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*
Institute for Brain and Immune Disorders, Minneapolis Medical Research Foundation, Minneapolis, MN 55404; and
College of Veterinary Medicine and
Medical School, University of Minnesota, Minneapolis, MN 55404
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokines, were found to inhibit CMV
expression, DNA synthesis, and replication. Treatment with certain
proinflammatory cytokines 24 h before CMV infection markedly
suppressed viral expression in astrocytes. TNF-
, IL-1, and
IFN-
all inhibited CMV expression (70 ± 4.2%, 65 ±
3.4%, and 82 ± 3.6% inhibition of viral expression,
respectively, n = 5). In contrast, no viral
suppression was observed following IL-6 treatment. Suppressive activity
was dependent on the addition of cytokines before CMV infection.
Cytokine pretreatment did not affect CMV entry into primary astrocytes,
and the observed cytokine-induced suppressive activity was not affected
by the NO synthase inhibitor
NG-monomethyl-L-arginine
(NGMA). Instead, the suppressive effect appeared to be
mediated through a mechanism involving inhibition of CMV major
immediate early promoter activity. These results support the hypothesis
that proinflammatory cytokines possess anti-CMV activity in brain
cells and may lead to new interventions for CMV encephalitis based upon
immunotherapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Published studies provide evidence that lymphocytes are able to mediate clearance of viral infections from the CNS without conventional MHC expression on target cells and without massive target cell death (2). Soluble factors, derived from both endogenous brain cells and infiltrating lymphocytes may possess antiviral activity, and the blood-brain barrier may inhibit diffusion and draining of soluble antiviral factors released by specific effector T cells (3, 4). In several animal models, it appears that CD4+ lymphocytes are required to overcome viral CNS infections, and mounting evidence suggests that cytokines play a major role in the blockade of intracerebral viral spread (5). However, little or nothing is known about the role of soluble factors in defense of the brain against human CMV disease.
We have recently found that primary human astrocytes, the predominant cell type in the brain, support productive, cytopathic CMV replication (6). The purpose of the present study was to characterize the regulatory effects of cytokines on CMV expression in acutely infected, primary human astrocytes. We tested the hypothesis that cytokines mediate nonlytic suppression of human CMV in these brain cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human proinflammatory cytokines (IFN-, TNF-,
IL-1, and IL-6), antiinflammatory cytokines (IL-4, IL-10, IL-13, and
TGF-1), and -chemokines (RANTES, macrophage inflammatory protein
(MIP)3-1), as well
as cytokine-specific polyclonal neutralizing Abs (goat) were obtained
from R&D Systems (Minneapolis, MN). Abs to glial fibrillary acidic
protein (GFAP) were obtained from DAKO (Carpinteria, CA).
Preparation of astrocyte cultures
Human fetal brain tissue was obtained from 16- to 22-week-old abortuses under a protocol approved by our Institutional Human Subjects Research Committee. Purified human fetal astrocyte cultures (>99% GFAP positive) were prepared using previously described methods (7), with minor modifications. In brief, brain tissues were cleared of meninges, minced into small fragments, and incubated with 0.25% trypsin (Sigma, St. Louis, MO) in Ca2+- and Mg2+-free Hanks’ saline (Life Technologies, Grand Island, NY) for 30 min at 37°C with gentle shaking. An equal volume of medium containing 10% FBS was added to inactivate the trypsin, and tissues were centrifuged at 1200 rpm for 5 min. After two washings with HBSS (Life Technologies), tissue fragments were replaced with fresh medium and triturated for 15 to 20 passages through a sterile Pasteur pipette. The cell suspension was then seeded into 75-cm2 flasks at a density of 80 to 100 x 106 cells/flask in a high glucose DMEM (Sigma) containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml). These flasks were then incubated at 37°C in a water-saturated, 10% CO2 incubator. After 72 h incubation, medium was changed with fresh DMEM containing 10% FBS. Culture medium was changed once a week thereafter. On day 21, flasks where shaken at 180 to 200 rpm for 16 h, followed by washing with the Ca2+- and Mg2+-free HBSS containing 0.125% trypsin for 30 min at 37°C, followed by addition of 10% FBS-containing medium. After centrifugation, the cell suspension was seeded into a new flask in DMEM containing 10% FBS, and this culture medium was changed 24 h later. This subculturing procedure was repeated three times at a weekly interval. Finally, highly enriched astrocytes (>99% stain with anti-GFAP Ab) were seeded into 24-well plates for experimentation.
Quantitiation of viral expression
The -galactosidase-expressing recombinant human CMV RC256
(constructed in the laboratory of Edward Mocarski, Stanford University)
was obtained from the American Type Culture Collection (ATCC, Manassas,
VA) and propagated on human foreskin fibroblasts (HFF). RC256 contains
the lacZ reporter gene under the control of one copy of the
major early promoter and possesses replication properties that are
equivalent to wild-type CMV (8). Cytokine-treated as well
as untreated astrocyte cultures were infected (multiplicity of
infection (MOI) = 2.5) for 72 h. Infected astrocytes were
pelleted, resuspended in 100 µL PBS, and subjected to three
freeze-thaw cycles.
5-bromo-4-chloro-3-indolyl--D-galactoside
(CPRG; Boehringer Mannheim, Indianapolis, IN) was used as a substrate
at a concentration of 1 mg/ml for quantitation of -galactosidase
activity in the lysates, indicative of viral expression.
Assays for viral replication
Human CMV strain AD169 (ATCC) was grown and titered by 50% tissue culture infectious dose assays (TCID50) on HFF. For determination of viral titers from treated and untreated astrocyte cultures, the infected cells (MOI = 1) were subjected to three cycles of freeze-thaw lysis, and 4-fold serial dilutions were plated onto HFF. The cultures were assessed for cytopathic effect after 14 days to determine TCID50 titers.
Assays for CMV entry into astrocytes and DNA synthesis
DNA extracted from CMV AD169-infected, cytokine-treated, as well as untreated astrocytes, at 6 h postinfection were used to determine the effect of cytokines on viral entry. DNA extracted from astrocytes at 72 h postinfection was used to determine the effect of cytokine treatment on CMV DNA synthesis. Samples from both time points were analyzed by quantitative PCR using the CMV-Quant Kit according to the manufacturer’s instructions (BioSource International, Camarillo, CA).
Measurement of NO production
Supernatants from cytokine-treated and untreated astrocytes were assayed for nitrite content, as a reflection of NO, in both the presence and absence of CMV, by Griess reagent (0.1% naphthylethylene diamine dihydrochloride and 1% sulfanilamide plus 2.5% phosphoric acid in equal volumes), as previously described (9).
Western blot analysis
Preparation of cell lysates, electrophoresis, and protein transfer were performed using standard procedures. Briefly, 1 x 106 cytokine-treated or untreated astrocytes were resuspended in 2 vol of lysis buffer, boiled in Laemmli sample buffer, and loaded onto a 12% SDS-polyacrylamide gel. The gels were transferred onto nitrocellulose paper (Micron Separations, Westborough, MA) and probed for CMV immediate early (IE) protein expression using a rabbit anti-IE72 (R8575, 1:1000, provided by J. Nelson, Portland, OR) Ab. The corresponding IE72 protein was detected using a chemiluminescence detection kit according to the manufacturer’s instructions (Boehringer Mannhiem, Indianapolis, IN).
Northern blot analysis
RNA extraction and preparation of IE-specific probes were performed using standard procedures. Briefly, 2 x 106 astrocytes were pretreated with cytokines for 72 h and subsequently infected with sucrose gradient-purified CMV (AD169). RNA was extracted from the infected astrocytes 3 h postinfection using guanidium isothiocyanate as previously described (10). Ten micrograms of total RNA was separated on 1.5% denaturing formaldehyde agarose gels and transferred to nylon membranes (Magna Graph; Micron Separation). The blots were probed for IE1-specific mRNA using a PCR amplification product of CMV IE1 exon-4 gene region as a probe. Primer sets used to amplify the exon-4 region were 5'-CTCCCCTGATGAGATTATGGCTTATG-3' (sense) and 5'-GGTGGCCAAAGTGTAGGCTACAATAG-3' (antisense). DNA extracted from AD169-infected astrocytes (100 ng) was amplified in a 100-µl PCR containing 10x PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.0 at 25°C), and 1% Triton X-100), 25 mM MgCl2, 10 mM deoxynucleoside triphosphate mixture, 5 U Taq DNA polymerase (Promega, Madison, WI) per milliliter with 0.22 µg/µl TaqStart Ab (Clontech, Palo Alto, CA), and 25 mM primers (sense and antisense) for 27 cycles using cycling conditions of 95°C for 60 s, 65°C for 35 s, and 72°C for 60 s, followed by extension at 72°C for 10 min. Amplified product was purified by electroelution and labeled with [32P]dATP by primer extension using random primers (Prime-it II Kit; Stratagene, La Jolla, CA). Expression of the housekeeping gene GADPH was used as a loading control. Primers used for production of the GAPDH probe were 5'-CCACCCATGGCAAATTCCATGGCA-3' (sense) and 5-TCTAGACGGCAGGTCAGGTCCACC-3' (antisense). Blots were hybridized overnight in 50% formamide at 42°C, then washed under high stringency conditions (0.1 x SSPE at 65°C), developed on a phosphoimager (Molecular Dynamics, Sunnyvale CA) and analyzed using ImageQuant software (Molecular Dynamics). Ratios of GAPDH to IE exon-4 band density were calculated for each treatment to determine the decrease in CMV mRNA expression post cytokine treatment.
Measurement of CMV IE promoter activity
Stocks of E1a-deleted CMV lacZ-containing adenovirus
(obtained from Jeff Albrecht, University of Minnesota) were grown and
titered on 293 cells. Astrocytes were infected with the recombinant
adenovirus at an MOI of 10, and cell extracts were assayed for
-galactosidase activity 72 h postinfection using CPRG.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment of highly purified, fully permissive human astrocytes
with selected proinflammatory cytokines for 24 h before infection
with human CMV was found to dramatically inhibit viral expression.
Addition of IL-1 (10 ng/ml) to astrocyte cultures was highly
suppressive of subsequent CMV infection (Fig. 1
), resulting in a 65 ± 3.4%
inhibition of -galactosidase activity, an indicator of viral
expression. A similar suppressive effect was observed following the
addition of TNF- (20 ng/ml), which reduced CMV expression by 70
± 4.2% (Fig. 1). Treatment of astrocyte cultures with IFN- (200
U/ml) also markedly reduced CMV expression by 82 ± 3.6% (Fig. 1). In contrast, pretreatment of astrocyte cultures with IL-6 (100
ng/ml) did not result in suppression of CMV expression (Fig. 1).
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1 (10 ng/ml), 24 h before CMV infection, had no
effect on viral expression (Fig. 1
). In addition, treatment of
astrocyte cultures with the -chemokines RANTES (100 ng/ml) and
MIP-1 (100 ng/ml), like antiinflammatory cytokines, had no effect on
viral expression (Fig. 1).
Treatment of astrocyte cultures with anti-IL-1,
anti-TNF-, or anti-IFN- Abs (10 µg/ml) 45 min before
addition of the corresponding cytokine blocked the cytokine-induced
suppressive effect on CMV expression (with -galactosidase levels of
159%, 144%, and 53% compared with untreated control cultures,
respectively), whereas 10 µg/ml of isotype control Abs had no effect.
Inclusion of each cytokine into the culture medium had no direct
cytotoxic effect on astrocyte viability at the highest concentration
used, as assessed by trypan blue dye exclusion (data not shown).
Inhibitory effects are cytokine concentration dependent
The doses of cytokines and chemokines used in the experiments
presented in Fig. 1 are high doses that have been reported to
confer distinct effects upon astrocytes in previous experiments
(7, 11, 12, 13, 14). Dose-response studies were conducted with
each of the three cytokines that suppressed expression of CMV. The
suppressive effects of cytokine treatment on CMV expression were found
to be dose dependent and reached maximal suppression at low
concentrations (1 pg/ml, 200 pg/ml, and 0.02 U/ml for IL-1, TNF-,
and IFN-, respectively, Fig. 2).
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Astrocytes are fully permissive for CMV replication
(6). To correlate levels of CMV -promoter activity with
viral replication and the production of infectious progeny, the
infected cultures were collected 7 days postinfection, and plated onto
HFF indicator cells for determination of viral titer by
TCID50 assay. IFN-, TNF-, and IL-1 were
all found to inhibit CMV replication with 75-, 60-, and 10-fold
reductions in TCID50 titers, respectively (Fig. 3). The cytokine doses used in these
experiments were the same as those used in Fig. 1 (i.e., IFN-, 200
U/ml; TNF-, 20 ng/ml; IL-1, 10 ng/ml; and IL-6, 100 ng/ml).
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Cytokine-treated and untreated astrocyte cultures were infected
with CMV (AD169), and viral DNA levels (number of viral genomes) were
quantified by PCR. To determine the effects of proinflammatory cytokine
treatment on viral entry, DNA levels were determined at 6 h
postinfection. The number of CMV genomes measured in cytokine-treated
astrocytes at this early time point (8.08 x
105, 8.46 x 105, and
2.74 x 106 copies for IFN-, TNF-, and
IL-1 treatment, respectively) was similar to that of astrocytes
without cytokine treatment (8.72 x 105
copies, Fig. 4). However, when viral DNA
levels were assessed at 72 h postinfection, the three
proinflammatory cytokines markedly inhibited progeny DNA synthesis:
1.40 x 107 copies of the CMV genome in
untreated astrocytes vs 7.42 x 105 genome
copies for IFN- (94.7% inhibition), 2.61 x
106 copies for TNF- (81.4% inhibition), and
6.49 x 106 copies for IL-1 (53.7%
inhibition) treatment, whereas treatment with IL-6 had no suppressive
effect (1.35 x 107 genome copies; Fig. 4).
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We next examined the effect of the timing of cytokine treatment on
the induction of an antiviral state in astrocytes. For IL-1,
TNF-, and IFN-, strong suppressive activity was dependent upon
addition of cytokines 72 or 24 h before CMV infection, whereas
simultaneous treatment had little or no suppressive effect (Fig. 5). Again, IL-6 pretreatment for either
72 or 24 h displayed no suppressive effect.
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To evaluate the influence of NO on viral expression, the level of
NO produced by astrocytes after cytokine treatment, with and without
CMV infection, was examined. We found that CMV infection of astrocytes
did not itself induce NO (Table I). Of
the three inhibitory cytokines tested, only IL-1 had the ability to
induce NO in astrocytes (52 ± 4.8 µM
NO3-, Table I). Addition
of the NO synthase inhibitor
NG-monomethyl-L-arginine
(NGMA) blocked IL-1-induced astrocyte NO
production (13 ± 3.5 µM
NO3- vs 48 ± 4.7
µM without NGMA), but did not block the
suppressive effects of cytokine treatment on viral expression
(0.75 ± 0.06 OD units vs 0.68 ± 0.08 OD units). Also, the
addition of NGMA to either untreated or TNF--
or IFN--treated, CMV-infected astrocyte cultures had no effect on
viral expression (Table I). Similar results were obtained in two
independent experiments using astrocytes isolated from different brain
specimens. These data provide evidence that the antiviral effects of
proinflammatory cytokines on astrocytes are not mediated through NO
production.
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Western blot analysis of cytokine-treated, and untreated astrocyte
lysates for IE72 protein expression following CMV infection was
performed to determine whether cytokine treatment inhibits IE
expression. Astrocytes were incubated with each proinflammatory
cytokine for 72 h, infected with AD169, and analyzed for IE72
protein levels 24 h postinfection. Infected astrocytes pretreated
with IL-1 , TNF-, or IFN- displayed greatly reduced CMV IE72
levels when compared with untreated controls, whereas cells treated
with IL-6 displayed IE protein levels similar to control cells (Fig. 6).
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To determine whether the effects of proinflammatory cytokines were
mediated at the level of CMV IE1 transcription, RNA from
cytokine-treated and untreated infected astrocyte cultures were
analyzed using an IE exon 4-specific probe. Astrocytes were pretreated
with either IL-1, TNF-, IFN-, or IL-6 72 h before
infection with AD169, and RNA was extracted 3 h postinfection.
Infected astrocytes that were pretreated with TNF- and IFN-
showed markedly decreased IE mRNA transcription, whereas IL-6 treatment
did not influence viral expression compared with untreated controls
(51.2 ± 7.7%, 49.2 ± 9.9%, and 0.9 ± 9.5%,
respectively, based on densitometry, n = 3).
IL-1-treated cells, however, showed increased IE1 band density in
Northern blots (163 ± 8.7%), consistent with the increase in
levels of viral entry following IL-1 treatment (Fig. 6, B
and C).
Cytokine treatment inhibits CMV major IE promoter (MIEP) activity in primary human astrocytes
To determine whether the suppressive effects of proinflammatory
cytokine treatment were due to an inhibition of the activity of CMV
regulatory DNA elements, a replication-defective adenovirus vector was
used to transduce a CMV-MIEP-lacZ construct into our primary
astrocyte cultures. This adenovirus approach was used because primary
brain cells are difficult to efficiently transfect. Transduction of the
construct into untreated astrocytes resulted in high -galactosidase
activity, due to CMV MIEP activity (Fig. 7). This signal was strongly reduced when
the construct was transduced following pretreatment with
proinflammatory cytokines, but not with IL-6 (Fig. 6). Pretreatment for
24 h with IFN-, TNF-, or IL-1 was found to inhibit CMV
MIEP activity by 76%, 70%,and 55%, respectively. Less of a
suppressive effect was observed following simultaneous cytokine
treatment and adenovirus infection (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-1, have been clearly implicated in neuronal damage
(15, 16, 17, 18, 19), little is known about the regulatory effect of
these cytokines on viral infections of the CNS or whether they possess
beneficial, antiviral effects in brain cells.
In response to viral infection, cytokines are secreted within the CNS
from both activated glial cells and T lymphocyte infiltrates
(20). IFN- is a T lymphocyte-produced cytokine that is
well known for its antiviral activity, including effects against CMV
(21, 22, 23). The antiviral activity of TNF- has also
previously been reported (24, 25, 26, 27), and IL-1 treatment
decreases replication of murine CMV in vitro (28).
However, the antiviral effects of particular cytokines depend on
receptors present on specific infected cell types, hence the importance
of using primary cells. We have previously reported on the antiviral
activity of proinflammatory cytokines on
HIV-1SF162 expression in both mixed
glial/neuronal and purified microglial cell cultures (29).
Davignon et al. (30) have shown that CMV IE-1
peptide-specific CD4+ T cell clones inhibit viral
replication in U373 MG astrocytoma cells, and this suppression can be
mimicked through synergistic interaction of IFN- and TNF-. In the
present study, the proinflammatory cytokines IFN-, TNF-, and
IL-1, but not IL-6, were found to markedly suppress CMV expression,
replication, DNA synthesis, and MIEP activity in purified cultures of
primary human astrocytes. In addition, pretreatment of primary
astrocytes with the antiinflammatory cytokines IL-4, IL-10, IL-13, and
TGF-1, or the -chemokines RANTES and MIP-1, were found not to
affect viral expression.
The production and release of antiviral cytokines by glial cells may be
beneficial to neighboring cells through protecting them from infection
(i.e., bystander effect). Although work in our laboratory has
demonstrated that IL-1, IL-6, and TNF- are produced by activated
glial cells, we have been unable to detect IFN- release (using a
sensitive ELISA assay; R&D Systems) from primary human microglial cell,
astrocyte, or neuronal cultures in response to stimulation with LPS,
IL-1, or TNF- (our unpublished data). Thus, infiltrating T
lymphocytes would appear to be a major source of this potent antiviral
cytokine in the CNS. In a healthy state, the CNS is devoid of
identifiable leukocytes; however, during immune activation within the
body, T lymphocytes have been shown to traffic through many organs,
including the brain (31). Because CMV encephalitis usually
occurs only in patients with profound defects in T cell numbers or
function, the loss of the capacity of these cells to infiltrate the
brain and produce IFN- may explain the predisposition of these
patients to CMV brain infection.
Within the CNS, IL-6 has been reported to possess antiinflammatory as
well as proinflammatory effects (32). In our study, IL-6
was not found to have the same profound anti-CMV effects in
astrocytes as the other proinflammatory cytokines. It has recently been
reported that IL-6 alone fails to initiate intracellular signaling
events within astrocytes, but soluble IL-6 receptor restores the
signaling function (33). Therefore, it appears likely that
primary human astrocytes lack sufficient IL-6 receptors to transduce
signals that induce an antiviral state, with corresponding resistance
to CMV expression, following IL-6 treatment. A similar explanation
could underlie the lack of antiviral activity of the -chemokines
RANTES and MIP-1, although the presence or absence of receptors for
these cytokines on astrocytes has not been defined.
Using quantitative PCR, we determined the number of viral genome copies present in infected astrocyte cultures that had been treated with inhibitory cytokines. At a 6-h postinfection time point (i.e., before viral DNA synthesis), we found similar numbers of viral genomes in cytokine-treated and untreated astrocytes, thus suggesting that the suppressive effect of cytokine treatment occurs at a postviral entry step. At 72 h postinfection, markedly less progeny viral DNA was detected in the cytokine-treated astrocytes. Taken together, these data indicate that the three antiviral proinflammatory cytokines exert their suppressive effects at a step between entry of the viral genome and DNA synthesis.
Although NO has been shown to play a role in the inhibition of
replication of many viruses in response to cytokine treatment
(34, 35), the cytokine-mediated inhibition of CMV in
primary human astrocytes reported here was found to be NO independent.
CMV has recently been reported to block NO production in
IL-1/IFN--stimulated human retinal pigment epithelial cells
(23). In contrast to these findings, in the present study
we found that CMV infection does not block IL-1-induced NO
production in astrocytes. Furthermore, the NO synthase inhibitor
NGMA blocked IL-1-induced astrocyte NO
production but did not block the antiviral effects of cytokine
treatment on viral expression. Thus, the suppressive effects of
proinflammatory cytokine treatment of astrocytes are not mediated
through NO production.
Using murine CMV, IFN- treatment has been reported to directly
inhibit IE gene expression in fibroblasts (36, 37). Our
finding of cytokine-mediated inhibition of the CMV IE
promoter-lacZ construct in a recombinant adenovirus
indicates that the antiviral mechanism in astrocytes, at least in part,
involves inhibition of MIEP activity. The Northern blot analysis
demonstrated that IFN- and TNF- also inhibit activity of the CMV
MIEP when positioned at its normal locus within the context of the
viral genome. Based upon these Northern blots, it appears that IL-1 may
mediate its suppressive effect through additional mechanisms. These
data, along with those obtained by quantitative PCR and Western blot,
indicate that the block in CMV expression is not at the level of viral
entry, but rather at the level of viral gene expression.
The molecular basis for the cytokine-mediated viral inhibition reported
here may involve inhibition of the activation of cellular transcription
factors that bind to the CMV IE promoter and trans-activate
transcription. In the murine CMV system, for example, IFN- inhibits
IE gene expression by down-regulating activity of the NF-
B
transcription factor (38). Multiple binding sites for this
transcription factor have also been identified in the human CMV IE
promoter/enhancer region, and NF-B is activated during CMV infection
of HFF (39, 40). We have previously found that TNF- and
IFN-, as well as IL-1, activate NF-B in primary human
astrocytes (41). Additionally, we have found that NF-B
is activated in primary astrocytes in response to CMV infection (our
unpublished data). It is possible that 24 h pretreatment of
astrocytes with proinflammatory cytokines desensitizes the cell to
subsequent CMV-induced NF-B activation, leading to decreased IE
promoter activity and decreased viral expression. This desensitization
could be mediated through increased nuclear levels of newly synthesized
I-B following treatment with inhibitory, proinflammatory
cytokines (42).
In summary, the findings of this study demonstrate that the
proinflammatory cytokines IL-1, TNF-, and IFN- induce an
antiviral state in human astrocytes that results in suppression of CMV
expression and suggest that cytokines play a role in host defense of
the CNS against CMV. These findings may lead to the development of
immune-based therapies for management of CMV encephalitis in
immunocompromised patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James R. Lokensgard, Minneapolis Medical Research Foundation, 914 South 8th Street, Building D-3, Minneapolis, MN 55404. E-mail address:
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; GFAP, glial fibrillary acidic protein; NGMA, NG-monomethyl-L-arginine; CPRG, 5-bromo-4-chloro-3-indolyl--D-galactoside; TCID50, 50% tissue culture infectious dose; HFF, human foreskin fibroblasts; MOI, multiplicity of infection; IE, immediate early; MIEP, major IE promoter.
Received for publication March 19, 1999. Accepted for publication October 26, 1999.
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