Differences in cytokine and chemokine responses during neurological disease induced by polytropic murine retroviruses Map to separate regions of the viral envelope gene

Abstract

Infection of the central nervous system (CNS) by several viruses can lead to upregulation of proinflammatory cytokines and chemokines. In immunocompetent adults, these molecules induce prominent inflammatory infiltrates. However, with immunosuppressive retroviruses, such as human immunodeficiency virus (HIV), little CNS inflammation is observed yet proinflammatory cytokines and chemokines are still upregulated in some patients and may mediate pathogenesis. The present study examined expression of cytokines and chemokines in brain tissue of neonatal mice infected with virulent (Fr98) and avirulent (Fr54) polytropic murine retroviruses. While both viruses infect microglia and endothelia primarily in the white matter areas of the CNS, only Fr98 induces clinical CNS disease. The pathology consists of gliosis with minimal morphological changes and no inflammation, similar to HIV. In the present experiments, mice infected with Fr98 had increased cerebellar mRNA levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), TNF-beta, and interleukin-1 alpha and chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta, monocyte chemoattractant protein 1 (MCP-1), gamma-interferon-inducible protein 10 (IP-10), and RANTES compared to mice infected with Fr54 or mock-infected controls. The increased expression of these genes occurred prior to the development of clinical symptoms, suggesting that these cytokines and chemokines might be involved in induction of neuropathogenesis. Two separate regions of the Fr98 envelope gene are associated with neurovirulence. CNS disease associated with the N-terminal portion of the Fr98 env gene was preceded by upregulation of cytokines and chemokines. In contrast, disease associated with the central region of the Fr98 env gene showed no upregulation of cytokines or chemokines and thus did not require increased expression of these genes for disease induction.

FIG. 1

Cytokine and chemokine mRNA expression in the cerebellum of mice infected with Fr54 or Fr98. Mice were infected i.p. with 10⁴ FFU of either Fr98 or Fr54 at 24 to 48 h after birth. Cerebellums were removed at 14 days postinfection. Total RNA was purified from the cerebellum and analyzed for cytokine mRNA expression using the RNase protection assay. RNase protection assay results from two mice from each infected group are shown.

FIG. 2

Kinetics of cytokine mRNA expression in the cerebellum of mice infected with Fr54 or Fr98. Mice were infected i.p. with 10⁴ FFU of either Fr98 or Fr54 at 24 to 48 h after birth. Cerebellums were removed at the times indicated postinfection. Total RNA was purified from the cerebellum and analyzed for cytokine mRNA expression using the RNase protection assay. Results are expressed as the ratio of cytokine RNA to L32 (housekeeping gene) RNA for each sample. Results are the average of three to six mice per data point. Mock- and FB29-infected mice were also analyzed as a control for cytokine expression at day 14 postinfection. The arrow at day 14 indicates time of onset of clinical ataxia in Fr98-infected mice.

FIG. 3

Kinetics of chemokine mRNA expression in the cerebellum of mice infected with Fr54 or Fr98. Total cerebellar RNA was analyzed for chemokine expression using the RNase protection assay described in Fig. 1. The specificity of MCP-1 and IP-10 expression by the RNase protection assay (11) was confirmed by a probe set containing only MCP-1, L32, and GAPDH or IP-10, L32, and GAPDH. Results are the average for three to six mice per data point. Mock- and FB29-infected mice were also analyzed as a control for cytokine expression at day 14 postinfection.

FIG. 4

Cytokine and chemokine mRNA expression in the cerebrum of mice infected with Fr98 or Fr54 or mock infected at 14 days postinfection. Total cerebrum RNA was analyzed for chemokine expression using the RNase protection assay described in Fig. 1. Results are the average for three mice per data point.

FIG. 5

Cytokine and chemokine mRNA expression in the cerebellum of mice infected with SE. (A) Cerebellums were removed from Fr98-, SE-, or mock-infected mice at 14 days postinfection. At this time point, only Fr98-infected mice had clinical symptoms of disease. Total RNA was analyzed for cytokine and chemokine mRNA expression as described in the legend to Fig. 1. Results are the average for four to six mice per group. (B) Cerebellums were removed from SE-infected mice after the development of clinical symptoms and were compared to those of age-matched but nonsymptomatic SE-infected mice or mock-infected mice. Results are the average for four to eight mice per group.

FIG. 6

Cytokine and chemokine mRNA expression in the cerebellum of mice infected with EC. (A) Cerebellums were removed from Fr98-, EC-, or mock-infected mice at 14 days postinfection. Only Fr98-infected mice had clinical symptoms of disease. Total RNA was analyzed for cytokine and chemokine mRNA expression as described in the legend to Fig. 1. Results are the average for four to six mice per group. Although the increase in IP-10 expression at 2 weeks postinfection in EC-infected mice is significant, this could be due to the very low values for the mock-infected control at this time point. (B) Cerebellums were removed from EC-infected mice after the development of clinical symptoms and compared to those of age-matched but nonsymptomatic mice infected with the nonvirulent virus EC-1, which differs from EC by two amino acids in the envelope gene. RNAs from the cerebellum of age-matched mock-infected mice were used as additional negative controls. Results are the average for four to eight mice per group.

FIG. 7

Correlation between envelope sequences and cytokine and chemokine expression. The viral genomes of Fr98, Fr54, SE, and EC are shown. Black bars indicate Fr98 envelope gene sequences, hatched bars indicate Fr54 envelope gene sequences, and white bars indicate FB29 sequences. Development of clinical signs of ataxia, seizures, and death occurred at approximately 2 weeks in Fr98-infected mice, 4 to 10 weeks in SE-infected mice, and 3 to 5 weeks in EC-infected mice. High virus load indicates that viral p30 expression in the cerebellum at the time of clinical disease was two- to threefold higher than that in mice with low viral loads (30). Upregulated expression of cytokine and chemokine mRNA was determined by comparison to mock-infected controls as described in Fig. 1 through 5.