Functional significance of globotriaosyl ceramide in interferon-alpha(2)/type 1 interferon receptor-mediated antiviral activity

The N-terminus of the type 1 interferon receptor subunit, IFNAR1, has high amino acid sequence similarity to the receptor binding B subunit of the Escherichia coli-derived verotoxin 1, VT1. The glycolipid, globotriaosyl ceramide (Gb(3): Gal alpha(1) --> 4 Gal beta 1 --> 4 Glu beta 1 --> 1 Cer) is the specific cell receptor for VT1. Gb(3)-deficient variant cells selected for VT resistance are cross-resistant to interferon-alpha (IFN-alpha)-mediated antiproliferative activity. The association of eIFNAR1 with Gal alpha 1 --> 4 Gal containing glycolipids has been previously shown to be important for the receptor-mediated IFN-alpha signal transduction for growth inhibition. The crucial role of Gb(3) for the signal transduction of IFN-alpha-mediated antiviral activity is now reported. IFN-alpha-mediated antiviral activity, nuclear translocation of activated Stat1, and increased expression of PKR were defective in Gb(3)-deficient vero mutant cells, although the surface expression of IFNAR1 was unaltered. The VT1B subunit was found to inhibit IFN-alpha-mediated antiviral activity, Stat1 nuclear translocation and PKR upregulation. Unlike VT1 cytotoxicity, IFN-alpha-induced Stat1 nuclear translocation was not inhibited when RME was prevented, suggesting that the accessory function of Gb(3) occurs at the plasma membrane. IFN-alpha antiviral activity was also studied in Gb(3)-positive MRC-5 cells, which are resistant to IFN-alpha growth inhibition, partially resistant to VT1 but still remain fully sensitive to IFN-alpha antiviral activity, and two astrocytoma cell lines expressing different Gb(3) fatty acid isoforms. In both systems, long chain fatty acid-containing Gb(3) isoforms, which are less effective to mediate VT1 cytotoxicity, were found to correlate with higher IFN-alpha-mediated antiviral activity. Inhibition of Gb(3) synthesis in toto prevented IFN-alpha antiviral activity in all cells. We propose that the long chain Gb(3) fatty isoforms preferentially remain in the plasma membrane, and by associating with IFNAR1, mediate IFN-alpha antiviral signaling, whereas short chain Gb(3) fatty acid isoforms are preferentially internalized to mediate VT1 cytotoxicity and IFNAR1-dependent IFN-alpha growth inhibition.     

Disorders in the immune responses of T- and B-cells in mice administered intravenous verotoxin 2

To obtain a better insight into the pathogenesis of verotoxin-producing Escherichia coli (VTEC)-associated diseases, we explored the effect of verotoxin 2 (VT2) on the immune response in mice. The distribution of lymphocyte phenotypes and the lymphocyte immune response were examined after intravenous administration of VT2 to mice. Among the peripheral lymphocytes and splenocytes of 4-week-old C57BL/6 mice, there was first of all a decrease in T-cells, which began 24 h after intravenous administration of VT2 (50 ng/kg, lethal dose). The CD4+ cell subpopulations of the peripheral blood and spleen were significantly decreased at 24 h, while the B220+ splenocyte subpopulation was markedly decreased at 45 h after VT2 administration. In the thymus, a decrease in CD4+CD8+ cells was predominantly observed near death. Interestingly, in E. coli lipopolysaccharide (LPS)-responder mouse strains (C57BL/6 and C3H/HeN) cotreated with LPS, the susceptibility to VT2 was enhanced, and the increase in B220+ cells induced by LPS alone was suppressed. Furthermore, splenocytes from C57BL/6 mice treated with VT2 (50 ng/kg) 6-24 h earlier reduced LPS-induced proliferative responses to 50-52% of that in control cells, indicating that the effect of VT2 on the immunoresponse seen in vivo may be negatively exerted on the proliferation of the cells. In addition, the number of splenocytes that produced anti-sheep red blood cell antibody was decreased in mice treated with VT2. These results suggest that VTEC infection may eliminate CD4+ and CD8+ T-cells and B-cells by affecting their survival and proliferative responses, leading to reduced antibody production.     

Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity

In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.     

Target cells for Friend virus-induced erythroid bursts in vitro

Erythropoietin (Epo) acts on mouse bone marrow cells in vitro in plasma clot or methyl cellulose culture systems to induce the formation of single erythroid colonies, or clusters of erythroid colonies termed bursts. Our laboratory has recently reported the observation that infection of mouse bone marrow cells in vitro with the polycythemia-inducing strain of Friend virus (FV) resulted in the formation of erythroid bursts after 5 days in plasma clot culture in the absence of added Epo. We have now used this system to characterize the target cells for this FV-induced erythroid transformation. The greatest number of FV bursts were observed when marrow cells were obtained from mice whose erythropoiesis had been stimulated by bleeding or phenylhydrazine treatment. Bleeding also resulted in an increase in the number of FV bursts following the infection of spleen cells in vitro. Hypertransfusion of mice, which results in decreased erythropoiesis, yielded a reduced number of FV bursts in vitro, as did prior treatment with actinomycin D. Cell separation studies using velocity sedimentation at unit gravity showed that the cells, which give rise to FV bursts, sedimented with a modal sedimentation velocity between 5.1–8.5 mm/hr. The Epo-dependent colony-forming unit erythroid (CFU-E), which gives rise to a single erythroid colony, also sediments with a modal velocity between 5.1–8.5 mm/hr, while the Epo-dependent day 8 burst-forming unit erythroid (day 8 BFU-E) sediments with a modal velocity between 3.0–6.0 mm/hr. A 20 min incubation of marrow cells with high specific activity ³H-thymidine, prior to virus infection, resulted in a 75–80% reduction in the number of FV bursts. Mixing cells from the upper portion of the gradient, which yielded no FV bursts, with cells from an area in which high numbers of FV bursts were observed did not result in the inhibition of burst formation. These experiments indicate that the primary target cells for FV bursts in vitro are most probably erythroid precursor cells that have matured beyond the day 8 BFU-E and are closely related to the CFU-E.     

Diagnosis of Shiga toxin producing Escherichia coli infection, contribution of genetic amplification technique

There has been no culture method of choice for detecting non-O157 Shiga toxin-producing Escherichia coli strains (STEC) because of their biochemical diversity The aim of this study was the assessment of verotoxin gene detection (VT1/VT2) within STEC PCR compared with the Vero cells cytotoxicity among O157 and non-O157 STEC serotypes. Stool cultures were performed on Tryptic Soy Broth and sorbitol MacConkey agar with cefixitime and tellurite supplements which were identified as Escherichia coli (E. coli) by BBL crystal. Further identifications were performed including verotoxin production assessment by Vero cells cytotoxicity assay, PCR for specific VT1/VT2 genotyping, and isolates were plated on blood agar and tested for enterohemolysis. Vero cells cytotoxicity assay revealed that 58 of E. coli isolates (71.6%) were STEC. In PCR, 33 (56.9%) of the 58 strains were positive for the VT2 gene, 24 (41.4%) were positive for the VT1 gene and one isolate was positive for both genes. In comparison to Vero cells cytotoxicity, the sensitivity, specificity of PCR were 100%. In comparative study between verotoxin assessment by Vero cells cytotoxicity and enterohemolytic activity, concordance positive results between both were 53 (91.4%). The most common serogroups of STEC were O157 (33%) and O26 (20%). From this study we can conclude that enterohemolysin production can be used as surrogate marker for STEC. The most rapid and promising approach for detection of STEC is by molecular method.     

Effect of erythrocyte breakdown products on mast cells and erythropoietin formation]

Experiments were conducted on CBA mice and albino rats. A study was made of the effect of erythrocyte destruction products (EDP) on the content of hemopoietic colony-forming units (CFU), differentiation of stem cells and the erythropoietin production. It was shown that 3 or 4 EDP injections to normal mice or to lethally irradiated (1000 rad) mice after the transplantation of bone marrow cells caused no changes in the CFU level of stem cells differentiation. In case of a daily (for 3 days) administration of EDP to mice before the irradiation (1000 rad) and bone marrow transplantation there was observed an increase of the colonies count in the recipients' spleen on account of the erythroid colonies. EDP injection caused no changes in the erythropoietic activity of the blood serum. A possible role of erythrocyte destruction products in the mechanism of erythropoiesis autoregulation is discussed.     

Cyclin A2 regulates erythrocyte morphology and numbers

Cyclin A2 is an essential gene for development and in haematopoietic stem cells and therefore its functions in definitive erythropoiesis have not been investigated. We have ablated cyclin A2 in committed erythroid progenitors in vivo using erythropoietin receptor promoter-driven Cre, which revealed its critical role in regulating erythrocyte morphology and numbers. Erythroid-specific cyclin A2 knockout mice are viable but displayed increased mean erythrocyte volume and reduced erythrocyte counts, as well as increased frequency of erythrocytes containing Howell-Jolly bodies. Erythroblasts lacking cyclin A2 displayed defective enucleation, resulting in reduced production of enucleated erythrocytes and increased frequencies of erythrocytes containing nuclear remnants. Deletion of the Cdk inhibitor p27^Kip1 but not Cdk2, ameliorated the erythroid defects resulting from deficiency of cyclin A2, confirming the critical role of cyclin A2/Cdk activity in erythroid development. Loss of cyclin A2 in bone marrow cells in semisolid culture prevented the formation of BFU-E but not CFU-E colonies, uncovering its essential role in BFU-E function. Our data unveils the critical functions of cyclin A2 in regulating mammalian erythropoiesis.     

Capping and receptor-mediated endocytosis of cell-bound verotoxin (shiga-like toxin) 1: Chemical identification of an amino acid in the B subunit necessary for efficient receptor glycolipid binding and cellular internalization

The glycolipid globotriaosylceramide (Gb₃) is the plasma membrane receptor that mediates the internalization of verotoxin (VT1) into susceptible cells by capping and receptor-mediated endocytosis (RME). Internalization of fluorescein isothiocyanate-conjugated holotoxin into Daudi lymphoma cells was found to be slower than the pentameric receptor binding B subunit alone, suggesting that the A subunit may interact with the membrane to compromise the lateral mobility of the receptor bound B subunit. 3-D reconstruction of fluorescent images by confocal microscopy confirmed the complete internalization of holotoxin. VT1 internalization and cytotoxicity was inhibited by monodansyl cadavarine, which supports a role for clathrin coated pits in the RME of VT1. Biotinylation of the B subunit (in contrast to fluorescein labelling) was found to prevent toxin internalization. This effect correlated with reduced binding of Gb₃ and reduced cytotoxicity in vitro. By cleavage of the B subunit at the single tryptophan residue, the reduced Gb₃ binding and lack of cellular internalization was shown to be due to the biotinylation of lysine 53 in the VT1 B subunit. This residue was not labelled with fluorescein isothiocyanate in the native protein. This conclusion was confirmed by the finding that biotinylation of VT2c (which contains lys 53) prevented glycolipid receptor binding, whereas biotinylation of VT2e (in which lys 53 is substituted by ile) had no effect. © 1994 Wiley-Liss, Inc.     

Inhibition of Vero cell cytotoxic activity in Escherichia coli O157:H7 lysates by globotriaosylceramide, Gb3, from bovine milk

In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.     

Induction of commitment of murine erythroleukemia cells (TSA8) to CFU-E with DMSO

The commitment of novel mouse erythroleukemic (MEL) cells (TSA8) to colony-forming units of erythroid (CFU-E) by dimethylsulfoxide (DMSO) was investigated. After exposure to the inducer in liquid culture, the cells were transferred to a semi-solid culture to examine their ability to form erythroid colonies which were dependent on erythropoietin. Exposure to DMSO for 2 days is optimum for CFU-E type colony formation and colonies induced in this manner are equivalent to CFU-E. The induction occurred in a synchronous manner. Partly stained colonies appeared prior to CFU-E formation and are thought to be a result of asymmetric cell division. Appearance of these partly stained colonies suggested that the number of erythropoietin receptors is important in the complete responsiveness to erythropoietin. TSA8 cells constitute a suitable model system in which to analyse the mechanism of commitment in early erythropoiesis.     

A quantitative study on the recovery of rat marrow erythropoietic cells after experimental suppression of erythropoiesis by repeated doses of cyclophosphamide

Rat erythropoiesis was experimentally depressed by repeated doses of cyclophosphamide (CY) or by restriction of food consumption and the diagnostic value of quantitative haematology examinations of rat marrow erythroid morphology was evaluated. A slight depression of rat erythropoiesis (following a dose of 5 mg/kg/day) as well as a higher one (15 mg/kg/day) and the following recovery were determined by a quantitation of marrow erythroid morphology and by peripheral reticulocyte counts, yet a lower validity of peripheral erythrocyte and marrow relative erythroid cell counts was obtained; erythrocyte counts did not change even after a high depletion of marrow erythroid cells. The restriction of food consumption led to a similar depression of rat marrow erythropoiesis as that observed after CY administration. Our data may suggest that the quantitation of marrow erythroid morphology and peripheral reticulocyte counts are desirable in haematological examinations in preclinical safety evaluations performed on rats provided a new drug administered in repeated doses can be cytotoxic.     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo

The mitochondrial transporter ATP binding cassette mitochondrial erythroid (ABC-me/ABCB10) is highly induced during erythroid differentiation by GATA-1 and its overexpression increases hemoglobin production rates in vitro. However, the role of ABC-me in erythropoiesis in vivo is unknown. Here we report for the first time that erythrocyte development in mice requires ABC-me. ABC-me-/- mice die at day 12.5 of gestation, showing nearly complete eradication of primitive erythropoiesis and lack of hemoglobinized cells at day 10.5. ABC-me-/- erythroid cells fail to differentiate because they exhibit a marked increase in apoptosis, both in vivo and ex vivo. Erythroid precursors are particularly sensitive to oxidative stress and ABC-me in the heart and its yeast ortholog multidrug resistance-like 1 have been shown to protect against oxidative stress. Thus, we hypothesized that increased apoptosis in ABC-me-/- erythroid precursors was caused by oxidative stress. Within this context, ABC-me deletion causes an increase in mitochondrial superoxide production and protein carbonylation in erythroid precursors. Furthermore, treatment of ABC-me-/- erythroid progenitors with the mitochondrial antioxidant MnTBAP (superoxide dismutase 2 mimetic) supports survival, ex vivo differentiation and increased hemoglobin production. Altogether, our findings demonstrate that ABC-me is essential for erythropoiesis in vivo.     

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.     

Indicators of erythrocyte formation and degradation in rats with either vitamin A or iron deficiency

Vitamin A deficiency produces anemia and altered iron status. In this study with rats we tested two hypotheses regarding vitamin A deficiency: (1) that it impairs erythropoiesis, leading to an increased red cell turnover, and (2) that it inhibits the glycosylation of transferrin. Erythropoietic activity was assessed indirectly by determining the myeloid:erythroid ratio in bone marrow smears, the number of erythroid colonies in the red pulp of spleen, the blood reticulocyte index, and zinc protoporphyrin and plasma transferrin receptor concentrations. Transferrin glycosylation was assessed by measuring the sialic acid content of transferrin. The effects of vitamin A deficiency were compared with those of iron deficiency. Iron deficiency produced anemia and low iron levels in organs. Vitamin A deficiency produced low levels of plasma and hepatic retinol, and it induced decreased plasma total iron-binding capacity and raised iron levels in tibia and spleen. Short- but not long-term iron deficiency reduced the number of erythroid colonies in spleen; vitamin A deficiency had no influence. Neither iron nor vitamin A deficiency influenced the myeloid:erythroid ratio in bone marrow smears and the blood reticulocyte production. Plasma transferrin receptor and erythrocyte zinc protoporphyrin concentrations were not affected by vitamin A deficiency but increased with iron deficiency. Vitamin A deficiency did not stimulate erythrocyte breakdown, as indicated by unaltered plasma lactate dehydrogenase activity and reduced plasma total bilirubin levels. Both vitamin A and iron deficiencies raised the proportion of multiple sialylated transferrins in plasma. Thus, we have not found evidence that vitamin A deficiency affects erythropoiesis and erythrocyte turnover. The iron accumulation in spleen and bone marrow may be related to reduced iron transport due to inhibition of transferrin synthesis rather than inhibition of transferrin sialylation.     

Inhibitory effect of intestinal anti-Gb3 IgA antibody on verotoxin-induced cytotoxicity

The effect of intestinal IgA antibody against the receptor for verotoxin (VT), globotriaosylceramide (Gb3), on VT-mediated cytotoxicity was examined. Intestinal IgA antibodies against Gb3 were prepared by oral immunization of mice with Gb3 and adjuvant monophosphoryl lipid A (MPL)-containing liposomes composed of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylserine and cholesterol (1 : 1 : 2, molar ratio) (PS-liposome). Oral administration with Gb3 and MPL-containing PS-liposome induced significant IgA responses to Gb3 in the intestinal lavage fluid in all mice tested. Furthermore, anti-Gb3 IgA antibodies in the lavage fluid effectively inhibited the cytotoxicity of VT2 to Vero cells in a dose-dependent manner. These results suggest that anti-Gb3 IgA antibodies produced in the intestinal tract, upon oral immunization with Gb3-containing liposome, function as inhibitors against VT and also indicate the potential usefulness of oral PS-liposome vaccines containing MPL for the induction of a protective mucosal immune response against intestinal diseases.     

STUDIES OF HEMATOPOIESIS IN MURINE SPLEENS: SIGNIFICANCE OF HEMATOPOIETIC COLONIES FORMED ON THE SURFACE OF SPLEENS

Conies of hematopoietic tissue are formed in spleens of lethally irradiated mice by the injection of small numbers of hematopoietic cells. Some of these colonies appear as surface colonies, others can be identified only in serial sections of the spleen. The present studies have related the number and cellular composition of total hematopoietic colonies in the murine spleen to their visual recognition on the splenic surface. These studies demonstrate that only 50% of the total colonies in a spleen are recognized as surface colonies and that of those colonies on the surface, approximately 80% contain erythroid elements. At least four factors play important roles in the recognition of hematopoietic colonies as splenic surface colonies: (1) dose of repopulating cells or hematopoietic stem cells injected into the irradiated animal; (2) location of colonies within the spleen; (3) size of colonies; and (4) cellular content of the colonies. These studies demonstrate that surface colony formation reflects primarily erythropoiesis and not total hematopoiesis.     

Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: relevance to the cell-lineage models of globin switching

The cellular control of the switch from embryonic to fetal globin formation in man was investigated with studies of globin expression in erythroid cells of 35- to 56-day-old embryos. Analyses of globins synthesized in vivo and in cultures of erythroid progenitors (burst-forming units, BFUe) showed that cells of the yolk sac (primitive) erythropoiesis, in addition to embryonic chains, produced fetal and adult globins and that cells of the definitive (liver) erythropoiesis, in addition to fetal and adult globins, produce embryonic globins. That embryonic, fetal, and adult globins were coexpressed by cells of the same lineage was documented by analysis of globin chains in single BFUe colonies: all 67 yolk sac-origin BFUe colonies and 42 of 43 liver-origin BFUe colonies synthesized epsilon-, gamma-, and beta-chains. These data showed that during the switch from embryonic to adult globin formation, embryonic and definitive globin chains are coexpressed in the primitive, as well as in the definitive, erythroid cells. Such results are compatible with the postulate that the switch from embryonic to fetal globin synthesis represents a time-dependent change in programs of progenitor cells rather than a change in hemopoietic cell lineages.     

Stimulation of erythropoiesis in teratocarcinoma cells by erythropoietin and “Friend inducers”

Murine teratocarcinoma cells (PCC3/A1) formed erythroid cells in the form of blood islands when they were grown in organ culture. Addition of dimethyl sulfoxide (DMSO), N′N-dimethylacetamide and erythropoietin enhanced the formation of blood islands. An additive stimulatory effect was observed when expiants were incubated with DMSO and erythropoietin. In all of these cultures, the formed erythroblasts showed the characteristics of primitive erythroid cells, regardless of the nature of treatment. Small, enucleated red cells were occasionally observed. These results are compared with the characteristics of erythropoiesis in normal adults, embryos and in murine erythroleukemia.     

Zidovudine (AZT) treatment suppresses granulocyte-monocyte colony stimulating factor receptor type alpha (GM-CSFR alpha) gene expression in murine bone marrow cells

In vitro exposure of murine bone marrow cells to increasing concentrations of zidovudine (AZT, 0.1-50 microM) had a concentration dependent suppressive effect on the growth of granulocyte-monocyte colony forming unit (CFU-GM) derived colonies. In our previous published study, the mechanism of AZT-induced suppression of erythroid colony forming unit (CFU-E) derived colonies was linked to a decrease in erythropoitin receptor (Epo-R) gene expression. In this study, we have observed that AZT exposure also induced a concentration dependent suppressive effect (35-90%) on GM-CSF receptor type alpha (GM-CSFR alpha) gene expression. The suppression of GM-CSFR alpha mRNA expression was specific, since AZT caused a much lower decrease (15-22%) on the IL-3 receptor type alpha (IL-3R alpha) message level, and had an insignificant effect on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and c-myc message levels. Erythropoietin (Epo) therapy has been used for reversal of AZT induced erythroid toxicity. Exposure to increasing concentrations (10-500 U/ml) of GM-CSF was unable to override the suppressive effect of AZT on CFU-GM derived colonies, however, treatment in combination with IL-3 (10-250 U/ml) ameliorated the suppressive effects of AZT on CFU-GM and on GM-CSFR alpha and IL-3R alpha gene expression. These findings suggest a mechanism via which AZT may suppress granulocyte-monocyte specific differentiation in murine bone marrow cells. These data also suggest that a combination of GM-CSF and IL-3 may be a superior therapeutic intervention for AZT-induced neutropenia.