Mature bone marrow erythroid burst-forming units do not require T cells for induction of erythropoietin-dependent differentiation.

Cell-cell interactions between mature T cells and peripheral blood null cells induce erythropoietin-stimulated differentiation of peripheral blood-derived erythroid progenitors. By the use of complement-fixing cytolytic murine hybridoma and antibody uniquely reactive with mature T lymphocytes, this dependence of immature peripheral blood erythroid burst-forming unit (BFU-E) differentiation upon mature T cells or a T cell conditioned medium is confirmed. By using the same antibody, it is demonstrated that the differentiation of mature bone marrow BFU-E does not require either mature T cells or lymphocyte mitogenic factor. These findings do not preclude the presence in the bone marrow of other cells, perhaps even immature T cells, that influence erythropoietin-dependent erythroid differentiation of mature marrow BFU-E.

Human erythroid burst-forming unit: T-cell requirement for proliferation in vitro.

Human mononuclear leukocytes were fractionated into populations of null, T and B cells by immunoabsorbent column chromatography followed by E-rosette formation and purification of T cells by differential centrifugation and osmotic lysis. The unfractionated and fractionated cell populations were first separately cultured for 14 days in plasma clots in the presence of two international units erythropoietin. Typical erythroid burst-forming unit (BFU-E)-derived colonies grew in the unfractionated cell cultures but not from T- or B-cell cultures. BFU-E colonies grew in null cell cultures but most of the colonies were small and variably hemoglobinized with less than three subcolonies. When intact T cells were added to null cells and cocultured, many typical large BFU-E colonies with more than 10 well homogenized subcolonies appeared. Increasing numbers of large BFU-E colonies in null cell cultures were induced by stepwise addition of T cells but not by the addition of B cells. A conditioned medium in which T cells had been induced to divide by tetanus toxoid substituted for intact T cells in this T-cell-dependent BFU-E colony formation observed in null cells. These findings demonstrate that the BFU-E, a committeded erythroid stem cell, resides in the null cell fraction of peripheral blood, but its proliferative capacity and differentiation in vitro requires a soluble product of T cells. Such experiments now permit a new approach to the assessment of various disorders of erythropoiesis. Erythroid hypoplasia in a particular case may be due to dysfunction of the committed precursor cell or to a failure of a helper effect induced by T cells.

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle.

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum-stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.

Leukocyte oxidase: defective activity in chronic granulomatous disease.

The intact leukocytes of two children with chronic granulomatous disease fail to reduce nitroblue tetrazolium during phagocytosis. This is due to defective operation of an oxidase of reduced nicotinamide adenine dinucleotide that is insensitive to cyanide and that indirectly stimulates the oxidation of glucose-6-phosphate in leukocytes. Such leukocytes undergo no increase in oxygen consumption or in activity of the hexose monophosphate shunt during phagocytosis, although lactate production is normal. The addition of nitroblue tetrazolium to a leukocyte suspension appears to provide a sensitive diagnostic screening test for this disease.

Beta thalassemia trait: detection at birth.

The synthesis of alpha, beta, and gamma chains in samples of cord blood was measured by the incorporation of leucine labeled with carbon-14 into these chains. In a newborn affected with beta thalassemia trait, the presence of one beta thalassemia gene was revealed on the 1st day of life by the lower specific radioactivity of the beta chain.

Human IL-3 and GM-CSF act synergistically in stimulating hematopoiesis in primates.

Interleukin-3 (IL-3) is a member of a family of growth factors, each of which supports the proliferation and development of hematopoietic precursors in culture. Although the biologic effects of the different hematopoietic growth factors have been well documented in different culture systems, it has only recently become possible to study the activities of these molecules in vivo. In comparison with the later acting hematopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor, IL-3 elicited a delayed and relatively modest leukocytosis when continuously infused intravenously in primates. The IL-3 infusion, however, greatly potentiated the responsiveness of the animal to subsequent administration of a low dose of GM-CSF. These results suggest that IL-3 expands an early cell population in vivo that subsequently requires the action of a later acting factor such as GM-CSF to complete its development. Optimal stimulation of hematopoiesis may be achieved with combinations of hematopoietic growth factors.

Human recombinant granulocyte-macrophage colony-stimulating factor: a multilineage hematopoietin.

Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was tested for its ability to induce colony formation in human bone marrow that had been enriched for progenitor cells. In addition to its expected granulocyte-monocyte colony-stimulating activity, the recombinant GM-CSF had burst-promoting activity for erythroid burst-forming units and also stimulated colonies derived from multipotent (mixed) progenitors. In contrast, recombinant erythroid-potentiating activity did not stimulate erythroid progenitors. The experiments prove that human GM-CSF has multilineage colony-stimulating activity.

Mild thalassemia: the result of interactions of alpha and beta thalassemia genes.

Homozygous thalassemia is due to inherited unbalanced synthesis of the alpha- or beta-chains of hemoglobin. Clinical severity may be in part related to the extent of alpha:beta imbalance. Two families are presented that illustrate this concept. Thalassemia in these individuals was evaluated by clinical and genetic criteria. The relative rates of alpha- and beta-chain synthesis in their reticulocytes were estimated by the extent of incorporation of 1-leucine-U-(14)C into the chains. Unusual combinations of clinical and hematological data and biosynthetic ratios were obtained in certain individuals which indicated the presence of combinations of alpha- and beta-thalassemia genes. The propositus of the first family had mild Cooley's anemia and was believed to have one alpha- as well as two beta-thalassemia genes. Presumably the alpha-thalassemia gene interfered with alpha-chain production which lead to less accumulation of alpha-chains and a reduced rate of intramedullary and peripheral hemolysis. In the second family two individuals were believed to have an alpha-thalassemia, a "silent carrier," and a beta-thalassemia gene. Despite the fact that they appeared to have the genotype of hemoglobin H disease, their cells contained no hemoglobin H and had a normal lifespan presumably because excess beta-chain production was inhibited by the beta-thalessemia gene. These family studies suggest that the alpha:beta imbalance observed in thalassemia may be favorably influenced by combinations of alpha- and beta-thalassemia genes.

Stages in the incorporation of fatty acids into red blood cells.

Mature human erythrocytes were incubated with (14)C-labeled palmitic acid bound to crystalline human albumin. Energy-dependent incorporation of the labeled palmitic acid into cell membrane phospholipids occurred, and various stages in this incorporation were defined. Initially the palmitic acid was rapidy transferred from the albumin to a "superficial" membrane pool of free fatty acid (F-1), which was removable when the cells were washed with defatted albumin. This process was independent of red cell metabolism. The labeled fatty acid then passed into a second "deeper" membrane pool of free fatty acids (F-2), which was not extractable with albumin. This process was energy-dependent and proceeded at a slower rate than the initial transfer from albumin to F-1. Ultimately the labeled fatty acid was incorporated into phosphatides (PL). This process also was dependent upon cellular metabolism. The kinetics of pulse label studies suggest that the processes observed were sequential and that precursor-product relationships exist between the F-1 and F-2 pools and the F-2 and PL pools. [Formula: see text] From the size and specific activities of these pools, calculations of the extent of phospholipid turnover were made. An approximate figure of 2% /hr or 30 nmoles/ml of packed red blood cells per hr was obtained. The figure was further calculated to represent an energy cost to the red blood cell of approximately 5% of the energy available from glycolysis.

Exercise-induced hemolysis in xerocytosis. Erythrocyte dehydration and shear sensitivity.

A patient with xerocytosis was found to have swimming-induced intravascular hemolysis and shortening of erythrocyte life-span. In a microviscometer, xerocytes were more susceptible than normal erythrocytes to hemolysis by shear stress. Fractionation of normal and abnormal cells on discontinuous Stractan density gradients revealed that increasingly dehydrated cells were increasingly more shear sensitive. This sensitivity was partially corrected by rehydrating xerocytic erythrocytes by means of the cation-ionophore nystatin in a high potassium buffer. Conversely, normal erythrocytes were rendered shear sensitive by dehydrating them with nystatin in a low potassium buffer. This effect of dehydration was entirely reversible if normal cells were dehydrated for less than 4 h but was only partially reversed after more prolonged dehydration. It is likely that dehydration of erythrocytes results in shear sensitivity primarily because of concentration of cell contents and reduced cellular deformability. With prolonged dehydration, secondary membrane changes may potentiate the primary effect. This increased shear sensitivity of dehydrated cells may explain atraumatic exercise-induced hemolysis in xerocytosis as cardiac output is shifted to vessels of exercising muscles with small diameters and high shear rates.

Heterogeneity of DNA deletion in gamma delta beta-thalassemia.

By restriction endonuclease mapping, gene cloning, and DNA sequencing we have determined the region of DNA that is deleted in a family with gamma delta beta-thalassemia. The deletion removes the linked epsilon, gamma-, and delta-globin structural genes and terminates within the coding portion of the beta-globin gene. Since the extent of DNA deletion in this family differs from that reported in another family, we conclude that gamma delta beta-thalassemia is heterogeneous at the molecular level.

Failure of nitro blue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease.

The leukocytes of patients with chronic granulomatous disease (CGD) may be identified by their failure to reduce Nitro Blue Tetrazolium (NBT) during phagocytosis. This reaction, normally detected in the phagocytic vacuole, is absent or delayed in CGD monocytes and eosinophils as well as in neutrophils, even though sonicates of normal and CGD leukocytes contain equal activities of a cyanide insensitive enzyme system capable of reduction of NBT in the presence of pyridine nucleotide. Enlargement of CGD phagocytic vacuoles appears to be inhibited. Histochemical estimates of the rate of release of alkaline phosphatase are normal in CGD cells. Peroxidase activity is released from CGD cells, but the rate appears to be somewhat slower than normal in some cases. The latter observation may be explained by the increased intensity of the peroxidase stain in resting and phagocytizing CGD cells. The severity of the defect in NBT reduction within the phagocytic vacuoles of the leukocytes of patients and carriers is more variable than was previously appreciated. Some female carriers have profoundly reduced dye reduction and others are nearly indistinguishable from normal. Three brothers with CGD demonstrated significant, albeit delayed, NBT reduction in phagocytic vacuoles during prolonged incubation of their leukocytes. No obvious relationship exists, however, between the rate of reduction of NBT in vacuoles and the clinical severity of the disease.

Globin chain synthesis in the alpha thalassemia syndromes.

Whole blood samples of patients with various forms of alpha thalassemia including hemoglobin H disease, alpha thalassemia trait, and the "silent carrier" state were incubated with leucine-(14)C for definition of relative rates of production of alpha and beta chains in these disorders. The chains were separated by carboxymethyl cellulose chromatography in the presence of 8 M urea and dithiothreitol. Their absorptions at 280 mmu were determined and their radioactivities measured in a liquid scintillation spectrometer. After correction for differences in extinction coefficients, the specific activities of the widely separated alpha and beta peaks were determined. In 11 nonthalassemic individuals, the alpha/beta specific activity ratios were found to be 1.02+/-0.07; in nine patients with alpha thalassemia trait, 0.77+/-0.05; in six patients with hemoglobin H disease, 0.41+/-0.11; and in four "silent carriers," 0.88 with a range of 0.82-0.95. The results show that in peripheral blood, alpha chain production relative to beta chain production is indeed limited in the alpha thalassemia syndromes. Hemoglobin H disease results from doubly heterozygous inheritance of a gene resulting in moderate depression of alpha chain production (alpha thalassemia trait) and a gene resulting in very mild depression of alpha chain production (the "silent carrier" syndrome."

Amino acid oxidase of leukocytes in relation to H 2 O 2 -mediated bacterial killing.

D-Amino acid oxidase and L-amino acid oxidase have been measured in sucrose homogenates of polymorphonuclear leukocytes (PMN) obtained from guinea pigs and humans. Subcellular distribution patterns and studies on latency indicate that these oxidases are soluble enzymes. Their hydrogen peroxide-generating capacity was verified. Chronic granulomatous disease PMN, which lack a respiratory burst and fail to generate H(2)O(2) during phagocytosis and do not kill catalase positive bacteria, had peroxide-generating amino acid oxidase activity equal to that found in PMN homogenates from patients with bacterial infections. The precise metabolic and bactericidal role of amino acid oxidases in PMN remains uncertain.

Energy metabolism in human erythrocytes. I. Effects of sodium fluoride.

Exposure of red cells to fluoride produces a variety of metabolic alterations, most of which are based upon the secondary effects of enolase inhibition, which reduces pyruvate synthesis and interferes with the regeneration of diphosphopyridine nucleotide (NAD). Adenosine triphosphate (ATP) is consumed in the hexokinase and phosphofructokinase reactions but is not regenerated since the deficiency of NAD limits glyceraldehyde phosphate dehydrogenase. ATP depletion in the presence of fluoride and calcium induces a massive loss of cations and water. Of the other known sites of ATP utilization, membrane-bound ATPase is inhibited by fluoride, but the incorporation of fatty acids into membrane phospholipids is unaffected until ATP is depleted. The addition of methylene blue to fluoride-treated red cells regenerates NAD, permitting triose oxidation and the generation of 3-phosphoglycerate and 2,3-diphosphoglycerate. Enolase inhibition is then partially overcome by mass action, and sufficient glycolysis proceeds to maintain the concentration of ATP. This in turn prevents the massive cation and water loss, and permits membrane phospholipid renewal to proceed. Membrane ATPase activity is not restored by the oxidant so that normal cation leakage remains unopposed by cation pumping in red cells exposed to the combination of fluoride and methylene blue.

Correction of metabolic deficiencies in the leukocytes of patients with chronic granulomatous disease.

Polymorphonuclear leukocytes from patients with chronic granulomatous disease (CGD) exhibit metabolic and bactericidal deficiencies that may be the result of inadequate production of H(2)O(2). A hydrogen peroxide-generating system was, therefore, inserted into CGD leukocytes. This was accomplished by allowing the cells to phagocytize latex spherules coated with glucose oxidase. This produced an amelioration in the known metabolic deficiencies of these cells during phagocytosis: (a) intracellular (catalatic) formate oxidation dependent upon hydrogen peroxide production was enhanced fourfold; and (b) hexose monophosphate shunt activity, which other workers have shown to be at least partially dependent upon the availability of H(2)O(2), was markedly stimulated. These data strengthen the evidence that the fundamental metabolic lesion in CGD cells during phagocytosis is indeed deficient production of hydrogen peroxide, probably, as previously shown, due to diminished oxidase for reduced nicotinamide adenine dinucleotide.

Oxidant injury of caucasian glucose-6-phosphate dehydrogenase-deficient red blood cells by phagocytosing leukocytes during infection.

Patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency of red blood cells (RBC) may develop sudden hemolytic anemia during infection. Since phagocytizing polymorphonuclear leukocytes (PMN) are known to generate hydrogen peroxide, we explored the influence of this oxidant product of PMN on juxtaposed G6PD-deficient and normal RBC. The oxidant stress induced by phagocytosis depleted G6PD-deficient RBC of reduced glutathione (GSH) and this was associated with rapid removal of these cells from the circulation by the liver and spleen. No such effect was observed on normal RBC. Phagocytizing chronic granulomatous disease (CGD) PMN which lack hydrogen peroxide generation, failed to diminish GSH level in G6PD-deficient RBC. Thus, PMN can pose as a source of oxidant damage to G6PD-deficient RBC due to hydrogen peroxide generated during phagocytosis.

Changes in fatty acid metabolism after erythrocyte peroxidation: stimulation of a membrane repair process.

To study certain membrane repair processes in human erythrocytes, vitamin E-deficient cells were incubated with hydrogen peroxide. The incorporation of exogenous fatty acid and the transfer of fatty acid from phosphatidylcholine and neutral lipid into phosphatidylethanolamine were examined using radioactive fatty acids. Hydrogen peroxide stimulated the incorporation of fatty acid into all membrane phospholipids. The specific activity of phosphatidylethanolamine was increased disproportionately. The lipids of the membranes of erythrocytes were labeled with saturated and unsaturated fatty acid. When these erythrocytes were subsequently incubated with hydrogen peroxide, both types of fatty acid were transferred from superficial erythrocyte neutral lipids into phosphatidylethanolamine. However, the unsaturated fatty acids of phosphatidylethanolamine were subsequently altered by hydrogen peroxide, whereas the saturated fatty acids were not. The cumulative effect of these processes was a relative decrease in unsaturated fatty acid and an increase in saturated fatty acid in the phosphatidylethanolamine of the erythrocyte membrane. The net effect of these events represents the operation of repair processes which distort the usual fatty acid composition of erythrocyte membranes in the presence of H(2)O(2). This distortion may contribute to membrane permeability changes which occur during peroxide exposure and which precede the eventual hemolysis of these cells.

Equal synthesis of - and -globin chains in erythroid precursors in heterozygous -thalassemia.

In patients with heterozygous beta-thalassemia, the beta/alpha synthetic ratio in marrow erythroid cells incubated in vitro is 1, whereas in reticulocytes the ratio is 0.5. These ratios reflect the equal synthesis of the two chains on the polyribosomes of the bone marrow and unequal synthesis on the polyribosomes of the peripheral blood reticulocytes. alpha- and beta-chain synthesis is also equal in marrow cells in vivo. Equal synthesis is probably due both to a decrease in alpha-chain synthesis and an increase in beta-chain synthesis in bone marrow erythroid cells and may contribute to the absence of overt hemolysis due to excess alpha-globin chain accumulation in heterozygous beta-thalassemia.

Identification of three accessory cell populations in human bone marrow with erythroid burst-promoting properties.

Several laboratories have demonstrated a requirement for burst-promoting activity (BPA), a product of T cells, or T cell/monocyte collaboration in the induction of differentiation of peripheral blood erythroid burst-forming units (BFU-E) in vitro. The physiologic significance of this finding is brought into question by patients with severe mature T cell deficiency who have normal in vivo erythropoiesis. The studies described here were designed to determine whether the burst-promoting effects of marrow T cells and adherent cells are similar to those of peripheral blood, to define whether a third population of marrow cells is capable of production of BPA, and to describe the BPA requirements of immature and mature marrow erythroid progenitors. To that end we prepared adherence- and E-depleted low-density peripheral blood mononuclear cells as a source of BFU-E and demonstrated that their optimal erythropoietin-induced differentiation requires BPA. We then determined that both bone marrow and peripheral blood T cells and monocytes could provide the necessary BPA to induce their erythropoietin dependent differentiation. BPA production by T cells was sensitive to irradiation, but that of the whole bone marrow low-density population was considerably less sensitive. This in itself demonstrated that BPA production in marrow is not T cell dependent. We further demonstrated a potent, albeit infrequent, third population of BPA-producing marrow cells. These proved to be nonadherent, E receptor-negative, granulocyte antigen-negative, and gamma-Fc receptor-positive. Finally, we separated all of these BPA-producing cells from marrow erythroid progenitors and concentrated the latter into a population in which they comprised 6% of the cells. With this population we demonstrated that both immature (BFU-E) and mature (colony-forming units [CFU-E]) erythroid progenitors require BPA in addition to erythropoietin to induce them to form erythroid colonies in vitro. These results may explain the normal erythropoiesis found in patients with mature T cell deficiency. Though the differentiation of both BFU-E and CFU-E requires BPA, this need can be met by a special class of nonadherent, radioresistant, E receptor-negative, granulocyte antigen-negative, and gamma-Fc-positive cells.