A model of hemopoietic stress in a lactate dehydrogenase mouse mutant with hemolytic anemia

A codominantly inherited mutation of the lactate dehydrogenase (LDH) in the C3H mouse causes a severe hemolytic anemia in homozygous mutants, whereas viability and fertility are close to normal. Investigation of multipotent hemopoietic stem cells (CFU-S), myeloid (GM-CFC) and erythroid progenitors (BFU-E, CFU-E) in femur and spleen indicates a general shift from bone marrow to splenic hemopoiesis. In terms of total body hemopoiesis, however, the BFU-E pool is 1.4- and the CFU-E pool 19-fold enlarged, whereas CFU-S and GM-CFC show little or no deviation from normal. It is concluded that this mouse mutant is an appropriate model of long-term hemopoietic stress showing that compensation in this severe hemolytic anemia is achieved primarily by an increase of the number of the most mature erythroid progenitors.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.     

Comparative histopathology of mice infected with the 17XL and 17XNL strains of Plasmodium yoelii

Plasmodium yoelii 17XL was used to investigate the mechanism of Plasmodium falciparum-caused cerebral malaria, although its histological effect on other mouse organs is still unclear. Here, histological examination was performed on mice infected with P. yoelii 17XL; the effect of P. yoelii 17XL infection on anemia and body weight loss, as well as its lesions in the brain, liver, kidney, lung, and spleen, also was investigated. Plasmodium yoelii 17XL-infected red blood cells were sequestered in the microcirculation of the brain and in the kidney. Compared with the nonlethal P. yoelii 17XNL strain, infection by P. yoelii 17XL caused substantial pulmonary edema, severe anemia, and significant body weight loss. Although P. yoelii 17XNL and 17XL produced a similar focal necrosis in the mouse liver, infection of P. yoelii 17XL induced coalescing of red and white pulp. Mortality caused by P. yoelii 17XL may be due to cerebral malaria, as well as respiratory distress syndrome and severe anemia. Plasmodium yoelii 17XL-infected rodent malaria seems to be a useful model for investigating severe malaria caused by P. falciparum.     

Erythropoietic progenitors capable of colony formation in culture: state of differentiation

The differentiated state of mouse erythropoietic progenitor cells (CFU-E), detected by their ability to form erythropoietin-dependent colonies in vitro, has been investigated. Transfusion-induced plethora was found to reduce the population size of CFU-E in both spleen and femoral marrow, which indicates that a significant number of CFU-E arise by differentiation processes that are themselves erythropoietin-dependent. Individual spleen colonies were found to be heterogeneous in their content of CFU-E, and the numbers of CFU-E per colony were not correlated either positively or negatively with the numbers of granulocyte-macrophage progenitors (CFU-C) present in the same colonies. The absence of a negative correlation between CFU-E and CFU-C indicates that the erythropoietic and granulopoietic pathways of differentiation are not mutually exclusive within individual spleen colonies. The numbers of CFU-E per spleen colony were also found to vary independently of the numbers of pluripotent stem cells (CFU-S) per colony; in contrast, as found previously, the numbers of CFU-C and CFU-S per colony were positively correlated. These results indicate that more randomizing events separate CFU-E from CFU-S than separate CFU-C from CFU-S, and are consistent with the view that CFU-E occupy a position on the erythropoietic pathway of differentiation that is more remote from the pluripotent stem cells than is the corresponding position of CFU-C on the granulopoietic pathway.     

Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells

We have systematically studied the effect on hemopoietic colony-forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30-60 min incubation with a 5 microM concentration of stain. Flow-cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony-forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU-M, a megakaryocyte colony-forming cell; CFU-E, a relatively differentiated erythroid precursor; BFU-E, a primitive erythroid precursor; CFU-GM, a granulocyte-macrophage precursor; and CFU-S, the spleen colony-forming cell or hemopoietic stem cell. A staining procedure involving a 30-min exposure to 5 microM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU-M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.     

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria.

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weight ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection, in parallel to the parasitaemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that the polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.     

Association of intermediate T cell receptor cells, mainly their NK1.1(-) subset, with protection from malaria

Mice were infected with Plasmodium (P.) yoelii blood-stage parasites. Both the liver and spleen were the sites of inflammation during malarial infection at the beginning of day 7. The major expanding cells were found to be NK1.1(-) intermediate alphabetaTCR (alphabetaTCR(int)) in the liver and spleen, although the population of NK1.1(+) alphabetaTCR(int) cells remained constant or slightly increased. These TCR(int) cells are of extrathymic origin or are generated by an alternative intrathymic pathway and are distinguished from conventional T cells of thymic origin. During malarial infection, the population of conventional T cells did not increase at all. TCR(int) cells purified from the liver of mice which had recovered from P. yoelii infection protected mice from malaria when they were transferred into 6.5-Gy-irradiated mice. Interestingly, the immunity against malaria seemed to disappear as a function of time after recovery, namely, mice which had recovered from malaria 1 year previously again became susceptible to malarial infection. The present results suggest that TCR(int) cells are intimately associated with protection against malarial infection and, therefore, that mice which had recovered from malaria 1 year previously lost such immunity.     

Self-renewal capacity of murine hemopoietic stem cells under internal contamination with239Pu and241Am

Summary The self-renewal capacity of murine pluripotent hemopoietic stem cells (CFU-S) of vertebral bone marrow was studied under conditions of short-term and long-term internal contamination with²³⁹Pu or²⁴¹Am in female mice. Measurement of the CFU-S self-renewal capacity was carried out using double transplantation assay. To evaluate the production of differentiated progeny of stem cells average erythroblast numbers/visible spleen colony and⁵⁹Fe-uptake/colony were computed. The marrow cellularity/vertebra and the number of CFU-S/vertebra were decreased and affected more by²³⁹Pu than by²⁴¹Am. The production of erythroblasts per a single CFU-S and the⁵⁹Fe-uptake/colony were reduced, similarly the numbers of secondary spleen colonies and of secondary CFU-S in primary colonies. The above changes resulting from impaired functions of surviving CFU-S were more serious with²⁴¹Am than with²³⁹Pu. The biological effects of plutonium and americium appeared independent of the phase of contamination.     

Erythropoietin sensitivity as a differentiation marker in the hemopoietic system: Studies of three erythropoietic colony responses in culture

A time course study of the sequential appearance of erythropoietin-dependent colonies and bursts (derived from CFU-E and BFU-E, respectively) was performed on mouse hemopoietic cells cultured in methyl cellulose containing 2-mercaptoethanol. A new type of small, short-lived burst was found to be apparent by the third day in culture. By the sixth day most of these bursts had lysed. At the same time, differentiating erythroblasts began to be detectable in the large, late appearing bursts described previously. These two types of burst, differing from each other and from CFU-E derived colonies both in their ultimate size and morphology, as well as in their time course of appearance and lysis, were compared in other ways. It was found that early burst formation required about 100 times more erythropoietin than that needed to stimulate CFU-E. On the other hand, early burst formation required less than one-quarter of the amount of erythropoietin needed to obtain the large, late appearing bursts. Comparison of the distribution of early burst progenitors relative to pluripotent stem cells (CFU-S) in individual spleen colonies gave a correlation coefficient that was also intermediate between that obtained comparing CFU-S with CFU-E and that obtained comparing CFU-S with the progenitors of late bursts. These results suggest that decreasing proliferative capacity is associated with progressively increasing erythropoietin responsiveness as primitive erythropoietic progenitors move from a position close to pluripotent stem cells through several differentiation steps to reach a stage just prior to the onset of detectable hemoglobin synthesis.     

Nude mice bearing human CSF-producing tumor: analysis of hemopoietic factor(s) acting on primitive stem cells

Previously, we serially transplanted tumors that produced colony-stimulating factor (CSF) into nude mice, who developed marked granulocytosis along with tumor growth; their leukocyte counts reaching approximately one million per cu mm. The numbers of CFU-GM, CFU-E, CFU-Meg, CFU-S and BFU-E were increased in nude mice bearing CSF-producing tumor. We here report that tumor-conditioned medium (TCM) derived from the CSF-producing tumors had colony-stimulating activity (CSA) and burst-promoting activity (BPA) when normal murine spleen cells as well as normal human bone marrow cells were the target cells. The activity of TCM supported multilineage colony formation in 5-fluorouracil (5-FU)-treated mouse spleen cells, in which only the primitive population of stem cells was reserved. No interleukin-3 (IL-3) activity was detected in TCM when assayed using the IL-3 dependent cell lines. We conclude that the factor in TCM acts on pluripotent stem cells and on the early progenitor stage of various cell lineages. It is distinct from IL-3.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

Hematopoiesis in the rat: quantitation of hematopoietic progenitors and the response to iron deficiency anemia

To determine the quantitative effects of iron deficiency on erythropoiesis and to assess the response of erythroid progenitors to sustained anemia, we developed quantitative assays for various hematopoietic progenitors in the adult, Sprague-Dawley rat including erythroid colony- and burst-forming cells (CFU-E and BFU-E), granulocyte/macrophage colony-forming cells (CFU-GM), and megakaryocytic colony-forming cells (CFU-Meg). CFU-E were cultured in methylcellulose and grew best in the presence of fetal calf serum. CFU-GM, BFU-E, and CFU-Meg grew better in normal rat plasma and required the presence of pokeweed mitogen-stimulated rat spleen cell conditioned medium. The numbers of progenitors and nucleated erythroblasts in total marrow were estimated by the ratios of radioactivity in the humerus to the total skeleton as determined by radioiron dilution. The numbers of progenitors and erythroblasts in the spleen were measured by simple dilution. Sustained anemia was brought about through chronic iron deficiency. The response to iron deficiency anemia (IDA) was monitored by the numbers of the various progenitors and their cell cycle characteristics as measured by the tritiated thymidine suicide technique. With IDA, the number of CFU-F in the body (marrow plus spleen) was increased to 3.5 times control, whereas the numbers of BFU-E and CFU-GM were unchanged. There was no difference in the percentage of CFU-E, BFU-E, and CFU-GM in DNA synthesis (68%, 19.4%, and 18.8%, respectively). With iron therapy of IDA, CFU-E numbers in marrow began to decrease by day 1 and fell in a manner reciprocal to changes in the hematocrit. Marrow and spleen erythroblasts, 1.7 times control in IDA, increased further to 3.9 times control by the fourth day after iron administration. There was no change in BFU-E or CFU-GM numbers in response to iron repletion, although the fraction of progenitors increased in the spleen. Thus, IDA does not limit the increase in CFU-E seen with anemia, but does restrict erythroid maturation. Furthermore, the increase in CFU-E and the state of chronic anemia occur without detectable changes in the number of cell cycle state of the more primitive BFU-E.     

Radiosensitivity and proliferative activity of vertebral CFU-S surviving in mice injected with oncogenic activities of 239Pu or 241Am

Survival, radiosensitivity and capability to produce differentiated progeny were followed in CFU-S from lumbar vertebrae of mice injected with 198.6 kBq 239Pu/kg or 208.6 kBq 241Am/kg. The CFU-S assay and 59Fe uptake into spleen colonies were used. The number of CFU-S from treated mice was significantly lower than in controls. Higher radiosensitivity of CFU-S from 239Pu- or 241Am-treated mice was demonstrated using additional exposure to 0.5 Gy X-rays 1, 24, 48, 72 hrs after cell transplantation and expressed more precisely by survival curves obtained 1 hr after the marrow cell injection. The effect of 239Pu on CFU-S was characterized by Do 0.58 Gy (n = 0.91) and that of 241Am by Do 0.64 Gy (n = 0.91); corresponding control values were Do 0.89 Gy, n = 1.11. Lower iron utilization due not only to the decreased CFU-S numbers, but also to the defective production of erythroid cells per one CFU-S was found. Complexity of radiation effect on hemopoietic stem cells was demonstrated by the present study.     

大豆异黄酮对辐射小鼠造血系统损伤防护作用的实验研究

研究大豆异黄酮(SoybeanIsoflavone,SI)对辐射损伤小鼠造血系统功能的影响。雄性昆明小鼠照射前补充SI,经4Gyγ射线照射,观察补充SI对电离辐射损伤小鼠内源性脾结节(CFU-S)数、骨髓细胞粒、巨噬系细胞集落形成单位(CFU-GM)数、骨髓细胞DNA含量及外周血淋巴细胞DNA损伤程度的影响。结果显示SI提高了辐射损伤小鼠CFU-S数,增加了骨髓有核细胞CFU-GM数,降低外周血淋巴细胞DNA损伤程度,对骨髓细胞DNA含量也有一定的增加趋势。表明SI可以提高辐射损伤小鼠造血干祖细胞的增殖能力,减轻辐射对骨髓细胞和外周血淋巴细胞DNA的损害,SI对辐射小鼠的造血系统有一定的防护作用。     

Functional state of the hematopoietic system in different stages of CCL4-induced liver fibrosis in mice]

The state of hemopoietic system at different stages of CCL4-induced liver fibrosis in mice and its reaction to stimuli was examined. At early stages of liver fibrosis (2 weeks of CCL4-administration) we observed a disinhibition of granulo-monocytopoiesis. This is manifested in an increase of the number of immature granulocytes, monocytes/macrophages and CFU-GM in the bone marrow. After 24 h of zymosan injection in this stage an increase of both bone marrow CFU-GM and CFU-E, and the rate of serum colony stimulating activity (CSA) occurred. On the contrary, in advanced liver fibrosis (16 weeks of CCL4 administration), the rate of both granulo-monocytopoiesis and erythropoiesis were decreased as compared with the preceding stage of the process and the response to zymosan became perverted: the number of CFU-GM was paradoxically decreased, while the number of CFU-E remained practically unchanged. The rate of serum CSA increased very poorly too. Thus, in advanced liver fibrosis the hemopoietic system is functionally defective and this factor may be important in development of fibrotic process in the liver.     

The effect of interleukin-17 on hematopoietic cells and cytokine release in mouse spleen

To evaluate whether the response of hematopoietic cells to interleukin-17 (IL-17) depends on the tissue microenvironment in which hematopoiesis occurs, the influence of recombinant mouse IL-17 on spleen hematopoietic cells and cytokine release was assessed in normal mice in vitro and in vivo. In vitro, IL-17 did not significantly affect the growth of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) derived colonies. A single injection of IL-17 in vivo exhibited stimulatory effects on hematopoietic cells from both granulocytic and erythroid lineages. The increased number of metamyelocytes 48 h after treatment imply to the IL-17-induced stimulation of granulopoiesis. The number of BFU-E was increased at 24 h, while the number of CFU-E increased 6 h and 24 h after treatment. Since the same treatment in the bone marrow decreased the number of CFU-E, it may be concluded that the local microenvironment plays an important role in IL-17-mediated effects on CFU-E. IL-17 increased the release of IL-6 both in vitro and in vivo, but showed tendency to suppress the constitutive secretion of IL-10 by spleen cells. Our results suggest the complexity of target cell response and interplay of secondary induced cytokines by IL-17 in different hematopoietic organs.     

Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation,differentiation and leukemogenic capacity

Long-term cultures of proliferating hematopoietic stem cells derived from bone marrow permit the study of the interaction between murine leukemia virus (MuLV) infection and the proliferation and differentiation of stem cells. We have used this system to analyze the replication of different biological variants of MuLV in bone marrow cells; the effect of MuLV infection upon pluripotent stem cell (CFU-S) proliferation; and the effect of MuLV on differentiation of CFU-S along different hematopoietic pathways. Two MuLV variants were studied in detail: the Moloney strain of lymphatic leukemia virus (Mol-MuLV) and the erythroleukemic Friend virus complex (FLV) consisting of the lymphoid leukemia helper virus and the defective spleen focus-forming virus (SFFV). Mol-MuLV and its sarcoma virus pseudotype, MSV(Mol-MuLV), replicate efficiently in the bone marrow cultures; however, CFU-S are lost more readily than in uninfected cultures, and the cultures are soon represented by a majority population of mononuclear macrophages. On the other hand, infection with FLV produces a prolonged survival of the spleen colony-forming cells, CFU-S, and CFU-C (the committed granulocytic precursor cells). Production of erythroleukemogenic SFFV is maintained in these cultures for more than 40 weeks. No erythroblastic differentiation was observed in vitro, however, neither erythroblast precursor cells (CFU-E) nor hemoglobin-producing cells could be detected. This suggests that the target cell for FLV is an earlier precursor cell.     

小鼠胎肝基质细胞造血刺激因子体外生物活性研究

本实验对基质细胞造血刺激因子－１（ＳＨＦ－１）的体外生物活性进行了研究。结果表明,ＳＨＦ－１可刺激小鼠骨髓ＣＦＵ－Ｅ、ＢＦＵ－Ｅ、ＣＦＵ－ＧＭ、ＣＦＵ－Ｍｉｘ集落的形成,它产生的这些广泛造血刺激作用是其自身所具活性的直接影响。正常小鼠骨髓细胞与ＳＨＦ－１在体外孵育４ｈ,其中ＣＦＵ－Ｓ的自杀率可提高约１０％,显示它对造血干细胞也有诱导增殖作用。     

Effects of Trichinella spiralis infections upon bone marrow stem cells in BALB/c mice

Trichinella spiralis infections provoke a variety of responses in the host, some of which involve stem cell proliferation and myeloid cell maturation, increases in the mast cell precursor cell populations, and maturation and eosinopoiesis. Very little is known about the influence of T. spiralis upon bone marrow stem cells and splenic colony formation. In the present communication we report that T. spiralis infection in mice stimulates the generation of colony-forming units in the spleen (CFU-S). Passive transfer of bone marrow cells from uninfected BALB/c mice to X-irradiated (650 R) T. spiralis-infected recipients resulted in a significant increase of CFU-S at 14 and 24 days postinfection. Passive transfer of bone marrow cells from T. spiralis-infected mice to X-irradiated uninfected mice also resulted in increased numbers of CFU-S in the donor mice at 24 days postinfection. These findings strongly suggest that T. spiralis infection conditions the microenvironment in the spleen which stimulates CFU-S.     

Isolation of murine pluripotent hemopoietic stem cells in the Go phase

A method to purify pluripotent hemopoietic stem cells in the Go phase from mouse bone marrow was established. Bone marrow cells from 5-fluorouracil (5-FU)-treated mice were fractionated by Percoll density gradient. The cells with density between 1.063 and 1.075 were further separated into wheat germ agglutinin (WGA)-positive and -negative cells using fluorescent-activated cell sorter (FACS) after staining with fluorescein isothiocyanate-conjugated WGA (FITC-WGA). An assay for spleen colony-forming units (CFU-S) revealed that the WGA-positive cells (1 X 10(6)) produced 1380 CFU-S (about 150 times of the number in the original bone marrow cells) on day 12 (but no CFU-S on day 8), whereas the WGA-negative cells produced no CFU-S. Thus, the stem cells in the Go phase are found to be enriched 150 times in 5-FU-treated WGA-positive cells.