Tumor necrosis factor alpha directly and indirectly regulates hematopoietic progenitor cell proliferation: role of colony-stimulating factor receptor modulation.

Tumor necrosis factor alpha (TNF-alpha) has been shown to both stimulate and inhibit the proliferation of hematopoietic progenitor cells (HPCs) in vitro, but its mechanisms of action are not known. We demonstrate that the direct effects of TNF-alpha on murine bone marrow progenitors are only inhibitory and mediated at least in part through downmodulation of colony-stimulating factor receptor (CSF-R) expression. The stimulatory effects of TNF-alpha are indirectly mediated through production of hematopoietic growth factors, which subsequently results in increased granulocyte-macrophage CSF and interleukin 3 receptor expression. In addition, the effects of TNF-alpha (stimulatory or inhibitory) are strictly dependent on the particular CSF stimulating growth as well as the concentration of TNF-alpha present in culture. A model is proposed to explain how TNF-alpha might directly and indirectly regulate HPC growth through modulation of CSF-R expression.

Transforming growth factor beta 1 selectively regulates early murine hematopoietic progenitors and inhibits the growth of IL-3-dependent myeloid leukemia cell lines.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to be associated with active centers of hematopoiesis and lymphopoiesis in the developing fetus. Therefore, the effects of TGF-beta 1 on mouse hematopoiesis were studied. TGF-beta 1 is a potent inhibitor of IL-3-induced bone marrow proliferation, but it does not inhibit the proliferation induced by granulocyte/macrophage, colony-stimulating factor (CSF), granulocyte CSF, and erythropoietin (Epo). TGF-beta 1 also inhibits IL-3-induced multipotential colony formation of bone marrow cells in soft agar, which includes early erythroid differentiation, while Epo-induced terminal differentiation is unaffected. In addition, IL-3-induced granulocyte/macrophage colonies were inhibited; however, small clusters of differentiated myeloid cells were consistently seen in cultures containing IL-3 and TGF-beta 1. Thus, TGF-beta 1 selectively inhibits early hematopoietic progenitor growth and differentiation but not more mature progenitors. TGF-beta 1 is also a potent inhibitor of IL-3-dependent and -independent myelomonocytic leukemic cell growth, while the more mature erythroid and macrophage leukemias are insensitive. Therefore, TGF-beta 1 functions as a selective regulator of differentiating normal hematopoietic cells, and suppresses myeloid leukemic cell growth.

Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action.

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1-induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1.

Recombinant transforming growth factor beta 1 and beta 2 protect mice from acutely lethal doses of 5-fluorouracil and doxorubicin.

Transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2 can reversibly inhibit the proliferation of hematopoietic progenitor cells in vivo, leading us to hypothesize that such quiescent progenitors might be more resistant to high doses of cell cycle active chemotherapeutic drugs, thereby allowing dose intensification of such agents. Initial studies showed that whereas administration of TGF-beta 1 or TGF-beta 2 did not prevent death in normal mice treated with high doses of 5-fluorouracil (5-FU), those mice that received TGF-beta 2 did exhibit the beginning of a hematologic recovery by day 11 after administration of 5-FU, and were preferentially rescued by a suboptimal number of transplanted bone marrow cells. Subsequently, it was found that the administration of TGF-beta 2 protected recovering progenitor cells from high concentrations of 5-FU in vitro. This protection coincided with the finding that significantly more progenitors for colony-forming unit-culture (CFU-c) and CFU-granulocyte, erythroid, megakaryocyte, macrophage (GEMM) were removed from S-phase by TGF-beta in mice undergoing hematopoietic recovery than in normal mice. Further studies showed that the administration of TGF-beta protected up to 90% of these mice undergoing hematologic recovery from a rechallenge in vivo with high dose 5-FU, while survival in mice not given TGF-beta was < 40%. Pretreatment of mice with TGF-beta 1 or TGF-beta 2 also protected 70-80% of mice from lethal doses of the noncycle active chemotherapeutic drug, doxorubicin hydrochloride (DXR). These results demonstrate that TGF-beta can protect mice from both the lethal hematopoietic toxicity of 5-FU, as well as the nonhematopoietic toxicity of DXR. This report thus shows that a negative regulator of hematopoiesis can be successfully used systemically to mediate chemoprotection in vivo.

Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells.

Raf-1 is a 74-kD serine/threonine kinase located in the cell cytoplasm that is activated by phosphorylation in cells stimulated with a variety of mitogens and growth factors, including hematopoietic growth factors. Using c-raf antisense oligonucleotides to block Raf-1 expression, we have established that Raf-1 is required for hematopoietic growth factor-induced proliferation of murine cell lines stimulated by growth factors whose receptors are members of several different structural classes: (a) the hematopoietin receptor family, including interleukin (IL)-2, IL-3, IL-4, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor (GM-CSF), and erythropoietin; (b) the tyrosine kinase receptor class, including Steel factor and CSF-1; and (c) IL-6, leukemia inhibitory factor, and oncostatin M, whose receptors include the gp130 receptor subunit. Although results of previous experiments had suggested that IL-4 does not phosphorylate or activate the Raf-1 kinase, c-raf antisense oligonucleotides inhibited IL-4-induced proliferation of both myeloid and T cell lines, and IL-4 activated Raf-1 kinase activity in an IL-4-dependent myeloid cell line. In colony assays, c-raf antisense oligonucleotides completely inhibited colony formation of unseparated normal murine bone marrow cells stimulated with either IL-3 or CSF-1 and partially inhibited cells stimulated with GM-CSF. In addition, c-raf antisense oligonucleotides completely inhibited both IL-3- and GM-CSF-induced colony formation of CD34+ purified human progenitors stimulated with these same growth factors. Thus, Raf-1 is required for growth factor-induced proliferation of leukemic murine progenitor cell lines and normal murine and human bone marrow-derived progenitor cells regardless of the growth factor used to stimulate cell growth.

Mobilization of long-term reconstituting hematopoietic stem cells in mice by recombinant human interleukin 7.

Administration of recombinant human interleukin 7 (rh)IL-7 to mice has been reported by our group to increase the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakarocyte macrophage) from the bone marrow to peripheral organs (blood, spleen[s], and liver). We now report that IL-7 also stimulates a sixfold increase in the number of more primitive CFU-S day 8 (CFU-S8) and day 12 (CFU-S12) in the peripheral blood leukocytes (PBL) of mice treated with rhIL-7 for 7 d. Moreover, > 90% of lethally irradiated recipient mice that received PBL from rhIL-7-treated donor mice have survived for > 6 mo whereas none of the recipient mice that received an equal number of PBL from diluent-treated donors survived. Flow cytometry analysis at 3 and 6 mo after transplantation revealed complete trilineage (T, B, and myelomonocytic cell) repopulation of bone marrow, thymus, and spleen by blood-borne stem/progenitor cells obtained from rhIL-7-treated donor mice. Thus, IL-7 may prove valuable for mobilizing pluripotent stem cells with long-term repopulating activity from the bone marrow to the peripheral blood for the purpose of gene modification and/or autologous or allogeneic stem cell transplantation.

TET2 binds the androgen receptor and loss is associated with prostate cancer.

Genetic alterations associated with prostate cancer (PCa) may be identified by sequencing metastatic tumour genomes to identify molecular markers at this lethal stage of disease. Previously, we characterized somatic alterations in metastatic tumours in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2), which is altered in 5-15% of myeloid, kidney, colon and PCas. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma. We perform fine-mapping of PCa risk across TET2 using genotypes from the PEGASUS case-control cohort and identify six new risk variants in introns 1 and 2. Oligonucleotides containing two risk variants are bound by the transcription factor octamer-binding protein 1 (Oct1/POU2F1) and TET2 and Oct1 expression are positively correlated in prostate tumours. TET2 is expressed in normal prostate tissue and reduced in a subset of tumours from the Cancer Genome Atlas (TCGA). Small interfering RNA-mediated TET2 knockdown (KD) increases LNCaP cell proliferation, migration and wound healing, verifying loss drives a cancer phenotype. Endogenous TET2 bound the androgen receptor (AR) and AR-coactivator proteins in LNCaP cell extracts, and TET2 KD increases prostate-specific antigen (KLK3/PSA) expression. Published data reveal TET2 binding sites and hydroxymethylcytosine proximal to KLK3. A gene co-expression network identified using TCGA prostate tumour RNA-sequencing identifies co-regulated cancer genes associated with 2-oxoglutarate (2-OG) and succinate metabolism, including TET2, lysine demethylase (KDM) KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1/2, SDHA/B, and FH. The co-expression signature is conserved across 31 TCGA cancers suggesting a putative role for TET2 as an energy sensor (of 2-OG) that modifies aspects of androgen-AR signalling. Decreased TET2 mRNA expression in TCGA PCa tumours is strongly associated with reduced patient survival, indicating reduced expression in tumours may be an informative biomarker of disease progression and perhaps metastatic disease.

Antineoplastic dolastatins: potent inhibitors of hematopoietic progenitor cells.

Dolastatins 10 and 15, isolated from the shell-less marine mollusk Dolabella auricularia, are potent antineoplastic agents with unknown myelotoxic effects in vivo. The goal of this study was to determine whether the dolastatins inhibit the proliferation of normal hematopoietic progenitor cells. Assays to test inhibition of colony formation and of cell proliferation were performed in vitro with bone marrow cell preparations enriched for progenitor cells and with progenitor cell lines, respectively, using varying drug concentrations and exposure times. Dolastatins 10 and 15 both inhibited human and murine bone marrow cell colony formation in a concentration-dependent manner, with the concentration required for half maximal inhibition ranging from 0.1 to 1 pg/mL for dolastatin 10 and from 10 to 100 pg/mL for dolastatin 15. These concentrations are 25-fold to 100-fold lower than the concentration required for antineoplastic activity. Complete inhibition of human bone marrow cell colony formation was observed at concentrations of 10-100 pg/mL for dolastatin 10 and 1000-10,000 pg/mL for dolastatin 15. Committed progenitor cells and multipotential progenitor cells were similarly inhibited. The magnitude of inhibition of human hematopoietic cell colony formation was dependent on pre-exposure time to dolastatins 10 and 15, with a reversible effect up to 8 hours and with a 24-hour preincubation resulting in maximal (100%) and irreversible inhibition. Dolastatin 10 at a concentration of 10-100 pg/mL limited the proliferation of six human and four murine hematopoietic progenitor cell lines, as measured by tritiated thymidine incorporation, to between 34% and 83% of that occurring in the absence of the drug. These results indicate that the dolastatins are potent inhibitors of normal hematopoietic progenitor cell proliferation.

Prevention of acute chemotherapy-induced death in mice by recombinant human interleukin 1: protection from hematological and nonhematological toxicities.

Previous studies have demonstrated that interleukin 1 (IL-1) can protect most mice from the acute lethal toxicity mediated by high doses of radiation and/or some chemotherapeutic drugs. The results presented herein demonstrate that the pretreatment of mice with recombinant human interleukin 1 alpha (rhIL-1 alpha) protects mice from the lethal effects of several myelotoxic chemotherapeutic drugs, including 5-fluorouracil (5FUra), cyclophosphamide, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II), and 1,3-bis-(2-chloroethyl)-1-nitrosourea. However, pretreatment with rhIL-1 alpha was not effective against the acute lethal toxicity generated by doxorubicin and cisplatin. The chemoprotective effects appear to be at least partially due to myeloprotection/restoration, since the recovery of myeloid colony-forming units and the total cellularity in the bone marrow and spleen were accelerated in the rhIL-1 alpha-pretreated mice. However, the chemoprotective effects of rhIL-1 alpha are apparently not limited to myeloprotection, since pretreatment with rhIL-1 alpha protects mice against the lethal toxicity of both 5FUra and cyclophosphamide, yet bone marrow transplants rescue mice treated with 5FUra but not those treated with cyclophosphamide. The chemoprotective effects of rhIL-1 alpha may be at least partially indirect, since the efficacy of chemoprotection by rhIL-1 alpha is reduced in athymic mice, and interleukin 6, but not tumor necrosis factor alpha, can substitute for rhIL-1 alpha in chemoprotection from 5FUra.

Introduction of v-abl oncogene induces monocytic differentiation of an IL-3-dependent myeloid progenitor cell line.

There are a variety of murine hematopoietic progenitor cell lines which are differentiation arrested, but still require growth factors such as interleukin-3 for their continued growth and survival. While oncogenes such as v-myc and v-abl have been demonstrated to abrogate the requirement for exogenous growth factors, none have been shown to have an effect on the differentiation of these cell lines. In this report, we demonstrate that the introduction and expression of Abelson murine leukemia virus into a myeloblast progenitor cell line can promote further differentiation along the monocytic lineage. There is a marked alteration in cell morphology, the acquisition of Mac-1 antigen expression, the induction of nonspecific esterase expression and the induced ability to phagocytize opsonized zymosan. Thus, the expression of Abelson murine leukemia virus protein in interleukin-3-dependent hematopoietic progenitors can provide differentiation-inducing signals in cells which are arrested in differentiation. The potential role of Abelson murine leukemia virus gene products in normal hematopoietic cell differentiation and in transformation is discussed.

Retroviral v-myc infection of primary fetal liver cells: transformation of monocytes in vitro.

Oncogenes carried by retroviruses can alter the growth properties of many cell types. We examined the molecular mechanism by which a retrovirus containing one or a combination of oncogenes can transform and immortalize hematopoietic cells. Murine fetal liver cells were used as an enriched source of early hematopoietic cell progenitors; the cells were infected with a series of recombinant murine retroviruses capable of expressing the avian v-myc, v-H-ras and v-raf oncogenes. Three factor-independent cell lines were obtained: FL-ras/myc, FL-J2 (v-raf/v-myc) and FL-myc, a unique cell line generated using a single oncogene. Cytochemical, morphologic and phenotypic analyses indicated that these cell lines were of the monocyte lineage. Southern and Northern blot analyses revealed that the three cell lines had integrated viral DNA and were expressing the mRNA transcripts corresponding to these viral oncogenes. To examine the mechanism of factor independence, supernatants from these cell lines were tested for CSF-1 activity. Supernatants from FL-myc and FL-ras/myc cells were shown to contain CSF-1 activity and Northern blot analysis of the three cell lines revealed the presence of mRNA transcripts for the CSF-1 and c-fms genes. It is possible that the growth factor independence of these cell lines is related to the development of autocrine-induced proliferation.

The effect of c-raf antisense oligonucleotides on growth factor-induced proliferation of hematopoietic cells.

While it is well established that Raf-1 kinase is activated by phosphorylation in growth factor-dependent hematopoietic cell lines stimulated with a variety of hematopoietic growth factors, little is known about the biological effects of Raf-1 activation on normal hematopoietic cells. Therefore, we examined the requirement for Raf-1 in growth factor-regulated proliferation and differentiation of hematopoietic cells using c-faf antisense oligonucleotide. Raf-1 required for the proliferation of growth factor dependent cell lines stimulated by IL-2, IL-3, G-CSF, GM-CSF and EPO that bind to the hematopoietin class of receptors. Raf-1 is also required for the proliferation of cell lines stimulated by growth factors that use the tyrosine kinase containing receptor class, including SLF and CSF-1. In addition, Raf-1 is also required for IL-6, LIF- and OSM-induced proliferation whose receptors share the gp 130 subunit. In contrast to previous results which demonstrated that IL-4 could not activate Raf-1 kinase, c-raf antisense oligonucleotides also inhibited IL-4-induced proliferation of T cell and myeloid cell lines. Using normal hematopoietic cells, c-raf antisense oligonucleotides completely suppressed the colony formation of murine hematopoietic progenitors in response to single growth factors, such as IL-3, CSF-1 or GM-CSF. Further, c-raf antisense oligonucleotides inhibited the growth of murine progenitors stimulated with synergistic combinations of growth factors (required for primitive progenitor growth) including two, three and four factor combinations. In comparison to murine hematopoietic cells, c-raf antisense oligonucleotides also inhibited both IL-3 and GM-CSF-induced colony formation of CD 34+ purified human progenitors. In addition, Raf-1 is required for the synergistic response of CD 34+ human bone marrow progenitors to multiple cytokines; however, this effect was only observed when additional antisense oligonucleotides were added to the cultures at day 7 of a 14 day assay. Finally, Raf-1 is required for the synergistic response of human Mo-7e cells and of normal human fetal liver cells to five factor combinations. Thus, Raf-1 is required to transduce growth factor-induced proliferative signals in factor-dependent progenitor cells lines for all known classes of hematopoietic growth factor receptors, and is required for the growth of normal murine and human bone marrow-derived progenitors.

Engraftment of SCID mice by human bone marrow hematopoietic cells cultured in vitro: an in vivo model for human gene transfer.

Demonstration of the ability of fresh human hematopoietic cells to engraft severe combined immuno-deficient (scid) mice has provided an in vivo assay for expansion and maturation of early human progenitor cells. However, engraftment of cultured hematopoietic cells has been difficult to achieve. We wished to further develop this model as an in vivo assay for efficiency of retroviral gene transfer and expression in the differentiated progeny of adult human bone marrow progenitor cells. Human bone marrow cells were cultured in vitro for six days under conditions suitable for infection by retroviral vectors prior to transfer to irradiated scid mice. Cultured human bone marrow cells introduced by both intravenous (i.v.) and intraperitoneal (i.p.) injection persisted in the bone marrow, spleen and peritoneum of recipient animals up to four weeks after transfer. Following irradiation scid mice receiving cultured human bone marrow cells by either i.v. or i.p. routes demonstrated engraftment of the bone marrow and spleen as determined by the growth of human hematopoietic progenitors in soft agar. By flow cytometric analysis human cells were also detected in the peritoneum of mice receiving cultured human bone marrow cells i.p. These results suggest that the transfer of cultured human bone marrow cells to scid mice with the subsequent engraftment of these cells in the bone marrow, spleen and peritoneum of recipients can routinely occur. This provides an in vivo model for retroviral gene transfer to human cells.

Hematopoietic growth factors and glucocorticoids synergize to mimic the effects of IL-1 on granulocyte differentiation and IL-1 receptor induction on bone marrow cells in vivo.

The mechanisms by which interleukin-1 (IL-1) stimulates hematopoiesis are not clear. We have previously shown that in vivo administration of IL-1 indirectly increases IL-1 receptor (IL-1R) expression on both immature and mature bone marrow (BM) cells, partly due to IL-1-induced hematopoietic growth factor (HGF) production. Because IL-1 also stimulates the hypothalamic pituitary-adrenal axis resulting in the production of glucocorticoids (GC), we assessed whether in vivo treatment with HGF and glucocorticoids upregulates IL-1R. Administration of IL-1 to adrenalectomized mice reduces by 53% IL-specific binding on light density bone marrow (LDBM) cells compared to sham-operated mice. The administration of dexamethasone (dex) alone induced only a slight increase in IL-1R expression but synergized with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), IL-3 and IL-6 to upregulate IL-1R expression. Flow cytometry analysis using the RB6-8C5 antibody, which is differentially expressed on myeloid cells, indicated that combined G-CSF and dex treatment acts to promote increased numbers of differentiated myeloid progenitors in the bone marrow. Autoradiographic analysis confirmed that while G-CSF and dex increased IL-1R expression on all myeloid cells, it was particularly pronounced for myelocytes, promyelocytes and metamyelocytes. These results suggest that the ability of IL-1 to enhance granulocyte differentiation in vivo is partly due to its ability to induce a cascade of cytokines and steroids which in turn regulate IL-1 receptor expression.

Direct synergistic effects of IL-4 and IL-11 on proliferation of primitive hematopoietic progenitor cells.

The present studies have investigated, for the first time, the synergistic effects of interleukin-4 (IL-4) and IL-11 on the growth of single murine bone marrow progenitor cells. These studies suggest that IL-4 and IL-11 are synergistic hematopoietic growth factors, enhancing colony formation of bone marrow progenitors from normal mice in the presence of colony-stimulating factors or stem cell factor, whereas neither IL-4 nor IL-11, alone or in combination, resulted in colony formation. However, in the presence of a neutralizing anti-TGF-beta antibody, IL-11 plus IL-4 induced clonal growth of primitive Lin-Sca1+ progenitors. Furthermore, here we report several observations extending the knowledge about IL-4 and IL-11 as synergistic factors. In addition to the established ability of IL-11 to enhance IL-3- and GM-CSF-induced colony formation, IL-11 also enhanced the number of G-CSF- and CSF-1-stimulated colonies of mature (Lin-) and primitive (Lin-Sca-1+) hematopoietic progenitors cultured at the single-cell level. In contrast, IL-4 bifunctionally regulated the growth of Lin- progenitors, whereas the growth of single Lin-Sca=1+ progenitors was unaffected or enhanced in the presence of IL-4. Finally, IL-4 and IL-11, in combination, potently synergized to enhance the high-proliferative-potential colony-forming cell colony formation of Lin-Sca-1+ progenitors in response to all four CSFs and to SCF.

The growth response of Lin-Thy-1+ hematopoietic progenitors to cytokines is determined by the balance between synergy of multiple stimulators and negative cooperation of multiple inhibitors.

The present studies investigated the balance of positive and negative growth signals in direct regulation of hematopoiesis. Interleukin-3 (IL-3) combined with Steel factor (SLF) optimally stimulated proliferation of Lin-Thy-1+ murine bone marrow progenitors in single-cell assays, and that proliferation was inhibited more than 90% by transforming growth factor-beta 1 (TGF-beta 1). Colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, or IL-6 as a third stimulatory growth factor was incapable of counteracting the TGF-beta 1-mediated inhibition of IL-3-plus-SLF-stimulated growth, while G-CSF slightly enhanced the number of TGF-beta 1-resistant clones. As a fourth factor, only IL-1 could partially overcome the TGF-beta 1-induced growth inhibition. While the presence of a cocktail of five additional stimulatory growth factors did not enhanced the frequency of single Lin-Thy-1+ progenitors proliferating in response to IL-3 plus SLF, the number of responding progenitors in the presence of TGF-beta 1 was enhanced nine-fold. Furthermore, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not macrophage inflammatory protein-1 alpha (MIP-1 alpha), cooperated with TGF-beta 1 to reverse the proliferative effects of multiple stimulatory cytokines, resulting in 76% inhibition. Thus, the direct effects of single inhibitory factors on hematopoietic progenitor cell growth can be reversed by multiple stimulatory growth factors, and negative growth factors can directly cooperate to suppress progenitor cell growth stimulated by multiple positive-acting factors.

Engraftment of ex vivo expanded and cycling human cord blood hematopoietic progenitor cells in SCID mice.

The ability of human hematopoietic cells to engraft SCID mice provides a useful model in which to study the efficiency of retroviral gene transfer and expression in primitive stem cells. In this regard, it is necessary to determine whether SCID mice can be engrafted by cycling human hematopoietic progenitor cells. Human cord blood cells from 12 different donors were cultured in vitro for 6 days with interleukin-3 and stem cell factor. Phenotypic analysis indicated that hematopoietic cells were induced to cycle and the number of progenitors was expanded, thus making them targets for retroviral gene transfer. The cells were then transferred to SCID mice. Human hematopoietic progenitor cell engraftment was assessed up to 7 weeks later by growth of human progenitor cells in soft agar. After in vitro culture under conditions used for retroviral gene transfer, human cord blood hematopoietic cells engrafted the bone marrow and spleen of SCID mice. Interestingly, cultured cord blood cells engrafted after intraperitoneal but not after intravenous injection. Furthermore, engraftment of cord blood cells was observed in mice receiving no irradiation before transfer of the human cells, suggesting that competition for space in the marrow is not a limiting factor when these cells have been cultured. Administration of human cytokines after transfer of human cord blood cells to SCID mice was also not required for engraftment. Thus, engraftment of SCID mice with human hematopoietic cells cultured under conditions suitable for gene transfer may provide an in vivo assay for gene transfer to early human hematopoietic progenitor cells.

Increased granulopoiesis after sequential administration of transforming growth factor-beta 1 and granulocyte-macrophage colony-stimulating factor.

Transforming growth factor-beta 1 (TGF-beta 1) is an inhibitor of the growth and differentiation of immature hematopoietic progenitors in vitro; however, we have demonstrated that TGF-beta 1 can promote granulopoiesis in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. We therefore examined the effect of the combined administration of TGF-beta 1 and GM-CSF in vivo. First, TGF-beta 1 enhanced the specific binding of GM-CSF (2.0-fold) on bone marrow cells, reaching a maximum 40 hours after injection, while the specific binding of interleukin-3 (IL-3) was unaffected. Using GM-CSF-specific binding to determine the optimal regimen for cytokine administration in vivo, we found that the administration of TGF-beta 1 and GM-CSF in sequence increased myelopoiesis. Total numbers of colony-forming units-granulocyte/macrophage (CFU-GM) and myeloblasts per femur were increased above the level obtained with the simultaneous injection of TGF-beta 1 plus GM-CSF, GM-CSF alone or TGF-beta 1 alone. Further, the sequential administration of TGF-beta 1 and GM-CSF resulted in enhanced numbers of mature granulocytes in both the bone marrow and peripheral blood. In contrast, the sequential combination of TGF-beta 1 and GM-CSF did not enhance the numbers or increase the recovery of erythroid cells in the bone marrow. These results show that TGF-beta 1 in vivo as in vitro has a multifunctional effect on bone marrow progenitors, and by using an optimal combination of TGF-beta 1 and GM-CSF in vivo, one can selectively increase both the central and peripheral granulopoietic compartments.

Distinct and direct synergistic effects of IL-1 and IL-6 on proliferation and differentiation of primitive murine hematopoietic progenitor cells in vitro.

While interleukin-1 (IL-1) and IL-6 have been demonstrated to synergize with colony-stimulating factors (CSFs) and stem cell factor (SCF) to stimulate myeloid colony formation of primitive hematopoietic progenitor cells, it has not yet been established whether these effects are directly mediated. In the present study, direct effects of IL-1 and IL-6 were examined on primitive Lin-Sca-1+ murine bone-marrow progenitor cells that were cultured and plated individually. IL-1 and IL-6 showed not only overlapping, but also distinct, patterns of direct synergy. While IL-1 or IL-6 had no proliferative effects as single growth factors, IL-1, in combination with granulocyte-macrophage CSF (GM-CSF), IL-3, CSF-1, and SCF, but not granulocyte-CSF (G-CSF), enhanced the cloning frequency of Lin-Sca-1+ progenitors three- to five-fold, whereas IL-6 increased the cloning frequency in response to all four CSFs and SCF two- to seven-fold. In all cases, the size of the colonies observed were increased as well. Furthermore, the combined action of IL-1 and IL-6 resulted in additive or synergistic enhancement of CSF- and SCF-stimulated colony formation of Lin-Sca-1+ high proliferative potential colony-forming cells (HPP-CFCs). Finally, IL-6, but not IL-1, enhanced the number of immature blast cells observed in CSF- and SCF-stimulated cultures.