Mobilization of haemopoietic stem cells (CFU) into the peripheral blood of the mouse; effects of endotoxin and other compounds.

Factors affecting the circulation of haemopoietic stem cells (CFU) in the peripheral blood of mice were investigated. I.v. injection of sublethal doses of endotoxin, trypsin and proteinase appeared to raise the number of CFU per ml blood from about 30-40 to about 300-400 or more within 10 min. The effect was smaller when smaller doses of the substances were injected. After this initial rise the number of circulating cells returned to normal in a few hours. Following endotoxin there was a second rise which started 2-3 days after injection and attained a peak on the 6th-7th day. The first rise is explained as a mobilization of stem cells from their normal microenvironments into the blood stream ; the second rise is considered to reflect proliferation of CFUs in the haemopoietic tissues. The spleen seems to be acting as an organ capturing CFUs from the blood and not as a source adding stem cells to the blood. The early mobilization of CFU after endotoxin injection did not coincide with a mobilization of neutrophils. The number of circulating band cells was increased during the first hours. The importance of ‘open sites’ in the haemopoietic tissue for capturing CFUs was studied by emptying these sites through a lethal X-irradiation and injecting normal bone marrow cells. When a greater number of syngeneic bone marrow cells was injected intravenously, the level of circulating CFU in irradiated mice was slightly lower than the level in unirradiated mice during the first hours. I N T R O D U C T I O N For several years it has been known that haemopoietic stem cells can be present in the peripheral blood of the mouse. Experimental evidence for the existence of circulating stem cells has been provided by Goodman & Hodgson (1962). These authors were able to promote Correspondence: D r 0. Vos, Department of Cell Biology and Genetics, Rotterdam Medical Faculty, Dr Molewaterplein 50, Rotterdam, The Netherlands. 467 468 0. Vos, W. A . Buurmari and R. E. Ploeniacher survival of lethally irradiated mice by injection of leucocytes from blood of normal donors and they could demonstrate that donor-type erythrocytes and granulocytes were produced in long-term survivors. More direct evidence for circulating stem cells was given by Hanks (19641, Hellman (1965) and Robinson, Commerford & Bateman (1965). In these publications a migration of stem cells from shielded bone marrow towards the spleen was described. Homing of these migrating cells in the spleen could be demonstrated with the endogenous spleen colony technique. Hellman & Grate (1968) observed that an initial rapid migration from the shielded marrow was followed by a slower outflow and they interpreted their findings 'as evidence of there being in the marrow a small pool of colony forming units having easy access to the blood'. They postulated that in addition to the 'small pool of colony-forming units having rapid interchange between blood and bone marrow' there was a slow release of cells from a bound bone marrow stem cell pool. Barnes & Loutit (1967a) and Hellman & Grate (1968) determined that 1 ml of peripheral blood of a normal mouse contains about 10-30 colony forming units (CFU). Although variations in the level of circulating CFU before (Barker, 1970) and after birth (Barnes & Loutit, 1967b) have been described and substances affecting the number of circulating stem cells have been published (Barnes & Loutit, 1967a, b), little is known about the dynamics and the regulatory mechanism for stem cell circulation and migration. Fliedner (personal communication) has made the suggestion that stem cells for bone marrow transplantation might be obtained from peripheral blood with an IBM cell separator. In this way bone marrow puncture of the donor could be avoided. Since the number of stem cells in the blood is low, an elevation of the level of circulating cells would improve the applicability of such a procedure. In the present paper fluctuations in the level of circulating CFU after injection of endotoxin and other substances will be described. The observations will be discussed in relation to dynamics of the stem cell pool and the mechanisms which may regulate it. M A T E R I A L S A N D M E T H O D S Male and virgin female CBA/Rij and F, (C57BL/Rij x CBA/Rij) mice were used. Mice employed as donors were 1&16 weeks old in all experiments. F, male recipients were 10-16 weeks, F, females 4-8 months and CBA male or female recipients 4-10 months old. Weights varied between 20 and 32 g. X-irradiation was performed with a 250 kV Philips X-ray machine; physical constants and other radiation details are described in a previous publication (Vos, 1967). CBA and F1 recipient mice were irradiated with a lethal dose of 770 and 825 rad respectively. These lethal doses were different because in former experiments it had been shown that F, mice were slightly more resistant to X-irradiation than CBA mice. Saltiionella ryphosa and Escherickia coli endot oxin (Bacto-lipopolysaccharide prepared by the Boivin method) solutions were made in Tyrode shortly before use. When not further specified in the text endotoxin from S. r ~ p l ~ o s a was used. Similarly proteinase (crystalline bacterial proteinase, Novo Industry A/s Copenhagen, Denmark), trypsin (1 : 300, Nutritional Biochemicals Corporation) and other solutions were made shortly before injection. 0.5 ml of a solution was injected into the tail vein or intraperitoneally. Each blood sample used for the CFU assay was obtained from the suborbital plexus of three ether anaesthetized mice. 2.5 ml blood was collected in calibrated glass tubes containing 10 ml Tyrode. Clotting was prevented by adding 5 1U heparin to the Tyrode. If necessary the Mobilization of haemopoietic stem cells 469 blood sample was further diluted with Tyrode before it was injected into the tail vein of a lethally irradiated recipient. The dilution which was injected contained 2-20 spleen colony forming cells per 0.5 ml. The correct dilution was estimated from results obtained in previously performed pilot experiments. From each blood sample two dilutions differing with a factor of 2 in blood concentration were made and 0.5 ml of each dilution was injected into ten recipients. Hence the number of CFU per ml blood of each sample was measured by counting colonies in spleens of twenty recipient mice. Blood used for leucocyte counts was obtained from the suborbital plexus. For each leucocyte count another mouse was used. The total number of white blood cells was determined with a Model B Coulter electronic particle counter. The percentage of neutrophil granulocytes was determined in May Griinwald-Giemsa stained blood smears. Bone marrow and spleen cell suspensions were made as described earlier (Vos, 1967). Liver and lung suspensions were made in a similar way as spleen suspensions. The organs were cut in small pieces with scissors and mashed through a nylon sieve. Three dilutions of each suspension were made and 0.5 ml of each dilution was injected into the tail vein of ten mice. Cell suspensions from blood, bone marrow or other organs were injected within a few hours after irradiation of the recipients. For lung and liver suspensions the maximal amount which could be injected was often restricted by the occurrence of death from embolism in the injected mice. The spleens were removed 8 days after injection and fixed in Bouin’s fluid. Colonies were counted under the low power objective of a stereomicroscope. Splenectomy was performed under Nembutal anaesthesia. Sham-splenectomized mice were treated exactly as splenectomized mice, except for the removal of the spleen.