Role of splenic stroma in the action of bacterial lipopolysaccharides on radiation mortality: a study in mice carrying the Slj allele

Abstract. Slj/+ mice display a slight macrocytic anaemia due to a defect in their haemopoietic organ stroma. They have a deficient endogenous spleen colony (CFU‐end) formation following sublethal doses of gamma‐radiation compared with their normal +/+ littermates, which is likely to be due to the low pre‐irradiation CFU‐S content of the Slj/+ spleen. CFU‐S in these congenic mice do not differ in their sensitivity to gamma‐irradiation or stem cell‐activating factor. While injection of +/+ mice with 10 μg of lipopolysaccharide‐W (LPS) one day prior to irradiation led to a substantial increase in their survival, the survival of Slj/+ mice was only slightly increased. Irradiation induced a similar dose‐related reduction in the numbers of CFU‐S in the spleen and femora of LPS‐injected Slj/+ mice compared to similarly treated +/+ mice when measured directly after irradiation. At Day 9 after irradiation, injection of LPS led to a significantly higher CFU‐end formation and higher numbers of CFU‐S and nucleated cells in the Slj/+ spleens compared to LPS‐injected +/+ mice. No such differences in the radioprotective effect of LPS were observed in the +/+ and Slj/+ mice with respect to the splenic and femoral 59Fe‐incorporation and the femoral CFU‐S numbers at Day 9. These data strongly suggest a contribution by immigrating CFU‐S to the CFU‐S numbers and endogenous colony formation in at least the Slj/+ spleen after LPS injection and subsequent sublethal irradiation. The observations also imply that the splenic organ stroma may play a mediatory role in the radioprotective action of LPS. In addition, the data represent an extreme example of a lack of correlation between animal survival and haemopoietic parameters. Caution should be taken when applying endogenous colony counts as a means of screening potential anti‐radiation drugs. Copyrigh

Characteristics of the Cfu‐S Population In Mice Carrying the SlJ Allele

Abstract Abstract. A tentative characterization of haemopoietic stem cells with respect to their organ distribution, seeding fraction and colony formation in the spleen, radiosen‐sitivity and humoral regulation was attempted in mice heterozygous for the mutant allele SlJ and in their normal littermates. SlJ/+ mice were characterized by a deficient CFU‐s content of the blood and spleen and had slightly lower femoral CFU‐s numbers. This CFU‐s distribution could not be explained by differences in seeding efficiency ‘f’ between CFU‐s of SlJ/+ and +/+ origin in lethally irradiated recipients used in the CFU‐s assay. the seeding fraction of CFU‐s of +/+ origin did not differ in +/+ and SlJ/+ recipients. However, in irradiated SIJ/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFU‐s of both +/+ and SlJ/+ origin. the serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in SlJ/+ mice, but significantly increased in Sl/Sld mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in SlJ/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFU‐s from spleen and bone marrow had a similar survival following in‐vitro y irradiation. Femurs and spleens of both SlJ/+ and +/+ origin were implanted into both SlJ/+ and +/+ hosts. Six weeks later the SlJ/+ grafts contained less CFU‐s than the +/+ grafts. These data show that the splenic stroma of SlJ/+ mice is not defective in its capacity to lodge injected CFU‐s but is deficient in its ability to maintain CFU‐s under ‘steady‐state’ conditions and stimulate their colony formation in a ‘perturbed state’. Some of the characteristics of SlJ/+ mice segregate them from Sl/Sld mice, i.e. a deficient splenic CFU‐s content, normal seeding fractions ‘f’ of CFU‐s from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. the study of the SlJ trait may be a useful extension of the current Sl/Sld model for exploration of hereditary defects in haematopoietic stroma. Copyrigh

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. Copyrigh

In vitro frequency analysis of spleen colony-forming and marrow-repopulating hemopoietic stem cells in the mouse

An assay is described for Day-12 spleen colony-forming cells (CFU-S-12) and hemopoietic stem cells with marrow-repopulating ability (MRA) in the mouse using a miniaturized stroma-dependent bone marrow culture assay in vitro. Bone marrow cells are grown in liquid culture in microtiter wells, and the resulting adherent stromal layers are depleted of all hemopoietic activity by 20 Gy gamma irradiation. Subsequently, single cell suspensions containing stem cells are overlaid in a range of concentrations, and the presence of one or more emerging phase nonrefractive cell clones (cobblestone areas) in a single well scored as positive. The frequencies of cobblestone area-forming cells (CAFC) are then calculated by employing Poisson statistics. It is shown that the CAFC Day-10 and CAFC Day-28 frequencies closely correlate with those of CFU-S-12 and MRA cells, respectively

Sensitivity of murine haemopoietic stem cell populations to X-rays and I MeV fission neutrons in vitro and in vivo under hypoxic I. Conditions

The radiosensitivity of primitive haemopoietic stem cells that repopulate the bone marrow with precursors of granulocytes and macrophages (MRA[CFU-C]), mature stem cells capable of forming spleen colonies in lethally irradiated recipients (CFU-S-7) and colony-forming units in culture (CFU-C) were determined in vitro and under hypoxic conditions in vivo for 1 MeV fission neutrons and 300 kV X-rays. The obtained D0's were compared with previously observed D0's after irradiation in vivo under normal oxic conditions. With 1 MeV fission neutron irradiation no significant difference in radiosensitivity of the cell populations was observed between normal in vivo irradiation and in vitro irradiation. With 300 kV X-rays a lower radiosensitivity for all three cell populations was observed after in vitro compared to in vivo irradiation. In vivo irradiation with fission neutrons under hypoxic conditions led to a small decrease in radiosensitivity. The obtained oxygen enhancement ratio (OER) for fission neutrons varied from 1.2 for MRA[CFU-C] to 1.5 for CFU-C. After in vivo irradiation with 300 kV X-rays under hypoxic conditions much higher OERs were observed. An OER= 1.8 was obtained for CFU-S and for MRA[CFU-C] and for CFU-C OER 3.0 and 2.9 were observed. These results indicate that the radioresistance of primitive haemopietic stem cells (MRA[CFU-C]) compared to mature stem cells (CFU-S-7) is mainly due to intrinsic factors and not to differences in localization or oxygenation between primitive and mature stem cells

Regulation of haemopoietic stem‐cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Slj allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone‐marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU‐s) proliferation in Slj/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU‐e and CFU‐G/M) kinetics after TBI and +/+ bone‐marrow transplantation in Slj/+ and +/+ mice. the Slj/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Slj/+ spleen colonies contained the same number of BFU‐E and CFU‐G/M as colonies from +/+ spleens, while their CFU‐s content was increased. On day 10 post‐transplantation, the macroscopic ‘missing’ colonies could be detected at the microscopic level. These small colonies contained far fewer CFU‐s than the macroscopic detectable colonies. Analysis of CFU‐s proliferation‐inducing activities in control and post‐LPS sera revealed that Slj/+ mice are normal in their ability to produce and to respond to humoral stem‐cell regulators. We postulate that Slj/+ mice have a normal number of splenic stromal ‘niches’ for colony formation. However, 35% of these niches is defective in its proliferative support. Copyrigh