Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D.

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.

Activation of hemoglobin C synthesis in sheep marrow culture.

Erythropoietin preferentially stimulates hemoglobin C synthesis in suspension cultures of marrow cells from sheep homozygous for hemoglobin A; the amount of synthesis is dependent on the dose of erythropoietin and is blocked by antiserum to erythropoietin. The results provide the first in vitro evidence that erythropoietin mediates the hemoglobin A --> C "switch" in sheep and indicate that bone marrow cultures may be used to investigate the mechanisms involved in the preferential gene activation characteristic of the hemoglobin A --> C system.

Interleukin 1 stimulates fibroblasts to synthesize granulocyte-macrophage and granulocyte colony-stimulating factors. Mechanism for the hematopoietic response to inflammation.

IL-1 is a family of polypeptides which play a critical role in the inflammatory response. Characteristics of this response include an enhanced release of bone marrow neutrophils, activation of circulating and tissue-phase phagocytes, and enhanced production of neutrophils and monocytes. We have sought to understand the hematopoietic response to acute and chronic inflammatory states on a cellular and molecular level. Colony-stimulating factors (CSFs) are glycoproteins involved in the production and activation of neutrophils and monocytes in vitro and in vivo. We have found that quiescent dermal fibroblasts constitutively release granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), and macrophage CSF in culture, and that picomolar concentrations of the inflammatory mediator IL-1 stimulate by at least fivefold the transcription and release of GM-CSF and G-CSF. These findings establish the role of IL-1 in the hematopoietic response to inflammation through the stimulation of the production and release of GM-CSF and G-CSF.

Modulation of in vitro erythropoiesis. The influence of beta-adrenergic agonists on erythroid colony formation.

Canine marrow erythroid colony growth is enhanced by agents linked to the adenyl cyclase/cyclic AMP (cAMP) system, including cAMP, a phosphodieterase inhibitor (RO-20-1724), cholera enterotoxin, and beta-adrenergic agonists. The adrenergic effect is mediated by receptors having beta2-subspecificity. These receptors are distinct from putative receptors for erythropoietin and those acted upon by cholera enterotoxin. In addition, the population of cells most responsive to beta-agonists is distinct from the majority of erythropoientin-responsive cells, perhaps representing a subpopulation of this class of cell. This demonstration of an adenyl cyclase-linked mechanism regulating mammalian erythroid colony growth provides a model for the modulation by other hormones or small molecules of in vitro and, perhaps, in vivo erythropoiesis.

The influence of thyroid hormones on in vitro erythropoiesis. Mediation by a receptor with beta adrenergic properties.

The erythropoietic effect of various thyroid hormones has been studied using erythroid colony formation by canine marrow cells. Although erythropoietin was required for colony growth, physiologic levels of thyroid hormones significantly enhanced colony numbers. The order of potency of the thyroid compounds in their in vitro erythropoietic effect parallels their known calorigenic potency in vivo, suggesting that the in vitro effect is physiologically relevant. A series of studies linked the mechanism of thyroid action to adrenergic receptors on responsive cells. Propranolol, a global beta-blocker, inhibited thyroid hormone-responsive erythroid colonies. When adrenergic antagonists having different blocking characteristics were added to culture, the thyroid hormone effect was blocked by those compounds having beta(2)-subspecificity. Velocity sedimentation analysis showed that the peak of colony-forming cells which respond to thyroid hormone and the adrenergic agonist, isoproterenol, sedimented at an identical rate (7.54 mm/h), which is slower than the major peak of colony-forming cells responding to erythropoietin alone (8.62 mm/h). These results demonstrate thyroid hormonal enhancement of in vitro erythroid colony growth which appears mediated by a receptor with beta(2)-adrenergic properties. The data suggest that changes in hormone-target cell interaction may occur during states of abnormal thyroid function.

Modulation of in vitro erythropoiesis. Studies with euthyroid and hypothyroid dogs.

The interactions of adrenergic agonists and thyroid hormones on the growth of erythroid colony-forming units were studied in cultures of dog marrow before and after the establishment of hypothyroidism. Erythroid colony growth in cultures form euthyroid dogs was enhanced by isoproterenol and other adrenergic agonists having beta 2-receptor specificity. With hypothyroidism, however, this responsiveness was lost, and sensitivity to alpha-agonists, such as phenylephrine and norepinephrine, was acquired. This alteration in receptor specificity appeared to be dependent upon thyroid hormone and was rapidly reversible. Preincubation of marrow cells from hypothyroid animals with thyroid hormone resulted in the reappearance of responsiveness to beta-adrenergic agonists and the loss of sensitivity to alpha-agonists. These findings are in agreement with previous suggestions that beta-adrenergic receptor activity is modulated by thyroid hormone levels and demonstrate that the specificity of adrenergic modulations of erythropoiesis in culture may accurately reflect the thyroid status of the intact animal.

Erythrocytosis associated with hemoglobin Rainier: oxygen equilibria and marrow regulation.

Hemoglobin Rainier (beta(145) tyrosine-->histidine) is an abnormal hemoglobin associated with increased oxygen affinity, decreased heme-heme interaction, presence of a Bohr effect, and erythrocytosis, but without obvious clinical sequelae. Regulation of erythropoiesis was studied in affected members of families having either hemoglobin Rainier or Yakima, abnormal hemoglobins associated with erythrocytosis. Apart from the elevated but stable hemoglobin concentration and red cell mass, parameters of red cell production in the subjects were normal. Initially normal values of erythropoietin excretion were increased by phlebotomy indicating a significant hypoxic stress at an otherwise normal hematocrit. This stress led to increased reticulocyte production and an eventual return to the prephlebotomy hematocrit. The erythrocytosis in carriers of hemoglobins Rainer and Yakima appears to be secondary to the increased oxygen affinity and this, with the response to phlebotomy, is consistent with the postulate that the renal sensor tissue regulating erythropoietin production is primarily influenced by the oxygen tensions of venous rather than arterial blood.

Hematopoiesis in the grey collie dog: studies of the regulation of erythropoiesis.

Hematopoiesis in the grey collie dog undergoes periodic fluctuations which involve reticulocytes, granulocytes, platelets, lymphocytes, and monocytes. This syndrome is inherited in an autosomal recessive manner and can be transmitted or abolished by appropriate bone marrow transplantation experiments, thus demonstrating this to be a primary marrow defect. Investigation of humoral regulation in this setting indicates that serum erythropoietin (ESF) also undergoes cyclic fluctuation and that shortly after the increase and peak in serum ESF levels recognizable red cell precursors appear in the marrow. Erythropoiesis in the grey collie is reciprocally related to the blood O2 carrying capacity. With phlebotomy, ESF activity and reticulocytes increase but continue to cycle, while hypertransfusion eliminates reticulocyte production completely. Neither phlebotomy nor hypertransfusion alter the underlying cycle time (11-12 days) nor influence the peaks of peripheral blood granulocytes. Thus, in these experiments, no direct evidence of competition between reticulocyte and granulocyte production is observed. In vitro studies of canine hemoglobin synthesis fail to demonstrate evidence of an inhibitor to ESF. These results indicate that periodic fluctuation of serum ESF is an integral part of the grey collie syndrome and are most consistent with some form of feedback regulation of ESF production.

Evidence for the involvement of B lymphoid cells in polycythemia vera and essential thrombocythemia.

Previous studies with the X-chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) as a marker of cellular mosaicism demonstrated that polycythemia vera (PV) and essential thrombocythemia (ET) are clonal disorders of hematopoietic stem cells that can differentiate to erythrocytes, granulocytes, and platelets. To determine if the involved stem cells could also differentiate along the B-lymphoid pathway, we studied one woman with PV and one woman with ET. Of 117 Epstein-Barr virus-transformed B-lymphoblastoid lines expressing a single G6PD derived from the patient with PV, 108 expressed G6PD type A, the type characteristic of the abnormal clone. The ratio of 108:9 was significantly different from the one to one ratio predicted for this patient, which suggested that at least some circulating progenitors for B-lymphoid cell lines differentiate from the stem cell involved by the disease. Results obtained from the patient with ET were similar--104 of the 109 lymphoblastoid lines monotypic for G6PD expression displayed the enzyme type found in the abnormal clone of marrow cells. Therefore, in these patients, PV and ET, like chronic myelogenous leukemia, involve a stem cell pluripotent for the lymphoid as well as the myeloid series.

Polycythemia vera. The in vitro response of normal and abnormal stem cell lines to erythropoietin.

Bone marrow cells from two glucose-6-phosphate dehydrogenase (G-6-PD) heterozygotes with polycythemia vera were cultured to determine whether progenitors which wre not of the polycythemia vera clone were present, and, if present, which cell lines contributed to the increase in erythroid colonies observed in response to added erythropoietin (ESF). To accomplish this, the G-6-PD isoenzyme activity of individual erythroid colonies was determined. All of the erythroid colonies analyzed in cultures without added ESF, contained the G-6-PD isoenzyme type characteristic of the abnormal clone. With higher ESF concentrations in the culture, however, there was an increase in the colonies that were not of the polycythemia vera clone. Analysis of the ratio of the various types of colonies indicated that normal and polycythemia vera cells are capable of responding to ESF in vitro. In selected patients, this technique permits analysis of the ratios of normal to abnormal cells during the course of the disease, in response to therapy and during late complications, such as myelofibrosis or leukemic transformation.

Hemoglobin olympia ( 20 valine leads to methionine): an electrophoretically silent variant associated with high oxygen affinity and erythrocytosis.

In a family with erythrocytosis, electrophoretic and chromatographic studies failed to demonstrate a hemoglobin variant. However, the oxygen dissociation curves of affected individuals were shifted to the left of normal and this shift persisted when oxygen equilibria were studied in 2.3-diphosphoglycerate-stripped hemolysates. A mutant hemoglobin was evidently present in the red blood cells of the affected persons and was responsible for the increased oxygen affinity and erythrocytosis. Specific staining of tryptic peptide maps of beta-chains from the propositus showed that peptide betaT(3) was positive for a sulfur-containing amino acid. Amino acid analysis yielded a composition identical to that of normal betaT(3), except that there were 2.6 residues of valine and 0.4 residues of methionine (normal composition: Val = 3.0, Met = 0). This suggested that the beta-chains of affected individuals consisted of a mixture of two kinds of chains, 40% of which had a methionyl residue in betaT(3). Structural studies of isolated cyanogen bromide fragments demonstrated unequivocally that, in the abnormal beta-chains, valine in position 20 is replaced by methionine. The new hemoglobin mutant is designated hemoglobin Olympia (beta20 (B2) valine --> methionine).

Erythrocyte function and marrow regulation in hemoglobin Bethesda (beta-145 histidine).

Hemoglobin Bethesda (beta145 histidine) is one of the two mutants known to affect the penultimate hemoglobin tyrosines. The result of this substitution is extreme disorganization of the oxygenation function of the molecule. Red cells containing 45% Hb Bethesda and 55% Hb A have increased oxygen affinity but, paradoxically, a normal Bohr effect. As is usually seen with other hemoglobins with increased oxygen affinity, Hb Bethesda clinically is manifest in heterozygotes by erythrocytosis. Red cell production in affected individuals is erythropoietin dependent. The reciprocal interdependence of oxygen delivery and effective erythropoiesis was documented by alterations in erythropoietin excretion, quantitative iron kinetics, and reticulocyte production in response to phlebotomy-induced reduction in the oxygen-carrying capacity.

Pancytopenia as a clonal disorder of a multipotent hematopoietic stem cell.

Hematopoiesis was investigated in a 14-yr-old girl who had a 2-yr history of stable asymptomatic pancytopenia and who was also heterozygous at the structural locus for glucose-6-phosphate dehydrogenase (G-6-PD). There was no morphologic or cytogenetic evidence for preleukemia and no suggestion of Fanconi anemia. In the skin and sheep erythrocytes-rosetted T lymphocytes, the ratio of G-6-PD A/B activities was 1:1. However, only type B activity was found in peripheral blood erythrocytes, granulocytes, and platelets. Most erythroid bursts and all granulocyte/macrophage colonies formed in methylcellulose culture were derived from the abnormal clone. These findings demonstrate that (a) some cases of pancytopenia are stem cell diseases that apparently develop clonally; (b) circulating differentiated cells originate from this clone; (c) despite a hypoproliferative anemia, the in vivo expression of presumably normal (nonclonal) progenitors is suppressed. In this patient, the relationship between clonal dominance and possible malignancy may be assessed prospectively.

The anemia of chronic renal failure in sheep. Response to erythropoietin-rich plasma in vivo.

The hypoproliferative anemia in chronic renal failure has been assumed to be the result of decreased erythropoietin (Ep) production by the damaged kidney and of the shortening of erythrocyte survival. However, many in vitro studies suggest that erythropoietic inhibitors in uremic plasma may contribute to the anemia. To determine the in vivo relevance of uremic inhibitors, increasing amounts of Ep as Ep-rich plasma were infused into six uremic sheep, and their erythropoietic responses were compared with those of nine normal sheep receiving similar amounts of Ep-rich plasma. Three sheep were studied in both normal and uremic states. Ep-rich plasma was obtained from phenylhydrazine- and phlebotomy-induced anemic sheep. Stable uremia was created by subtotal nephrectomy. Erythropoiesis was quantitated by reticulocyte response, ferrokinetics (plasma iron turnover and marrow transit time), and by hemoglobin C synthesis. Ep-rich plasma stimulated erythropoiesis similarly in uremic and normal sheep, regardless of the degree of uremia. Nondialyzed uremic sheep responded as well as dialyzed animals. The anemia was corrected in the uremic dialyzed animals. The anemia was corrected in the uremic sheep after 15-40 daily infusions of Ep-rich plasma, the total dosage depending on the severity of the anemia. Polycythemia was induced when the infusions were continued. Reticulocytes, plasma iron turnover, and erythrocyte mass changes increased as the amount of Ep-rich plasma was increased. These dose-response effects, coupled with the identical erythropoietic response in normal and uremic sheep given the same amount of Ep-rich plasma, imply that there are no physiologically significant erythropoietic inhibitors in uremia.

Chronic myelogenous leukemia: in vitro studies of hematopoietic regulation in a patient undergoing intensive chemotherapy.

A patient heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase and with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) was treated with combination chemotherapy and had a partial loss of Philadelphia chromosome accompanied by partial restoration of nonclonal hematopoiesis as determined by glucose-6-phosphate dehydrogenase. Studies of in vitro hematopoiesis were performed after chemotherapy to evaluate the influences of neoplastic stem cells on normal cells and to determine whether there were physical and cell kinetic differences between leukemic stem cells and their normal counterparts. The data revealed the following: (a) The frequencies of normal committed granulocytic stem cells (CFU-C) and erythroid stem cells (BFU-E) in blood did not differ from the frequencies in marrow. (b) Normal late erythroid progenitors (CFU-E) were found at a significantly lower frequency that the more primitive BFU-E. Calculations indicated that not only was there a decrease in CFU-E production by normal BFU-E, but there was also abnormal clonal expansion of CML BFU-E (CFU-E:BFU-E ratio for normal progenitors was 1.1, whereas for the CML clone it was 11.5). (c) No increase in frequency of normal CFU-C was found after marrow cells were exposed to high specific activity tritiated thymidine. (d) Normal CFU-C and those from the CML clone were not separable on the basis of density. (e) The frequency of normal BFU-E was consistently greater than that of CFU-C, suggesting that regulatory differences influence the commitment of normal progenitors to the two pathways.

Feline glucose-6-phosphate dehydrogenase cellular mosaicism. Application to the study of retrovirus-induced pure red cell aplasia.

Neoplasms result from the uncontrolled proliferation of abnormal or transformed cells. The early stages of this process are difficult to study because of the lack of sensitive and specific markers of clonal evolution in an experimental system. We have developed a cat model using cellular mosaicism for glucose-6-phosphate dehydrogenase (G-6-PD). Our findings confirm that the structural locus for feline G-6-PD is on the X-chromosome and demonstrate that it is randomly inactivated in somatic cells. Heterozygous cats have balanced ratios of G-6-PD enzyme types in peripheral blood cells and hematopoietic progenitors that remain stable over time. In our initial studies, we used the model to analyze the events surrounding marrow failure experimentally induced by selected strains of feline leukemia virus (FeLV). Two G-6-PD heterozygous cats, one F1 male hybrid and one domestic cat were infected with FeLV (C or KT) and developed pure red cell aplasia (PRCA). Colonies arising from the more mature erythroid colony-forming cell were not detected in marrow culture of anemic animals although erythroid bursts persisted, suggesting that the differentiation of early erythroid progenitors (BFU-E) was inhibited in vivo. The ratio of G-6-PD types in hematopoietic progenitors and peripheral blood cells from the heterozygous cats did not change when the animals developed PRCA. Thus, the anemia did not result from the clonal expansion of a transformed myeloid stem cell. With this experimental approach, one may prospectively assess clonal evolution and cellular interactions in other FeLV-induced diseases.

Mechanisms of thrombocytopenia in chronic autoimmune thrombocytopenic purpura. Evidence of both impaired platelet production and increased platelet clearance.

Mechanisms of thrombocytopenia were studied in 38 patients with mild to moderately severe chronic autoimmune thrombocytopenia (AITP). 51Cr and 111In-labeled autologous platelet turnover studies and in vitro analysis of committed megakaryocyte progenitors (CFU-Meg) were used as independent measures of platelet production. Autologous 111In-labeled platelet localization studies were performed to assess platelet clearance. Although there was no increase in the frequency of marrow CFU-Meg, a specific increase in the CFU-Meg [3H]TdR suicide rate was seen which was inversely correlated with the platelet count (P less than 0.001). Platelet turnover studies showed significant numbers of patients had inappropriate thrombopoietic responses to their reduced platelet counts. Platelet-associated antibody levels correlated inversely with platelet turnover suggesting that antiplatelet antibody impairs platelet production. The circulating platelet count was best predicted by an index relating platelet production (i.e., turnover) to the spleen-liver platelet clearance that correlated directly with platelet survival (P less than 0.001). In summary, both depressed platelet production and increased platelet clearance by the liver and spleen contribute to the thrombocytopenia of AITP.

Polycythemia vera. Physical separation of normal and neoplastic committed granulocyte-macrophage progenitors.

In previous studies of two patients with polycythemia vera (PV) who were heterozygous at the X-linked locus for glucose-6-phosphate dehydrogenase (G6PD), only A type enzyme was found in nonlymphoid blood cells. However, some erythroid and granulocytic colonies grown in vitro were type B and therefore arose from presumably normal progenitors. One patient had enough type B colonies (8%) that studies of the physical characteristics of normal and PV clonal colony-forming cells could be undertaken. When marrow cells were separated by velocity sedimentation at unit gravity, most PV clonal granulocyte-macrophage progenitors (CFU-C) (type A G6PD) sedimented between 6.4 and 7.2 mm/h, whereas most residual normal, type B CFU-C sedimented less than or equal to 5.9 mm/h (P = 0.04)., When blood cells were separated over a discontinuous buoyant density gradient, PV clonal CFU-C equilibrated at densities < 1.065 g/ml, whereas residual normal CFU-C were found greater than or equal to 1.065 g/ml (P < 0.01). PV clonal and residual normal erythroid burst-forming progenitors were not separable by either method. Thus PV clonal CFU-C are larger and less dense cells than are residual normal CFU-C.

Polycythemia vera. Increased expression of normal committed granulocytic stem cells in vitro after exposure of marrow to tritiated thymidine.

In previous studies of two patients with polycythemia vera (PV) and heterozygous at the X-linked locus for glucose-6-phosphate dehydrogenase (G-6-PD), only type A isoenzyme was found in non-lymphoid hematopoietic cells. However, some granulocytic and erythrocytic colonies grown in vitro had type B G-6-PD and therefore arose from presumably normal progenitors. In this study we exposed marrow cells from these same two patients to high-specific activity tritiated thymidine (3HTdR) before culture to kill cells actively synthesizing DNA. Individual granulocytic colonies were plucked and tested for G-6-PD after 14 d of culture. The frequency of type B colonies rose after exposure to 3HTdR from 8/101 to 11/36 in patient 1 and from 0/32 to 6/31 in patient 2 (P less than 0.003). No increase in the frequency of normal erythroid bursts after 3HTdR exposure was seen, implying that in PV, early granulopoiesis, and erythropoiesis are regulated differently. The results demonstrated that only type A granulocytic colonies, arising from the abnormal clone, were removed by the 3HTdR. In addition, for patient 2, statistical analysis indicated there was an absolute increase in normal granulocytic colonies detected in culture. Thus, PV clonal colony-forming units in culture (CFU-C) cycle more rapidly than do normal CFU-C and may suppress proliferation of normal CFU-C in vitro.

Polycythemia vera. Further in vitro studies of hematopoietic regulation.

Further in vitro studies of hematopoietic regulation were carried out in two patients with polycythemia vera who were also heterozygotes (Gd(B)/Gd(A)) for glucose-6-phosphate-dehydrogenase (G-6-PD). While only G-6-PD type A was detectable in circulating erythrocytes, granulocytes and platelets, cultures of peripheral blood and marrow from one patient revealed a substantial number of G-6-PD type B erythroid burst-forming units (BFU-E) and granulocyte/macrophage colony-forming units. Detailed analysis demonstrated: (a) where detectable, normal BFU-E and granulocyte/macrophage colony-forming units were found with similar frequencies; (b) the same frequencies for normal progenitors characterized cultures of peripheral blood and marrow; (c) inhibition of normal erythroid differentiation between BFU-E and the more mature erythroid colony-forming unit; (d) a decline in the prevalence of normal colony-forming units with time, suggesting that disease progression is associated with further suppression of normal hematopoiesis by products of the abnormal clone.