Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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Resident cells
of the respiratory and gastrointestinal tracts, including epithelial
and fibroblast cells, are the initial sites of entry for many viral
pathogens. We investigated the role that these cells play in the
inflammatory process in response to infection with reovirus 1/L. In
A549 human bronchial or HT-29 human colonic epithelial cells,
interferon (IFN)-, regulated on activation T cell expressed and
secreted (RANTES), IFN-
-inducible protein (IP)-10, and interleukin-8
were upregulated regardless of whether cells were infected with
replication-competent or replication-deficient reovirus 1/L. However,
in CCD-34Lu human lung fibroblast cells, IFN-
, IP-10, and RANTES
were expressed only after infection with replication-competent reovirus
1/L. Expression of interleukin-8 in CCD-34Lu fibroblast cells was viral
replication independent. This differential expression of IFN-
,
RANTES, and IP-10 was shown to be due to the lack of induction of IFN
regulatory factor-1 and -2 in CCD-34Lu fibroblast cells treated
with replication-deficient reovirus 1/L. We have shown that cytokine
and/or chemokine expression may not be dependent on viral replication.
Therefore, treatment of viral infections with inhibitors of replication
may not effectively alleviate inflammatory mediators because most viral
infections result in the generation of replication-competent and
replication-deficient virions in vivo.
proinflammatory signaling; inflammation; transcription factors; interferon-
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INTRODUCTION |
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ALTHOUGH ORGANIZED EPITHELIAL SURFACES were traditionally thought of as a nonspecific physical barrier against invading pathogens, it has been increasingly appreciated that resident mucosal cells directly participate in the response elicited by infectious agents. Airway epithelial cells have the capacity to recruit inflammatory cells to the airways via the release of chemoattractants, to direct inflammatory cell migration across the epithelium via the expression of cell surface molecules such as intercellular adhesion molecules, and to regulate inflammatory cell activity via the release of cytokines (1, 32, 41, 51). Each of these steps amplifies the inflammatory response, establishing the importance of airway epithelial cells in the modulation of airway inflammatory disease.
Chemokines comprise a family of small proteins that have been
implicated in the selective recruitment of leukocyte subsets (11,
36). The -chemokine (C-C) family chemoattracts monocytes, eosinophils, basophils, and T cells. Members include monocyte chemoattractant protein (MCP)-1, MCP-3, MCP-4 (54),
regulated on activation normal T cell expressed and secreted (RANTES)
(23), and eotaxin (34). The
-chemokine
(C-X-C) family can be divided into two subfamilies: one that is
chemoattractant for neutrophils and includes interleukin (IL)-8 and one
that is a potent chemoattractant for lymphocytes and includes
interferon (IFN)-
inducible protein (IP)-10 (15, 40).
Members of both families of chemokines are expressed in epithelial
cells after exposure to infectious agents. In the case of viruses,
infection of cultured or primary human bronchial epithelial cells with
a number of viruses increased IL-8 and RANTES expression in either a
viral replication-dependent or -independent fashion (2, 4, 8, 10,
17, 18, 20, 28, 44, 45, 47, 59). In addition to epithelial
cells, other resident cells, including fibroblasts, can also actively participate in the response to infectious or noninfectious agents. For
example, in response to infection with vaccinia virus in vitro, a
significant increase in IL-6 was observed in human skin fibroblast cells (42). Similarly, MRC-5, a human fetal lung
fibroblast cell line, expressed IL-6 after rhinovirus infection in
vitro in a viral replication-dependent fashion (59).
Reovirus is a double-strand RNA virus that has been isolated from the
gastrointestinal and respiratory tracts of both humans and animals
(56). Reovirus infection is typically lytic in nature, but
chronic infections have also been described both in vitro and in vivo
(19, 56). The ability of reovirus to cause persistent or
chronic infection of cells may be attributed to either mutations in the
viral genome itself or may be dependent on characteristics of the host
cell (19). Our laboratory (5, 6, 35, 52, 53)
has used reovirus 1/L infection of either the gut or the lung in mice
to investigate immunity at mucosal surfaces. Reovirus 1/L infection
induced both humoral- and cell-mediated immune responses at these
mucosal surfaces. Although the lymphocytic cellular response in all of
these models has been well characterized, the role that resident
mucosal cell populations play in the infection and disease process has
not yet been fully investigated. To examine the hypothesis that airway
epithelial and resident fibroblast cells are involved in the regulation
of the inflammatory response to reovirus 1/L, we analyzed the
expression of a panel of cytokines and chemokines in reovirus
1/L-infected A549 human bronchial epithelial cells and CCD-34Lu human
lung fibroblast cells. Because reovirus is a natural pathogen of both
the lung and the gut, HT-29 human colonic epithelial cells were
included to probe whether epithelial cells obtained from the gut
responded in a manner similar to epithelial cells obtained from the
lung. A previous study by Hamamdzic et al. (26)
has shown that although infection of A549 or HT-29 epithelial cells
with reovirus 1/L resulted in a lytic infection, infection of CCD-34Lu
fibroblast cells led to a persistent infection. The viability of
reovirus-infected A549 cells significantly decreased after 24 h of
infection, whereas the viability of HT-29 cells decreased after 72 h of culture. This decrease in viability paralleled an increase in
reovirus mRNA expression. The viability of CCD-34Lu cells was never
affected by reovirus infection, and reovirus replication was not
observed in CCD-34Lu cells until 2 days postinfection. All three cell
lines also produced similar levels of IL-8 in a viral
replication-independent fashion and time frame that paralleled reovirus
mRNA expression (26). However, CCD-34Lu fibroblast cells
that were chronically infected by reovirus 1/L exhibited a prolonged
expression of IL-8, suggesting that this mucosal population may be
involved in the generation of inflammatory responses after the
resolution of the initial lytic infection of the epithelium (26). Here, we describe the triggering of cell
type-specific proinflammatory cytokines, including the induction of the
T cell-specific chemoattractant proteins IP-10 and RANTES,
after reovirus 1/L infection and demonstrate the importance of IFN-
as an autocrine factor regulating this response. These studies suggest
that prolonged expression of chemokines by chronically infected cells
may influence the immunopathogenic consequences of the infection and
that treatment of viral infections with inhibitors of replication may
not effectively alleviate the production of inflammatory mediators.
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METHODS |
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Virus. Reovirus 1/L was originally obtained from Dr. Wolfgang Joklik (Duke University School of Medicine, Durham, NC). Third-passage gradient-purified stocks were obtained by recloning and amplifying parental stocks on L-929 fibroblast cells (American Type Culture Collection, Manassas, VA) (26). After the purification of new stocks, infectious viral titers were obtained by limiting dilution on L-929 monolayers. A replication-deficient preparation of reovirus 1/L was produced by exposure of the infectious virions to an ultraviolet (UV) light source (15 cm for 15 min). This UV-inactivated preparation did not contain infectious virions as determined by titration on L-929 fibroblast cells (<5 plaque-forming units).
Cell culture and infection protocol. The cell lines A549 (human type II bronchial epithelial), HT-29 (human colonic epithelial), and CCD-34Lu (human lung fibroblast) were purchased from the American Type Culture Collection. Each cell line was grown as a monolayer on tissue culture plates (Corning, Corning, NY) in DMEM (high glucose 25 mM) that contained 10% fetal bovine serum (FBS; Sigma, St. Louis, MO), 2 mM L-glutamine, 100 U/ml of penicillin, and 100 mg/ml of streptomycin and was kept at 37°C in an atmosphere of 5% CO2. Experimental interventions were initiated when cells were ~80% confluent. Monolayers in P100 tissue culture dishes were infected by the addition of 1 ml of 0.5% gelatin (Difco Laboratories, Detroit, MI) dissolved in PBS (gel-saline) that contained either reovirus 1/L at a multiplicity of infection (MOI) of 10 or an equivalent inoculum (at a MOI of 10) of UV-inactivated reovirus 1/L; then the cultures were incubated for 1 h at room temperature. After infection, 9 ml of DMEM were added, and the cells were incubated at 37°C in an atmosphere of 5% CO2 after which culture supernatants, cellular RNA, total cellular proteins, or nuclear protein extracts were collected over a 24-h time course for A549 and HT-29 epithelial cells or a 7-day time course for CCD-34Lu fibroblast cells.
RNA preparation and RNase protection assay.
Total cellular RNA was isolated from the cells by guanidinium
denaturation with TRI Reagent (Molecular Research Center, Cincinnati, OH). The riboquant multiprobe RNase protection assay (RPA) human template sets hCK-3 and hCK-5 were purchased from PharMingen (San Diego, CA). The template set hCK-3 contained probes for the cytokines tumor necrosis factor (TNF)-, TNF-
, lymphotoxin-
, IFN-
,
IFN-
, transforming growth factor (TGF)-
1, TGF-
2, and TGF-
3
as well as probes for the control genes glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and L32. The template set hCK-5 contained probes
for the chemokines lymphotactin, RANTES, IP-10, macrophage inhibitory protein (MIP)-1
, MIP-1
, MCP-1, IL-8, and I-309 and probes for the
control genes GAPDH and L32. Radiolabeled RNA transcripts from the
multiprobe sets were generated with an in vitro transcription kit
(PharMingen). Radiolabeled RNA transcripts were then hybridized to
total cellular RNA (10 µg) for 16 h at 55°C followed by RNase digestion with a RPA kit (PharMingen). Samples were precipitated and
separated on 5% denaturing polyacrylamide gels (Amresco, Solon, Ohio).
Gels were then dried and either exposed to Fuji RX film at
70°C
with DuPont Cronex Quanta III intensifying screens for 1-5 days or
analyzed with a Molecular Dynamics phosphorimaging system (Molecular
Dynamics, Sunnyvale, CA).
Western blot analysis.
Cell monolayers were washed twice with PBS (4°C), harvested by
scraping with a rubber policeman (4°C), and pelleted by
centrifugation at 1,200 rpm for 5 min at 4°C. Cells were then lysed
in 300 µl of lysis buffer [PBS, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS, 1 mg/ml of aprotinin, 10 mg/ml of
phenylmethylsulfonyl fluoride (PMSF), and 100 mM sodium
orthovanadate]. The protein concentration was determined with a
modified Lowry procedure (Sigma). Forty micrograms of cell lysate were
run on a premade 12% Tris-glycine denaturing acrylamide gel (Novex,
San Diego, CA). The proteins were transferred to enhanced
chemiluminescence-blotting nitrocellulose membranes (0.45 µm;
Amersham, Arlington Heights, IL) with a semidry transfer system (Novex)
for 2 h at 100 mA. The blots were incubated for 16 h at 4°C
in Blotto blocking solution (PBS with 5% dry milk) followed by
incubation with a rabbit anti-human polyclonal IFN-/
receptor (R)
antibody (residues 314-331 of the precursor form of the human
IFN-
/
R; Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h at
room temperature. The receptor antibody is non-cross-reactive with the
IFN-
R or the IFN-
R. Blots were then washed three times for 15 min
in 1× PBS with 0.05% Tween 20 and incubated with a goat anti-rabbit
IgG-horseradish peroxidase antibody (Santa Cruz Biotechnology) for
1 h at room temperature. The blots were then washed three times
for 15 min in PBS with 0.05% Tween 20 and incubated in enhanced
chemiluminescence developing substrate (Amersham). The blots were
exposed to Fuji RX film for 2-10 min. The size of the detected
protein was determined by comparison to high molecular mass standard
markers (range 15-220 kDa; Amersham).
ELISA.
Tissue culture supernatants were collected and stored at 70°C.
One hundred microliters of each tissue culture supernatant were
analyzed for human IP-10, RANTES, and IFN-
proteins. All samples
were analyzed in duplicate. The concentration of human IP-10 was
determined with a cytokine sandwich ELISA method. 4D5/A7/C5, a purified
mouse anti-human IP-10 monoclonal antibody (PharMingen) at a
concentration of 2 µg/ml, was used as the capture antibody. 6D4/D6/G2, a biotin-conjugated mouse anti-human IP-10 antibody (PharMingen) at a concentration of 1 µg/ml, was used as the detection antibody. A standard curve was plotted with recombinant human IP-10
(R&D Systems, Minneapolis, MN) at concentrations ranging between 0 and
2,500 ng/ml. A human IFN-
ELISA kit was used to quantitate IFN-
protein (Biosource International, Camarillo, CA). Human RANTES protein
concentration was determined by the University of Maryland Cytokine
Core Laboratory (Baltimore, MD).
Preparation of nuclear extracts and electrophoretic mobility
shift assay.
Cell monolayers were detached with a rubber policeman and resuspended
in 1 ml of PBS. Cells were centrifuged at 4,000 rpm at 4°C for 5 min
and resuspended in hypotonic buffer [10 mM HEPES, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM PMSF, and 0.5 mM dithiothreitol (DTT)]. After incubation on ice for 10 min, the cells were homogenized with a 1-ml insulin syringe and centrifuged for 15 min at 4,000 rpm at
4°C to pellet the nuclei. Nuclear pellets were resuspended in
low-salt buffer (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2,
25 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT) followed by dropwise addition of a high-salt buffer (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 1 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM
DTT). Nuclei were then extracted for 30 min at 4°C by agitation
followed by centrifugation at 14,000 rpm for 30 min. The supernatant
was collected and dialyzed in 20 mM HEPES, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT for a minimum of 1 h. The
supernatant was then centrifuged at 14,000 rpm for 20 min and stored in
aliquots at 70°C. The protein concentration of the nuclear extracts
was determined with a modified Lowry procedure (Sigma).
Flow cytometric analysis.
CCD-34Lu fibroblast cells were infected with either reovirus 1/L
or UV-inactivated reovirus 1/L as described in Cell
culture and infection protocol or were treated with 100 IU of
IFN- (Sigma) for the indicated time period. Cells were then released
from the plates by incubation with 5 ml of versene (GIBCO BRL, Life
Technologies, Gaithersburg, MD) for 10 min at 37°C, collected, and
washed twice with PBS containing 2% FBS and 0.002% sodium azide
(Sigma). Cells (1 × 106) were incubated in the
presence of a FITC-labeled polyclonal anti-human major
histocompatibility complex (MHC) I antibody (clone G42-2.6, 5 µg/ml; PharMingen) for 30 min at 4°C. Cells were
then washed three times with PBS and analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Data were analyzed with
CELL-QUEST software (Becton Dickinson).
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RESULTS |
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IFN- expression is induced by reovirus 1/L in
fibroblast cells in a viral replication-dependent fashion and in
epithelial cells in a viral replication-independent fashion. A549,
HT-29, and CCD-34Lu cells were infected with either
replication-competent (viable, MOI of 10) or replication-deficient
(UV-inactivated, equivalent MOI of 10) reovirus 1/L, harvested, and
probed for cytokine gene expression at the mRNA level by RPA analysis
at the indicated time points. In both A549 and HT-29 epithelial cells, IFN-
was induced by infection with either replication-competent (viable) or -deficient (UV-inactivated) reovirus 1/L (Fig.
1, A and
B). Furthermore, induction of IFN-
gene
expression in both A549 and HT-29 epithelial cells was more pronounced
in cells infected with replication-deficient (UV-inactivated) reovirus
1/L than in cells infected with replication-competent (viable) reovirus (Fig. 1, A and B). An IFN-
-specific ELISA was
performed on the culture supernatants at the indicated time points to
investigate whether the increase in IFN-
mRNA expression was
accompanied by an increase in IFN-
protein secretion. Infection of
both A549 and HT-29 epithelial cells with either replication-competent
(viable) or replication-deficient (UV-inactivated) reovirus 1/L
resulted in an increase of IFN-
protein secretion during the 24-h
time course (Fig. 1, A and B). Similar to what
was observed with the IFN-
mRNA level, a greater amount of IFN-
protein was observed after infection of either A549 or HT-29 epithelial
cells with replication-deficient (UV-inactivated) reovirus 1/L (Fig. 1,
A and B).
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In contrast to epithelial cells, in CCD-34Lu fibroblast cells, IFN-
gene expression was induced by reovirus 1/L in a viral replication-dependent fashion (Fig. 1C). IFN-
was not
expressed in control uninfected CCD-34Lu fibroblast cells (Fig.
1C) or CCD-34Lu fibroblast cells infected with
replication-deficient (UV-inactivated) reovirus 1/L. Similarly, in
CCD-34Lu fibroblast cells, secretion of IFN-
protein was only
observed after infection with viable reovirus 1/L (Fig. 1C).
This increase in IFN-
protein was observed beginning on day
3 postinfection, 1 day after significant detection of IFN-
mRNA
(Fig. 1C). All three cell lines constitutively expressed TGF-
1 and TGF-
2, and this expression was not altered by infection with either replication-competent (viable) or replication-deficient (UV-inactivated) reovirus 1/L (Fig. 1).
RANTES and IP-10 are induced by reovirus 1/L in fibroblast cells in
a viral replication-dependent fashion and in epithelial cells in a
viral replication-independent fashion.
In both A549 and HT-29 epithelial cells, IL-8, RANTES, and IP-10 mRNAs,
although not detected or detected at low levels in control uninfected
cells, were induced after infection with either replication-competent
(viable) or replication-deficient (UV-inactivated) reovirus 1/L (Fig.
2, A and B)
(26). Although independent of reovirus 1/L replication,
IP-10 and RANTES gene expression were more pronounced in cells infected
with replication-deficient (UV-inactivated) reovirus 1/L (Fig. 2,
A and B). Infection of both A549 and HT-29 epithelial cells with either replication-competent (viable) or replication-deficient (UV-inactivated) reovirus 1/L also resulted in an
increase in expression of RANTES and IP-10 protein secretion during the
24-h time course (Fig. 2, A and B). However,
although there was a small increase in IP-10 protein secretion in HT-29 epithelial cells infected with replication-deficient (UV-inactivated) reovirus 1/L, uninfected HT-29 epithelial cells constitutively expressed IP-10 protein over the 24-h time course (Fig. 2B).
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RANTES and IP-10 expression are regulated by IFN- in both
epithelial and fibroblast cells.
IFN-
has been reported to act as an autocrine factor that
upregulates the expression of the chemokines RANTES and IP-10
(40). To determine whether the differential expression of
RANTES and IP-10 that was observed in CCD-34Lu cells was not due to a
modulation of IFN-
/
R after infection with replication-competent
(viable) or replication-deficient (UV-inactivated) reovirus, the
expression of the IFN-
/
R in A549 and HT-29 epithelial cells and
CCD-34Lu fibroblast cells was determined by Western blot analysis after incubation with either replication-competent (viable) or
replication-deficient (UV-inactivated) reovirus 1/L. All three cell
lines constitutively expressed the IFN-
/
R, and this expression
was not significantly modulated by infection with either
replication-competent (viable) or replication-deficient
(UV-inactivated) reovirus 1/L (Fig. 3).
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MHC class I expression is modulated by IFN- or infection with
reovirus 1/L in a viral replication-dependent fashion in fibroblast
cells.
To determine whether virally induced IFN-
upregulates surface
MHC class I expression, CCD-34Lu cells were infected with
replication-competent (viable) reovirus 1/L, which resulted in the
production of IFN-
, or with replication-deficient (UV-inactivated)
reovirus 1/L, which did not. Because one effect of IFN-
is its
ability to upregulate surface MHC class I expression (15),
treatment with IFN-
was used as a positive control for the induction
of MHC class I expression. CCD-34Lu fibroblast cells were treated with
either viable (MOI of 10) or UV-inactivated (equivalent MOI of 10)
reovirus 1/L for 4 days or with 100 U/ml of IFN-
for 16 h, and
cell surface expression of MHC class I was evaluated by flow cytometry.
Treatment of CCD-34Lu fibroblast cells with IFN-
increased MHC class
I expression by 3.5-fold from a mean channel fluorescence intensity of
141 for control cells to 496 for IFN-
-treated cells (Fig.
5A). However, only infection
of CCD-34Lu fibroblast cells with replication-competent (viable)
reovirus 1/L and not infection with replication-deficient (UV-inactivated) reovirus 1/L resulted in an increase in MHC class I
expression (mean channel fluorescence intensity of 105 for control cells to 175 for treatment with replication-competent reovirus 1/L;
Fig. 5B).
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NF-B is activated by reovirus 1/L in a viral
replication-independent fashion in epithelial and fibroblast cells,
whereas IRF-1/2 are activated by reovirus 1/L in a viral
replication-dependent fashion in fibroblast cells.
The transcription factor NF-
B plays a critical role in the
transcriptional activation of a number of proinflammatory cytokines and
chemokines including IL-8, RANTES, and IP-10 (3, 55). To
determine whether NF-
B was translocated to the nucleus after infection with either replication-competent (viable) or
replication-deficient (UV-inactivated) reovirus 1/L, DNA binding
studies were performed with nuclear extracts obtained at the indicated
time points. In A549 epithelial cells, activation of NF-
B occurred
within 3 h after infection with either replication-competent
(viable) or replication-deficient (UV-inactivated) reovirus 1/L (Fig.
6A). In both HT-29 epithelial
and CCD-34Lu fibroblast cells, NF-
B was constitutively activated
(Fig. 6, B and C). This activity was maintained
after infection with either replication-competent (viable) or
replication-deficient (UV-inactivated) reovirus 1/L (Fig. 6,
B and C).
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DISCUSSION |
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This study investigated the role that resident mucosal (epithelial and fibroblast) cells from the respiratory tract may play in the inflammatory process. We used reovirus 1/L-infected A549 (bronchial epithelial) and CCD-34Lu (lung fibroblast) cells to examine the hypothesis that lung epithelial and/or fibroblast cells can participate in the inflammatory response after infection by reovirus 1/L. A previous study by Hamamdzic et al. (26) has shown that although infection of A549 epithelial cells with reovirus 1/L resulted in a lytic infection, infection of CCD-34Lu fibroblast cells led to a persistent infection. Both cell lines also produced IL-8 in a viral replication-independent fashion and time frame that paralleled reovirus mRNA expression (26). Chronically infected CCD-34Lu fibroblast cells exhibited a prolonged expression of IL-8, suggesting that this mucosal population may be involved in the generation of inflammatory responses after the resolution of the initial lytic infection of the epithelium (26).
Because epithelial and fibroblast cells release a number of cytokines
in response to various stimuli (32), we extended our initial observations to include the analysis of a panel of additional cytokines and chemokines. We infected cells with either
replication-competent or -deficient reovirus 1/L because there have
been reports that expression of cytokine genes can be viral replication
dependent (2, 4, 17) or independent (18, 30,
45). Reovirus 1/L infection induced the expression of IL-8,
IFN-, IP-10, and RANTES in A549 epithelial cells in a viral
replication-independent fashion. This induction of IL-8, IFN-
,
IP-10, and RANTES was more pronounced in those cells exposed to
replication-deficient reovirus 1/L. This phenomenon is in agreement
with a previous study (31) that showed that IFN production
by mouse fibroblast L cells infected with reovirus 1/L was not
dependent on viral replication. Furthermore, IFN induction in L cells
was increased up to 200-fold and was more rapid in onset when elicited
by replication-deficient reovirus 1/L (31). Our findings
are also consistent with those of others who reported increased IL-8
expression from lung epithelial cells in response to respiratory
syncytial virus (2, 4, 17, 18, 20), rhinovirus (44,
47, 59), rotavirus (45), and influenza virus
(10). In all of these models, IL-8 production was not
regulated by cytokine production but was thought to be directly related
to the viral infection, with some models that were independent of viral
replication and some that required viral replication (2, 10, 18,
20, 45). Lung epithelial cells have also been reported to
express RANTES in either a viral replication-dependent (8)
or -independent fashion (28) after infection with
rhinovirus (44, 59), rotavirus (8), or
respiratory syncytial virus (28) in vitro.
In contrast to A549 epithelial cells, CCD-34Lu fibroblast cells
expressed IFN-, IP-10, and RANTES in a viral replication-dependent fashion. Infection with replication-competent reovirus 1/L, but not
exposure to replication-deficient reovirus 1/L, induced the expression
of IFN-
, IP-10, and RANTES. Hamamdzic et al. (26) previously demonstrated that CCD-34Lu fibroblast cells were
persistently infected by reovirus 1/L and that prolonged induction of
IL-8 was observed. Here we also demonstrated prolonged expression of IFN-
, IP-10, and RANTES. Similar to our finding with CCD-34Lu fibroblast cells, SC1 mouse embryo fibroblasts persistently infected with reovirus released IFN-
(12). Our findings
complement those of others who have demonstrated that fibroblasts can
also actively participate in the response to infectious or
noninfectious agents. In response to infection with either vaccinia or
rhinovirus in vitro, IL-6 expression that was dependent on viral
replication (59) was observed in human fibroblasts
(43, 59). Alternatively, exposure of lung or dermal
fibroblasts to T helper type 1 and 2 cytokines stimulated the
production of eosinophil-activating chemokines (49). IL-4
preferentially stimulated either human lung or dermal fibroblasts to
secrete eotaxin, a potent eosinophil chemoattractant (38,
49), whereas TNF-
stimulated lung fibroblasts to express both
eotaxin and RANTES (49).
Because reovirus is a natural isolate of both the gastrointestinal and
respiratory tracts, HT-29 human colonic epithelial cells were included
in this study to probe whether epithelial cells obtained from the gut
responded to reovirus 1/L infection in a manner similar to epithelial
cells obtained from the lung. Reovirus causes a transient infection of
the gut epithelium of adult immunocompetent mice without any overt
clinical symptoms (9). However, colonic epithelial cells
may respond to infection or injury with chemokine secretion (30,
57) and thus serve as an early signal in immune and inflammatory
reactions. HT-29 colonic epithelial cells expressed IL-8, RANTES,
IP-10, and IFN- in a viral replication-independent fashion. Similar
to this study, rotavirus infection of HT-29 cells also induced
increased secretion of IL-8, growth-related protein-
, and RANTES
that required viral replication (8). Activated colonic
epithelial cells have also been shown to produce chemokines such as
IL-8, MCP-1, and RANTES in response to cytokine activation,
bacterial infection, and protozoan invasion (14, 29, 30, 33,
57). IL-8 and RANTES have been found to be the most potent
chemoattractants for intestinal intraepithelial lymphocytes (7,
13). Therefore, colonic epithelial cells may play an active role
in the recruitment of cells during the inflammatory response
(30). To support a role for epithelial cells in disease
processes, proinflammatory cytokines such as IL-1, IL-6,
granulocyte-macrophage colony-stimulating factor, and TNF-
have been
reported to be increased in serum, cultures of biopsy tissues, and
isolated lamina propria lymphocytes from inflammatory bowel disease
patients (24, 25, 37).
In addition to having antiviral activity, IFN- has been shown to act
as an autocrine factor to induce the expression of the chemokines
RANTES and IP-10 (20). This autocrine loop may be one
likely scenario to explain the differential expression of RANTES and
IP-10 in CCD-34Lu fibroblast cells infected with replication-competent versus replication-deficient reovirus 1/L because we have demonstrated constitutive expression of IFN-
/
R in control and infected cells. In A549 and HT-29 epithelial cells, we have shown that IFN-
is induced in a replication-independent fashion and that treatment with
IFN-
induced the expression of RANTES in A549 lung epithelial cells and both RANTES and IP-10 in HT-29 gut epithelial cells. IFN-
did not induce the expression of IP-10 in A549 lung epithelial cells, suggesting a direct involvement of reovirus in the induction of
IP-10 in this cell line. Therefore, the data support the hypothesis that RANTES and possibly IP-10 induction in epithelial cells may be
regulated by IFN-
in an autocrine fashion. In CCD-34Lu fibroblast cells, IFN-
is expressed in a replication-dependent fashion. Therefore, the absence of IFN-
gene induction in CCD-34Lu fibroblast cells after infection with replication-deficient reovirus 1/L may
explain the failure of UV-inactivated reovirus 1/L to induce RANTES and
IP-10. In support of this hypothesis, IFN-
treatment of CCD-34Lu
fibroblast cells resulted in the induction of both IP-10
and RANTES. The observation that the inhibition of
lipopolysaccharide-induced IP-10 in macrophages by IL-10 was indirect
and dependent on the immediate inhibition of lipopolysaccharide-induced
type I IFNs supports IFN-
as an autocrine factor that induces the
expression of IP-10 and RANTES (48).
Viral induction of IFN- requires NF-
B (22). However,
NF-
B is not the only protein that plays a critical role in the viral induction of IFN-
. Positive regulatory domains of the IFN-
gene interact synergistically to achieve maximal levels of viral induction in most cell types, and these regions serve as high-affinity binding sites for a number of IRFs (27, 42, 50). A549 and HT-29 epithelial cells expressed IRF-1/2 and IRF-3 in a viral
replication-independent fashion. Therefore, the expression of IFN-
,
as well as the expression of the IFN-
-inducible genes RANTES and
IP-10, was observed in a viral replication-independent fashion.
However, in CCD-34Lu fibroblast cells, the expression of IRF-1/2, but
not of IRF-3, was viral replication dependent. Therefore, CCD-34Lu
fibroblast cells infected with replication-deficient reovirus 1/L were
unable to express IFN-
and the IFN-
-inducible genes RANTES and
IP-10. Thus IRF-3 in combination with NF-
B is not sufficient to
induce IFN-
, RANTES, and IP-10 expression in CCD-34Lu fibroblast
cells infected with replication-deficient reovirus 1/L. Additionally, we found that activator protein-1 and NF-IL-6 were also
induced in a viral replication-independent fashion (data not shown).
Because both epithelial and fibroblast cells expressed NF-
B,
activator protein-1, and NF-IL-6 after viral infection, it is not
surprising that IL-8 was expressed after infection with either
replication-competent or replication-deficient reovirus 1/L. Although
we have found a complex pattern of transcription factor expression in
these studies, further studies aimed at probing for a specific NF-
B subunit(s) will help to further identify the molecular mechanisms responsible for cytokine production in this system. It has been suggested that the expression of the Rel B subunit of NF-
B in fibroblast cells is associated with the regulation of multiple cytokines, in part, by regulating overall NF-
B activity (46, 58). Although we have shown that uninfected CCD-34Lu lung
fibroblast cells express NF-
B, it is possible that viral infection
results in an alteration of the NF-
B subunit composition, with a
resultant change in cytokine expression patterns.
Resident mucosal cells may be differentially infected by the same viral pathogen, leading to cell type-specific proinflammatory signaling. These cell type-specific responses may be of interest because it is conceivable that a persistent infection may be clinically undetectable and, if untreated, may lead to more serious disease states. Our laboratory (5, 52, 53) has shown that infection of the respiratory tract with reovirus 1/L results in a spectrum of immunopathology. In these model systems, we have observed the expression of a number of chemokines including RANTES, IP-10, and MCP-1 from whole lung mRNA after reovirus 1/L infection and before inflammatory cell infiltration, suggesting that these chemokines may be the product of resident lung epithelial and/or fibroblast cells (London L, Majeski EI, Altman-Hamamdzic S, Enockson C, Harley RA, and London SD, unpublished observations). In addition, the C-X-C chemokine MIP-2 was also expressed in vivo within 6 h after reovirus infection in a rat lung pneumonia model system (16). Thus chemokine expression and the increased expression of class I MHC on respiratory epithelial and fibroblast cells by reovirus 1/L infection could be an important regulatory factor of the immune response at respiratory mucosal surfaces. The potential for reovirus 1/L to induce cytokine production in a viral replication-independent fashion would represent a mechanism for viral-induced inflammation that may not be affected by the use of antiviral therapies targeted at the inhibition of viral replication. Therefore, the balance of infectious versus defective viruses generated during an infection in vivo may be a critical determinant of the pathology observed.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the Medical University of South Carolina (MUSC) and the Hollings Cancer Center for their support of the MUSC Analytical Flow Cytometry Facility.
![]() |
FOOTNOTES |
---|
* D. Hamamdzic and T. Phillips-Dorsett contributed equally to this work.
This work was supported by National Institute of Allergy and Infectious Diseases Grants R01-AI-40175 and R21-AI-A40175 (both to L. London), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46964 (to S. D. London), and National Institute of Dental and Craniofacial Research DE-00378 (to S. D. London); and a grant from the American Lung Association (to L. London).
Address for reprint requests and other correspondence: L. London, Dept. of Microbiology and Immunology, Medical Univ. of South Carolina, 173 Ashley Ave., PO Box 250504, Charleston, SC 29425 (E-mail: londonl{at}musc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 April 2000; accepted in final form 10 July 2000.
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