Arthur A. Axelrad

Inherited resistance to N- and B-tropic murine leukemia viruses in vitro: Evidence that congenic mouse strains SIM and SIM.R differ at the Fv-1 locus

Abstract The congenic mouse strains SIM and SIM.R, known to differ at a locus that determines susceptibility or resistance to Friend leukemia virus in vivo, were examined for their responses to murine leukemia viruses of different tropism. Responses in vitro were studied by the XC cell plaque assay method in mouse embryo cultures prepared from the 2 strains. With N-tropic viruses, SIM cells were found about 1,000-fold more sensitive than SIM.R cells; with a B-tropic virus SIM.R cells were 100- to 1000-fold more sensitive than SIM cells. The sensitivities of SIM and SIM.R cells to an NB-tropic virus were approximately equal. Similar results were obtained in vivo by the spleen focus assay method with N- and NB-tropic Friend viruses. Cells of reciprocal (SIM × SIM.R) F1 hybrids were found resistant to both N- and B-tropic viruses. The specificity of the response to viral tropism, and the dominance and relative nature of the resistance indicate that the locus at which SIM and SIM.R strains differ is identical with the Fv-1 locus. Infection of SIM mouse embryo cells with an N-tropic virus was found to follow single-hit kinetics; the kinetics of SIM.R infection with the same virus were multi-hit. These findings are consistent with the hypothesis that Fv-1 controls relative resistance to murine leukemia viruses through its effect on the kinetics of infection by these viruses. We have developed continuous cell lines from the SIM and SIM.R strains; after 25 passages these have retained the same susceptibility and resistance to N-, B-, and NB-tropic viruses as the mouse embryo cells from which they were derived.

Fv-2 locus controls the proportion of erythropoietic progenitor cells (BFU-E) synthesizing DNA in normal mice

We have found that Fv-2 on chromosome 9 of the mouse, the locus originally identified as a major determinant of host susceptibility (Fv-2s) or resistance (Fv-2r) to Friend leukemia virus in mice, also functions in uninfected animals, where it determines whether a high (Fv-2s) or low (Fv-2r) proportion of erythropoietic progenitor cells BFU-E are normally engaged in DNA synthesis. Adult mice belonging to five inbred strains of genotype Fv-2rr, two inbred strains and three congenic strains of genotype Fv-2ss, two kinds of F1 hybrid of genotype Fv-2rs, and appropriate controls were given high specific activity 3H--thymidine intravenously for 1 hr and their bone marrow and spleen cells were assayed for surviving BFU-E at 7 days and CFU-E at 2 days in plasma culture. A high proportion of BFU-E in all Fv-2ss and Fv-2rs mice were killed, but all or nearly all of the BFU-E in Fv-2rr mice survived exposure to 3H--thymidine. The allelic difference of Fv-2 had no significant effect on the proportion of other progenitor or stem cells (CFU-E, CFU-C or CFU-S) normally undergoing DNA synthesis in the hemopoietic tissues, or on the hemoglobin concentration, red blood cells, hematocrit, total of differential white blood cell counts in the peripheral blood of these animals. While a few or no BFU-E were killed by 3H--thymidine in adult B6 (Fv-2rr) mice, a high proportion of BFU-E were killed by 3H--thymidine in these animals when they were less than 7 weeks old. Bleeding of adult B6 mice or lethal irradiation followed by repopulation by syngeneic bone marrow cells rendered a high proportion of their BFU-E vulnerable to 3H--thymidine. BFU-E of adult B6 mice which in vivo were unaffected by 3H--thymidine were rapidly killed when exposed to 3H--thymidine in vitro. Fv-2 thus seems to act at or near the G1-S or G0-S boundary to influence the rate or probability of transition between the nonDNA-synthesizing and the DNA-synthesizing states of BFU-E. The gene that controls susceptibility or resistance to a murine erythroleukemia virus appears also to be a regulatory gene that controls the proliferative behaviour of normal cells at a specific stage of erythropoietic differentiation.

Friend virus replication in normal and immunosuppressed C57BL/6 Mice

Young adult C57BL/6 mice are resistant to the replication of Friend virus. We show here that this resistance is not absolute. In 7-week old C57BL/6 mice injected with NB-tropic Friend virus iv, high titers of SFFV could be recovered from the spleen at 8 days after infection but by 21 days, no virus was detectable. A single dose of anti-Thy 1.2 monoclonal antibody iv before FV infection permitted continued replication of SFFV in these animals. This finding strongly supports the hypothesis that SFFV replication in C57BL/6 mice is restricted by a T cell-mediated immune response.

Friend virus replication as a function of age.

We have investigated the effect of age on the replication of Friend spleen focus-forming virus (SFFV). Recovery of SFFV from the spleens of four strains of mice was determined following intravenous infection with NB-tropic Friend virus (FV) complex at ages ranging from 6 to 134 weeks. In C57BL/6 mice, the virus did not replicate in adults up to 40 weeks of age, but beyond that there was a steep exponential increase with age in the amounts of SFFV recoverable. In C3H/He mice, which replicate the virus as young adults, the amount of SFFV recovered was 6-fold greater in old than in young mice. Recovery of virus was biphasic with age in SJL mice; in A strain mice no consistent change with age was noted. In C57BL/6 mice, reconstitution of lethally irradiated recipients with syngeneic marrow cells, followed by i.v. infection with FV, showed that the amounts of SFFV recovered depended on the age of the recipient. The present work shows that Friend SFFV replication is a sensitive indicator and can be used as a tool for the investigation of aging processes. The mechanisms responsible for the age-dependent change in regulation of virus replication and for the polymorphism remain to be determined.

Superoxide Dismutase as an Inhibitor of Erythroid Progenitor Cell Cyclinga

C57Bl/6 (B6) mice and mice of a congeneic strain, B6S, differ in the proportions of erythroid progenitor cells (BFU-E) typically seen in DNA synthesis in in vivo cell suicide assays, and bone marrow supernatants (MS) prepared from B6 mice can inhibit BFU-E cycling in vitro. Using in vitro BFU-E DNA synthesis assays and a model system of BFU-E in culture (DA-1 cells) as screening methods for the detection of inhibitors of BFU-E cycling, we have purified the protein that is apparently responsible for the inhibitory effects of MS on progenitor cells and that is also an antagonist of the stimulatory effects of interleukin-3 (IL-3) on DA-1 cell proliferation in culture. We have identified this protein as the Cu,Zn-containing form of the antioxidant enzyme superoxide dismutase (SOD), which is normally present in large amounts in erythrocytes. MS from B6S mice does not inhibit BFU-E DNA synthesis. However, measurements of SOD activity showed no differences between B6 and B6S mice; thus the difference between the effects of B6S-MS and B6-MS is not due to differences in the levels of SOD present. The inhibitory effects of SOD on BFU-E in vitro are opposed by the stimulatory effects of IL-3 in a dose-dependent manner, and similar interactions between stimulatory and inhibitory factors also appear to determine the effects of mouse-derived preparations on erythroid cells. If the interactions seen in vitro are applicable to the state in vivo, SOD may be a constitutive inhibitor of erythroid progenitor cell cycling in mice, acting in opposition to stimulatory factors whose expression varies in response to genetic and physiological influences.

Culture Systems In Vitro for the Assay of Erythrocytic and Megakaryocytic Progenitors

The production of erythrocytic colonies from murine fetal liver cells seeded in semisolid plasma culture medium was first accomplished in 1971.1 The plasma culture system was then improved2 and is now widely used along with the methylcellulose system3 for the assay of the erythropoietic progenitors, CFU-e and BFU-e, of adult as well as fetal hemopoietic tissues of several species including man.

Superoxide dismutase specifically inhibits erythroid cell DNA synthesis and proliferation.

The antioxidant enzyme superoxide dismutase (SOD) was previously shown to inhibit both the proliferation of murine erythroid DA-1 cells growing in the presence of Interleukin-3 (IL-3) and the DNA synthesis of marrow erythroid progenitor cells (BFU-E) in vitro. We show here that the inhibition of marrow cell DNA synthesis by SOD is specific for BFU-E and erythroid precursors (CFU-E), with other myeloid progenitors (CFU-GM) and stem cells (CFU-S) being unaffected, and IL-3 blocks the inhibitory effects of SOD on BFU-E in a dose-dependent manner. Extending earlier observations on the effects of SOD on cell proliferation, it was found that SOD was capable of inhibiting DA-1 cell proliferation supported by either IL-3 or erythropoietin (epo), but had no effect on IL-3 dependent FDCP-1 cells, nor on epo-dependent HCD-57 cells. Of several murine erythroleukemia cell lines tested, only those transformed with Friend SFFVa virus were inhibited by SOD, while those transformed with Friend SFFVp or MuLV virus were not affected. These results show that the effects of SOD are not antagonistic to particular growth factors but rather the inhibition is specific for erythroid cells, and cells of the proper stage can be inhibited even if they have been transformed to factor independence.

Immunosuppression after injection of friend virus into C57BL/6 mice of different ages

Injection with Friend virus (FV) causes immunosuppression in young and old C57BL/6 mice, i.e. it occurs whether or not the virus replicates very briefly or for a long period. There are only minor age-related differences in the extent of immunosuppression, except that suppression appears to persist somewhat longer in old than in young animals.