Differential activity of recombinant colony-stimulating factors in supporting proliferation of human peripheral blood and bone marrow myeloid progenitors in culture

Unlike bone marrow progenitor cells, human myeloid progenitors isolated from peripheral blood do not form colonies in semi-solid medium in the presence of rhG-CSF, rhM-CSF or rhIL-6, but do form colonies containing neutrophils, macrophages, eosinophils, basophils or mixed neutrophilic-macrophages colonies in the presence of rhIL-3 or rhGM-CSF. Priming of blood progenitors by culturing them for several days in the presence of rhGM-CSF resulted in a dramatic increase in the frequency of cells that proliferate in response to G-CSF and IL-6 and form neutrophilic granulocytic colonies. Suspension cultures maintained in the presence of IL-3 yielded increased numbers of clonogenic cells responsive to GM-CSF and G-CSF, but not to M-CSF or IL-6. rhIL-6 did not directly stimulate colony formation of peripheral blood progenitors but did prime them to respond to G-CSF. These results are consistent with a hierarchical model of granulocytic differentiation in which circulating progenitors proceed sequentially through a programme of changing growth factor sensitivity with the following sequence: IL-3, GM-CSF, IL-6 and/or G-CSF.     

Synergism of interleukin-12 and interleukin-3 on development of hematopoietic progenitors

Abstract: The recently cloned cytotoxic lymphocyte maturation factor [CLMF] also called NK cell stimulatory factor [NKSF] or interleukin-12 [IL-12] has been described as a growth factor for mature lymphoid cells. The present study investigated whether purified recombinant human IL-12 could stimulate CFU colony growth. Source of progenitor cells were peripheral blood cells depleted of adherent, CD2- and CD56-positive cells. RhIL-12 was investigated either alone or in combination with rhIL-3, rhIL-6 and rhGM-CSF. RhIL-12 alone did not support colony formation of myeloid or erythroid progenitors. RhIL-12 in combination with rhIL-3 increased the numbers of BFU-E and CFU-GM. No synergism or additive effect was seen with the combination of rhIL-12 and rhGM-CSF or rhIL-12 and rhIL-6. An additive increase in the number of granulocytic colonies was observed when rhIL-3, rhIL-6 and rhGM-CSF were used together with rhIL-12. Our result therefore suggest that, in addition to being a potent lymphopoietic stimulator, IL-12 acts synergistically with IL-3 in enhancing the sensitivity of hemopoietic progenitors to IL-3.     

The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines.

beta-Glucans stimulate leukocyte anti-infective activity, enhance murine hematopoietic recovery following bone marrow injury and mobilize murine progenitor cells from bone marrow. This study evaluated the in vitro hematopoietic potential of the beta-glucan, PGG-glucan, on human bone marrow mononuclear cells (BMMC) and CD34+ BMMC compared with protein cytokines. In the presence of submaximal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 0.5 ng/ml), PGG-glucan significantly increased BMMC myeloid colony formation comparable to the increase observed with either interleukin-3 (rhIL-3) or stem cell factor (rhSCF). Moreover, the addition of PGG-glucan to cultures containing GM-CSF + IL-3 or GM-CSF + SCF significantly augmented granulocyte-macrophage colony production above baseline, demonstrating that PGG-glucan acts independently of those early-acting cytokines and can enhance their activity in an additive manner. Anti-PGG-glucan monoclonal antibody specifically abrogated the growth-enhancing effect of added PGG-glucan in a saturable manner and other control carbohydrate polymers failed to affect colony formation. Further, PGG-glucan was not associated with induction of IL-6, GM-CSF production and removal of accessory cells by CD34+ cell isolation did not alter the PGG-glucan effect. These data demonstrate that PGG-glucan acts on committed myeloid progenitors to enhance human hematopoietic activity by a mechanism of direct action independent of IL-3 or SCF and independent of secondary cytokine stimulation.     

Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages.

The cDNA for human stem cell factor (hSCF) has been cloned and expressed in mammalian and bacterial hosts and recombinant protein purified. We have examined the stimulatory effect of recombinant human SCF (rhSCF) on human bone marrow cells alone and in combination with recombinant human colony stimulating factors (CSFs) and erythropoietin (rhEpo). RhSCF alone resulted in no significant colony formation, however, in the presence of rhGM-CSF, rhG-CSF or rhIL-3, rhSCF stimulated a synergistic increase in colony numbers. In addition, increased colony size was stimulated by all combinations. The morphology of cells in the colonies obtained with the CSFs plus rhSCF was identical to the morphology obtained with rhGM-CSF, rhG-CSF or rhIL-3 alone. RhEpo also synergised with rhSCF to stimulate the formation of large compact hemoglobinized colonies which stained positive for spectrin and transferrin receptor and had a morphological appearance consistent with normoblasts. RhSCF stimulation of low density non-adherent, antibody depleted, CD34+ cells suggests that rhSCF directly stimulates progenitor cells capable of myeloid and erythroid differentiation.     

Prevalence of the primitive megakaryocyte progenitors (BFU-meg) in adult human peripheral blood.

The in vitro growth of early (megakaryocyte burst-forming units, BFU-meg) and late (megakaryocyte colony-forming units, CFU-meg) megakaryocyte (meg) progenitors has been evaluated in normal adult human peripheral blood (PB). All the experiments were carried out using CD34+ cells, which were assayed in a serum-free fibrinclot assay. PB BFU-meg were morphologically characterized as plurifocal aggregates containing greater than 50 cells/colony, distinct from unifocal CFU-meg, in a limiting dilution assay. At variance with PB CFU-meg, PB BFU-meg were unaffected by the complement-mediated cytotoxicity with anti-HLA-DR. The optimal source of colony-stimulating activity for PB BFU-meg growth was recombinant human interleukin 3 (rhIL-3; 100 U/ml), which supported a significantly higher number of BFU-meg in comparison with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 200 U/ml, p = 0.043). Combinations of rhIL-3 (100 U/ml) plus rhGM-CSF (200 U/ml), rhIL-3 plus recombinant human interleukin 6 (rhIL-6; 100 U plus 100 U/ml) or rhIL-3 plus rhGM-CSF plus rhIL-6 (100 U plus 200 U/ml plus 100 U/ml) failed to further increase the number of PB BFU-meg with respect to rhIL-3 (100 U/ml) alone. Both PB BFU-meg and CFU-meg were markedly inhibited, in a dose-dependent fashion, by increasing doses of human purified transforming growth factor-beta 1 (TGF-beta 1) (from 0.001 to 10 ng/ml). Finally, the CFU-meg/BFU-meg ratio in PB (0.52) was significantly different from that of normal bone marrow (2.3), clearly indicating that adult human peripheral blood predominantly carries primitive megakaryocytic progenitors.     

Inhibition of human granulocyte-macrophage colony formation by interleukin 2-treated lymphocytes

The effects of interleukin 2 (IL-2)-treated lymphocytes on human myeloid progenitors (CFU-GM) were studied. When peripheral blood mononuclear cells (PBMC) were cultured for 3 days with recombinant IL-2, they developed lymphokine-activated killer (LAK) activity against normal bone marrow cells, and also suppressed colony formation by CFU-GM. Suppression of CFU-GM was found to be mediated mainly by natural killer (NK) cells, and to a lesser degree by T cells according to the results showing that isolated NK cells and T cells exhibited strong and moderate suppressor function, respectively. Since the levels of LAK activity and of CFU-GM inhibitory activity were not parallel in each individual, inhibition of CFU-GM does not seem to be due to a direct lytic action by LAK cells. This possibility was supported by our finding that the supernatant of IL-2-treated PBMC contained factor(s) that inhibited CFU-GM colony formation.     

Recombinant human interleukin 6 is a potent inducer of the acute phase response and elevates the blood platelets in nonhuman primates

Human interleukin 6 (IL-6) produced by molecular cloning was administered to nonhuman primates to assess its biological activities in vivo. Rhesus monkeys were treated s.c. with recombinant human (rh) IL-6 at 3 and 30 micrograms/kg body weight/day for 11 days, followed by the administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) at 5.5 micrograms/kg/day for 5 days. Serum levels of positively regulated acute phase proteins (APP) (C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin) increased, whereas negatively regulated APP (prealbumin) decreased in response to rhIL-6 treatment in a dose-dependent manner. Platelet counts rose after a latent period of 4-5 days following the start of rhIL-6 treatment, resulting in a maximum twofold increase above normal levels 2-3 days after the termination of the rhIL-6 treatment. Recombinant human IL-6 treatment induced a two to threefold rise in myeloid progenitor blood cell levels. The subsequent administration of rhGM-CSF to rhIL-6-pretreated animals did not increase the progenitor cell levels in blood above those found with rhGM-CSF treatment alone, indicating that rhIL-6 compared to recombinant human interleukin 3 (rhIL-3) has a minor proliferative effect on hematopoietic precursors in vivo. In conclusion, rhIL-6 was shown to be a potent stimulator of APP and was able to increase the number of platelets in circulation in nonhuman primates.     

Regulation of human eosinophil precursor production by cytokines: a comparison of recombinant human interleukin-1 (rhIL-1), rhIL-3, rhIL-5, rhIL-6, and rh granulocyte-macrophage colony-stimulating factor

The effect of a panel of recombinant human (rh) cytokines on the generation of human eosinophil precursors was assessed using a two-step culture technique. Normal human bone marrow was preincubated with different cytokine combinations in liquid culture before assessment of the number of eosinophil progenitors, which give rise to eosinophil colony-forming units (CFU-Eo) on secondary semi-solid culture with either interleukin-5 (IL-5), IL-3, or granulocyte-macrophage colony-stimulating factor. rhIL-3 or rhGM-CSF, but not rhIL-5, increased the number of CFU-Eo. CFU-Eo production by rhIL-3 or rhGMCSF was maximal after 7 days' preincubation. Neither rhIL-1 or rhIL-6 acted on eosinophil precursors, either alone or in combination with rhIL-5, rhIL-3, or rhGM-CSF. A similar spectrum of activity of the cytokines was demonstrated whether rhIL-5, rhIL-3, or rhGM-CSF was used in the secondary cultures as the eosinophil CSF. However, rhIL-3 induced relatively more rhIL-5-responsive CFU-Eo than rhIL-3-responsive CFU-Eo, suggesting that rhIL-3 is pushing progenitors into an rhIL-5-responsive compartment.     

Inhibitory effect of granulocyte-macrophage colony-stimulating factor therapy on the generation of natural killer cells.

We investigated the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) therapy on the natural killer (NK) cell lineage in patients with aplastic anemia and myelodysplastic syndrome. Selected bone marrow (BM) cells were prepared by the elimination of nylon wool-adherent cells and mature T and NK cells from BM cells. The frequency of BM NK progenitors relative to BM cells selected was significantly decreased 4 weeks after the start of rhGM-CSF therapy (P less than .01), while the peripheral blood NK cell count and NK activity were also significantly decreased (P less than .05). A return to the pretreatment levels was seen 4 weeks after the cessation of treatment in all cases. No suppressive effect was noted in the patients who received rhG-CSF therapy. These results suggest that rhGM-CSF therapy suppresses the generation of NK cells from human BM NK progenitors.     

Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture.

The cDNA for canine stem cell factor (cSCF, c-kit ligand) was cloned and expressed in Escherichia coli. The recombinant protein (rcSCF), 165 amino acids in length, is very similar structurally to the soluble form of previously cloned and sequenced rodent and human SCFs. The biological effects of rcSCF were studied in a day-10 granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay and in long-term liquid bone marrow culture of non-adherent hematopoietic cells in the absence of a stromal underlayer. Synergism in the stimulation of growth of CFU-GM was demonstrated between rcSCF and both recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and naturally occurring colony-stimulating activity present in the serum of a neutropenic dog. Alone, rcSCF was nonstimulatory for committed marrow precursors in methylcellulose cultures and had minimal effect on hematopoietic progenitor cell survival in stromaless, liquid cultures. When rcSCF was combined with phytohemagglutinin-stimulated canine lymphocyte-conditioned medium (PHA-LCM) or rh interleukin 6 (IL-6), with or without rhGM-CSF, CFU-GM survived for up to 5 weeks. The combination of rcSCF and rhGM-CSF, without rhIL-6, led to an early increase in CFU-GM in liquid cultures that declined more rapidly than in flasks that included rhIL-6. Survival of progenitor cells was negligible beyond 1 week in flasks with growth factor combinations lacking rcSCF. Sustained production of nonadherent cells in long-term cultures also was dependent on rcSCF in combination with canine PHA-LCM or recombinant human growth factors. It appears that rcSCF, like that from rodent and primate species, has the ability to influence the survival and proliferation of CFU-GM, and perhaps earlier progenitor cells, in hematopoietic tissues. In a long-term liquid culture system in which growth factor production by stromal cells is limited, rcSCF possesses a unique ability to maintain the viability of progenitor cells for up to 5 weeks.     

Effect of granulocyte-macrophage colony-stimulating factor and interleukin-3 on interleukin-8 production by human neutrophils and monocytes

Interleukin-8 (IL-8) is a major neutrophil chemoattractant and functional stimulant that is induced by IL-1, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS). We report that recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhIL-3 are also potent inducers of IL-8 messenger RNA (mRNA) accumulation and protein secretion by normal peripheral blood monocytes. Neutrophils produce IL-8 in response to GM-CSF but not to IL- 3. In contrast, recombinant human granulocyte-CSF (rhG-CSF), at concentrations as high as 100 ng/mL, does not induce IL-8 in either cell type. rhGM-CSF also induces IL-8 mRNA expression and IL-8 protein in the promonocytic cell line, U-937, whereas rhG-CSF does not. IL-8 secretion by monocytes was stimulated within 2 hours after incubation with rhGM-CSF or rhIL-3. Stimulation of neutrophils with rhGM-CSF resulted in an increase in cell-associated IL-8 at 4 hours. At 24 hours, cell-associated IL-8 levels declined, whereas secreted IL-8 levels increased. In contrast, virtually all IL-8 induced in monocytes appeared as secreted protein. Neither rhGM-CSF nor rhIL-3 induced detectable secretion of IL-1, TNF alpha, or IL-6 protein by monocytes. rhGM-CSF, and to a lesser degree rhIL-3, potently stimulated IL-8 secretion in cultures of heparinized whole blood, whereas rhG-CSF had no significant effect on IL-8 secretion. Induction of IL-8 by GM-CSF may be physiologically important in enhancing the acute inflammatory response.     

Recombinant human interleukin-3 expands the pool of circulating hematopoietic progenitor cells in primates--synergism with recombinant human granulocyte/macrophage colony-stimulating factor

The in vivo effect of recombinant human interleukin-3 (rhIL-3) on peripheral blood (PB) levels of hematopoietic progenitor cells was studied in nonhuman primates. Subcutaneous administration of 33 micrograms/kg/d of rhIL-3 for 11 to 14 days to rhesus monkeys slightly raised leukocyte counts (twofold) and substantially expanded the pool of circulating stem cells in the second week of treatment. At the end of rhIL-3 administration, PB levels of granulocyte/macrophage colony-forming units (CFU-GM) increased by a mean of 12-fold; burst-forming units-erythroid (BFU-E) by ninefold; CFU-mix, by 12-fold; and CFU-megakaryocyte (Mk), by 13-fold as compared with their respective pretreatment values. Subsequent administration of recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF; 5.5 micrograms/kg/d for 5 days) to rhIL-3-pretreated animals further expanded the PB stem cell compartment leading to maximum levels of CFU-GM that were in average much more increased (63-fold) than CFU-GM levels under rhIL-3 (14-fold) or rhGM-CSF (12-fold) alone. This hitherto unknown effect of rhIL-3 on the pool of circulating progenitors, particularly in synergy with rhGM-CSF, may facilitate harvest of hematopoietic progenitor cells from PB for stem cell transplantation.     

A comparison of treatment of canine cyclic hematopoiesis with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF interleukin-3, and canine G-CSF.

Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG-CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.     

Effect of interleukin 6 (IL-6) on the differentiation and proliferation of murine and human hemopoietic progenitors

We examined the effect of human recombinant (r) interleukin 6 (IL-6) on the differentiation of murine and human hemopoietic progenitors. Human IL-6 supported colony formation by murine bone marrow cells. These colonies consisted of neutrophils and macrophages. Recombinant IL-6 was able to support multilineage colony formation by spleen cells from 5-fluorouracil (5-FU)-treated mice. These colonies consisted of greater than 1 x 10(4) cells. Differential counts revealed large colonies exhibiting different combinations of cell lineages: neutrophils, macrophages, eosinophils, mast cells, and megakaryocytes. However, when blast cell colonies supported by interleukin 3 were replated into secondary dishes containing IL-6, they could differentiate into only neutrophils and macrophages. Single cells transferred from blast cell colonies formed only neutrophil/macrophage colonies. These results indicate that IL-6 had a direct effect on the growth and development of murine granulocyte-macrophage progenitors at a late stage and a significant effect on multipotential hemopoietic precursors that might be indirect through other cells. By contrast, human rIL-6 did not support colony formation by human bone marrow mononuclear cells. IL-6 may not show an independent activity for human hemopoiesis of myeloid lineage. However, the synergistic activity of IL-6 remains to be clarified.     

Study of early hematopoietic precursors in human cord blood.

Human cord blood is a source of transplantable stem cells. These stem cells express the antigen CD34, are resistant to treatment with 4-hydroperoxycyclophosphamide (CD34+/4-HCres), and do not give rise to colonies when plated in clonogenic assays. We studied the number of CD34+ cells present in cord blood and developed a two-step assay for early precursors (pre-colony-forming units, pre-CFU) capable of giving rise to committed progenitors. In this assay CD34+/4-HCres cord blood cells were cultured in suspension with different growth factors. After 7 days in suspension the remaining cells were plated in clonogenic assays, for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and mixed lineage colony-forming units (CFU-MIX), in the presence of pure factors or a combination of recombinant human (rh) interleukin 3 (IL-3) and medium conditioned by the PU34 primate cell line. Pre-CFU for all precursors were identified. These pre-CFU developed into committed progenitors in response to rhIL-3. The combinations of rhIL-3 plus rh interleukin 1 (IL-1) or rhIL-3 plus rh interleukin 6 (IL-6) did not enhance recovery of progenitors. The developing CFU-GM were responsive to rh granulocyte-macrophage colony-stimulating factor (GM-CSF) and rh granulocyte colony-stimulating factor (G-CSF) but much less so to rhIL-3. BFU-E and CFU-MIX developed in suspension but could only be detected when cells were replated in the presence of a combination of rhIL-3 and PU34 but not rhIL-3 alone. This assay may be useful in evaluating the number of early hematopoietic precursors present in cord blood samples and in defining growth factor combinations that could enhance hematopoietic recovery after cord blood stem cell transplants.     

Effect of stem cell factor (c-kit ligand) on clonogenic leukemic precursor cells: Synergy with other hematopoietic growth factors

Using clonogenic assay we investigated the effect of stem cell factor (SCF) on the in vitro growth of clonogenic precursor cells from acute myeloid leukemia (AML) and myelodys-plastic syndromes (MDS) in the presence or absence of recombinant human erythropoietin (rhEpo) or recombinant human granulocyte colony-stimulating factor (rhG-CSF). SCF as a single factor did not induce significant colony formation, and even in the presence of rhEpo or rhG-CSF it very weakly stimulated erythroid colony formation and was rarely capable of inducing myeloid colony formation by clonogenic leukemic cells. In culture dishes supplemented with SCF, both myeloid and erythroid colony formations were dramatically enhanced in MDS, regarding both colony number and size. Cotony-formation abilities by MDS progenitors were improved following costimulation with SCF and rhEpo. These results suggest that SCF may have a therapeutic role in restoring hematopoiesis in patients with MDS. © 1994 Wiley-Liss, Inc.     

Suppressive effect of granulocyte-macrophage colony-stimulating factor on the generation of natural killer cells in vitro.

We investigated the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human granulocyte-CSF (rhG-CSF) on the generation of natural killer (NK) cells in vitro. NK cells were cultured from selected human bone marrow cells obtained after the elimination of mature T and NK cells. rhGM-CSF significantly suppressed the generation of CD56+ cells and NK activity (P less than .01) in a dose-dependent manner. The generation of large granular lymphocytes (LGL) was also suppressed in the presence of rhGM-CSF (P less than .01). In contrast, rhG-CSF had no effect on LGL (P greater than .05). Both rhGM-CSF and rhG-CSF had no influence on the CD56+ cell count in the peripheral blood. These results suggest that rhGM-CSF suppresses the in vitro generation of NK cells.     

Effect of recombinant hemopoietic growth factors on human megakaryocyte colony formation in serum-free cultures

The effects of recombinant hemopoietic factors on the clonal growth of human megakaryocyte progenitors were explored using serum-free cultures of nonadherent and T-cell-depleted marrow cells. Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) supported megakaryocyte colony formation in a dose-dependent manner, the activity being lower than that of recombinant interleukin 3 (rIL-3). Recombinant IL-3 and rGM-CSF acted synergistically on megakaryocyte colony formation when rGM-CSF was added to cultures containing suboptimal concentrations of rIL-3. However, the number and size of colonies did not increase with rGM-CSF when cultures were plated with an optimal dose of rIL-3. Recombinant erythropoietin (rEpo) by itself did not stimulate the growth of megakaryocyte progenitors. Recombinant Epo did, however, produce a significant increase in the number and size of megakaryocyte colonies in the presence of rIL-3 or rGM-CSF. Other factors, including recombinant granulocyte colony-stimulating factor, recombinant interleukin 1 alpha, recombinant interleukin 4, and recombinant interleukin 6 showed no capacity to generate or enhance megakaryocyte colony formation when added to cultures alone or in combination with varying concentrations of rIL-3. These results show that rIL-3, rGM-CSF, and rEpo affect human megakaryocytopoiesis by themselves or by interacting with each other.     

c-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides.

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM-CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.     

Chronic childhood neutropenia: studies on the in vitro clonogenicity of bone marrow myeloid progenitors

Bone marrow mononuclear cells (MNC) from 6 pediatric patients with chronic neutropenia were tested for myeloid colony formation upon stimulation with the supernatant of the 5637 cell line or with recombinant granulocyte-macrophage colony-stimulating factor or interleukin 3. Heterogeneous patterns of response of myeloid progenitors were observed in the individual patients, with no colony growth in 2 cases and abnormalities of colony formation or composition in two additional cases. Morphologic and surface marker analyses showed that the bone marrow of some patients contained an excess of lymphocytes with an altered subset distribution. In order to investigate whether or not there was a relationship between the latter abnormality and the observed clonogenic defects, marrow MNC were tested for myeloid colony formation before and after lymphocyte depletion. No evidence for a cell-mediated suppression of colony growth was obtained; likewise, patient sera failed to inhibit colony formation by normal bone marrow myeloid progenitors. Taken together, these data make it unlikely that, in our cases, immunologic mechanisms were responsible for the pathogenesis of chronic neutropenia.