Influence of PI-3K/Akt pathway on Wnt signalling in regulating myeloid progenitor cell proliferation. Evidence for a role of autocrine/paracrine Wnt regulation

The regulation of myeloid progenitor cell (granulocyte-macrophage colony-forming units, CFU-GM) proliferation/differentiation by the Wnt and phosphatidylinositol-3 kinase (PI-3K) pathways was investigated using a colony-replating assay. The PI-3K pathway promoted differentiation of interleukin-3 (IL-3)-stimulated myelopoiesis via Akt, because inhibition of the PI-3K/Akt pathway with LY294002 or SH-5 increased proliferation. The involvement of canonical and non-canonical Wnt pathways was investigated using Wnt3a and Wnt5a respectively. Addition of the recombinant Wnts to IL-3 increased CFU-GM proliferation. Dkk-1, when combined with the Wnt proteins, abrogated the effects of Wnt3a but not Wnt5a. Surprisingly, the addition of Dkk-1 to LY294002 or SH-5 blocked their proliferative effects. We hypothesized that increased proliferation induced by PI-3K/Akt inhibitors was not mediated by downstream activation of the Wnt pathway but by induced endogenous production/release of Wnt proteins. The addition of SH-5 to IL-3 created an autocrine Wnt loop in CD34⁺ cells, resulting in the phosphorylation of lipoprotein-receptor-related-protein 6. Furthermore, the addition of medium conditioned by CD34⁺ cells cultured in IL-3 + SH-5 to IL-3 increased CFU-GM proliferation. This effect was abrogated by Dkk-1, suggesting that a Wnt in the conditioned medium increased proliferation. In summary, IL-3 via the PI-3K pathway promoted differentiation of myeloid progenitor cells through a decrease of endogenous Wnt production/release.     

Phosphatidylinositol-3 kinase inhibitors reproduce the selective antiproliferative effects of imatinib on chronic myeloid leukaemia progenitor cells

We investigated the role of the phosphatidylinositol-3 kinase (PI-3K) pathway in regulating the proliferation of primary chronic myeloid leukaemia (CML) progenitor cells by using imatinib to inhibit the activity of p210(Bcr-Abl). The effect of imatinib on the expression of PI-3K pathway proteins was investigated by kinase assays and Western blotting; PI-3K was inhibited by wortmannin or LY294002, Jak2 by AG490 and farnesylation by FTI II; progenitor cell proliferation (self-renewal) was measured by growing myeloid colonies in vitro, then replating them to observe secondary colony formation. Suppression of p210(Bcr-Abl) with imatinib indirectly suppressed the activity of PI-3K and its downstream targets (Erk, Akt and p70S6 kinase), thereby implicating the PI-3K pathway in p210(Bcr-Abl)-mediated signalling in primary CML progenitor cells. The PI-3K inhibitors, wortmannin and LY294002 reproduced the differential effects of imatinib on normal and CML progenitor cell proliferation in vitro by increasing normal cell (P = 0.001) and reducing CML cell proliferation (P = 0.0003). This differential effect was attributable to dysregulated signalling by granulocyte colony-stimulating factor in CML. The responses of individual patient's cells to wortmannin correlated with their responses to imatinib (P = 0.004) but not their responses to AG490 (Jak2 kinase inhibitor) or FTI II (farnesyltransferase inhibitor). Individual responses to wortmannin also correlated with responses to interferon alpha (IFNalpha) (P = 0.016). Imatinib-resistant K562 cells were sensitive to LY294002. Inhibition of the PI-3K pathway may be common to imatinib and IFNalpha and reflect dysregulated cytokine signalling. As imatinib-resistant cells remained sensitive to wortmannin and LY294002, targeting the PI-3K pathway may provide an alternative therapy for imatinib-resistant patients.     

Expansion of granulocyte colony-stimulating factor/chemotherapy-mobilized CD34+ hematopoietic progenitors: role of granulocyte-macrophage colony-stimulating factor/erythropoietin hybrid protein (MEN11303) and interleukin-15

Ex vivo stroma-free static liquid cultures of granulocyte colony-stimulating factor (G-CSF)/chemotherapy-mobilized CD34+ cells were established from patients with epithelial solid tumors. Different culture conditions were generated by adding G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (Flt3), megakaryocyte growth and development factor (Peg-rHuMGDF), GM-CSF/erythropoietin (EPO) hybrid protein (MEN11303), and interleukin-15 (IL-15) to the basic stem cell factor (SCF) + interleukin-3 (IL-3) + EPO combination. This study showed that, among the nine different combinations tested in our 5% autologous plasma-containing cultures, only those containing IL-3/SCF/Flt3/MEN11303 and IL-3/SCF/Flt3/MEN11303/IL-15 significantly expanded colony-forming unit granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), long-term culture-initiating cells (LTC-IC), CD34+, and CD34+/CD38- cells after 14 days of culture. Particularly, the addition of IL-15 to IL-3/SCF/Flt3/MEN11303 combination produced a significant increase of LTC-IC, with an average 26-fold amplification as compared to input cells, without any detrimental effect on CFU-GM and BFU-E expansion. This combination also produced a statistically significant 3.6-fold expansion of primitive CD34+/CD38- cells. Moreover, this study confirms the previously described erythropoietic effect of MEN11303, which, in our experience, was the only factor capable of expanding BFU-E. Compared to equimolar concentrations of GM-CSF and EPO, MEN11303 hybrid protein showed a significantly higher capacity of expanding CFU-GM, BFU-E, LTC-IC, CD34+, and CD34+/CD38- cells when these cytokines were tested in combination with IL-3/SCF/Flt3. These cultures indicated that Peg-rHuMGDF addition to IL-3/SCF/EPO/Flt3 does not affect CFU-GM and BFU-E expansion but, unlike G-CSF or GM-CSF, it does not decrease the ability of Flt3 to expand primitive LTC-IC. These studies indicate that, starting from G-CSF/chemotherapy-mobilized CD34+ cells, concomitant expansion of primitive LTC-IC, CFU-GM, BFU-E, CD34+, and CD34+/CD38- cells is feasible in simple stroma-free static liquid cultures, provided IL-3/SCF/Flt3/MEN11303/IL-15 combination is used as expanding cocktail in the presence of 5% autologous plasma.     

Phosphatidylinositol 3-kinase is involved in the protection of primary cultured human erythroid precursor cells from apoptosis.

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34(+) cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 micromol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V-binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.     

Divergent effects of interleukin-4 (IL-4) on the granulocyte colony-stimulating factor and IL-3-supported myeloid colony formation from normal and leukemic bone marrow cells.

Human recombinant interleukin-4 (IL-4) was studied for its effects on myeloid progenitor cells from normal and leukemic bone marrow cells in the presence and absence of additional growth factors. IL-4 itself did not support myeloid cluster or colony formation (CFU-GM). However, cultures supplied with IL-4 (300 U/mL) and IL-3 demonstrated a significant decline in myeloid colony numbers (CFU-GM) compared with the effects of IL-3 alone: (48 +/- 27 v 88 +/- 27 CFU-GM/10(5) MNC). In contrast, IL-4 augmented the G-CSF-supported CFU-GM: (80 +/- 31 v 148 +/- 52 CFU-GM/10(5) MNC). The effects of IL-4 were not mediated by accessory cells because similar results were obtained with and without T-cell, B-cell, or adherent depleted cell fractions. Morphologic analysis of clusters (day 7) and the colonies (day 14) demonstrated that IL-4 enhanced myeloid colony formation in the presence of G-CSF, whereas the cultures supplied with IL-3 and IL-4 did not show a lineage-restricted decline of CFU-GM. A heterogeneity in growth response was observed in the leukemic counterpart. With the 3H-thymidine proliferation assay, IL-4 augmented the G-CSF-induced proliferation of acute myeloid leukemic (AML) cells in 4 of the 12 cases, while the IL-3-supported proliferation was antagonized in 3 of the 12 cases. In the blast colony assay, IL-4 suppressed the IL-3-supported AML-CFU in the majority of cases, but enhanced the G-CSF stimulated AML-CFU in 3 of 6 cases. These data demonstrate divergent effects of IL-4 on the normal myeloid progenitor cell in the presence of IL-3 or G-CSF, while a variability in responsiveness is observed in the leukemic counterpart.     

Influence of combinations of cytokines on proliferation of isolated single cell-sorted human bone marrow hematopoietic progenitor cells in the absence and presence of serum.

Human mast cell growth factor (MGF, a c-kit ligand) and colony stimulating factors (Epo, GM-CSF, G-CSF, IL-3) were assessed in the absence or presence of serum for stimulation in semi-solid medium of single CD34 , CD34 HLA-DR+, or CD34 HLA-DR+CD33- cells sorted per microtiter well. The % of wells containing CFU-GM and erythroid containing (BFU-E and CFU-GEMM) colonies increased in proportion to the number of cytokines added. In the presence of serum, 1, to 4 cytokine combinations resulted in respective increases in cloning efficiencies of 10 to 21.0, 19.5 to 31.5, 35.8 to 42.9, and 46.3 to 60.0%. MGF had little effect by itself, but did act in combination with CSFs to enhance numbers and size of the colonies from isolated single cells. High cloning efficiencies were also obtained in the absence of serum when multiple cytokines were used. The results demonstrate that MGF and CSFs can act directly on the proliferation of single hematopoietic progenitor cells in the absence of accessory cells and serum.     

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.     

Activation of phosphatidylinositol 3-kinase is important for erythropoietin-induced erythropoiesis from CD34(+) hematopoietic progenitor cells

OBJECTIVE: Several transducing molecules, including JAK2, STAT5, MAP kinases, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma1, and PKC are activated by interaction between erythropoietin (EPO) and the EPO receptor. The aim of this was to examine the relative involvement of PI3K in the development of glycophorin A (GPA)(+) erythroid cells from normal hematopoietic progenitor cells. MATERIALS AND METHODS: CD34(+) hematopoietic progenitor cells or subpopulations obtained by FACS sorting were cultured in serum-free medium containing EPO with or without inhibitors for PI3K, p38, MEK, or PKC for various time periods before phenotypic analysis or detection of apoptosis by flow cytometry, cell cycle analysis, high-resolution tracking of cell division, Western blot analysis, or Akt kinase assay were performed. RESULTS: The PI3K inhibitor LY294002 completely counteracted the EPO-induced proliferation of CD34(+) progenitor cells and CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 also highly suppressed the expanded erythropoiesis induced by the combined action of EPO and stem cell factor. The profound inhibitory effect of LY294002 on proliferation was caused by its induction of cell cycle arrest in the G(0)/G(1) phase of the cell cycle. Some cells acquired GPA expression before they went through cell division. This was completely blocked by LY294002, implying an inhibitory effect on maturation. In addition, LY294002 completely blocked the viability-enhancing effect of EPO in CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 and various inhibitors of PKC completely suppressed the EPO-induced increase in the activity of Akt kinase, a direct downstream target of PI3K. CONCLUSIONS: Our results point to an important role for PI3K in mediating EPO-induced survival, proliferation, and possibly maturation of early erythroid progenitors.     

The effect of interleukin-12 in ex-vivo expansion of human haemopoietic progenitors

Summary. We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL-12. In cultures of low density cells, cytokine combinations including IL-12 were associated to a greater proliferation (up to 6.7 ± 2.5 CFU-GM fold expansion). However, in cultures of purified CD34⁺ cells the more efficient cytokine combination (147 ± 49 CFU-GM fold expansion) was SCF, IL-3, IL-11 and MlP-la, and the addition of IL-12 did not further enhance expansion of progenitors.
These results indicate that accessory cells, lost in CD34⁺ cell purification, could be in part responsible for IL-12 effect on progenitor cell proliferation. In CD34⁺ cell cultures the addition of IL-12 led to CD19 mRNA generation, suggesting that IL-12 acts on haemopoietic cells with both myeloid and lymphoid potential.     

Comparison of megakaryopoiesis in vitro of paired peripheral blood progenitor cells and bone marrow harvested during remission in patients with acute myeloid leukaemia

We have studied paired peripheral blood progenitor cells (PBPC) and bone marrow (BM) samples from 12 acute myeloid leukaemia (AML) patients following intensive chemotherapy, and assessed direct granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), megakaryocyte CFU (CFU-Mk) numbers and the production of CD61+ (platelet glycoprotein IIIa) cells in suspension culture in response to various haemopoietic growth factor combinations. We found that CFU-GM and BFU-E numbers per 105 mononuclear cells were similar in both AML PBPC and BM harvests; CFU-Mk numbers, however, were significantly higher in PBPC than BM. In addition, the higher total white cell count of the PBPC harvests meant that PBPC have much higher numbers of total progenitors per collection. CD61+ cell numbers in suspension cultures of AML PBPC and BM were lower than those of harvested normal marrow. However, response to pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) both alone and in combination with other growth factors was qualitatively similar to that of normal BM. As with normal BM, response to PEGrHuMGDF alone did not increase further with addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 6 (IL-6) or erythropoietin (EPO) in the AML PBPC and BM. Further responses over PEGrHuMGDF alone were seen when added with stem cell factor (SCF) or with a combination of SCF + IL-3 + EPO in both AML PBPC and BM cultures; however, the magnitude of the response was greater in the PBPC cultures. Response to PEGrHuMGDF + IL-3 was seen in the PBPC cultures but not in the AML BM. These data suggest that, in AML patients, there are proportionally more megakaryocyte progenitor cells in the mobilized PBPC than in the BM harvests, which would explain the more rapid platelet recovery following PBPC autografts.     

Activation of Human Neutrophils by Granulocyte Colony-Stimulating Factor,Granulocyte-Macrophage Colony-Stimulating Factor,and Tumor Necrosis Factor α: Role of Phosphatidylinositol 3-Kinase

Stimulation of human neutrophils with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or tumor necrosis factor alpha (TNF) resulted in phosphorylation of Akt, the potency being GM-CSF > G-CSF = TNF, which was inhibited by wortmannin. The findings indicated that phosphatidylinositol 3-kinase (PI3K) is activated by these cytokines. The possible participation of PI3K in activation of neutrophil functions induced by these cytokines was explored with PI3K inhibitors (wortmannin and LY294002). Superoxide release and adherence induced by GM-CSF or TNF were inhibited by PI3K inhibitors. Actin reorganization and morphological changes induced by G-CSF or GM-CSF were also inhibited by wortmannin, whereas these responses induced by TNF were unaffected by wortmannin. These findings suggested that PI3K is differentially involved in cytokine-mediated activation of neutrophil functions depending on the cytokines used. The results also showed that activation of extracellular signal-regulated kinase, but not p38 mitogen-activated protein kinase, induced by these cytokines is partly mediated by PI3K activation.     

Peripheral blood progenitor cell mobilisation alters myeloid, but not erythroid, progenitor cell self-renewal kinetics

Transplantation of progenitor cells which have been mobilised into the bloodstream (PBPC) following the administration of G-CSF results in more rapid neutrophil recovery than transplantation of bone marrow (BM). The reasons for the accelerated neutrophil engraftment are not clear, but would be explained by increased self-replication of myeloid progenitor cells (CFU-GM). We have used a CFU-GM replating assay to investigate myeloid progenitor self-replication, and quantification of subcolony formation during erythroid burst formation to quantify erythroid progenitor self-renewal. Secondary colony formation by CFU-GM, grown from PBPC and then replated was increased compared with secondary colony formation by BM CFU-GM (P = 0.0001); erythroid subcolony formation was not altered. There was no difference between the replating abilities of PBPC CFU-GM derived from allogeneic donors (normal individuals) and autologous donors (patients with malignant disease) although differences were found between subgroups of autologous donors. The increased replication of PBPC could not be accounted for by a reduction in progenitor cell apoptosis; PBPC CFU-GM contained slightly fewer apoptotic CD34+ cells than BM CFU-GM. The increased replication by PBPC CFU-GM was reversible because it declined when CFU-GM colonies were passaged through three sequential CFU-GM replating cycles. This decline in self-replication was more rapid than the decline seen in replated BM CFU-GM. The self-replication of PBPC CFU-GM, and subcolony formation by BFU-E could be further enhanced by exposure to cytokines in vitro. We conclude that mobilisation alters the replication kinetics of myeloid, but not of erythroid, progenitor cells, that mobilisation-induced events are of limited duration and that in vitro exposure to cytokines may modify PBPC progenitor cell kinetics.     

Activin A suppresses proliferation of interleukin-3-responsive granulocyte-macrophage colony-forming progenitors and stimulates proliferation and differentiation of interleukin-3-responsive erythroid burst-forming progenitors in the peripheral blood

We examined the effects of activin A on the proliferation and differentiation of immature hematopoietic progenitors prepared from peripheral blood (PB) using methylcellulose and liquid-suspension culture. In a kinetic analysis, colony formation by PB granulocyte- macrophage colony-forming unit (CFU-GM) was delayed in a dose-dependent manner by the addition of activin A only when stimulated with interleukin-3 (IL-3), but not when stimulated with granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF) plus G-CSF. DNA-synthesizing CFU-GM was increased by IL-3, but this effect was abolished by activin A. In contrast, PB erythroid burst-forming unit (BFU-E) was accelerated by the addition of activin A only when exposed to IL-3 plus erythropoietin (Epo), but not when exposed to Epo or Epo plus SCF. DNA- synthesizing BFU-E was increased by IL-3 and activin A, alone and additively in combination. In a mixed culture of myeloid and erythroid progenitors, activin A increased the numbers of BFU-E and CFU-Mix colonies at concentrations of 1 and 10 ng/mL and decreased the number of CFU-GM colonies in a dose-dependent manner. However, in a liquid- suspension culture of erythroid progenitors, activin A decreased total cell count and the percentage of hemoglobin-containing cells only when cells were exposed to IL-3 plus Epo. These results indicate that activin A suppresses the proliferation of IL-3-responsive CFU-GM progenitors and stimulates the proliferation and differentiation of IL- 3-responsive BFU-E progenitors, and suggest that activin A acts as a commitment factor of immature hematopoietic progenitors for erythroid differentiation.     

Association of p72 tyrosine kinase with Stat factors and its activation by interleukin-3, interleukin-6, and granulocyte colony-stimulating factor

Hematopoietic cytokines, including interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF), induce the proliferation, differentiation, and activation of hematopoietic lineage cells. These cytokines activate the Jak/Stat-mediated signal transduction pathway that is important in the biologic activities of these cytokines. In this study, we showed that hematopoietic cytokines, such as IL-3, IL-6, and G-CSF, all induced tyrosine-phosphorylation of Stat family proteins and Stat-associated 150-kD and 72-kD molecules in hematopoietic lineage cell lines. Furthermore, we showed that the 72-kD molecule had tyrosine kinase activity. The tyrosine kinase activity of the 72-kD molecule was enhanced by the stimulation through an IL-6 signal transducer, gp130, that was shared among the receptors for the IL-6-related cytokine subfamily, such as leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotrophic factor. Because 72-kD tyrosine kinase was distinct from Syk, Tec, and Btk and coimmunoprecipitated with anti-Stat antiserum, we termed it Stat- associated 72-kD tyrosine kinase (p72sak). p72sak may directly activate Stat family proteins or other signal transducing molecules for IL-3, G- CSF, and the IL-6-related cytokine subfamily.     

In vitro growth of human fetal CD34+ cells in the presence of various combinations of recombinant cytokines under serum-free culture conditions

Summary. CD34⁺ cells were purified from midtrimester human fetal blood and adult bone marrow samples and seeded in serum-free fibrin-clot cultures in order to evaluate the number and the responsiveness to recombinant cytokines of pluripotent (CFU-GEMM), erythroid (BFU-E), megakaryocyte (BFU-meg and CFU-meg) and granulocyte/macrophage (CFU-GM) haemopoietic progenitor cells.
The number of the different haemopoietic progenitors/1 × 10³ CD34⁺ cells, except CFU-meg, was significantly higher in fetal blood than in adult bone marrow in cultures stimulated by any combination of cytokines including interleukin-3 (IL-3), granulocyte/macrophage colony stimulating factor (GM-CSF) or stem cell factor (SCF) plus erythropoietin (Epo). Nevertheless, whereas adult BFU-E showed a maximal growth in the presence of Epo plus IL-3 or Epo plus SCF, fetal BFU-E showed an optimal growth in the presence of Epo alone, the sensitivity of fetal BFU-E to suboptimal concentrations of Epo being approximately 10–15-fold higher than that of adult BFU-E. Addition of optimal concentrations of IL-3, GM-CSF or SCF, alone or in various combinations, to Epocontaining cultures induced a significant increase in both the number and size of fetal CFU-GEMM, and CFU-GM, and a parallel decrease of fetal BFU-E. Finally, SCF potently syner-gized with IL-3 in increasing the growth of both classes of fetal megakaryocyte progenitors, BFU-meg and CFU-meg.     

Disparate regulation of human fetal erythropoiesis by the microenvironments of the liver and bone marrow

The liver and the bone marrow (BM) are the major organs that support hematopoiesis in the human fetus. Although both tissues contain the spectrum of hematopoietic cells, erythropoiesis dominates the liver. Previous studies suggested that a unique responsiveness of fetal burst-forming units erythroid (BFU-E) to erythropoietin (EPO) obviates the need for cytokines with burst-promoting activity (BPA) in fetal erythropoiesis. This potential regulatory mechanism whereby fetal erythropoiesis is enhanced was further investigated. Fluorescence-activated cell sorting was used to isolate liver and BM progenitors based on their levels of CD34 and CD38 expression. The most mature population of CD34+ lineage (Lin-) cells was also the most prevalent of the three subpopulations and contained BFU-E responsive to EPO alone under serum-deprived conditions. Kit ligand (KL) also strongly synergized with EPO in stimulating the growth of these BFU-E. An intermediate subset of CD34++CD38+Lin- cells contained erythroid progenitors responsive to EPO alone, but also displayed synergism between EPO and KL, granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin (IL)-3, demonstrating that erythroid progenitors that respond to cytokines with BPA do exist in fetal tissues as in the adult BM. Candidate stem cells (CD34++CD38-Lin- cells) did not respond to EPO. Synergisms among KL, GM-CSF, and IL-3, and to a lesser extent granulocyte colony-stimulating factor (G-CSF) and FLK-2/FLT-3 ligand (FL), supported the growth of primitive multipotent progenitors that became responsive to EPO. These data define the limits of EPO activity in fetal erythropoiesis to cells that express CD38 and demonstrate the potential for various cytokine interactions to be involved in regulating fetal erythropoiesis. Furthermore, a comparison of the responses of liver and BM erythroid progenitors revealed similarity in their responses to cytokines but a difference in the frequency of BFU-E among the three subpopulations examined. A higher frequency of BFU-E among the intermediate and late progenitor subsets in the liver indicates that regulatory factors acting on stem cells and their immediate progeny are partially responsible for the high content of erythropoiesis in the liver. These data implicate a critical role for the microenvironments of the liver and BM in regulating the disparate levels of erythropoiesis in these tissues.     

An inhibitor of PI3-K differentially affects proliferation and IL-6 protein secretion in normal and leukemic myeloid cells depending on the stage of differentiation

In this study, we examined the involvement of the phosphatidylinositol 3-kinase (PI3-K) and p70S6 kinase signal transduction pathway in the interleukin-1(IL-1)-mediated proliferation and cytokine production by normal and leukemic myeloid cells.Total AML blast populations, early progenitor (CD34(+)/CD36(-)) cells, and more differentiated (CD34(-)/CD36(+)) cells were treated with the PI3-K inhibitor Ly294002 and p70S6K inhibitor rapamycin. The effects on proliferation, IL-6 protein secretion, and intracellular signaling cascades were determined and compared with normal CD34(+) cells and monocytes.The function of the PI3-K pathway was dependent on the differentiation state of the AML cell population. In immature blasts, the IL-1-induced proliferation was strongly inhibited by Ly294002 and rapamycin, without a distinct effect on IL-6 protein production. In contrast, in mature monocytic blast cells inhibition of the PI3-K signaling route had a stimulatory effect on IL-6 protein secretion. Interestingly, these findings were not specifically linked to the malignant counterpart but were also observed with normal CD34(+) sorted cells vs mature monocytes. Evidence is provided that the Ly294002-induced increase in IL-6 protein secretion is linked to the cAMP dependent signaling pathway and not to changes in the phosphorylation of ERK or p38. However, although the enhanced IL-6 protein secretion is cAMP dependent, it was not found to be mediated by protein kinase A (PKA) or by the GTP-ase Rap1.This study indicates that inhibition of the PI3-K signaling pathway has an inhibitory effect on cell proliferation but a stimulatory effect on IL-6 expression mediated by a cAMP-dependent but PKA-independent route.     

Effects of immunosuppressants, FK506, deoxyspergualin, and cyclosporine A on immature human hematopoiesis

Effects of the immunosuppressants, FK506, deoxyspergualin (DSG), and cyclosporine A (CsA) on the growth of human hematopoietic progenitor cells were tested in the presence of interleukin-3 (IL-3) with purified bone marrow and blood cells as targets in methylcellulose culture. FK506 had a significant stimulatory effect on the growth of colony- forming units/granulocyte-macrophage (CFU-GM) and burst-forming units/erythroid (BFU-E) from peripheral blood and cord blood cells but not from bone marrow cells. Neither DSG nor CsA had an effect on any type of target cell. Liquid-suspension-limiting dilution assay with IL- 3 showed that FK506 directly stimulated the growth of blood progenitors in a dose-dependent manner with single-hit kinetics. Liquid-suspension preincubation of blood cells with FK506 before culture in methylcellulose induced a significant increase in the amount of IL-3- supported growth of CFU-GM and BFU-E, whereas initial preincubation with IL-3 and subsequent culture with FK506 plus IL-3 exerted its stimulatory effect only on BFU-E. These data suggest that the stimulation of hematopoietic progenitor cells by FK506 occurs at a very early stage of maturation and diminishes with further myeloid development.     

Potential use of human stem cell factor as adjunctive therapy for human immunodeficiency virus-related cytopenias.

Hematopoietic dysfunction with peripheral cytopenias is a common complication of human immunodeficiency virus (HIV) infection. Symptomatic anemia is the most common cytopenia and occurs in the presence and absence of myelosuppressive drug therapy such as zidovudine. Drug-induced neutropenia and immune thrombocytopenia are also frequent and occur in up to 50% of acquired immunodeficiency syndrome (AIDS) patients. Attempts to reduce the impact of bone marrow failure have focused on dose reduction of zidovudine, ganciclovir, and chemotherapy, and the use of recombinant hematopoietic hormones such as erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF). Despite these maneuvers, approximately 30% of patients with AIDS receiving zidovudine will become transfusion-dependent. This has led to investigations of other cytokines that may increase blood cell formation. The recent identification of decreased number and proliferation of hematopoietic progenitors in patients with HIV infection suggests that agents which have activity on progenitor cell pools may have clinical utility. We demonstrate that human stem cell factor (HuSCF) increases burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte-monocyte (CFU-GM), and CFU-Mix formation in vitro in normal and HIV-infected individuals. HuSCF also decreases the sensitivity of BFU-E to inhibition by zidovudine without altering HIV replication in lymphocytes or monocytes, altering peripheral blood mononuclear cell proliferation to phytohemagglutinin (PHA) and interleukin-2 (IL-2) or altering the effectiveness of zidovudine or dideoxyinosine in inhibiting HIV replication in lymphocytes or monocytes. These studies suggest that HuSCF may have clinical utility in HIV infection as an adjunctive treatment for HIV-related cytopenias.