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The Journal of Neuroscience, January 15, 2003, 23(2):481-492
Interleukin-6 Protects Anterior Horn Neurons from Lethal
Virus-Induced Injury
Kevin D.
Pavelko1,
Charles L.
Howe1,
Kristen
M.
Drescher2,
Jeff D.
Gamez1,
Aaron J.
Johnson1,
Tao
Wei3,
Richard M.
Ransohoff3, and
Moses
Rodriguez1
1 Departments of Immunology and Neurology, Mayo Clinic,
Rochester, Minnesota 55905, 2 Department of Medical
Microbiology and Immunology, Creighton University, Omaha, Nebraska
68178, and 3 Department of Neurosciences, Lerner Research
Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
We evaluated the role of interleukin-6 (IL-6) in neuronal injury
after CNS infection.
IL-6 / and
IL-6+/+ mice of resistant major histocompatibility
complex (MHC) H-2b haplotype intracerebrally
infected with Theiler's virus cleared the infection normally without
development of viral persistence, lethal neuronal infection, or late
phase demyelination. In contrast, infection of
IL-6/ mice
on a susceptible H-2q haplotype resulted in frequent
deaths and severe neurologic deficits within 2 weeks of infection as
compared with infected IL-6+/+
H-2q littermate controls. Morphologic analysis
demonstrated dramatic injury to anterior horn neurons of
IL-6/
H-2q mice at 12 d after infection. Infectious
viral titers in the CNS (brain and spinal cord combined) were
equivalent between
IL-6/
H-2q and IL-6+/+
H-2q mice. In contrast, more viral RNA was detected
in the spinal cord of
IL-6/ mice
compared with IL-6+/+ H-2q mice.
Virus antigen was localized predominantly to anterior horn cells in
infected
IL-6/
H-2q mice. IL-6 deletion did not affect the humoral
response directed against virus, nor did it affect the expression of
CD4, CD8, MHC class I, or MHC class II in the CNS. Importantly,
IL-6 was expressed by astrocytes of infected IL-6+/+
mice but not in astrocytes of
IL-6/ mice
or uninfected IL-6+/+ mice. Furthermore, expression
of various chemokines was robust at 12 d after infection in both
H-2b and H-2q
IL-6/
mice, indicating that intrinsic CNS inflammatory responses did not
depend on the presence of IL-6. Finally, in vitro
analysis of virus-induced death in neuroblastoma-spinal cord-34
motor neurons and primary anterior horn cell neurons showed that IL-6
exerted a neuroprotective effect. These data support the hypothesis
that IL-6 plays a critical role in protecting specific populations of
neurons from irreversible injury.
Key words:
Theiler's murine encephalomyelitis virus; interleukin-6; neuron; chemokine; CNS; multiple sclerosis
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Introduction |
A major area of investigation
in neurobiology is directed at understanding factors that participate
in neuronal survival versus death. A number of neurotrophic growth
factors have been identified that promote either neuronal survival or
differentiation (Carlson et al., 1999 ; Middleton et al., 2000; Strelau
et al., 2000). Less attention has been given to the role of cytokines,
because these soluble factors are primarily thought of as immune
modulators. We chose to study the role of interleukin-6 (IL-6) in
neuronal survival because its signal transducing protein, gp130, is
also used by well characterized neurotrophic factors such as ciliary neurotrophic factor (Kopf et al., 1994). IL-6 binds to a specific receptor that subsequently induces homodimerization of gp130, ultimately leading to activation of the Janus kinase/signal transducer and activator of transcription (STAT) signaling pathway. This results in tyrosine phosphorylation of acute phase response
factor/STAT3 and its translocation to the nucleus (Akira, 1997).
This signaling cascade is shared by several IL-6-related cytokines,
including CNTF, leukemia inhibitory factor, oncostatin-M, and
IL-11.
We examined the role of IL-6 in a viral model of CNS
infection that localizes predominantly to neurons early in the course of disease. Intracerebral injection of Theiler's murine
encephalomyelitis virus (TMEV), a picornavirus, induces a
characteristic disease course in the CNS of mice. During the first week
of infection, the virus replicates primarily in neurons of the
hippocampus, striatum, cortex, and anterior horn of the spinal cord and
then is rapidly cleared from the CNS. In addition, distinct arms of the
immune system play a critical role in protecting these cell populations
from viral-induced pathology (Drescher et al., 1999). Oligodendrocytes
and macrophages are also infected early (Njenga et al., 1997), and in
animals of susceptible haplotypes, the virus persists in these cells,
particularly in the spinal cord white matter and brain stem. To examine
the role of IL-6 in TMEV-induced neuropathogenesis, we used mice with a
specific disruption in the IL-6 gene. The original
IL-6/
mice were generated on a B6/129 background [major
histocompatibility complex (MHC) haplotype
H-2b] that is resistant to TMEV
persistence and subsequent demyelination. We also generated a line of
mice with the MHC haplotype H-2q to
address the contribution of IL-6 in mice that develop viral persistence. These mice exhibited decreased survival and increased brain and spinal cord pathology as compared with infected littermate controls. In addition, we found that IL-6 is critical for the prevention of lethal infection of anterior horn motor neurons in
vivo and is able to protect neuroblastoma-spinal cord
(NSC)-34 motor neurons and primary spinal motor neurons from
virus-induced cell death in vitro. Together, these data
support the hypothesis that IL-6 is necessary for prevention of neuron
injury after virus infection of the CNS.
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Materials and Methods |
Virus. The Daniel's strain of TMEV was used for all
experiments (Lipton, 1975).
Mice. B6;129S-IL-6tm1Kopf mice
were obtained from Jackson Laboratories (Bar Harbor, ME). These mice
have a targeted disruption of the IL-6 gene on chromosome 5 and are
homozygous for MHC class-I H-2b.
IL-6/
H-2q mice were generated by backcrossing
the IL-6 knock-outs to B10.Q-H2q/SgJ mice
(Jackson Laboratories). F1 mice were mated to
obtain F2 breeding pairs negative for the
wild-type IL-6 gene and H-2b. Mice were
screened by two different PCR assays. One reaction was performed to
detect either the neo cassette used to disrupt the IL-6 gene or the
wild-type gene, and the other was performed to specifically detect the
neo cassette (Kopf et al., 1994). The first reaction used primers that
flanked the neo cassette on both the forward
(5'-TTCCATCCAGTTGCCTTCTTGG-3') and downstream
(5'-TTCTCATTTCCACGATTTCCCAG-3') ends. This reaction resulted in
either a 174 bp product for the wild-type allele or a 1314 bp product
that included the neo cassette. Mice that were positive for both
alleles indicated heterozygosity and were discarded. Preliminary
IL-6+/+ and
IL-6/
mice were further screened for detection of the neo cassette by using
an internal 3' primer (5'-CCGGAGAACCTGCGTGCAATCC-3') with the former 5'
primer. This reaction resulted in either a positive 380 bp fragment,
indicating the null allele, or in no product, indicating the absence of
the neo cassette. Those animals that were
IL-6/
and IL-6+/+ were further screened by
FACS using an antibody to H-2b (BD
PharMingen, San Diego, CA). Mice that were negative for
H-2b were used as breeders to establish
the H-2q line of mice. All offspring were
screened by both assays to confirm genotype. All experiments were
controlled by comparing
IL-6/
H-2q mice with littermate
IL-6+/+ H-2q mice.
Infection and harvesting of the CNS for morphology. At 4-6
weeks of age, mice were intracerebrally infected with 2 × 105 plaque-forming units (pfu) of TMEV in
a total volume of 10 µl. At various times after infection (or when
moribund), mice were perfused via intracardiac puncture with 50 ml of
Trump's fixative. Spinal cords and brains were removed and postfixed
for 24-48 hr in Trump's fixative in preparation for morphologic analysis.
Spinal cord morphometry. Spinal cords were removed from
spinal columns and cut into 1 mm coronal blocks; every third block was
osmicated and embedded in glycol methacrylate (Rodriguez et al., 1986a,
1991a,b). Sections (2 µm) were prepared and stained with a modified
erichrome/cresyl violet stain (Pierce and Rodriguez, 1989).
Morphological analysis was performed on 12-15 sections per mouse as
described previously (Rodriguez et al., 1986a, 1991a,b). Briefly, each
quadrant from every coronal section from each mouse was graded for the
presence or absence of gray matter disease, meningeal inflammation, and
demyelination. The score was expressed as the percentage of spinal cord
quadrants examined with the pathologic abnormality. A maximum score of
100 indicated that there was a particular pathologic abnormality in
every quadrant of all spinal cord sections of a given mouse. All
grading was performed without knowledge of the experimental group.
Additional spinal cord blocks were embedded in paraffin for immunocytochemistry.
Brain pathology. Brain pathology was assessed at day 12 post-infection (p.i.) using our previously described technique
(Drescher et al., 1999). After perfusion with Trump's fixative, two
coronal cuts were made in the intact brain at the time of removal from the skull (one section through the optic chiasm and a second section through the infundibulum). As a guide we used the atlas of the mouse
brain and spinal cord corresponding to sections 220 and 350 (Sidman et
al., 1971). This resulted in three blocks that were then embedded in
paraffin. This allowed for systematic analysis of the pathology of the
cortex, corpus callosum, hippocampus, brainstem, striatum, and
cerebellum. The resulting slides were then stained with hematoxylin and
eosin. Pathologic scores were assigned without knowledge of
experimental group to the following areas of the brain: cortex, corpus
callosum, hippocampus, brainstem, striatum, and cerebellum. Each area
of the brain was graded on a four-point scale as follows: 0 = no
pathology; 1 = no tissue destruction but only minimal
inflammation; 2 = early tissue destruction (loss of architecture)
and moderate inflammation; 3 = definite tissue destruction
(demyelination, parenchymal damage, cell death, neurophagia, neuronal
vacuolation); 4 = necrosis (complete loss of all tissue elements
with associated cellular debris). Meningeal inflammation was assessed
and graded as follows: 0 = no inflammation; 1 = one cell
layer of inflammation; 2 = two cell layers of inflammation; 3 = three cell layers of inflammation; 4 = four or more cell layers of inflammation. The area with maximal extent of tissue damage was used
for assessment of each brain region.
Clinical disease assessment. Mice were assessed clinically
by three criteria: appearance, activity, and paralysis. A score for
each criterion was given ranging from 0 (no disease) to 3 (severe
disease). For appearance, 1 indicated minimal change in coat, 2 indicated a moderate change (scruffy appearance), and 3 indicated a
severe change (incontinence and stained coat). For activity, 1 indicated decreased spontaneous movements (minimal ataxia), 2 indicated
moderate slowing (minimal spontaneous movements), and 3 indicated
severe slowing (no spontaneous movement). For paralysis, 0.5 indicated
a spastic extremity, 1 indicated a paralyzed extremity, 1.5 indicated
two or more spastic extremities, 2 indicated two paralyzed extremities
(unable to walk), 2.5 indicated no righting response, and 3 indicated
three or four paralyzed extremities (moribund). The total score for
each mouse was the cumulative total from each criterion (maximum of 9).
Survival analysis. Mice were monitored throughout the time
of infection, and deaths were recorded at the end of each week.
Virus-specific antibody isotype ELISA. Whole blood was
collected from mice at time they were killed, and sera was
isolated and stored at  80°C. Total serum IgGs and IgMs against TMEV
were assessed by ELISA as described previously (Njenga et al., 1996). Virus was adsorbed to 96-well plates (Immulon II; Dynatech
Laboratories, Chantilly, VA) and then blocked with 1% bovine serum
albumin (BSA) (Sigma St. Louis, MO) in PBS. Serial serum dilutions were
made in 0.2% BSA/PBS and added in triplicate. Biotinylated anti-mouse IgG or IgM secondary antibodies were used for detection (Jackson ImmunoResearch Labs, Westbury, NY). Signals were amplified with streptavidin-labeled alkaline-phosphatase (Jackson ImmunoResearch Labs)
and detected using p-nitrophenyl phosphate as the substrate. Absorbances were read at 405 nm and plotted against serum dilution factors.
Virus neutralization assay. Virus-neutralizing antibodies
were assessed as described previously (Rodriguez et al., 2000). Briefly, TMEV was diluted to contain 100 pfu per sample and then mixed
with an equal volume of serial twofold dilutions of heat-inactivated sera from infected mice. After incubation on ice for 30 min, this mixture was assayed for infectivity by plaque assay on L2 cells. Data
were expressed as the titer that neutralized 90% of the virus.
Virus plaque assay. Virus titers in brain and spinal cords
were performed at various days after TMEV infection. Assays were performed as described (Rodriguez et al., 1986a). Briefly, brain and
spinal cords were homogenized to yield a 10% w/v homogenate in DMEM
(BioWhittaker, Walkersville, MD). Samples were sonicated, clarified by
centrifugation, and stored at 70°C until the time of plaque assay.
The assay was performed on L2 cells without knowledge of mouse strain.
All dilutions were done in triplicate. Data are presented as
plaque-forming units per gram of CNS tissue.
Immunostaining for virus antigen. Immunocytochemistry was
performed on paraffin-embedded sections as described previously (Drescher et al., 1998). Slides were deparaffinized in xylene and
rehydrated through an ethanol series (absolute, 95, 70, 50%). Virus
antigen staining was performed using polyclonal antisera to TMEV
(Rodriguez et al., 1993), which reacts strongly with the capsid
proteins of TMEV. After incubation with a secondary biotinylated antibody (Vector Laboratories, Burlingame, CA), immunoreactivity was
detected using the avidin-biotin immunoperoxidase technique (Vector
Laboratories). The reaction was developed using Hanker-Yates reagent
with hydrogen peroxide as the substrate (Polysciences, Warrington, PA).
Slides were lightly counterstained with Mayer's hematoxylin. The
number of virus antigen-positive cells was expressed per area of gray
matter or white matter in the spinal cord.
Northern hybridization. Brains and cords were removed from
12 d infected mice, and RNA was isolated using RNA-Stat. Ten
micrograms of total RNA were applied to a formaldehyde gel and blotted
onto nitrocellulose. Blots were probed with random
primer-generated 32P-labeled cDNA probe
specific for either glyceraldehyde-3'-phosphate dehydrogenase (GAPDH)
(a housekeeping gene) or VP2, one of the TMEV capsid proteins. A
Storm Phosphorimager was used to measure the amount of
radiolabeled probe bound to the specific RNAs.
Immunostaining for CD4, CD8, MHC class I, MHC class II, and
IL-6. Frozen sections were prepared by embedding brain and spinal cord tissue from
IL-6/
and IL-6+/+ in OCT embedding compound. The
brain was cut into three coronal blocks, and the spinal cord was
removed in three longitudinal sections. The blocks were then frozen and
stored in liquid nitrogen until immunostaining for CD4, CD8, MHC class
I, MHC class II, and IL-6 was performed. Frozen sections (10 µm) of
the spinal cord and brain were cut and placed on Superfrost plus slides
(Fisher Scientific, Houston, TX) and allowed to air dry. Slides were
fixed in 95% ethanol at 20°C for 20 min and then washed twice with PBS for 5 min each. Sections were then blocked with avidin and biotin
(Vector) for 10 min each and then washed with PBS. Primary antibodies
for CD4, CD8, IL-6 (PharMingen), and antibodies to MHC class I (Y3) and
MHC class II (10216) were used to identify antigens. Detection of
primary antibodies was performed by using the appropriate
biotin-labeled secondary antibody, and detection was performed using
avidin-biotin complex methodology (Vector Labs) and Hanker-Yates
(Polysciences). After development, slides were lightly counterstained
with hematoxylin, dehydrated, and coverslipped.
Flow cytometric analysis. Cells
(106) isolated from the brains of two
IL-6+/+ H-2q
and two
IL-6/
H-2q mice were independently stained with
anti-CD8 allophycocyanin (APC), anti-CD4 PerCp, and anti-B220
phycoerythrin (PE) on ice for 20 min. In two parallel experiments with
the same brain sample, cells were stained with anti-CD8 APC, anti-CD4
PerCp, and either anti-PanNK PE or anti-Mac1 PE. All antibodies are
available from PharMingen. Samples were then washed twice with FACS
buffer (1% BSA and 2% sodium azide), resuspended in cold PBS, and
fixed in 1% paraformaldehyde. Samples were analyzed on a Becton
Dickinson FACScan instrument (Mountain View, CA) using Win MDI software (Scripps, La Jolla, CA). To calculate the relative frequency of lymphocyte subsets to one another in a particular brain cell isolate, all cell types were gated, counted, and compared with the CD4+ T-cell
compartment. Using the CD4+ T-cell compartment as the standard enabled
us to estimate the relative frequency of each immune cell compartment
across three separate experiments performed on a single brain sample.
Expression of chemokines. The brain and spinal cord were
removed from animals on various days after TMEV infection. The tissues were frozen in liquid nitrogen, chilled in isopentane, and stored in
liquid nitrogen. Two 30 µm cryostat sections of each tissue per
animal were stored in sterile tubes at 80°C. Trizol (Invitrogen, Gaithersburg, MD) was added (500 µl for spinal cord sections and 700 µl for brain sections), and RNA was precipitated with isopropanol, using 1 ml of 20 mg/ml glycogen (Roche, Indianapolis, IN) as a carrier
at 20°C overnight. RNA concentration was determined by spectrophotometry, and 1 mg of RNA was DNase treated (Invitrogen) according to the manufacturer's instruction. First-strand cDNA was
synthesized using 1 mg of DNase-treated RNA, oligo-dT primers, and
superscript II (Invitrogen), according to the manufacturer's instruction.
Generation of standard curves. The fragments of mouse
MCP-1/chemokine ligand (CCL)2 (~400 bp), IP-10/CXCL10
(~600 bp), and RANTES/CCL5 (~400 bp) transcripts were
amplified in RT-PCR reactions using gene-specific primers.
The primer pair sequences were as follows: MCP-1 forward,
5'-ATCCCAATGAGTAGGCTGGAGAGC-3', backward, 3'-AAGGCATCACAGTCCGAGTCACAC-5'; IP-10, forward,
5'-CAACCCAAGTGCTGCC-3', backward, 3'-GGGAATTCACCATGGCTTGACCA-5';
RANTES, forward, 5'-TTTGCCTACCTCTCC-CTAGAGCTG-3', backward,
3'-ATGCCGATTTTCCCAGGACC-5'. The PCR products were subcloned into the
PCR 2.1 vector (Original TA cloning kit; Invitrogen, Carlsbad, CA)
following the TA cloning kit protocol. The plasmid DNA was quantified
by spectrophotometry. Five serial 10-fold dilutions of plasmid DNA
(from 2000 fg to 0.2 fg per reaction) were prepared, amplified by PCR,
and labeled with SYBR Green (Roche), which yields a bright
fluorescence on binding of double-stranded nucleic acids; this
fluorescence abruptly diminishes on denaturation of DNA strands during
melting-curve analysis. PCR and analysis to generate standard curves
were performed in 20 ml reactions in glass capillaries, using a
LightCycler (Roche) and LightCycler 3 software. For each reaction,
melting-curve analysis was used to detect the synthesis of nonspecific
products. Negative controls (omitting input cDNA) were also used in
each PCR run to confirm the specificity of PCR products. PCR standard
curves were linear across serial 10-fold dilutions, and the melting
curve analysis indicated synthesis of a single homogeneous product of
expected melting temperature.
Standard curves were generated with each set of samples. The
reactions were done in 20 ml capillaries containing 2.5 mM
Mg2+, 0.2 mM of each forward
and backward primer (identical with those used to generate the plasmid
DNA template for standard curve), 1× DNA Master SYBR Green
(LightCycler-DNA Master SYBR Green I kit, Roche), and 2 ml cDNA.
Reaction conditions for PCR were as follows: denature at 95°C for 1 min; 40 cycles of amplification by denaturing at 95°C for 15 sec,
annealing at 60°C for 5 sec, and extending at 72°C for 15 sec. The
accumulation of products was monitored by SYBR Green fluorescence at
the completion of each cycle. Analysis was performed on the LightCycler
3 software, and results are expressed as the crossing point at which
accumulation of PCR products became exponential. Using the standard
curves, this value was converted to femtograms of target. Reaction
conditions for melting curve analyses were denaturation to 95°C at
20°C/sec without plateau phase, annealing at 65°C for 15 sec,
denaturation to 95°C at 0.1°C/sec, with continuous monitoring of
SYBR Green fluorescence.
In vitro protection assay. NSC-34 motor
neurons (kindly provided by Dr. Neil Cashman, University of Toronto,
Toronto, Ontario) were grown in DMEM supplemented with 10% FCS
and 1% Pen-Strep. After several propagation passages, cells were
switched to a differentiation media consisting of DME/F12 (50:50)
supplemented with 1% FCS, 1% nonessential amino acids, and 1%
Pen-Strep (Cashman et al., 1992; Eggett et al., 2000). After
several passages under these conditions, the cells exhibited neurites
and were considered to be differentiated to a motor neuron
phenotype. Such differentiated cells were plated to 70%
confluency on 12-well plates, grown overnight to obtain >80%
confluency, and then infected with 1.5 pfu of TMEV per cell. At the
time of infection, some cells were treated with various concentrations
of IL-6 or left untreated. After an overnight incubation, cell survival
was measured using a standard MTT assay.
For generation of primary motor neurons, spinal cords were dissected
into Hibernate A media supplemented with B27 and then minced with a
razor blade and digested in Hibernate A containing papain. After
trituration, motor neurons were isolated by centrifugation through an
OptiPrep step gradient. Cells were plated on poly-D-lysine and grown in Neurobasal A media containing B27 supplement
and b-FGF (10 ng/ml). Morphologically, after 2 weeks in culture,
the predominant cell type appeared to be motor neurons, and these cells
exhibited large, complex neurite networks. Cultures were analyzed for
the effect of IL-6 on survival as described above for NSC-34 cells.
Statistics. Data were analyzed using either the
Student's t test for normally distributed data or the
Mann-Whitney rank sum test for data that were not normally
distributed. Proportional data were evaluated using the z
test. The level for significance was set as p < 0.05 for all tests.
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Results |
IL-6 is expressed primarily in astrocytes after Theiler's
virus infection
Immunohistochemistry was used to examine the distribution of IL-6
in the brain and spinal cord. In noninfected
IL-6+/+ H-2b
mice there was no expression of IL-6 in the brain or spinal cord (Fig.
1A,B).
After 7 d of Theiler's virus infection there was a dramatic
upregulation of IL-6 immunoreactivity observed primarily in astrocytes
of the brain and spinal cord (Fig. 1C,D). As
expected, IL-6/
H-2b mice infected with virus showed no
significant IL-6 expression in the brain or spinal cord (Fig.
1E,F).
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Figure 1.
Immunoperoxidase staining for IL-6 antigen using
an antibody specific for IL-6. There was no staining for IL-6 in the
brain (A) or spinal cord
(B) of noninfected mice. C,
Prominent staining for IL-6 primarily in astrocytes from the brain of
an infected IL-6+/+ mouse. D,
Scattered positive staining for IL-6 in the spinal cord of an infected
IL-6+/+ mouse. Staining is present in both the gray
(g) and white (wh) matter. By
morphology these cells appear to be either astrocytes or microglia.
E,
IL-6/
H-2b mouse shows only minimal background staining in
the brain. F, Absence of IL-6 expression in the spinal
cord of an infected
IL-6/
mouse.
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IL-6/
mice with a normally resistant H-2b MHC remain
resistant to persistent TMEV demyelination and neurologic deficits
Injection of Theiler's virus into the CNS of mice results in two
distinct disease phenotypes. Mice of the resistant
H-2b,k,d haplotypes develop acute
encephalitis at 7-10 d after infection, with virus replication
restricted primarily to the hippocampus and striatum (Rodriguez et al.,
1993). The virus is then rapidly cleared such that persistence does not
develop and demyelination does not ensue. In contrast, mice of
susceptible H-2s,v,q,u,r haplotypes
develop similar acute encephalitis, but this is followed by incomplete
clearance of the virus and subsequent chronic demyelinating disease in
the spinal cord beginning 35-45 d after infection (Rodriguez and
David, 1985). The mechanism of resistance to virus persistence and
demyelination is dependent on the development of a rapid virus-specific class I-restricted cytotoxic lymphocyte response directed against viral
capsid antigen (Lin et al., 1995). We tested whether
genetic disruption of IL-6 would convert normally resistant
H-2b mice to a susceptible phenotype.
Theiler's virus-infected
IL-6/
H-2b mice had similar survival rates
compared with IL-6+/+ control mice (Fig.
2A). Only 2 of 19 IL-6/
H-2b mice died within 2 weeks of infection
as compared with 0 of 15 IL-6+/+
H-2b mice. In addition both the
IL-6/
H-2b and
IL-6+/+ H-2b
mice showed no clinical or neurologic deficits. Analysis of the spinal
cord at 12 d after infection showed slightly more gray matter
disease in
IL-6/
H-2b mice as compared with
IL-6+/+ H-2b
mice, but this was not statistically significant
(Table 1). Only small areas of
demyelination were observed in both groups of mice. By 45 d after
infection the virus was cleared, and minimal or no pathologic
abnormalities were observed in the spinal cord of six
IL-6/
H-2b mice and eight
IL-6+/+ mice. We conclude that genetic
deletion of IL-6 had no effect on the normal resistance to chronic
virus persistence and demyelination observed in
H-2b mice.
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Figure 2.
A, Survival analysis of
IL-6/
H-2b, IL-6+/+
H-2b,
IL-6/
H-2q, and IL-6+/+
H-2q mice infected intracranially with Theiler's
virus. There was a statistically significant decrease in survival in
IL-6-deficient mice of susceptible MHC haplotype
H-2q as compared with H-2q mice
with normal expression of IL-6 (p < 0.05 by
z test). In contrast, mice of normally resistant MHC
haplotype (H-2b) showed no significant decrease in
survival regardless of IL-6 expression. B, Clinical
scores of IL-6+/+ H-2q mice and
IL-6/
H-2q mice after Theiler's virus infection. Scores
were obtained on day 12 after infection. Sixteen of 18 IL-6/
H-2q mice showed neurologic deficits or were
moribund. In contrast, 9 of 10 IL-6+/+
H-2q mice were clinically normal. This difference in
clinical scores between
IL-6/
H-2q and IL-6+/+
H-2q mice was statistically significant by rank sum
(p < 0.001). Both
IL-6/
H-2b and IL-6+/+
H-2b mice remained clinically normal for the 45 d of observation after virus infection.
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IL-6 disruption in susceptible H-2q mice results
in marked clinical deficits and early death after Theiler's virus
infection
To address the function of IL-6 in animals of susceptible
haplotype, we crossed
IL-6/
H-2b mice to B10.Q mice. An
F2 generation was produced, and animals homozygous for
IL-6/
and H-2q were selected. These mice were
intercrossed to generate a line of
IL-6/
H-2q mice and a line of
IL-6+/+ H-2q
mice. After Theiler's virus infection,
IL-6/
H-2q mice showed a dramatic decrease in
survival compared with IL-6+/+
H-2q mice. Seventeen of 29 IL-6/
H-2q mice died by 2 weeks after infection
(Fig. 2A). Most deaths occurred during the first
12 d after infection. This implies that animals were likely dying
as a result of the early neuronal disease seen with this model. In
contrast, only 3 of 23 IL-6+/+
H-2q mice were dead by 2 weeks
(p < 0.05 by z test). These animals also showed major clinical deficits characterized by uncoordination, motor hindlimb weakness or paralysis, scruffy fur, and poor general appearance (Fig. 2B). Approximately 40% of the mice
survived until day 45, a time point traditionally used in this model to
determine the presence or absence of chronic demyelinating disease
(Rodriguez et al., 1986b). Those mice that survived the acute neuronal
disease exhibited significant improvement.
IL-6/
mice develop humoral immune responses directed against Theiler's
virus
IL-6 has been shown to play an important role in the
differentiation of B cells, and thus deficiency of this cytokine from birth could theoretically affect the protective humoral response directed against Theiler's virus and lead to reduced survival and
enhancement of clinical deficits (Tosato et al., 1988; Strestik et al.,
2001). To test this possibility we assessed antibody responses in the
serum by ELISA directed against purified virus antigens (Fig.
3). Serum IgM and IgG responses were
measured at 12 and 45 d after infection. At 12 d after
infection both
IL-6/
and IL-6+/+
H-2b mice had titered antibody response
against the virus. Similar IgM responses were observed in
IL-6/
H-2q mice compared with
IL-6+/+ H-2q
mice. To address this further, we analyzed virus-specific
neutralization using antiserum from
IL-6+/+ H-2q
and
IL-6/
H-2q mice at this time point. No
difference in virus neutralization between the groups was identified.
By 45 d after infection, both IL-6+/+
and
IL-6/
mice had higher serum IgG directed against the virus as compared with
IL-6+/+ H-2b
or
IL-6/
H-2b mice. We conclude from these
experiments that a deficit in IL-6 did not affect the normal humoral
response to Theiler's virus.
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Figure 3.
A, ELISA for serum IgM antibodies
(12 d after infection) directed against purified TMEV antigens in
IL-6/
H-2b, IL-6+/+
H-2b,
IL-6/
H-2q, and IL-6+/+
H-2q mice. Negative control is from mice not
infected with TMEV. Both
IL-6/ and
IL-6+/+ mice developed humoral antibody response to
the virus antigen.
IL-6/
H-2q mice had similar IgM responses at 12 d as
compared with the other experimental groups. B,
Virus-specific IgG ELISA at 12 d after infection also showed no
differences between groups. C, Virus neutralization
using antiserum from IL-6+/+ H-2q
and IL-6/
H-2q mice at 12 d after infection shows that
there was no difference between the groups. Each bar
represents one animal. Data are expressed as the log2
dilution of antiserum required to neutralize 90% of Theiler's virus
plaques in L2 cells. D, ELISA for serum
IgG at 45 d after infection. By 45 d, both
IL-6/ and
IL-6+/+ H-2q mice had higher
responses as compared with IL-6+/+ or
IL-6/
H-2b mice.
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IL-6-deficient mice after infection with Theiler's virus show
normal distribution of CD4 and CD8 T cells and expression of class I
and class II MHC antigens in the CNS
We next asked whether IL-6 deficiency in
H-2q mice altered the distribution of T
cells or the expression of MHC in the CNS after virus infection.
Immunostaining was performed on six
IL-6/
and three IL-6+/+ mice. CD4 and CD8 T
cells were observed in the spinal cord and brain of both strains of
mice (Fig. 4). As described previously (Lindsley and Rodriguez, 1989), CD4 cells were found mostly in a
perivascular location, whereas CD8 T cells were scattered away from
blood vessels throughout the parenchyma. Class I MHC immunostaining was
distributed within the lesion in blood vessels and in cells with
presumed glial morphology. Class II MHC immunostaining was expressed in
cells with morphology consistent with macrophages and microglia. No
difference in the distribution or intensity of the staining was
observed between
IL-6/
and IL-6+/+ mice. Previous experiments
using FACS have demonstrated that the relative frequency of CD4 cells
to CD8 and natural killer (NK) cells is an indicator of
infiltrating cell distributions in the brain of TMEV-infected mice
(Johnson et al., 2001). Using this methodology we found no differences
in CD8 cells, NK cells, or macrophages in the brains of infected
IL6/
H-2q mice compared with infected
IL6+/+ H-2q
mice. We therefore conclude that adaptive immunity in
IL6/
H-2q mice was not impaired.
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Figure 4.
Immunoperoxidase staining for CD4+ and CD8+ T
cells in the CNS of
IL-6/
H-2q and IL-6+/+
H-2q mice at 12 d after infection.
A, CD4+ cells are abundant in the brain of
IL-6/
H-2q mice. Some of the CD4+ cells are present in the
meninges of the brain (asterisk), whereas others are in
the parenchyma, frequently in a perivascular location
(arrowhead). B, CD4+ cells in the
brain of an IL-6+/+ mouse show a distribution
similar to that seen in an
IL-6/
mouse shown in A. C,
Serial section from that shown in A
(IL-6/
H-2q mouse) was stained for CD8+ T cells. Note that
the cells are distributed widely within the brain parenchyma
(arrow) and have a distinct distribution compared with CD4+
T cells. D, Serial section from that shown in
B (IL-6+/+ mouse) shows that the pattern
of CD8 staining is similar to that seen in C
(IL-6/
H-2q mouse). E, CD4-positive cells
are present almost exclusively in the gray matter of the spinal cord of
an IL-6/
H-2q mouse. Many of the CD4+ cells are clumped
together. F, Similar distribution of CD4+ cells in the
gray matter of an IL-6+/+ mouse as compared with
E. G, Serial section from that
shown in E demonstrates the localization of CD8+
T cells. These cells are more widely scattered in the gray matter as
compared with CD4+ T cells. H, Serial section from that
shown in F shows similar distribution of CD8+ T
cells in the spinal cord of an IL-6+/+ mouse as
compared with an
IL-6/
mouse. g, Gray matter; wh, white
matter.
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|
Chemokine expression is not changed in the CNS of IL-6-deficient
mice after infection
Previous studies have shown that antibody-mediated blockade of
IP-10 drastically increases the mortality of mice infected intracerebrally with mouse hepatitis virus
(Liu et al., 2000), leading to the proposal that chemokines serve as
essential sentinel molecules during the innate response of the CNS to
viral infection (Asensio and Campbell, 2001). Furthermore,
chemokine expression in models of innate immunity, such as the
cutaneous air pouch, were found to be contingent on IL-6 signaling
(Romano et al., 1997). Expression of chemokines during TMEV is tightly
associated with pathogenesis (Murray et al., 2000) and is independent
of the presence of CD4+ or CD8+ T cells (Ransohoff et al., 2001). Therefore, chemokines implicated in both antiviral responses (IP-10, RANTES) and wound repair (MCP-1) were monitored during TMEV disease in
H-2q and H-2b
mice that possessed or lacked IL-6. In susceptible and resistant mice,
expression of all three chemokines was at least as vigorous in
IL-6/
H-2q as observed in
IL-6+/+ H-2q
mice, demonstrating a lack of dependence on IL-6 (Fig.
5) (data not shown). In the resistant
H-2b strain, expression of all three
chemokines in both brain and spinal cords was virtually identical in
IL-6+/+ H-2b
mice and IL-6-null mice (data not shown). Susceptible
IL-6/
H-2q mice expressed more MCP-1 and RANTES
in the affected spinal cord than did
IL-6+/+ H-2q
mice (Fig. 5). As observed previously, expression of chemokines associated with an antiviral Th1 response (IP-10 and RANTES) was much
more robust than expression of MCP-1 (Ransohoff et al., 2001). These
results indicate that the innate response of the CNS to TMEV was not
directly governed by IL-6 or indirectly impaired in its absence.
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Figure 5.
Chemokine expression in
IL-6/
H-2q and IL-6+/+
H-2q mice after 12 d of TMEV infection in the
brain and spinal cord. A, MCP-1 expression was present
in both the spinal cord and brain of
IL-6/ and
IL-6+/+ mice. In addition, there was an increase in
MCP-1 expression in the spinal cord of
IL-6/
H-2q mice compared with IL-6+/+
H-2q mice. B, Similar results were
found with IP-10 expression. The IP-10 expression in
IL-6/
H-2q mice was increased compared with
IL-6+/+ H-2q mice.
C, RANTES expression was found in both the brain and
spinal cord of
IL-6/ and
IL-6+/+ mice. This level of expression was found to
be similar in both groups.
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|
IL-6/
H-2q mice show severe pathology in the gray matter
of the spinal cord at 12 d after infection
Given the absence of differences in innate and
adaptive immune responses in these animals, we undertook a detailed
morphologic analysis of
IL-6/
H-2q and
IL-6+/+ H-2q
mice at 12 d after infection to investigate the reason for the high death rate in the
IL-6/
H2q mice (Table 1). At this time point
many of the mice were clinically ill or moribund. We found a twofold
increase in the spinal cord quadrants with gray matter disease
(13.5 ± 3.8 in
IL-6/
H-2q mice as compared with 5.3 ± 1.0 in IL-6+/+
H-2q mice; p < 0.05 by
t test). In addition there was increased meningeal inflammation and early demyelination, although this did not reach significance. Multiple examples of anterior horn neurons undergoing cell death were also observed (Fig.
6B). Neurophagia, as
demonstrated by macrophages and other inflammatory cells engulfing
neuronal debris, was observed in
IL-6/
H-2q mice but not in the
IL-6+/+ H-2q
controls. By electron microscopy these dying neurons showed vacuolar changes in the cytoplasm without early changes in the nuclei (Fig. 6E). We were also able to study 10 IL-6/
H-2q and 10 IL-6+/+ H-2q
mice that survived to 45 d after infection.
IL-6+/+ H-2q
(Fig. 6C) and
IL-6/
H-2q (Fig. 6D) mice
showed demyelination in the spinal cord, and the extent and
distribution of demyelination were not different between the two groups
(Table 1). The extent of gray matter disease was greater, however, in
IL-6/
mice compared with IL-6+/+. The number of
quadrants demonstrating meningeal inflammation was reduced in
IL-6/
H-2q mice, indicating that one of the
primary reasons that
IL-6/
H-2q mice experienced marked neurologic
deficits and early deaths was likely caused by injury of anterior horn
cells in the gray matter of the spinal cord.
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Figure 6.
Pathologic analysis of spinal cord blocks embedded
in plastic. Sections shown in A-D were
postfixed in osmium and stained with modified erichrome/cresyl violet
stain. A, Anterior horn cells from an
IL-6+/+ H-2q mouse infected with
Theiler's virus for 12 d show normal morphology (900×).
B, Vacuolar changes (arrowheads) in
anterior horn cells from an
IL-6/
H-2q mouse infected for 12 d (900×).
C, Focal area of demyelination and inflammatory
infiltrates in the white matter of the spinal cord in an
IL-6+/+ H-2q mouse 45 d
after infection (300×). D, Similar area of
demyelination and inflammation in an
IL-6/
H-2q mouse 45 d after infection. (300×).
E, Electron microscopy of the gray matter of an
IL-6/
H-2q mouse infected for 12 d shows degenerating
neurons (arrowheads) surrounded by inflammatory cells
(asterisks) (2500×).
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|
Theiler's virus-infected
IL-6/
H-2q mice show more severe disease in the cortex of
the brain
We asked whether deficiency in IL-6 would predispose
specific populations of brain neurons to virus-induced injury. We
analyzed the brains of
IL-6/
H-2b, IL-6+/+
H-2b,
IL-6/
H-2q, and
IL-6+/+ H-2q
mice for severity of pathologic injury to areas of the brain (Fig.
7). We used a semiquantitative four-point
scale for analysis. On average, H-2q mice
showed greater severity of brain pathology than
H-2b mice regardless of IL-6 expression;
however, a higher proportion of
IL-6/
H-2q mice had severe disease in the cortex
as compared with IL-6+/+
H-2q mice (p < 0.05 by z test). Of interest, no major change in the severity of brain disease was observed in the cerebellum, brainstem, hippocampus, corpus callosum, or striatum, and the degree of meningeal inflammation was not altered. These results indicate that IL-6 was
necessary to protect specific populations of brain neurons against
virus-induced injury.
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Figure 7.
Pathologic analysis after 12 d of infection
of brain areas (cerebellum, brain stem, cortex, hippocampus, striatum,
corpus callosum, and meninges) of
IL-6/
H-2q mice (A),
IL-6+/+ H-2q mice
(B),
IL-6/
H-2b mice (C), and
IL-6/
H-2b mice (D). Pathology
qualitative scores from 0 to 4 are described in Materials and Methods.
Each circle represents one mouse. Note the increased
severity of brain pathology in H-2q mice as compared
with H-2b mice regardless of IL-6 expression. More
severe cortical disease was observed in
IL-6/
H-2q mice as compared with
IL-6+/+ H-2q mice after 12 d
of Theiler's virus infection (p < 0.05).
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IL-6/
H-2q mice propagate more virus infection in the gray
matter of the spinal cord
We evaluated the level of virus infectivity, RNA expression, and
virus antigen expression in IL-6+/+
H-2b,
IL-6/
H-2b, IL-6+/+
H-2q, and
IL-6/
H-2q mice at various time points after
infection. Viral plaque assays that measure virus infectivity showed
that H-2q mice on average replicated
100-fold more virus than H-2b mice
regardless of IL-6 expression (Fig.
8A). The number of
viral plaques per gram of CNS was not different between
IL-6/
and IL-6+/+ mice at 7 d after
infection when the entire CNS was assayed. To address more precisely
where virus was replicating, we used immunoperoxidase staining with an
antibody specific for virus antigen. The number of virus
antigen-positive cells at 12 d after infection was expressed as a
function of the area of either spinal cord white matter or gray matter
(Fig. 8B). In IL-6+/+
H-2q mice, the number of virus
antigen-positive cells was similar in both spinal cord white matter and
gray matter. In contrast, in
IL-6/
H-2q mice there was an eightfold increase
in the number of virus antigen-positive cells in the gray matter versus
the white matter. To confirm this observation we further measured
virus-specific RNA (Fig. 8C) by Northern blot. VP2 cDNA, one
of the major capsid proteins, was used to probe virus RNA as compared
with GAPDH as an internal standard. The data were expressed as the
ratio of VP2 to GAPDH. In IL-6+/+
H-2q mice the level of virus RNA was
similar in the brain and spinal cord at day 7 after infection. By day
12 the amount of virus RNA decreased dramatically in both the brain and
spinal cord. In
IL-6/
H-2q mice, similar levels of virus RNA
were observed in the brain and spinal cord at day 7, and these levels
were comparable with those observed in
IL-6+/+ H-2q
mice. In contrast, on day 12 after infection there was a 20-fold increase in virus-specific RNA in the spinal cord compared with the
brain (p < 0.05 by t test). In
addition, although it did not reach significance (NS by rank sum),
virus antigen in the gray matter of
IL-6/
H-2q mice was increased compared with
IL-6+/+ H-2q
mice. Both the analysis of virus antigen-positive cells and the Northern blot data support the conclusion that IL-6 deficiency allowed
for robust virus replication in the gray matter of the spinal cord.
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Figure 8.
Levels of virus infectivity, antigen
expression, and RNA expression in
IL-6/ and
IL-6+/+ mice. A, Virus infectivity as
measured by plaque assay is expressed as the number of plaque-forming
units per gram of total CNS (brain and spinal cord). On average there
was a ~100-fold increase in plaque-forming units in
H-2q mice compared with H-2b at
7 d after infection regardless of IL-6 expression.
B, The number of virus antigen-positive cells was
determined by immunoperoxidase staining and expressed per square
millimeters of spinal cord gray matter or white matter area. No
difference was observed in the number of virus antigen-positive cells
in the white matter when comparing
IL-6/
H-2q mice as compared with
IL-6+/+ mice. More virus antigen-positive cells were
observed in the spinal cord gray matter of
IL-6/
H-2q mice as compared with
IL-6+/+ H-2q mice; however, this
did not reach statistical significance. C, Levels of
viral capsid VP2 RNA message were analyzed by Northern blot as a
function of GAPDH message. In IL-6+/+
H-2q mice, the level of VP2 message was similar in
the brain and spinal cord on day 7, and both were decreased by 12 d after infection. In contrast, in
IL-6/
H-2q mice the level of VP2 message was similar in
the brain and spinal cord at 7 d after infection, whereas at
12 d a dramatic increase in VP2 message was observed in the spinal
cord (p < 0.05 by t
test).
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IL-6 deficiency permits prominent virus infection of anterior horn
motor neurons in the spinal cord
We examined which cells in the nervous system were expressing
virus antigen in IL-6+/+
H-2q and
IL-6/
H-2q mice 12 d after infection (Fig.
9). In the brain of
IL-6/
H-2q mice there was notable virus in the
cortex, hippocampus, and striatum; however, the most unique finding was
the localization of virus within the anterior horn cells of the spinal
cord. Virus antigen was localized exclusively to the cytoplasm of these
cells. In many of these cells the morphology was sufficiently intact to
identify the nucleoli. No examples of anterior horn motor neurons expressing virus antigen (analysis of 18 mice and 144 sections) were
identified in the control mice. IL-6+/+
H-2q mice showed similar virus antigen
staining in the brain as compared with
IL-6/
H-2q mice, with staining in the spinal
cord gray matter limited to virus that was scattered among and engulfed
by inflammatory cells. This provided strong evidence that the reason
for high morbidity and mortality in
IL-6/
H-2q mice was prominent infection of motor
neurons in the spinal cord.
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Figure 9.
Immunoperoxidase staining for virus antigen
using a polyclonal rabbit antiserum that reacts strongly with all the
structural viral capsid proteins of Theiler's virus (Rodriguez et al.,
1983). A, Low magnification (300×) image from the
cortex and hippocampus showing multiple antigen-positive cells in an
IL-6/
H-2q mouse infected with TMEV for 12 d.
B, A similar staining pattern was seen in the cortex and
hippocampus of the infected IL-6+/+
H-2q mice. C, Virus antigen within
anterior horn cells of the spinal cord of an
IL-6/
H-2q mouse. Virus is localized exclusively to the
cytoplasm of the neuron. No similar virus antigen-positive anterior
horn cell neurons were identified in infected
IL-6+/+ H-2q mice.
D, Virus antigen localized to the spinal cord gray
matter of an infected IL-6+/+
H-2q mouse. Virus antigen is associated primarily
with inflammatory cells and presumed macrophages.
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IL-6 protects motor neurons from virus-induced death
in vitro
NSC-34 cells, under appropriate culture conditions, display a well
characterized motor neuron phenotype (Cashman et al., 1992; Eggett et
al., 2000). We used these cells to directly test the hypothesis that
IL-6 protects motor neurons from virus-induced death. Infection with
1.5 pfu of TMEV per cell induced the death of 45% (±1.0%;
p < 0.05) of NSC-34 cells after 24 hr. Treatment with
1 ng/ml of IL-6 added at the time of infection rescued 10% (±2.9%;
p = 0.009 vs vehicle) more NSC-34 cells than vehicle
alone (Fig. 10A).
Likewise, 10 ng/ml of IL-6 rescued 21% (±2.7%; p = 0.002 vs vehicle) more cells, and treatment with 100 ng/ml of IL-6
rescued 30% (±2.3%; p = 0.002 vs vehicle) more cells
than vehicle only (Fig. 10A). Importantly, treatment
with IL-6 in the absence of DAV infection did not induce cell
proliferation at any concentration tested (p = 0.83 across all groups by ANOVA; data not shown).
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Figure 10.
IL-6 rescues NSC-34 motor neurons and primary
spinal motor neurons infected in culture with Theiler's virus.
A, NSC-34 cells were infected with 1.5 pfu of virus per
cell for 24 hr in the presence or absence of various concentrations of
IL-6. Treatment with 1 ng/ml of IL-6 added at the time of infection
rescued 10% (±2.9%; p = 0.009 vs vehicle) more
NSC-34 cells than vehicle alone. Similarly, 10 ng/ml of
IL-6 rescued 21% (±2.7%; p = 0.002 vs
vehicle) more cells, and treatment with 100 ng/ml IL-6 rescued
30% (±2.3%; p = 0.002 vs vehicle) more cells
than vehicle only. B, Primary spinal motor neurons were
infected with 1.5 pfu of virus per cell, and cultures were
concomitantly treated with 100 ng/ml of IL-6 or with vehicle. After 24 hr, cell survival was measured by MTT assay and normalized to
uninfected cultures. Although only 51.6 ± 0.5% of spinal motor
neurons survived in vehicle-treated cultures, IL-6-treated cultures
exhibited survival of 77.4 ± 7.8% (p = 0.042 vs vehicle) of motor neurons.
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|
On the basis of our findings in NSC-34 cells, we asked whether IL-6
protected primary motor neurons from DAV-induced cell death. Spinal
motor neurons were isolated from neonatal C57BL/6J mice and cultured
for 2 weeks in defined media. Cells were infected with 1.5 pfu of DAV
per cell and treated either with 100 ng/ml IL-6 or with vehicle. After
24 hr, cell survival was measured with the MTT assay and compared with
uninfected cultures. As shown in Figure 10B,
77.4 ± 7.8% (n = 3; p = 0.042 vs
vehicle) of motor neurons survived in the IL-6-treated DAV-infected
cultures, whereas treatment with only vehicle supported the survival of
51.6 ± 0.5% (n = 3) of motor neurons. Hence, we
conclude that the enhanced mortality and pathology observed in
IL-6/
H-2q mice were the result of a deficit in
neuroprotection normally afforded by the presence of IL-6 in astrocytes.
| |
Discussion |
Our results suggest that IL-6 is critical for protecting specific
neuronal populations in the spinal cord and brain from cell death
induced by infection with Theiler's virus. Disruption of IL-6 in
normally resistant H-2b mice had no effect
on virus-induced injury of neurons. These mice cleared the virus
infection normally, likely as a result of a vigorous antiviral class
I-restricted cytotoxic lymphocyte response (Borson et al., 1997;
Dethlefs et al., 1997). In contrast, when we crossed IL-6-deficient
mice with susceptible H-2q mice,
these animals showed early death and prominent infection in the gray
matter of the spinal cord. The severe virus-induced injury to anterior
horn motor neurons in
IL-6/
H-2q mice is the likely explanation for
the clinical phenotype that we observed. Of interest is the mechanism
by which IL-6, a multipotential cytokine with functions that are not
confined to the immune and hematopoietic systems, protects neurons from
injury (Hama et al., 1989; Ramsay et al., 1994; Zhong et al., 1999). We
have considered four possibilities for how IL-6 deficiency may result
in severe neuronal injury after virus infection.
The first possibility is that IL-6 deficiency affects the antiviral
humoral response such that neutralizing antibodies are not generated.
We addressed this hypothesis using virus-specific ELISA for both the
IgM and IgG response in mice with or without disruption of IL-6 in both
the H-2q and
H-2b haplotype. Although IL-6 has been
shown to exert a strong influence on maturation and differentiation of
B cells (Roldan and Brieva, 1991; Roldan et al., 1992; Kopf et al.,
1998), no effect was observed in the viral-specific antibody responses.
To explore this further we examined the neutralization of virus with
antiserum from
IL-6/
H-2q or
IL-6+/+ H-2q
mice infected with TMEV for 12 d. Titers confirmed that there were
no differences in the ability of the serum from either strain to
neutralize the virus. Our data indicate that the generation of a
neutralizing antibody response to TMEV is independent of IL-6.
The second possibility is that IL-6 deficiency altered the cellular
inflammatory response to virus injury (Sarawar et al., 1998; Wang et
al., 2000). IL-6, IL-1, and TNF can be released by activated monocytes.
These cytokines are part of the acute injury response to foreign
antigens and viral infection (Conn et al., 1995). IL-6 has been shown
to be crucial for recruitment of myelomonocytes and activation of glial
cells after focal cryo-injury of the frontoparietal cortex (Penkowa et
al., 1999).
IL-6/
mice have impaired leukocyte accumulation in target tissues as a result
of reduced in situ production of chemokines (Romano et al.,
1997), and are highly susceptible to Listeria monocytogenes, probably as a result of inefficient neutrophilia (Dalrymple et al.,
1995).
IL-6/
mice have reduced myocardial damage after infection of
encephalomyocarditis virus, and this is likely mediated via
modification of the immune response (Kanda et al., 1996). In the
gammaherpesvirus-68 model in mice, however, no differences
were observed in IL-6-deficient mice in regard to number or activation
status of leukocytes (Sarawar et al., 1998). Thus the IL-6 system may
or may not have a role in local inflammatory reactions. To address this
issue we examined CD4 and CD8 infiltrates, as well as class I and class
II expression in
IL-6/
H-2q mice as compared with
IL-6+/+ H-2q
mice at 12 d after infection. No differences were detected by immunoperoxidase staining or by FACS analysis. In addition, we analyzed
RNA expression of various chemokines in the CNS of these mice. At
12 d after infection, chemokine expression in the CNS of
susceptible mice that lacked IL-6 was extremely robust, demonstrating that innate responses to viral challenge were not deficient
in IL-6-null mice. This observation suggests that CNS cellular immune host defense is not contingent on the presence of IL-6 in
H-2q mice.
The third possibility is that IL-6-deficient mice develop the lethal
phenotype after virus infection because of a lack of antiviral
activity. IL-6 was initially discovered because of biologic effects
similar to interferon  ; however, previous in
vitro experiments treating Theiler's virus-infected cells with
various concentrations of IL-6 showed no direct antiviral
effect on the growth of this picornavirus (Rodriguez et al., 1994). In
addition, in the present experiments there were no differences in
infectious viral titers from the CNS of
IL-6/
mice as compared with IL-6+/+ mice;
however, more virus RNA replication was observed in the gray matter of
the spinal cord in
IL-6/
H-2q mice. Therefore, it is possible that
IL-6 is working to limit virus spread in the anterior horn.
We, however, favor the possibility that IL-6 is working to support the
survival of specific neuronal populations. Previous experiments on
primary CNS cultures from susceptible and resistant animals show no
difference in TMEV binding (Rubio et al., 1990). Therefore, we isolated
primary spinal motor neurons from C57BL/6 mice and tested whether IL-6
could protect these cells from TMEV infection. This data further
supported our data on NSC-34 motor neurons which indicate that IL-6
protects from cell death induced by infection with Theiler's virus,
and we hypothesize that IL-6 is functioning in a similar manner
in vivo to protect anterior horn motor neurons from
virus-induced death. Further support for this hypothesis is provided by
the finding that IL-6 enhances the survival of septal cholinergic
neurons and acetylcholinesterase-positive neurons in culture (Hama et
al., 1989). Of particular interest, IL-6 can rescue spinal motor
neurons from axotomy-induced cell death, and an IL-6/sIL-6R fusion
protein has been shown to promote neurite outgrowth and neuron survival
in cultured enteric neurons (Schafer et al., 1999). Likewise, in
vivo coadministration of IL-6 and soluble IL-6 receptor delays
progression of wobbler mouse motor neuron disease (Ikeda et al., 1996).
Importantly, IL-6 has been shown to be produced by astrocytes during
acute neurotropic coronavirus infection (Sun et al., 1995), and
reactive oxygen-free radicals can enhance the transcription of IL-6 by
astrocytes (Maeda et al., 1994). Thus, we propose that the release of
IL-6 from astrocytes exerts a neuroprotective effect that prevents the
lethal injury of anterior horn spinal motor neurons after infection
with Theiler's virus.
| |
FOOTNOTES |
Received May 24, 2002; revised Sept. 26, 2002; accepted Oct. 25, 2002.
This work was supported by National Institutes of Health Grants P01 NS
38468 (M.R.), R01 NS 32129 (M.R.), and 2RO1 NS 32151 (R.M.R.). We
gratefully acknowledge the gift of a LightCycler from the Cleveland
area Multiple Sclerosis Women's Committee.
Correspondence should be addressed to Dr. Moses Rodriguez,
Departments of Immunology and Neurology, Mayo Clinic, 200 First Street
SW, Rochester, MN 55905. E-mail:
rodriguez.moses{at}mayo.edu.
| |
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TOP
ABSTRACT
Introduction
