scholarly journals Long-term generation of human mast cells in serum-free cultures of CD34+ cord blood cells stimulated with stem cell factor and interleukin- 3

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3667-3674 ◽  
Author(s):  
B Durand ◽  
G Migliaccio ◽  
NS Yee ◽  
K Eddleman ◽  
T Huima-Byron ◽  
...  
Keyword(s):  
Mast Cell ◽  
Stem Cell ◽  
Growth Factors ◽  
Mast Cells ◽  
Cord Blood ◽  
Stem Cell Factor ◽  
Human Mast Cells ◽  
Differentiated Cells ◽  
Murine Fibroblasts

The generation of murine mast cells is supported by several cytokines, and mast cell lines are frequently established in long-term cultures of normal murine marrow cells. In contrast, growth of human mast cells was initially dependent on coculture with murine fibroblasts. The growth factor produced by murine fibroblasts and required to observe differentiation of human mast cells is attributable in part to stem cell factor (SCF). However, other factors are likely involved. We have previously shown that the combination of SCF and interleukin-3 (IL-3) efficiently sustains proliferation and differentiation of colony- forming cells (CFCs) from pre-CFC enriched from human umbilical cord blood by CD34+ selection. With periodic medium changes and the addition of fresh growth factors, five consecutive cultures of different cord blood samples gave rise to differentiated cells and CFCs for more than 2 months. Although differentiated cells continued to be generated for more than 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue positive, are karyotypically normal, are CD33+, CD34-, CD45+, c-kit-, and c-fms-, and die in the absence of either SCF or IL- 3. These cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentration of no less than 10(4) cells/mL. At refeedings, the cultures contain a high number (= 50%) of dead cells and have a doubling time ranging from 5 to 12 days. This suggests that subsets of the cell population die because of a requirement for a growth factor other than SCF or IL-3. These results indicate that the combination of cord blood progenitor and stem cells, plus a cocktail of growth factors including SCF and IL-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor- dependent cell lines. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia.

Regulatory effects of stem cell factor and interleukin-4 on adhesion of human mast cells to extracellular matrix proteins

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 966-972 ◽  
Author(s):  
Axel Lorentz ◽  
Detlef Schuppan ◽  
Andreas Gebert ◽  
Michael P. Manns ◽  
Stephan C. Bischoff
Keyword(s):  
Extracellular Matrix ◽  
Mast Cell ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Mast Cells ◽  
Stem Cell Factor ◽  
Collagen I ◽  
Interleukin 4 ◽  
Human Mast Cells ◽  
Ecm Proteins

Abstract Mast cells are inflammatory and immunoregulatory cells resident in tissues. They develop from bone marrow-derived progenitor cells that enter the tissue through the blood circulation. The specific localization and migration of mast cells in tissues is dependent on their interaction with extracellular matrix (ECM) proteins. Adhesion of human mast cells isolated from intestinal mucosa and cultured in the presence of stem cell factor (SCF) to ECM proteins is analyzed. It was observed that SCF is a unique cytokine enhancing mast cell adhesion to all tested ECM proteins (fibronectin, laminin, collagen I, III, IV, VI, XIV) up to 5-fold, particularly to fibronectin (54% ± 12% of mast cells) and to denatured collagens (40% ± 12% on cyanogen bromide-cleaved peptides of collagen I). Most noteworthy, preculture of mast cells with interleukin-4 (IL-4), in addition to SCF, reduced their potency to adhere to ECM proteins to one third compared to mast cells cultured with SCF alone. Mast cell adhesion was preferentially mediated by β1 integrins, and most cells expressed the ECM-binding integrins α2β1, α3β1, α4β1, α5β1, and αVβ3. SCF-induced mast cell adhesion was totally blocked by wortmannin and apigenin, indicating an involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase, and it was related to an up-regulation of the HUTS-21 β1 epitope, which is associated with an activated conformation of β1. In conclusion, these data indicate that SCF induces the adhesion of cultured mast cells to ECM proteins, whereas IL-4 may promote detachment from the ECM.

Interleukin-6 Directly Modulates Stem Cell Factor-Dependent Development of Human Mast Cells Derived From CD34+Cord Blood Cells

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 496-508 ◽  
Author(s):  
Tatsuya Kinoshita ◽  
Nobukuni Sawai ◽  
Eiko Hidaka ◽  
Tetsuji Yamashita ◽  
Kenichi Koike
Keyword(s):  
Mast Cell ◽  
Stem Cell ◽  
Mast Cells ◽  
Cord Blood ◽  
Interleukin 6 ◽  
Blood Cells ◽  
Stem Cell Factor ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Cord Blood Cells

In the present study, we attempted to clarify the effects of interleukin-6 (IL-6) on the growth and properties of human mast cells using cultured mast cells selectively generated by stem cell factor (SCF) from CD34+ cord blood cells. The addition of IL-6 to cultures containing mast cells resulted in a substantial reduction of the number of progenies grown by SCF in the liquid culture. This IL-6–mediated inhibition of mast cell growth may be due in part to the suppression at the precursor level, according to the results of a clonal cell culture assay. Moreover, a flow cytometric analysis showed that the cultured mast cells grown in the presence of SCF+IL-6 had decreased c-kit expression. The exposure of cultured mast cells to SCF+IL-6 also caused substantial increases in the cell size, frequency of chymase-positive cells, and intracellular histamine level compared with the values obtained with SCF alone. The flow cytometric analysis showed low but significant levels of expression of IL-6 receptor (IL-6R) and gp130 on the cultured mast cells grown with SCF. The addition of either anti–IL-6R antibody or anti-gp130 antibody abrogated the biological functions of IL-6. Although IL-4 exerted an effect similar to that of IL-6 on the cultured mast cells under stimulation with SCF, the results of comparative experiments suggest that the two cytokines use different regulatory mechanisms. Taken together, the present findings suggest that IL-6 modulates SCF-dependent human mast cell development directly via an IL-6R-gp130 system.

scholarly journals Interleukin-4 inhibits the expression of Kit and tryptase during stem cell factor-dependent development of human mast cells from fetal liver cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1519-1527 ◽  
Author(s):  
G Nilsson ◽  
U Miettinen ◽  
T Ishizaka ◽  
LK Ashman ◽  
AM Irani ◽  
...  
Keyword(s):  
Mast Cell ◽  
Stem Cell ◽  
Mast Cells ◽  
Stem Cell Factor ◽  
Fetal Liver ◽  
Liver Cells ◽  
Interleukin 4 ◽  
Human Mast Cells ◽  
Fetal Liver Cells

Abstract Although interleukin-4 (IL-4) in mice is known to augment the proliferation of mast cells and to modulate the expression of certain mast cell protease transcripts, its effect on human mast cells is less well understood. The current study examined the effects of recombinant human IL-4 (rhuIL-4) on stem cell factor (SCF)-dependent fetal liver- derived human mast cells in liquid culture. In no case did rhuIL-4 augment proliferation of mast cells. rhuIL-4 selectively inhibited certain aspects of the development of mast cells in cultures of fetal liver cells with rhuSCF. These include lower numbers and percentages of cells expressing tryptase and surface Kit, smaller cells, and lower contents of cells for tryptase, histamine, and Kit. Development of metachromasia was not attenuated. The downregulation of Kit, the surface receptor for SCF, is probably a critical factor, because cells lacking this molecule would not be able to respond to SCF. In contrast to mast cell progenitors, mast cells already developed in vitro from fetal liver cells are relatively resistant to rhuIL-4, but are still dependent for survival on the presence of rhuSCF.

Structural Specificity of the Biological Activity of Human and Murine Stem Cell Factor in Stress Erythropoiesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3395-3395
Author(s):  
Elena Masselli ◽  
Lilian Varricchio ◽  
Barbara Ghinassi ◽  
Anna Rita F Migliaccio
Keyword(s):  
Biological Activity ◽  
Stem Cell ◽  
Growth Factors ◽  
Mast Cells ◽  
Cord Blood ◽  
Stem Cell Factor ◽  
Conflicts Of Interest ◽  
Biological Activities ◽  
Biological Significance ◽  
Stress Erythropoiesis

Abstract Abstract 3395 Stem cell factor (SCF) is a cyokine produced by bone marrow stromal cells that upon engagement of its cognate receptor cKit and in combination with other hematopoietic growth factors initiates downstream phosphorylation events that generate hematopoietic progenitors and precursors of all types. SCF and cKit are encoded by highly conserved genes. The human (hSCF) and murine (mSCF) forms are highly homologous and are 82% identical at the amino acid levels. Both have been reported to sustain differentiation of human mast cells from cord blood (CB) but mast cells generated with mSCF remain immature (Mitzu et al, PNAS 90:735–739). In addition, anecdotic reports indicate that hSCF is 100-fold less active than mSCF on mouse cells while mSCF and hSCF are equally active on human cells. To clarify if structural differences between the two growth factors have biological significance, we compared cord blood (CB) mononuclear cells and CD34pos cells as the stem cell source for in vitro production of colony forming cells, CFU-GM and BFU-E, using established techniques, and for ex-vivo expansion of megakaryocytes (MK) (SCF + IL-3 + TPO) and erythroid cells (EB) under standard conditions (IL-3 and EPO) and conditions of stress (IL-3 + EPO + dexamethasone). In semisolid assay, hSCF and mSCF (20 ng/mL) sustained growth of similar numbers of CFU-GM (51±5 vs 44±5/2×105 MNC and 15.4±0.9 vs 13.3±2.0/500 CD34pos cells)- and BFU-E (8.5±0.5 vs 8.7±1.2/500 CD34pos cells)-derived colonies. By day 12 of MK expansion assay, hSCF and mSCF induced similar 2-1.6-fold increases over those observed with IL-3 and TPO alone. In addition, hSCF and mSCF-stimulated cultures contained similar number of MK as identified by morphology and CD41a/CD42b staining (∼40–60% CD41aposCD42bpos cells) (Figure 1). By day 13 of EB expansion culture without steroids, hSCF and mSCF generated similar numbers of EB (FI= ∼6–8 with both MNC and CD34pos cells), ∼60% of which had a mature CD36posCD235ahigh phenotype (Figure 1), but, by day 15 of EB expansion with steroids, hSCF induced significantly greater numbers of EB that mSCF (FI=154±45 vs 54±15 from MNC, and 13,200±740 vs 2,000±550 for CD34pos cells, respectively, p<0.01 in both cases). In addition, day 8 EB generated with mSCF were more mature than those generated with hSCF (86% vs 31% of CD36posCD235ahigh EB, Figure 1). Similar results were obtained in EB expansion cultures of AB MNC. To clarify the difference in biological activity of hSCF and mSCF in cultures containing steroids, expression of glucocorticoid receptor α (GRα) and hSCF and mSCF signaling in adult blood (AB) and CB EB generated with the two growth factors were compared. hSCF-generated EB expressed 2-times more GRα than those generated with mSCF (Figure 2) and the GRα content of hSCF-generated EB was reduced by 50% after 24 h exposure with mSCF. In addition, both transient (within 15 m) and sustained (after 2 h) ERK phosphorylation were observed only in EB stimulated with hSCF while both hSCF and mSCF induced low levels of STAT-5 phosphorylation but neither factor induced AKT phosphorylation. In summary, hSCF and mSCF have overlapping biological activities in culture conditions which mimic steady-state hematopoiesis (semisolid cultures, MK and EB expansion without dexamethasone) but hSCF was uniquely capable to sustain GRα expression and was more effective than mSCF in cultures which mimic stress erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

Signaling pathways activated in a unique mast cell line where interleukin-3 supports survival and stem cell factor is required for a proliferative response

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3655-3668 ◽  
Author(s):  
AM O'Farrell ◽  
M Ichihara ◽  
AL Mui ◽  
A Miyajima
Keyword(s):  
Mast Cell ◽  
Stem Cell ◽  
Mast Cells ◽  
Cell Line ◽  
Stem Cell Factor ◽  
Mapk Pathway ◽  
Interleukin 3 ◽  
Murine Mast Cell ◽  
Mast Cell Line

Stem cell factor (SCF) and interleukin-3 (IL-3) both act on several target hematopoietic populations, including mast cells. We have isolated a unique murine mast cell line, B6M, that is phenotypically similar to immature mast cells. For B6M cells, IL-3 is a survival factor and alone does not stimulate proliferation. SCF can stimulate proliferation of B6M cells, and together IL-3 and SCF synergize to stimulate optimal proliferation and long-term growth. A sustained induction of c-myc is observed only in the presence of SCF (with or without IL-3). In B6M cells, both IL-3 and SCF stimulate phosphorylation of Shc and activation of the Ras, Raf-1, MAPK pathway. Interestingly, IL-3 plus SCF synergistically activate MAPK. IL-3, but not SCF, leads to activation of Jak 2 and Stat 5 and induces pim-1 expression. From these data, we suggest that the induction of pim-1 and c-myc is independently regulated. Furthermore, IL-3-stimulated activation of the Jak 2/Stat 5 pathway, induction of pim-1, and activation of the Ras/MAPK pathway are insufficient to mediate proliferation of B6M cells. The unusual IL-3 response of B6M cells provides a useful model to dissect signals required for IL-3-stimulated survival and proliferation.

Thrombopoietin Augments Stem Cell Factor–Dependent Growth of Human Mast Cells From Bone Marrow Multipotential Hematopoietic Progenitors

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3703-3712 ◽  
Author(s):  
Nobukuni Sawai ◽  
Kenichi Koike ◽  
Hadija Hemed Mwamtemi ◽  
Tatsuya Kinoshita ◽  
Yumi Kurokawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Stem Cell ◽  
Mast Cells ◽  
Stem Cell Factor ◽  
Early Stage ◽  
Flow Cytometric Analysis ◽  
Immunocytochemical Staining ◽  
Cell Lineages ◽  
Human Mast Cells

Abstract The effects of thrombopoietin (TPO) and/or stem cell factor (SCF) on the development of human mast cells from CD34+ bone marrow (BM) cells were investigated using a serum-deprived liquid culture system. Mast cells were identified by measurement of intracellular histamine content, immunocytochemical staining, and flow cytometric analysis. Whereas SCF alone generated only a small number of tryptase+ cells, the addition of TPO to the culture containing SCF resulted in an apparent production of mast cells from 3 weeks until at least 15 weeks. Some of the cells reacted with an antichymase monoclonal antibody as well. Based on the effects of growth factor(s) on a later phase of the mast cell growth, TPO may stimulate an early stage of mast cell development in combination with SCF, whereas subsequent growth seems to be supported by SCF alone. Single-cell culture studies indicated that the CD34+CD38−c-kit+ cells and CD34+CD38+c-kit+ cells were responsible for the SCF + TPO–dependent mast cell production. Two-step culture assays clearly showed that mast cells originated from multilineage colony-forming cells that had potential to differentiate into neutrophil/mast cell lineages, neutrophil/macrophage/mast cell lineages, or neutrophil/macrophage/mast cell/erythroid lineages. These results suggest that TPO plays an important role in the development of human mast cells from CD34+ BM cells in concert with SCF, and provide direct evidence of the differentiation into the mast cell lineage of human multipotential BM-derived progenitors.

scholarly journals Interleukin-4 inhibits the expression of Kit and tryptase during stem cell factor-dependent development of human mast cells from fetal liver cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1519-1527 ◽  
Author(s):  
G Nilsson ◽  
U Miettinen ◽  
T Ishizaka ◽  
LK Ashman ◽  
AM Irani ◽  
...  
Keyword(s):  
Mast Cell ◽  
Stem Cell ◽  
Mast Cells ◽  
Stem Cell Factor ◽  
Fetal Liver ◽  
Liver Cells ◽  
Interleukin 4 ◽  
Human Mast Cells ◽  
Fetal Liver Cells

Although interleukin-4 (IL-4) in mice is known to augment the proliferation of mast cells and to modulate the expression of certain mast cell protease transcripts, its effect on human mast cells is less well understood. The current study examined the effects of recombinant human IL-4 (rhuIL-4) on stem cell factor (SCF)-dependent fetal liver- derived human mast cells in liquid culture. In no case did rhuIL-4 augment proliferation of mast cells. rhuIL-4 selectively inhibited certain aspects of the development of mast cells in cultures of fetal liver cells with rhuSCF. These include lower numbers and percentages of cells expressing tryptase and surface Kit, smaller cells, and lower contents of cells for tryptase, histamine, and Kit. Development of metachromasia was not attenuated. The downregulation of Kit, the surface receptor for SCF, is probably a critical factor, because cells lacking this molecule would not be able to respond to SCF. In contrast to mast cell progenitors, mast cells already developed in vitro from fetal liver cells are relatively resistant to rhuIL-4, but are still dependent for survival on the presence of rhuSCF.

scholarly journals Diamine Oxidase-Gold Ultrastructural Localization of Histamine in Human Skin Biopsies Containing Mast Cells Stimulated to Degranulate In Vivo by Exposure to Recombinant Human Stem Cell Factor

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 2893-2900 ◽  
Author(s):  
Ann M. Dvorak ◽  
John J. Costa ◽  
Ellen S. Morgan ◽  
Rita A. Monahan-Earley ◽  
Stephen J. Galli
Keyword(s):  
Mast Cell ◽  
Stem Cell ◽  
Mast Cells ◽  
Human Skin ◽  
Stem Cell Factor ◽  
Diamine Oxidase ◽  
Human Mast Cells ◽  
Skin Biopsies

AbstractStem cell factor (SCF ) has a major role in hematopoiesis and in the regulation of mast cell development and function. For example, recombinant human SCF (rhSCF ) can induce the development of human mast cells from precursor cells in vitro, stimulate mediator release from human skin mast cells in vitro, and promote both the development and functional activation of human skin mast cells in vivo. In the present study, we used a new ultrastructural enzyme-affinity method, employing diamine oxidase (DAO)-conjugated gold particles (DAO-gold), to detect histamine in skin biopsies obtained from patients with breast carcinomas who were receiving daily subcutaneous (SC) injections of rhSCF in a phase I study of this cytokine. We examined control biopsies obtained at sites remote from rhSCF injection as well as biopsies of rhSCF-injected skin that were obtained within 2 hours and 30 minutes of the SC injection of rhSCF at that site. The rhSCF-injected sites (which clinically exhibited a wheal-and-flare response), but not the control sites, contained mast cells undergoing regulated secretion by granule extrusion. The DAO-gold-affinity method detected histamine in electron-dense granules of mast cells in control and injected skin biopsies; however, the altered matrix of membrane-free, extruded mast cell granules was largely unreactive with DAO-gold. Notably, DAO-gold bound strongly to fibrin deposits and collagen fibers that were adjacent to degranulated mast cells. These findings represent the first morphologic evidence of histamine secretion by classical granule exocytosis in human mast cells in vivo.

Interleukin-6 Directly Modulates Stem Cell Factor-Dependent Development of Human Mast Cells Derived From CD34+Cord Blood Cells

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 496-508 ◽  
Author(s):  
Tatsuya Kinoshita ◽  
Nobukuni Sawai ◽  
Eiko Hidaka ◽  
Tetsuji Yamashita ◽  
Kenichi Koike
Keyword(s):  
Mast Cell ◽  
Stem Cell ◽  
Mast Cells ◽  
Cord Blood ◽  
Interleukin 6 ◽  
Blood Cells ◽  
Stem Cell Factor ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Cord Blood Cells

Abstract In the present study, we attempted to clarify the effects of interleukin-6 (IL-6) on the growth and properties of human mast cells using cultured mast cells selectively generated by stem cell factor (SCF) from CD34+ cord blood cells. The addition of IL-6 to cultures containing mast cells resulted in a substantial reduction of the number of progenies grown by SCF in the liquid culture. This IL-6–mediated inhibition of mast cell growth may be due in part to the suppression at the precursor level, according to the results of a clonal cell culture assay. Moreover, a flow cytometric analysis showed that the cultured mast cells grown in the presence of SCF+IL-6 had decreased c-kit expression. The exposure of cultured mast cells to SCF+IL-6 also caused substantial increases in the cell size, frequency of chymase-positive cells, and intracellular histamine level compared with the values obtained with SCF alone. The flow cytometric analysis showed low but significant levels of expression of IL-6 receptor (IL-6R) and gp130 on the cultured mast cells grown with SCF. The addition of either anti–IL-6R antibody or anti-gp130 antibody abrogated the biological functions of IL-6. Although IL-4 exerted an effect similar to that of IL-6 on the cultured mast cells under stimulation with SCF, the results of comparative experiments suggest that the two cytokines use different regulatory mechanisms. Taken together, the present findings suggest that IL-6 modulates SCF-dependent human mast cell development directly via an IL-6R-gp130 system.

Non-IgE–dependent activation of human lung– and cord blood–derived mast cells is induced by eosinophil major basic protein and modulated by the membrane form of stem cell factor

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1898-1904 ◽  
Author(s):  
Adrian M. Piliponsky ◽  
Gerald J. Gleich ◽  
Arnon Nagler ◽  
Ilan Bar ◽  
Francesca Levi-Schaffer
Keyword(s):  
Mast Cell ◽  
Stem Cell ◽  
Mast Cells ◽  
Cord Blood ◽  
Stem Cell Factor ◽  
Basic Protein ◽  
Human Lung ◽  
Late Phase ◽  
Major Basic Protein ◽  
Eosinophil Major Basic Protein

The allergic reaction begins with the antigen-induced aggregation of occupied high-affinity IgE receptors expressed on mast cell surface, their activation, and the release of proinflammatory mediators that cause the “early phase” of this process. In addition, mast cell activation induces the onset of a “late phase” reaction characterized by the tissue infiltration of inflammatory cells, mainly eosinophils. We have hypothesized that during the late phase mast cells interact with and are activated by eosinophils. Here we report that highly purified human lung mast cells became responsive to eosinophil major basic protein (MBP) when in coculture with human lung fibroblasts. In addition, cord blood–derived mast cells maintained in coculture with 3T3 fibroblasts released more histamine and prostaglandin D2 (PGD2) compared with cells maintained in suspension. The fibroblast-derived membrane form of stem cell factor (SCF) was found to be involved in the mast cell increased responsiveness to MBP. In fact, cord blood–derived mast cells cocultured with 3T3 in the presence of antisense for SCF or cocultured with fibroblasts that do not express the membrane form of SCF were inhibited in their histamine-releasing activity toward MBP. In addition, this form of SCF induced the expression of a pertussis toxin–sensitive Gi protein, Gi3 that interacts with MBP to trigger mast cell non-IgE–dependent activation in a manner similar to other cationic compounds such as compound 48/80. Mast cell responsiveness to eosinophil mediators is a potentially novel evidence for an alternative pathway of allergen-independent activation able to contribute to the perpetuation of allergy.

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