C-kit Ligand Research Articles

Interleukin 11.

IL-11 is a pleiotropic cytokine originally isolated from a bone marrow stromal cell line. It has been shown to share many activities with IL-6, namely to stimulate T cell-dependent B cell maturation, megakaryopoiesis and various stages of myeloid differentiation, but to inhibit adipogenesis. However, the activity of IL-11 on different stages of erythropoiesis in vitro clearly sets it apart form IL-6. IL-11 has little hematopoietic colony stimulatory activity of its own although it sustains terminal differentiation of the late erythroid progenitors CFU-E. In combination with IL-3, IL-11 has profound stimulatory effects on early multipotent hematopoietic progenitors (pre-CFCmulti), on multilineage colony-forming cells (CFCmulti), as well as on erythroid progenitors. The combination of IL-11 with the ligand for c-kit (KL) preferentially acts on early cells since it promotes the multiplication of pre-CFCmulti and stimulates highly proliferative erythroid progenitors that yields remarkable macroscopic erythroblast colonies in culture. The synergistic activity of IL-11 and KL, two stromal factors present in the bone marrow microenvironment, points to a pivotal role of IL-11 in early hematopoiesis. In vivo administration of recombinant human IL-11 elevates the number of circulating neutrophils and platelets and increased megakaryopoiesis in normal mice and primates.

Stem cell factor is a potent synergistic factor in hematopoiesis.

Stem cell factor (SCF), a ligand for c-kit, has a broad range of activities including effects on cells at or near the level of the multipotential stem cell as well as on committed cells. Preclinical studies show that SCF can protect against lethal irradiation, elicit multilineage responses in peripheral blood and bone marrow cellularity, and increase circulating peripheral blood progenitor cells (PBPC) in a dose-dependent manner. Recombinant human SCF has major clinical potential through its synergy with other factors, especially recombinant human granulocyte colony-stimulating factor, to enhance mobilization of PBPC.

Stem cell factor induces mast cell adhesion to fibronectin.

Stem cell factor (SCF) or c-kit ligand is a growth factor cytokine produced by stromal cells that is known to influence mast cell proliferation and differentiation. We hypothesized that SCF may also influence the adhesion of mast cells to connective tissue matrix. To examine this hypothesis, we stimulated MCP5/L mast cells or murine bone marrow-derived cultured mast cells (BMCMC) with either SCF or PMA and observed adhesion to fibronectin (FN). As expected, 80 to 90% of PMA-activated MCP5/L cells or BMCMC adhered to FN. In addition, SCF promoted MCP5/L cell or BMCMC adhesion to FN in a dose-response fashion with 50 to 60% of BMCMC adhering to FN at a concentration 10 ng/ml of SCF. BMCMC adhesion was observed with as little as 200 pg/ml of SCF. Adhesion of SCF stimulated BMCMC to FN did not require IL-3, but was dependent on the concentration of FN used to coat the assay surface. Mast cell adhesion in the presence of SCF appeared to occur through an integrin receptor as adhesion was calcium dependent and could be blocked by an RGD (Ang, Gly, Asp)-containing peptide. SCF did not directly mediate adhesion through interaction with c-kit, as FN-coated surfaces exposed to SCF before initiation of the adhesion assay did not promote adhesion in the absence of soluble SCF. Rather, SCF appeared to stimulate adhesion to FN by activating mast cells through its interaction with c-kit. Thus, antibody to SCF blocked adhesion, and rat and murine SCF stimulated BMCMC adhesion to FN, but human SCF, which does not bind to murine c-kit, did not stimulate adhesion. Genistein, which inhibits tyrosine kinase activity, partially inhibited SCF-induced adhesion. SCF thus stimulates mast cell adhesion and, because SCF is produced normally in tissues, it may be a major factor responsible for the adhesion of mast cells to connective tissue matrix under physiologic conditions.

Porcine stem cell factor/c-kit ligand: its molecular cloning and localization within the uterus.

Stem cell factor (SCF), or c-kit ligand, is a multipotent growth factor that has been implicated in an important role in various aspects of animal development, including maintenance of the viability of primordial germ cells. A porcine SCF (pSCF) cDNA was generated from porcine uterine endometrial mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR), and its nucleotide sequence was determined. The 952-bp pSCF cDNA contained an open reading frame encoding 274 amino acids. The deduced amino acid sequence of pSCF is approximately 86%, 83%, and 82%, identical to human, rat, and mouse SCFs, respectively; and it contains the four conserved cysteine residues and Asn-linked glycosylation sites. One additional amino acid was identified in pSCF, Glu130, which is not in the human (hSCF), rat (rSCF), or mouse (mSCF) sequences. Northern analysis of poly(A)+ RNA obtained from Day 16 pregnant endometrium revealed a transcript of approximately 6.5 kb. The size of this transcript is consistent with the size of full-length SCF mRNA, and the occurrence of alternatively spliced pSCF mRNAs were not detected by RT-PCR/Southern hybridization analysis of endometrial and ovarian total cellular RNA (tcRNA). Porcine SCF mRNA has been localized by in situ hybridization in porcine endometrial stromal tissue. Pregnancy does not appear to be a prerequisite for pSCF mRNA expression in endometrial tissue since it was detectable in tissue and tcRNA obtained from pregnant and nonpregnant gilts. The biological significance of uterine pSCF expression is currently unclear, but it probably participates in intracellular communication within the uterus.

C-kit ligand combined with GM-CSF and/or IL-3 can expand CD34+ hematopoietic progenitor subsets for several weeks in vitro.

It might be possible to facilitate engraftment after transplantation of purified hematopoietic progenitor cells if the cells are stimulated ex vivo prior to transplantation. The aim of this study was to analyze the potential of c-kit ligand (CKL) combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) to induce proliferation and differentiation of human bone marrow CD34+ cells in vitro. Particular attention was paid to the ability to expand populations that could maintain progenitor characteristics, i.e. CD34 expression and generation of colony forming cells (CFC), for a considerable period of time. Purified CD34+ cells were cultured in liquid medium for 42 days interrupted by immunophenotyping and CFC assays. In the presence of CKL combined with GM-CSF and/or IL-3, the total number of cells expressing CD34 increased significantly for several weeks after an initial decline. Further, CFC were continually recovered in these cultures. Based on the kinetics and the flow cytometry analysis, the expanding populations that continued to express CD34 probably originated from noncommitted, immature CD34+ cell subsets. CKL combined with GM-CSF and/or IL-3 also induced strong cell proliferation. The majority of the proliferating cells lost CD34 expression and acquired a series of mature myeloid cell surface markers associated with the monocytic, granulocytic and megakaryocytic lineages. These cells probably originated from committed CD34+ cell subsets. We conclude that CKL combined with GM-CSF and/or IL-3 can stimulate noncommitted, immature as well as committed CD34+ cell populations to expand and to differentiate. This property might be useful in a short-term ex vivo pretransplant stimulation of CD34+ cells in an attempt to facilitate rapid and stable engraftment after stem cell transplantation.

Development of human mast cells from their progenitors

Two types of human mast cells, which are morphologically similar to skin mast cells and lung mast cells, repectively, can be developed from pluripotent stem cells under different culture conditions. The major growth factor for mast-cell development is c-kit ligand, which induces mastocytosis in vivo. However, this cytokine is not sufficient for full maturation of the cells.

Insulin-Like Growth Factor-1 Potentiates Expansion of Interleukin-7–Dependent Pro-B Cells

Commitment to B-lymphocyte differentiation is characterized by expression of the B220 form of the common leukocyte antigen (Ly-5) and D-JH rearrangement of the Ig heavy chain gene complex. B-lineage progenitor cells, or pro-B cells, that have initiated Ig gene rearrangement, but do not express detectable Ig heavy or light chain protein, have recently been shown to retain substantial capacity for expansion in vitro in the presence of bone marrow (BM) stromal cells and interleukin-7 (IL-7). Although the potentiating effect of stromal cells on pro-B-cell proliferation can be partially attributed to the ligand for the proto-oncogene receptor c-kit (c-kit ligand [KL] or stem cell factor), several lines of evidence suggest that c-kit-mediated cell signalling is not required for pro-B-cell expansion. Previous studies from this laboratory demonstrated that insulin-like growth factor-1 (IGF-1) potentiated the proliferative effect of IL-7 on nonadherent cells from lymphoid long-term BM cultures in a manner similar to that shown for KL. To further delineate specific cell stages that respond to lymphopoietic cytokines, we derived continuously proliferating pro-B-cell lines from day-14 murine fetal liver in the presence of IL-7 and BM stromal cell clone S10. Initial expansion and continued proliferation of these pro-B-cell lines was absolutely dependent on the presence of both IL-7 and stromal cells. In the absence of KL, IL-7-stimulated proliferation of these cells in short-term cultures and addition of either recombinant IGF-1 or KL significantly potentiated this proliferative response. Although IGF-2 and insulin also potentiated the effect of IL-7, our data suggest that neither IGF-2 nor insulin represent normal regulators of intramyeloid lymphocyte development. IGF-1 and KL activate unique cascades of intracellular signalling events and inclusion of both cytokines in cultures of IL-7-stimulated pro-B cells resulted in additive potentiation of the proliferative response. Taken together, these results suggest that expansion of pro-B cells in vivo is maintained by at least three stromal cell-derived cytokines. IL-7 appears to be unique in delivering the primary proliferative signal for pro-B-cell expansion; however, both KL and IGF-1 potentiate the proliferative effect of IL-7 on these cells. The functional redundancy and additive effects of IGF-1 and KL as amplification signals for developing B-lineage cells underscore the essential nature of clonal expansion and diversification in development of immunocompetent lymphoid cells.

Insulin-like growth factor-1 potentiates expansion of interleukin-7- dependent pro-B cells

Commitment to B-lymphocyte differentiation is characterized by expression of the B220 form of the common leukocyte antigen (Ly-5) and D-JH rearrangement of the Ig heavy chain gene complex. B-lineage progenitor cells, or pro-B cells, that have initiated Ig gene rearrangement, but do not express detectable Ig heavy or light chain protein, have recently been shown to retain substantial capacity for expansion in vitro in the presence of bone marrow (BM) stromal cells and interleukin-7 (IL-7). Although the potentiating effect of stromal cells on pro-B-cell proliferation can be partially attributed to the ligand for the proto-oncogene receptor c-kit (c-kit ligand [KL] or stem cell factor), several lines of evidence suggest that c-kit-mediated cell signalling is not required for pro-B-cell expansion. Previous studies from this laboratory demonstrated that insulin-like growth factor-1 (IGF-1) potentiated the proliferative effect of IL-7 on nonadherent cells from lymphoid long-term BM cultures in a manner similar to that shown for KL. To further delineate specific cell stages that respond to lymphopoietic cytokines, we derived continuously proliferating pro-B-cell lines from day-14 murine fetal liver in the presence of IL-7 and BM stromal cell clone S10. Initial expansion and continued proliferation of these pro-B-cell lines was absolutely dependent on the presence of both IL-7 and stromal cells. In the absence of KL, IL-7-stimulated proliferation of these cells in short- term cultures and addition of either recombinant IGF-1 or KL significantly potentiated this proliferative response. Although IGF-2 and insulin also potentiated the effect of IL-7, our data suggest that neither IGF-2 nor insulin represent normal regulators of intramyeloid lymphocyte development. IGF-1 and KL activate unique cascades of intracellular signalling events and inclusion of both cytokines in cultures of IL-7-stimulated pro-B cells resulted in additive potentiation of the proliferative response. Taken together, these results suggest that expansion of pro-B cells in vivo is maintained by at least three stromal cell-derived cytokines. IL-7 appears to be unique in delivering the primary proliferative signal for pro-B-cell expansion; however, both KL and IGF-1 potentiate the proliferative effect of IL-7 on these cells. The functional redundancy and additive effects of IGF-1 and KL as amplification signals for developing B- lineage cells underscore the essential nature of clonal expansion and diversification in development of immunocompetent lymphoid cells.

Ultrastructural morphology of immature mast cells in sequential suspension cultures of human cord blood cells supplemented with c-kit ligand; distinction from mature basophilic leukocytes undergoing secretion in the same cultures

The ability to culture human mast cells and, thus, to determine their ontogeny and possible relationships to other lineages has been facilitated by new studies using cocultures of cord blood cells with mouse fibroblasts and recombinant human or murine c-kit ligand-supplemented suspension cultures of cord blood cells. In this study, we examined c-kit ligand-supplemented cord blood cell suspension cultures designed so that the effects of growth factor source, individual cord sample, and culture time (3-17 weeks) on the developing mast cell lineage could be individually evaluated. We found that human mast cells, basophils, neutrophils, eosinophils, macrophages, megakaryocytes, and endothelial cells were present in these cultures. The numbers of mast cells and their granules increased with culture time; mature basophils, present in quantity in 3-week cultures, decreased in number and released granule contents with increased culture times. The mast cell lineage developed similarly, regardless of which factor preparation was added to cultures, but considerable variability existed among individual donors from whom cord bloods were obtained. Unlike the mature, crystal-containing mast cells that regularly developed in fibroblast cord blood cocultures (Furitsu et al. [1989] Proc. Natl. Acad. Sci. USA 86, 10039-10043), human mast cells failed to attain full maturity in the suspension cultures examined here, regardless of individual cord sample, added growth factor, or culture time. Furthermore, unlike cells of the basophil lineage in which granule content release was regularly observed, morphologic evidence of secretion from human mast cells was absent. Instead, these cells were actively undergoing granule building as determined by the increasing numbers of granules and filling of these containers over culture time. Crystal granules never developed, even at the maximum culture time of 17 weeks. We conclude that fibroblasts are necessary and sufficient for the differentiation and maturation of human mast cells in vitro from their agranular precursors in cord blood but that soluble c-kit ligand is not.

Induction by IL-9 and suppression by IL-3 and IL-4 of the levels of chromosome 14-derived transcripts that encode late-expressed mouse mast cell proteases.

Abstract Immature, rIL-3-dependent mouse bone marrow-derived mast cells (BMMC) contain high steady-state levels of the mouse mast cell protease (mMCP) 5 transcript but undetectable levels of the mMCP-1, mMCP-2, or mMCP-4 transcripts even though all four of their genes reside at a locus on chromosome 14. These mast cells can be induced by recombinant c-kit ligand (rKL) to obtain high steady-state levels of the mMCP-4 transcript and by rIL-10 to obtain high steady-state levels of the mMCP-1 and mMCP-2 transcripts. rIL-3 and rKL both elicit the differentiation of progenitor cells into immature BMMC and then stimulate their proliferation. We now report that although rIL-9 alone has no effect on BMMC proliferation as assessed by their incorporation of [3H]thymidine, rIL-9 in combination with rKL enhances the long term viability of BMMC. Furthermore, rIL-9 in the presence of rKL stimulates mouse BMMC to undergo a phenotypic change by inducing accumulation of high steady-state levels of the mMCP-1 and mMCP-2 transcripts. In contrast, in BMMC, the presence of rIL-4 suppresses the rIL-9-induced accumulation of the mMCP-1 and mMCP-2 transcripts, the rIL-10-induced accumulation of the mMCP-1 and mMCP-2 transcripts, and the rKL-induced accumulation of the mMCP-4 transcript, but not the rIL-3-induced accumulation of the mMCP-5 transcript. The presence of rIL-3 also suppresses the rIL-9-induced accumulation of the mMCP-1 and mMCP-2 transcripts. Because of their counter-regulatory actions on the steady-state levels of transcripts that encode three late-expressed serine proteases in BALB/cJ mice, rIL-4 and rIL-3 both inhibit the final stages of differentiation and maturation of mast cells. Because rIL-4, unlike rIL-3, is neither an inducer of early-expressed proteases nor alone a proliferative factor for BMMC, the counterregulatory actions of rIL-3 and rIL-4 on differentiation and maturation of these mouse mast cells are independent of their other functions.

Effects of chronic treatment with the c-kit ligand, stem cell factor, on immunoglobulin E-dependent anaphylaxis in mice. Genetically mast cell-deficient Sl/Sld mice acquire anaphylactic responsiveness, but the congenic normal mice do not exhibit augmented responses.

We treated genetically mast cell-deficient WCB6F1-Sl/Sld mice and the congenic normal (WCB6F1(-)+/+) mice with the c-kit ligand recombinant rat stem cell factor164 (rrSCF164; 100 micrograms/kg per d, subcutaneously) or with vehicle for 21 d, then passively sensitized the mice with anti-dinitrophenol30-40 immunoglobulin E (IgE) antibodies, and 1 d later measured the changes in heart rate, pulmonary dynamic compliance, and pulmonary conductance, and assessed the death rates associated with intravenous challenge of these animals with specific antigen. rrSCF164 treatment induced the development of mast cells in Sl/Sld mice, and these mice exhibited tachycardia, but not death, after challenge with IgE and antigen. rrSCF164 treatment induced mast cell hyperplasia in +/+ mice, but the cardiopulmonary changes associated with passive anaphylaxis in these mice were virtually indistinguishable from those observed in control +/+ mice treated with vehicle instead of rrSCF164. Moreover, the highest dose of antigen challenge produced significantly fewer fatalities in rrSCF164-treated than in vehicle-treated +/+ mice (1/11 vs. 8/11, respectively, P < 0.01). Thus, in normal mice, chronic treatment with rrSCF164 induces mast cell hyperplasia but does not increase, and in certain respects diminishes, the severity of IgE-dependent anaphylactic reactions.

IL-3-dependent murine mast cells undergo apoptosis on removal of IL-3. Prevention of apoptosis by c-kit ligand.

It is well established that mast cell proliferation and maturation are regulated by two principle cytokines, IL-3 and the c-kit ligand stem cell factor (SCF). Little is known, however, how these two processes are negatively regulated and thus, how mast cell number is controlled in normal or pathologic processes. In this study we hypothesized that IL-3-dependent mast cells would undergo programmed cell death (apoptosis) on removal of IL-3 as was shown with other growth factor-dependent hemopoietic cells. Apoptotic changes were analyzed using light microscopy, fluorescent staining with acridine orange, flow cytometric analysis, and DNA electrophoresis. We could demonstrate that elimination of IL-3 from either primary bone marrow-derived cultured mast cell cultures (BMCMC) or from the growth factor-dependent mast cell line MCP5 resulted in the characteristic changes of apoptosis including condensed chromatin, fragmented nuclei, cellular vacuolization, typical pattern of propidium iodide or Hoechst 33342 uptake by flow cytometry, and the characteristic 200 bp "ladder" pattern of DNA cleavage. These events were prevented by SCF, an action that was in part mediated by tyrosine kinases, in that the tyrosine kinase inhibitor herbimycin inhibited the action of SCF in preventing apoptosis in IL-3-deprived cells. By using anti c-kit mAb and IL-3-dependent BMCMC obtained from W/Wv mice homozygous for mutation at the w locus that encodes the c-kit receptor, we could also show that SCF exerted its effect via c-kit. Neither dexamethasone nor cyclosporin A inhibited the "rescue" effect of SCF, suggesting that "rescue" was mediated by SCF and not through the induction of other cytokines. Thus, IL-3-dependent mast cells undergo apoptosis on removal of IL-3, an event that is prevented by the addition of SCF through its ligand c-kit, thus demonstrating how these principle mast cell growth factors may act in concert to regulate mast cell number under physiologic conditions.

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.

Expression of c-kit ligand in human keratinocytes

The c-kit ligand is expressed on tissue-anchored stromal cells. It plays an important role in the development of c-kit-bearing cells, such as haematopoietic cells, germ cells, mast cells and melanocytes. In the present study, we used the reverse transcriptase-mediated polymerase chain reaction (PCR) technique to investigate whether human keratinocytes are able to express c-kit ligand mRNA. Two sets of primers were designed to distinguish two types of c-kit ligand mRNA (full-length type and spliced type). One set was used to amplify an 882-bp DNA fragment from the full-length type, and a 798-bp DNA fragment from the spliced type. Another set was used to amplify a 375-bp DNA fragment from the full-length type only. A cDNA fragment corresponding to the full-length type mRNA was amplified from a cDNA preparation of cultured human keratinocytes as well as from epidermis obtained by the suction blister technique. This result indicates the spontaneous transcription of full-length type mRNA of the c-kit ligand in human keratinocytes.

Constitutive Expression of Steel Factor Gene by Human Stromal Cells

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

Constitutive expression of steel factor gene by human stromal cells

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

Expression of tyrosinase, tyrosinase-related protein 1 and 2, C-kit and C-kit ligand in vitro and in vivo

Expression of tyrosinase, tyrosinase-related protein 1 and 2, C-kit and C-kit ligand in vitro and in vivo

W-sash affects positive and negative elements controlling c-kit expression: ectopic c-kit expression at sites of kit-ligand expression affects melanogenesis.

The receptor tyrosine kinase c-kit and its cognate ligand KL are encoded at the white spotting (W) and steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl locus cause deficiencies in gametogenesis, melanogenesis and hematopoiesis (erythrocytes and mast cells). The W-sash mutation differs from most W mutations in that it affects primarily mast cells and melanogenesis but not other cellular targets of W and Sl mutations. Thus, Wsh/Wsh mice are fertile and not anemic, but they lack mast cells in their skin and intestine and are devoid of coat pigment. Heterozygotes are black with a broad white sash/belt in the lumbar region. In order to determine the basis for the phenotypes of W-sash mice, we investigated c-kit RNA and protein expression patterns in adult Wsh/Wsh mice and during embryonic development. We show that c-kit expression is absent in bone-marrow-derived Wsh/Wsh mast cells, the fetal and the adult lung, and the digestive tract at embryonic day 13 1/2 (E13 1/2), tissues that normally express c-kit. Unexpectedly, in E10 1/2 and 11 1/2d Wsh/Wsh embryos, we found c-kit expression in the dermatome of the somites, the mesenchyme around the otic vesicle and the floorplate of the neural tube, structures known to express the c-kit ligand in wild-type embryos. The ectopic c-kit expression in Wsh homozygous embryos does not affect c-kit ligand expression. The presumed Wsh/Wsh melanoblasts appeared to be normal and, at E10 1/2, similar numbers were found in normal and homozygous mutant embryos. At E13 1/2 +/+ embryos had a graded distribution of melanoblasts from cranial to caudal with a minimum in the lumbar region. Whereas E13 1/2 homozygous Wsh/Wsh embryos essentially lacked c-kit-positive cells in the skin, E13 1/2 heterozygous Wsh/+ embryos had reduced numbers of melanoblasts compared to +/+ with few or none in the lumbar region (future sash). It is proposed that ectopic c-kit expression in the somitic dermatome affects early melanogenesis in a dominant fashion. Molecular analysis of Wsh chromosomal DNA revealed a deletion or rearrangement in the vicinity of the c-kit gene. These results provide an explanation for the Wsh phenotype and have implications for the control of c-kit expression.

Self-renewal and differentiation of normal avian erythroid progenitor cells: Regulatory roles of the [formula omitted] and [formula omitted] receptors

The c- kit proto-oncogene product is a major regulator of early hematopoiesis in mice. We show here that the avian c-Kit protein, together with the c- erbB proto-oncogene product, regulates self-renewal and differentiation in two types of normal chick erythroid progenitors. A relatively frequent progenitor expressing only c-Kit transiently proliferated in response to avian c-Kit ligand (stem cell factor [SCF]). A second, rare progenitor coexpressed c-Kit and c-ErbB and was induced to long-term self-renewal by SCF or transforming growth factor α (TGFα), a c-ErbB ligand. In the absence of SFC or TFGα, both progenitors underwent erythropoietin (Epo)-dependent terminal differentiation with indistinguishable kinetics. Interestingly, Epo induced differentiation in the SCF progenitors even when SCF was present. In contrast, the c-ErbB-expressing, TGFα-induced progenitors continued to self-renew when treated with Epo plus the growth factors SCF, TGFα, or both. Expression of c-ErbB thus may be a dominant determinant for the sustained self-renewal of committed erythroid progenitors.

Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells, a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells, purified from umbilical cord blood by flow cytometry and cell sorting, were selected for this study because of their high content of clonogenic cells (34%), particularly multipotent progenitors (CFU-MIX, 12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF, IL-6, IL-3, and erythropoietin (Epo); (3) MGF, IL-6, granulocyte-macrophage colony- stimulating factor (GM-CSF)/IL-3 fusion protein (FP), macrophage colony- stimulating factor (M-CSF), and granulocyte-CSF (G-CSF); and (4) MGF, IL-6, FP, M-CSF, G-CSF, and Epo. Maximum numbers of erythroid progenitors (BFU-E, up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF, IL-6, IL-3, and Epo, whereas maximum levels of myeloid progenitors (CFU-C, up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF, IL-6, FP, M-CSF, and G-CSF. When MGF, IL-6, FP, M-CSF, G-CSF, and Epo were present, maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore, the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors, but rather had a permissive effect on the development of lineage-restricted clonogenic cells.