Infection with Salmonella enterica Serovar Typhimurium Leads to Increased Proportions of F4/80+ Red Pulp Macrophages and Decreased Proportions of B and T Lymphocytes in the Spleen

Infection of mice with Salmonella enterica serovar Typhimurium (Salmonella) causes systemic inflammatory disease and enlargement of the spleen (splenomegaly). Splenomegaly has been attributed to a general increase in the numbers of phagocytes, lymphocytes, as well as to the expansion of immature CD71⁺Ter119⁺ reticulocytes. The spleen is important for recycling senescent red blood cells (RBCs) and for the capture and eradication of blood-borne pathogens. Conservation of splenic tissue architecture, comprised of the white pulp (WP), marginal zone (MZ), and red pulp (RP) is essential for initiation of adaptive immune responses to captured pathogens. Using flow cytometry and four color immunofluorescence microscopy (IFM), we show that Salmonella-induced splenomegaly is characterized by drastic alterations of the splenic tissue architecture and cell population proportions, as well as in situ cell distributions. A major cause of splenomegaly appears to be the significant increase in immature RBC precursors and F4/80⁺ macrophages that are important for recycling of heme-associated iron. In contrast, the proportions of B220⁺, CD4⁺ and CD8⁺ lymphocytes, as well as MZ MOMA⁺ macrophages decrease significantly as infection progresses. Spleen tissue sections show visible tears and significantly altered tissue architecture with F4/80⁺ macrophages and RBCs expanding beyond the RP and taking over most of the spleen tissue. Additionally, F4/80⁺ macrophages actively phagocytose not only RBCs, but also lymphocytes, indicating that they may contribute to declining lymphocyte proportions during Salmonella infection. Understanding how these alterations of spleen microarchitecture impact the generation of adaptive immune responses to Salmonella has implications for understanding Salmonella pathogenesis and for the design of more effective Salmonella-based vaccines.     

Role of the spleen in the exaggerated polycythemic response to hypoxia in chronic mountain sickness in rats.

In a rat model of chronic mountain sickness, the excessive polycythemic response to hypoxic exposure is associated with profound splenic erythropoiesis. We studied the uptake and distribution of radioactive iron and red blood cell (RBC) morphology in intact and splenectomized rats over a 30-day hypoxic exposure. Retention of (59)Fe in the plasma was correlated with (59)Fe uptake by both spleen and marrow and the appearance of (59)Fe-labeled RBCs in the blood. (59)Fe uptake in both the spleen and the marrow paralleled the production of nucleated RBCs. Splenic (59)Fe uptake was approximately 10% of the total marrow uptake under normoxic conditions but increased to 60% of the total marrow uptake during hypoxic exposure. Peak splenic (59)Fe uptake and splenomegaly occurred at the most intense phase of erythropoiesis and coincided with the rapid appearance of (59)Fe-labeled RBCs in the blood. The bone marrow remains the most important erythropoietic organ under both resting and stimulated states, but inordinate splenic erythropoiesis in this rat strain accounts in large measure for the excessive polycythemia during the development of chronic mountain sickness in chronic hypoxia.     

Novel virulence properties of the Salmonella typhimurium virulence-associated plasmid: immune suppression and stimulation of splenomegaly

Mice infected subcutaneously with wild-type Salmonella typhimurium, SR11, developed a significant splenomegaly when compared with mice infected with an equal number of a plasmid-cured strain. Further, the bacterial load in the spleen at 14 days after infection, measured as colony-forming units per gram tissue, was significantly higher in mice infected with the parent strain than in mice infected with the plasmid-cured strain. These data confirm the previously reported plasmid-associated ability of Salmonella to multiply within the spleen. In addition, lymph node cells (LNC) from mice infected with the parent strain had a significantly reduced ability to proliferate in response to concanavalin A, a T-cell mitogen, and to heat-killed S. typhimurium cells when compared with LNC isolated from mice infected with the plasmid-cured strain. Finally, reintroduction of a functional Tn5-tagged 90-kb plasmid into a plasmid-free strain restored its capacity to cause a marked splenomegaly and to suppress lymph node cell proliferation in BALB/c mice. These data demonstrate that the 90-kb plasmid of highly virulent S. typhimurium strains mediates several novel pathogenic properties in infected mice: (1) enhancement of the ability of Salmonella to multiply within the spleen; (2) stimulation of a splenic inflammatory response as displayed by marked splenomegaly; and (3) a general suppression of lymphocyte responsiveness to both T-cell mitogens and specific Salmonella antigens.     

Plasmodium berghei: immunosuppression of the cell-mediated immune response induced by nonviable antigenic preparations

In this work, plasmodial antigens were examined for their ability to suppress the cellular immune response during lethal Plasmodium berghei infection. Splenic enlargement and the number and function of white spleen cells were assessed after injection of normal mice with irradiated parasitized erythrocytes (IPE) or with parasitized erythrocytes (PE) membranes. Both IPE and PE membranes caused splenomegaly and an increase in the number of splenic white cells with concurrent alteration of the relative proportions of T cells and macrophages. The percentage of T lymphocytes was fractionally diminished, but there was a marked increase in Lyt 2.2 positive (suppressor and cytotoxic) T subsets and in the number of splenic macrophage precursors. The pathological enlargement of the spleen was induced by various plasma membrane-derived antigens containing both proteins and carbohydrates. Splenocytes of mice injected with liposomes containing deoxycholate-treated PE or PE fractions showed both diminished interleukin 2 production and a decreased response to mitogen. It appears that some of the changes in the cellular immune response during P. berghei infection are a consequence of the massive provision of a wide spectrum of antigens, capable of suppressing the immune response. Thus, it may be appropriate to evaluate the possible negative effect of parasite epitopes that are candidates for vaccine.     

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.     

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.     

Fetal spleen stroma drives macrophage commitment

The role of the fetal spleen in hematopoeisis remains largely unknown. In this particular environment, we show that hematopoietic stem cells do not proliferate, but that they lose multipotency and differentiate exclusively into mature macrophages. B lymphocytes in the spleen derive from committed B cell precursors that are likely to have immigrated from the fetal liver. We developed fetal spleen stromal cell lines that are unique in their capacity to expand myeloid precursors, resulting in large numbers of mature macrophages. These lines secrete high levels of anti-inflammatory molecules. By phenotype, fetal splenic macrophages are reminiscent of their adult counterparts found in the red pulp. We postulate that F4/80(+) splenic macrophages participate in fetal erythropoiesis, as well as in the formation of the splenic architecture.     

A quantitative study on recovery from acute haemorrhage in adult laboratory rats. II. Bone marrow, spleen and liver morphology

We investigate bone marrow, spleen and liver morphology in adult laboratory rats following acute haemorrhage to establish their validity in preclinical studies, especially in safety drug evaluations. The most marked changes were found in the proportion of spleen erythroid nucleated cells. Higher E: M ratios and relative erythroid cell counts were also marked in the bone marrow. Their increase was not so intensive and disappeared earlier than the increase of spleen erythropoiesis. Variations in counts of marrow neutrophils and their precursors were not specific.     

Malarial anaemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria

It has been proposed that the basis of severe malarial anaemia, a major cause of morbidity and mortality in endemic areas, is multifactorial. Inappropriately low reticulocytosis is observed in malaria patients suggesting that insufficient erythropoiesis is a major factor. Clinical studies provide conflicting data concerning the production of adequate levels of erythropoietin (EPO) during malaria. Plasmodium chabaudi AS causes non-lethal infection in resistant C57BL/6 mice, and lethal infection in susceptible A/J mice. In P. chabaudi AS infected C57BL/6 and A/J mice, which experience varying degrees of severity of anaemia, kidney EPO production is appropriate to the severity of anaemia and is regulated by haematocrit level. Neutralisation of endogenous EPO during infection leads to lethal anaemia while timely administration of exogenous EPO rescues mice although reticulocytosis is suppressed in proportion to the parasitemia level. Characterisation of alterations in splenic erythroid compartments in naive and P. chabaudi AS infected A/J mice revealed that infection, with or without EPO treatment, leads to sub-optimal increases in TER119+ erythroblasts compared to EPO-treated naive mice. A lower percentage of TER119+ erythroblasts in infected mice undergo terminal differentiation to become mature haemoglobin-producing cells. Furthermore, there is a shift in transferrin receptor (CD71) expression from TER119+ cells to a non-erythroid population. Deficiencies in the number and maturation of TER119+ erythroblasts during infection coincide with blunted proliferation to EPO stimulation in vitro by splenocytes, although a high frequency express EPO receptor (EPOR). Together, these data suggest that during malaria, EPO-induced proliferation of early EPOR+ erythroid progenitors is suppressed, leading to sub-optimal generation of TER119+ erythroblasts. Moreover, a shift in CD71 expression may result in impaired terminal maturation of erythroblasts. Thus, suppressed proliferation, differentiation, and maturation of erythroid precursors in association with inadequate reticulocytosis may be the basis of insufficient erythropoiesis during malaria.     

Differential regulatory and compensatory responses in hematopoiesis/erythropoiesis in alpha- and beta-globin hemizygous mice

Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in alpha- and beta-globin hemizygous mice, lacking the two adult tandemly organized genes from either the alpha- or beta-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, beta-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for alpha-globin hemizygous mice. In contrast, however, beta-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of beta-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free alpha-chain than beta-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the beta-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in beta-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units approximately 100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units approximately 7-fold). This early compensatory mechanism was less pronounced in alpha-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for alpha- and beta-thalassemia.     

Salmonella flagellin induces bystander activation of splenic dendritic cells and hinders bacterial replication in vivo

Bacterial flagellin is a target of innate and adaptive immune responses during Salmonella infection. Intravenous injection of Salmonella flagellin into C57BL/6 mice induced rapid IL-6 production and increased expression of activation markers by splenic dendritic cells. CD11b(+), CD8alpha(+), and plasmacytoid dendritic cells each increased expression of CD86 and CD40 in response to flagellin stimulation, although CD11b(+) dendritic cells were more sensitive than the other subsets. In addition, flagellin caused the rapid redistribution of dendritic cells from the red pulp and marginal zone of the spleen into the T cell area of the white pulp. Purified splenic dendritic cells did not respond directly to flagellin, indicating that flagellin-mediated activation of splenic dendritic cells occurs via bystander activation. IL-6 production, increased expression of activation markers, and dendritic cell redistribution in the spleen were dependent on MyD88 expression by bone marrow-derived cells. Avoiding this innate immune response to flagellin is important for bacterial survival, because Salmonella-overexpressing recombinant flagellin was highly attenuated in vivo. These data indicate that flagellin-mediated activation of dendritic cells is rapid, mediated by bystander activation, and highly deleterious to bacterial survival.     

Increased Susceptibility to Salmonella Infection in Signal Regulatory Protein α-Deficient Mice

Recent studies have shed light on the connection between elevated erythropoetin production/spleen erythropoiesis and increased susceptibility to Salmonella infection. In this article, we provide another mouse model, the SIRPα-deficient (Sirpα(-/-)) mouse, that manifests increased erythropoiesis as well as heightened susceptibility to Salmonella infection. Sirpα(-/-) mice succumbed to systemic infection with attenuated Salmonella, possessing significantly higher bacterial loads in both the spleen and the liver. Moreover, Salmonella-specific Ab production and Ag-specific CD4 T cells were reduced in Sirpα(-/-) mice compared with wild-type controls. To further characterize the potential mechanism underlying SIRPα-dependent Ag-specific CD4 T cell priming, we demonstrate that lack of SIRPα expression on dendritic cells results in less efficient Ag processing and presentation in vitro. Collectively, these findings demonstrate an indispensable role of SIRPα for protective immunity to Salmonella infection.     

Plasmodium chabaudi AS: erythropoietic responses during infection in resistant and susceptible mice.

The course of anemia and the erythropoietic response in the bone marrow, spleen, and blood were studied during Plasmodium chabaudi AS infection in resistant C57BL/6 (B6) and susceptible A/J (A) mice. Infections in B6 mice were characterized by moderate levels of both parasitemia and anemia and survival. In contrast, A mice experienced high parasitemia, severe anemia, and high mortality rates. During the period of anemia, erythropoiesis, as measured by in vivo 59Fe incorporation, was significantly more depressed in bone marrow and more increased in the spleen in resistant B6 mice. The increase in splenic 59Fe incorporation was a function of the size of the spleen. Bone marrow CFU-E were decreased to 50% of control in both strains, while splenic CFU-E were increased twofold greater in B6 mice compared to those in A mice. However, the absolute numbers of CFU-E per spleen in the two strains were not significantly different during peak parasitemia. Bone marrow BFU-E were transiently increased before peak parasitemia whereas splenic BFU-E peaked during peak parasitemia. A mice had significantly lower numbers of BFU-E per spleen on all days except at peak parasitemia. The frequency of blood-borne BFU-E and plasma erythropoietin titers was increased earlier and to a greater extent in A mice. These results suggest that an impaired amplification of late-stage splenic erythropoiesis may be an important determinant in the severity of anemia and lethality of infection with P. chabaudi AS in A mice. Moreover, these results demonstrate that the defective amplification of splenic erythropoiesis in A mice is neither caused by a defect in the mobilization of BFU-E from the bone marrow to the spleen nor caused by a defect in erythropoietin production.     

Effects of dexamethasone on rat dendritic cell function.

Glucocorticoids have been reported to affect immunity at varying concentrations. While glucocorticoids have shown profound effects on innate immunity, their effects on rat dendritic cells have not been fully examined. In this study, we evaluated the effects of the synthetic glucocorticoid dexamethasone on cultured rat dendritic cells (DCs) from spleen and derived from bone marrow cells to determine whether responsiveness to dexamethasone varies between DCs from different organ sites. Cells were analyzed for expression of glucocorticoid receptor (GR), the primary receptor through which dexamethasone exerts its effects and was found to be primarily located in the cytoplasm of immature DCs. Bone marrow-derived DCs showed more sensitivity to dexamethasone treatment compared to splenic DCs. Dexamethasone treatment of LPS-matured DCs had profound dose-dependent effects on cytokine production. Dexamethasone treatment also led to a dose-dependent downregulation of expression of costimulatory molecules by mature DCs. Dexamethasone modified immature DC uptake of antigen (FITC-Dextran), with slightly higher numbers of splenic DCs taking up antigen compared to bone marrow-derived DCs. These data suggest that dexamethasone is able to similarly affect both bone marrow-derived and splenic DC function at the immature and mature DC states and could contribute to exacerbation of infection by hindering DC-mediated immune responses.     

Chromosomal protein synthesis during erythropoiesis in the mouse spleen

Induced erythropoiesis in the mouse spleen was employed to study chromosomal protein synthesis during erythroid cell development. Splenic erythropoiesis occurring after phenylhydrazine induced hemolysis can be divided into an early phase during which nuclear RNA polymerase activity and RNA production are maximal and a late phase in which hemoglobin synthesis and DNA accumulation are maximal. Chromatin was isolated from splenic tissue during both the early and late phases of erythropoiesis as well as from non-anemic animals. The total protein content of chromatin from the early erythroid phase was greater than that of chromatin from the late erythroid phase or from non-anemic controls. The increase was due to a coordinate increase in the concentration of both histone and nonhistone proteins. During late erythropoiesis, the concentration of each returned to pre-anemic levels. Total histone synthesis increased 2.6-fold during early erythropoiesis as compared with the pre-anemic state and remained elevated in late erythropoiesis. The increase in histone synthesis was due to an increase in the synthesis of all five major histone proteins. Nonhistone protein synthesis was more active than that of histones in the pre-anemic spleen and rose only slightly during early erythropoiesis, returning to preanemic levels during late erythropoiesis. Fractionation of nonhistone proteins on SDS-urea polyacrylamide gels revealed complex patterns with significant differences between the pattern of erythroid spleen non-histone proteins and that of the pre-anemic spleen. Analysis of the incorporation of ³H-valine into the non-histone proteins indicated that during early erythropoiesis there was a generalized increase in nonhistone protein synthesis. During the late erythroid phase, the decline in non-histone protein synthesis was most marked for the higher molecular weight proteins.     

Macrophages Support Splenic Erythropoiesis in 4T1 Tumor-Bearing Mice

Anemia is a common complication of cancer; a role of spleen in tumor-stress erythropoiesis has been suggested. However, the molecular mechanisms involved in the splenic erythropoiesis following tumor maintenance remain poorly understood. Here we show that tumor development blocks medullar erythropoiesis by granulocyte colony-stimulating factor (G-CSF) and then causes anemia in murine 4T1 breast tumor-bearing mice. Meanwhile, tumor-stress promotes splenic erythropoiesis. Splenectomy worsened tumor-induced anemia, and reduced tumor volume and tumor weight, indicating the essential role of spleen in tumor-stress erythropoiesis and tumor growth. Tumor progression of these mice led to increased amounts of bone morphogenetic protein 4 (BMP4) in spleen. The in vivo role of macrophages in splenic erythropoiesis under tumor-stress conditions was investigated. Macrophage depletion by injecting liposomal clodronate decreased the expression of BMP4, inhibited splenic erythropoiesis, aggravated the tumor-induced anemia and suppressed tumor growth. Our results provide insight that macrophages and BMP4 are positive regulators of splenic erythropoiesis in tumor pathological situations. These findings reveal that during the tumor-stress period, the microenvironment of the spleen is undergoing changes, which contributes to adopt a stress erythropoietic fate and supports the expansion and differentiation of stress erythroid progenitors, thereby replenishing red blood cells and promoting tumor growth.     

稳定表达促红细胞生或素的重组腺病毒的构建及其对大鼠的促红细胞生成作用

用EPO基因组基因构建了腺病毒质粒型载体psp1B/hEPO,该质粒含有以RSV-LTR为启动子的完整的EPO基因表达盒.单独转染CHO细胞,经暂态表达检测到EPO的表达。用psp1B/hEPO与腺病毒拯救型载体pBHG11共转染293细胞,获得了表达EPO的重组腺病毒AdhEPO.经Southern杂交证实AdhEPO中有EPO表达盒,ELISA检测到了EPO阳性表达.用5×108pfu的AdhEPO给大鼠作一次性肌肉注射,观察到了其促进大鼠红细胞生成的短期效应。在注射后第1,3,5,7,10d分别检测了大鼠的红细胞压积、血红蛋白含量和红细胞计数等指标,发现大鼠的红细胞数量显著提高。在第10d红细胞压积从46±4%上升至65±6%。证实了重组腺病毒AdhEPO具有潜在的临床应用价值,可用于贫血症的基因治疗。     

A mathematical model of erythropoiesis in mice and rats. Part 4: Differences between bone marrow and spleen

Abstract. In a preceding analysis we hypothesized that the most important parameter controlled by erythropoietic regulation in vivo is the degree of amplification (number of cell divisions) in the CFU-E and erythroblast cell stages. It was concluded that erythropoetic amplification in vivo is controlled according to a sigmoidal dose-response relationship with respect to the control parameter which is the haematocrit (or haemoglobin concentration). Here, this hypothesis is extended to include the differences in murine bone marrow and splenic erythropoiesis that are described and quantified by different dose-response relationships. Comparing several sets of experimental data with mathematical model simulations, this approach leads to the following conclusions: (i) in the unperturbed normal steady state at least one extra erythropoietic cell division takes place in the spleen compared with the bone marrow; (ii) a strong erythropoietic stimulus, such as severe bleeding or hypoxia, can induce five to six additional cell divisions in the spleen but only two to three additional divisions in the bone marrow; this results in a considerable increase in the spleen's contribution to erythropoiesis from about 10% in normal animals to over 40% during strong stimulation; (iii) under erythropoietic suppression, such as red cell transfusion, a similar number of cell divisions is skipped in both organs and the splenic contribution to erythropoiesis remains unchanged. In conclusion, the concept that bone marrow and spleen microenvironments differ in the dose-response relationship for erythropoietic regulation provides an explanation for the changing contribution of splenic murine erythropoiesis following a variety of experimental treatments.     

A mathematical model of erythropoiesis in mice and rats. Part 4: Differences between bone marrow and spleen

In a preceding analysis we hypothesized that the most important parameter controlled by erythropoietic regulation in vivo is the degree of amplification (number of cell divisions) in the CFU-E and erythroblast cell stages. It was concluded that erythropoietic amplification in vivo is controlled according to a sigmoidal dose-response relationship with respect to the control parameter which is the haematocrit (or haemoglobin concentration). Here, this hypothesis is extended to include the differences in murine bone marrow and splenic erythropoiesis that are described and quantified by different dose-response relationships. Comparing several sets of experimental data with mathematical model simulations, this approach leads to the following conclusions: (i) in the unperturbed normal steady state at least one extra erythropoietic cell division takes place in the spleen compared with the bone marrow; (ii) a strong erythropoietic stimulus, such as severe bleeding or hypoxia, can induce five to six additional cell divisions in the spleen but only two to three additional divisions in the bone marrow; this results in a considerable increase in the spleen's contribution to erythropoiesis from about 10% in normal animals to over 40% during strong stimulation; (iii) under erythropoietic suppression, such as red cell transfusion, a similar number of cell divisions is skipped in both organs and the splenic contribution to erythropoiesis remains unchanged. In conclusion, the concept that bone marrow and spleen microenvironments differ in the dose-response relationship for erythropoietic regulation provides an explanation for the changing contribution of splenic murine erythropoiesis following a variety of experimental treatments.     

Protection against malaria by anti-erythropoietin antibody due to suppression of erythropoiesis in the liver and at other sites

We have previously reported that erythropoiesis commences in the liver and spleen after malarial infection, and that newly generated erythrocytes in the liver are essential for infection of malarial parasites as well as continuation of infection. At this time, erythropoietin (EPO) is elevated in the serum. In the present study, we administered EPO or anti-EPO antibody into C57BL/6 (B6) mice to modulate the serum level of EPO. When mice were infected with a non-lethal strain (17NXL) of Plasmodium yoelii (blood-stage infection of 10(4) parasitized erythrocytes per mouse), parasitemia continued for 1 month, showing a peak at day 17. Daily injection of EPO (200 IU/day per mouse) from day five to day 14 prolonged parasitemia, whereas injection of anti-EPO antibody (1.5 mg/day per mouse) every second day from day five to day 28 decreased it. Erythropoiesis was confirmed in the liver, spleen and bone marrow by the appearance of nucleated erythrocytes (TER119+). When anti-EPO antibody was injected by the same protocol into mice infected with a lethal strain (17XL) of P. yoelii, all mice showed decreased parasitemia and recovered from the infection. These results suggest that the use of anti-EPO antibody after malarial infection may be of therapeutic value in severe cases of malaria.