John D. Lutton

Suppression of Erythroid-Colony Formation by Lymphocytes from Patients with Aplastic Anemia

To explore the possible role of cell-mediated suppression of erythropoiesis in acquired aplastic anemia, we studied the effect of peripheral blood lymphocytes from seven patients with aplastic anemia on erythroid-colony formation by normal human bone marrow in an in vitro plasma clot-culture system. Varying numbers of peripheral blood lymphocytes (0.5 to 6 X 10(5) cells) were cocultured with 6 X 10(5) normal bone-marrow cells in the presence of 2 IU of erythropoietin for seven days. Peripheral blood lymphocytes from five of the seven patients with aplastic anemia caused a 79 to 94 per cent reduction of erythroid-colony formation. Thus, at a peripheral blood lymphocyte concentration of 2 X 10(5) cells, the numbers of erythroid colonies formed (control vs. experimental) were 374 +/- 16 (S.E.M.) vs. 22 +/- 6, 48 +/- 4, 50 +/- 16, 80 +/- 4 and 27 +/- 3. These results suggest that some patients with aplastic anemia possess a population of lymphocytes capable of suppressing in vitro erythropoiesis.

Erythroid colony formation by polycythemia vera bone marrow in vitro. Dependence on erythropoietin.

In the plasma clot culture system both normal and polycythemia vera (PV) bone marrow cells respond to erythropoietin (Ep), giving rise to large numbers of colonies of erythroid cells. In PV, but not in normal individuals, the marrow produced endogenous erythroid colonies (EED) in the absence of exogenous Ep. The number of EEC formed varied from patient to patient comprising anywhere from 6 to 29% of the total number of colonies formed in the presence of Ep. Exposure, before use in culture, of fetal calf serum and citrated bovine plasma to the gammaglobulin fraction of rabbit anti-Ep serum followed by treatment with goat anti-rabbit gamma-globulin re sulted in a significant decrease in EEC formation. Addition of anti-Ep directly to the culture medium produced similar results. In addition, the production of EEC in response to added Ep was inhibited in the presence of anti-Ep. Addition of very small doses of highly purified Ep to anti-Ep-treated cultures resulted in the reappearance of a significantnumber of EEC formation in PV may be due to a population of erythroid-committed precursors that are abnormally sensitive to small concentrations of Ep which may be present in fetal calf serum and citrated plasma. Although the mechanism of formation of these cells is not known, it appears that the final steps in the formation of red cells derived from this clone of precursors is subject to the usual Ep control.

The role of haem biosynthetic and degradative enzymes in erythroid colony development: the effect of haemin

Summary. In vitro culture of murine bone marrow has proved to be a useful system for defining the haem biosynthetic and degradative enzymatic pathways during erythroid colony (CFU‐E) growth and development. Previous attempts to elucidate these pathways during erythropoiesis have been limited by the amount of biological material available as well as the sensitivity of specific enzyme assays. These conditions were overcome in the present study. In order to obtain an enrichment of CFU‐E, nonadherent mouse bone‐marrow cells were cultured in special culture plates using a modified methyl‐cellulose medium with erythropoietin. CFU‐E yields were increased approximately threefold. Utilizing the sensitive radiochemical assay, direct measurement of ALAS activity was determined in very early developing CFU‐E cultures as well as mature cultures. ALAS activity was found to reach a peak after 60 h of culture growth and then started to decline in activity. Cellular synthesis of haem was determined (with 14C ALA) and minor modifications of standard assays were also made in order to determine spectrophotometrically δ‐aminolaevulinic acid dehydratase (ALAD) and haem oxygenase activity in developing CFU‐E cultures. These assays were reproducible with as few as four or five culture plates. It was found that ALAD activity rose progressively after 36 h of culture growth and reached a plateau at about 60 h of growth. A continuous increase in 14C ALA incorporation into haem was seen at later hours of culture growth, suggesting that the induction of other haem enzymes beyond ALAS is still rate limiting in haem synthesis. Conversely, haem oxygenase activity declined up to 60 h of growth and was elevated at later culture periods without a subsequent increase in ALASA and ALAD. The observed increase in haem enzymes brought about by haemin was completely suppressed by addition of cycloheximide to the cultures. These results suggest that

The toxic effects of heavy metals on rat bone marrow in vitro erythropoiesis: Protective role of hemin and zinc

The effects of gold (Au), lead (Pb), and cadmium (Cd) on rat bone marrow in vitro erythropoiesis (CFUE) were studied. Au was found to be significantly toxic to CFUE growth at concentrations as low as 10(-9) M whereas Pb and Cd displayed toxicity at 10(-7) M. Addition of Pb plus Cd in combination had a greater toxic effect on CFUE growth than when the metals were added singly, and the toxic effect of Cd was reduced when Zinc (Zn) was added in combination to the cultures. When hemin (10(-6) M) was added to cultures containing 10(-6) M Au, Pb, or Cd, CFUE numbers were obtained that were equivalent to control cultures without hemin. Thus, hemin exerted a protective effect on erythropoiesis in the presence of otherwise toxic amounts of Au, Pb, and Cd. It is concluded that Au, Pb, and Cd have toxic effects on in vitro erythropoiesis and that this toxicity may be overcome in part by Zn or hemin. The possible involvement of the heme biosynthetic and degradative pathways is discussed with respect to these results.

Mechanisms of differentiation of U937 leukemic cells induced by GM-CSF and 1,25(OH)2 vitamin D3

The human monoblast cell line, U937, was employed to elucidate early events associated with differentiation induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1,25-dihydroxy-Vitamin D3 (VD3). Exposure of cells to a combination of GM-CSF and VD3 resulted in an up-regulation of c-fos mRNA within 1 h and a marked down-regulation of c-myc mRNA by 24 h and this was associated with a shift of cell population from the S phase to the G0 + G1 phase of the cell cycle by 18%. This was followed by a marked enhancement of monocyte-associated cell surface antigens [OKM1 (CD11b), LeuM3 (CD14), M77.7], as determined by monoclonal antibodies and flow cytometry. Functional characteristics such as nitroblue-tetrazolium reduction, alpha-naphthyl butyrate esterase activity, and phagocytic capability occurred. Cells treated with GM-CSF or VD3 alone showed only minor changes. These results demonstrate a potent synergistic effect of GM-CSF and VD3 on induction of U937 differentiation. This differentiation was partially blocked by H7, a protein kinase C (PKC) inhibitor. Changes in c-myc and c-fos mRNA expressions and a shift in cell cycle were shown to be early events in this process.

Inhibition of Human Adult and Fetal Heme Oxygenase by New Synthetic Heme Analogues

Heme oxygenase (HO) is the rate-limiting enzyme for heme degradation, and elevated levels of HO may be associated with a variety of pathologic disturbances. A limited number of HO inhibitors such as the metallo-porphyrins have been proposed as possible chemotherapeutic agents for the treatment of hyperbilirubinemia. We undertook the study of various natural newly synthesized heme analogues as possible inhibitors of HO in human adult and fetal liver microsomes. We investigated two compounds with substitutions at the 2 and 4 position of the porphyrin ring, iron deuteroporphyrin 2,4 disulfonic (1a) and iron deuteroporphyrin 2,4 bis glycol (1b), and two compounds with substitutions of aromatic groups on the methene bridges of the porphyrin molecule, meso-tetra-4-carboxyphenyl-porphine (2a) and meso-tetra-4-sulfonatophenyl-porphine (2b). When these heme analogues were incubated in the reaction media in the presence of heme, two of the analogues (1a) and (1b) inhibited the conversion of heme to bilirubin. This inhibition was 97% and 65% respectively for (1a) and (1b) when both were present in 30 μM concentrations. Both of these compounds exhibited competitive type inhibition. The kI for the more potent inhibitor, (1b), was determined to be 0.30 μM. Porphyrins with aromatic substitutions at the methene bridges (2a, 2b) did not inhibit the conversion of heme to bilirubin, even at relatively high concentrations. Furthermore, the specific activity of HO was significantly greater (5×) in fetal microsomes as contrasted with adult microsomes. Even though fetal microsomes had greater HO activity, 5 μM of compound (1b) caused a similar degree of inhibition in both adult and fetal preparations. These studies represent the discovery of a new class of inhibitors of HO, distinct in structure from known inhibitors, the metallo-porphyrins. These compounds thus represent potentially novel drugs in the treatment of hyperbilirubinemic states or research tools in understanding mechanisms of heme oxidation.

Adenovirus Mediated Alpha Interferon (IFN‐α) Gene Transfer into CD34+ Cells and CML Mononuclear Cells

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Interferon‐α (IFN‐α) has been used in the management of leukemia but its diverse adverse activities with multiple potential side effects, possibly unrelated to therapeutic targets, may negatively influence the ability of IFN‐α to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)‐IFN‐α gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN‐α in these cells. Ad‐cytomegalovirus promoter driven IFN‐α (AdCMV‐IFN‐α) at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture, and optimal transduction of CD34+ cells was achieved using 120 plaque forming units. Flow cytometric determinations revealed that there was no significant difference in cell viability for the 4 h or 24 h transfection periods. Immunoassay of IFN‐α produced by CD34+ cells shows that IFN‐α levels increased several fold in transfected cells. Transient expression of the IFN‐α gene did not suppress proliferation of CD34+ progenitors as indicated by BFU‐E or colony forming units‐granulocyte‐macrophage (CFU‐GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34+ harvested CFU‐GM progenitor cells demonstrated transient IFN‐α mRNA expression. Similarly, CML BMMNC were transfected with AdCMV‐IFN‐α under similar conditions as described for CD34+ cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN‐α at a substantial level. This in vitro data suggest that Ad‐mediated gene transfer of IFN‐α into hematopoietic stem cells can be achieved and that the IFN‐α gene can be translated into its specific mRNA in CD34 progenitor cells.

Physiologic Role of Heme and Cytochrome P-450 in Hematopoietic Cells:

Heme (ferroprotoporphyrin IX) is a ubiquitous molecule that serves as the prosthetic group of a variety of important hemoproteins that are essential for hemopoietic processes. Heme is involved in oxygen transport as the prosthetic group of hemoglobin, in prostaglandin synthesis as the prosthetic group of cyclooxygenase, in the enzymatic decomposition of H2O2 as the prosthetic group of catalase and peroxidase, and in the inactivation of oxygen molecules, as the prosthetic group of mitochondrial and microsomal cytochrome P-450. The latter refers to a family of isozymes for which heme serves as the prosthetic group that oxidizes a wide variety of structurally unrelated compounds, inactivates leukotrienes (leukotriene B4), and metabolizes arachidonic acid (AA) to bioactive metabolites, some of which are involved in the signal transduction process for hematopoietic growth factors. However, the effect of heme on erythropoiesis appears to be distinct from its direct involvement as a prosthetic group. The expression of specific heme metabolic enzymes determines the level of cellular heme that is necessary for proper erythropoiesis. This concept is supported by evidence that hereditary or experimental alterations in enzymatic or biosynthetic events are often accompanied by a disturbance in heme levels, and that treatment with drugs or inhibitors of heme synthesis may affect progenitor cells, resulting in altered growth and differentiation (1). It is becoming increasingly evident that the role of heme metabolic enzymes in the regulation of hematopoiesis has a dual nature, since metabolic enzymes appear to participate in the implementation of both stimulation and suppression of erythropoiesis (1, 2). Enhancement of erythropoiesis obtained with the growth factors interleukin 3 and erythropoietin (Epo) also results in increased levels of δ-aminolevulinic acid synthase (ALAS) and porphobilinogen deaminase (PBGD), the proposed rate-limiting enzymes in the heme biosynthetic pathway.

Gene transfer of alpha interferon into hematopoietic stem cells

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. IFN-alpha has been used in the management of leukemia, and gene transfer of the IFN-alpha gene into hematopoietic progenitor cells may have great potential for the treatment of chronic myelogenous leukemia (CML). Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal bone marrow hematopoietic CD34+ stem cells and the production of IFN-alpha protein by these cells. Ad-cytomegalovirus (CMV) promoter-driven IFN-alpha at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture. Optimal transduction of CD34+ cells with the AdCMV-IFN-alpha construct was achieved using 120 plaque forming units (pfu). Flow cytometric determinations revealed that there was no significant difference in CD34+ cell viability for the 8 or 12-h transfection periods. Immunoassay of IFN-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells and this was not seen in CD34+ cells transfected with the heme oxygenase gene (HO-1). These in vitro data suggest that adenovirus-mediated gene transfer of IFN-alpha into hematopoietic stem cells can be achieved and that the IFN-alpha protein is produced by viable CD34 progenitor cells.

Comparative Effects of Heme and Metalloporphyrins on Interferon-γ-Mediated Pathways in Monocytic Cells (THP-1)

Abstract Previous results have demonstrated links between cell-mediated immunity, interferon (IFN)-γ and neopterin production with heme, porphyrins, and iron metabolism. In this study, we compared the effects of heme, several metalloporphyrins, protoporphyrin IX, and iron on the signal or IFN-γ-mediated pathways, such as the expression of major histocompatability complex class II antigens, neopterin formation, and the degradation of tryptophan. Using the human monocytic cell line, THP-1, we found that heme, Zn-mesoporphyrin, Zn-deuteroporphyrin, Co-protoporphyrin, and iron reduced the efficiency of the IFN-γ signal. In addition, Zn-mesoporphyrin almost fully inhibited IFN-γ-induced degradation of tryptophan by the heme protein, indoleamine 2,3-dioxygenase. In contrast, tin-protoporphyrin enhanced the IFN-γ effects as seen by increased neopterin production, enhanced tryptophan degradation, and elevated HLA-DR antigen expression on cells. These effects are considered to be due to the action of heme, metalloporphyrins, iron, or heme byproducts on the IFN-γ signal, rather than to direct effects on IFN-γ-induced enzymatic pathways. Heme and metalloporphyrins were previously shown to affect heme oxygenase activity, T cell growth, and lipid peroxidation and to modulate interleukin 2 activity. These pathways are also known to be influenced by IFN-γ, and our data suggest that heme and metalloporphyrins may directly modulate the efficiency of the IFN-γ signal.