Inhibition of erythropoiesis by a soluble factor in murine malaria.

To study the cellular mechanisms involved in the ineffective erythropoiesis associated with malaria, an in vitro proliferative assay was used to measure the response to erythropoietin (Epo) of erythroid progenitor cells from malaria-infected mice. In this assay, spleen (SP) cells from phenylhydrazine (PHZ)-treated mice (PHZ-SP), enriched for erythroid progenitor cells, respond to Epo in a dose-dependent manner. Despite a similar degree of anemia, SP and bone marrow (BM) cells from Plasmodium berghei- or P. vinckei-infected mice did not show a significant response to Epo in this assay. When SP or BM cells from malaria-infected mice were added to cultures of SP or BM cells from PHZ-treated mice the response to Epo of these cells was significantly inhibited. Removal of parasitized red blood cells (pRBC) from SP cells of P. berghei-infected mice had no effect on the ability of the cells to inhibit the response to Epo. Adherent SP cells and SP cells positive for the Mac-1 antigen, from malaria-infected mice, were shown to be enriched for cells that could inhibit the response to Epo. Cell-free conditioned media (CM) prepared from SP cells of P. berghei- or P. vinckei-infected mice or from normal SP cells incubated with pRBC were also able to inhibit the response to Epo of SP cells from PHZ-treated mice. These investigations have shown that during the course of malaria infection, cells appear in the SP and BM capable of inhibiting, via soluble mediators, the response to Epo of erythroid progenitor cells. The cells responsible are probably macrophages. The nature of the factor(s) and its mechanism of action are not known. Through the ability to inhibit erythropoiesis, soluble factors may, in part, mediate the anemia associated with malaria.

Inhibition by interleukin-1 of the action of erythropoietin on erythroid precursors and its possible role in the pathogenesis of hypoplastic anaemias.

Highly purified and cloned preparations of interleukin-1 (IL-1) were found to antagonize the capacity of erythropoietin (Epo) to stimulate the proliferation of mouse spleen and bone marrow erythroid precursor cells (EPC) in culture. Cloned murine IL-1 and purified and cloned human IL-1 alpha and IL-1 beta were approximately equipotent in this assay. IL-1 inhibited the proliferation response of EPC even when added as long as 17 h after Epo, suggesting that IL-1 does not affect binding of Epo to receptors or biochemical events following shortly thereafter. Indomethacin did not influence the inhibitory effect of IL-1 on Epo-induced proliferation, and PGE2 had no demonstrable effect on the process. Tumor-necrosis factor-alpha and interferons beta 1, and gamma did not affect Epo-induced proliferation. It is suggested that IL-1 mediated antagonism of the effects of Epo on erythroid precursors is a factor in the pathogenesis of many types of hypoplastic anaemia, including those associated with infections, rheumatoid arthritis and systemic lupus erythematosus, giant-cell arteritis, graft-versus-host disease and disorders associated with lymphocyte-mediated suppression of erythropoiesis.

Murine malaria decreases hemopoietic stem cells.

The causes of anemia and immunosuppression, major outcomes of malaria, are not well established. This study was undertaken to investigate whether erythropoietin (EP) production is adequate and whether the hemopoietic stem cells (CFU-S) were affected during the course of infection. Groups of female Balb/c mice infected with Plasmodium vinckei vinckei, Plasmodium berghei, or Plasmodium chabaudi adami were exposed to five hours of simulated altitude equivalent to 22,000 ft. Plasma samples were collected for EP bioassay and radioimmunoassay (RIA). Using radioiron incorporation as an index of erythropoiesis, differences in response to infection with different species of plasmodia were observed. In general, decreases in erythropoietic activity were observed in bone marrow and spleen as the infection progressed and continued to be depressed after apparent resolution of a nonlethal infection with P. chabaudi adami. Marrow from infected and control femurs were tested for CFU-S content using the spleen colony assay. The cellularity and CFU-S content of the femoral marrow decrease as the parasitemia increases. All three species of plasmodia stimulate EP production during peak parasitemias, indicating that adequate amounts of EP are available to the erythron during malarial infection. Depletion of CFU-S and probable lack of compensatory turnover of CFU-S may contribute to the disease characteristics of malaria.

Neuraminidase increases DNA synthesis of spleen cells induced by native and asialylated erythropoietin.

The uptake of 3H-thymidine by a suspension of spleen cells, obtained from mice made anemic by phenylhydrazine injections, is increased above the values obtained with native human or mouse erythropoietin (Ep) if the hormone is enzymatically asialylated. Preparations of human Ep asialylated by mild acid hydrolysis stimulated DNA synthesis to a lesser extent than the native hormone; however, when neuraminidase was added to the culture medium, the stimulation exhibited by the asialylated Ep was not significantly different from the stimulation shown by the native hormone. The DNA synthesis of these cells is increased even more if neuraminidase and the asialylated hormone are added to the cell suspension. The asialylated Eps are not biologically active in vivo. The sialic acid moieties of Ep and of the cells responding to Ep are involved in the in vitro response of spleen cells to the hormone, possibly by making Ep receptors available to interact with the hormone.

Growth of murine bone marrow adherent stromal cells in culture without hydrocortisone in a low oxygen environment.

Successful culture of hematopoietic cells depends upon the growth of stromal cells. The growth of bone marrow stromal cells is best achieved when hydrocortisone is added to the culture medium. However, the hydrocortisone requirement is eliminated if the cultures are grown in an atmosphere of 5% CO2, 5% O2, and 90% N2 rather than 5% CO2 in air, which is more common.

Serum lipid and lipoprotein concentrations following exposure to ozone.

The effects of exposure to ozone (O3) on concentrations of serum lipids and lipoproteins were investigated. Male and female guinea pigs were exposed to O3 at 1 ppm for two weeks. Serum concentrations of cholesterol, triglycerides, low density (LDL) and very low density (VLDL) lipoproteins were elevated after O3 exposure, particularly in males. During O3 exposure the food intake per day decreased (for a constant body weight), suggesting that metabolic rate and possibly basal metabolic rate was lower. Lung wet weights increased during O3 exposure by 87% for males and 45% for females. When individual lung weight/body weight ratios were correlated with cholesterol and LDL values from the same animal, a high correlation is found for males (r = 0.81, P less than 0.05), suggesting that there may be a relationship between lipoprotein elevations and lung damage for males. Because elevated concentrations of lipids and lipoproteins in humans increase the risk of coronary heart disease (CHD), the lipoprotein results suggest that an epidemiological study of the incidence of CHD with metropolitan O3 levels may be warranted.

Response of colony-forming units-spleen to heavy charged particles.

Survival of colony-forming units-spleen (CFU-S) was measured after single doses of photons or heavy charged particles from the BEVALAC. The purposes were to define the radiosensitivity to heavy ions used medically and to evaluate relationships between relative biological effectiveness (RBE) and dose-averaged linear energy transfer (LET infinity). In in vitro irradiation experiments. CFU-S suspensions were exposed to 220 kVp X rays or to 20Ne (372 MeV/micron) or 40Ar (447 MeV/micron) particles in the plateau portion of the Bragg curve. In in vivo irradiation experiments, donor mice from which CFU-S were harvested were exposed to 12C (400 MeV/micron). 20Ne (400 or 670 MeV/micron), or 40Ar (570 MeV/micron) particles in Bragg peaks spread to 4 or 10 cm by spiral ridge filters. Based on RBE at 10 survival, the maximum RBE of 2.1 was observed for 40Ar particles characterized by an LET infinity of approximately 100 keV/micron. Lower RBEs were determined at lower or higher estimated values of LET infinity and ranged from 1.1 for low energy 40Ar particles to 1.5-1.6 for low energy 12C and 20Ne. The responses of CFU-S are compared with responses of other model systems to heavy charged particles and with the reported sensitivity of CFU-S to neutrons of various energies. The maximum RBE reported here, 2.1 for high energy 40Ar particles, is somewhat lower than values reported for fission-spectrum neutrons, and is appreciably lower than values for monoenergetic 0.43-1.8 MeV neutrons. Low energy 12C and 20Ne particles have RBEs in the range of values reported for 14.7 MeV neutrons.

Comparison of the effectiveness of bone marrow and spleen stem cells for platelet repopulation in lethally irradiated mice.

To determine whether stem cells from spleen differ from those of bone marrow in their ability to support platelet repopulation after lethal irradiation we compared normal, splenectomized and plethoric (3 weeks exposure to CO) spleen cell recipients with similar groups of mice reconstituted with comparable numbers of bone marrow stem cells. Female LAF1 mice were given 825-950 R from a 60Co source and were transplanted with 0, 0.5, 1, 2, or 4 x 10(6) bone marrow or 10-64 x 10(6) spleen cells. CFUs content of the transplant was calculated from day 9 splenic nodules in mice receiving 5 x 10(4) bone marrow or 1.0-1.6 x 10(6) spleen cells. Blood volumes, platelet counts and hematocrits were determined on day 12. Total circulating platelets increased with increasing stem cell dose (range 50-1000 CFUs) after both types of transplant. Plethoric mice always had lower platelet levels than controls even when corrected for expanded blood volume. There was little difference between values for normal and splenectomized mice. Platelet production per CFUs in all groups except splenectomized spleen cell recipients declined with large transplants presumably because of feedback inhibition. Although the number of splenic megakaryocytes in spleen cell recipients was approximately 2.5 times as great as in bone marrow recipients, platelet levels were significantly higher only in normal and splenectomized mice receiving more than 300 CFUs. No differences were found between the two types of transplant when plethoric hosts were used.

Erythropoietin complexed with the F(ab')2 fragment of anti-erythropoietin retains its biologic activity.

Evidence is presented that the F(ab')2 fragment of rabbit anti-erythropoietin forms immune complexes with erythropoietin. The in vivo biologic activity of the hormone remains when complexed to the antibody fragment, whereas the hormone has no in vivo biologic activity when complexed with the intact antibody. It is concluded that the effector functions of the Fc fraction of the antibody molecule play an important role in the ability of the intact antibody to neutralize the in vivo biologic activity of the hormone. It is proposed that this role may relate to differences in the rate of catabolism of the different antibody-hormone complexes. It is also concluded that the biologically active sites of the hormone and its antigenic determinants do not correspond.

Decreased surface charge and accelerated senescence of red blood cells following neuraminidase treatment.

Female LAF1 mice were given single or repeated injections of V. cholerae N'ase and the effects on circulating RBC surface charge and life span were determined. Intravenous injection of N'ase caused a rapid decrease in RBC surface charge of approximately 14 percent, and survival of such treated cells was reduced by approximately one fifth by virtue of an acceleration of senescence. When RBC's were treated in vitro with N'ase, a comparable (14 to 17 percent) reduction in surface charge was seen. Such cells, when injected into intact mice, showed a similar acceleration of senescence. When N'ase was injected intravenously into splenectomized mice, RBC survival was similar to that of controls. Intravenous injection of N'ase 1 hour before injection of labeled RBC's did not alter RBC survival nor did it accelerate the clearance of carbon particles by the RES. These results indicate that N'ase accelerates senescence in treated mouse erythrocytes by acting on the RBC's and not by activating the RES. Absence of this effect in splenectomized mice implicates the spleen as the sensor of the induced alterations in surface charge. These results and those recently reported for treated RBC's in the dog, rat, rabbit, and man suggest that at least a portion of the phenomenon of RBC senescence may be related to the loss of RBC surface charge.

Adenosine, AMP, cyclic AMP, theophylline and the action and production of erythropoietin.

Erythropoiesis, as measured by the uptake of 59Fe into plethoric mice, is stimulated by adenosine, AMP, cyclic AMP, and dibutyryl cyclic AMP, but not by cytidine, its nucleotides or cyclic GMP. This stimulation is erythropoietin dependent, because it is prevented by anti-erythropoietin. Theophylline neither stimulates erythropoiesis nor potentiates the action of erythropoietin on bone marrow cells in plethoric mice. Theophylline does potentiate the production of erythropoietin in rats following a frief hypoxic exposure but does not cause a similar increase in mice.