Modulation of murine hemopoiesis in vivo by a synthetic hemoregulatory pentapeptide (HP 5b)

A synthetic pentapeptide analogous to an inhibitory factor associated with human granulocytes was tested in vivo on female C3H mice. The relative and absolute numbers of myelopoietic and erythropoietic cells in the bone marrow were measured following injections as well as the continuous infusion of the pentapeptide in dose ranges between 10(-8)M and 10(-4)M (0.12 micrograms to 1.2 mg/mouse). In low doses, the pentapeptide reduced the number of myelopoietic cells in the bone marrow, and this was accompanied by reduced numbers of granulocytes and monocytes and peripheral blood. Elevated doses also decreased erythropoiesis. In contrast, continuous infusion of 14 micrograms/h for 19 days seemed to make the myelopoietic cells refractory to further action. A regulatory function of the pentapeptide is proposed.     

Hemopoietic colony studies. VI. Increased eosinophil-containing colonies obtained by antigen pre-treatment of irradiated mice reconstituted with bone marrow cells

The cellular response to an intraperitoneal injection of antigen (tetanus toxoid) was studied in reconstituted animals in order to determine the mechanism of control of eosinophil granulocytopoiesis. Antigen treatment of the marrow cell donors did not consistently increase the number of spleen and bone marrow colonies in recipient animals or change the percentage of eosinophil or other hemopoietic colony types. Antigen pre-treatment of the irradiated recipients increased the percentage of eosinophil-containing colonies in the spleen and femoral bone marrow without significantly changing the total number of either spleen or marrow colonies. Antigen treatment of both the bone marrow cell donor and recipient produced a further increase in the percentage of eosinophil-containing colonies in the marrow cavity, but not in the spleen. Antigen treatment of the irradiated recipient increased the number of eosinophilic cells (but not the total number of cells) in both the peritoneal cavity and the bone marrow. Antigen treatment of both the marrow donor and recipient produced a further increase in the number of eosinophilic cells in the peritoneal cavity, but not in a single femur. Since antigen treatment of the marrow recipient, or recipient and donor, but not of the marrow donor alone, results in increased eosinophilic cell and colony numbers, the effect of antigen appears to be mediated through some host factor(s), perhaps the eosinophilic hemopoietic inducing microenvironment (HIM), rather than directly on the hemopoietic stem cells.     

Differences between exogenous and endogenous hemopoietic spleen colonies

Histologic examination of the spleens in RFM/Un mice killed 6 to 9 days after 350 to 800 R whole-body x-irradiation revealed hemopoietic colonies, the numbers of which decreased exponentially with increasing radiation dose. In such animals, myelocytic colonies were the predominant type on the sixth to the eighth day. However, they decreased in number with time, being fewer than erythropoietic colonies by the ninth day after irradiation. In C57BL mice, erythropoietic colonies were relatively more numerous, markedly predominating on both the eighth and the thirteenth days. RFM/Un mice injected with nonirradiated syngeneic bone marrow cells within 24 hours after 750 R developed colonies, predominantly of erythropoietic and undifferentiated types, the numbers of which were proportional to the numbers of marrow cells injected. The number of colonies formed from exogenous marrow cells increased slightly between the sixth and ninth days after inoculation, possibly because of a greater likelihood of counting them due to an increase in their size.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

The effect of cadmium on the survivability of colony-forming hematopoietic cells in mice exposed to x-ray irradiation]

It has been reported elsewhere that in addition to enhancing the expression of metallothionein genes, the previous injection of cadmium salts into sublethally X-irradiated mice increases by 10 times the number of endogenous spleen colonies. To understand the mechanism of the strong radioprotective cadmium effect donors and recipients were treated separately. It is shown that the survival of exogenous bone marrow colony-forming cells in lethally irradiated recipient remains at the control level independently of the donor cadmium treatment, whereas the injection of cadmium nitrate to recipient mice leads to the stimulation of colony formation by 1.7-1.8 times. The data allow to conclude that the cadmium effect on the survival of colony-forming hemopoietic murine cells after X-irradiation is not mediated by the enhanced expression of metallothionein genes.     

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.     

Hemopoietic progenitor cells of W anemic mice studied in vivo and in vitro

The proliferation and differentiation of hemopoietic cells from genetically anemic W^v/W^x,W/W^v, and W^v/W^v mice, and from nonanemic carrier W/+, W^b/+, and W^v/+ mice have been evaluated in vivo by transplantation techniques and in vitro by the agar gel culture method. Marrow from anemic and carrier mice contained progenitor cells which were decreased in number and formed small, often rudimentary, colonies in the spleens of irradiated recipient mice. Proliferation and differentiation of both erythropoietic and leukopoietic progenitor cells were delayed and reduced, but erythropoiesis was more severely affected than leukopoiesis. The severity of the hemopoietic impairment was gene-dose dependent. The W gene effect on leukopoietic progenitor cells was not secondary to anemia or to abnormal erythropoiesis. The marrow cells of anemic and carrier mice which form colonies of granulocytic and mononuclear cells in vitro were neither decreased in number nor impaired in proliferation and differentiation. Hypertransfusion of red blood cells increased the frequency of in vitro colony-forming cells, but not that of in vivo progenitor cells. The data demonstrate that colony-forming cells which proliferate in the agar gel cultures in vitro are distinct from the in vivo colony-forming cells and suggest that the former are primitive members of the granulocytic cell line. Perhaps in vitro CFU are in an intermediate stage of differentiation between in vivo CFU and myeloblasts, analogous to that which has been suggested for the erythropoietin-sensitive cell in the red cell series. W mutant alleles appear to act, therefore, at or very near the beginning of hemopoietic differentiation.     

The effect of erythropoietin on the growth and development of spleen colony-forming cells

The progressive growth and development of spleen colonies was studied in heavily irradiated host mice in which erythropoiesis was modified by various procedures. Erythropoietic activity in non-polycythemic hosts bearing spleen colonies was not increased by injections of exogenous erythropoietin. Detectable levels of erythropoietin were found in the heavily irradiated host mice suggesting that the failure of exogenous erythropoietin to modify erythropoiesis was because the host mice were already maximally stimulated by the high endogenous erythropoietin levels. Spleen colonies do not become erythroid in polycythemic mice. The injection of exogenous erythropoietin into heavily irradiated polycythemic hosts did not decrease the total number of spleen colonies produced by a given bone marrow transplant, as would be expected if erythropoietin acted directly on the colony-forming cells. Comparison of growth curves for colony-forming cells in the spleens of polycythemic hosts either receiving or not receiving erythropoietin indicated that the overall doubling time of colony-forming cells during the first ten days after transplantation was not changed by the daily injection of erythropoietin. These experiments are consistent with the concept that erythropoietin is necessary for the development of erythroid colonies. Erythropoietin acts upon some progeny of the colony-forming cell rather than the colony-forming cell itself.     

Transient erythropoietic spleen colonies: effects of erythropoietin in normal and genetically anemic W/Wv mice.

Properties of the cells (TE-CFU) that give rise within four to six days to transient endogenous erythropoietic spleen colonies in irradiated mice have been investigated. The results obtained indicate that (1) erythropoietic maturation within such colonies is highly erythropoietin-dependent, (2) the population size of TE-CFU is not erythropoietin-dependent, (3) initial exposure to a high dose of erythropoietin followed by continuing exposure to lower doses is required for maximal efficiency of colony formation by TE-CFU, (4) successful transplantation of TE-CFU has not been achieved, but they appear among the progeny of transplanted hemopoietic cells, (5) TE-CFU are defective in mice of genotype W/Wv. These findings are consistent with the view that the TE-CFU assay detects a class of early erythropoietin-sensitive progenitor cells committed to erythropoietic diffferentiation, rather than "abortive" colony formation by pluripotent stem cells.     

Factors influencing hematopoietic spleen colony formation in irradiated mice. IV. The effect of erythropoietic stimuli

A variety of erythropoietic stimuli influenced the number of endogenous spleen colonies in irradiated mice and the number of transplantable colony forming cells in the spleen and marrow of unirradiated mice. Bleeding was the most effective stimulus. Bleeding before irradiation resulted in a 30-fold increase in endogenous spleen colonies and in increases in spleen weight, spleen iron and iododeoxyuridine uptake and volume of packed red cells ten days after irradiation. Bleeding unirradiated mice produced a 10-fold increase in the number of transplantable colony forming cells in the spleen and a slight decrease in the total number in the humerus. Bleeding before irradiation resulted in a significant reduction in 30-day post irradiation deaths, an effect abolished by splenectomy. Plasma from bled mice induced an increase in endogenous colonies when injected before irradiation into normal mice. Injection of erythropoietin, testosterone or testosterone plus cobalt induced effects which were, in general, qualitatively similar to those of bleeding, although they were less effective quantitatively. Except for a slight effect induced by ten injections of erythropoietin, post-irradiation stimulation in normal mice proved ineffective. Erythropoietin increased colony numbers and spleen iron uptake when given after irradiation to hypertransfused mice. The results of these studies do not support the concept that the colony forming cell and the erythropoietin sensitive cell are separate entities.     

Evaluation of the marrow culture on the macrophage layer covering intraperitoneally implanted membrane as an assay for hemopoietic progenitor cells

When cellulose acetate membranes are implanted into abdominal cavity of mice they turn into a foreign body overgrown with macrophages. Such macrophage layer has been shown by other authors to be able to support the growth of hemopoietic colonies formed by intraperitoneally injected hemopoietic cells. This study confirms and extends this observation by showing that both granulopoietic and erythropoietic colonies may be observed. The number of colonies grown is in linear correlation with that of injected hemopoietic cells. The frequency of erythropoietic colonies was greatly enhanced by blood letting of the host mice. Colony forming cells were most numerous in the bone marrow then in the spleen and peripheral blood and hardly in the thymus. Prior irradiation of the host mice was essential for obtaining colony growth and the optimal dose was determined to be 6.0 Gy. This technique opens the way to studies into hemopoietic progenitor cells for laboratories having no sophisticated tissue culture equipment and where necessary reagents are easily available.     

In vitro production of CFU-S and cells with erythropoiesis repopulating ability by 5-fluorouracil treated mouse bone marrow

Mouse bone marrow, obtained from donors three days after treatment with 5-fluorouracil, had a very low ability to form macroscopic spleen colonies in irradiated mice at 10 days after transplantation of the cells (CFU-S10); such marrow also had no detectable erythropoiesis repopulating ability but did have near normal marrow repopulating ability and spleen megakaryocyte repopulating ability. Incubation of this marrow in vitro for 7 days with medium containing growth factor preparations (a) pregnant mouse uterus extract plus human spleen conditioned medium or (b) mouse spleen conditioned medium, resulted in marked increases in CFU-S10 and in cells with erythropoietic repopulating ability together with maintenance of cells with marrow repopulating ability. These responses were not observed in cultures with control medium alone. Spleen megakaryocyte repopulating ability was also maintained in the presence of the factor preparations.     

Formation of hematopoietic colonies of the donor type in the bone marrow of irradiated chickens after transplantation of the cells of the yolk sac and hindlimb of the quail embryo]

The ability of yolk sac and primary bone marrow cells of the quail to form hemopoietic colonies at 6 hours of incubation (i. e. before establishment of circulation) was studied in the bone marrow of 3-week sublethally irradiated chickens. The experiments were based on the possibility of differentiating between quail and chicken cells from the natural cell marker (Pheulgen-positive nucleolus). The number of hemopoietic colonies produced by cells transplanted from the primary bone marrow was three times greater than that consequent on transplantation of yolk sac cells. With the given dose of irradiation the bone marrow shows about 75% exogenous (quail) and 25% endogenous (chicken) hemopoietic colonies.     

Deficient Fe59 and I-125 deoxyuridine uptake by lympho-hemopoietic cell transplants engaged in homograft reactions

Erythropoietic activity of spleen cell grafts was measured (Fe⁵⁹ uptake) in X-irradiated recipient mice under conditions in which these grafts were engaged in homograft reactions against allogeneic target cells or in graft-versus-host reactions. Such Fe⁵⁹ incorporation was greatly reduced at 7 to 10 days after graft implantation relative to that of control grafts. This reduced erythropoiesis did not occur when the spleen cell graft was immunologically incompetent. Transplantation of bone marrow-lymph node cell mixtures also resulted in a relative decline in Fe⁵⁹ uptake, but only when minimal numbers (10⁵ to 10⁶) of marrow cells were injected. The incorporation of I¹²⁵ UdR in the spleen of irradiated recipients was used to assess cellular proliferation. Incorporation of this label was reduced when measured 7–10 days after implantation of the lympho-hemopoietic cell graft, but reached a peak at five days—the latter indicating stimulated lymphopoiesis. These data are consistent with the concept of depletion of a pluripotent stem cell pool (limited in size under these experimental conditions) due to excessive and concurrent functional demands for erythropoiesis and lymphopoiesis. An alternative explanation would involve cytotoxic effects on hemopoietic elements present in the milieu of the immunologic reaction.     

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.     

PROPERTIES OF HAEMOPOIETIC STEM CELLS IN PHENYLHYDRAZINE TREATED MICE

Recovery of erythropoiesis was fast in Balb/c mice irradiated 700 R 5 days after initiation of phenylhydrazine treatment and took place predominantly in the spleen, which showed numerous large frequently confluent endogenous colonies. Post irradiation phenylhydrazine induced anaemia did not accelerate recovery of erythropoiesis; it did, however, produce a slight but significant rise in endogenous colony formation.
Radiosensitivity of spleen CFU-S from phenylhydrazine treated mice was similar to that of CFU-S in normal mouse spleen.
Spleen CFU-S in mice 5 days after initiation of phenylhydrazine treatment were sensitive to the lethal action of Hydroxyurea, while bone marrow CFU-S were not.
The self-renewal capacity of CFU-S in the endogenously repopulated spleen of phenylhydrazine pretreated 700 R X-irradiated mice was low when compared to that of spleen exogenously repopulated by cells from normal mouse bone marrow, normal and phenylhydrazine treated mouse spleen. CFU circulating in blood of phenylhydrazine treated mice had a low self-renewal capacity.
The marked strain differences in self-renewal capacity of spleen CFU-S, and of the capacity of spleen CFU-S to increase by proliferation are discussed.     

Effects of lymphocytes from the thymus and lymph nodes on differentiation of hemopoietic spleen colonies in irradiated mice

Hemopoietic colonies were counted macroscopically and microscopically in spleens of hybrid mice seven or eight days after they had been irradiated and given parental bone marrow in donor-host combinations exhibiting poor growth. Colonies counted microscopically were classified as to differentiation pathway. Lymphocytes from the thymus or lymph nodes were injected into some recipients at several different dosages and lymphocyte: bone marrow (L:B) ratios. In confirmation of earlier work it was found that thymocytes increased the number and size of colonies in recipients of marrow. A shift of differentiation toward granulopoiesis was also seen when thymocytes were given, although erythropoietic colonies were still the most frequently seen type except at very high L:B ratios. Lymph node lymphocytes shifted the pattern more markedly toward granulopoiesis, even at low L:B ratios. When lymphocytes from either source were given without marrow, only a few colonies could be found in recipients, and if differentiated they were almost exclusively granulopoietic. Irradiation (900 R) of lymphocyte donors reversed the shift so that a normal pattern of differentiation, like that resulting from marrow alone, was seen; irradiated lymphocytes were nonetheless capable of augmenting the size and total number of hemopoietic colonies.     

Comparison of response to stem cell differentiation signals between normal and autoimmune mouse strains

Normal DBA/2 and autoimmune NZB mice were studied with regard to signals eliciting differentiation and division of bone marrow stem cells. Irradiated (NZB X DBA/2)F1 mice were repopulated with various combinations of T-depleted bone marrow from NZB and DBA/2 mice. In response to the repopulation signal of irradiation, recipients of autoimmune NZB marrow initially demonstrated expansion of LY-5+ lymphoid and hemopoietic cells, particularly of the B cell lineage. The greater the proportion of NZB marrow, the higher the percentage of lymphoid cells observed 2 wk post-repopulation. B cells (ThB-positive cells) were increased in disproportionate numbers in recipients of NZB marrow, even those that had received as little as 20% NZB bone marrow cells. However, by 2 mo, the initially observed increase in lymphoid cells in recipients of NZB marrow was no longer observed. Up to 6 mo post-repopulation, cytogenetic analysis revealed that irradiated recipients were repopulated in the same proportion of DBA/2: NZB as was in the injected marrow. Endogenous colony formation assays indicated that recipients of 100% NZB, 80% NZB, and 20% NZB marrow all had greater numbers of splenic endogenous colonies than did recipients of DBA/2 marrow alone. These studies indicated that autoimmune NZB marrow repopulated irradiated mice in the proportion in which it was injected, but there was a disproportionate early increase in cells of the B lineage as well as a disproportionate increase in splenic colony formation.     

Comparative studies on the characteristics of proliferation and differentiation of spleen colony-forming cells

Using a single spleen colony transplantation technique and sex chromosome typing as a natural cytogenetic marker, most spleen colony-forming cells (CFC) in adult bone marrow or fetal livers of inbred LACA or C57 mice re-established hemopoiesis in lethally irradiated mice when the spleen colonies were sampled at 13 days after transplantation. However, most of the spleen colony-forming cells in the peripheral blood of normal mice possess little potential for proliferation and are less efficient in the re-establishment of hemopoiesis in lethally irradiated mice. The CFC population is heterogeneous in the mice. From the subsequent retransplantation of colonies from colony-forming cells in the peripheral blood, the simple assessment of spleen colony-forming units (CFU-s) content, based on the number of splenic colonies, does not reliably represent the content of hemopoietic stem cells.     

Defective transient endogenous spleen colony formation in S1/S1d mice.

WCB6F1 mice of the genotype S1/S1d did not form transient 5-day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE-CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.