| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
- and ß-Chemokine Expression in the Central Nervous System During Mouse Hepatitis Virus-Induced Demyelinating Disease1
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-irradiated mice with MHV does not result in demyelination,
even though these animals exhibit high viral titers in the CNS (2, 6).
Furthermore, adoptive transfer of spleen cells to SCID or
-irradiated animals results in active demyelination (2, 6). Taken
together, these studies suggest an immunologic basis for demyelination
in MHV-infected mice.
Maintenance of a chronic inflammatory response appears to be an
important aspect contributing to demyelination in MHV-infected spinal
cords. A prominent feature of MHV-induced demyelination is the presence
of inflammatory mononuclear cell infiltrates, including T lymphocytes
and monocytes associated with demyelinating lesions (4). Sun et al. (7)
have demonstrated recently the expression of the proinflammatory
cytokines TNF-
, IL-1ß, and IL-6 by astrocytes in the spinal cords
of chronically infected mice. These cytokines may act individually or
in concert to recruit cells, such as T lymphocytes and monocytes, to
sites of viral infection, which then participate in demyelination (7).
However, the soluble signals that regulate CNS invasion by inflammatory
cells are likely to consist of multiple factors that have yet to be
fully defined, and remain a key question in understanding the pathology
of chronic demyelination in MHV-infected mice.
The chemokines represent a family of low m.w., proinflammatory
cytokines that are divided into two major subfamilies based on
structural and functional criteria (8, 9). The -family is
structurally characterized by a conserved C-X-C motif in the amino
terminus of the protein, with the X representing an amino acid
separating two cysteine residues, while the ß-chemokine family
contains two cysteines that are adjacent, i.e., C-C (8, 9). Recently, a
third chemokine, lymphotactin, has been identified (10). The individual
subfamilies of chemokines have been shown to selectively attract
distinct leukocyte populations during periods of inflammation (8). In
general, the -chemokines function in attracting neutrophils, yet
have limited effect on T lymphocytes or monocytes. However, there are
exceptions to this rule, as CRG-2 (the murine homologue of the human
chemokine IP-10) has been demonstrated to be chemotactic for activated
T cells and monocytes (11). The ß-chemokines predominantly attract T
cells, monocytes, and macrophages, but have only limited effect on
neutrophils (8). Lymphotactin appears to be strictly lymphocyte
specific (10). Recent work has shown that chemokine production is
associated with inflammatory pathology in CNS diseases, including
models of demyelination such as EAE (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). We have investigated
production of chemokine mRNA transcripts in the CNS of mice suffering
from both acute and chronic MHV infection to more fully define factors
that control inflammation and demyelination in this model.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The MHV strain V5A13.1 was derived from the wild-type MHV-4, as previously described (22). Infection of susceptible strains of mice with the neuroattenuated MHV-V5A13.1 results in a chronic demyelinating disease in susceptible animals (22). Viral titers from brains of infected animals were determined by plaque assay on the DBT astrocytoma cell line, as previously described (22, 23, 24). Age-matched (5–7 wk) male C57BL/6 mice (H-2b background) were used for all studies described. Following anesthetization by inhalation of methoxyflurane (Pitman-Moore, Washington Crossing, NJ), mice were injected intercranially with 30 µl of 10 PFU of MHV-V5A13.1 suspended in sterile saline. Control animals were injected with sterile saline alone. Animals were killed by methoxyflurane anesthesia at days 2, 3, 7, 15, 21, and 35 p.i., and brains and spinal cords were removed. One-half of each brain of all killed animals was used for plaque assay to determine viral burden. The remaining half brain as well as spinal cords were either fixed in 10% normal buffered Formalin for paraffin embedding, frozen in O.C.T. compound (Miles, Elkhart, IN), or stored at -70°C for RNA isolation.
Cell culture and viral infection
Primary cultures of cerebral cortical astrocytes were prepared from newborn C57BL/6 mice (1–2 days old). Forebrains were removed aseptically from the skull, the meninges were excised carefully under a dissecting microscope, and the neocortex was dissected. The cells were dissociated by being passed through needles of decreasing gauge sizes (16, 19, and 25 gauge) two or three times with a 10-ml syringe. Trypsin was not used for tissue dissociation. The cells were seeded at a density of 105 cells/cm2 on six-well plates in DMEM containing 10% FCS and 25 mM glucose in a final volume of 2 ml/well and incubated at 37°C in an atmosphere containing 5% CO2 at 95% humidity. The culture medium was renewed 3 to 4 days after seeding and subsequently twice per week. These conditions yielded astrocyte cultures containing >90% GFAP-immunoreactive cells and <1% Mac-1-positive cells. Confluent monolayers of cells were infected with increasing concentrations of MHV-V5A13.1: 0, 0.1, 0.5, 1, 5, 10, and 50 PFU/ml. In addition, 10 and 50 PFU of virus was UV inactivated and added to separate monolayers. Virus was allowed to adsorb for 1 h, at which time monolayers were washed and replaced with 2 ml fresh medium. Infected monolayers were incubated for 24 h, at which time total RNA was extracted using TRIzol reagent (Life Technologies) and 10 µg used for RNase protection assay (RPA) (described below).
RNase protection assay
Total RNA was extracted from brain and spinal cords using the
TRIzol reagent, as previously described (24). For RPA, a multiprobe set
was used that detected the following chemokine transcripts:
-chemokines; MIP-2 and CRG-2; ß-chemokines; C10, RANTES,
MCP-1, MCP-3, MIP-1, MIP-1ß, and T cell
activation-3; and lymphotactin. A probe for L32 was included in the
probe set to verify consistency in RNA loading and assay performance.
Details on the construction and performance of the multiprobe chemokine
RPA set have been described recently (25). RPA analysis was performed
on 10 µg total RNA using a previously described protocol (25, 26, 27).
Following separation by PAGE, protected 32P-labeled probe
fragments were visualized by film autoradiography. Controls included
the probe set hybridized to transfer RNA only and to transfer RNA plus
an equimolar pool of synthetic-sense RNAs complementary to the probe
set. For quantification, autoradiographs were scanned (Scanjet 4C/T;
Hewlett Packard, San Jose, CA) and band density was assessed with
National Institutes of Health image 1.57 software.
Reverse-transcription PCR
The antisense riboprobe used to detect CRG-2 mRNA was
derived by RT-PCR amplification of cDNA generated from total RNA
isolated from the brain of an MHV-V5A13.1-infected mouse at day 7
p.i. (24). Oligonucleotide primers for CRG-2 amplification were as
follows: 5'-CAG CAC CAT GAA CCC AAG TGC and 5'-GCT GGT CAC CTT TCA GAA
GAC C. PCR amplification for the oligonucleotide primers was performed
using an automated Perkin-Elmer (Norwalk, CT) model 480 DNA
thermocycler with the following profile: step 1, initial denaturation
at 94°C for 45 s; step 2, annealing at 60°C for 45 s; and
step 3, extension at 72°C for 2 min. Steps 1 to 3 were repeated 34
times for a total of 35 cycles. A final extension at 72°C of 7 min
was performed. The PCR amplicon was subjected to gel electrophoresis on
a 1.5% agarose gel and visualized by ethidium bromide staining (0.5
µg/ml). For CRG-2, the expected 450-bp fragment was cut out of
the gel and extracted using the Gene Clean II system (Bio 101, San
Diego, CA). This fragment was cloned into the pCR Script
SK+ vector (Stratagene, La Jolla, CA), and sequence
analysis identified 
95% nucleotide identity with mouse
CRG-2 (28).
In situ hybridization
The protocol for in situ hybridization of brain and spinal cord sections as well as the description of the riboprobe for MHV has been described previously (24). The CRG-2 antisense riboprobe was generated by linearization with the NotI restriction enzyme, while the control sense probe was generated using the EcoRI restriction enzyme. The [35S]UTP-radiolabeled RNA probes were derived by in vitro transcription reaction with an RNA transcription kit (Stratagene). Upon completion of the in situ procedure, the slides were dehydrated and dried. Next, the slides were dipped in a Kodak NTB2 nuclear emulsion at 42°C and exposed at 4°C for 2 to 4 wk in a desiccator. The slides were developed and fixed with Kodak D-19 developer and fixer, counterstained with hematoxylin and eosin Y solutions, dehydrated, and mounted.
Immunohistochemistry
Abs used for immunohistochemical detection of cellular Ags are as follows (diluted in PBS): anti-bovine GFAP (rabbit polyclonal; Dakopatts, Carpinteria, CA), 1/1000; anti-CD4 (rat mAb L3T4; PharMingen), 1/100; anti-CD8 (rat mAb Ly-2 and Ly-3; PharMingen, San Diego, CA), 1/200; and anti-Mac-1 (rat mAb M170). In all cases, a biotinylated secondary Ab was used (1/300; Vector Laboratories, Burlingame, CA). Staining for GFAP was performed on brain and spinal cord sections fixed in 10% normal buffered Formalin and embedded in paraffin according to previously described protocols (24, 27). Immunohistochemical analyses for CD4, CD8, and Mac-1 Ags were performed on frozen sections fixed in acetone at -20°C. The ABC Elite (Vector Laboratories) staining system was used according to manufacturer’s instructions, and either VIP or diaminobenzidine was used as a chromagen. All slides were counterstained with hematoxylin.
Combined immunohistochemistry and in situ hybridization
For studies designed to colocalize cellular Ags with in situ signals for CRG-2 mRNA, slight modifications in the procedures were implemented. In all cases, immunohistochemical analysis preceded in situ hybridization. PBS used for dilution of Abs as well as in washing steps was Diethyl pyrocarbonate treated to reduce RNase contamination and loss of in situ signal. For studies done on frozen sections, the slides were dried overnight in a desiccator and fixed in 4% paraformaldehyde for 10 min at 4°C before beginning the immunohistochemistry/in situ procedure. Following application of diaminobenzidine, slides were washed twice in PBS and then prehybridized in hybridization buffer for 1 h at 42°C. Following this incubation, the linearized 35S-labeled riboprobe was added to the sections and the standard in situ hybridization procedure was followed. Upon development, the slides were counterstained in hematoxylin only, then dehydrated and mounted.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Infection of C57BL/6 mice with MHV-V5A13.1 resulted in an acute
viral encephalomyelitis with approximately 10% mortality. The
surviving animals developed the clinical characteristics of MHV-induced
demyelination, e.g., awkward gait and hind-limb paralysis between days
15 and 35 p.i. Virus could not be isolated from the majority of
infected mice by day 15 p.i., as determined by plaque assay (limit
of detection 
100 PFU/g tissue). Examination of brains and spinal
cords of MHV-infected animals at day 35 p.i. revealed extensive
demyelination with persistent viral RNA associated with white matter
tracts (Fig. 1
). CD4+
and CD8+ T cell subsets as well as Mac-1-positive cells
were closely associated with white matter lesions in spinal cords of
mice (Fig. 2). A summary of these
clinical and pathologic observations is shown in Table I.
|
|
|
In attempt to identify chemokines expressed temporally during
early and late stages of MHV infection, RNA was extracted from the
brains and spinal cords of infected animals and subjected to RPA using
a probe set designed to detect 10 different chemokine transcripts (25).
As shown in Figure 3, chemokine mRNA
transcripts were detected at various times following infection with
MHV. At day 3 p.i., a transcript for the -chemokine CRG-2
was expressed abundantly in the brain. In addition to CRG-2,
transcripts for the ß-chemokines MCP-1, MCP-3,
MIP-2, and RANTES were also detected, although at much
lower levels. Very low chemokine expression was observed at day 3 in
the spinal cord. A dramatic increase in mRNA expression of the
ß-chemokines MIP-1ß, MCP-1, MCP-3, and RANTES
as well as CRG-2 was observed in the brains and spinal cords of
animals by day 7 p.i., which coincided with the increased numbers
of inflammatory cell in the brain and spinal cord at this time. By day
35 p.i., surviving animals had cleared infectious virus (but not
viral RNA), and RANTES, CRG-2, and MIP-1ß mRNAs were
the predominant chemokines in brains and spinal cords.
|
was scanned, and the density of individual
chemokine bands was measured and the data presented as a ratio of
chemokine band intensity to the L32 control. Brains and spinal cords
from mice at each time point were analyzed separately, and the data
presented in Figure 3, B and C, represent the
average signal intensity for each chemokine following infection. There
was an approximately 80-fold increase in CRG-2 mRNA as compared
with sham-infected controls at day 3 p.i. in the brain, while
there was approximately a 25-fold increase in CRG-2 expression in
the spinal cord (Fig. 3, B and C). A
20-fold increase in MCP-1 was observed in the brain at this time,
followed by increases of approximately 15-fold for MCP-3 and
MIP-1ß and an 8-fold increase in RANTES expression. By day
7 p.i., the average level of CRG-2 in the brain dropped
slightly as compared with day 3, although it remained the predominant
chemokine transcript to be expressed in the brain. Compared with day 3,
there was a dramatic increase in chemokine mRNAs, with 2- to 4-fold
increases in MCP-1, MCP-3, and MIP-1ß in the brain.
RANTES expression in the brain was approximately 4-fold higher at
day 7 as compared with day 3. An increase in chemokine mRNAs was
observed in the spinal cord of MHV-infected mice, with an approximate
4-fold increase in CRG-2 expression as compared with day 3, while
there was a 3- to 8-fold increase in the expression of MCP-1,
MIP-1ß, MCP-3, and RANTES. By day 35 p.i., a drop
in levels of chemokine mRNAs occurred in both brains and spinal cords.
RANTES, CRG-2, and MIP-1ß are the predominant
chemokines expressed in these tissues at this time point. RANTES
was expressed at slightly higher levels than CRG-2, which, in
turn, was expressed at higher levels as compared with
MIP-1ß. CRG-2 expression and MHV infection
We examined the expression of CRG-2 in more detail because
this particular chemokine was expressed both early (day 2) and late
(day 35) following infection with MHV. In situ hybridization analysis
using antisense probes for CRG-2 and MHV was performed on
sequential sagittal sections of brain and spinal cord to determine the
spatial relationship between viral RNA and CRG-2 expression. The
data in Figure 4 demonstrate the
colocalization of viral RNA with CRG-2 mRNA at days 2 and 7. By
day 21 p.i., both viral RNA and CRG-2 mRNA could be detected in
the brain; however, there was not the strict colocalization as was
observed at days 2 and 7. Examination of spinal cords at day 35
p.i. revealed expression of CRG-2 mRNA in white matter tracts
associated with areas of demyelination (Fig. 5). Positive cells were found
predominantly on the edge of demyelinating lesions.
|
|
Resident glia that have been shown to express CRG-2 include
microglia and astrocytes (13, 28, 29). Double labeling using an
antisense riboprobe for CRG-2 mRNA and a polyclonal Ab to either
GFAP (specific for astrocytes) or Mac-1 (recognizes
microglia/macrophage) was performed to determine whether these cell
types were responsible for production for CRG-2 mRNA following
infection with MHV. These experiments revealed that during acute stage
of infection, e.g., day 7, both astrocytes and Mac-1-positive cells
were found to express CRG-2 mRNA; however, astrocytes were the
predominant cell type expressing this chemokine transcript (Fig. 6). During early and late stage of
disease (day 3 and day 35), only astrocytes were found to express
CRG-2 in both brain and spinal cord.
|
The in vivo data indicated that astrocytes were the predominant
cell type expressing CRG-2 mRNA at both early and late stages of
infection. We wished to extend these findings and study the chemokine
transcript profile by primary astrocytes following in vitro infection
with MHV-V5A13.1. Primary astrocytes were cultured from the brains of
1- to 2-day-old C57BL/6 mice and infected with increasing
concentrations of virus. In addition, separate monolayers were infected
with UV-inactivated virus to determine whether MHV replication was
required for expression of chemokines. Total RNA was isolated from the
cell monolayers following a 24-h incubation and subjected to RPA with
the chemokine probe set. The results presented in Figure 7A indicate that virally
infected astrocytes expressed transcripts for MIP-2, MCP-3,
MIP-1ß, MCP-1, and CRG-2. Low level expression of
MIP-1ß was evident even in control monolayer cultures,
suggesting that isolation of cells may have resulted in slight
activation of the cells. These results are interesting in that the
chemokine profile of MHV-infected astrocytes is similar to what was
observed in MHV-infected animals, with CRG-2, MCP-1,
MIP-1ß, MCP-3, and MIP-2 being expressed both in vivo
and in vitro. A notable difference was that RANTES was not
expressed by MHV-infected astrocytes in vitro, while it was detected in
the brains and spinal cords of MHV-infected mice. Quantitation of the
signal intensities reveal that at MHV concentrations lower than 10
PFU/ml, MIP-1ß was the prominent chemokine expressed (Fig. 7B). Increasing MHV to 50 PFU/ml resulted in a shift
in expression, with CRG-2 being expressed at the highest levels,
with approximately a sixfold increase over medium control, followed by
MCP-1, MIP-1ß, MIP-2, and MCP-3. UV inactivation
of virus resulted in approximately a two- to fourfold decrease in
expression of all chemokine transcripts. However, chemokine expression
was not reduced to the levels observed in cells incubated with medium
only, suggesting that astrocyte expression of chemokine genes was not
entirely dependent upon MHV replication.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1ß (31, 32, 33). Recent studies have
defined a unique family of structurally related small, chemotactic
cytokines termed chemokines. The chemokines have been shown to direct
the specific migration of distinct leukocyte populations during periods
of inflammation in wide variety of disease processes including trauma
(16, 34, 35), autoimmune disorders (12, 13, 14), and microbial infection,
including viral disease (36, 37, 38, 39, 40, 41). Cook and colleagues (42) demonstrated
that homozygous MIP-1 knockout mice were resistant to
Coxsackievirus-induced myocarditis, suggesting an important role for
this chemokine in mediating virus-induced inflammation in vivo.
Sasseville et al. (43) have shown elevated expression of the
ß-chemokines MCP-3, MIP-1 and MIP-1ß, RANTES, and the
-chemokine CRG-2/IP-10 in SIV-induced encephalitis, suggesting that
these chemokines could be involved in leukocyte recruitment to the
brain in SIV-infected rhesus macaque monkeys. These observations lend
support to the idea that chemokines play an essential role in the
development of inflammation in response to viral infection.
Production of chemokines in EAE has also been suggested to play an
important role in both the acute as well as chronic stages of disease.
Multiple chemokines have been shown to be up-regulated preceding and
during the acute stage of EAE (14, 16, 17). Glabinski et al. (13)
recently have shown coordinate up-regulation of MCP-1, MIP-1, KC,
RANTES, and CRG-2/IP-10 in the brain and spinal cord during spontaneous
relapses of chronic EAE. These data suggest that during the course of
this autoimmune demyelinating disease, there is strict regulation of
chemokine expression, which in turn regulates CNS inflammation and
disease process.
In this study, we have analyzed the time course and cellular source of
chemokine gene expression in an animal model of virus-induced
demyelination. Both - and ß-chemokines were expressed during
subacute, acute, and chronic stages of disease in the brains and spinal
cords of MHV-V5A13.1-infected animals. As early as day 2, the
-chemokine CRG-2 was expressed in the brain, and a limited
number of inflammatory cells was present. However, by day 7 p.i.,
the animals were suffering from an acute viral encephalomyelitis
characterized by widespread dissemination of virus throughout the brain
as well as spinal cord, and a massive inflammatory response that
included T cells (both CD4+ and CD8+ T cell)
and macrophages. This particular time point also reflected an enhanced
expression of mRNA transcripts for the ß-chemokines MCP-1,
MCP-3, MIP-1ß, and RANTES, in addition to CRG-2.
By day 35 p.i., the animals had cleared the majority of infectious
virus, yet viral RNA persisted in white matter tracts of the brain and
spinal cord. Clinically, the animals exhibited awkward mobility and, in
more extreme cases, hind-limb paralysis. Histologic examination of the
brain and spinal cords revealed extensive white matter demyelination.
Although present in limited numbers, inflammatory cells were observed
in the brains and spinal cords of these animals. Transcripts for
RANTES, CRG-2, and MIP-1ß mRNA were the predominant
chemokines expressed at this time. These studies indicated that
chemokine expression was regulated during different phases of
MHV-induced disease.
A correlation between chemokine mRNA transcripts and protein expression was not examined in this study. Therefore, while the data presented in this study convincingly show chemokine gene expression in the CNS following MHV infection, it remains a possibility that certain chemokine transcripts may not be expressed at the protein level. However, the fact that chronic infiltration by lymphocytes into the CNS of infected animals is observed suggests that chemoattractants are being expressed and influencing inflammation. Current studies in our laboratory are investigating chemokine protein expression in the CNS of MHV-infected mice during disease.
The expression of CRG-2 was examined in more detail because 1) it
was expressed at all stages of MHV infection examined, 2) CRG-2
mRNA strictly colocalized with areas of viral RNA in brain at days 2
and 7 p.i., and 3) CRG-2 mRNA is associated topographically
with areas of demyelination. Together, these observations suggest a
role for this particular chemokine in MHV-induced CNS disease. In
addition, CRG-2 is the predominant chemokine expressed in SIV
encephalitis (43) and lymphocytic choriomeningitis (25). Thus,
prominent expression in diverse viral infection of the CNS suggests an
important role in the CNS host response to viral infection. The in vivo
function of CRG-2 during MHV-induced CNS disease is not presently
known. CRG-2 is a potent in vitro chemoattractant for activated T
lymphocytes as well as monocytes (11). Recent studies have shown that
CRG-2 is expressed during the acute and chronic stages of EAE,
suggesting that it contributes to the disease process (13, 29). This is
supported by observations that have shown that blocking CRG-2
expression by intrathecal administration of antisense oligonucleotides
resulted in a significant decrease in the severity of EAE (44). A
single chemokine may exert an essential, nonredundant role in the
development of inflammatory disease, as evidenced by studies by Karpus
et al. (45), which showed that anti-MIP-1 Abs delivered in vivo
blocked EAE. Additional studies have demonstrated that in vivo
neutralization of select chemokines has a profound influence on the
recruitment of T lymphocytes and monocytes to sites of delayed
hypersensitivity reactions, pulmonary granulomas, and interstitial
pneumonia and fibrosis (46, 47, 48, 49).
The cytokine IFN- has been shown to be a potent agonist in
activating both microglia and astrocytes to express and secrete CRG-2
(44). Intravenous injection of mice with IFN- results in
tissue-specific expression of CRG-2 (36, 50). Although IFN-
mRNA is present during the acute phase of infection, i.e., day 7
p.i., we were not able to detect any IFN- at either day 3 or day
35 p.i., yet CRG-2 mRNA was readily detectable at these time
points, suggesting that IFN- is not strictly required for CRG-2
expression. This is consistent with a recent study by Amichay and
colleagues (36), which demonstrated that induction of CRG-2
following infection with various microbes including vaccinia virus was
not completely dependent on IFN- expression. In addition, a recent
study by Asensio and Campbell (25) has demonstrated that CRG-2 is
expressed in the brains of IFN- knockout mice during lymphocytic
choriomeningitis. The majority of CRG-2-positive astrocytes were
not infected with MHV, as determined by in situ hybridization
experiments, suggesting that astrocyte expression of CRG-2 may be
a secondary event preceded by either cytokine/chemokine production by
MHV-infected cells that then function in both an autocrine and/or
paracrine manner to activate neighboring, uninfected astrocytes to
express CRG-2.
The fact that CRG-2 mRNA is expressed predominantly as early as
day 2 in the absence of a prominent inflammatory infiltrate suggests
that MHV infection alone is sufficient to trigger CRG-2 expression
by resident astrocytes. Therefore, it is reasonable to suggest that
CRG-2 is a key signal in initiating the inflammatory response to
MHV infection of the CNS. By day 7 p.i., there is a dramatic
up-regulation in mRNAs for the chemokines MCP-1, MCP-3,
MIP-1ß, and RANTES as well as CRG-2. Furthermore, in
infections with a related MHV strain, increased expression of the
cytokines IFN- and TNF- was also present at this time, which may
potentiate expression of certain chemokines (24, 27). The increased
expression of cytokines and chemokines observed is most likely a
reflection of the massive influx of CD4+ and
CD8+ T cells and macrophages in response to the high viral
burden at this stage of the infection. Infiltrating mononuclear cells,
in addition to resident glia, are most likely responsible for the
production of these chemokines.
The ß-chemokines appear to be the predominant chemokine family
expressed in the CNS during inflammatory disease, including EAE and SIV
encephalitis (13, 14, 43). We have shown that ß-chemokines are
expressed predominantly during the acute phase response to MHV
infection of the CNS. However, during both early (2 days) and late (35
days), the -chemokine CRG-2 is a predominant chemokine
expressed in the brain and spinal cord. The significance of these
observations is not clear. The ß-chemokines tend to target T
lymphocytes and macrophages, which are the major immune cell
participants in various autoimmune disorders as well as in microbial
infections. In contrast, the -chemokines generally attract
neutrophils during inflammation. However, CRG-2 is an
-chemokine that has been shown to be chemotactic for T lymphocytes
and monocytes (11). Therefore, chronic expression of CRG-2 in the
CNS of MHV-infected animals may aid in attracting T cells and
macrophages to sites of viral persistence.
Studies examining chemokines and CNS inflammatory disease have demonstrated that astrocytes are a primary source of chemokines. The in vivo results presented in this study indicate that astrocytes were the predominant cell type to express CRG-2 mRNA during the subacute, acute, and chronic phases of MHV infection. Mac-1-positive cells were also found to express CRG-2 mRNA at day 7 p.i., which represented the height of the inflammatory response, with CD4+ and CD8+ T cells present in the brain and spinal cord as well as increased expression of chemokines and cytokines. In such an environment, the presence of these activating factors appears to be sufficient to stimulate CRG-2 expression by Mac-1-positive cells. The majority of such cells most likely represent microglia and macrophages; however, other cell types, such as neutrophils and NK cells, also express the Mac-1 Ag. Previous work by Williamson et al. (51) has demonstrated that NK cells are present in the brain at day 7 following infection with MHV; therefore, it is possible that some CRG-2-positive cells are NK cells. However, the majority of Mac-1-positive cells had the morphology of macrophage and/or microglia, suggesting that these cell types were responsible for CRG-2 expression.
In vitro studies with astrocytes treated with MHV indicate that a similar chemokine profile exists as compared with the in vivo chemokine profile obtained from MHV-infected mice. The chemokines CRG-2, MCP-3, MIP-1ß, MCP-1, and MIP-2 were expressed in MHV-infected brains and spinal cords as well as MHV-infected astrocytes. In contrast, RANTES is expressed in MHV-infected animals and not in astrocytes exposed to MHV. At the highest concentration of MHV (50 PFU/ml), CRG-2 was the predominant chemokine expressed. Similarly, CRG-2 was the predominant chemokine observed in MHV-infected brains and spinal cords during the acute phase of the disease and was expressed at day 35 in brains and spinal cords. In both circumstances, astrocytes were the primary cell type responsible for CRG-2 expression. UV inactivation of virus resulted in a marked decrease in chemokine gene expression, suggesting that active viral replication contributes to expression of chemokine genes. However, there was not complete reduction in expression, indicating that chemokine expression was not entirely dependent upon MHV replication. Similar results were reported in a recent study by Vanguri and Farber (49), which examined the relationship between chemokine expression by astrocytes and NDV. Expression of CRG-2 by astrocytes was dependent on the concentration of NDV. In addition, UV-inactivated NDV was also able to induce expression of CRG-2 by astrocytes. Together, these results suggest that astrocytes may be exquisitely sensitive to exposure to infectious agents such as viruses, and as a result are activated to send an emergency signal, i.e., chemokines that result in the recruitment of effector cells to sites of infection. An unresolved question regarding MHV-induced chronic demyelination is the state of the persistent viral RNA present in the CNS and what role it has in contributing to chronic inflammation. Our results indicate chemokine expression is not dependent upon MHV replication. These results may be extended to suggest that even if persistent virus is present in the CNS, yet is unable to replicate, i.e., it is defective/translationally inactive, it may still be stimulating chemokine expression, which in turn is contributing to chronic demyelinating disease by attracting effector cells into the CNS to sites of viral RNA.
The results presented in this study clearly show that viral infection of the CNS results in an orchestrated cascade of chemokine gene expression. These results indicate that the regulation of chemokine expression contributes to the initiation and maintenance of the chronic inflammatory response that is observed in MHV-infected mice. Production of chemokines, notably CRG-2, by resident astrocytes may aid in attracting T cells and microglia/macrophage to areas in which MHV is present, and these cells participate in the demyelination process through, as of yet, undiscovered mechanisms.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Thomas E. Lane, Department of Neuropharmacology CVN8, The Scripps Research Institute, La Jolla, CA 90034. E-mail address:
3 Abbreviations used in this paper: MHV, mouse hepatitis virus; CNS, central nervous system; CRG, cytokine-response gene; EAE, experimental autoimmune encephalomyelitis; GFAP, glial fibrillary acidic glycoprotein; IP-10, interferon-inducible protein-10; Mac, macrophage; MCP, macrophage-chemoattractant protein; MIP, macrophage-inflammatory protein; MS, multiple sclerosis; NDV, Newcastle disease virus; PFU, plaque-forming unit; p.i., postinfection; RPA, RNase protection assay.
Received for publication July 10, 1997. Accepted for publication October 3, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
with patterns of tissue expression that suggest nonredundant roles in vivo. J. Immunol. 157:4511.[Abstract]
for an inflammatory response to viral infection. Science 269:1583. in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155:5003.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  K. B. Walsh, M. B. Lodoen, R. A. Edwards, L. L. Lanier, and T. E. Lane Evidence for Differential Roles for NKG2D Receptor Signaling in Innate Host Defense against Coronavirus-Induced Neurological and Liver Disease J. Virol., March 15, 2008; 82(6): 3021 - 3030. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B. Walsh, L. L. Lanier, and T. E. Lane NKG2D Receptor Signaling Enhances Cytolytic Activity by Virus-Specific CD8+ T Cells: Evidence for a Protective Role in Virus-Induced Encephalitis J. Virol., March 15, 2008; 82(6): 3031 - 3044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Miura, E. A. Travanty, L. Oko, H. Bielefeldt-Ohmann, S. R. Weiss, N. Beauchemin, and K. V. Holmes The Spike Glycoprotein of Murine Coronavirus MHV-JHM Mediates Receptor-Independent Infection and Spread in the Central Nervous Systems of Ceacam1a / Mice J. Virol., January 15, 2008; 82(2): 755 - 763. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. B. Walsh, R. A. Edwards, K. M. Romero, M. V. Kotlajich, S. A. Stohlman, and T. E. Lane Expression of CXC Chemokine Ligand 10 from the Mouse Hepatitis Virus Genome Results in Protection from Viral-Induced Neurological and Liver Disease J. Immunol., July 15, 2007; 179(2): 1155 - 1165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. N. Stiles, J. L. Hardison, C. S. Schaumburg, L. M. Whitman, and T. E. Lane T Cell Antiviral Effector Function Is Not Dependent on CXCL10 Following Murine Coronavirus Infection J. Immunol., December 15, 2006; 177(12): 8372 - 8380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Samuel and M. S. Diamond Pathogenesis of West Nile Virus Infection: a Balance between Virulence, Innate and Adaptive Immunity, and Viral Evasion J. Virol., October 1, 2006; 80(19): 9349 - 9360. [Full Text] [PDF]
|
|
|
|
|
|
M.-F. Hsieh, S.-L. Lai, J.-P. Chen, J.-M. Sung, Y.-L. Lin, B. A. Wu-Hsieh, C. Gerard, A. Luster, and F. Liao Both CXCR3 and CXCL10/IFN-Inducible Protein 10 Are Required for Resistance to Primary Infection by Dengue Virus J. Immunol., August 1, 2006; 177(3): 1855 - 1863. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Gonzalez, C. C. Bergmann, C. Ramakrishna, D. R. Hinton, R. Atkinson, J. Hoskin, W. B. Macklin, and S. A. Stohlman Inhibition of Interferon-{gamma} Signaling in Oligodendroglia Delays Coronavirus Clearance without Altering Demyelination Am. J. Pathol., March 1, 2006; 168(3): 796 - 804. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Hardison, R. A. Wrightsman, P. M. Carpenter, T. E. Lane, and J. E. Manning The Chemokines CXCL9 and CXCL10 Promote a Protective Immune Response but Do Not Contribute to Cardiac Inflammation following Infection with Trypanosoma cruzi Infect. Immun., January 1, 2006; 74(1): 125 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Hardison, R. A. Wrightsman, P. M. Carpenter, W. A. Kuziel, T. E. Lane, and J. E. Manning The CC Chemokine Receptor 5 Is Important in Control of Parasite Replication and Acute Cardiac Inflammation following Infection with Trypanosoma cruzi Infect. Immun., January 1, 2006; 74(1): 135 - 143. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Weiss and S. Navas-Martin Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus Microbiol. Mol. Biol. Rev., December 1, 2005; 69(4): 635 - 664. [Abstract] [Full Text] [PDF]
|
|
|
