Phenotypic and functional changes induced in hematopoietic stem/progenitor cells after gamma-ray radiation exposure.

Ionizing radiation (IR) exposure causes rapid and acute bone marrow (BM) suppression that is reversible for nonlethal doses. Evidence is accumulating that IR can also provoke long-lasting residual hematopoietic injury. To better understand these effects, we analyzed phenotypic and functional changes in the stem/progenitor compartment of irradiated mice over a 10-week period. We found that hematopoietic stem cells (HSCs) identified by their repopulating ability continued to segregate within the Hoechst dye excluding "side population (SP)" early after IR exposure. However, transient phenotypic changes were observed within this cell population: Sca-1 (S) and c-Kit (K) expression levels were increased and severely reduced, respectively, with a concurrent increase in the proportion of SP(SK) cells positive for established indicators of the presence of HSCs: CD150 and CD105. Ten weeks after IR exposure, expression of Sca-1 and c-Kit at the SP cell surface returned to control levels, and BM cellularity of irradiated mice was restored. However, the c-Kit(+)Sca-1(+)Lin(-/low) (KSL) stem/progenitor compartment displayed major phenotypic modifications, including an increase and a severe decrease in the frequencies of CD150(+)Flk2(-) and CD150(-)Flk2(+) cells, respectively. CD150(+) KSL cells also showed impaired reconstituting ability, an increased tendency to apoptosis, and accrued DNA damage. Finally, 15 weeks after exposure, irradiated mice, but not age-matched controls, allowed engraftment and significant hematopoietic contribution from transplanted congenic HSCs without additional host conditioning. These results provide novel insight in our understanding of immediate and delayed IR-induced hematopoietic injury and highlight similarities between HSCs of young irradiated and old mice.

Low-dose X-irradiation and 4-hydroperoxycyclophosphamide distinguishes three Lyt-1+ lymph node T-cell subtypes that respond to specific antigen in vitro.

We investigated whether preculture exposure to low-dose X-irradiation and culture treatment with 4-hydroperoxycyclophosphamide (4-HPCY), a synthetic derivative of cyclophosphamide (CY), might distinguish subtypes of thymic-dependent (T) lymphocytes that respond to specific antigen in vitro. Lymph node (LN) cells were obtained from mice pretreated with CY and immunized with aggregated (A) human IgG (HGG) in Freund's complete adjuvant (CFA), and proliferation was assessed by incorporation of tritiated thymidine. Primed LN cells were untreated or exposed to low-dose irradiation before being cultured in medium alone and in medium containing 4-HPCY. The results show that these agents (irradiation and 4-HPCY) distinguished, in a dose-dependent manner, subtypes of T-cells which contribute to the specific antigen-stimulated proliferative response in vitro. For LN T-cells and LN Lyt-1+ T-cells, 20-25 rads and 1.0 microM 4-HPCY inactivated non-overlapping cell subtypes that respectively accounted for 26% and 28% of the response to HGG. The remaining 46% of HGG-responding cells were not affected by either agent. Although similar cell subtypes were discerned in unseparated LN cells, it required use of higher agent-doses. Cell cycle analysis revealed that treatment with irradiation, 4-HPCY, and the combination (both agents) caused S-phase arrest of 29%, 30%, and 55% of HGG-responding cells, respectively. Thus, identification of these cell subtypes could not be attributed to agent-mediated inactivation of HGG-responding cells that might be in exclusively different phases of the cell cycle.

[Nonidentity of Lyt phenotypes and the radiosensitivity of early and late producers of macrophage migration inhibitory factor in reaction to H-2 complex antigens]. / Neidentichnost' Lyt-fenotipov i radiochuvstvitel'nosti "rannikh" i "pozdnikh" produtsentov faktora podavleniia migratsii makrofagov pri reaktsii na antigeny kompleksa H-2.

MIF production induced at different times after intravenous immunization of mice with irradiated allogeneic splenic cells showed different sensitivity to the treatment with anti-Lyt-antibodies and to gamma-irradiation. The "early" MIF producers induced several hours after alloimmunization were sensitive to irradiation at a dose of 500 rad and to the treatment with anti-Lyt-1- and anti-Lyt-2-antibodies and complement, while the "late" MIF producers which appeared 21 days after alloimmunization were resistant to irradiation at doses of 500 and 1500 rad and to the treatment with anti-Lyt-2-antibodies but sensitive to anti-Lyt-1-antibodies. It is supposed that the "early" MIF producers of the Lyt-1+2+ phenotype are immature precursors of T cells which, in contradistinction to the "late" MIF producers of the Lyt-1+2+ phenotype, are activated and produce MIF without proliferation after a twofold contact with antigen.

Low dose radiosensitivity of alloimmune cytotoxic T cells.

Low dose radiosensitivity of in vitro generated alloimmune murine cytotoxic T lymphocytes (CTL) was studied. It appears that a subset of CTL exists that can be killed with 10-25 rad of x rays. These radiosensitive CTL are Lyt-1,2+ T lymphocytes. Analyses of cytotoxicity by chromium release assays indicate that the radiosensitive CTL are present in responder spleen cell cultures from all strains of mice tested. The generation of these effector cells is most pronounced in animals of the C57BL background. The mechanism of low dose radiosensitivity appears to be interphase death.