Variations in erythropoiesis throughout a lifetime. Studies in a high-leukaemic mouse strain, the AKR/O strain, and a non-leukaemic strain, the WLO strain

We have studied the development of some haematological variables: erythropoiesis stimulating factor(s) (ESF), investigated with an in vitro cell culture assay; and the content of bone marrow and spleen erythroid colony forming unit(s) (CFU-E) and erythroid burst forming unit(s) (BFU-E) throughout the lifetime of 2 different mouse strains: the high-leukaemic, retrovirus infected AKR/O strain, and the non-leukaemic WLO strain. During the recovery phase of the postnatal anaemia, a peak in plasma ESF occurs in both strains. In young adult mice of both strains another peak in plasma ESF occurs at 70-110 days of age, associated with an increased number of bone marrow CFU-E, in a period when packed cell volume (PCV) remains stable. As the animals grow older PCV decreases, whereas plasma ESF and bone marrow CFU-E concentration increase. These results, together with in vitro dose-response studies, suggest reduced sensitivity to erythropoietin (Epo) of the ageing erythron. Throughout, the AKR/O strain has higher levels of plasma ESF and bone marrow CFU-E concentrations than the WLO strain, indicating both a reduced Epo responsiveness and some degree of ineffective erythropoiesis in the AKR/O strain. At all ages the AKR/O strain has a high concentration of Epo independent bone marrow CFU-E, possibly caused by the virus infection of precursor cells.     

Isolation of an erythropoietic stimulating factor from the serum of anemic chicks

A factor stimulating fowl erythropoiesis in vitro has been isolated from the blood of anemic chick by gel filtration and ion exchange chromatography. This purification procedure yields a preparation whose specific activity is enriched 44 times and which has been relieved of toxic factors present in the crude anemic serum. In vitro erythropoiesis requires addition into the culture medium of erythropoietic stimulating factor and of another factor present in the serum of anemic as well as non-anemic chick. This last factor is lost during purification of the erythropoietic stimulating factor and is not present in fetal calf serum.     

An erythropoietic stimulating factor similar to erythropoietin released by macrophages after treatment with silica

An erythropoietic stimulating factor (ESF) can be detected in the supernatant from fetal liver and adult bone marrow and spleen cells when preincubated with the macrophage-specific cytotoxic agent, silica. Stimulation is observed in 12-day fetal liver CFU-E cultures in the absence of added erythropoietin (Ep). The concentration of ESF in the supernatant added to CFU-E cultures is dependent on the preincubated cell dose and the volume added. The stimulating activity is abolished when mice are hypertransfused and increased above normal values when mice are bled. A concentrated silica-treated spleen supernatant was able to stimulate erythropoiesis in the polycythemic mouse bioassay. It is concluded that the ESF is similar, if not identical, to Ep.     

In vitro development of chicken erythropoietin-sensitive cells

Addition of anemic chick serum to cultures of chicken marrow in plasma clots induces the development of erythrocytic colonies. Mammal erythropoietin shows no effect on cultures of chicken marrow. These observations confirm the existence of an erythropoiesis stimulating factor specific for birds, with properties similar to those of mammalian erythropoietin.     

Erythropoietin-like factor production by young chick blastoderms.

When grown in liquid culture, chick blastoderm cells release an erythropoietic stimulating factor (ESF) whose properties in vitro and in vivo are very similar to those of erythropoietin found in the serum of anemic adult chick. The role of this factor in the induction of the first blood islands of the chick embryo is discussed.     

Changes in the nature of the differentiation of the erythroid stem cell in the bone marrow of mice as affected by the peritoneal cells of donors subjected to bloodletting

The influence of the peritoneal cells unstimulated by an inflammatory agent (PCs) on the bone marrow erythropoiesis in the CBA mice was studied in the normal conditions and after the massive haemorrhage. The PCs obtained from the intact or anemic donors (2, 4 and 18 h after the haemorrhage) were introduced intraperitoneally into the intact syngeneic recipients. The massive haemorrhage induces a special type of differentiation of erythroid cells in bone marrow, "reserve erythropoiesis", which is characterized by a decrease, within four days, in the proliferation of basophilic proerythrocytes, stimulation of proliferation of polychromatophilic proerythrocytes and entry of oxyphilic proerythrocytes into mitosis. The transplantation of the PCs from the anemic donors induced the "reserve erythropoiesis" in the bone marrow of the recipients. The transplantation of the PCs from the intact donors did not induce the "reserve erythropoiesis" and appeared to inhibit erythropoiesis via lengthening the time of mitoses in polychromatophilic and basophilic proerythrocytes.     

The role of prostaglandins in the production of erythropoietin (ESF) by the kidney. II. Effects of indomethacin on erythropoietin production following hypoxia in dogs

In order to determine the sequence of events leading to the increase in ESF production following renal hypoxia, the possible role of prostaglandins in the generation of ESF by the kidney was assessed. ESF production following exposure to hypoxia and indomethacin (I) treatment were studied in dogs. Plasma ESF titers were measured in carotid artery blood before the hypoxic stimulus, and after 1, 3 and 5 hours exposure. It was found that plasma ESF levels were significantly elevated in 6 dogs exposed to an hypoxic stimulus while anesthetized with pentobarbital by breathing an atmosphere of reduced oxygen (8% oxygen, 5% carbon dioxide and 87% nitrogen) for 3 and 5 hours. On the other hand, 6 dogs exposed to this hypoxic stimulus and pretreated with indomethacin (5 mg/kg orally 2 x), a drug which inhibits prostaglandins synthesis, did not show a significant increase in plasma ESF levels after exposure to hypoxia as compared to controls. These results indicate that renal prostaglandins play a role in ESF production after an hypoxic stimulus.     

Effect of Trypanosoma brucei brucei on erythropoiesis in infected rats

Anemia generated from African trypanosome infection is considered an important symptom in humans and in domestic animals. In order to recover from anemia, the process of erythropoiesis is essential. Erythropoiesis is affected by erythropoietin (EPO), an erythropoietic hormone, supplying iron and inflammatory and proinflammatory cytokines. However, the role of these factors in erythropoiesis during African trypanosome infection remains unclear. In the present study, we analyze how erythropoiesis is altered in anemic Trypanosoma brucei brucei (interleukin-tat 1.4 strain [ILS])-infected rats. We report that the packed cell volume (PCV) of blood from ILS-infected rats was significantly lower 4 days after infection, whereas the number of reticulocytes, as an index of erythropoiesis, did not increase. The level of EPO mRNA in ILS-infected rats did not increase from the third day to the sixth day after infection, the same time that the PCV decreased. Kidney cells of uninfected rats cultured with ILS trypanosome strain for 8 hr in vitro decreased EPO mRNA levels. Treatment of both ILS and cobalt chloride mimicked hypoxia, which restrained the EPO-production-promoting effect of the cobalt. Messenger RNA levels of β-globin and transferrin receptor, as markers of erythropoiesis in the bone marrow, also decreased in ILS-infected rats. Levels of hepcidin mRNA, which controls the supply of iron to the marrow in liver, were increased in ILS-infected rats; however, the concentration of serum iron did not change. Furthermore, mRNA levels of interleukin-12, interferon-γ, tumor necrosis factor-α, and macrophage migration inhibitory factor in the spleen, factors that have the potential to restrain erythropoiesis in bone marrow, were elevated in the ILS-infected rats. These results suggest that ILS infection in rats affect erythropoiesis, which responds by decreasing EPO production and restraining its function in the bone marrow.     

Changes in hemopoietic and regulator levels in mice during fatal or nonfatal malarial infections. I. Erythropoietic populations

Erythroid precursors BFU-E and CFU-E and erythroblasts (ERB) were monitored in the marrow and spleen of mice during fatal or nonfatal malaria. Transient depletions of marrow CFU-E and ERB without modification of BFU-E or erythropoietin (Epo) levels were found as early events in fatal infections. Before anemia development, erythropoiesis was reduced in the bone marrow but increased in the spleen. During the anemic phase, for comparable levels of anemia, plasma Epo levels were elevated to a similar degree in fatal and nonfatal malaria. In the bone marrow, CFU-E increased twofold and BFU-E were usually reduced as expected in severe anemia. ERB populations increased but remained below or within normal values, suggesting an impairment of marrow erythropoiesis related to early events following infection. In contrast, in the spleen, ERB production was strongly simulated but amplification of ERB, CFU-E, and BFU-E populations was 2.5-fold lower in fatal than in nonfatal malaria. The results suggest that a defect in amplification of splenic erythropoiesis is a crucial determinant of the fatal outcome of malarial infection. This may have been mediated by a defective stem cell migration or multiplication. Some evidence obtained during recovery stages suggested that a factor(s) other than Epo may control splenic erythropoiesis during the anemia associated with malaria.     

Relationships between blood platelet and erythrocyte formation

The rate of thrombopoiesis was measured by platelet incorporation of ³⁵S-sulfate in mice having markedly stimulated or suppressed erythropoiesis, and compared to controls. Twenty-four hr exposure to 0.4 atm in a low pressure chamber caused increased platelet incorporation of ³⁵S, while exposure for 2 to 5 days caused reductions in both ³⁵S uptake and platelet counts at time of sacrifice. These values were progressively reduced with the longer periods of hypoxia. Two units of human urinary erythropoietin (ESF) were injected twice daily for 1 to 5 days and also caused reduced platelet ³⁵S uptake in mice given the ESF for 2, 4, or 5 days. Mice rendered polycythemic by hypertransfusion or previous exposure to low barometric pressure had increased platelet ³⁵S uptake and increased platelet counts. One possible explanation is that the severe hypoxia or the administration of ESF strongly stimulated erythropoiesis, but might have inhibited thrombopoiesis by depleting megakaryocyte precursors in the bone marrow.     

Iron metabolism in iron-deficient male quail

1. Male quails submitted 20 and 120 days to a low iron diet (7 ppm) were compared to female laying quails, exposed for 30 days to the same low iron regime, in order to compare the response of the iron metabolic control under a single (erythropoiesis) or a doubled (erythropoiesis and egg formation) iron demand. 2. Iron deposit in storage organs, the classical hematology and the intestinal iron absorption were analyzed in these animals. 3. In males, after 120 days, the iron deposits were reduced 50 and 75%, but hematological values (hematocrit and hemoglobin concentration) were normal, although in laying quails, after 30 days, an anemic condition was evident in both blood parameters and iron deposits, provoking an iron deficient erythropoiesis. 4. The enhancement of the intestinal iron uptake, confirms the anemic character of these birds.     

In vitro effects of ascorbic acid and dehydroascorbic acid on diphosphopyridine nucleotide diaphorase in toad testis.

The enzyme diphosphopyridine nucleotide diaphorase (DPND) was demonstrated histochemically in both the tubular and Leydig cells of the toad testis. Addition of 200 mug of dehydroascorbic acid (DHA) to 100 mg of testicular slices in the incubating medium increased the activity of DPND, while a similar dose of ascorbic acid failed to do so. The evidence indicates that DHA is involved in the oxidation of reduced DPND in toad testis.     

An experimental study on the pathway of iron transfer from macrophages to erythrocytes in rat liver

In order to reveal the pathway of iron release from macrophages, a 59Fe-labelled ferric hydroxide-potassium polyvinyl sulfate complex (Fe-PVS) was injected intravenously into anemic rats and the level of radioactivity in the liver, spleen, bone marrow, blood plasma and red blood cells (RBC) was estimated at various time intervals after the injection. Histochemical observation of ferric iron and ferritin in the liver was also made on anemic rats treated using unlabelled Fe-PVS. Fe-PVS injection promoted the recovery of anemia causing a rapid increase in the RBC number, with activated erythropoiesis occurring in the spleen and bone marrow. Soon after the injection, most of the radio iron was found in the liver with a small amount in the circulating erythrocytes, bone marrow and spleen. The iron level in the liver decreased gradually with a rapid increase in the iron level of the erythrocytes which reached a very high level 6 days after the 59Fe-PVS injection. Histochemical observations showed a heavy deposition of ferritin in the Kupffer cells 3 days after Fe-PVS injection. This deposition was minimized after 6 days with an increase in the level of ferritin in the parenchymal cells in the central area of acini. The level of radioferritin estimated biochemically in the nonparenchymal cell fractions of the liver revealed that the level dropped by about one third approximately 3.5 days after the Fe-PVS injection, showing the stimulated ferritin release at this stage. Results indicate that Kupffer cells in the liver play an important role in ferritin synthesis from the phagocytized iron compounds and that the iron is supplied for erythroid cell proliferation.     

Age-related expression of PEDF/EPC-1 in human endometrial stromal fibroblasts: implications for interactive senescence

Aging is the major risk factor for many cancers, and age-related changes in the tissue microenvironment can facilitate tumor growth. This study uses human endometrial cells to begin to test the hypothesis that age-related changes in pigment epithelium-derived factor/early population doubling cDNA-1 (PEDF/EPC-1) levels create an environment that is more permissive to tumor growth. Endometrial stromal fibroblasts (ESF) are the predominant cell type in the human endometrium and exert regulatory control over the glandular epithelial cells, which are the source of most tumors. As ESF age in vitro, their ability to regulate appropriate growth and differentiation of epithelial cells declines. Endometrial epithelial cells in primary culture expressed relatively low levels of PEDF/EPC-1 mRNA. In contrast, early passage quiescent ESF from adult donors produce higher levels of the 1.5-kb PEDF/EPC-1 mRNA and 50-kDa secreted protein than epithelial cells. As ESF age in vitro the relative abundance of PEDF/EPC-1 mRNA declines, as does the level of PEDF/EPC-1 protein secreted into cell culture medium. Treatment with PEDF/EPC-1 protein had no effect on ESF proliferation but did inhibit anchorage-dependent and anchorage-independent proliferation of endometrial carcinoma cells in a dose- and time-dependent manner. These findings imply that an age-related loss of PEDF/EPC-1 expression by ESF could eliminate a negative regulator of cancer cell growth and, thereby, contribute to the age-related increase in cancer incidence.     

NPY- and CGRP-like immunoreactive nerve fibers in the testis and mesorchium of the toad (Bufo arenarum)

The presence and distribution of peptidergic nerve fibers were studied in the testis and mesorchium of the toad by means of immunohistochemistry. Cryostat sections of the testis and whole-mount preparations of mesorchia were immunostained with antisera to calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY). After leaving the mesorchium CGRP-immunoreactive (IR) fibers were seen predominantly running in between the seminiferous tubules. In addition, a small population of CGRP-IR nerve fibers formed thin plexuses around blood vessels. Conversely, NPY-like immunoreactivity predominated in nerve fibers that formed dense plexuses around vessels both in the mesorchium and testis. Additionally, some single NPY-IR nerve fibers could be seen in both structures studied. The functional significance of these peptidergic systems in the testis of the toad remains to be analyzed.     

Increase in hematocrit, hemoglobin and red cell mass in normal mice after treatment with cyclic AMP (38543).

Chronic treatment of normal mice with either dibutyryl cyclic AMP or erythropoietin produced elevations in the hematocrit, hemoglobin concentration and red cell mass when compared to these same hematological parameters in untreated mice. Dibutyryl cyclic AMP increased red cell mass by 46% while ESF treatment resulted in a 56% increase in red cell mass. These studies confirm earlier reports of the effects of cyclic AMP in increasing radioactive iron incorporation into red cells and further indicate that this change is associated with an absolute increase renal cyclic AMP concentrations probably stimulate erythropoiesis as a consequence of increased kidney production of erythropoietin.     

Identification of ovotransferrin as a heme-, colony- and burst-stimulating factor in chick erythroid cell cultures

Clonal growth of erythroid cells from bone marrow of 2-day-old chicks in fibrin clots under different culture conditions has been used as independent assays for heme-, colony- and burst-stimulating activities found in anemic chicken plasma. The properties of the heme-stimulating activity analyzed by gel filtration, isoelectrofocusing, resistance to heat denaturation, ammonium sulfate precipitation, DEAE-chromatography or HAP-chromatography, suggested serum transferrin as the heme-stimulating factor(s). Heme-, colony- and burst-stimulating factor(s) from anemic chicken plasma did not separate from each other by gel filtration or isoelectrofocusing. Ovotransferrin from egg white also showed heme-, colony- and burst-stimulating activities by the assay employed even after further purification by limited trypsin digestion, electrophoresis, hydroxylapatite chromatography or fractionation by ConA-Sepharose chromatography.     

Digitalis-like compounds in the toad Bufo viridis: tissue and plasma levels and significance in osmotic stress.

Digitalis-like compounds (DLC), constituents of animal tissues, are possible regulators of the Na+, K(+)-ATPase implicated in water and salt homeostasis. The distribution of DLC in the toad (Bufo viridis) was determined following methanol extraction and partial purification. DLC highest levels were found in the skin but it was also detected in the plasma and many internal organs. Short term (hours) exposure of the toad to hypertonic shock (1.5% NaCl) induced an increase in plasma osmolarity due to an increase in Na+ and Cl- levels. This treatment induced a transient, three fold, increase of DLC levels in the brain and transient reduction of its levels in the ventral skin. Acclimation of the toads to burrowing conditions for six weeks resulted in an increase in plasma osmolarity due to a large increase in plasma urea with a small increase in ion concentrations. Under these conditions DLC levels in the dorsal skin increased by 100% without alteration of its levels in the plasma, brain and ventral skin. DLC levels in the toad brain of control animals, showed a significant dependence on season, being highest in the summer and lowest in the winter. DLC levels in the skin peaked in May while the levels in the plasma were season independent. The changes in DLC levels induced by the short- as well as long-term perturbations in the animal environmental salinity together with the seasonal differences suggest that DLC in the toad is involved in water and salt homeostasis of these animals, but may also participate in other unknown functions.     

Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis

The Krüppel-like factor 1 (KLF1) and KLF2 positively regulate embryonic β-globin expression and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1(-/-) KLF2(-/-) double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1(-/-), and KLF1(-/-) KLF2(-/-) mice. Among these, the gene for Myc (c-Myc) emerged as a central node in the most significant gene network. The expression of the Myc gene is synergistically regulated by KLF1 and KLF2, and both factors bind the Myc promoters. To characterize the role of Myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia, analogous to KLF1(-/-) KLF2(-/-) embryos. In the absence of Myc, circulating erythroid cells do not show the normal increase in α- and β-like globin gene expression but, interestingly, have accelerated erythroid cell maturation between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate Myc to control the primitive erythropoietic program.     

Release of erythropoietin from macrophages by treatment with silica

An erythropoietic stimulating factor (ESF) can be shown to be released from preincubated macrophage-containing cell suspensions from mice by the macrophage-specific, cytotoxic agent, silica. A concentrated silica treated spleen cell supernatant containing ESF is shown to cause a dose dependent increase in ⁵⁹ Fe incorporation into red blood cells using the in vivo polycythemic mouse bioassay. The ESF from the same supernatant can also be neutralized by anti-erythropoietin. A second concentrated supernatant fractionated using wheat germ lectin-Sepharose 6MB and compared to either unfractionated or fractionated step 111 erythropoietin (Ep), tested in vitro using the erythroid colony-forming technique and 12-day fetal liver as target cells, indicates parallelism of all linear dose-response lines. This, together with the in vivo data, strongly suggests that the ESF released from macrophages treated with silica is, in fact, Ep. Substituting Ca²⁺ ions for fetal calf serum in the preincubation procedure results in the same activity being released compared to the presence of 1% or 20% fetal calf serum.