C-kit Ligand Research Articles

Post-transcriptional Stabilization by Interleukin-1β of Interleukin-6 mRNA Induced by c-kit Ligand and Interleukin-10 in Mouse Bone Marrow-derived Mast Cells

We demostrate that a specific combination of cytokines elicits high levels of interleukin (IL)-6 gene expression in mast cells and define the cellular mechanisms of the exogenous cytokine action. The addition of c-kit ligand (KL) and IL-10 to IL-3-derived mouse bone marrow mast cells (BMMC) elicited an approximately 2-fold increase in steady-state IL-6 mRNA levels that peaked after 0.5 h and was followed by the release of approximately 0.2 ng of IL-6/10(6) cells by 5-7 h. The addition of IL-1beta to KL + IL-10 elicited a prolonged approximately 12-fold increase in the level of IL-6 mRNA by 3-5 h and an approximately 50-fold increase in the level of IL-6 protein released by 7 h. As determined by nuclear run-on analysis, KL + IL-10 stimulated IL-6 gene transcription within 0.5 h, and the addition of IL-1beta did not increase transcription. Instead, IL-1beta slowed by approximately 8-fold the decay of IL-6 mRNA as compared to its decay in BMMC stimulated with KL + IL-10 alone. The exposure of BMMC to cycloheximide 0.5 h before the addition of the three exogenous cytokines inhibited by approximately 50% the level of IL-6 mRNA generated but did not inhibit the effects of KL + IL-10, indicating that IL-1beta induces the synthesis of a protein that stabilizes IL-6 mRNA. The stabilization of IL-6 mRNA was inhibited by the addition of actinomycin D at 0.5 but not 3 h after BMMC were stimulated with IL-1beta in combination with KL + IL-10, suggesting that once transcribed, the stabilizing protein is long-lived. The addition of cycloheximide to BMMC after stimulation with KL + IL-10 with or without IL-1beta increased the levels of steady-state IL-6 mRNA compared to levels in cells without drug, indicating that in addition to stimulating IL-6 transcription, KL + IL-10 induces a protein factor that destabilizes IL-6 mRNA. Thus, there exists a novel Fcepsilon receptor type I-independent mechanism by which a mast cell can provide substantial amounts of IL-6 protein in response to the synergistic action of KL and IL-10 to induce IL-6 gene transcription, and IL-1beta to stabilize otherwise short-lived IL-6 transcripts.

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200–10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.

Thrombopoietin Stimulates Megakaryocytopoiesis, Myelopoiesis, and Expansion of CD34+ Progenitor Cells From Single CD34+Thy-1+Lin- Primitive Progenitor Cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy-1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony-forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.

Topical tretinoin increases dermal mast cells, induces epidermal mast cell growth factor (c-kit ligand) and modulates its distribution in hairless mice

Topical tretinoin increases dermal mast cells, induces epidermal mast cell growth factor (c-kit ligand) and modulates its distribution in hairless mice

Combined Signaling Through Interleukin-7 Receptors and flt3 But Not c-kit Potently and Selectively Promotes B-Cell Commitment and Differentiation From Uncommitted Murine Bone Marrow Progenitor Cells

Multiple cytokines can synergize to stimulate the in vitro proliferation and exclusive myeloid differentiation of multipotent bone marrow progenitor cells. The ligand for c-kit (stem cell factor [SCF]) plays a key role in stimulating myeloid and erythroid cell production of primitive hematopoietic progenitors. SCF in combination with interleukin-7 (IL-7) can also stimulate the combined myeloid and B-cell differentiation of uncommitted hematopoietic progenitor cells as well as the growth of early B-cell progenitor cells, although the involvement of c-kit in early B lymphopoiesis remains controversial. In the present study, the flt3-ligand (FL), which, in combination with other cytokines, has overlapping activities with SCF on myeloid cell production from uncommitted progenitors, was investigated for its ability to induce selective stroma-independent B-cell commitment from uncommitted Lin-Sca-1+ bone marrow progenitor cells. IL-7 alone did not induce any clonal growth and FL alone gave rise to a few clusters (< 50 cells) but no colonies (> 50 cells), whereas the combined stimulation with FL and IL-7 resulted in clonal growth of 10% of Lin-Sca-1+ bone marrow cells. After 12 days of incubation of Lin-Sca-1+ cells in FL + IL-7, an almost 400-fold increase in cell production was observed. Phenotyping showed that greater than 99% of the cells produced were of the B-cell lineage, in that they expressed B220, but not cell surface markers specific for myeloid, erythroid, or T-cell lineages. Furthermore, the cells did not express cytoplasmic mu-heavy chain (cmu) or surface IgM, but were positive for CD24 (heat stable antigen [HSA]) and CD43 (leukosialin), suggesting that the cells produced were blocked at a late pro-B-cell stage. Interestingly, although all FL + IL-7-responsive Lin-Sca-1+ progenitor cells and the resulting pro-B cells expressed c-kit, FL + IL-7 was much more potent (62-fold) than SCF + IL-7 in stimulating production of cells of the B-cell lineage. In addition, whereas FL + IL-7 selectively stimulated the production of pro-B cells, SCF + IL-7 predominantly stimulated the production of mature granulocytes. Replating studies showed that FL + IL-7-responsive Lin-Sca-1+ progenitors were not committed to the B-cell lineage, because after 2 days of incubation in FL + IL-7, 80% of the progenitors retained a myeloid potential. As much as 27% of the FL + IL-7-responsive progenitors remained uncommitted after 7 days of incubation, but all had committed to the B-cell lineage after 10 days of incubation in FL + IL-7. These results show that FL much more potently and selectively than SCF synergizes with IL-7 to enhance B-cell commitment and development from uncommitted progenitor cells.

Mammalian oocyte growth in vitro is stimulated by soluble factor(s) produced by preantral granulosa cells and by Sertoli cells.

We have evaluated the possibility that mouse oocyte growth in vitro could be achieved under the influence of soluble compound(s) released by different somatic cell types. For this purpose, zona-free denuded oocytes from 12-day-old mice were cultured on monolayers of NIH-3T3 fibroblasts, which are able to establish gap junctional communications with them, in the presence or absence of media conditioned by preantral granulosa cells or by Sertoli cells, plated at increasing concentrations from 0.3-1 x 10(6) ml-1 cells. After 3 days, no increase in vitellus diameter was recorded from fibroblast-coupled oocytes maintained in culture medium or in the presence of media conditioned by 0.3 x 10(6) ml-1 Sertoli cells. By contrast, increasing proportions of coupled oocytes grew, provided the continuous presence of media conditioned by 0.5 or 1 x 10(5) ml-1 Sertoli cells, or by 0.3, 0.5, and 1 x 10(5) ml-1 preantral granulosa cells. Since the ligand of c-kit, the growth factor KL, promotes the growth in vitro of oocytes cultured in follicles from 8-day-old mice, an antibody against mouse KL was used to evaluate whether in our culture conditions KL might also be responsible for the growth of oocytes from 12-day-old mice. No inhibition of growth was evident in oocytes cultured directly on preantral granulosa or Sertoli-cell monolayers. Furthermore, the growth of fibroblast-coupled oocytes cultured in media conditioned by preantral granulosa cells was not significantly affected by the presence of this antibody during culture. By contrast, a high percentage of oocytes cultured on fibroblasts in the presence of media conditioned by Sertoli cells showed a significant inhibition of growth and no metabolic cooperativity. It was concluded that, besides KL, other bioactive factor(s) released by either preantral granulosa or Sertoli cells can induce a significant stimulation of mouse oocyte growth in vitro.

Activity of the ligand for c-mpl, thrombopoietin, in early haemopoiesis.

We examined the role of the ligand for c-mpl. thrombopoietin (TPO). in murine early haemopoiesis. using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) supported colony formation by marrow cells of 5-fluorouracil (5-FU)-treated mice whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL-3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL-3-dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL-3 to act on early multipotential haemopoietic progenitors.

C-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-Myc and resultant depression of proliferation.

Topical tretinoin increases dermal mast cells, induces epidermal mast cell growth factor (c-kit ligand) and modulates its distribution in hairless mice.

In previous studies we have noted that mast cells are increased in tretinoin-treated photoaged hairless mouse skin. Because UV radiation is known to increase mast cell numbers, we were interested in whether tretinoin alone would modulate the mast cell population in unirradiated mice. Animals were treated topically with 0.05% tretinoin, 5 days a week, for 2, 4, 6, 8 and 10 weeks. Untreated and vehicle controls were included. Biopsies were processed for light microscopy and stained with toluidine blue. Mast cells in the upper and lower dermis were scored separately under high magnification. After 2 weeks of tretinoin, mast cells in the upper dermis were significantly increased, as indicated by the appearance of small, moderately metachromatically granulated cells near the dermal-epidermal junction. Mast cells in the lower dermis, the site of a granulomatous reaction, were large, densely granular and significantly increased after 6 weeks of treatment. Immunohistochemical evaluation for mast cell growth factor (MGF) revealed a marked increase in keratinocyte cytoplasmic staining by week 2. After 4-6 weeks, membrane-associated or intercellular staining was evident. Cells in the upper dermis also showed membrane reactivity for MGF. By 8-10 weeks, epidermal MGF reactivity had dissipated in the more basal keratinocytes. These findings show that topical tretinoin can induce epidermal MGF along with an associated mast cell hyperplasia. It is suggested that the two populations of dermal mast cells may have different functions.

Stem-cell factor regulates the expression of cyclin A and retinoblastoma gene product in the growth and differentiation pathway of human megakaryocytic cells.

The biological effects of c-kit ligand (stem-cell factor: SCF) on an immortalized human megakaryocytic cell line (CMK) was evaluated using methods including the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, surface marker analysis, DNA cell-cycle analysis and immunoblotting. SCF stimulated the growth of CMK cells. Incubation with SCF resulted in increased expression of IIb/IIIa platelet-related glycoprotein (gpIIb, IIIa), indicating enhanced differentiation of CMK cells. Treatment of CMK cells with SCF resulted in a decrease in the subpopulation in the G1 phase, with a reciprocal increase in those in the S phase and the G2 + M phase. Moreover, SCF significantly increased cellular expression of cyclin A, a regulatory subunit of cyclin-dependent protein kinase (CDK), and the ratio of phosphorylated/dephosphorylated retinoblastoma gene product (RB protein). These results suggest that SCF stimulates the growth and differentiation of megakaryocytic cells possibly through mechanisms related to the activation of cell-cycle-dependent serine/threonine kinase and inactivation of the nuclear tumor-suppressor gene product.

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

Regulation of colony forming cell generation by flt-3 ligand.

The recently cloned ligand for the flt-3/flk-2 receptor was examined for its effect on colony formation by subpopulations of CD34+ cells including the least mature CD34+lin-CD38- small-medium lymphocyte-sized cell population. Flt-3 ligand (flt-3l) had little or no effect when added alone to cells. Isolated CD34+lin+ cells formed increased numbers of colony-forming cells (CFC) when flt-3l was added together with IL-3, IL-6, G-CSF, GM-CSF or c-kit ligand (KL), or with the combination of IL-3 and KL. Significant increases in CFC formation from CD34+lin- cells were consistently seen when flt-3l was added to the IL-3 and KL combination, with variable effects observed when it was added to individual growth factors. Studies of the generation of CFC from CD34+lin- cells in liquid cultures showed that cultures containing IL-3 and KL continued to produce CFC after 3 weeks of culture, whereas cultures with IL-3, KL and flt-3l produced few CFC past 2 weeks of culture. Flt-3l alone or the combination of IL-3 and KL did not stimulate significant growth of CD34+lin-CD38- small-medium lymphocyte-sized cells, although these cells reproducibly generated CFC when grown in the combination of IL-1 beta, IL-3, IL-6, G-CSF, GM-CSF and KL. Addition of flt-3l to either IL-3 and KL or to a combination of growth factors induced increased CFC in three of four experiments. These data therefore demonstrate a role for flt-3l in the induction of myelopoiesis by haemopoietic precursors, including the least mature subpopulation population of CD34+ cells.

Stem cell factor and stromal cell co-culture prevent apoptosis in a subculture of the megakaryoblastic cell line, UT-7

The megakaryoblastic cell line, UT-7, is dependent for its growth upon interleukin-3 (IL-3), erythropoietin, or granulocyte-macrophage colony stimulating factor (GM-CSF). A subculture of this line can be maintained in recombinant human c-kit ligand [stem cell factor (SCF)] at 100 ng/ml without requirement for other growth factors. Removal of this subculture from SCF results in rapid loss of viability and decreased proliferation. Cells grown in SCF also can be maintained in GM-CSF but not vice versa. In this work, we have characterized the SCF dependence of this UT-7 subculture. Stem cell factor removal results in apoptosis and a decline in viability which can be restored partially by re-addition of SCF, GM-CSF, or co-culture with adherent marrow stromal cells. Apoptosis in the factor-starved UT-7 population has been documented by light microscopy, electron microscopy and DNA analysis, showing the typical 180 base pair laddering characteristic of apoptosis. To quantitate the degree of apoptosis in the cell populations, and to assess whether apoptosis decreased with re-exposure of starved cells to growth factors or stroma, we utilized flow cytometry. This confirmed that exposure of previously factor-starved cells to stroma decreased the percentage of cells undergoing apoptosis. Co-culture with an SCF-deficient murine stromal cell line was also able to prevent apoptosis, suggesting contribution of other stromal cell factors. Experiments performed using trans-well inserts which do not allow cell passage, showed greatest viability of cells in contact with stroma, but viability was also improved in cells cultured in the presence of, but not in contact with, stromal cells compared to those cultured above plastic, suggesting a role for soluble stroma-produced substances. These data demonstrate that SCF alone can prevent apoptosis in cells dependent upon its presence for proliferation. Also, marrow stromal cells can serve as a partial substitute for growth factor in the prevention of apoptosis in these cells, probably due to constitutive presentation of SCF and other hematopoietic growth factors in both soluble and surface-bound forms.

Mast cells expressing chymase but not tryptase can be derived by culturing human progenitors in conditioned medium obtained from a human mastocytosis cell strain with c-kit ligand.

Human mast cells (MC) were derived from umbilical cord blood and bone marrow progenitors cultured in the presence of a conditioned medium from a human mastocytosis cell strain and recombinant human kit ligand. MCs were studied using a sequential double immunoenzymatic analysis to determine the heterogeneity of expression of tryptase and chymase, two MC-specific proteases. The conditioned medium and kit ligand promoted the development of distinct MC subtypes from bone marrow and umbilical cord blood progenitors. kit ligand induced MC from umbilical cord blood predominantly of two immunophenotypes, MCT positive for tryptase but negative for chymase, and MCTC positive for tryptase and chymase. In contrast, the conditioned medium induced MC subtype MCTC and a third subtype, MCC, positive for chymase but negative for tryptase from both bone marrow and umbilical cord blood progenitors. In situ hybridization analyses confirmed the existence of a chymase-positive, tryptase-negative human MC in the culture. In umbilical cord blood cultures supplemented with both conditioned medium and kit ligand, the number of MCC cells was up-regulated from 2.7 to 34.0% of the total cells in the culture. In contrast, the number of MCT cells declined from 54.3 to 21.5% on day 49 of culture. In bone marrow cultures supplemented with conditioned medium alone, the MCC subtype represented 100% of the MCs on day 10 of culture. This study clearly demonstrates that a third type of MC, MCC, expressing chymase without concomitant expression of tryptase can be induced in vitro from normal human progenitors. In addition, it shows that tryptase and chymase, two MC-specific proteases, can be differentially expressed in in vitro derived human MCs by changing the cytokine combination of the culture.

In vitro characterization of fetal hematopoietic stem cells: Range and kinetics of cell production from individual stem cells

We have developed methods for detailed characterization of the proliferation kinetics and lineage potential of single human hematopoietic progenitor cells in an in vitro culture system. Fetal bone marrow CD34(hi)/lin(-) cells were cultured at one cell per well in the presence of c-kit ligand (KL), interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) on a murine stroma cell monolayer. Individual wells were scored for growth between 1 and 10 weeks of culture and analyzed by flow cytometry for lineage composition. A wide variation in time (1 to 8 weeks) was observed before initial cell division, even in the presence of cytokines promoting cell division in primitive progenitors. Eleven percent of the plated cells eventually produced a confluent culture well of approximately 20,000 progeny. Confluent wells were harvested and individually analyzed by flow cytometry for cell surface phenotype. Forty-eight percent of confluent wells contained primitive progenitors (CD34(+)lin(-)), 16% contained B-lymphoid cells (CD19(+) or CD10(+)), and 100% contained cells committed to the myelo-erythroid lineage (CD33(+)). CD34(+)/lin(-) cells from confluent wells were replated at one cell per well in secondary culture and the analysis repeated. One of 216 original single cells plated produced populations of B-lymphoid cells, myeloid cells, and primitive progenitors (CD34(+)/lin(-)) which persisted through two expansion cycles. We estimate that more than 36 million cells can be produced from a single cell under these culture conditions. A very small percentage of the CD34(hi)/lin(-) population (about 1%) was responsible for the majority of subsequent cell production. Our estimate of stem cell content in fetal bone marrow, defined by self-renewal as well as both B-lymphoid and myeloid differentiation from one cell, is approximately 1/13,000. This assay system provides direct in vitro measurements of the expected characteristics of hematopoietic stem cells (high proliferation potential, multilineage potential, self-renewal, and quiescence), and is therefore well suited to assessment of stem cell activity within various cell populations.

Dendritic Cells and Macrophages Can Mature Independently From a Human Bone Marrow-Derived, Post-Colony-Forming Unit Intermediate

CD34+ precursors in normal human bone marrow (BM) generate large numbers of dendritic cells alongside macrophages and granulocytic precursors when cultured for 12 to 14 days in c-kit ligand, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). This study reports an intermediate cell type that develops by day 6, and has the potential to differentiate into either macrophages or dendritic cells. When the d6 progeny are depleted of mature macrophages and residual CD34+ precursors, a discrete CD14+ HLA-DR+ population persists in addition to immunostimulatory CD14- HLA-DR() dendritic cells. Half of the CD14+ HLA-DR+ population is in cell cycle (Ki-67+), but colony-forming units (CFUs) are no longer detectable. The calls are c-fms+, but lack myeloperoxidase and nonspecific esterase. They also possess substantial phagocytic and allostimulatory activity. These post-CFU, CD14+ HLA-DR+ intermediates develop into typical macrophages when recultured in the absence of exogenous cytokines. M-CSF supports up to approximately 2.5-fold expansion of macrophage progeny. In contrast, the combination of GM-CSF and TNF-alpha supports quantitative differentiation into dendritic cells, lacking c-fms, CD14, and other macrophage properties, and expressing HLA-DR, CD1a, CD83, CD80, CD86, and potent allostimulatory activity. Therefore, normal CD34+ BM precursors can generate a post-CFU bipotential intermediate in the presence of c-kit ligand, GM-CSF, and TNF-alpha. This intermediate cell type will develop along the dendritic cell pathway when macrophages are removed and GM-CSF and TNF-alpha are provided. Alternatively, it can differentiate along a macrophage pathway when recultured with or without M-CSF.

Thrombopoietin Primes Human Platelet Aggregation Induced by Shear Stress and by Multiple Agonists

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine-diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.

The transcription factors c-myb and GATA-2 act independently in the regulation of normal hematopoiesis.

The transcription factors c-myb and GATA-2 are both required for blood cell development in vivo and in vitro. However, very little is known on their mechanism(s) of action and whether they impact on complementary or overlapping pathways of hematopoietic proliferation and differentiation. We report here that embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually or in combination, underwent hematopoietic commitment and differentiation in the absence of added hematopoietic growth factors but that stimulation with c-kit and flt-3 ligands enhanced colony formation only in the c-myb transfectants. This enhancement correlated with c-kit and flt-3 surface receptor up-regulation in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid colony-forming ability in methyl cellulose; however, introduction of a full-length GATA-2 or c-myb cDNA, respectively, rescued the hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells expressed c-kit and flt-3 surface receptors and formed increased numbers of hematopoietic colonies upon stimulation with the cognate ligands. These results are in agreement with previous studies indicating a fundamental role of c-myb and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that GATA-2 and c-myb exert their roles in hematopoietic gene regulation through distinct mechanisms of action in nonoverlapping pathways.

Stem cell factor (c-kit ligand) influences eosinophil recruitment and histamine levels in allergic airway inflammation.

The increased reactivity of mast cells during allergic airway inflammation has been linked to several aspects of pulmonary disease. A primary inducer of mast cell differentiation, proliferation, and activation has been identified as c-kit ligand or stem cell factor (SCF). In the present study, we used an established murine model of allergic eosinophilic airway inflammation to examine the role of SCF during an Ag-specific airway response. Initial data demonstrates increased SCF protein production at 8 h postchallenge in both lungs and serum of allergen-challenged, but not vehicle-challenged, mice. The immunolocalization of SCF in Ag-challenged lungs suggested that macrophage populations were the primary source of SCF, while epithelial cell regions also stained positive. Intense immunohistochemical staining of macrophages in bronchoalveolar lavage samples recovered from Ag-sensitized mice indicate that these cells may be a significant source of SCF in the lungs. Alveolar macrophages from the airways of normal mice stimulated with either TNF (0.1-10 ng/ml) or IL-4 (10 ng/ml) produced significant levels of SCF. Furthermore, neutralization studies demonstrated that the inhibition of airway SCF during allergen challenge significantly decreased eosinophil, but not neutrophil, infiltration throughout the response. Furthermore, when mice were treated with anti-SCF Ab, histamine levels were significantly reduced at 8 h postchallenge, the time of significant SCF production. Together, these data indicate that the production of SCF during Ag-induced lung inflammation by alveolar macrophages can play a significant role in the subsequent recruitment of eosinophils, possibly via mast cell activation and degranulation.