Reovirus triggers cell type-specific proinflammatory responses dependent on the autocrine action of IFN-beta

Damir Hamamdzic*, Taetia Phillips-Dorsett*, Sanja Altman-Hamamdzic, Steven D. London, and Lucille London

Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29425


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-beta , regulated on activation T cell expressed and secreted (RANTES), IFN-gamma -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-beta , 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-beta , 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-beta


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 alpha -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)-gamma 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-beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-alpha , TNF-beta , lymphotoxin-beta , IFN-beta , IFN-gamma , transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 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)-1alpha , MIP-1beta , 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-alpha /beta receptor (R) antibody (residues 314-331 of the precursor form of the human IFN-alpha /beta R; Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h at room temperature. The receptor antibody is non-cross-reactive with the IFN-alpha R or the IFN-gamma 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-beta 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-beta ELISA kit was used to quantitate IFN-beta 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).

The double-strand oligonucleotide probe for nuclear factor (NF)-kappa B was synthesized based on the sequence of the NF-kappa B binding site in the IL-8 gene promoter (21). The 5' to 3' sequence of the oligonucleotide for NF-kappa B was AGTTGAGGGGACTTTCCCAGGC. The double-strand oligonucleotide probe for IFN regulatory factor-1 and -2 (IRF-1/2) was synthesized based on the binding sequence located within the IFN-beta gene promoter (50). The 5' to 3' sequence for this probe was GAAAACTGAAAGGGAGAAGTGAAAGT. This probe does not distinguish between IRF-1 and IRF-2 protein binding (27). T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [gamma -32P]ATP (New England Nuclear, Boston, MA) were used to end label the oligonucleotide probes.

For gel shift assays, 5 µg of nuclear extract were incubated with 5 mg/ml of BSA on ice for 30 min. Binding reaction buffer (12% glycerol, 12 mM HEPES, 4 mM Tris · HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, and 1 mM DTT), 2 µg of poly[d(I-C)] (Roche Molecular Biochemicals, Indianapolis, IN), and labeled oligonucleotides (0.3 ng, 25,000 counts/min) were then added to the samples that were incubated, with shaking, for an additional 20 min at room temperature. Nonspecific binding was determined by the addition of a 100-fold excess of unlabeled oligonucleotide as competitor DNA. Transcription factor-DNA complexes were resolved by electrophoresis on 6% nondenaturing polyacrylamide gels (Amresco). The gels were then dried and either exposed to Fuji RX film at -70°C with a DuPont Cronex Quanta III intensifying screen for 1-5 days or analyzed with a Molecular Dynamics phosphorimaging system.

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-beta (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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IFN-beta 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-beta 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-beta 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-beta -specific ELISA was performed on the culture supernatants at the indicated time points to investigate whether the increase in IFN-beta mRNA expression was accompanied by an increase in IFN-beta 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-beta protein secretion during the 24-h time course (Fig. 1, A and B). Similar to what was observed with the IFN-beta mRNA level, a greater amount of IFN-beta 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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Fig. 1.   Cytokine mRNA and protein expression after infection with reovirus (Reo) 1/L. A: A549 human type II bronchial epithelial cells. B: HT-29 human colonic epithelial cells. C: CCD-34Lu human lung fibroblast cells. A-C, top: cell monolayers were infected with either replication-competent Reo 1/L at a multiplicity of infection (MOI) of 10 or an equivalent dose (MOI of 10) of UV-inactivated Reo 1/L. mRNA was harvested at the indicated time points and probed by RNase protection assay (RPA) with the hCK-3 cytokine multiprobe template set. Left, full-length RNA probe set. Drawn lines, observed protected fragments. mRNA expression of the control gene L32 revealed equal loading of samples. TNF, tumor necrosis factor; Lt, lymphotoxin; IFN, interferon; TGF, transforming growth factor; UV, ultraviolet. Data represent 4 independent experiments. A-C, bottom: detection of IFN-beta protein by ELISA in the medium of cells infected with either replication-competent or replication-deficient Reo 1/L. , Control uninfected cells; , cells infected with replication-competent Reo 1/L; black-triangle, cells infected with replication-deficient Reo 1/L. Values are means ± SD from 3 independent experiments performed in duplicate.

In contrast to epithelial cells, in CCD-34Lu fibroblast cells, IFN-beta gene expression was induced by reovirus 1/L in a viral replication-dependent fashion (Fig. 1C). IFN-beta 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-beta protein was only observed after infection with viable reovirus 1/L (Fig. 1C). This increase in IFN-beta protein was observed beginning on day 3 postinfection, 1 day after significant detection of IFN-beta mRNA (Fig. 1C). All three cell lines constitutively expressed TGF-beta 1 and TGF-beta 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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Fig. 2.   Chemokine mRNA and protein expression after infection with either replication-competent or replication-deficient Reo 1/L. A: A549 human type II bronchial epithelial cells. B: HT-29 human colonic epithelial cells. C: CCD-34Lu human lung fibroblast cells. A-C, top: cell monolayers were infected with either replication-competent Reo 1/L at a MOI of 10 or an equivalent dose (MOI of 10) of UV-inactivated Reo 1/L. mRNA was harvested at the indicated time points and probed by RPA with the hCK-5 chemokine multiprobe template set. Left, full-length RNA probe set. Drawn lines, observed protected fragments. mRNA expression of the control gene L32 revealed equal loading of samples. Ltn, lymphotactin; RANTES, regulated on activation T cell expressed and secreted; IP-10, IFN-gamma -inducible protein-10; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; IL, interleukin. Data represent 4 independent experiments. A-C, middle and bottom: detection of RANTES and IP-10, respectively, by ELISA in the medium of cells infected with either replication-competent or replication-deficient Reo 1/L. , Control uninfected cells; , cells infected with replication-competent Reo 1/L; black-triangle, cells infected with replication-deficient Reo 1/L. Values are means ± SD from 3 independent experiments performed in duplicate.

In contrast to A549 and HT-29 epithelial cells, RANTES and IP-10 gene expression were only observed after the infection of CCD-34Lu fibroblast cells with replication-competent (viable) reovirus 1/L (Fig. 2C). The appearance of IP-10-specific mRNA was detected on day 1 postinfection and was present through day 7, whereas RANTES-specific mRNA was first detected on day 2 postinfection and was present through day 7 (Fig. 2C). This was accompanied by secretion of RANTES and IP-10 protein by CCD-34Lu fibroblast cells only after infection with replication-competent (viable) reovirus 1/L (Fig. 2C). IL-8 mRNA was constitutively expressed in CCD-34Lu fibroblast cells but was upregulated from day 3 through day 7 regardless of the replication potential of reovirus 1/L (Fig. 2C) (26).

RANTES and IP-10 expression are regulated by IFN-beta in both epithelial and fibroblast cells. IFN-beta 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-alpha /beta R after infection with replication-competent (viable) or replication-deficient (UV-inactivated) reovirus, the expression of the IFN-alpha /beta 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-alpha /beta 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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Fig. 3.   Expression of IFN-alpha /beta receptor protein by Western analysis after infection with either replication-competent or replication-deficient Reo 1/L. A: A549 human type II bronchial epithelial cells. B: HT-29 human colonic epithelial cells. C: CCD-34Lu human lung fibroblast cells. At the indicated time points, whole cell protein extracts were prepared for Western analysis and probed with a rabbit anti-human polyclonal IFN-alpha /beta receptor antibody. Data represent 2 independent experiments.

Although it is known that RANTES and IP-10 can be induced by IFN-beta , to determine whether infection with reovirus 1/L results in the production of IFN-beta and the subsequent induction of RANTES and IP-10 by virus-induced IFN-beta , it was necessary to determine whether IFN-beta can induce IP-10 and RANTES in these cell lines. To determine whether treatment with IFN-beta caused an upregulation of the chemokines IP-10 and RANTES, all three cell lines were treated with human IFN-beta (100 IU for 16 h) and analyzed for RANTES and IP-10 mRNA expression (Fig. 4). Treatment with IFN-beta induced the expression of RANTES mRNA in A549 epithelial (Fig. 4A), HT-29 epithelial (Fig. 4B), and CCD-34Lu fibroblast (Fig. 4C) cells. Treatment with IFN-beta induced the expression of IP-10 mRNA in HT-29 epithelial (Fig. 4B) or CCD-34Lu fibroblast (Fig. 4C) cells but not in A549 epithelial cells (Fig. 4A).


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Fig. 4.   Effect of IFN-beta treatment on chemokine mRNA expression. A: A549 human type II bronchial epithelial cells. B: HT-29 human colonic epithelial cells. C: CCD-34Lu human lung fibroblast cells. Cell monolayers were treated with 100 IU of IFN-beta . At the indicated time points, mRNA was harvested and probed by RPA with the hCK-5 chemokine multiprobe template set. Left, full-length RNA probe set. Drawn lines, observed protected fragments. mRNA expression of the control gene L32 revealed equal loading of samples. Data represent 3 independent experiments.

MHC class I expression is modulated by IFN-beta or infection with reovirus 1/L in a viral replication-dependent fashion in fibroblast cells. To determine whether virally induced IFN-beta 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-beta , or with replication-deficient (UV-inactivated) reovirus 1/L, which did not. Because one effect of IFN-beta is its ability to upregulate surface MHC class I expression (15), treatment with IFN-beta 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-beta for 16 h, and cell surface expression of MHC class I was evaluated by flow cytometry. Treatment of CCD-34Lu fibroblast cells with IFN-beta increased MHC class I expression by 3.5-fold from a mean channel fluorescence intensity of 141 for control cells to 496 for IFN-beta -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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Fig. 5.   Modulation of major histocompatibility complex (MHC) class I expression after treatment with IFN-beta or infection with replication-competent (viable) or replication-deficient (UV-inactivated) Reo 1/L. CCD-34Lu cell monolayers were treated with 100 IU/ml of IFN-beta for 16 h (A) or infected with Reo 1/L at a MOI of 10 or an equivalent dose of a MOI of 10 of UV-inactivated Reo 1/L for 4 days (B). Cell surface expression of human MHC class I molecules was determined by flow cytometry with a FITC-conjugated antibody. Dashed line, control untreated cells; thick solid line, cells treated with either IFN-beta (A) or viable Reo 1/L (B); thin solid line, cells treated with UV-inactivated Reo 1/L (B). Data represent 4 independent experiments.

NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa 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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Fig. 6.   Nuclear factor (NF)-kappa B activation after Reo 1/L infection. A: A549 human type II bronchial epithelial cells. B: HT-29 human colonic epithelial cells. C: CCD-34Lu human lung fibroblast cells. At the indicated time points, nuclear extracts were prepared for electrophoretic mobility shift assay (EMSA). Arrows, position of the NF-kappa B binding activity and the free probe. NS, nonspecific binding activity in the presence of 100-fold excess unlabeled oligonucleotide. Data represent 4 independent experiments.

Because IFN-beta expression was differentially expressed in CCD-34Lu fibroblast cells infected with replication-competent (viable) versus replication-deficient (UV-inactivated) reovirus 1/L, we investigated the transcription factors IFR-1/2 because expression of these factors is required for activation of IFN-beta gene expression (27, 42, 50). IRF-1/2 expression was activated in both A549 and HT-29 epithelial cells after infection with either replication-competent (viable) or replication-deficient (UV-inactivated) reovirus 1/L (Fig. 7, A and B). However, only infection of CCD-34Lu fibroblast cells with replication-competent (viable) reovirus activated the expression of IRF-1/2 (Fig. 7C). In addition to the expression of IRF-1/2, we also investigated the expression of IRF-3 after infection with either replication-competent (viable) or replication-deficient (UV-inactivated) reovirus 1/L. IRF-3 was constitutively expressed and upregulated in all three cell lines after infection with either replication-competent (viable) or replication-deficient (UV-inactivated) reovirus 1/L (data not shown).


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Fig. 7.   IFN regulatory factor-1 and -2 (IRF-1/2) activation after Reo 1/L infection. A: A549 human type II bronchial epithelial cells. B: HT-29 human colonic epithelial cells. C: CCD-34Lu human lung fibroblast cells. At the indicated time points, nuclear extracts were prepared for EMSA. Arrows, position of the IRF-1/2 binding activity and the free probe. NS, nonspecific binding activity in the presence of 100-fold excess of unlabeled oligonucleotide. Data represent 2 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta , IP-10, and RANTES in A549 epithelial cells in a viral replication-independent fashion. This induction of IL-8, IFN-beta , 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-beta , 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-beta , 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-beta , IP-10, and RANTES. Similar to our finding with CCD-34Lu fibroblast cells, SC1 mouse embryo fibroblasts persistently infected with reovirus released IFN-beta (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-alpha 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-beta 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-alpha , 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-alpha 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-beta 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-alpha /beta R in control and infected cells. In A549 and HT-29 epithelial cells, we have shown that IFN-beta is induced in a replication-independent fashion and that treatment with IFN-beta induced the expression of RANTES in A549 lung epithelial cells and both RANTES and IP-10 in HT-29 gut epithelial cells. IFN-beta 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-beta in an autocrine fashion. In CCD-34Lu fibroblast cells, IFN-beta is expressed in a replication-dependent fashion. Therefore, the absence of IFN-beta 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-beta 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-beta as an autocrine factor that induces the expression of IP-10 and RANTES (48).

Viral induction of IFN-beta requires NF-kappa B (22). However, NF-kappa B is not the only protein that plays a critical role in the viral induction of IFN-beta . Positive regulatory domains of the IFN-beta 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-beta , as well as the expression of the IFN-beta -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-beta and the IFN-beta -inducible genes RANTES and IP-10. Thus IRF-3 in combination with NF-kappa B is not sufficient to induce IFN-beta , 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-kappa 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-kappa 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-kappa B in fibroblast cells is associated with the regulation of multiple cytokines, in part, by regulating overall NF-kappa B activity (46, 58). Although we have shown that uninfected CCD-34Lu lung fibroblast cells express NF-kappa B, it is possible that viral infection results in an alteration of the NF-kappa 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.


    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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