Congenital erythrocytosis with elevated erythropoietin level: an incorrectly set "erythrostat"?

PURPOSE: A child who was extensively evaluated for polycythemia is reported. Polycythemia, or erythrocytosis, is seen rarely in children. The mechanisms for congenital and/or familial erythrocytosis are discussed. PATIENT AND METHODS: A 10 1/2-year-old white girl was referred for evaluation of polycythemia, which was detected incidentally during an emergency room visit for a febrile illness. She underwent extensive evaluation to determine the cause of the polycythemia. The literature was reviewed to determine the occurrence of congenital and/or familial erythrocytosis in children and its various causes. RESULTS: Despite extensive evaluation, no specific cause of the erythrocytosis could be determined in our patient. The erythrocytosis appeared to be secondary to an inappropriately elevated serum erythropoietin concentration. Serum erythropoietin rose further after phlebotomy, suggesting nonautonomous hypersecretion. After a review of the literature, we hypothesize that she had an inappropriate erythropoietin expression related to an abnormality in the renal oxygen-sensing mechanism governing erythropoietin synthesis. DISCUSSION: A discussion of congenital and familial erythrocytosis is presented, and a review of the literature regarding the possible mechanisms causing erythrocytosis is included.

Effects of hematopoietin-1 and interleukin 1 activities on early hematopoietic cells of the bone marrow.

Hematopoietin-1 (H-1) was purified from the human cell line 5637 and two amino acid sequences were observed in the preparation. One sequence was identical to that of interleukin 1 alpha (IL 1 alpha) and the other to that of IL 1 beta. The action of recombinant IL 1 alpha and other hematopoietic growth factors was studied using (a) a high proliferative potential colony-forming cell assay that uses primitive hematopoietic precursors from bone marrow, and (b) a spleen colony-forming unit assay. The results indicate that the IL 1 alpha target cell population is different than the target cell populations of IL 3, granulocyte-macrophage colony-stimulating factor; that IL 1 alpha in combination with mononuclear phagocyte colony-stimulating factor provides a proliferative stimulus; and that IL 1 alpha has at least a survival-enhancing and possibly proliferation-inducing effect on primitive hematopoietic stem cells.

Bone marrow transplantation for constitutional pure red cell aplasia.

Constitutional pure red cell aplasia (CPRCA) is a syndrome of failed erythropoiesis usually diagnosed within the first year of life. Four patients with CPRCA received transplants with marrow from their HLA-identical, mixed lymphocyte culture-nonreactive siblings. All patients were resistant to corticosteroid therapy and were dependent on regular red cell transfusions for at least 5 years. Three patients were conditioned with procarbazine, antithymocyte globulin, cyclophosphamide, and busulfan, and one was conditioned with antithymocyte serum, cyclophosphamide, and busulfan. Three patients promptly had successful engraftments with establishment of donor hematopoiesis. One patient initially rejected his graft but received a successful retransplant. All patients are currently alive with Karnofsky performance scores of 100 and normal erythropoiesis of donor origin. Despite a history of multiple transfusions, bone marrow transplantation is a potentially curative therapy for patients with CPRCA.

Measurement of human erythroid burst-promoting activity by a specific cell culture assay.

A two-stage cell culture assay specific for human erythroid burst-promoting activity (BPA) is described. Human peripheral blood mononuclear cells were cultured in suspension with or without a BPA test sample for two days, then transferred to methylcellulose medium with added erythropoietin (EPO) and incubated for ten more days, and finally BFU-E-derived colonies were scored. An increase in number of colonies due to the presence of BPA was observed that was proportional to the concentration of BPA in the test sample. This response was linear with respect to number of cells plated between 2 and 5 X 10(5)/ml. The system was standardized with a partially purified human urinary BPA preparation. Dose responses to urinary protein preparations, plasma, and serum were parallel. The assay system was found to be nonresponsive to highly purified EPO and to bacterial endotoxin. It was determined that BPA action was confined to the suspension culture stage of the assay, while EPO presence was an absolute requirement during methylcellulose culture. In the two-stage assay optimal amounts of BPA caused up to 358% increases of BFU-E-derived colonies; the same amounts of BPA added to conventional methylcellulose cultures caused only up to 54% increases over the number of colonies obtained with EPO alone. Plasma and serum BPA levels of hematologically normal and abnormal individuals showed no correlation with EPO levels and hemoglobin (Hb) concentrations. This seems to rule out the possibility that BPA elaboration is regulated by oxygen availability or the amount of EPO circulating in an organism.

The role of erythropoietin, thrombopoietic stimulating factor, and myeloid colony-stimulating factors on murine megakaryocyte colony formation.

Various growth factors including purified erythropoietin, colony-stimulating factor (CSF-1), and granulocyte-macrophage CSF (GM-CSF) were tested for their ability to stimulate megakaryocytopoiesis. Four separate preparations of erythropoietin were tested in highly defined cell culture medium. One unit of purified material stimulated small but significant numbers of megakaryocyte colonies, both in serum-containing and in serum-free cultures. All other erythropoietin preparations failed to induce megakaryocyte colony formation. Purified erythropoietin showed no synergistic activity with either WEHI-3 cell conditioned medium (WEHI-3CM, a source of both megakaryocyte CSF and megakaryocyte-potentiating activity) or P388D1 cell conditioned medium (P388D1CM, a preparation containing megakaryocyte potentiator). Partially purified thrombopoietic stimulatory factor did not stimulate directly megakaryocyte colony formation, but acted together with WEHI-3CM, augmenting the number of clonable progenitors detected. Optimal activity was observed at 12-25 micrograms protein per plate. Myeloid growth factors (CSF-1 and GM-CSF) were inactive in the murine megakaryocyte assay. The data show lineage specificity for the myeloid stimulators, but a purified erythropoietin preparation was found to stimulate a small level of megakaryocytopoiesis.

A clinical trial of prostaglandin E2 to increase erythropoiesis in anemia of end stage renal disease. A preliminary report.

Prostaglandin E2 is known to stimulate erythropoiesis by different mechanisms. A clinical trial of prostaglandin E2 to stimulate erythropoiesis in four patients with anemia of end stage renal disease resulted in an increment in peripheral blood Burst Forming Units-Erythroid (BFU-E). This increase in erythroid progenitors returned to baseline with cessation of therapy. A significant increase in serum erythropoietin (EPO) activity was demonstrated in one patient and was noticeable in another. Side effects mainly consisted of local pain at the site of the infusion and vomiting.

The relationship between human spleen and blood erythroid burstforming units (BFU-E).

The influence of splenectomy on erythroid burst colony formation by peripheral blood mononuclear cells from 10 patients (four with hereditary spherocytosis, two with beta-thalassaemia major, two with Hodgkin's disease and two with idiopathic thrombocytopenic purpura) was studied. In every instance splenectomy was followed by a lowering of blood BFU-E. The post-splenectomy levels ranged from 0 to 30% of the preoperative levels. Mononuclear cells from the spleens of eight patients were cultured and found to contain numerous BFU-E. The total quantity of BFU-E in the whole blood and in the spleen of the patients was generally of the same order of magnitude. The number of splenic BFU-E did not correlate with spleen size. Splenic BFU-E differed from peripheral blood BFU-E in that they were more sensitive to erythropoietin (Ep) and in that they failed to respond to burst promoting activity (BPA) produced by preincubating the spleen mononuclear cells with phytohaemagglutinin M (PHA). In contrast, media conditioned by PHA-treated spleen cells contained BPA active on peripheral blood BFU-E from normal individuals. These data suggest that the spleen may have an influence on the numbers and functional properties of BFU-E.

Comparison of the effects of crude and purified erythropoietin on the synthesis of hemoglobin in rat bone marrow cell cultures.

The stimulatory effects of crude (148 U/mg protein) and purified (15,920 U/mg protein) human urinary erythropoietin (Ep) on heme and globin synthesis of rat bone marrow cells in short term (2-3 day) suspension culture were compared. Freshly isolated, unfractionated marrow cells responded equally well to both Ep preparations. Velocity sedimentation at unit gravity was used to separate the marrow cells into a fast-moving (population I) and a slow-moving (population II) fraction. Of the 2 preparations tested, only crude Ep was found to stimulate heme and globin synthesis of population I cells. Population II cells were unresponsive to both Ep preparations. Preincubation of the rat marrow cells in the absence of Ep for 48 h resulted in the disappearance of most of population II, the remaining cells only responded to the crude Ep preparation. Direct remixing experiments involving various proportions of population I and II cells also showed that addition of the purified Ep preparation could only bring about an increase in hemoglobin synthesis in those cultures which contained a sufficient number of population II cells. Cultures deficient in population II cells to which the crude Ep preparation was added did not show this requirement, suggesting that this preparation contained a component which could substitute for the presence of population II cells. This demonstrates that the action of Ep and of a second factor (or of a cell to cell signal) is required to increase hemoglobin synthesis in short term rat marrow cell cultures.

Effect of parathyroid hormone on erythropoiesis.

Inhibitors of erythropoiesis have been found in the blood of uremic patients but their nature has not been identified. These patients have excess blood levels of parathyroid hormone (PTH) and it is possible that PTH inhibits erythropoiesis. The present study was undertaken to examine the effect of intact PTH molecules and some of its fragments on human peripheral blood and mouse bone marrow burst-forming units-erythroid (BFU-E), on mouse bone marrow erythroid colony-forming unit (CFU-E), and granulocyte macrophage progenitors (CFU-GM), and evaluate the interaction between PTH and erythropoietin (Ep) on human BFU-E. Intact PTH (1-84 bPTH) in concentrations (7.5-30 U/ml;) comparable to those found in blood of uremic patients produced marked and significant (P less than 0.01) inhibition of BFU-E and mouse marrow GFU-GM, but not of mouse marrow CFU-E. Inactivation of 1-84 bPTH abolished its action on erythropoiesis. Increasing the concentration of Ep in the media from 0.67 to 1.9 U/ml overcame the inhibitory effect of 1-84 bPTH on BFU-E. The N-terminal fragment of PTH (1-34 bPTH) and 53-84 hPTH had no effect on BFU-E. The results demonstrate that (a) either the intact PTH molecule or a C-terminal fragment(s) bigger than 53-84 moiety exerts the inhibitory effect on erythropoiesis, and (b) adequate amounts of Ep can overcome this action of PTH. The data provide one possible pathway for the participation of excess PTH in the genesis of the anemia of uremia.

Inhibitory effects of interferon on mouse megakaryocytic progenitor cells in culture.

Mouse bone marrow cells were grown in plasma clots, megakaryocyte formation was stimulated with human urinary erythropoietin (Ep). The expression of megakaryocyte colony forming units (CFUM) and of single megakaryocyte forming units (M) was evaluated after seven days in culture. Usually ten times more M than CFUM were found. Megakaryocytic colony formation showed a linear dependence on cell dose from 0.5 to 2 X 10(5) cells/clot. The colony size frequency distribution exhibited a single peak in the two-cell size class, followed by a continuous decrease, suggesting that cell division may occur asynchronously in cells making up a colony. Mouse L cell interferon (IF) in doses from 10 to 1000 U/ml was included in clots together with Ep. This resulted in a biphasic, dose dependent reduction of colony formation and of single megakaryocyte formation. At all doses tested CFUM were more sensitive to IF than M. These observations are best explained by a differential inhibitory effect of IF on replication of two or more classes of megakaryocytic progenitor cells.

Constancy of erythroid burst forming unit (BFUE) levels in the blood of hematologically normal individuals.

Repeated determination of circulating blood BFUE levels in 7 hematologically normal individuals, during a 6 to 16 month period, showed their respective levels to be maintained within narrow limits (coefficient of variation did not exceed 15.1%). However, males differed from each other 3-fold and females as much as 12-fold in the number of BFUE/ml blood. These differences may reflect various sizes of the early erythroid progenitor compartment, which in the presence of different levels of humoral regulators of erythropoiesis are capable of maintaining a normal rate of red cell production. Our findings suggest that the observation of changes of the blood BFUE level of individuals, over a period of time, may be indicative of a change in their erythropoiesis and may shed some light on the physiological role of the peripheral blood BFUE.

Biological properties of a human urinary protein fraction with burst promoting activity.

Urinary proteins from patients with iron deficiency anemia and acquired aplastic anemia were fractionated by chromatography on QAE-Sephadex A-50 and Sephadex G-25. Fractions containing erythroid burst promoting activity (code named regulatory protein RP) were separated from erythropoietin. Mouse bone marrow cells were preincubated for one day in suspension culture, in the presence or absence of RP, transferred to a methylcellulose based system and incubated for six more days with erythropoietin (EPO). It was found that the presence of RP in the preincubation medium had a 2 to 4 fold enhancing effect on subsequent erythroid burst colony formation. However, when RP was added to methylcellulose based cultures simultaneously with EPO, the erythroid burst response was reduced or abolished. Addition of RP to marrow cell suspension cultures increased the number of self replicating, pluripotent (erythroid/granuloid, E/G ratio = 3) spleen colony forming units (CFU-S) found at the end of 2 days incubation 3-5 fold over their number in control cultures incubated without the factor. In marked contrast to this, addition of EPO to the cultures caused an increased persistence of CFU-S with a predominantly erythroid commitment (E/G ratio = 19) and a low self replicating ability, as measured by retransplantation of spleen cells into secondary recipients. These observations are compatible with the presence in RP of a factor, or factors, capable of maintaining the size of certain early precursor cell compartments.

Human erythroid burst-promoting activity produced by phytohemagglutinin-stimulated, radioresistant peripheral blood mononuclear cells.

The regulation of erythroid burst-colony formation was studied in cultures of human peripheral blood mononuclear cells. Numbers of erythropoietin-stimulated colonies obtainable from the cells in response to various treatments were compared. One-day preincubation of the cells with phytohemagglutinin (PHA) doubled the yield of colonies. Irradiation of the cells with 3000 rad eliminated their ability to form erythroid bursts, but did not impair the ability of PHA-treated cells to enhance burst formation when added to a fresh batch of cells. This was due to a humoral factor, since media conditioned by PHA-treated washed cells were as effective as the cells themselves. When cells were separated into subpopulations by an adherence procedure and according to their ability to form rosettes with sheep red blood cells, it was found that the PHA-dependent burst-promoting activity released into the medium originated in a nonadherent, nonrosetting (T-cell depleted) cell population.

Suppressive effect of interferon on erythroid cell proliferation.

The effect of interferon on mouse and human in vitro erythropoiesis was investigated using the mouse marrow CFU-E and the human blood BFU-E systems, respectively. Homologous interferons were found to have a marked inhibitory effect on mouse CFU-E and human BFU-E proliferation. This inhibitory effect was dose related and independent of the erythropoietin concentration used. The effective concentration of human interferon was within the range observed in humans following viral infections. It is suggested that interferon may play a role in the mechanism of the acute erythroblastopenic crisis observed at times in patients with chronic anemia following viral infections.

Suppression of mouse erythroid colony formation by L1210 leukemia cells.

The effect of L1210 transplantable leukemic cells on in vitro formation of erythroid colonies from CD2F1 mouse bone marrow progenitor cells (CFU-E) was investigated. Clonal cell culture was carried out by a methylcellulose technique. Human urinary erythropoietin served as the stimulator. After 44 hours of incubation aggregates of eight or more erythroid cells were scored as colonies. The number of CFU-E which could be demonstrated in marrow cells from mice that had been injected intravenously 6 days before with 5 x 10(4) L1210 cells was far below that obtained from normal marrow cells. When 1.3 x 10(5) marrow cells from leukemic mice or L1210 ascites cells were cultured with an equal amount of normal cells, the number of CFU-E expressed was reduced by 51% and by 86%, respectively, relative to controls with normal cells only. Neither lethally irradiated L1210 cells (4500 rad) nor L1210 cell conditioned media suppressed erythroid colony formation. It is suggested that in L1210 leukemia erythropoiesis is decreased because of a cell-to-cell inhibitory action of the leukemia cells on CFU-E.

A beneficial effect of the in situ kidney on in vitro marrow erythropoiesis in chronic renal failure.

The effect of the in situ kidney on transfusion requirements and in vitro erythropoiesis was investigated in 20 patients with end stage renal disease undergoing hemodialysis. 6 of the 12 patients with in situ kidneys did not require transfusion, whereas the other 6 had an average monthly transfusion requirement of 277 ml of sedimented RBCs. All 8 anephric patients required transfusions with an average requirement of 352 ml of sedimented RBCs per month. Serum erythropoietin activity was inappropriately low for the degree of anemia in all but 1 patient, and bone marrow was uniformly hypocellular. Marrow cells from patients with in situ kidneys exhibited a greater response to erythropoietin than marrow cells from their anephric counterparts. The response was not improved by hemodialysis.