STUDIES ON THE MECHANISM OF HAEMOPOIETIC STEM CELL (CFUs) MOBILIZATION

A variety of substances can mobilize haemopoietic stem cells (CFUs) into the peripheral blood. In this study the involvement of the complement system in the mobilization process was investigated. Pretreatment of mice with the complement-activating factor of cobra venom (CoF), which lowered the serum C3 levels to 10–25% of the normal value, could completely prevent CFUs mobilization induced by high doses of CoF, endotoxin (ET) from Salmonella typhosa, inulin, zymosan and the proteolytic enzymes proteinase and trypsin. On the other hand, mobilization induced by the polyanions dextran sulphate and the copolymer of polymethacrylic acid and styrene could not be prevented, or at least affected only slightly. There appears to be a relationship between the extent of decomplementation by CoF and the extent of CFUs mobilization induced by ET. The results indicate that certain agents mobilize CFUs via the complement system, whereas other agents induce CFUs mobilization independent of the availability of complement components.     

MULTIPLICATION OF HAEMOPOIETIC COLONY FORMING UNITS (CFU) IN MICE AFTER X-IRRADIATION AND BONE MARROW TRANSPLANTATION

Kinetics of the multiplication of haemopoietic CFUs was studied in lethally irradiated mice receiving various numbers of syngeneic bone marrow cells. After transplantation of a small number of bone marrow cells, the growth rate of CFU in femoral bone marrow appeared to decrease after about 10 days after transplantation, before the normal level of CFU in the femur was attained. In the spleen it was found that the overshoot which was observed about 10 days after transplantation of a large number of bone marrow cells is smaller or absent when a small number of cells is transplanted. Experiments dealing with transplantation of 50 x 10⁶ bone marrow cells 0, 4 or 10 days after a lethal irradiation indicated that the decline in growth rate of CFUs about 10 days after irradiation could not be attributed to environmental changes in the host.
The results are explained by the hypothesis that a previous excessive proliferation of CFUs diminishes the growth rate thereafter. This hypothesis is supported by experiments in which 50 x 10⁶ bone marrow cells derived from normal mice or from syngeneic chimaeras were transplanted. The slowest growth rate was observed when bone marrow that had been subjected to the most excessive proliferation in the weeks preceding the experiment was transplanted.     

THE INFLUENCE OF DIETARY PROTEIN DEFICIENCY ON HAEMOPOIETIC CELLS IN THE MOUSE

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1 -4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs–although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18 %) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstitutive role during the refeeding process.     

FURTHER STUDIES ON MOBILIZATION OF CFUs

Mobilization of CFUs from haemopoietic tissues into circulation was studied after injection of different bacterial lipopolysaccharides (LPS), zymosan, phytohaemagglutinin (PHA), concanavalin A (Con A), trypsin and di-isopropyl-fluorophosphate-inhibited trypsin. All bacterial LPS used gave an increase of CFUs in the peripheral blood at 1 h after i.v. injection. Some variation in activity could not be excluded. As with Salmonella typhosa LPS, zymosan gave an increase in circulating CFUs during the first few hr and a second peak a few days later. After injection of zymosan as well as S. typhosa LPS the second peak in the blood was accompanied by a large increase in CFUs numbers in the spleen. PHA gave an immediate mobilization of CFUs, but the mobilization after injection of Con A during the first few hr occurred more slowly. After injection of S. typhosa LPS, zymosan and PHA the blood C3 level was found to be depressed considerably. This might indicate that the complement system is involved in the early mobilization of CFUs. Dexamethasone, a synthetic hormone which has been reported to give sequestration of several cell types in the bone marrow, did not inhibit the early and late mobilization of CFUs which normally occurs after injection of S. typhosa LPS.     

Studies on the mechanism of haemopoietic stem cell (CFUs) mobilization. A role of the complement system.

A variety of substances can mobilize haemopoietic stem cells (CFUs) into the peripheral blood. In this study the involvement of the complement system in the mobilization process was investigated. Pretreatment of mice with the complement-activating factor of cobra venom (CoF), which lowered the serum C3 levels to 10-25% of the normal value, could completely prevent CFUs mobilization induced by high doses of CoF, endotoxin (ET) from Salmonella typhosa, inulin, zymosan and the proteolytic enzymes proteinase and trypsin. On the other hand, mobilization induced by the polyanions dextran sulphate and the copolymer of polymethacrylic acid and styrene could not be prevented, or at least affected only slightly. There appears to be a relationship between the extent of decomplementation by CoF and the extent of CFUs mobilization induced by ET. The results indicate that certain agents mobilize CFUs via the complement system, whereas other agents induce CFUs mobilization independent of the availability of complement components.     

Enhanced Post-Irradiation Recovery of the Haemopoietic System In Animals Pretreated With A Variety of Cytotoxic Agents

It is known that pretreatment of mice with bacterial endotoxin and certain stathmokinetic agents between 1 and 3 days prior to exposure to ionizing radiation reduce radiation lethality. In this communication it is shown that pretreatment with cytosine arabinoside, methotrexate, nortestosterone and chlorambucil reduces radiation (1000 rad) induced lethality. This reduction can be ascribed to enhanced regeneration of the haemopoietic system in pretreated animals and not to increased survival of colony-forming cells (CFU) in these animals. Regeneration of CFUs was underway within 24 hr after 900 rad in the pretreated mice but did not start until day 3 in mice treated with γ radiation only. Two agents, namely radiation itself (either 75 or 150 rad) and busulphan (10 mg/kg) did not reduce the lethal effects of subsequent γ irradiation nor enhance the regeneration of CFUs, even though radiation, like the protective cytosine arabinoside, induces early CFUs proliferation. The administration of nucleoside precursors of DNA enhanced regrowth of haemopoietic stem cells to an extent comparable with that of the most effective pretreatment, cytosine arabinoside. It is postulated that drugs like cytosine arabinoside operate by causing cell death, providing a source of DNA that can enhance the regrowth of surviving stem cells in the bone marrow.     

A COMPARATIVE STUDY OF THE REPOPULATING POTENTIAL OF GRAFTS FROM VARIOUS HAEMOPOIETIC SOURCES: CFU REPOPULATION

The growth rate of the CFU populations in spleens and femora has been studied in irradiated mice injected with cell suspensions, containing equivalent number of CFU, from various sources. The doubling times are shown to be dependent upon the source of the cells. Grafts of bone marrow, spleen and foetal liver cells produced doubling times in the spleen of approximately 25, 19 and 16 hr respectively. Grafts of marrow-derived and spleen-derived spleen colony cells both gave rise to CFU doubling times lower than those of the corresponding primary grafts (approx. 33 and 26 hr respectively in the spleen). In the case of both bone marrow and spleen grafts the CFU population growth was shown to be independent of the size of the graft. A hypothesis is advanced which invokes at least a dual population of CFU, having different doubling times, different seeding capacities in the haemopoietic tissues following i.v. injection and present in different proportions in the various haemopoietic tissues.     

Effect of Hyperthyroidism On Haemopoietic Stem Cell Kinetics In Mice

Changes in the pool of haemopoietic colony-forming units (CFUs) of bone marrow and spleen were studied in experiments with mice fed dried thyroid gland (TH) for 21 days, and during the 13 days that followed feeding. After HU treatment, the number of CFUs in DNA synthesis was estimated. As early as the second day of TH treatment, the pool of CFUs is gradually increased, leading to an increase in the total number of splenic and bone marrow CFUs persisting after TH treatment for the period examined. Simultaneously, the numbers of nucleated cells in the bone marrow and spleen are increased. During TH feeding and following its termination, the total number of erythrocytes and the haematocrit values did not change significantly, whereas an increased number of leucocytes was observed in the peripheral blood after TH treatment. Elevation of the proliferative activity of CFUs occurred early in the period of TH treatment, with the maximum attained by end of the first week of TH feeding. This suggests a rapid response of the haemopoietic stem cell compartment to the administration of TH hormones. the participation of humoral factors controlling CFUs compartments in the mechanism of the stimulatory effect of TH hormones on haemopoietic stem cells is discussed.     

Genetic Control of Lipopolysaccharide-Induced Mobilization of Cfus Dissociation Between Early and Delayed Mobilization of Cfus In Complement C5-Deficient Mice and Lps Non-Responder Mice

Lipopolysaccharide (LPS)-induced mobilization of CFUs from haemopoietic tissues into the circulation has a biphasic pattern. the first rise occurs within 30 min of LPS injection, the second 4–7 days later. This second rise coincides with an increase of the CFUs number in the spleen from about 3000 to about 50,000. We have investigated the relationship between the two peaks by making use of complement C5-deficient mouse strains and the LPS non-responder mouse strains C3H/HeJ and C57BL/10ScCr. These latter two strains lack a serologically identifiable structure (‘LPS-receptor’) which is present in all LPS-responder strains. After injection of eleven different mouse strains with LPS, the numbers of circulating CFUs increased rapidly in all strains, except in the C5-deficient A/J, AKR/J, DBA/2J and B10.D2/oSn mice. On the other hand, the delayed LPS-induced accumulation of CFUs in blood and spleen occurred in all mouse strains tested, including the C5-deficient strains, but not in the LPS non-responder strains C3H/HeJ and C57BL/10ScCr. These results show that (a) early LPS-induced mobilization of CFUs is dependent on the availability of C5, in contrast to the delayed CFUs accumulation in blood and spleen, (b) the presence of the LPS receptor is not required for early CFUs mobilization by LPS and (c) recognition of the mobilizing agent by a specific receptor is required for the delayed accumulation of CFUs in blood and spleen.     

THE f NUMBER OF PRIMARY TRANSPLANTED SPLENIC COLONY-FORMING CELLS

The proportion of murine haemopoietic stem cells that settled in the spleen, after transplanting spleen cells into lethally-irradiated recipient mice, was found by comparing the number of spleen colonies obtained by transplanting a whole spleen with an estimate of the total number of colony-forming units (CFU) present in the intact spleen. the latter number was estimated assuming that endogenous spleen colonies were produced from surviving spleen-derived CFU which exhibited the same survival parameters as transplanted CFU.
Account was taken of the post-irradiation loss of CFU from the spleen in the endogenous assay, which was found to be a reasonably constant factor for doses higher than about 100 rad X-rays.
The total measured number of CFU/spleen from transplantation was less than the number calculated in the intact spleen by a factor, the primary f number, of 0.03 ± 0.02.     

Further studies on mobilization of CFUs.

Mobilization of CFUs from haemopoietic tissues into circulation was studied after injection of different bacterial lipopolysaccharides (LPS), zymosan, phytohaemagglutinin (PHA), concanavalin A (Con A), trypsin and di-isopropyl-fluorophosphate-inhibited trypsin. All bacterial LPS used gave an increase of CFUs in the peripheral blood at 1 h after i.v. injection. Some variation in activity could not be excluded. As with Salmonella typhosa LPS, zymosan gave an increase in circulating CFUs during the first few hr and a second peak a few days later. After injection of zymosan as well as S. typhosa LPS the second peak in the blood was accompanied by a large increase in CFUs numbers in the spleen. PHA gave an immediate mobilization of CFUs, but the mobilization after injection of Con A during the first few hr occurred more slowly. After injection of S. typhosa LPS, zymosan and PHA the blood C3 level was found to be depressed considerably. This might indicate that the complement system is involved in the early mobilization of CFUs. Dexamethasone, a synthetic hormone which has been reported to give sequestration of several cell types in the bone marrow, did not inhibit the early and late mobilization of CFUs which normally occurs after injection of S. typhosa LPS.     

THE ROLE OF THE SPLEEN IN THE REPOPULATION OF THE HAEMOPOIETIC SYSTEM OF HEAVILY-IRRADIATED MICE

The respective role of the spleen or of the bone marrow in the regeneration of the haemopoietic progenitor compartment of heavily-irradiated mice has been investigated. Splenectomy was used to this end in animals injected with exogenous isogenic cells or regenerating from endogenous spleen or marrow cells. Analysis of the data as a function of time shows that the presence of the spleen affects marrow CFU repopulation only at the early post-irradiation stages. The expansion of the marrow progenitor pool proceeds, however, rather independently of the spleen and marrow CFU remain eventually as the main source of haemopoietic cells in the surviving mice. Thus the reaction of the spleen may be envisaged as a fast, important but transient contribution to the overall haemopoietic function of heavily-irradiated animals.     

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.     

The influence of dietary protein deficiency on haemopoietic cells in the mouse.

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1-4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs--although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18%) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstructive role during the refeeding process.     

Bone Marrow Response to Damage Induced By Hydroxyurea Or Colchicine

The extent of bone marrow damage caused by the administration of single or repeated doses of either hydroxyurea (1000 mg/kg b.w.) or colchicine (1 mg/kg b.w.) are comparable. This conclusion is based on serial studies of bone marrow cellularity and of the CFUc numbers in the bone marrow. the proliferation response of the pluripotential haemopoietic stem cells, determined by the cells forming colonies in the spleen of lethally irradiated mice (CFUs) markedly differs if the bone marrow damage is caused by hydroxyurea or colchicine. While hydroxyurea administration stimulates a large proportion of the resting G₀ cells into the cell cycle, the damage induced by colchicine is followed by only a mild increase in the CFUs proliferation rate. The seeding efficiency of the spleen colony technique has been determined after both hydroxyurea and colchicine administration. This parameter, important for the estimation of the number of the pluripotential haemopoietic stem cells in blood forming organs, is significantly affected by hydroxyurea administration, but also by repeated injections of colchicine. Following a single dose of hydroxyurea, the time-course of the CFUs numbers, which were corrected for the change in the seeding efficiency, shows an overshoot occurring after 18–20 hr. At the other time periods, the number of pluripotential haemopoietic stem cells is little affected by a single hydroxyurea injection. This poses a question about the nature of the stimulus, which after hydroxyurea administration triggers the CFUs from the resting G₀ state into the cell cycle. There is evidence that this stimulus is probably not represented by the damage caused to the various intensively proliferating cell populations of the bone marrow. This evidence is based on experiments which show that colchicine induced damage, of a degree similar to that after hydroxyurea, does not stimulate the CFUs proliferation rate to an extent comparable to hydroxyurea. The possibility that colchicine could block CFUs in the G₀ state or that it could interfere with the progress of CFUs through the G₁ and S phases of the cell cycle have been ruled out by experiments which demonstrated that colchicine (1 mg/kg b.w.), administered 10 min before hydroxyurea, does not reduce the number of CFUs triggered into the cell cycle as the consequence of hydroxyurea administration.     

STUDY ON THE PROLIFERATIVE STATE OF HAEMOPOIETIC STEM CELLS (CFU)

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUJ in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU_S (endo-CFU,) and exogenous CFU_S (exo-CFU_s) are identical. After irradiation (650 R) the surviving endo-CFU_s begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3′,5′-monophosphate shortly after marrow cell graft, triggers the transplanted CFU_S into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea.     

KINETICS OF HAEMOPOIETIC RECOVERY IN ENDOTOXIN-TREATED MICE

Kinetics of mouse spleen colony forming units were studied after intra-peritoneal injection of 1 μ/g body weight bacterial endotoxin S. typhosa. When these mice were used as unirradiated and sublethally irradiated donors, it was possible to study the effect of the endotoxin injection upon the cells. Use of the treated mice as irradiated recipients of normal cells gave information about the host effect. In treated unirradiated mice, the total nucleated cell and the CFU counts were disturbed, and 2 days later a large fraction of the CFU were found in the DNA synthesis (S) phase. This meant that injection of endotoxin generated factors affecting the kinetics of the CFU and triggering the resting CFU into the proliferative cycle. If then the mice were given supralethal irradiation and used as recipients of normal bone marrow cells, more CFU seeded to the spleen as compared to normal recipients; but the dip and the growth rate of the CFU were not changed. Hence the endotoxin-generated factors had been eliminated in 2 days. A total body sublethal irradiation by 400 rad X-ray 2 days after endotoxin injection reduced the post-irradiation dip in the recovery curve of the CFU, indicating that though the factors affecting the cell kinetics had been eliminated, the cycling CFU behaved like a growing population. During the first week, the growth rate of the CFU remained the same as in control irradiated mice. The growth rate of the spleen CFU of the endotoxin-treated mice slowed down during the second week, and their self-replicating ability was low. Fluctuations in the DNA synthesizing fraction of the spleen CFU suggested a variability in the ratio of the length of the S phase and the cell generation time.     

ORGANIZATION OF HAEMOPOIETIC STEM CELLS: THE GENERATION-AGE HYPOTHESIS

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 12 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half, to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

Complement Split Product C5a Mediates the Lipopolysaccharide-Induced Mobilization of Cfu-S and Haemopoietic Progenitor Cells, But Not the Mobilization Induced By Proteolytic Enzymes

Abstract. Intravenous (i.v.) injection of mice with lipopolysaccharide (LPS), and the proteolytic enzymes trypsin and proteinase, mobilizes pluripotent haemopoietic stem cells (CFU-s) as well as granulocyte-macrophage progenitor cells (GM-CFU) and the early progenitors of the erythroid lineage (E-BFU) from the haemopoietic tissues into the peripheral blood. We investigated the involvement of the complement (C) system in this process. It appeared that the early mobilization induced by LPS and other activators of the alternative complement pathway, such as Listeria monocytogenes (Lm) and zymosan, but not that induced by the proteolytic enzymes, was absent in C5-deficient mice. the mobilization by C activators in these mice could be restored by injection of C5-sufficient serum, suggesting a critical role for C5.
The manner in which C5 was involved in the C activation-mediated stem cell mobilization was studied using a serum transfer system. C5-sufficient serum, activated in vitro by incubation with Lm and subsequently liberated from the bacteria, caused mobilization in both C5-sufficient and C5-deficient mice. C5-deficient serum was not able to do so. the resistance of the mobilizing principle to heat treatment (56°C, 30 min) strongly suggests that it is identical with the C5 split product C5a, or an in vivo derivative of C5a. This conclusion was reinforced by the observation that a single injection of purified rat C5a into C5-deficient mice also induced mobilization of CFU-s.     

Study on the proliferative state of haemopoietic stem cells (CFU).

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUs) in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rated of endogenous CFUs (endo-CFUs) and exogenous CFUs (exo-CFUs) are identical. After irradiation (650 R) the surviving endo-CFUs begin to proliferate immediately. By contrast exo-CFUs transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosin 3',5'-monophosphate shortly after marrow cell graft, triggers the transplanted CFUs into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFUs become to be increasingly sensitive to hydroxyurea.