Critical role for protein tyrosine phosphatase SHP-1 in controlling infection of central nervous system glia and demyelination by Theiler's murine encephalomyelitis virus

Abstract

We previously characterized the expression and function of the protein tyrosine phosphatase SHP-1 in the glia of the central nervous system (CNS). In the present study, we describe the role of SHP-1 in virus infection of glia and virus-induced demyelination in the CNS. For in vivo studies, SHP-1-deficient mice and their normal littermates received an intracerebral inoculation of an attenuated strain of Theiler's murine encephalomyelitis virus (TMEV). At various times after infection, virus replication, TMEV antigen expression, and demyelination were monitored. It was found that the CNS of SHP-1-deficient mice uniquely displayed demyelination and contained substantially higher levels of virus than did that of normal littermate mice. Many infected astrocytes and oligodendrocytes were detected in both brains and spinal cords of SHP-1-deficient but not normal littermate mice, showing that the virus replicated and spread at a much higher rate in the glia of SHP-1-deficient animals. To ascertain whether the lack of SHP-1 in the glia was primarily responsible for these differences, glial samples from these mice were cultured in vitro and infected with TMEV. As in vivo, infected astrocytes and oligodendrocytes of SHP-1-deficient mice were much more numerous and produced more virus than did those of normal littermate mice. These findings indicate that SHP-1 is a critical factor in controlling virus replication in the CNS glia and virus-induced demyelination.

FIG. 1.

Focal demyelinating lesions in dorsal cervical spinal cords of moth-eaten (me/me) mice at 5 days after i.c. inoculation with the attenuated BeAn 8386 strain of TMEV. Five-micrometer paraffin sections were stained with rat monoclonal antibody to MBP and labeled with FITC-conjugated secondary antibody. Sections were photographed with color film by double exposure under both FITC (green) and red fluorescence filter sets. Green fluorescence labels MBP. Red profiles above the background in panel D indicate autofluorescent red blood cells in focal hemorrhagic lesions in regions of demyelination in the parenchyma of the spinal cord. The dorsal funiculus of the cervical spinal cord is shown in either TMEV-infected (B and D) or sham-infected (A and C) normal littermate mice (A and B) and me/me mice (C and D).

FIG. 2.

Demyelination, virus infection, and inflammation in the ventral cervical spinal cords of me/me mice 5 days after intracerebral inoculation with TMEV. Immunohistochemical staining of MBP (A) and TMEV (B) and H+E staining (C) in adjacent 5-μm paraffin sections are shown.

FIG. 3.

Double immunofluorescence of TMEV (A, C, and E; FITC) and PLP (B, D, and F; TRITC) in the ventral cervical spinal cords of me/me (A to D) mice 5 days after inoculation with TMEV. (A and B) Doubly labeled cells (arrows) in the ventral funiculus at the interface between the white matter and the gray matter of the medial nuclei. A large motoneuron in the gray matter also contains TMEV antigen (arrowhead). (C and D) Doubly labeled cells (arrows) in a demyelinated region of the ventral funiculus adjacent to the ventral median fissure (∗). (E and F) No TMEV antigens are seen in cervical spinal cords of +/− animals infected with TMEV.

FIG. 4.

TMEV infection of GFAP-positive astrocytes in moth-eaten mouse cervical spinal cords (A and B) and corpus callosa (C and D) 5 days after inoculation with TMEV. Arrows indicate astrocytes doubly labeled for TMEV (FITC) and GFAP (TRITC). The arrowheads in panels A and B indicate TMEV-infected neurons in the spinal cord gray matter that are not stained for GFAP. (E and F) Normal littermate mouse cervical spinal cords show no TMEV-infected cells.

FIG. 5.

Double immunofluorescence of MBP (TRITC; red) and TMEV (FITC; green) in the brain 5 days after inoculation with TMEV. Five-micrometer paraffin sections were stained with rat monoclonal antibody to MBP and labeled with TRITC-conjugated secondary antibody. TMEV antibodies were detected with FITC-conjugated antibodies. Sections were photographed with color film by double exposure of the same frame under both FITC and TRITC filter sets. (A) ×400 magnification of the corpus callosum (lower) at the interface with the cerebral cortex (upper). (B) Higher magnification (×630) of an area similar to that in panel A but centered at the interface, where myelination is sparse, to allow resolution of doubly labeled cells (brownish yellow cell bodies indicated by arrows).

FIG. 6.

TMEV titers in brains and spinal cords of infected me^v/me^v and me/me mice and normal littermate (+/− Control) mice. Brains (A and C) and spinal cords (B and D) were harvested from paralyzed viable moth-eaten (me^v/me^v) (A and B) or moth-eaten (me/me) (C and D) animals along with age-matched sham-infected normal littermate animals (+/− Control). Error bars indicate standard errors of the means. Numbers in the histograms are for control PFU per gram of brain or cord where these cannot be read from the ordinate. Differences in the means between normal littermate mice and either moth-eaten or viable moth-eaten mice were significant (P < 0004) for both brains and spinal cords.

FIG. 7.

Frequency of oligodendrocyte infection in vitro. (A) Double-immunofluorescence analysis of TMEV infection of O1 antigen-positive oligodendrocytes in moth-eaten and normal littermate mice 2 days after inoculation. Individual cells plated on glass chamber slides were labeled for TMEV (TRITC), and O1 antigens (FITC) were photographed at a ×630 magnification. (B) Histogram of doubly labeled TMEV-positive, O1 antigen-positive oligodendrocytes counted in random fields at a ×120 magnification in +/−and me/me cultures infected for 2 days with TMEV. O1 antigen-positive oligodendrocytes (TMEV positive plus TMEV negative) were present at the same densities in the two samples. Error bars indicate standard errors of the means.

FIG. 8.

Double-immunofluorescence analysis of TMEV infection of GFAP-positive astrocytes in moth-eaten and normal littermate mice 2 days after inoculation. Glial cultures of +/− mice (A to D) or me/me mice (E to H) were either inoculated with TMEV (C, D, G, and H) or sham inoculated (A, B, E, and F). The left panels were stained for TMEV antigens, and the corresponding fields represented in the right panels were stained for GFAP.

FIG. 9.

TMEV titers in vitro. Glial cultures of either me/me mice or normal littermate mice (+/− Control) were inoculated with TMEV. Virus was harvested in both supernatants (released) and cell lysates (cell associated) at 3 and 6 days after infection (P.I.) and quantified by plaque assays. Histograms indicate the mean PFU per milliliter in supernatants and PFU per cell based on protein content in cell lysates. Statistical differences between specimens were measured by Student's t test (one tailed). Each experiment was performed in triplicate. Error bars indicate standard errors of the means. Differences in the means between normal littermate (+/− Control) and moth-eaten (me/me) glial cultures were significant at each time after infection for both released and cell-associated viruses (P < 0.001).