Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential.

BACKGROUND AND AIMS: Mesenchymal stem cells from bone marrow (MSCs) may have the potential to differentiate in vitro and in vivo into hepatocytes. We investigated whether transplanted human MSCs (hMSCs) may engraft the liver of non-obese diabetic severe combined immuno-deficient (NOD/SCID) mice and differentiate into cells of hepatic lineage. METHODS: Ex vivo expanded, highly purified and functionally active hMSCs from bone marrow were transplanted (caudal vein) in sublethally irradiated NOD/SCID mice that were either exposed or not to acute liver injury or submitted to a protocol of chronic injury (single or chronic intraperitoneal injection of CCl(4), respectively). Chimeric livers were analysed for expression of human transcripts and antigens. RESULTS: Liver engraftment of cells of human origin was very low in normal and acutely injured NOD/SCID mice with significantly higher numbers found in chronically injured livers. However, hepatocellular differentiation was relatively rare, limited to a low number of cells (ranging from less than 0.1% to 0.23%) as confirmed by very low or not detectable levels of human transcripts for alpha-fetoprotein, CK18, CK19 and albumin in either normal or injured livers. Finally, a significant number of cells of human origin exhibited a myofibroblast-like morphology. CONCLUSIONS: Transplanted hMSCs have the potential to migrate into normal and injured liver parenchyma, particularly under conditions of chronic injury, but differentiation into hepatocyte-like cells is a rare event and pro-fibrogenic potential of hMSC transplant should be not under-evaluated.

Platelet activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis.

Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS.

Kinetics of human hemopoietic cells after in vivo administration of granulocyte-macrophage colony-stimulating factor.

The kinetic changes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) on hemopoietic cells were assessed in physiological conditions by administering GM-CSF (8 micrograms/kg per d) for 3 d to nine patients with solid tumors and normal bone marrow (BM), before chemotherapy. GM-CSF increased the number of circulating granulocytes and monocytes; platelets, erythrocytes, lymphocyte number, and subsets were unmodified. GM-CSF increased the percentage of BM S phase BFU-E (from 32 +/- 7 to 79 +/- 16%), day 14 colony-forming unit granulocyte-macrophage (CFU-GM) (from 43 +/- 20 to 82 +/- 11%) and day 7 CFU-GM (from 41 +/- 14 to 56 +/- 20%). The percentage of BM myeloblasts, promyelocytes, and myelocytes in S phase increased from 26 +/- 14 to 41 +/- 6%, and that of erythroblasts increased from 25 +/- 12 to 30 +/- 12%. This suggests that GM-CSF activates both erythroid and granulomonopoietic progenitors but that, among the morphologically recognizable BM precursors, only the granulomonopoietic lineage is a direct target of the molecule. GM-CSF increased the birth rate of cycling cells from 1.3 to 3.4 cells %/h and decreased the duration of the S phase from 14.3 to 9.1 h and the cell cycle time from 86 to 26 h. After treatment discontinuation, the number of circulating granulocytes and monocytes rapidly fell. The proportion of S phase BM cells dropped to values lower than pretreatment levels, suggesting a period of relative refractoriness to cell cycle-active antineoplastic agents.

Opposite effect of tumor necrosis factor alpha on granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor-dependent growth of normal and leukemic hemopoietic progenitors.

The effect of recombinant human tumor necrosis factor alpha (TNF-alpha) on normal and chronic myeloid leukemia granulocyte-macrophage progenitors (CFU-GM) growing in semisolid agar cultures in the presence of recombinant granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor was studied. Granulocyte-macrophage colony-stimulating factor-dependent growth of normal and chronic myeloid leukemia bone marrow CFU-GM was greatly enhanced by TNF-alpha at doses of 0.1 to 100 units/ml. Growth enhancement included neutrophil, eosinophil, and monocyte-macrophage colonies and clusters at 7 and 14 days of culture. Since similar results were achieved with highly enriched progenitor cell populations, devoid of accessory cells, an indirect effect on CFU-GM growth through the release by accessory cells of other cytokines upon TNF-alpha stimulation was thus ruled out. By contrast, the same doses of TNF-alpha inhibited the growth of normal CFU-GM in granulocyte colony-stimulating factor-dependent cultures. Taken together, our findings indicate that the final effect of TNF-alpha on normal bone marrow granulocyte-macrophage progenitor growth is dependent on the specific growth factor interacting with it, and that both normal and chronic myeloid leukemia CFU-GM are equally responsive to the combined effects of TNF-alpha and a given colony-stimulating factor.

Expression of HLA class II (DR, DQ) determinants by normal and chronic myeloid leukemia granulocyte/monocyte progenitors.

It has been suggested that the expression of certain HLA class II antigens stemming from three distinct loci (DR, DP, and DQ) is important not only in the regulation of the immune response but also on the response of hemopoietic precursors to factors inhibiting myelopoiesis. Changes in the expression of DR antigens may be involved in the pathogenesis of altered cell proliferation in chronic myeloid leukemia, since they result in decreased sensitivity of the colony forming units, granulocyte-macrophage to prostaglandin E and acidic isoferritins. In studies using monoclonal antibodies against monomorphic DR or DQ determinants, in a complement-dependent cytotoxic assay, it was found that nearly all normal and chronic myeloid leukemia bone marrow colony forming units, granulocyte-macrophage express DR antigens. The dose response curve was similar for both normal and leukemic precursors. Leukemic peripheral blood precursors were more sensitive than were normal peripheral blood precursors. Normal colony forming units, granulocyte-macrophage did not express DQ antigens, whereas these were expressed in varying quantities by leukemic cells. This study shows that, in the patients we studied, leukemic cells express DR antigens in amounts comparable to normal. In addition, varying amounts of DQ antigens may be observed on leukemic but not on normal progenitors, perhaps as a consequence of an increase in the number of antigens also expressed by normal cells, though in an amount below the detection threshold of cytotoxicity techniques.

Platelet-activating factor (PAF) induces the early tyrosine phosphorylation of focal adhesion kinase (p125FAK) in human endothelial cells.

Platelet-activating factor (PAF) is a potent activator of angiogenesis and controls the motility and the shape of vascular endothelium. The mechanism(s) whereby PAF exerts its action are in part known. Here we report that the biological active (R)PAF enantiomer administrated to cultured endothelial cells induces the early phosphorylation in tyrosine residues of focal adhesion kinase (p125FAX) and paxillin, two molecules involved in the early signaling and cytoskeleton assembly in cells that undergo integrin-mediated adhesion or are challenged by neuropeptides or lysophosphatidic acid. The phenomenon is rapidly turned on, lasts for a few minutes and is adhesion-independent indicating that the chain of events induced by (R)PAF, including p125FAK activation, precedes adhesion. The inhibitory effect of WEB2086, a PAF receptor antagonist, and the lack of activity exerted by the (S)PAF enantiomer, indicate that (R)PAF-mediated p125FAK activation, is PAF receptor-dependent. Calphostin C, an inhibitor of protein kinase C blocks the effect of (R)PAF on p125FAK phosphorylation suggesting that protein kinase C activation is up-stream the activation of this tyrosine kinase. When endothelial cells are exposed to a substratum that allows adhesion and spreading. (R)PAF-stimulated cells, change their adhesive phenotype and start migrating. Inhibitors of tyrosine kinases, like 3-(1,4,-dihydroxytetralyl) methylen-2-oxindole and herbimycin A, reduce the cells migration, the transendothelial flux of albumin and the enhancement of p125FAK activity induced by (R)PAF. The observation that increased tyrosine phosphorylation of p125FAK and its ensuring association with focal adhesion occurs rapidly upon (R)PAF challenge indicates that this signaling molecule has a primary and independent role also in the signaling cascade initiated by (R)PAF.

Studies on the mechanism of interleukin 1 stimulation of platelet activating factor synthesis in human endothelial cells in culture.

Interleukin 1 promotes the conversion of the biologically inactive lyso-platelet activating factor (lyso-PAF) to the bioactive platelet activating factor (PAF) by an acetylation reaction in cultured human endothelial cells. After 2 h stimulation with interleukin 1, 1-O-alkyl-2-lysoglycero-3-phosphocholine (GPC): acetyl CoA acetyltransferase is activated, reaching a plateau after 6 h and then declining to the basal value within 24 h. This time course is comparable to that of PAF production. These cells are able to incorporate [3H]acetate and [3H]lyso-PAF into PAF. Synthetized [3H]PAF is then catabolized in [3H]alkylacyl phosphoglycerides. 1-O-alkyl-2-acetylglycerol: CDP-choline cholinephosphotransferase and 1-O-alkyl-2-acetyl-GPC: acetylhydrolase activities are both present in endothelial cells, but are not activated under our conditions of stimuli. These findings indicate that interleukin 1 induces the PAF synthesis by a deacylation/reacetylation mechanism into human endothelial cells.

Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts.

The stem cell tyrosine kinase 1 (STK1) protein is the human homologue of the murine FLT3 gene product, a receptor belonging to the FMS/KIT family. FLT3 and KIT with their ligands control the growth and differentiation of early human hemopoietic cells. In the present study, 16 cases of acute myeloid leukemia (AML) were examined by flow cytometry for cell surface expression of FLT3 and KIT receptors. All cases were also tested for their proliferative response to human FLT3 ligand (FL) and KIT ligand (KL) and for colony formation in the presence of single or associated cytokines. Among 16 AML cases tested, 10/16 expressed FLT3 receptor and 12/16 expressed KIT receptor, without any correlation with FAB subtype. FL and KL stimulated the proliferation of leukemic blasts in 11/16 AML cases (including five FLT3 or KIT receptor-negative cases), with an additive effect when added simultaneously. By contrast, some receptor-expressing AMLs did not display significant proliferative responses to their respective ligands. FL and KL as single factors induced or significantly increased the colony formation by clonogenic precursor cells respectively in eight and six of 13 cases tested. In some cases growth factor association significantly enhanced colony growth. Taken together these observations provide evidence that the pattern of FLT3 and KIT receptor expression is extremely variable among the AMLs and that receptor presence is not necessarily combined with proliferative and clonogenic response or vice versa.

Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.

Cord blood (CB) is an attractive alternative to bone marrow or peripheral blood as a source of transplantable hematopoietic tissue. However, because of the reduced volume, the stem cell content is limited; therefore its use as a graft for adult patients might require ex vivo manipulations. Two systems have been described that identify these stem cell populations in vitro in both mice and humans: (1) the long-term culture-initiating cells (LTC-IC), thus named because of their ability to support the growth of hematopoietic colonies (colony-forming cell (CFC)) for 5-6 weeks when co-cultured on stromal layers; (2) the generation of hematopoietic progenitors (CFC) from stroma-free liquid cultures for extended periods of time, which provides further indirect evidence of the presence of primitive stem cells. Both systems detect largely overlapping but not identical populations of stem cells. Thus the identification of the growth factor requirements for the maintenance and amplification of both systems is relevant. On this basis, analysis of the effects of 18 cytokine combinations on stroma-free liquid cultures of CB CD34+ cells, showed that: (1) after 7- and 14 day-incubation periods, several growth factor combinations expanded the LTC-IC pool to a similar extent; as compared to the LTC-IC, the generation of CFC was not impressive; (2) time-course analysis of the LTC-IC expansion demonstrated that, by extending the incubation period, only a few growth factor combinations, containing FL, TPO, KL and IL6, could support a further, increasingly greater LTC-IC expansion (up to 270000-fold of the initial value). In similar culture conditions, CFC production underwent continuous expansion, which persisted for over 7 months and reached values of one million-fold of the initial value. The simultaneous presence of FL and TPO was both necessary and sufficient to support this phenomenon. The addition of KL+/-IL6 did not appear to substantially modify the extent of LTC-IC expansion; nevertheless, it played an important role in sustaining an even more massive and prolonged output of CFU-GM, CFU-Mk and BFU/CFU-GEMM (up to 100 million-fold); (3) the presence of IL3 was found to be negative, in that it inhibited both the extent of LTC-IC expansion and the long-term generation of CFC. Thus, FL and TPO appear as two unique growth factors that preferentially support the self-renewal of primitive stem cells; the additional presence of KL and IL6 seems to enhance the proliferative potential of at least one subpopulation of daughter stem cells, which may follow three differentiation pathways. Far from being definitive, our data demonstrated that massive stem cell expansion, in cord blood, can be obtained in reasonably well-defined culture conditions. This could represent an initial step towards larger scale cultures for transplantation and gene therapy protocols.

Stem cell factor improvement of proliferation and maintenance of hemopoietic progenitors in myelodysplastic syndromes.

Human recombinant stem cell factor (rSCF) was tested for its capability of improving the defective growth of hemopoietic progenitors in 28 cases of myelodysplastic syndromes (MDS). In vitro growth and response to rSCF were quite variable. However, in most cases, rSCF stimulated CFU-GM growth induced by rG-CSF, rGM-CSF, rIL-3, 5637 conditioned medium (50-1400% enhancement). rSCF effect was slightly more evident on day 14 CFU-GM and in the presence of rIL-3. BFU-E growth induced by rEPO or rIL-3 + rEPO was enhanced by rSCF in about 50% of cases, in linear correlation with the levels of patients' hemoglobin. rSCF did not increase CFU-E growth, whereas it slightly stimulated CFU-Mk in 33% of the cases. EPO, SCF and, particularly, their combination, enhanced the recovery of normal CFU-E and BFU-E after 7 days of liquid culture. This was less evident in cultures of MDS patients. Conversely, CFU-GM generation in long term liquid cultures, although highly variable, was stimulated by rSCF and, above all, by rSCF + rG-CSF, similarly to what was observed with normal bone marrow samples. SCF seems to enhance in vitro erythropoiesis only in MDS cases presenting without severe anemia. It has little effect on megakaryocytopoiesis, while it seems to be more active on CFU-GM growth and maintenance.

In vivo effect of human granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophil GM-CSF receptors.

The effect of in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophils GM-CSF receptor, was investigated in patients with neoplastic diseases and normal hematopoiesis. Patients were divided into two groups. Group A received a single dose of rhGM-CSF (5 micrograms/kg/day) and receptor studies were performed 90 min and 48 h after treatment. Group B received three doses, administered subcutaneously every 24 h and receptor studies were performed 90 min after first injection and 24 h after the last. Before treatment neutrophils only displayed high-affinity receptors (KD 85 +/- 53 pM; number of receptors/cell 1318 +/- 567). The first injection of rhGM-CSF produced a transient leucopenia and the internalization of GM-CSF receptor on neutrophils in both groups of patients: 90 min after s.c. administration receptors could not be detected with conventional binding studies. In group A patients, 48 h after a single dose of rhGM-CSF, receptors, albeit with a decreased affinity (KD = 240 +/- 131 pM; number of receptors/cell 783 +/- 494) were again expressed. In group B patients, 24 h after the last rhGM-CSF injection, low intermediate affinity receptors not present before treatment appeared (KD 720 +/- 175 pM; number of receptor/cell 1222 +/- 179). They were associated with a low number of high affinity receptors (KD = 9 +/- 4 pM; number of receptors/cell 106 +/- 44). These observations indicate that more than one type of GM-CSF receptor may exist on neutrophils. It may be suggested that in vivo the regulation of the GM-CSF receptor is different from that in vitro and is related to the presence of the cytokine in patient blood.

In vivo effect of granulocyte-macrophage colony-stimulating factor on the kinetics of human acute myeloid leukemia cells.

Granulocyte-macrophage colony-stimulating factor, (GM-CSF) was given at 8 micrograms/kg daily by continuous i.v. infusion for 72 h to six patients with acute myeloid leukemia (AML) in expansion and one with chronic myeloid leukemia in blastic crisis to determine whether it was possible to augment the proliferative activity of the neoplastic population. The percentage of marrow blasts in S phase (labeling index, LI) was increased in five patients (1.3-, 1.5-, 1.9-, 2.3- and 3.2-fold change). The increase in LI was similar 24 and 48 h after beginning GM-CSF. The RNA Index also increased in patients who showed an increased LI, suggesting that GM-CSF had recruited quiescent neoplastic cells into the cell cycle. Forty eight hours after beginning GM-CSF, chemotherapy was started. The fate of S phase cells, labeled in vivo with bromodeoxyuridine (BrdU) immediately before cytostatic treatment, was monitored. BrdU positive cells were identified by fluorescent antibody for up to 28 days. A preferential killing of BrdU (S phase) cells was observed in 5/7 patients who obtained a complete remission, whereas this was not apparent in the two patients who achieved only a partial remission. Chemotherapy induced a rapid and profound aplasia; its duration, however, was not significantly different from that observed in historical controls. GM-CSF may have a potential role in the treatment of AML, as this study shows that it recruits leukemic cells into the cell cycle without adversely prolonging aplasia after cycle-specific therapy.

Megakaryocyte growth and development factor (MGDF)-induced acute leukemia cell proliferation and clonal growth is associated with functional c-mpl.

The effects of human recombinant megakaryocyte growth and development factor (MGDF) (also known as thrombopoietin (TPO)), alone or in combination with other growth factors, on the proliferation and on the clonal growth of clonogenic progenitors from 24 acute myeloblastic leukemia (AML) patients were evaluated. A significant proliferative response to MGDF alone (proliferation index > 1.5) was observed in nine of 23 cases; the responding cases belonged to all FAB subtypes. However, the greatest response (proliferation index > 7) was found in one M6 and in one M7 case. MGDF also enhanced interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), c-kit ligand (KL) and FLT3 ligand (FL) stimulated blast cell proliferation. MGDF as a single factor induced or significantly enhanced colony formation by clonogenic precursor cells in 12 of 14 AML cases. MGDF strongly increased KL-induced leukemic colony growth in seven cases, whereas it only moderately enhanced IL-3- or GM-CSF-induced colony growth. The analysis of tyrosine phosphorylated protein(s) upon MGDF stimulation in fresh AML cells was also performed. The results demonstrated a band of approximately 90 kDa phosphorylated protein(s) upon MGDF stimulation in AML responsive cases, but not in unresponsive ones. Taken together the present findings suggest that, in a consistent proportion of AML cases, MGDF stimulates blast cell growth and induces tyrosine protein phosphorylation.

In vitro reappearance of myeloid progenitors killed by mafosfamide.

Cyclophosphamide derivatives active in vitro, such as mafosfamide, are potentially capable of reducing the number of leukemic cells remaining in marrow explanted for autografting. Although this treatment kills nearly all the committed hemopoietic progenitors, it does not prevent the reinstatement of hemopoiesis after chemoradiotherapy. This points to the persistence of more immature hemopoietic progenitors not detectable with current semi-solid culture techniques and resistant to cytotoxic treatment. Treatment of normal marrow with 80-140 micrograms/ml mafosfamide is followed in medium-term cultures by a gradual and dose-dependent reduction in total cellularity, whereas granulomonocyte progenitors (CFU-GM), virtually absent at the start of the culture, progressively reappear. The quantity of progenitors present after day 14 in liquid culture is, however, less in the treated marrows than in the controls, and the reappearance of CFU-GM is inversely related to the mafosfamide dose. In addition, the recovery of the more immature (day-14) CFU-GM is greater than that of the more mature (day 7) CFU-GM.

Responsiveness of highly enriched CFU-GM subpopulations from bone marrow, peripheral blood, and cord blood to hemopoietic growth inhibitors.

Human early and late granulocyte-monocyte progenitors (granulocyte-macrophage colony-forming units, CFU-GM), depleted of accessory cells, were physically separated using an antimyeloid monoclonal antibody (DS1.1). They were separately cultured at optimal growth conditions and tested for responsiveness to prostaglandin E2 (PGE2), recombinant tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta-1 (TGF beta 1). Late (DS1.1+) CFU-GM displayed the highest sensitivity to PGE2 and TNF alpha, the first significant inhibition being evident at 10(-9)M PGE2 and 1 U/ml TNF alpha. Conversely, their growth was stimulated (211%-217%) by 0.25-2.5 ng/ml TGF beta 1. Early (DS1.1-) marrow CFU-GM evidenced a lower sensitivity to PGE2 and TNF alpha. Their growth, however, was inhibited by 0.25-2.5 ng/ml TGF beta 1. Early CFU-GM constitute the totality of peripheral blood myeloid progenitors. Cord blood CFU-GM were also demonstrated here to be entirely DS1.1-. Both adult and cord blood CFU-GM displayed the highest resistance to PGE2 and TNF alpha. By contrast, they showed the maximum sensitivity to growth inhibition by TGF beta 1, active at 0.025-0.25 ng/ml. For the first time, therefore, highly purified subsets of human CFU-GM were separated that displayed a different responsiveness to well-defined growth-regulatory molecules. Our results indicate that TGF beta 1 has a dual activity; it is inhibitory on early and stimulatory on late CFU-GM, whereas PGE2 and TNF alpha preferentially inhibit late CFU-GM growth.

The effects of human FLT3 ligand on in vitro human megakaryocytopoiesis.

The human homolog of the murine flt3/flk2 gene product is a tyrosine kinase receptor that plays a role in regulating the proliferation and differentiation of cells in the hematopoietic system. Using a plasma-clot clonal assay and a long-term bone marrow culture (LTBMC) system, we studied the effects of the recently cloned human flt3 ligand (FL) alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or stem cell factor (c-kit ligand [KL]) on human megakaryocytopoiesis. The effects of FL on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. FL alone had no megakaryocytic colony-stimulating activity (MK-CSA), but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. FL synergized with IL-3 at the level of both CFU-MK and BFU-MK and with GM-CSF and KL at the level of CFU-MK. Although FL alone exhibited a limited potential in sustaining long-term megakaryocytopoiesis in vitro, it synergistically augmented the ability of IL-3 and KL, alone or in association, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that FL plays a significant role in this process by amplifying the MK-CSA of GM-CSF, IL-3, and KL.

Interaction of transforming growth factor-beta 1 with hemopoietic growth factors in the regulation of human normal and leukemic myelopoiesis.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on the growth of normal and chronic myeloid leukemia (CML) granulo-monopoietic progenitors (CFU-GM) and erythroid progenitors (BFU-E) of different origins and degrees of maturation. In the presence of the supernatant of the 5637 cell line, used as a source of growth factors, TGF-beta 1 stimulates the growth of day-7 CFU-GM from Ficoll-isolated normal bone marrow cells. Maximum stimulation (172% of controls) is observed with 2.5 ng/ml TGF-beta. The results with a highly enriched progenitor cell population stimulated by recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage CSF (rGM-CSF) were similar, suggesting a direct effect of TGF-beta 1 on hemopoietic progenitors. In contrast to this stimulatory effect of TGF-beta 1 on normal day-7 bone marrow CFU-GM, TGF-beta 1 does not affect the growth of day-14 CFU-GM. The growth of normal bone marrow BFU-E is strongly inhibited. In the majority of cases (11/15) of CML, bone marrow day-7 CFU-GM growth is inhibited by TGF-beta 1. In few cases (4/15) leukemic progenitors respond to TGF-beta 1 as normal cells. TGF-beta 1 always inhibits the growth of day-14 bone marrow CFU-GM from CML patients.

An immunoenzyme technique for the identification of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors using digoxigenated-GM-CSF.

A method for detecting granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors has been devised using human macrophages and a GM-CSF/IL-3-dependent human megakaryoblastic leukemia cell line (M-07e). Recognition of the factor-binding site was accomplished by linking recombinant human (rh) unglycosylated GM-CSF previously labeled with digoxigenated compounds. Digoxigenates were able to link amino and sulphydryl groups of the soluble factor and an immunoperoxidase technique using monoclonal anti-digoxigenin antibody was employed to demonstrate the interaction. To support morphological data cross-linking analysis was performed with M-07e cells using digoxigenated-rh-GM-CSF. Macrophages and M-07e cells incubated with digoxigenated-rh-GM-CSF showed intense positivity by the immunoperoxidase technique. In cross-linking, M07e cells showed a 96 kDa band corresponding to receptor plus bound factor. This technique permits a high degree of specificity in the detection of GM-CSF receptors with good morphological preservation of cellular detail.

In-vitro effect of retinoic acid on normal and chronic myeloid leukemia granulopoiesis.

The effect of increasing concentrations of retinoic acid (RA) on the in-vitro proliferation of normal and chronic myeloid leukemia (CML) granulo-monocyte precursors (CFU-GM) was studied. 10(-7)M RA added to semisolid cultures stimulated the growth of day 14 but not of day 7 normal CFU-GM, whereas in CML the growth of both populations was either unchanged or inhibited. Five-day and 10-day preincubation of normal bone marrow cells with RA augmented the number of day 14 CFU-GM (by up to 187% with 10(-6) M RA), whereas there was a marked decrease when CML cells were used. Total cellularity was not much affected, though a slight increase in liquid normal bone marrow cultures and a slight fall in CML cultures could be detected. These data point to a difference in the response to RA of normal and CML precursors. They may offer of preclinical basis for its employment to delay the blastic progression of CML.

Effect of interferon-gamma on HLA class II antigen expression and sensitivity to prostaglandin E1 by normal and leukemic myeloid progenitors.

Chronic myelogenous leukemia (CML) granulo-monocyte committed progenitors (CFU-GM) are markedly less sensitive than normal progenitors to the inhibitory action of prostaglandin E (PGE). This phenomenon has been ascribed to their abnormal expression of HLA class II (mainly DR) determinants. Since interferon gamma (IFN-gamma) is a potent inducer of the expression of HLA class II (DR and to a lesser extent DQ) antigens, we have sought to determine the extent to which this agent can modulate both the antigenic pattern of normal and leukemic progenitors and their sensitivity to PGE 1. 72-h preincubation of normal and CML bone marrow cells with or without IFN-gamma does not significantly change DR and DQ expression by CFU-GM. Pre-incubation for 72 h with and without IFN-gamma produces the following changes in PGE 1 sensitivity: (1) normal CFU-GM lose some sensitivity to PGE 1. This is only marginally counteracted by the presence of IFN-gamma. (2) CML CFU-GM, preincubated with IFN-gamma regain a significant sensitivity to high concentrations of PGE 1. Our data confirm the expression of DR molecules on normal and leukemic progenitors. They also show that, although incubation with IFN-gamma for 72 h in a liquid culture system does not significantly affect the expression of HLA class II molecules by progenitor cells, it may increase their sensitivity to PGE, particularly in the case of CML CFU-GM. Thus expression of HLA class II antigens and sensitivity to PGE may be dissociated.