Willie W. Smith

Effect of endotoxin on the kinetics of hemopoietic colony-forming cells in irradiated mice.

Hemopoietic recovery in irradiated animals is hastened by the action of bacterial endotoxin given as a single injection either before or shortly after irradiation (1). Bone marrow cellularity, peripheral blood granulocyte, erythrocyte, platelet, and lymphocyte concentrations, and survival are favorably affected and, in the mouse, the number of endogenous spleen colonies is increased (2-4). The fact that endotoxin is effective when given after irradiation rules out several modes of action. These include chemical protection, hypoxia, increased numbers of stem cells at the time of irradiation, and decreased sensitivity of the stem cells to the immediate killing action of radiation. Among the possibilities remaining are recovery of cells that otherwise would die on the resumption of mitosis, early resumption of mitosis, and shortening of the cell cycle. Means of exploring such possibilities are provided by the technique for enumerating colony-forming units (CFU) developed by Till and McCulloch (5-7). Suspensions containing hemopoietic cells are injected into recipient mice whose endogenous spleen colony formation has been suppressed by lethal irradiation and the resulting spleen colonies are counted 10 days later. The transplanted colonyforming units have many of the characteristics of stem cells (8), and in the present experiments we assume that their enumeration provides an assay of stem cells. The experiments presented here concern population changes in colony-forming units in spleen and femoral marrow resulting from an injection of Salmonella typhosa endotoxin 24 hours before or a few minutes after irradiation with 400 rads.

Effects of Bacterial Endotoxin on the Occurrence of Spleen Colonies in Irradiated Mice

It is generally agreed that survival in the midlethal radiation dose range depends principally on hemopoietic recovery. Administration of endotoxin as a single injection either before or shortly after irradiation in the mouse hastens recovery of granulocytes, erythrocytes, platelets, and lymphocytes (1, 2), increases resistance to infection (3), and reduces mortality (4). In contrast, multiple injections given before irradiation cause no advance in leukocyte recovery or increase in survival (4, 5), even though such injections in nonirradiated mice cause greater increases in spleen weight and resistance to bacterial challenge than a single injection. Till and McCulloch have observed that nodules seen in the spleen 10 days after irradiation are erythroid, granulocytic, megakaryocytic, or mixed (6) and that their number is related to radiation dose and survival (7, 8). The present experiments show that a single injection of endotoxin increases splenic nodules as well as granulocyte count and survival. In contrast, the multiple injections increase the number of splenic nodules (though to a lesser extent) without promoting marrow recovery or survival.

Induced Changes in Transplantability of Hemopoietic Colony Forming Cells

Summary Hemopoietic colony forming cells can be estimated from the assay of CFU, provided the transplantation fraction is known. The fraction for first transplantation cannot be determined by the present methods and has been assumed to be the same as the measurable fraction obtained from a second transplantation. Fractions of CFU recovered on third transplantation were found to be the same as on second, supporting this assumption. Transplantability was altered by treatment of donor mice with irradiation, endotoxin, and VLB. Thus, the loss in CFC between minutes after and 1 day after irradiation was less than the loss in CFU. Pretreatment of irradiated mice with endotoxin or VLB caused a greater reduction in the transplantation fraction than radiation alone. Thus, 1 day after irradiation, differences in CFC values between control and treated mice were even greater than the differences in CFU values previously reported. Clearly, changes in transplantability can be induced and this must be taken into account in the interpretation of effects of various treatments on hemopoietic colony forming cell populations.

ESTIMATION OF RADIATION DOSE-REDUCTION FACTOR FOR

As recovery progresses in irradiated mice, colonies of hemopoietic cells become grossly visible on the spleen, the number of colonies being inversely related to radiation dose (1). In studying characteristics of the colony-forming units, Till, McCulloch, and others most frequently employ donor cells injected into lethally irradiated recipients (for example, 2, 3). For some purposes, however, it is appropriate to study colonies arising endogenously. It seemed to us that the endogenous colony counts might be used to advantage in estimating dose-reduction factors (DRF) for radioprotective agents. Mortality as a basis for DRF estimation has the obvious disadvantage that death is a remote effect of irradiation, subject to conditions which may be difficult or impossible to control. M\oreover, the period of approximately 30 days needed for completion of a test is considerably longer than the time required for protection to become apparent. Although granulocyte and lymphocyte counts in peripheral blood may be used for DRF estimation as early as 3 or 4 days after irradiation (4), these also are subject to extraneous influences, both physiological and technical. Reduction in the number of colony-forming units (CFU) would appear to be a more direct effect of irradiation than either death of the animal or leucopenia, and hence a preferable end point. Meaningful counts in untreated mice can be made over a dose range approximately equal in extent to the range yielding 1 % to 99 % mortality and have the advantage of being completed 10 days after irradiation. The distribution of endogenous colony counts is quite skewed, however, and a transformation is needed if the counts are to be used for DRF estimation.

beta

Damage in the precursors of blood cells due to incorporation of tritiated thymidine was evaluated in vivo. The parameter measured was the speed of recovery of peripheral blood cells after isologous marrow transplantation in irradiated mice. Donor animals received a total of either 20 or 40 mu C of H/ sup 3/-thymidine per gm of body weight. A delay in recovery was demonstrable only with small inocula, even after the very large doses of triated thymidine were administered. (auth)

-MERCAPTOETHYLAMINE BY ENDOGENOUS SPLEEN COLONY COUNTS

Summary Intraperitoneal injections of cortisone, or subcutaneous injections of ACTH, failed to increase survival time or number of irradiated mice surviving.

Toxicity of Tritiated Thymidine to Bone Marrow Transplants

The recovery of bone marrow and splenic hemopoiesis was studied in irradiated mice given colchicine either 2 days before or shortly after wholebody irradiation. The preirradiation treatment accelerates both marrow and splenic regeneration, with resulting increased survival due to early recovery of normal granulocyte levels. Postirradiation treatment accelerates splenic erythropoietic regeneration, but fails to improve marrow regeneration, resulting in persistent granulocytopenia and in death rates similar to that found in the controls. Colony-forming units, as assayed by Till and McCullochs method, responded similarly. They recovered earlier in the marrow of the pretreated animals, but failed to do so in the marrow of mice that received colchicine after irradiation. In contrast, the time of recovery of splenic CFU was advanced with postirradiation treatment as well as with treatment given 2 days before irradiation. The observations presented add to the mounting evidence that either committed and noncommi...

Failure of Cortisone or ACTH to Reduce Mortality in Irradiated Mice

Summary The net effect of a sufficiently cold environment is to lower the resistance of mice to the lethal action of X-rays. Subjection to a 10° or 30°C environment for 2 weeks prior to irradiation favors survival of irradiated mice maintained in those environments. Mice kept in a 30° environment that die following irradiation frequently show fatty changes in the liver.

KINETICS OF COLCHICINE-INDUCED HEMOPOIETIC RECOVERY IN IRRADIATED MICE.

Effects of radiation on survival and proliferation of stem cells were compared in weanling and adult mice, and the observed differences related to the differences in granulocyte count and 30-day LD...

Effect of Environmental Temperature on the Response of Mice to Whole-Body Roentgen Radiation

Summary Exposure to radiation, especially in the lethal range, caused a marked increase in sensitivity of mice to S. typhosa endotoxin. This was most pronounced on the third day after irradiation. At that time the endotoxin LD50 in mice given 1000 rads was 0.5 μg compared to a control value of 480 μg per 20 g mouse. Shielding the hind legs was without effect on endotoxin LD50 (2.3 μg for 850 r, 200 KV X-rays). Shielding the back, including the adrenals, was partially effective (LD50, 55 μg). Treatment with cortisone had an effect comparable to that of shielding the back. Shielding the front of the animal, including practically all of the intestinal tract, was very effective in spite of the fact that the adrenals were irradiated (LD50, 410 μg). Shielding a small portion of the abdomen was also quite effective in reducing endotoxin sensitivity in the irradiated mice (LD50, 165 μg).