Stem cell concepts in myelodysplastic syndromes: lessons and challenges.

According to the cancer stem cell (CSC) hypothesis, CSCs are the only cancer cells that can give rise to and sustain all cells that constitute a cancer as they possess inherent or acquired self-renewal potential, and their elimination is required and potentially sufficient to achieve a cure. Whilst establishing CSC identity remains challenging in most cancers, studies of low-intermediate risk myelodysplastic syndromes (MDS), other chronic myeloid malignancies and clonal haematopoiesis of indeterminant potential (CHIP) strongly support that the primary target cell usually resides in the rare haematopoietic stem cell (HSC) compartment. This probably reflects the unique self-renewal potential of HSCs in normal human haematopoiesis, combined with the somatic initiating genomic driver lesion not conferring extensive self-renewal potential to downstream progenitor cells. Mutational 'fate mapping' further supports that HSCs are the only disease-propagating cells in low-intermediate risk MDS, but that MDS-propagating potential might be extended to progenitors upon disease progression. The clinical importance of MDS stem cells has been highlighted through the demonstration of selective persistence of MDS stem cells in patients at complete remission in response to therapy. This implies that MDS stem cells might possess unique resistance mechanisms responsible for relapses following otherwise efficient treatments. Specific surveillance of MDS stem cells should be considered to assess the efficiency of therapies and as an early indicator of emerging relapses in patients in clinical remission. Moreover, further molecular characterization of purified MDS stem cells should facilitate identification and validation of improved and more stem cell-specific therapies for MDS.

Absence of Fms-like tyrosine kinase 3 ligand (Flt3L) signalling protects against collagen-induced arthritis.

OBJECTIVE: Comprehending the mechanisms that regulate activation of autoreactive T cells and B cell antibody production is fundamental for understanding the breakdown in self-tolerance and development of autoimmunity. Here we studied the role of Fms-like tyrosine kinase 3 ligand (Flt3L) signalling in the pathogenesis of collagen-induced arthritis (CIA). METHODS: CIA was induced in mice lacking Flt3L (Flt3L(-/-)) and wild-type (WT) littermates (C57/BL6, 8-10 weeks old). Mice were killed in the initial phase (acute phase: experiment 1) and late phase (chronic phase: experiment 2) of the disease. Arthritis severity was assessed using a semiquantitative scoring system (0-4), and histological analysis of cellular infiltration, cartilage destruction and peptidoglycan loss was performed. Phenotypic and functional analysis of T and B cells, FoxP3 expression, activation and lymphocyte costimulatory markers, and cytokine production were performed ex vivo by flow cytometry in lymph nodes. Serum collagen type II (CII)-specific antibodies were measured by ELISA. RESULTS: Flt3L(-/-) mice showed a marked decrease in clinical arthritis scores and incidence of arthritis in both acute and chronic phases of CIA compared with WT mice. Moreover, decreased synovial inflammation and joint destruction was observed. Both the magnitude and quality of T cell responses were altered in Flt3L(-/-). In the acute phase, the amount of CII-specific IgG2a antibodies was lower in Flt3L(-/-) than WT mice. CONCLUSIONS: These results strongly suggest a role for Flt3L signalling in the development of arthritis.

Non-destructive evaluation of chlorophyll content in quinoa and amaranth leaves by simple and multiple regression analysis of RGB image components.

Leaf chlorophyll content provides valuable information about physiological status of plants; it is directly linked to photosynthetic potential and primary production. In vitro assessment by wet chemical extraction is the standard method for leaf chlorophyll determination. This measurement is expensive, laborious, and time consuming. Over the years alternative methods, rapid and non-destructive, have been explored. The aim of this work was to evaluate the applicability of a fast and non-invasive field method for estimation of chlorophyll content in quinoa and amaranth leaves based on RGB components analysis of digital images acquired with a standard SLR camera. Digital images of leaves from different genotypes of quinoa and amaranth were acquired directly in the field. Mean values of each RGB component were evaluated via image analysis software and correlated to leaf chlorophyll provided by standard laboratory procedure. Single and multiple regression models using RGB color components as independent variables have been tested and validated. The performance of the proposed method was compared to that of the widely used non-destructive SPAD method. Sensitivity of the best regression models for different genotypes of quinoa and amaranth was also checked. Color data acquisition of the leaves in the field with a digital camera was quick, more effective, and lower cost than SPAD. The proposed RGB models provided better correlation (highest R (2)) and prediction (lowest RMSEP) of the true value of foliar chlorophyll content and had a lower amount of noise in the whole range of chlorophyll studied compared with SPAD and other leaf image processing based models when applied to quinoa and amaranth.

Effects of quinoa hull meal on piglet performance and intestinal epithelial physiology.

Saponin-containing feed additives have shown positive effects on pig performance. Quinoa hull has high saponin content and may be of interest as a feed additive. This study aimed to evaluate quinoa hull meal (QHM) as a feed additive in a pig diet. The effects of QHM were assessed for three dosages of South American (SA) origin (100, 300 and 500 mg/kg) and one dosage of Danish (DK) quinoa (300 mg/kg). In addition, the effect of dietary SA-QHM and SA-QHM-extract on jejunal epithelial physiology was studied ex vivo in Ussing chambers. The experiment included 400 piglets weaned at 28 ± 2 days of age and the experimental period was 4 weeks. Piglets were weighed initially and finally and feed intake registered. The ex vivo studies were performed with epithelium from 40 pigs receiving control or SA-QHM. Epithelium from each pig was placed into eight Ussing chambers, where four concentrations of SA-QHM-extract were added. Epithelial permeability, Na(+)-dependent glucose transport and serotonin (5-HT) and theophylline-induced secretion were measured. The results showed that QHM had no influence on piglet's growth (p = 0.41) or feed intake (p = 0.17). In spite of a large difference in saponin content between SA-QHM and DK-QHM (28.7% and 2.0% w/w respectively) the source did not affect pig performance. The ex vivo studies revealed no effect (p > 0.05) of adding QHM-extract into the medium. The permeability and glucose induced absorption were highest (p = 0.003 and p = 0.04 respectively) in epithelium from pigs that consumed 100 or 300 mg/kg SA-QHM. The secretory response to 5-HT was not affected (p = 0.59) by dietary treatments, but the theophylline-induced secretion decreased (p = 0.02) with increasing dietary SA-QHM. The changes in epithelial physiology measured ex vivo did not affect animal performance in vivo in this study.

Cytotoxic lymphocyte maturation factor (interleukin 12) is a synergistic growth factor for hematopoietic stem cells.

The recently cloned cytotoxic lymphocyte maturation factor (interleukin 12 [IL-12]) has been described as a growth factor for mature lymphocytes. The present study investigated whether purified recombinant murine IL-12 (rMuIL-12) also could affect the proliferation of primitive bone marrow progenitor cells. Using a population of Lin-Sca-1+ murine bone marrow stem cells, we now demonstrate that IL-12 is a potent synergistic factor for primitive hematopoietic stem cells. The synergy of IL-12 was observed in single-cell cloning assays, demonstrating that its effects are directly mediated. Specifically, IL-12 enhanced stem cell factor-induced myelopoiesis of Lin-Sca-1+ cells sevenfold, and synergized with colony-stimulating factors (CSFs) to induce proliferation of Lin-Sca-1+ stem cells. IL-12 increased the number of responding progenitor cells as well as the size of the colonies formed. IL-12 also increased colony formation of high proliferative potential colony-forming cells with multiple CSF combinations. The effects of IL-12 were concentration dependent with a 50% effective dose of 2-20 and 20-200 ng/ml, resulting in maximum stimulation. Furthermore, a neutralizing anti-IL-12 antibody blocked the synergistic effects of rMuIL-12. In addition, IL-12 was found to have synergistic effects on more committed bone marrow progenitors as well. Our results therefore suggest that in addition to being a potent lymphopoietic stimulator, IL-12 is a regulator of the growth of hematopoietic stem cells and their myeloid progeny.

Novel role of interleukin 7 in myelopoiesis: stimulation of primitive murine hematopoietic progenitor cells.

Interleukin 7 (IL-7) has been demonstrated to be an important regulator of the growth of B and T cell precursors as well as mature T cells, whereas IL-7 has been reported to have no direct myeloproliferative effects. Here we show that IL-7 potently and directly enhances colony stimulating factor-induced myeloid colony formation from Lin-Sca-1+ murine bone marrow progenitor cells, increasing the cloning frequency up to ninefold and cell numbers up to 50-fold, without affecting their ability to differentiate along the myeloid lineages In contrast, IL-7 has no effect on proliferation of committed Lin- myeloid progenitors. Thus, in addition to its established lymphopoietic potential, this study implicates a novel role of IL-7 in early myelopoiesis.

The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors.

The recently cloned murine flt3 ligand (FL) was studied for its ability to stimulate the growth of primitive (Lin-Sca-1+) and more committed (Lin-Sca-1-) murine bone marrow progenitor cells, alone and in combination with other hematopoietic growth factors (HGFs). Whereas FL was a weak proliferative stimulator alone, it potently synergized with a number of other HGFs, including all four colony-stimulating factor (CSF), interleukin (IL) 6, IL-11, IL-12, and stem cell factor (SCF), to promote the colony formation of Lin-Sca-1+, but not Lin-Sca-1- or erythroid progenitor cells. The synergistic activity of FL was concentration dependent, with maximum stimulation occurring at 250 ng/ml, and was observed when cells were plated at a concentration of one cell per culture, suggesting that its effects are directly mediated. 2 wk of treatment with FL in combination with IL-3 or SCF resulted in the production of a high proportion of mature myeloid cells (granulocytes and macrophages), whereas the combination of FL with G-CSF, IL-11, or IL-12 resulted predominantly in the formation of cells with an immature blast cell appearance. Accordingly, FL in combination with G-CSF or IL-11 expanded the number of progenitors more than 40-fold after 2 wk incubation. Thus, FL emerges as a potent synergistic HGF, that in combination with numerous other HGFs, can directly stimulate the proliferation, myeloid differentiation, and expansion of primitive hematopoietic progenitor cells.

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.

Interleukin 13: novel role in direct regulation of proliferation and differentiation of primitive hematopoietic progenitor cells.

The recently cloned interleukin 13 (IL-13) shares most investigated biological activities on B lymphocytes and monocytes with IL-4. In this study we investigated for the first time the potential role of IL-13 in the regulation of the growth of hematopoietic progenitor cells. IL-13 enhanced stem cell factor (SCF)-induced proliferation of Lin-Sca-1+ bone marrow progenitor cells more potently than IL-4. The effect of IL-13 was purely synergistic, since IL-13 alone stimulated no colony formation. Single cell experiments suggested that the synergistic effect of IL-13 on Lin-Sca-1+ progenitors was directly mediated. In contrast, IL-13 had no synergistic activity on SCF-induced proliferation of the more mature Lin-Sca-1- progenitor cells. Thus, the cloning frequency in response to SCF + IL-13 was at least 20-fold higher in the Lin-Sca-1+ than the Lin-Sca-1- progenitor cell population. Furthermore, IL-13 but not IL-4 synergistically enhanced colony formation of Lin-Sca-1+ progenitors in response to granulocyte/macrophage colony-stimulating factor (GM-CSF) (threefold), whereas both IL-4 and IL-13 enhanced G-CSF-induced colony formation (threefold), and neither of the two significantly affected CSF-1 and IL-3-induced proliferation. Finally, whereas stimulation of Lin-Sca-1+ progenitors by SCF + G-CSF resulted in the formation of 90% granulocytes, the addition of IL-13 resulted in the production of macrophages exclusively. This novel effect on differentiation was directly mediated, shared with IL-4, and could not be observed on Lin-Sca-1- progenitor cells. Collectively, these findings indicate a novel role of IL-13 in early myelopoiesis, partially overlapping but also different from that of IL-4.

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.

Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation.

Multipotent self-renewing hematopoietic stem cells (HSCs) are responsible for reconstitution of all blood cell lineages. Whereas growth stimulatory cytokines have been demonstrated to promote HSC self-renewal, the potential role of negative regulators remains elusive. Receptors for tumor necrosis factor (TNF) and Fas ligand have been implicated as regulators of steady-state hematopoiesis, and if overexpressed mediate bone marrow failure. However, it has been proposed that hematopoietic progenitors rather than stem cells might be targeted by Fas activation. Here, murine Lin(-)Sca1(+)c-kit(+) stem cells revealed little or no constitutive expression of Fas and failed to respond to an agonistic anti-Fas antibody. However, if induced to undergo self-renewal in the presence of TNF-alpha, the entire short and long-term repopulating HSC pool acquired Fas expression at high levels and concomitant activation of Fas suppressed in vitro growth of Lin(-)Sca1(+)c-kit(+) cells cultured at the single cell level. Moreover, Lin(-)Sca1(+)c-kit(+) stem cells undergoing self-renewal divisions in vitro were severely and irreversibly compromised in their short- and long-term multilineage reconstituting ability if activated by TNF-alpha or through Fas, providing the first evidence for negative regulators of HSC self-renewal.

All-trans- and 9-cis-retinoic acid: potent direct inhibitors of primitive murine hematopoietic progenitors in vitro.

Retinoic acid (RA) stimulates the clonal proliferation of mature bone marrow progenitor cells and inhibits the growth of leukemic progenitors, whereas its effects on normal primitive hematopoietic progenitors have not yet been investigated. This study investigated the ability of all-trans- and 9-cis-RA to modulate the proliferation and differentiation of murine Lin-Sca-1+ bone marrow progenitor cells. Both RA isoforms inhibited in a reversible and dose-dependent fashion, the proliferation of multi- but not single-factor responsive Lin-Sca-1+ progenitor cells. The 50% effective dose was 10 nM for both all-trans- and 9-cis-RA. Maximum inhibition was observed at 100-1,000 nM RA, resulting in a 50-75% reduction in the number of proliferative clones. Lin-Sca-1+ cells with high proliferative potential were preferentially inhibited by RA, resulting in a 80-100% inhibition depending on the hematopoietic growth factors stimulating their growth. The inhibitory effects of RA were directly mediated on the target cell, since the effects were observed at the single cell level. Furthermore, autocrine transforming growth factor beta (TGF-beta) production can probably not account for the observed inhibitory effects of RA, since a TGF-beta neutralizing antibody did not block RA-induced inhibition. Whereas RA, in general, is a differentiation-inducing agent, treatment of Lin-Sca-1+ progenitors resulted in the accumulation of an increased fraction of blasts and immature myeloid cells. Thus, RA inhibits the proliferation as well as differentiation of normal primitive hematopoietic progenitor cells.

Hypermethylated SUPERMAN epigenetic alleles in arabidopsis.

Mutations in the SUPERMAN gene affect flower development in Arabidopsis. Seven heritable but unstable sup epi-alleles (the clark kent alleles) are associated with nearly identical patterns of excess cytosine methylation within the SUP gene and a decreased level of SUP RNA. Revertants of these alleles are largely demethylated at the SUP locus and have restored levels of SUP RNA. A transgenic Arabidopsis line carrying an antisense methyltransferase gene, which shows an overall decrease in genomic cytosine methylation, also contains a hypermethylated sup allele. Thus, disruption of methylation systems may yield more complex outcomes than expected and can result in methylation defects at known genes. The clark kent alleles differ from the antisense line because they do not show a general decrease in genomic methylation.

Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation.

Epigenetic silenced alleles of the Arabidopsis SUPERMAN locus (the clark kent alleles) are associated with dense hypermethylation at noncanonical cytosines (CpXpG and asymmetric sites, where X = A, T, C, or G). A genetic screen for suppressors of a hypermethylated clark kent mutant identified nine loss-of-function alleles of CHROMOMETHYLASE3 (CMT3), a novel cytosine methyltransferase homolog. These cmt3 mutants display a wild-type morphology but exhibit decreased CpXpG methylation of the SUP gene and of other sequences throughout the genome. They also show reactivated expression of endogenous retrotransposon sequences. These results show that a non-CpG DNA methyltransferase is responsible for maintaining epigenetic gene silencing.

Gene silencing: Maintaining methylation patterns.

Recent studies of an Arabidopsis gene family have shown that inverted repeats can be potent silencers of other identical sequences in the genome, causing them to become stably methylated at cytosine residues. From mutations affecting this process we are beginning to understand how methylation patterns are maintained.

Ectopic hypermethylation of flower-specific genes in Arabidopsis.

BACKGROUND: Arabidopsis mutations causing genome-wide hypomethylation are viable but display a number of specific developmental abnormalities, including some that resemble known floral homeotic mutations. We previously showed that one of the developmental abnormalities present in an antisense-METHYLTRANSFERASEI (METI) transgenic line resulted from ectopic hypermethylation of the SUPERMAN gene. RESULTS: Here, we investigate the extent to which hypermethylation of SUPERMAN occurs in several hypomethylation mutants, and describe methylation effects at a second gene, AGAMOUS. SUPERMAN gene hypermethylation occurred at a high frequency in several mutants that cause overall decreases in genomic DNA methylation. The hypermethylation pattern was largely similar in the different mutant backgrounds. Genetic analysis suggests that hypermethylation most likely arose either during meiosis or somatically in small sectors of the plant. A second floral development gene, AGAMOUS, also became hypermethylated and silenced in an Arabidopsis antisense-METI line. CONCLUSIONS: These results suggest that ectopic hypermethylation of specific genes in mutant backgrounds that show overall decreases in methylation may be a widespread phenomenon that could explain many of the developmental defects seen in Arabidopsis methylation mutants. This resembles a phenomenon seen in cancer cells, which can simultaneously show genome-wide hypomethylation and hypermethylation of specific genes. Comparison of the methylated sequences in SUPERMAN and AGAMOUS suggests that hypermethylation could involve DNA secondary structures formed by pyrimidine-rich sequences.

Tumor necrosis factor-alpha inhibits stem cell factor-induced proliferation of human bone marrow progenitor cells in vitro. Role of p55 and p75 tumor necrosis factor receptors.

Stem cell factor (SCF), a key regulator of hematopoiesis, potently synergizes with a number of hematopoietic growth factors. However, little is known about growth factors capable of inhibiting the actions of SCF. TNF-alpha has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-alpha and SCF. Here, we demonstrate that TNF-alpha potently and directly inhibits SCF-stimulated proliferation of CD34+ hematopoietic progenitor cells. Furthermore, TNF-alpha blocked all colony formation stimulated by SCF in combination with granulocyte colony-stimulating factor (CSF) or CSF-1. The synergistic effect of SCF observed in combination with GM-CSF or IL-3 was also inhibited by TNF-alpha, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNF-alpha were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-alpha downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well.

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.

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.

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.