactive mastocytosis and SMCD; however, SMCD usually reveals dyspoietic mast cells and other dyspoietic bone marrow elements. In addition, SMCD is frequently (45%) associated with myeloproliferative disorders (MPDs) (17%) and myelodysplastic syndromes (MDSs) (28%). Thus, it has been suggested that SMCD may represent one aspect of a hematologic disorder that involves multiple bone marrow lineages. Objective.—To perform a systematic evaluation of MPDs and MDSs without SMCD for dyspoietic mast cells. Design.—A total of 55 MPDs or MDSs were reviewed, including 20 cytogenetically proven chronic myeloid leukemias, 6 essential thrombocythemias, 2 polycythemia veras, 21 cytogenetically proven MDSs, and 6 chronic myelomonocytic leukemias. Cases of idiopathic myelofibrosis were not included due to lack of spicules. The bone marrow aspirates were reviewed for an increase in mast cells (1+ to 3+), dyspoietic features within mast cells (decreased cytoplasmic granularity, uneven granule distribution), and a predominance of fusiform mast cells. Results.—All cases, except 2 MDSs, had evaluable bone marrow spicules. Of interest, the MPDs were significantly more associated with increased and dyspoietic mast cells (57% and 61%, respectively) than were the MDSs (11% and 4%, respectively). The 2 polycythemia veras and 6 chronic myelomonocytic leukemias did not reveal increased or dyspoietic mast cells. Conclusions.—These findings indicate that MPDs (chronic myeloid leukemia and essential thrombocythemia) frequently contain neoplastic mast cells as the spectrum of abnormal bone marrow cells. This feature, in conjunction with other parameters, may possibly be useful in the differential diagnosis of MPDs and MDSs. Our findings, compared with the previously reported findings in SMCD, suggest that SMCD may be more closely related to MPDs than to MDSs.

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Development of a lethal mast cell disease in mice reconstituted with bone marrow cells expressing the v-erbB oncogene

Blood ◽

10.1182/blood.v79.12.3145.3145 ◽

1992 ◽

Vol 79 (12) ◽

pp. 3145-3158 ◽

Cited By ~ 2

Author(s):

T von Ruden ◽

S Kandels ◽

T Radaszkiewicz ◽

A Ullrich ◽

EF Wagner

Keyword(s):

Bone Marrow ◽

Animal Model ◽

Mast Cell ◽

Mast Cells ◽

Bone Marrow Cells ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

Cell Disease ◽

Mast Cell Disease ◽

Marrow Cells

Abstract An animal model for malignant mastocytosis is described in mice reconstituted with bone marrow cells expressing the v-erbB oncogene. The lethal mast cell disease is characterized by massive infiltration of bone marrow, spleen, and several other visceral organs by connective tissue mast cells, which normally reside in the skin and the peritoneal cavity. As is frequently found in malignant mastocytosis, the v-erbB- induced mast cell disease was accompanied in some primary recipients by an acute myelogenous leukemia (AML) that killed all secondary recipients regardless of whether the AML was already evident in the primary host. The infiltrating mast cells stained strongly positive with berberine sulfate, suggesting that they were terminally differentiated and in vitro they showed only a weak proliferative capacity. The leukemias were clonal but apparently of different origin than the malignant mast cells, implying the transformation of two independent cell populations. Leukemic cells expressed various myeloid- specific markers as well as the B220 antigen, normally associated with the B-cell lineage. However, the Ig heavy chain genes were still in germ line configuration. In culture, these cells proliferated in the absence of exogenous growth factors and had the capacity to differentiate into mature myeloid cells. Preliminary experiments suggest that v-erbB may use parts of a signal transduction pathway normally coupled to the c-kit receptor. The v-erbB-induced malignant mast cell disease should provide a useful animal model for elucidating the cause for malignant mastocytosis in humans and to explore possible therapeutic strategies.

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Development of a lethal mast cell disease in mice reconstituted with bone marrow cells expressing the v-erbB oncogene

Blood ◽

10.1182/blood.v79.12.3145.bloodjournal79123145 ◽

1992 ◽

Vol 79 (12) ◽

pp. 3145-3158 ◽

Cited By ~ 9

Author(s):

T von Ruden ◽

S Kandels ◽

T Radaszkiewicz ◽

A Ullrich ◽

EF Wagner

Keyword(s):

Bone Marrow ◽

Animal Model ◽

Mast Cell ◽

Mast Cells ◽

Bone Marrow Cells ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

Cell Disease ◽

Mast Cell Disease ◽

Marrow Cells

An animal model for malignant mastocytosis is described in mice reconstituted with bone marrow cells expressing the v-erbB oncogene. The lethal mast cell disease is characterized by massive infiltration of bone marrow, spleen, and several other visceral organs by connective tissue mast cells, which normally reside in the skin and the peritoneal cavity. As is frequently found in malignant mastocytosis, the v-erbB- induced mast cell disease was accompanied in some primary recipients by an acute myelogenous leukemia (AML) that killed all secondary recipients regardless of whether the AML was already evident in the primary host. The infiltrating mast cells stained strongly positive with berberine sulfate, suggesting that they were terminally differentiated and in vitro they showed only a weak proliferative capacity. The leukemias were clonal but apparently of different origin than the malignant mast cells, implying the transformation of two independent cell populations. Leukemic cells expressed various myeloid- specific markers as well as the B220 antigen, normally associated with the B-cell lineage. However, the Ig heavy chain genes were still in germ line configuration. In culture, these cells proliferated in the absence of exogenous growth factors and had the capacity to differentiate into mature myeloid cells. Preliminary experiments suggest that v-erbB may use parts of a signal transduction pathway normally coupled to the c-kit receptor. The v-erbB-induced malignant mast cell disease should provide a useful animal model for elucidating the cause for malignant mastocytosis in humans and to explore possible therapeutic strategies.

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Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

Blood ◽

10.1182/blood.v71.3.573.573 ◽

1988 ◽

Vol 71 (3) ◽

pp. 573-580 ◽

Cited By ~ 25

Author(s):

Y Kanakura ◽

A Kuriu ◽

N Waki ◽

T Nakano ◽

H Asai ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Peritoneal Cavity ◽

Bone Marrow Cells ◽

Water Injection ◽

Lymphoid Cells ◽

Distilled Water ◽

Peritoneal Mast Cells ◽

Chediak Higashi Syndrome

Abstract Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. “Large” mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas “medium” and “small” mast cell colonies are produced by morphologically identifiable mast cells (M-CFU- Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU- Mast from the bloodstream and to inhibit the differentiation of L-CFU- Mast to M-CFU-Mast.

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Rescue of W-associated mast cell defects in W/Wv bone marrow cells by ectopic expression of normal and mutant epidermal growth factor receptors

Blood ◽

10.1182/blood.v82.5.1463.1463 ◽

1993 ◽

Vol 82 (5) ◽

pp. 1463-1470

Author(s):

T von Ruden ◽

L Stingl ◽

A Ullrich ◽

EF Wagner

Keyword(s):

Signal Transduction ◽

Bone Marrow ◽

Mast Cell ◽

Growth Factor ◽

Mast Cells ◽

Epidermal Growth Factor ◽

Bone Marrow Cells ◽

Ectopic Expression ◽

Tissue Type ◽

Epidermal Growth

Abstract The normal human epidermal growth factor receptor (EGF-R) (HERc), a chimeric EGF-R/v-erbB (HERerbB) receptor, and the ligand-independent oncogenic EGF-R variant (v-erbB) were used to correct the mast cell defects in W/Wv bone marrow (BM) cells. In culture, all three receptor molecules transduced functional mitogenic signals in infected interleukin-3 (IL-3)-dependent bone marrow-derived mast cells (BMMCs) and enabled their differentiation into safranin-positive mast cells resembling connective tissue-type mast cells (CTMCs). Furthermore, expression of these receptors restored the capacity of W/Wv BMMCs to colonize the peritoneal cavity of mast cell-deficient W/Wv mice where they differentiated to safranin-positive cells with similar frequencies as wild-type BMMCs. These experiments show that expression of normal and mutant EGF-Rs in W/Wv BM cells is able to complement the function of the c-kit-encoded Steel factor receptor (SLF-R) in mast cell development. We conclude that signal transduction by normal and mutant EGF-Rs in murine hematopoietic cells apparently involves components also used by the SLF-R, which suggests that these receptors use overlapping pathways for signal transduction.

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Kit Signaling Regulates Mitf Expression in Mastocytosis.

Blood ◽

10.1182/blood.v108.11.3601.3601 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3601-3601

Author(s):

Youl-Nam Lee ◽

Pierre Noel ◽

Amir Shahlaee ◽

Melody Carter ◽

Reuben Kapur ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Systemic Mastocytosis ◽

Tyrosine Kinase Receptor ◽

Bone Marrow Biopsies ◽

Activating Mutations ◽

Cell Assays ◽

Mast Cell Disease ◽

Mitf Expression

Abstract Mastocytosis is a heterogeneous disease arising from abnormal proliferation of mast cells. Activating mutations in codon D816 of the tyrosine kinase receptor, c-kit, are found in the majority of adult patients with systemic mastocytosis, an aggressive form of the disease. Constitutive activation of the Kit signaling pathway is critical to the transformed phenotype, and thus understanding how this pathway regulates downstream events is of great importance. A number of transcription factors are also essential to mast cell development, including the Microphthalmia-associated transcription factor (Mitf). We examined Mitf expression in bone marrow biopsies from nine patients with systemic mastocytosis by immunohistochemistry; we found that Mitf is highly expressed in all cases with the D816V mutation. In contrast, Mitf is not highly expressed in non-malignant mast cells in the bone marrow from patients with aplastic anemia and leukemia, suggesting thatMitf expression is regulated by Kit-dependent signalsMitf may play a role in the transformed phenotype of mastocytosis.We show that in normal mast cells, Kit signaling markedly upregulates Mitf expression. In both normal and malignant mast cells, pharmacologic inhibitors of Kit, and the downstream kinase, PI3K, block Mitf expression. To examine whether Mitf is required for transformed phenotype from constitutive Kit signaling in mast cells, we have used a shRNA-expressing lentivirus to knockdown Mitf expression in mastocytosis cell lines. We found that silencing of Mitf markedly impaired growth in proliferation and colony forming cell assays. This work demonstrates a link between two critical factors, Kit and Mitf, in the development of malignant mast cell disease.

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

Blood ◽

10.1182/blood.v114.22.77.77 ◽

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.

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Mucosal mast cells and nematode infection: strain-specific differences in mast cell precursor frequency revisited

Journal of Helminthology ◽

10.1079/joh2002160 ◽

2003 ◽

Vol 77 (2) ◽

pp. 155-161 ◽

Cited By ~ 9

Author(s):

J.K. Brown ◽

S.H. Wright ◽

H.R.P. Miller

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Mast Cells ◽

Transforming Growth Factor ◽

Bone Marrow Cells ◽

Trichinella Spiralis ◽

Cell Precursor ◽

Mucosal Mast Cells ◽

Bone Marrow Cultures ◽

Marrow Cultures

AbstractMucosal mast cells (MMC) play an important role in the immune response against selected species of intestinal nematode. The kinetics with which different strains of inbred mice resolve infection withTrichinella spiraliscorrelates with their ability to mount MMC responses in the intestinal mucosa. Homologues of MMC that express and constitutively secrete abundant amounts of the granule chymase, mouse mast cell protease-1 (mMCP-1), can be generatedin vitrofrom bone marrow cultures supplemented with interleukins-3 and -9, stem cell factor and transforming growth factor-β1. Using the enhanced growth characteristics of these MMC homologues, a novel limiting dilution assay for mast cell precursor (MCp) frequency has been developed. The assay is highly specific, in that cultures containing mast cells are identified with mMCP-1 specific antibody, and almost three-fold more sensitive than previously published systems. MCp frequencies were compared in BALB/c and C57/BL10 strains of mice that, respectively, respond rapidly and slowly to infection withT. spiralis. MCp frequency (1/378 bone marrow cells) was significantly greater (P<0.05) in BALB/c than C57/BL10 mice (frequency: 1/751). Similarly the rate of growth of MMC homologues and the production of mMCP-1 was significantly (P<0.05) greater in BALB/c than in C57/BL10 bone marrow cultures.

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