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in the Central Nervous System of Mice Protects Against Lethal Neurotropic Viral Infection but Induces Inflammation and Neurodegeneration1
*
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037;
Department of Veterinary Microbiology, Division of Immunology, Swedish University of Agricultural Sciences, Uppsala, Sweden;
Astra Arcus AB, Sodertälje, Sweden; and
§
Department of Pathology, University of San Diego, San Diego, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, appear to have complex and
broad-ranging actions in the central nervous system (CNS) that may
result in protection or injury. To better understand these issues, we
generated transgenic mice that produce IFN-1 chronically
from astrocytes. These glial fibrillary acidic protein-IFN-
transgenic mice developed a progressive inflammatory encephalopathy,
with marked calcium mineralization, meninoencephalitis, gliosis, and
neurodegeneration. Many features of this murine encephalopathy
resembled those found in certain human encephalopathies of unknown
etiology; these diseases, exemplified by Aicardi-Goutières
syndrome and some viral encephalopathies, show increased intrathecal
production of IFN-. Our data suggest that IFN- overproduction may
be the primary factor initiating these human diseases. Following
intracerebral infection with lymphocytic choriomeningitis virus, glial
fibrillary acidic protein-IFN- mice had significantly increased
survival rates associated with markedly reduced virus titers and immune
pathology in the brain but normal peripheral CTL responses. Therefore,
the production of IFN- in the CNS can be a two-edged sword that on
the one hand confers protection against a lethal viral infection but on
the other causes significant injury to the brain. These transgenic mice
provide a novel animal model in which to further evaluate the
mechanisms that underlie the diverse actions of type I IFNs in the
intact CNS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-ß) and type II (IFN-
), respectively. In humans,
at least 15 IFN- subtypes have been identified, whereas there is
only one gene encoding IFN-ß. In vivo, the IFN-s are produced
predominantly by leukocytes (in particular dendritic cells and
macrophages), whereas IFN-ß is produced by a large variety of cells,
including fibroblasts, epithelial cells, and macrophages. Expression of
these type I IFNs, which share a common receptor and have closely
overlapping biologic actions, can be induced by many factors but
particularly by infection with viruses. Type II IFN or IFN- is
produced by activated T lymphocytes and NK cells and binds to a unique
receptor. Although sharing many overlapping actions with type I IFNs,
IFN- also possesses several activities that are unique and relate
particularly to its immunoregulatory functions.
In addition to their antiviral actions, IFNs may profoundly affect a
variety of functions (1, 2, 3, 4), including cellular metabolism, growth and
differentiation, immune function, and tumor development. IFNs exert
their biologic effects by binding to specific high affinity cell
surface receptors, which then signal a cascade of cellular responses
associated with changes in gene expression that lead to an altered
cellular phenotype (5). A number of genes are known to be induced in
cells following their exposure to IFNs, including 2',5'-oligoadenylate
synthetase (2',5'-OAS),3 the
MX gene, ß2-microglobulin, MHC class I and II (induced by
IFN- only) molecules, and elF-2 protein kinase. Induction of some
of these genes is highly specific for IFNs and has led to their use as
surrogate markers for the presence of IFNs in different disease states.
The protein products of some of these IFN-response genes (e.g.,
2',5'-OAS, elF-2 protein kinase, and MX protein) are known to
inhibit viral replication and therefore may contribute directly to the
antiviral state conferred on uninfected cells exposed to IFNs. Other
protein products of IFN-response genes (e.g., MHC molecules) do not
affect viral replication directly but enhance recognition and killing
of virus-infected cells by antiviral immune processes. IFN production
during viral infection in vivo is therefore an essential factor in the
host antiviral response normally being induced within hours of systemic
viral infection and decreasing with resolution of the infectious state
(5, 6).
Expression of the type I IFN genes may be induced in the central
nervous system (CNS) in different viral infections, e.g., lymphocytic
choriomeningitis virus (LCMV) infection in mice (7). However, the
cellular sources for IFN production in the CNS are poorly defined.
Glial cells (astrocytes and microglia) have been shown to produce type
I IFNs in vitro, suggesting that these cells might provide a local
source of IFN production to defend the brain against viral insult (8, 9). There is evidence to suggest that the type I IFNs may be toxic to
the brain. Thus, when used in systemic pharmacologic therapy, IFN-
can produce a flu-like syndrome; behavioral and cognitive impairments;
and, in rare circumstances, cerebral coma and death (10, 11). In
addition, increased intrathecal IFN- has been associated with
certain familial encephalopathies, including the
Aicardi-Goutières syndrome (12, 13, 14) and Cree encephalitis (15)
(P. Lebon and D. Black, unpublished observations). However, whether
IFN- has a cause and effect role in the development of these and
other neurologic disorders is not clear. Therefore, the aim of the
present study was to generate transgenic mice (termed GIFN mice) with
astrocyte-targeted expression of IFN- in which we could explore 1)
the neurologic impact of IFN- expression in the intact CNS and 2)
the antiviral properties of local IFN- production following
intracerebral infection of the transgenic mice with LCMV.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fusion gene and production of transgenic mice
The methods used for the construction of the
GFAP-IFN-1 fusion gene and the generation and screening
of transgenic mice were similar to those described previously for the
generation of GFAP-IL-6 and GFAP-IL-3 transgenic mice (16, 17).
Briefly, a murine IFN-1 cDNA (a kind gift of Dr. E.
Zwarthoff, Erasmus University, Rotterdam, the Netherlands) containing
the entire coding region was inserted between an upstream SV40 intron
and a downstream SV40 polyadenylation sequence, at the SalI
site within the first exon of the modified GFAP expression vector. The
GFAP-IFN- transgene was microinjected into the germline of
C56BL/6J x BALB/c F1 hybrid mice, and transgenic
offspring were screened for integration by tail DNA dot blot
hybridization using a 32P-labeled SV40 polyadenylation
sequence fragment as probe.
RNA isolation and analysis
Organs were removed and immediately frozen in liquid nitrogen,
and poly(A)+ RNA was isolated as described previously (18).
Total RNA was extracted directly from frozen tissue or astrocyte
cultures with TRIZOL reagent (Life Technologies, Grand Island, NY) used
according to the manufacturer’s instructions. RNase protection assay
for IFN- was performed as described previously (7). For Northern
blot hybridization, poly(A)+ RNA was denatured,
electrophoresed in 1% agarose/2.2 M formaldehyde gels, transferred to
nylon membranes, and hybridized overnight at 45°C with
32P-labeled cDNA probes. Probes used were: a 2-kb
EcoRI fragment of murine 2',5'-OAS cDNA (cDNA kindly
provided by Dr. Bryan Williams, Cleveland Clinic Foundation, Cleveland,
OH), a 0.6-kb KpnI-SacI fragment of a murine MHC
class I (H2Db) cDNA (provided by Dr. P. Petersen, Scripps
Research Institute), a 0.932-kb fragment of murine MHC class II
(Iab) chain (19), and a 0.26-kb fragment of the murine
ß-actin gene (20).
Astrocyte cultures and determination of IFN- production and
activity
Astrocytes were prepared from cerebrum and cerebellum of
individual neonatal GIFN-39 transgenic mice (see Results) or
wild-type (wt) littermates as described previously (21). The cells were
seeded in 25-cm2 Falcon culture flasks (Becton Dickinson,
Franklin Lakes, NJ) in complete medium and incubated at 37°C in an
atmosphere of 5% CO2 with 95% humidity. After 2 days in
culture, conditioned medium was collected for assay of secreted
IFN-. Immunoreactive IFN- was measured by dissociation-enhanced
lanthanide fluoroimmunoassay (7). Briefly, microplates were coated with
sheep anti-mouse IFN- IgG (a kind gift of Dr. Michael Tovey,
Centre National de la Recherche Scientifique, Paris, France), incubated
with samples or IFN- standards for 2 h, and then incubated for
1 h with a rat anti-murine IFN- mAb (0.2 mg/ml; clone
4E-1; Seikagaku America, Rockville, MD) labeled with europium
lanthanide chelate (Wallac Oy, Turku, Finland). After adding
enhancement solution (Wallac), fluorescence was measured in a 1234
DELFIA (dissociation-enhanced lanthanide fluoroimmunoassay) Research
Fluorimeter (Wallac). A laboratory murine IFN- standard was
routinely used consisting of the medium of culture supernatants of
splenocytes stimulated by Sendai virus. This was calibrated against a
National Institutes of Health murine IFN- standard (batch Ga
02-901-511). The biologic assay for IFN- used a conventional
cytopathic effect inhibition assay using crystal violet staining of L
cells following infection with vesicular stomatitis virus (22).
In situ hybridization
Brains were removed, and one hemisphere was fixed overnight in
ice-cold 4% paraformaldehyde in PBS (pH 7.4). Paraffin-embedded
sagittal sections (8 mm) were processed for in situ hybridization as
described previously (23). 35S-labeled cRNA to
IFN-1, MHC class I, 2',5'-OAS, and viral nucleoprotein
(NP), respectively, were used as probes. After hybridization and
posthybridization washes, sections were exposed to autoradiographic
film for 5 to 15 days.
Histology and immunostains
For routine histology, organs were removed, fixed in 4% buffered paraformaldehyde and embedded in paraffin, and stained with hematoxylin and eosin. An alizarin red S technique modified from Dahl (24) was used for identification of calcium. Frozen sagittal brain sections (9 mm) fixed in 95% ethanol were incubated in alizarin red S (Sigma, St. Louis, MO) staining solution, rinsed with acid/ethanol, counterstained with Mayer’s hematoxylin (Sigma), and examined by brightfield microscopy.
Analysis of patterns of neurodegeneration was conducted as described previously (16, 25) by immunolabeling free-floating vibratome sections with Abs against synaptophysin (Boehringer Mannheim, Indianapolis, IN), a synaptic marker; microtubule-associated protein 2 (MAP2), a dendritic marker (Boehringer Mannheim); GAP 43 (Sigma), a marker of sprouting and regeneration; parvalbumin and calbindin (Sigma), markers of interneurons; and ChAT (Chemicon), a marker for cholinergic neurons. Briefly, sections labeled with MAP2 and synaptophysin were recognized with FITC and analyzed with the confocal microscope, whereas the rest were developed with 3',3'-diaminobenzidine (Sigma) and analyzed with Quantimet 570C software (16, 25). For statistical analysis, one-way ANOVA with Dunnett’s t test was used.
For other immunostains, brains were removed from mice, embedded in OCT compound (Sakura Finetek, Torrance, CA) and snap frozen in liquid nitrogen. Sagittal sections (9 µm) cut with the aid of a cryomicrotome were air dried and stored at -70°C. Immediately before staining, sections were fixed in cold (-20°C) methanol:acetone (1:1) solution for 1 min and then incubated for 20 min in PBS containing 2% FCS (to reduce nonspecific reactivity). Sections were incubated with rat mAb to Mac-1 (TIB126; American Type Culture Collection, Manassas, VA), CD4 (L3T4; PharMingen, San Diego, CA), CD8a (Ly-2; PharMingen), B220 (CD45R, PharMingen), ICAM-1 (YN1/1; kindly provided by Dr. F. Takei, Toronto, Canada), and VCAM-1 (CD106; PharMingen), respectively. Bound Ab was detected with a biotinylated anti-rat mAb (Southern Biotechnology Associates, Birmingham, AL), followed by strepavidin-labeled horseradish peroxidase (Boehringer Mannheim). Staining employed 3',3'-diaminobenzidine as substrate. Before mounting, sections were counterstained with Mayer’s hematoxylin.
Electron microscopic analysis
Transgenic mice and controls were killed by intracardiac perfusion with a 3% phosphate-buffered glutaraldehyde solution while under deep anesthesia. Blocks for electron microscopy were taken from the cerebral and cerebellar hemispheres. They were postfixed in 1% phosphate-buffered osmium tetraoxide, dehydrated in serial alcohol solutions, and embedded in araldite resin. Thick sections were cut with a Porter Blum microtome and stained with either methylene blue or paraphenylene diamine. Thin sections from selected blocks were cut with diamond knives using a Sorvall ultramicrotome and stained with uranyl acetate and lead citrate before examination by electron microscopy.
LCMV infection
Because GIFN-39 mice exhibited spontaneous seizure activity, only GIFN-12 mice (see Results) were used in these experiments. At the age used, GIFN-12 mice exhibited minimal neuropathologic alterations; in particular, calcium mineralization and meningoencephalitis were absent. GIFN-12 transgenic mice (6–8 wk old) and wt littermates were inoculated intracranially (i.c.) with 25 µl of serum-free DMEM containing 30 plaque-forming units of LCMV-Arm 53b stock obtained from a triple plaque-purified clone subsequently passaged in BHK cells (26). At indicated times postinfection, mice from each group were killed, and the brain and the spleen were removed and immediately frozen in liquid nitrogen and stored at -70°C pending measurement of LCMV titers. Frozen organs were weighed, homogenized in tissue culture medium M199 containing 10% FCS, and immediately centrifuged to remove debris. Infectious LCMV present in the clarified homogenate was quantitated by plaque assay on Vero cell monolayers as described previously (27). At day 5 postinfection, an additional three surviving infected wt or GIFN-12 mice were killed, and brains were removed and fixed in ice-cold 4% buffered paraformaldehyde solution. After overnight fixation, the tissues were dehydrated, embedded in paraffin, and processed for in situ hybridization and histologic examination as described above.
CTL assays
wt and GIFN-12 mice (both MHC haplotype H-2b/d) were infected with LCMV as described above and killed at day 4 or day 6 postinfection. In addition, as a positive control, BALB/c (MHC haplotype H-2d) or wt and GIFN-12 mice were infected (5 x 106 plaque-forming units) i.p. with LCMV and killed at day 7 postinfection. Single-cell suspensions from spleen were prepared in supplemented (2 mM glutamine, 50 µM 2-ME, 50 µg/ml streptomycin, and 50 U/ml penicillin) DMEM containing 10% FBS (Life Technologies). For the determination of T cell cytotoxicity, a standard 6-h 51Cr release assay, as described by Byrne et al. (28), was performed. BALB/c17 (H-2d) and L929 (H-2k) cell lines were infected with LCMV-Arm.53b at a multiplicity of injection of 0.1, and 48 h later they were used as target cells. The effector splenocytes were incubated in triplicate, with target cells at E:T cell ratios of 50:1 and 25:1. The percentage of specific lysis was calculated as 100 x (cpm release by CTL - cpm spontaneous release)/(cpm total release - cpm spontaneous release).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mice
Two stable lines of mice (termed GIFN-39 and GIFN-12) positive for
IFN- transgene integration were generated showing (see below)
moderate and low levels of transgene-encoded IFN- expression,
respectively. A number of mice from the GIFN-39 line were found to
exhibit physical differences from their wt littermates, in that they
were runted, ataxic, moribund, and susceptible to seizures beginning as
early as 4 wk of age, and they progressed to death at 5 to 10 mo of
age. Mice from the GIFN-12 line had a similar but much less severe
phenotype seen in some older (8–12 mo old) mice that exhibited loss of
weight and became moribund but in which seizures were rarely observed.
CNS- and astrocyte-restricted expression IFN- in GFAP-IFN-
mice
Transgene-encoded expression of IFN- was demonstrated in the
CNS of both GFAP-IFN- lines. By RNase protection assay, there was no
detectable expression of IFN-1 mRNA in the brain of wt
(nontransgenic) control littermates (Fig. 1
a). However, IFN- mRNA was
found to be expressed in the brain of GIFN-39 mice and, at somewhat
lower levels, in GIFN-12 mice. Similar to wt control mice, in the GIFN
mice from both lines, expression of the IFN- mRNA was not detectable
in the peripheral organs, spleen, kidney, and liver. In situ
hybridization analysis (see Fig. 5b) revealed IFN- mRNA
expression detectable in the cerebellum, neocortex, and thalamus of
GIFN-39 mice. Expression of IFN- mRNA was not detectable by this
method in brain from either GIFN-12 or wt mice. Astrocyte cultures
isolated from the cerebrum and cerebellum of GIFN-39 mice produced
significant levels of immunoreactive and bioactive IFN-. In
contrast, supernatants obtained from similarly prepared cultures from
wt mice were found to contain little detectable IFN- activity (Fig. 1b).
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mice
A striking neuropathologic finding in the GIFN mice was the
progressive accumulation of mineral deposits in the cerebellum and
basal ganglia of 4- to 6-mo-old GIFN-39 and 10- to 12-mo-old GIFN-12
mice. The presence of insoluble calcium in these inclusions was
confirmed by alizarin red S staining (Fig. 2, c and d).
Scanning electron microscopy with energy-dispersive x-ray microanalysis
further indicated that these inclusions were composed almost entirely
of calcium and coprecipitating phosphorus (data not shown). In its
advanced stages, calcification in the basal ganglia was reflected, at a
microscopic level, by massive deposition of material obliterating much
of the parenchyma and extending across the midline into the adjacent
basal ganglia region (Fig. 2d). Calcium mineralization in
the granular layer of the cerebellum consisted of concentric, often
laminated deposits (Fig. 2c). In the GIFN-39 mice, calcium
mineralization was observed in perivascular regions as well as in
vessel walls as early as 4 wk after birth. Although some of the
deposits could be localized to the cytoplasm of individual endothelial
cells, these aggregates also formed within the neuropil and were
flanked by clumps of glial filaments, suggesting they may have
originated within astrocytes.
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by astrocytes on
neuronal structure was also analyzed. To evaluate the overall integrity
of the synaptodendritic organization, sections were immunolabeled with
Abs against MAP2 and synaptophysin. Confocal analysis of the sections
showed that in 8- to 12-mo-old GIFN-12 mice, there was a moderate
decrease in synaptophysin and MAP2 immunoreactivity in the cortex and
hippocampus. In contrast, in 4- to 6-mo-old GIFN-39 mice, there was a
widespread loss of these markers. In particular, the molecular layer of
the hippocampus and cortical areas receiving input from the basal
forebrain were affected (data not shown). These neurodegenerative
changes were accompanied by astrogliosis as revealed by the GFAP
immunostains (data not shown). Since these areas receive cholinergic
innervation, we analyzed the integrity of the cholinergic system by
immunolabeling serial sections with a choline acetyltransferase Ab
(Fig. 2
). This analysis showed a transgene dose-dependent decrease in
the number of cholinergic neurons in the septal nuclei and basal
forebrain (Fig. 2, i–k). Since these terminals are known to
express high levels of GAP 43, a molecule associated with neuronal
sprouting, sections immunolabeled with an Ab against this molecule
showed a profound loss of this marker in both the hippocampus and the
neocortex (Fig. 2, e–h). This indicates that degeneration
of cholinergic cells is accompanied by an inability to mount an
appropriate sprouting response. Evaluation of the integrity of the
interneurons and other neuronal populations was conducted using Abs
against parvalbumin and calbindin. These studies showed that both
calbindin- and parvalbumin-immunoreactive neurons in the cortex and
hippocampus were not altered in GIFN-12 mice but were reduced in
GIFN-39 mice (data not shown).
At the untrastructural level, electron-dense debris were present in the
walls of vessels, being located in pericyte cytoplasm as well as within
endothelia (Fig. 3c).
Ependymal cells lining the ventricles and on the epithelial surface of
the choroid plexus also showed abnormalities consisting of swelling and
irregular configuration of their cell surfaces. In areas of
inflammatory cell infiltration, astrocytes contained clusters of glial
filaments that stained intensely for GFAP (data not shown) and were
hypertrophied, suggesting that these cells were activated. Some
perivascular astrocytes contained masses of tubulovesicular
intracytoplasmic material identical with the fine structure of
so-called tubuloreticular or "lupus" inclusions (29, 30). These
inclusions had curvilinear profiles, and some were seen in astrocytes
adjacent to clumps of glial filaments (Fig. 3a). A second
type of inclusion was observed in the endoplasmic reticulum (ER) of
many cells and consisted of tubular infoldings of cisternae (Fig. 3d). These inclusions resembled "cylindrical confronting
cisternae" described in cells with tubuloreticular inclusions (30).
Another noteworthy change in the transgenic mice was a degeneration of
myelinated axons in the cerebellar white matter. At the light
microscopic level, this manifested as bubbly disintegration of the
myelin sheath, whereas electron microscopic examination revealed
swollen axons containing masses of organelles, most prominently
lysosomes, accumulated in a manner consistent with wallerian
degeneration. Some axons contained profiles resembling ER membrane
arranged concentrically inside the myelin sheath, obliterating the
axonal cytoplasm (Fig. 3e). Similar membranous complexes
appeared elsewhere in the neuropil (Fig. 3b). Abnormal
membrane formation was noted in the ER of many cells. Primary
demyelination was not observed in the tissue examined.
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mice revealed inflammation with discrete perivascular accumulations of
mononuclear cells (see below). No evidence of hemorrhage was found;
however, blood vessels were dilated, and endothelial cell hypertrophy
was apparent.
CNS-infiltrating inflammatory cells are predominantly lymphocytes
in GFAP-IFN- mice
The identity of the inflammatory cells present in the brain of the
GFAP-IFN- mice was determined by immunohistochemical staining with
specific Abs. Different leukocyte subsets were identified in the
perivascular cuffs, including CD4+ T cells (Fig. 4b), CD8+ T cells
(Fig. 4c), and B220+ B cells (Fig. 4d). In addition, CD4+ T cells were also
observed in many areas of the brain, including in the meninges, in the
dentate gyrus of the hippocampus, scattered throughout the brain
parenchyma, and in the choroid plexus of both older (6–12 mo old)
GIFN-12 and younger (1–6 mo old) GIFN-39 mice (data not shown). In
addition to the immune cells, a generalized activation of microglia was
observed throughout most of the brain, as revealed by increased
expression of the microglia/macrophage marker Mac-1 as well as by
marked hypertrophy of the microglia (Fig. 4f). During
inflammatory responses, multiple factors contribute to leukocyte
recruitment and trafficking through the CNS. Cell adhesion molecules,
such as ICAM-1 and VCAM-1, both members of the Ig superfamily, have
been implicated in this process (31, 32). In the GFAP-IFN-
transgenic mice, markedly increased expression of ICAM-1 (Fig. 4h) but not VCAM-1 (data not shown) by the cerebrovascular
endothelium was seen throughout the brain.
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mice
The cellular response to IFNs is known to involve the induction
and the synthesis of a number of proteins that directly or indirectly
mediate many actions of IFNs. We examined the expression of two
prototypic IFN- responsive genes, 2',5'-OAS and MHC class I. As
shown by Northern blot hybridization (Fig. 5a), compared with
nontransgenic controls, a transgene dose-dependent increase in the
expression of both of these genes was observed in the brain of
GFAP-IFN- transgenic mice. A slight increase in cerebral MHC class
II gene expression was also noted in the GIFN-39 mice. In peripheral
organs, small increases in the expression of these IFN-responsive genes
were seen variably in the spleen and the liver but not the kidney of
the GFAP-IFN- mice. By in situ hybridization (Fig. 5b), a
diffuse pattern of expression was observed throughout the brain for the
MHC class I and 2',5'-OAS RNA. This was particularly evident in the
GIFN-39 mice, with high levels of MHC class I RNA expressed in the
olfactory bulb, cortex, hippocampus, thalamus, and choroid plexus,
whereas expression of 2',5'-OAS RNA was highest in dentate gyrus,
choroid plexus, ependyma, and the granular cell layer of the
cerebellum.
Protection from LCMV with reduced viral loads and inflammation in
the CNS but normal CTL activity in GFAP-IFN- mice
IFN- is implicated in the host defense against virus infection.
To assess the antiviral actions of IFN- in vivo, 6- to 8-wk-old
GIFN-12 mice and wt littermates were inoculated i.c. with a normally
lethal dose of LCMV. By day 8 postinfection, 23 of 24 control
littermates had succumbed compared with 10 of 23 GIFN-12 mice (Fig. 6a). Surviving LCMV-infected
GFAP-IFN- mice remained alive at 28 days postinfection, at which
time no titerable virus or viral NP (determined by RNase protection
assay) could be detected in the brain (data not shown). Analysis of
viral titers in the brain at days 3 and 5 postinfection revealed
significantly reduced levels of LCMV in GIFN-12 mice compared with
controls (Fig. 6, b and c). In contrast, in
spleen at day 3 postinfection, LCMV titers were similar for the GIFN-12
and wt animals (Fig. 6d). By in situ hybridization, the
level and distribution of LCMV NP RNA was observed to be also
considerably reduced in the brain of infected GIFN-12 mice (Fig. 6e).
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). Although there was some killing of
uninfected and infected MHC-incompatible (H-2k) target
cells, which presumably reflected NK cell activity, increased MHC class
I-restricted (H-2d) lysis of LCMV-infected target cells was
observed at day 6 but not at day 4 (data not shown) postinfection in
both infected wt and transgenic mice. However, no significant
difference was observed in the efficiency of killing between the two
groups of animals, indicating that CTL responses developed normally in
the periphery of the LCMV-infected transgenic mice.
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a and b). In
contrast, in GFN-12 mice, the degree of mononuclear cell infiltration
in these brain regions was considerably less, with only modest numbers
of inflammatory cells being discernible (Fig. 8, c and
d). Thus, the reduction in viral load in the CNS of the
GIFN-12 mice was paralleled by a reduction in the severity of the
immune pathology.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the
CNS of transgenic mice was associated with the development of an
adult-onset, progressive inflammatory encephalopathy, with
marked calcium mineralization, meningoencephalitis, gliosis, and
neurodegeneration. A number of observations indicated that the genesis
of this neurologic disorder was directly attributable to the actions of
IFN-. First, expression of transgene-encoded IFN- RNA and
bioactive protein was demonstrated in the CNS. Second, both the
clinical severity and neuropathology correlated with the levels and
distribution of transgene-encoded IFN-. Third, cerebral expression
of the known IFN-responsive genes 2',5'-OAS and MHC class I were
significantly increased. Fourth, many of the clinical and pathologic
findings in the GFAP-IFN- mouse were distinct from those seen in
similarly developed transgenic mice expressing other cytokines under
the control of the murine GFAP promoter, including IL-6 (16), IL-3 (17)
and TGF-ß (34).
In many respects, the neuropathologic findings in the GFAP-IFN-
mouse recapitulate those found in a variety of human encephalopathies
in which there is elevated intrathecal production of type I IFN or
IFN-. These include the familial disorders such as
Aicardi-Goutières syndrome (12, 13, 14), Cree encephalitis (P. Lebon
and D. Black, unpublished observations), and some congenital viral
encephalopathies (35). Our findings, therefore, provide strong support
for the proposition (14) that IFN- is a primary causative factor in
these human disorders and establish the GFAP-IFN- transgenic mouse
as a unique animal model to dissect the molecular mechanisms involved
in the neuropathogenic actions of IFN-.
The cerebral calcification observed in the GFAP-IFN- mice and the
human disorders alluded to above may represent a specific response in
the brain to the presence of this cytokine. Pronounced calcium
mineralization was also found in the testis of mice with transgenic
expression of IFN-1 in this organ (36).
Although IFN- shares many overlapping properties with
IFN-, chronic expression of this cytokine by oligodendrocytes in
transgenic mice was not reported to be associated with cerebral
calcification (37, 38). However, the exact mechanisms involved in the
IFN--induced calcium mineralization process are unknown but likely
emanate from a severe disturbance in cellular calcium metabolism.
Whether this disturbance is due to the direct actions of IFN- or is
secondary to the other pathologic alterations is unclear. In the early
stages of calcification, cerebral blood vessels in the GFAP-IFN-
mice were a prominent site of calcium mineralization, indicating that
changes in cerebrovascular endothelium may be critical in the
development of the mineral deposits. Later, other cells likely
contribute, including astrocytes and neurons, as dense cytoplasmic
calcium deposits were clearly visible in these cells by electron
microscopy. At the subcellular level, we observed extensive morphologic
changes affecting the ER in endothelial cells, astrocytes, and neurons.
In some cases, massive expansion of the ER congested axonal processes
and could have been a cause of neuronal dysfunction and loss. The ER is
known to be a major target for IFN- action both in vitro and in
vivo, where morphologic alterations and inhibition of ER-associated
enzymes has been reported (29, 39, 40). Tubuloreticular inclusions and
cylindrical confronting cisternae are unusual structural formations
arising from the ER that are typically observed in leukocytes and
endothelial cells during viral infections (e.g., HIV infection and in
autoimmune diseases such as systemic lupus erythematosis (reviewed in
30 . These formations have also been observed in various
neurologic disorders including HIV encephalopathy (41) and Alzheimer’s
disease (42). The function of these structures is an enigma; however,
their formation is known to be induced specifically by IFN- (39).
Consistent with this, tubuloreticular inclusions and cylindrical
confronting cisternae were observed in astrocytes and cerebrovascular
endothelial cells in the GFAP-IFN- mice. The ER plays a central role
in regulating cytoplasmic calcium and, therefore, its perturbation by
chronic exposure to IFN- may contribute to the calcification
observed in the CNS of the GFAP-IFN- mice.
Type I IFNs are important immunoregulatory molecules that exert both
positive and negative effects on many limbs of the immune response
(reviewed in 43 , including modulation of B cell function and Ab
production, inhibition of T cell proliferation, enhancement of T cell
and NK cell cytotoxicity, and inhibition of delayed-type
hypersensitivity. The systemic administration of type I IFNs in some
patients with multiple sclerosis has proven to be an effective means of
therapy, the basis for which appears to involve, in part, the
counterregulatory immune actions of these cytokines (10, 44, 45). In
the present studies, the chronic, localized production of IFN- by
astrocytes was associated with the existence of a number of cellular
and molecular changes characteristic of a chronic inflammatory process.
Thus, at the cellular level, progressive meningoencephalitis involving
CD4+ and CD8+ T cells and B cells,
cerebrovascular endothelial cell activation, and marked gliosis with
both structural and functional activation of the astrocytes and
microglia were prominent, whereas at the molecular level, up-regulation
of both the MHC class I and ICAM-I genes was observed. The findings
therefore highlight a propensity of IFN- to promote the recruitment
of lymphocytes (particularly those belonging to the CD4+
subset) into the brain.
GFAP-IFN- mice exhibited significant changes in neuronal morphology
and loss of specific neurons, including cholinergic neurons; injury to
and loss of this population is also seen in Alzheimer’s disease. The
cause of these neuropathologic changes is likely to be complex,
involving direct actions of the transgene-encoded IFN-, as well as
other indirect factors. Thus, as indicated above, the presence of
IFN--associated morphologic structures within some neurons (i.e.,
tubuloreticular inclusions) suggests a direct toxicity of the cytokine.
Neuronal damage may have been further compounded by either seizure or
hypoxia-related injury. It is also conceivable that neuronal injury
could have been inflicted by the associated chronic cerebral
inflammatory response in these mice. In regard to this latter point,
chronic inflammation involving T cells caused pancreatic ß cell
destruction in transgenic mice in which IFN- expression was directed
to the islets of Langerhans under the transcriptional control of the
rat insulin promoter (46). Given the parallels between the GFAP-IFN-
mouse model and a number of human neurodegenerative disorders with
underlying chronic inflammation (e.g., Aicardi-Goutières syndrome
(14, 47) and HIV encephalitis (48)), the mechanisms for the neuronal
demise are of considerable interest and are amenable to more detailed
study in this transgenic animal model.
IFN- is a prototypic antiviral cytokine that is of crucial
importance for survival and recovery from acute viral infections (49, 50). The IFN- transgenic mice represent a useful tool from which to
further explore the antiviral actions of the type I IFN in vivo.
Previous studies using neutralizing Abs have established a pivotal role
for the type I IFNs in the pathogenesis of intracranial LCMV infection
(51, 52). Here we clearly established that preexisting astocyte
expression of IFN- in the CNS can afford a significant level of
protection against the lethal consequences of LCMV infection. This
protection was associated with a marked reduction in the levels of
infectious virus and distribution of LCMV NP in the brain. Consistent
with a direct antiviral effect of the transgene-encoded IFN-, the
reduced CNS LCMV load occurred early after infection before the
appearance of detectable CTL activity and infiltration of the brain
with mononuclear cells. The basis for the inhibitory effect of IFN-
on LCMV replication is not known. In general, the molecular mechanisms
involved in IFN- inhibition of viral replication are not well
understood (3, 50). IFN- is known to induce the expression of a
number of genes, including 2',5'-OAS, shown here to be elevated in the
brain of the GFAP-IFN- mice, that are capable of inhibiting
different steps in the viral replication pathway (53). In any event,
the GFAP-IFN- mice offer new opportunities for deciphering the
mechanism(s) for the antiviral actions of IFN- in vivo.
The anti-LCMV-specific CD8+ CTL response that
targets the brain after LCMV infection is responsible for the demise of
the host (33, 54). In the LCMV-infected GFAP-IFN- mice, the severity
of the immune pathology was considerably reduced and was therefore
likely to be a prime factor contributing to the increased survival of
these mice. The reduced immune pathology most likely is directly
consequent to the diminished LCMV load observed in the brain of the
transgenic mice. It has been suggested previously that the CNS viral
load plays a key role in the targeting CTLs to the CNS following
intracranial LCMV infection (51, 52). An alternative explanation for
the decreased immune pathology in the brain of the LCMV-infected
GFAP-IFN- mice is that it may have been due to suppression of the
antiviral immune response. However, this possibility seems unlikely.
First, surviving LCMV-infected GFAP-IFN- mice were able to clear the
virus from the spleen and the brain, indicating that the CTL response
was not impaired in these animals. Secondly, we observed that in
LCMV-infected GFAP-IFN- mice, CTL activity was comparable with
similarly infected wt control mice. Therefore, the GFAP-IFN- mice
are able to mount an effective anti-LCMV directed CTL response that
is not compromised by the presence of chronic IFN- production in the
CNS.
In conclusion, this report describes a novel transgenic model for the
chronic astrocyte-targeted expression of IFN-. The findings
underscore the pleiotropic nature of this cytokine, which caused a
spectrum of molecular and cellular CNS alterations and induced an
inflammatory encephalopathy. This disorder is remarkably similar to
certain human encephalopathies with documented elevations in
intrathecal IFN production and support the notion that IFN- is a
probable primary pathogenic agent in these human diseases. The findings
also highlight the concept that IFN- on the one hand can confer
protection against a lethal neurotropic virus infection, while on the
other, it can induce CNS injury and neurologic disease. Therefore,
these transgenic mice provide a unique model in which to further
evaluate the mechanisms that underlie the diverse actions of type I
IFNs in the intact CNS. Finally, the clinical implications of our
findings are wide ranging and include the possibility for anti-IFN
therapy for patients with encephalopathies involving the inappropriate
intrathecal production of IFN- and for better understanding and
prevention of the CNS toxicity that limits the effective therapeutic
use of type I IFNs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Iain L. Campbell, Department of Neuropharmacology, CVN-9, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail:
3 Abbreviations used in this paper: 2',5'-OAS, 2',5'-oligoadenylate synthetase; CNS, central nervous system; GFAP, glial fibrillary acidic protein; LCMV, lymphocytic choriomeningitis virus; NP, nucleoprotein; ER, endoplasmic reticulum; MAP2, microtubule-associated protein 2; wt, wild-type.
Received for publication April 6, 1998. Accepted for publication July 1, 1998.
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