Factors influencing hematopoietic spleen colony formation in irradiated mice. 3. The effect of repetitive irradiation upon proliferative ability of colony-forming cells.

Mice were irradiated repetitively at 6 wk intervals. The proliferative capacity of the hematopoietic stem cell compartment was studied after each irradiation and compared to that of age-matched controls which had been irradiated only once. Hematopoietic proliferation capacity was measured by determining the number of spleen colonies, splenic iron uptake, spleen weight, and volume of packed red cells 10 days after irradiation. 6 wk after the first irradiation, the hematopoietic compartment was apparently supranormal in size for, when such mice were again irradiated, their postirradiation hematopoiesis was in excess of that of the controls. Thereafter, there was a steady decline in regenerative capacity with each sequential irradiation. After the sixth irradiation, the number of spleen colonies and iron uptake were reduced to one-fifth of that of singly irradiated controls. A decline in body weight and an increase in irradiation mortality accompanied the decline in postirradiation hematopoiesis. The degree of measured decline in hematopoietic proliferative ability was less than that observed by other investigators who studied the effect of serial transplantation of cells upon their ability to proliferate. Furthermore, even after the sixth irradiation, a marked stimulation of postirradiation hematopoiesis was induced by bleeding the animals before irradiation.

Factors influencing hematopoietic spleen colony formation in irradiated mice. II. The effect of foreign materials.

Normal dog plasma and serum, human, rat, and Swiss-Webster mouse plasma, phytohemagglutinin, sheep red cells, mumps and influenza vaccine, fibrinogen, and endotoxin injected before irradiation led to an increased number of endogenously derived spleen colonies in irradiated mice. Spleen weight and uptake of radioactive iron and iododeoxyuridine into such spleens were also increased. The relationship between these parameters of splenic hematopoiesis was unchanged by plasma injection suggesting that, while the number of colonies was increased, the composition of individual colonies was unchanged. This conclusion was supported by studies on plethoric mice in which splenic erythropoiesis is abolished. Increased splenic hematopoiesis was accompanied by an increase in the volume of packed red blood cells 10 days after irradiation. The total volume of plasma injected, the number of days of plasma injection preceding irradiation, and the route of administration were all important variables influencing the effect of plasma injections. Crude fractions of human albumin and gamma globulin, cortisol, C57BL (maternal) and DBA (paternal) mouse plasma, and isogeneic plasma were without effect. The ineffectiveness of isogeneic and closely related allogeneic plasma rendered unlikely the hypothesis that this effect represented the presence of homeostatic hematopoietic regulating factors in plasma. The increased hematopoiesis induced with plasma appeared to be limited to the spleen, for increased bone marrow hematopoiesis was not detected. Certain observations suggested that the effect of plasma may not be due to an antigenic or an inflammatory effect. From current observations, it was unclear whether the increased colonies induced by plasma were representative of expansion of the colony-forming cell pool or of increased efficiency of growth of the fraction surviving irradiation.

Factors influencing hematopoietic spleen colony formation in irradiated mice. I. The normal pattern of endogenous colony formation.

Data pertaining to the endogenous mouse spleen colony system, 10 days postirradiation, are presented. The D(o) for visible colonies is 78 R, while that for 6 hr iron uptake over a range of 600-800 R is 50 R. The D(o) for spleen weight is 196 R and that for IUdR uptake is 193 R. These measurements increase with the age of the mouse. Hypertransfusion decreases spleen iron uptake and colony number. DF-(32)P and sodium sulfate-(35)S are not useful indicators of splenic hematopoiesis in this system. Visible hematopoietic colonies in the spleen are not produced by vinblastine, nitrogen mustard, methotrexate, or cyclophosphamide.

Ferritin metabolism in reticulated-siderocytes.

Reticulated-siderocytes (reticulocytes which contain siderotic granules), obtained from the circulation of pigs after vigorous phlebotomy, were incubated in vitro. A rapid disappearance of granules from the reticulocytes was observed over 24 hr. Simultaneously with the decrease in granules, soluble ferritin accumulated in the media and siderotic granules developed in monocytes. The disappearance of the granules from the reticulated-siderocytes was oxygen-dependent and the loss of granules and the accumulation of ferritin in the media were both prevented by the addition of cyanide or dinitrophenol. It is concluded that the ferritin aggregates in the granules of reticulated-siderocytes are dispersed intracellularly into soluble ferritin, that soluble ferritin is excreted from the cell, and that one or both of these steps is dependent upon oxidative metabolism. Blood monocytes are capable of taking up soluble ferritin from the media and converting this into siderotic granules. Thus, a reticulocyte to plasma to monocyte ferritin pathway has been described.

The role of superoxide anion radical in the reduction of ferritin iron by xanthine oxidase.

Superoxide dismutase exerted a pronounced inhibitory effect upon xanthine oxidase-mediated reduction of iron in ferritin, ferric chloride, or ferric ADP. Maximal inhibition was observed when the superoxide dismutase concentration was only about 1% of that found in normal porcine liver. These observations indicate that superoxide anion radical is an intermediate in the reduction of iron by xanthine oxidase in vitro but not in vivo.

Iron metabolism in copper-deficient swine.

The way in which iron is handled by the duodenal mucosa, the reticuloendothelial system, the hepatic parenchymal cell, and the normoblast was investigated in copper-deficient swine.Copper-deficient swine failed to absorb dietary iron at the normal rate. Increased amounts of stainable iron were observed in fixed sections of duodenum from such animals. When (59)iron was administered orally, the mucosa of copper-deficient animals extracted iron from the duodenal lumen at the normal rate, but the subsequent transfer to plasma was impaired.When intramuscular iron supplements were given to copper-deficient pigs, increased amounts of iron were found in the reticuloendothelial system, the hepatic parenchymal cells, and in normoblasts (sideroblasts). Hypoferremia was observed in the early stages of copper deficiency, even though iron stores were normal or increased. When red cells that were damaged by prolonged storage were administered, the reticuloendothelial system failed to extract and transfer the erythrocyte iron to the plasma at the normal rate. Administration of copper to copper-deficient animals with normal iron stores resulted in a prompt increase in the plasma iron. The observed abnormalities in iron metabolism are best explained by an impaired ability of the duodenal mucosa, the reticuloendothelial system, and the hepatic parenchymal cell to release iron to the plasma. It is suggested that copper is essential to the normal release of iron from these tissues. This concept is compatible with the suggestion made by others that the transfer of iron from tissues to plasma requires the enzymatic oxidation of ferrous iron, and that the plasma copper protein, ceruloplasmin, is the enzyme (ferroxidase) which catalyzes the reaction. Because excessive amounts of iron were found in normoblasts, it is suggested that an additional defect in iron metabolism affects these cells and plays a major role in the development of anemia. As a result of the proposed defect, iron cannot be incorporated into hemoglobin and, instead, accumulates as nonhemoglobin iron.

Neutrophil kinetics in acute infection.

Neutrophil kinetics of acute experimental infection were studied with diisopropylfluorophosphate-(32)P labeling in 31 dogs inoculated intrabronchially with pneumococci. In vitro neutrophil labeling indicated a rapid transit time through the blood in early infections, with an elevated marginal granulocyte pool sometimes preceding an elevation of the circulating granulocyte pool. 13 hr after infection, the circulating and total blood granulocyte pools were increased but the rate of neutrophil transit through the blood was normal. During the recovery from infection there was a marked prolongation of neutrophil blood transit time, suggesting virtually complete cessation of bone marrow release of neutrophils into the blood. Labeling of neutrophils in vivo indicated an increased rate of emptying of the bone marrow storage pool proportional to the severity of infection as measured by the fever index. The change in the blood ratio of nonsegmented to segmented neutrophils was a much more accurate index of the severity of infection than the blood granulocyte concentration, correlating significantly with the fever index.

Association of HLA-linked hemochromatosis with idiopathic refractory sideroblastic anemia.

Five of seven patients with idiopathic refractory sideroblastic anemia carried an HLA-A3 alloantigen (relative risk, 7.3; P = 0.02). The significance of this association was strengthened by study of two pedigrees. An abnormality in iron metabolism was found in two siblings who had an HLA-A3,B14 haplotype in common with the first proband. A second proband with idiopathic refractory sideroblastic anemia had clinically manifest hemochromatosis. His brother had clinically manifest hemochromatosis but not sideroblastic anemia. This proband and his brother shared only the HLA-A3,B12 haplotype. Our findings infer that patients with idiopathic refractory sideroblastic anemia carry a single allele for hemochromatosis, that this allele accounts for the increased iron loading in this form of anemia, and that clinically manifest hemochromatosis may develop in an occasional patient with only one allele for hemochromatosis in the presence of the sideroblastic factor.

The role of ceruloplasmin in iron metabolism.

The importance of ceruloplasmin in iron metabolism was studied in swine made hypoceruloplasminemic by copper deprivation. When the plasma ceruloplasmin level fell below 1% of normal, cell-to-plasma iron flow became sufficiently impaired to cause hypoferremia, even though total body iron stores were normal. When ceruloplasmin was administered to such animals, plasma iron increased immediately and continued to rise at a rate proportional to the logarithm of the ceruloplasmin dose. The administration of inorganic copper induced increases in plasma iron only after ceruloplasmin appeared in the circulation. Thus, ceruloplasmin appeared to be essential to the normal movement of iron from cells to plasma. Studies designed to define the mechanism of action of ceruloplasmin were based on the in vitro observation that ceruloplasmin behaves as an enzyme (ferroxidase) that catalyzes oxidation of ferrous iron. Retention of injected ferrous iron in the plasma of ceruloplasmin-deficient swine was significantly less than that of ferric iron, reflecting impaired transferrin iron binding. Rat ceruloplasmin, which has little ferroxidase activity, was much less effective than porcine or human ceruloplasmin in inducing increases in plasma iron. These observations suggest that ceruloplasmin acts by virtue of its ferroxidase activity. Eight patients with Wilson's disease were evaluated in order to investigate iron metabolism in a disorder characterized by reduced ceruloplasmin levels. Evidence of iron deficiency was found in six of these, and in five of the six, plasma ceruloplasmin was less than 5% of normal. In comparison, the two patients without evidence of iron deficiency had ceruloplasmin levels of 11 and 18% of normal. It is suggested that iron deficiency tends to occur in those patients with Wilson's disease who have the severest degrees of hypoceruloplasminemia, possibly because of defective transfer of iron from intestinal mucosal cells to plasma.

Leukokinetic studies. 13. A non-steady-state kinetic evaluation of the mechanism of cortisone-induced granulocytosis.

The mechanism by which adrenocortical steroids induce granulocytosis in man has been investigated using granulocytes labeled with radioactive diisopropylfluorophosphate. After an intravenous injection of 200 mg of cortisol was given to five normal subjects, the mean value for the total blood granulocyte pool increased from 79 to 138 x 10(7) cells per kg of body weight and reflected an increase in the size of both the circulating granulocyte pool and the marginal granulocyte pool. When granulocytes in the circulation were labeled with diisopropylfluorophosphate and granulocytosis was induced later by the intravenous administration of cortisol, the rate of decline of granulocyte specific activity was increased, indicating that the blood pool was being diluted at an accelerated rate by unlabeled cells entering from the bone marrow. The rate of egress of granulocytes from the blood pool to an inflammatory exudate was studied by the "skin window" technique. After the administration of cortisol, there was a mean reduction in the cellularity of induced inflammatory exudates of 75%. However, this reduction in cellularity varied considerably from subject to subject (45-98%). From these studies we can infer that steroids induce an absolute granulocytosis by decreasing the rate of egress of cells from the total blood granulocyte pool as well as by increasing the influx of cells from the bone marrow. By model simulation studies of the non-steady state induced by cortisol injection, it has been possible to quantitate these rate changes. In the present studies cortisol injection resulted in a mean decrease in blood granulocyte egress of 74% (1-99%) and a mean increase in cell inflow of 450% (300-750%).

Genetic mapping of the hemochromatosis locus on chromosome six.

The purpose of this paper is to report a pedigree with hereditary hemochromatosis in which a recombination between the HLA-A and B loci occurred. Both the maternal and paternal HLA-A3, B7 haplotypes were carrying an allele for hemochromatosis. The A3, B12 haplotype of the proband was a recombinant of the maternal A3, B7 and A28, B12 haplotypes. The hemochromatosis locus segregated with the HLA-A locus and not with the HLA-B locus. Thus, the hemochromatosis locus maps in close proximity to the HLA-A locus.