Late-onset chronic inflammatory encephalopathy in immune-competent and severe combined immune-deficient (SCID) mice with astrocyte-targeted expression of tumor necrosis factor

Abstract

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.

Figure 1.

Analysis of TNF-α expression. A: Transgene versus endogenous TNF-α expression was distinguished using target-specific probes generated as described in Materials and Methods. Organs were removed from control mice (C) or presymptomatic (P) and symptomatic (S) GT-2 mice. Total RNA (10 μg) was then used for analysis by RPA. B: In situ hybridization and immunohistochemistry was performed as described in Materials and Methods. In situ hybridization was used to detect TNF-α and combined with immunohistochemical staining for GFAP. Co-localization of TNF-α RNA and GFAP protein is shown for astrocytes (arrows) in the cerebellum of a GT-8 transgenic mouse. Original magnification, ×500. C: Immunohistochemical stain for TNF-α demonstrates expression of this protein in cells (arrow) with astrocyte morphology in the cerebellum of a GT-8 transgenic mouse. Original magnification, ×500.

Figure 2.

Routine histological analysis of the CNS. Luxol fast blue stains showing normal morphology of cerebellum and spinal cord is shown in A and D, respectively. Original magnification, ×125. In contrast, large areas of perivascular and parenchymal infiltration as seen in cerebellum (B) or spinal cord (E) from symptomatic GT-8 or GT-2 transgenic mice, respectively. Original magnification, ×125. Higher magnification (×500) view of same sections in B and E, showing dense well organized follicular-like structures (C, arrows) and a perivascular infiltrate (F, arrows).

Figure 3.

Active demyelination in the spinal cord. A and B: CNPase immunohistochemical stain on control (A) and GT-2 (B) spinal cord sections. Original magnification, ×312. C and D: Oil red O stain and immunohistochemistry was performed as described in Materials and Methods. Oil red O stain of neutral fat in wild-type control (C) and GT-2 (D) spinal cord. Original magnification, ×312.

Figure 4.

A: Transverse section from the midthoracic spinal cord of a wild-type (control) mouse showing normal white matter. Note the compact appearance of the myelinated fibers and the absence of inflammatory cells from meninges and parenchyma (araldite-embedded 1-μm-thick section stained with methylene blue. Original magnification, ×200. B: Transverse section of spinal cord from a symptomatic GT-2 mouse with inflammatory changes in the meninges and cord parenchyma. A cluster of foamy macrophages is present at the center of the picture. C: Section from a severely affected region in which the meninges are thickened and infiltrated by mononuclear cells. Within the densely infiltrated spinal cord tissue normal structures are effaced and individual demyelinated axons (arrowheads) as well as small groups of demyelinated axons are present. Original magnification, ×200. Note the greatly swollen axonal profiles (arrows) indicating Wallerian degeneration in groups of axons beneath the inflamed meninges at the top of the picture. D: Electron micrograph illustrating primary demyelination in a group of contiguous axons (arrows) from the severely inflamed region. The axoplasm appears quite normal, but the myelin has been completely removed. The nucleated cells may represent activated microglia having an elongated cytoplasm and scanty cytoplasm. Original magnification, ×5000. E: A foamy macrophage (m) packed with lipid droplets appears between a demyelinated axon (a, arrow) and a plasma cell (p) in a severely inflamed region. Original magnification, ×6000. F: A massively swollen axon is present and is surrounded by normal-appearing myelinated fibers. Note the accumulation of darkly staining organelles, including lysozomal profiles, mitochondria, and membranous debris within the affected axon. Original magnification, ×5500.

Figure 5.

Neurodegenerative changes in the CNS of symptomatic GT-8 transgenic mice. Immunohistochemical stains and their quantification were performed as described in Materials and Methods. A: Quantitative analysis of changes in parvalbumin (PV), calbindin (Calb), MAP-2 levels, and in DNA fragmentation (APO). B, upper panel: Parvalbumin-immunostained sections showing cerebellum from wild-type mice and presymptomatic or symptomatic GT-8 mice Original magnification, ×650. B, lower panel: Analysis of DNA fragmentation by TUNEL stain in the spinal cord from control mice and presymptomatic or symptomatic GT-2 mice. Original magnification, ×640.

Figure 6.

Immunophenotypic characterization of inflammatory lesions in the cerebellum of a symptomatic GT-8 mouse. Immunohistochemistry was performed as described in Materials and Methods. Comparison of a perivascular (A, C, E, G, I, and K) with a parenchymal lesion (B, D, F, H, J, and L) immunostained for CD45 (A and B), B220 (C and D), CD4 (E and F), Mac-1 (G and H), CD8 (I and J), and neutrophils (K and L).

Figure 7.

Flow cytofluorimetric analysis of mononuclear cells isolated from the lymph node of wild-type control or spinal cord of symptomatic GT-2 mice. A: Characterization of lymphocyte phenotypes. Expression of CD44, CD62L, and CD69 by CD4⁺, CD8⁺, or B220 cells in control (open histogram) or transgenic (solid histogram ) mice. Similar patterns of expression were seen in the cerebellum of symptomatic mice from the GT-8 line. Infiltrating T and B cells were not detected in the CNS of control mice. B: CD45 levels of FcR-positive cells isolated from control (solid histogram) or from transgenic (open histogram) mice. Note that the x axis is in log scale for both A and B.

Figure 8.

Expression of cellular adhesion molecules in the spinal cord. Frozen sections (10 μm) were prepared and immunostained with specific monoclonal antibodies against murine ICAM-1, VCAM-1, MAdCAM, or endoglin as described in Materials and Methods. All panels are the same original magnification (×500).

Figure 9.

Cytokine and chemokine expression in the CNS of GFAP-TNFα mice (GT-2). Total RNA from CNS tissue from control (c), presymptomatic GT-2 (p), or symptomatic (s) GT-2 was analyzed for cytokine (A) and chemokine (B) expression as described in Materials and Methods. Principal areas with altered cytokine and chemokine expression co-localized with transgene expression.

Figure 10.

Immunophenotypic characterization of inflammatory lesions in GFAP-TNFα ^SCID/SCID mice. Frozen sections (10 μm) of brain from transgenic (A–F) or wild-type (G and H) mice were immunostained with monoclonal antibodies to identify leukocytes (CD45; A and G), macrophage/microglia (Mac-1; B and H), T lymphocytes (CD4 and CD8; C and D, respectively), B lymphocytes (E), and MHC class II (F).