Protection against murine leukemia virus-induced spongiform myeloencephalopathy in mice overexpressing Bcl-2 but not in mice deficient for interleukin-6, inducible nitric oxide synthetase, ICE, Fas, Fas ligand, or TNF-R1 genes

Abstract

Some murine leukemia viruses (MuLVs), among them Cas-Br-E and ts-1 MuLVs, are neurovirulent, inducing spongiform myeloencephalopathy and hind limb paralysis in susceptible mice. It has been shown that the env gene of these viruses harbors the determinant of neurovirulence. It appears that neuronal loss occurs by an indirect mechanism, since the target motor neurons have not been found to be infected. However, the pathogenesis of the disease remains unclear. Several lymphokines, cytokines, and other cellular effectors have been found to be aberrantly expressed in the brains of infected mice, but whether these are required for the development of the neurodegenerative lesions is not known. In an effort to identify the specific effectors which are indeed required for the initiation and/or development of spongiform myeloencephalopathy, we inoculated gene-deficient (knockout [KO]) mice with ts-1 MuLV. We show here that interleukin-6 (IL-6), inducible nitric oxide synthetase (iNOS), ICE, Fas, Fas ligand (FasL), and TNF-R1 KO mice still develop signs of disease. However, transgenic mice overexpressing Bcl-2 in neurons (NSE/Bcl-2) were largely protected from hind limb paralysis and had less-severe spongiform lesions. These results indicate that motor neuron death occurs in this disease at least in part by a Bcl-2-inhibitable pathway not requiring the ICE, iNOS, Fas/FasL, TNF-R1, and IL-6 gene products.

FIG. 1.

Incidence of paralysis in IL-6 KO mice. (A) Newborn IL-6^+/− or IL-6^−/− littermate mice were inoculated with ts-1 MuLV and observed for the development of paralysis. (B) Pathological assessment of the CNS of IL-6^+/− and IL-6^−/− mice. Spongiosis developed comparably in both heterozygote (left) and homozygote (right) mice.

FIG. 1.

Incidence of paralysis in IL-6 KO mice. (A) Newborn IL-6^+/− or IL-6^−/− littermate mice were inoculated with ts-1 MuLV and observed for the development of paralysis. (B) Pathological assessment of the CNS of IL-6^+/− and IL-6^−/− mice. Spongiosis developed comparably in both heterozygote (left) and homozygote (right) mice.

FIG. 2.

Incidence of paralysis in iNOS, ICE, Fas, FasL, and TNF-R1 KO mice. Heterozygote and homozygote newborn littermate mice from each strain were inoculated with ts-1 MuLV and observed for the development of paralysis. (A) iNOS; (B) ICE; (C) Fas; (D) FasL; and (E) TNF-R1.

FIG. 3.

Incidence and extent of paralysis in NSE/Bcl-2 Tg mice. (A and B) Newborn Tg and non-Tg control littermates were inoculated with ts-1 MuLV and observed for the development of paralysis. In Experiment 1 (A), the mice were observed for up to 6 months. Data are presented as cumulative incidence (left) and Kaplan-Meir analysis (with log-rank posttest) (right) (P < 0.00001). (B) A second experiment (Experiment 2) was carried out but was terminated at 2.5 months. For comparison, the data from Experiment 1 at 3 months were replotted. The numbers below the groups represent the numbers of paralyzed mice out of the total numbers of mice under experimentation. (C through E) Quantitation of spongiform lesions in the brain stems of ts-1 MuLV-inoculated NSE/Bcl-2 Tg and non-Tg mice. Three separate brain stem areas (C) exhibiting spongiform changes were assessed for the total number of vacuoles per mm² of tissue surface area (D) and the percentage of tissue area occupied by vacuoles (E). Note that both indices of disease demonstrated a statistically significant reduction in the extent of lesions in Tg mice: (D) non-Tg (n = 7), 218.8 ± 17.6; Tg (n = 12), 131.8 ± 15.4; (E) non-Tg (n = 7), 4.72 ± 0.37; Tg (n = 12), 3.01 ± 0.40 (means ± SEMs).

FIG. 4.

Assessment of virus replication in NSE/Bcl-2 Tg mice. (A) In situ hybridization was performed with sense and antisense U3 LTR-specific probes on CNS brain stem sections of ts-1 MuLV-infected adult mice at the time of sacrifice. Note that most infected cells have the appearance of microglial cells, as reported previously. (B) Quantitation of the number of ts-1 MuLV-infected cells from the brain stems of Tg and non-Tg mice. Animals of the same groups as those presented in Fig. 3D were studied. The number of MuLV-infected cells per mm² of tissues exhibiting spongiform lesions was counted. No statistically significant difference in the number of infected cells was observed between Tg and non-Tg animals: non-Tg (n = 10), 75.8 ± 7.8; Tg (n = 15), 93.6 ± 7.1 (means ± SEMs). (C) Quantitation of infectious ts-1 MuLV in CNS, spleen, thymus, and serum of 6-day-old NSE/Bcl-2 Tg and non-Tg mice infected as newborns (≤36 h). PFUs are given as means + SEMs. No statistically significant differences could be determined between any of the groups of tissues analyzed (P > 0.05, as determined by Student's t test).

FIG. 4.

Assessment of virus replication in NSE/Bcl-2 Tg mice. (A) In situ hybridization was performed with sense and antisense U3 LTR-specific probes on CNS brain stem sections of ts-1 MuLV-infected adult mice at the time of sacrifice. Note that most infected cells have the appearance of microglial cells, as reported previously. (B) Quantitation of the number of ts-1 MuLV-infected cells from the brain stems of Tg and non-Tg mice. Animals of the same groups as those presented in Fig. 3D were studied. The number of MuLV-infected cells per mm² of tissues exhibiting spongiform lesions was counted. No statistically significant difference in the number of infected cells was observed between Tg and non-Tg animals: non-Tg (n = 10), 75.8 ± 7.8; Tg (n = 15), 93.6 ± 7.1 (means ± SEMs). (C) Quantitation of infectious ts-1 MuLV in CNS, spleen, thymus, and serum of 6-day-old NSE/Bcl-2 Tg and non-Tg mice infected as newborns (≤36 h). PFUs are given as means + SEMs. No statistically significant differences could be determined between any of the groups of tissues analyzed (P > 0.05, as determined by Student's t test).