IFN-beta mediates coordinate expression of antigen-processing genes in RSV-infected pulmonary epithelial cells

Mohammad Jamaluddin1, Shaofei Wang1, Roberto P. Garofalo2,3, Todd Elliott2, Antonella Casola2, Samuel Baron3, and Allan R. Brasier1,4

Departments of 1 Medicine, 2 Pediatrics, and 3 Microbiology and Immunology and 4 Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555-1060


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) clear respiratory tract infections caused by the pneumovirus respiratory syncytial virus (RSV) and also mediate vaccine-induced pulmonary injury. Herein we examined the mechanism for RSV-induced MHC class I presentation. Like infectious viruses, conditioned medium from RSV-infected cells (RSV-CM) induces naive cells to coordinately express a gene cluster encoding the transporter associated with antigen presentation 1 (TAP1) and low molecular mass protein (LMP) 2 and LMP7. Neutralization of RSV-CM with antibodies to interferon (IFN)-beta largely blocked TAP1/LMP2/LMP7 expression, whereas anti-interleukin-1 antibodies were without effect, and recombinant IFN-beta increased TAP1/LMP2/LMP7 expression to levels produced by RSV-CM. LMP2, LMP7, and TAP1 expression were required for MHC class I upregulation because the irreversible proteasome inhibitor lactacystin or transfection with a competitive TAP1 inhibitor blocked inducible class I expression. We conclude that RSV infection coordinately increases MHC class I expression and proteasome activity through the paracrine action of IFN-beta to induce expression of the TAP1/LMP2/LMP7 locus, an event that may be important in the initiation of CTL-mediated lung injury.

respiratory syncytial virus; pulmonary inflammation; major histocompatibility complex class II locus; ABC transporter; 26S proteasome; interferon-beta


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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RESPIRATORY SYNCYTIAL VIRUS (RSV) infection is the major cause of epidemic bronchiolitis and pneumonia, accounting for the hospitalization of 100,000 children yearly and 2,000 deaths in the United States alone (42). These inflammatory processes have been linked to chronic sequelae (airway hyperreactivity). Characteristic pathological findings of lower respiratory tract RSV infection include epithelial cell necrosis and sloughing in lower airways, intense peribronchial mononuclear infiltrates, edema, and luminal plugging by mucus and necrotic material (9, 24). Although some of these cytopathic responses may be due to direct effects of the virus, RSV-induced lung injury is also mediated by immunological mechanisms because 1) bronchiolitis occurs late in the course of primary RSV infection at a time when virus is being cleared (38); 2) children with wheezing have high titers of virus-specific IgE and greater lymphoproliferative responses to RSV (50, 51); and 3) children immunized with formalin-inactivated RSV develop severe respiratory disease when subsequently naturally infected with RSV (31).

Primary RSV infection activates antibody- and cell-mediated immune responses (reviewed in Refs. 20 and 38). Neutralizing antibodies appear to have only a minor role in viral clearance; importantly, in patients lacking cell-mediated immunity, prolonged viral shedding and severe disease occur (33 and reviewed in Ref. 38). Although T cell responses are essential for viral clearance, exuberant cytotoxic T lymphocyte (CTL) activity appears to mediate pulmonary injury in severe primary infection and disease augmentation by vaccines. For example, passive transfer of RSV-specific CD8+ CTLs to RSV-infected BALB/c mice allowed clearance of the primary infection, but surprisingly, recipients developed fatal acute respiratory disease (8). Additionally, others have shown that although both CD4+ and CD8+ CTLs are involved in terminating RSV replication, CD8+ CTLs appear to have a major role in causing illness (23). Together, these observations strongly support the involvement of CD8+ T cells in RSV-induced airway disease (8, 23).

CD8+ CTLs destroy virus-infected cells when the viral antigenic peptides are presented on the cell surface by major histocompatibility complex (MHC) class I molecules (reviewed in Ref. 26). The MHC class I complex is a heterodimer of a membrane-anchored heavy chain and a soluble light chain (beta 2-microglobulin) that complexes with antigenic peptide in the endoplasmic reticulum (ER). Peptides presented by the MHC class I molecule are produced by cytosolic proteolysis and are subsequently transported into the ER. Because a significant reduction in MHC class I expression occurs after either exposure to specific proteasome inhibitors or "knock-out" experiments where mice are made deficient in catalytic proteasome beta -subunits, the processing of intracellular peptides for MHC class I presentation is believed to be mediated largely by the proteasome (14, 47). A subsequent step in peptide presentation is the transport of peptide from cytosol to ER where assembly of peptides with MHC class I takes place. It is now established that two genes in the MHC class II locus encode peptides, termed transporter associated with antigen presentation (TAP) 1 and TAP2, which are responsible for shuttling the peptides across the ER membrane (11, 35, 43, 46 and reviewed in Ref. 13).

Herein we examined whether RSV infection induced expression of the MHC class II TAP1/LMP2/LMP7 locus, a region encoding key components of peptide transport and proteasome subunits. Conditioned medium from RSV-infected A549 cells (RSV-CM) when added to naive A549 cells induced the coordinate expression of the TAP1/LMP2/LMP7 locus. The effect of RSV-CM, rich in biologically active interferon (IFN)-beta , was largely blocked by neutralizing IFN-beta antibodies, indicating that IFN-beta is the major inducer present in RSV-CM. The requirement of TAP1/LMP2/LMP7-encoded gene products was indicated through the effect of specific proteasome and TAP inhibitors, which inhibit viral-induced MHC class I expression. These data indicate a role of epithelium-derived IFN-beta in mediating MHC class I expression and cell-mediated RSV immunopathology through coordinate expression of the TAP1/LMP2/LMP7 locus.


    MATERIALS AND METHODS
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Cell culture and treatment. Human A549 pulmonary type II epithelial cells [American Type Culture Collection (ATCC)] were grown in DMEM with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a 5% CO2 incubator (21, 29). Primary cultures of small airway epithelial (SAE) cells were obtained from Clonetics (San Diego, CA) and cultured according to the supplier's recommendations. Recombinant human IFN-beta (Biosource International) was added to the culture medium at the indicated concentrations for 12 h.

Preparation of RSV. The human Long strain of RSV (A2) was grown in HEP-2 cells and purified by polyethylene glycol precipitation followed by centrifugation on a 35-65% discontinuous sucrose gradient as described (24). The virus titer of the purified RSV (pRSV) pool, as determined by a methyl-cellulose plaque assay (25), was eight to nine log plaque-forming units per milliliter. Virus pools were divided into aliquots, quick-frozen, and stored at -70°C until used.

RSV-CM. RSV-CM was prepared by infecting monolayers of A549 cells with pRSV at a multiplicity of infection (MOI) of 1 in DMEM with 2% FBS for 48 h. The supernatant was collected, centrifuged at 3,000 g, exposed to ultraviolet light to inactivate the live virus, quick-frozen, and stored at -70°C until used (19). When indicated, RSV-CM was used to stimulate fresh cells at a 1-25% (vol/vol) concentration for various times. For antibody neutralization, 20 µl of RSV-CM were mixed with either 15 µg of rabbit anti-human IFN-beta antibody (Chemicon International), 30 µg of anti-human interleukin (IL)-1alpha antibody (R&D Systems), or 30 µg of preimmune serum (PI) in a total volume of 2 ml of culture medium and incubated for 2 h at 37°C. For vaccinia virus-mediated expression, cells were infected with wild-type vaccinia virus (VV-TK-) or recombinant (VV-ICP47) virus at MOI of 0.1 for times indicated.

IFN-beta bioassay. IFN-beta activity was determined using a virus plaque reduction assay, as described previously (13, 40). Briefly, serial dilutions of the culture supernatant were assessed for their ability to inhibit vesicular stomatitis virus-induced cytopathic effect on WISH cells. This assay is standardized with the international reference IFN of known activity.

Flow cytometry. Cell surface expression of MHC class I was analyzed by flow cytometry as described (19) using monoclonal antibodies (MAbs) specific for a common epitope on HLA-A, -B, and -C (MAb W6/32, ATCC). Briefly, adherent A549 cells were dislodged by short treatment with trypsin-0.1 M EDTA, washed twice in phosphate-buffered saline (PBS), and counted. Approximately 5 × 105 viable cells were stained with either 1 µg of anti-class I MHC (W6/32) or irrelevant MAbs of the same isotype in 100 µl of fluorescence-activated cell sorter (FACS) staining buffer (1% fetal calf serum in PBS) at 4°C for 30 min. The cells were washed three times in FACS buffer and incubated with 2 µg of FITC-conjugated anti-mouse F(ab')2 antibody (Tago) at 4°C for 30 min. Washed cells were fixed in 1% paraformaldehyde, and 10,000 cells per treatment were analyzed for fluorescence by single-color flow cytometry on a FACScan analyzer (Becton Dickinson).

cDNA probes. A549 mRNA was reverse transcribed using Superscript II and oligo(dT) as a primer according to the manufacturer's recommendations (GIBCO BRL). Partial cDNAs encoding TAP1, low molecular mass protein (LMP) 2 and LMP7 were amplified using A549 cDNA as a template. The 322-nucleotide fragment of TAP1 (GenBank accession number X66401) was amplified using the primer pair TAP1 (antisense) 5'-ATGAAGCTTCATTCTGGAGCATCTGCAGGAGCCTG-3' and TAP1 (sense) 5'-GGCTGGATCCCAGCTGTCAGGGGGTCAGC-3' and cloned into the pCRII plasmid (Invitrogen). The 250-nucleotide fragment of LMP2 (accession number X66401) was amplified using the primer pair LMP2 (sense) 5'-CTGGGATCCATGCTGACTCGACAGCCTTTTGC-3' and LMP2 (antisense) 5'-GAAGAAGCTTCACTCATCATAGAATTTTGGCAGTTCATT-3' and cloned into pCRII (Invitrogen). The 280-nucleotide fragment of LMP7 (accession number X66401) was amplified using the primer pair LMP7 (antisense) 5'-ACCAAGCTTATTGATTGGCTTCCCGGTACTG-3' and LMP7 (sense) 5'-CAAGGATCCTACTACGTGGATGAACATGG-3' and cloned into the plasmid pCRII (Invitrogen). All cDNAs were sequenced to confirm authenticity. Probes were radiolabeled using asymmetric PCR amplification of the cDNA insert with the corresponding antisense primer and purified by gel filtration chromatography (49). The 566-bp fragment of human MHC-I cDNA (accession number M11886) was generated by PCR using A549 cell cDNA by the primer pair 5'-GCAAGGATTACATCGCCCTG AACGAG-3' (sense) and 5'-CATCATAGCGGTGACCACAGCTCCAA-3' (antisense) obtained from Clontech (Palo Alto, CA). Radiolabeled probe was made by asymmetric PCR (using the antisense primer) and the 566-bp fragment gel-purified before hybridization.

Northern blot. Total cellular RNA was extracted from A549 cells by acid guanidium-phenol extraction (RNAzol B, TelTest). RNA (30 µg) was denatured, fractionated by electrophoresis on a 1.2% agarose-formaldehyde gel, capillary transferred to a nitrocellulose membrane (Zeta-Probe GT, Bio-Rad), and prehybridized as described previously (28, 48). The membrane was sequentially hybridized with 1-2 × 106 counts/min of 32P-TAP1, -LMP2, and -LMP7 cDNA probes or MHC-I probe at 50°C overnight in 5% sodium dodecyl sulfate (SDS) hybridization buffer. The membrane was washed with a buffer containing 5% SDS and 1× saline-sodium citrate (0.15 M NaCl and 0.015 M sodium citrate) for 20 min at room temperature followed by 30 min at 50°C. Internal control hybridization was performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S RNA (49). The membrane was exposed to XAR film (Kodak) for 24-48 h at -70°C and quantified by exposure to PhosphorImager cassette (Molecular Dynamics).

Western immunoblot. Cytoplasmic proteins (200 µg) from RSV-infected or IFN-gamma -treated cells were separated in 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane by electroblotting. The membrane was blocked in 5% nonfat dry milk and probed with TAP1 antibody (Rockland, Gilbertsville, PA) or LMP7 antibody (Affiniti Research Products, Exeter, UK). Immunocomplexes were detected by horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (Amersham). As internal control, the same membrane was probed with MAb to beta -actin (Sigma, St. Louis, Mo).

26S proteasome assay. Chymotryptic activity of the 26S proteasome was measured in cytoplasmic A549 cell extracts as described previously (25, 28, 41). Cytoplasmic protein (150 µg) was added to assay buffer (20 mM Tris-Cl, pH 8.0, 1 mM ATP, and 2 mM MgCl2) in the presence of the fluorogenic synthetic peptide substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (AMC; final concentration 60 µM; Sigma) in a final volume of 1 ml. The tubes were incubated at 30°C for 30 min, after which the reaction was terminated by the addition of 1 ml of cold ethanol and the lysate was spun at 12,000 g for 10 min at 4°C. Fluorescence product in the supernatant was measured in a fluorometer at 440-nm emission (Iex, 380 nm) and activity was normalized to AMC standards. Chymotryptic activity was depleted by ultracentrifugation at 150,000 g for 26 h at 4°C (conditions where 26S proteasome is sedimented, leaving other proteases in solution) (25).


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Class I MHC expression in RSV-infected A549 cells is a paracrine effect. Respiratory epithelial cells are the primary sites of RSV infection and replication. After exposure to RSV, human alveolar type II-like epithelial cells (A549) synthesize an identical pattern of chemokines and viral structural proteins and exhibit viral cytopathic effects (syncytia formation) typical of primary cells (20, 21, 32). In addition, RSV infection enhances cell surface class I MHC expression (19) and proteasome activity in A549 cells (28) through an unknown mechanism. We reasoned that the changes in MHC class I and proteasome activity may be linked. The recent mapping of the MHC locus on the short arm of human chromosome 6 [6p21.3, (5, 6)] intrigued us, especially an 800-kb region that contained a gene cluster involved in proteasome processing and peptide transport. Here, the gene encoding the peptide transporter TAP1 and the gene encoding inducible proteasome subunit LMP2 are simultaneously under control of a bidirectional promoter. The TAP1/LMP2 transcriptional unit is closely linked with a separate transcription unit encoding another inducible proteasome subunit, LMP7. Because TAP1 is required for peptide transport into the ER (2, 36) and LMP2 and LMP7 are incorporated into the proteasome at the expense of other constitutive subunits to alter its substrate specificity (12, 17), we investigated whether TAP1, LMP2, and LMP7 gene expression is altered after RSV infection. In uninfected cells, low levels of 2.8-kb TAP1, 0.9 kb LMP2, and 1.3 kb LMP7 transcripts could be detected (Fig. 1A). After 6 h of RSV infection, a significant induction of TAP1 and LMP2 expression was observed that continued to increase until a peak at 24 h, indicating a rapid and sustained activation of the bidirectional TAP1/LMP2 promoter (Fig. 1A). The LMP7 gene was expressed at a greater level in uninfected cells and its magnitude of induction was less (Fig. 1A). Nevertheless, LMP7 expression was activated with similar kinetics as TAP1/LMP2 where induction could be detected as early as 6 h and maximum expression occurred at 24 h (Fig. 1A). At peak levels of expression (24 h of RSV infection), there was a 28-fold, 30-fold, and 3-fold increase in TAP1, LMP2, and LMP7 expression, respectively, compared with that in uninfected cells (0 h). At 36 h (and later time points), the steady-state levels of TAP1, LMP2, and LMP7 transcripts fell. This appeared not to be entirely due to cytopathic viral effects as the levels of 18S RNA remained constant during this time.


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Fig. 1.   A: respiratory syncytial virus (RSV) infection induces expression of TAP1/LMP2/LMP7 in A549 cells. A549 cells were infected with RSV [multiplicity of infection (MOI) of 1] for 0-36 h. Total RNA was isolated and the steady-state level of transporter associated with antigen presentation (TAP) 1, low molecular mass protein (LMP) 2 and LMP7 transcripts were determined by Northern blot. Shown is an autoradiographic exposure following hybridization with specific probes. The blot was probed with 18S rRNA probe to determine equal loading of samples. B: induction of TAP1/LMP2/LMP7 expression in small airway epithelial (SAE) cells by RSV infection. TAP1, LMP2, and LMP7 transcripts were detected by Northern blot (see METHODS). The blot was hybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to show equal loading of samples. C: inducible expression of MHC-I transcripts in A549 cells by RSV infection. A549 cells were infected with RSV for times indicated, and major histocompatibility complex (MHC)-I expression was determined by Northern blot. Shown is an autoradiographic exposure following hybridization with MHC-I or GAPDH probe. D: effect of conditioned medium from RSV-infected cells (RSV-CM) on TAP1/LMP2/LMP7 expression. A549 cells were treated with RSV-CM (25% vol/vol) for 0-48 h, and transcript abundance was measured by Northern blot. 18S RNA hybridization is shown as internal control.

To show that TAP1/LMP2/LMP7 expression by RSV infection also occurs in primary SAE cells, SAE cells were infected with RSV for 0-24 h and gene expression was measured by Northern blot (Fig. 1B). RSV infection resulted in increased expression of TAP1/LMP2/LMP7 transcripts in a similar time-dependent manner as seen in A549 cells. Like that seen for 18S transcripts, the expression of housekeeping GAPDH mRNA also was not altered by RSV infection. Previously, we showed that RSV induced cell surface MHC-I expression in A549 cells by flow cytometry. To show that the cell surface expression of peptide-bound MHC-I by RSV is not entirely dependent on presynthesized MHC-I protein rather than RSV-induced MHC-I transcription leading to enhanced MHC-I synthesis, the steady-state level of MHC-I mRNA was measured by Northern blot in RSV-infected A549 cells. MHC-I mRNA abundance detectably increased 12 h after RSV infection (Fig. 1C) and further increased 24 h after infection. These data indicate coordinate induction of antigen processing and presentation genes by RSV infection in A549 and SAE cells.

To separate whether RSV replication directly or secretion of paracrine factors secondarily were responsible for TAP1/LMP2/LMP7 expression, we tested the effect of ultraviolet-inactivated RSV-CM on naive A549 cells. Cells were incubated with RSV-CM (25% vol/vol) for different periods of time (0-48 h) before analysis of TAP1/LMP2/LMP7 expression by Northern blot. In unstimulated cells, low levels of 2.8-kb TAP1, 0.9-kb LMP2, and 1.3-kb LMP7 transcripts could be detected (Fig. 1D). After treatment (24 h), RSV-CM increased TAP1, LMP2, and LMP7 transcript abundance, peaking at ninefold, ninefold, and fourfold, respectively (Fig. 1D).

To show that RSV increased TAP1 and LMP7 protein levels in infected A549 cells, Western immunoblot was done using specific antibodies to TAP1 and LMP7. There was a low level of both TAP1 and LMP7 protein detected in uninfected cells that increased in a time-dependent manner after either RSV infection or IFN-gamma stimulation in parallel with their changes in mRNA (Fig. 2).


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Fig. 2.   RSV infection increases TAP1 and LMP7 protein in A549 cells. Cytoplasmic extracts from RSV-infected or interferon (IFN)-gamma -treated cells were analyzed, and TAP1 and LMP7 were detected by Western immunoblot. beta -Actin staining represents equal loading of samples.

To determine the dose of RSV-CM required to enhance the expression of these genes, cells were challenged with different amounts of RSV-CM (prepared from 48-h infected cells) for 12 h. As low as 1% (vol/vol) RSV-CM was able to induce the expression of TAP1, LMP2, and LMP7 transcripts, with an apparent saturation at ~5% (Fig. 3A). These data indicate that RSV-CM contains an extremely potent factor sufficient for induction of TAP1/LMP2/LMP7 expression.


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Fig. 3.   A: RSV-CM increases TAP1/LMP2/LMP7 expression in a dose-dependent manner. A549 cells were incubated with different concentrations of RSV-CM (% vol/vol) for 12 h. Shown is the autoradiogram of TAP1, LMP2, and LMP7 expression by Northern blot. Hybridization of 18S RNA represents internal control. One-half maximal stimulation is seen between 1 and 2% (vol/vol) RSV-CM and 90% maximal stimulation is seen at 5% RSV-CM. B: IFN-beta induces the expression of TAP1/LMP2/LMP7. A549 cells were stimulated with increasing doses of human recombinant IFN-beta for 6 h before harvest and assay. TAP1, LMP2, and LMP7 mRNA levels were measured by Northern blot. 18S RNA hybridization signal is shown as internal control.

RSV-CM contains biologically active IFN-beta . To determine whether IFN-like bioactivity was present in the RSV-CM and, if so, its kinetics of secretion, we analyzed IFN activity in A549 supernatants after RSV infection for different lengths of time (0-48 h). A plaque reduction assay of vesicular stomatitis virus infection of WISH cells (40) indicated that RSV infection rapidly induced IFN production in a time-dependent manner. IFN activity was first detectable at 120 IU/ml (3 h after infection, n = 2 experiments), rose to 780 IU/ml (6 h after infection), and peaked at 12,000 IU/ml (12 h after infection, data not shown). This activity was entirely ascribed to IFN-beta by the ability of neutralizing antibodies to block its effect. We noted that the RSV-induced increase in IFN-beta production within 3 h of RSV infection was a time preceding TAP1/LMP2/LMP7 expression (compare with Fig. 1).

To compare the effect of RSV-CM with that of recombinant IFN-beta (rIFN-beta ), we treated A549 cells with various doses of rIFN-beta . We observed a broad dose-response curve, with detectable stimulation of TAP1/LMP2/LMP7 expression at 125 U/ml that did not appear to saturate until 500 U/ml (Fig. 3B); importantly, this maximal stimulatory concentration was reached in RSV-CM within 6 h after RSV infection. The maximal effect of rIFN-beta resulted in an average 20-fold induction of TAP1 and LMP2 steady-state mRNA levels after 6 h of stimulation. Similarly, LMP7 expression was detectable after 6-h stimulation and peaked after 12 h. These data indicate that bioactive IFN-beta is present in RSV-CM at sufficient concentrations to active TAP1/LMP2/LMP7 expression.

In addition to paracrine IFN-beta secretion RSV infection also induces IL-1alpha secretion at concentrations sufficient to induce paracrine synthesis of intercellular adhesion molecule-1 (39) and may also have a role in MHC class I expression. To determine whether IL-1alpha could induce TAP1/LMP2/LMP7 expression, we incubated A549 cells with and without human recombinant IL-1alpha (4 ng/ml), IFN-beta (1,000 U/ml), or both for 12 h (Fig. 4). Either alone or in combination with IFN-beta , IL-1alpha had no detectable effect on the abundance of TAP1, LMP2, or LMP7 transcripts. These data suggest that IFN-beta probably represents the major paracrine factor responsible for the coordinate expression of the TAP1/LMP2/LMP7 locus in RSV-infected epithelial cells.


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Fig. 4.   Effect of recombinant IFN-beta and interleukin (IL)-1alpha . A549 cells were treated with either IFN-beta (1,000 U/ml) or IL-1alpha (4 ng/ml) alone or in combination for 12 h. Induction of TAP1, LMP2, and LMP7 was determined by Northern blot. 18S RNA hybridization is shown as internal control (Cont).

Effect of neutralizing IFN-beta and IL-1alpha in RSV-CM. To determine the role of IFN-beta in RSV-CM-induced TAP1/LMP2/LMP7 expression, RSV-CM treated with excess amounts of neutralizing antibodies to IFN-beta or IL-1alpha was used to stimulate naive A549 cells for 12 h (Fig. 5). In this experiment, untreated RSV-CM (1%) induced TAP1/LMP2/LMP7 expression where the individual steady-state mRNA abundance was increased five- to sevenfold over untreated control. RSV-CM treated with PI induced TAP1/LMP2/LMP7 expression indistinguishably from untreated RSV-CM. However, RSV-CM neutralized with anti-IFN-beta was markedly, but not completely, reduced in its ability to induce TAP1/LMP2/LMP7 expression. By contrast, RSV-CM neutralized with anti-IL-1alpha did not inhibit TAP1/LMP2/LMP7 expression. Consistent with the effects observed with recombinant hormones, RSV-CM neutralized with a combination of anti-IFN-beta and anti-IL-1alpha antibodies showed no effect greater than that of RSV-CM treated with anti-IFN-beta alone. As a control, IFN-beta bioactivity in RSV-CM after antibody neutralization with anti-IFN-beta was measured and confirmed to be <50 U/ml, levels below those needed for its effect (see Fig. 3). To confirm that IL-1alpha in RSV-CM was neutralized by anti-IL-1alpha treatment, we measured IL-1alpha bioactivity in RSV-CM by a nuclear factor-kappa B (NF-kappa B) activation assay, where RSV-CM neutralized with anti-IL-1alpha (but not the preimmune control) failed to activate NF-kappa B complex (data not shown). These data indicate that IFN-beta , but not IL-1alpha in RSV-CM, is predominantly responsible for TAP1/LMP2/LMP7 expression. However, the inability of anti-IFN-beta to completely inhibit RSV-CM induction of TAP1/LMP2/LMP7 expression perhaps indicates the potential presence of additional virus-specific or paracrine factors that have an additional role in the induction of TAP1/LMP2/LMP7 genes (see DISCUSSION).


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Fig. 5.   RSV-CM-induced expression of TAP1/LMP2/LMP7 is IFN-beta dependent. RSV-CM (48 h) was pretreated with rabbit anti-human IFN-beta antibody (anti-IFN-beta ), anti-human IL-1alpha antibody (anti-IL-1alpha ) or preimmune serum (PI) in a total volume of 2 ml of culture medium. Naive A549 cells were then treated with antibody pretreated RSV-CM (1% vol/vol) for 12 h, and transcript levels were measured by Northern blot. 18S RNA hybridization signal is shown as internal control. Addition of anti-IFN-beta antibody reduced the TAP1 mRNA expression level to 46.8 ± 6.62%, LMP2 mRNA to 36.6 ± 0.77%, and LMP7 mRNA to 58.5 ± 2.95% compared with untreated RSV-CM.

Effect of IFN-beta on inducible 26S proteasome activity. LMP2 and LMP7 constitute inducible beta -catalytic subunits of 26S proteasome that are preferentially incorporated into the 26S proteasome at the expense of constitutive subunits and alter proteasome activity (12, 16-18). To establish whether IFN-beta and RSV-CM could activate 26S proteasome activity after cell stimulation, we measured cytosolic chymotryptic activity using a fluorogenic assay that specifically detects activity of the 26S proteasome (25, 28). Both rIFN-beta and RSV-CM increased chymotryptic activity significantly over control A549 cytoplasmic extracts (Fig. 6). Furthermore, to examine whether the RSV-CM-induced increase in chymotryptic activity was due to IFN-beta , both rIFN-beta and RSV-CM were neutralized with anti-IFN-beta antibodies and used to stimulate cells. Anti-IFN-beta completely abrogated chymotryptic activity in cells treated with rIFN-beta and significantly reduced that induced by RSV-CM (the inability to completely block the RSV-CM effect in this particular experiment is probably due to slight differences in biological activity of IFN-beta in various RSV-CM preparations). On the other hand, treatment with anti-IL-1alpha antibodies did not reduce RSV-CM-stimulated chymotryptic activity, indicating that IFN-beta in the RSV-CM largely mediates the increase in 26S proteasome activity.


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Fig. 6.   Cytosolic 26S proteasome activity in stimulated A549 cells was mediated by IFN-beta . A549 cells were treated with recombinant IFN-beta (1,000 U/ml), before and after neutralization with anti-IFN-beta or RSV-CM (1% vol/vol) before and after neutralization with anti-IFN-beta or anti-IL-1alpha antibodies. Chymotryptic activity was measured in cytoplasmic extract of A549 cells using a specific fluorogenic assay for hydrolysis of Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (28). *P < 0.01, RSV-CM vs. RSV-CM + anti-IFN-beta . **P < 0.001, IFN-beta vs. IFN-beta  + anti-IFN-beta .

To confirm the requirement of the 26S proteasome in MHC class I expression, cells were treated with a specific irreversible 26S proteasome inhibitor, lactacystin (15, 25). Lactacystin-pretreated cells were infected with RSV and failed to express cell surface MHC class I after RSV infection (Fig. 7), indicating an absolute requirement for proteasome activity in RSV-inducible MHC-I upregulation.


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Fig. 7.   Inhibition of RSV-induced MHC class I upregulation by proteasome inhibitor lactacystin. A549 cells were left untreated or infected with RSV or RSV + lactacystin (50 µM) for 24 h, and MHC class I expression was measured by immunofluorescence.

Inhibition of MHC class I expression by herpes virus ICP47. Recently, a herpes virus-derived peptide, ICP47, was found to strongly inhibit MHC class I antigen presentation to CD8+ T lymphocytes (22, 27, 53). Both in vivo and in vitro studies with recombinant ICP47 demonstrated that ICP47 competes with peptide binding to TAP and blocks peptide transport into the ER (1, 45). To determine whether the MHC class I upregulation following RSV infection is TAP dependent, we used a recombinant vaccinia virus expressing ICP47 (VV-ICP47). Control and RSV-infected A549 cells (for 30 h) were then infected with VV-ICP47 or control vaccinia TK- (VV-TK-) for 6 h before harvest. Cells were harvested, stained with anti-MHC class I antibodies, and subjected to FACS analysis. In uninfected cells, VV-ICP47 inhibited 50% of the basal MHC class I expression (Fig. 8). In contrast, control VV-TK- did not inhibit basal MHC class I expression but rather stimulated it. In RSV-infected cells, VV-TK- partially inhibited MHC class I expression as a nonspecific effect, but VV-ICP47 had a much higher inhibitory effect on the expression of MHC class I. These data indicate that in A549 cells, both basal and RSV-induced enhanced expression of MHC class I are greatly dependent on TAP function.


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Fig. 8.   Inhibition of RSV-induced MHC class I upregulation by the TAP inhibitor ICP47. A549 cells were infected with RSV at MOI of 1. Control or 24-h RSV-infected cells were treated with either wild-type (VV-TK-) or recombinant vaccinia virus expressing human herpes virus protein ICP47 (VV-ICP47) at MOI of 0.1. Six hours later, all cells were harvested and MHC class I expression measured by flow cytometry.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

Airway epithelial cells infected with the mucosal pathogen RSV express a number of homeostatic and inflammatory genes that serve to initiate and sustain the immune response. Clinically, RSV-induced severe lower respiratory tract infection and vaccine-augmented disease initiate lung damage through immunopathogenic mechanisms in which CD8+ CTLs appear to have an important role. CD8+ CTLs sample cells for the presence of foreign intracellular proteins through antigens presented on the cell surface in the context of MHC class I molecules. In this study, we demonstrate that RSV infection induces expression of the MHC class II locus containing the TAP1/LMP2/LMP7 genes that encode products important for the sequential peptide processing and translocation involved in class I MHC antigen presentation. Our most important finding is that these genes and the proteins that they encode are coordinately induced by RSV infection and their induction is in part mediated by a secreted paracrine factor IFN-beta . We further showed that the kinetics of TAP1/LMP2/LMP7 expression by RSV is similar in both normal SAE and transformed type II alveolar cells (A549), indicating that induction of MHC-processing genes is a general response of the RSV-infected respiratory epithelium. To our knowledge, this is the first report of MHC class I antigen-processing gene expression by respiratory viruses.

Inducible expression of MHC class II TAP1/LMP2/LMP7 locus by RSV. Assembly of mature MHC class I complex requires the combined presence of a cytoplasmic protease activity to generate peptides that will ultimately associate with MHC class I binding groove and specific transporters that allow these cytoplasmic peptides to cross the ER membrane into its lumen where MHC class I assembly takes place. The requirement for inducible TAP1 expression has been demonstrated in mutant cell lines lacking MHC genes; these cells have grossly impaired class I assembly and presentation (30). In MHC II-deficient cells, antigen presentation is restored on transfection with cDNAs encoding TAP1, indicating that TAP1 is a necessary component of peptide transport (3, 34). The requirement of the 26S proteasome in MHC class I presentation has been shown either using specific proteasome inhibitors to block cell surface MHC class I (41) or in transgenic animals with homozygous deletion of LMP2 or LMP7 genes. Mice deficient in LMP7 have 25-45% reduction in class I levels on its cell surface (14). MHC class I deficiency is restored to normal levels after exogenous addition of peptides, indicating that peptide supply is the limiting factor in LMP7-deficient mice. LMP2-deficient mice apparently have a more subtle defect in proteasome specificity where impaired cleavage of specific antigens with hydrophobic and basic residues is seen. LMP2-deficient mice have significantly impaired CTL precursor response when infected with influenza but not with Sendai virus, indicating that LMP2 may be involved in processing distinct viral epitopes (26). Herein we show that RSV induces the expression of TAP1, LMP2, and LMP7 with kinetics that precede the appearance of cell surface MHC class I molecules and is dependent on the paracrine secretion of IFN-beta . Whether LMP2, LMP7, or both are required for processing of specific RSV antigens will require additional experimentation where selective downregulation of individual proteasome subunits is achieved.

The TAP1/LMP2/LMP7 locus is contained in a short 800-kb stretch of the short arm of chromosome 6 that is the most highly polymorphic region on the human genome identified thus far (6). Transcription of TAP1 and LMP2 is regulated by a inducible bidirectional promoter (52). The TAP1/LMP2 promoter is a "TATA-less" promoter whose activity is controlled by inducible NF-kappa B and IFN response elements (ISREs). In theory, both IL-1 (via NF-kappa B) and IFNs (through the ISRE) should be activators of TAP1/LMP2 expression. Our data indicate that IFN-beta is a potent activator of TAP1/LMP2 expression, whereas IL-1 is not. The inability of IL-1 to activate TAP1/LMP2 is surprising because RSV-CM and IL-1 potently activate NF-kappa B (data not shown) and RSV induces expression of other cytokine promoters through an NF-kappa B-dependent mechanism (7, 21). Together, these observations indicate the ISRE is an important inducible TAP1/LMP2 enhancer. Controlled by a distinct promoter, the LMP7 transcription unit contains several putative ISREs, one at the 5'-end and another downstream in the 4th intron. The regulation and relative importance of these putative sites have not been investigated. Finally, although LMP7 is subject to differential RNA splicing (where exon 1 is deleted in certain B cells), our data indicate that only one transcript is present in epithelial cells.

Secretion of type I IFNs in mucosal pathogen defense. IFN-alpha and -beta , type I IFNs, are a family of secreted cytokines inducibly expressed as an early response to viral infection of eukaryotic cells, including fibroblasts, leukocytes, and epithelial cells. The actions of this alpha -helical bundle class of cytokines is mediated through a common receptor, where its activation confers resistance to viruses, cell growth and differentiation, MHC class I expression, and stimulation of natural killer (NK) cells. As an illustration of its importance, mice deficient in type I IFN action, lacking a common subunit of the type I IFN receptor (IFNAR1), are phenotypically normal but die on exposure to vesicular stomatitis virus at nonlethal doses (44). Our data indicate that RSV infection-dependent upregulation of MHC class I is mediated primarily through the paracrine effects of IFN-beta . This statement is supported by the following observations: 1) the detection of bioactive IFN-beta in RSV-CM at times preceding TAP1/LMP2/LMP7 expression, activity of which is neutralized specifically by anti-IFN-beta antibodies, 2) the ability of RSV-CM to induce TAP1/LMP2/LMP7 expression is blocked by 70% following treatment with anti-IFN-beta antibodies, and 3) the ability of recombinant IFN-beta to induce TAP1/LMP2/LMP7 expression with a similar magnitude as RSV-CM. Although IFN-beta appears to be the predominant activity in RSV-CM, we note that IFN-beta stimulation is transient, whereas RSV-CM stimulation is prolonged, and after IFN-beta neutralization, some additional stimulation remains. RSV-CM is rich in a panoply of cytokines such as granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IL-6, and others (4, 21, 37), which could participate in MHC class I upregulation. The identity of additional paracrine mediators will require further investigation.

Importance of MHC class I in RSV immunopathology. Primary RSV infection results in the initial recruitment of CD4-CD8- cells (with possible NK activity) into the lung and later, the recruitment of CD8+ T cells. Passive transfer of RSV-specific CD8+ CTLs to mice has been shown to clear RSV from the lungs but can also intensify disease manifestations, pulmonary pathology, and death (8, 23). These observations point to the involvement of CD8+ T cells in both clearance of RSV and initiation of airway pathology. CD8+ cells respond to presentation of antigenic peptides associated with class I MHC; enhanced class I MHC expression increases their susceptibility to CD8+-mediated lysis (10). Thus through the paracrine effect of IFN-beta secretion, MHC class I upregulation is likely to be an important mechanism underlying immunopathological response to RSV.

In summary, immunopathogenic mechanism for RSV-induced lung disease appears to be at least in part mediated through CD8+ CTLs. Our study uncovers some important and relevant molecular events in this process and suggests that paracrine secretion of IFN-beta activates an inducible MHC class II locus to coordinate peptide processing and transport of MHC class I-associated peptides. By understanding these events in more detail, it will be possible to develop therapeutic agents for the control of RSV-induced lung diseases.


    ACKNOWLEDGEMENTS

We thank Dr. Jonathan Yewdell (Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases) for the gift of the VV-ICP47, Dr. Barry Rouse (Dept. of Microbiology, University of Tennessee, Knoxville) for the VV-TK- vector, and Dr. Victor Reyes (University of Texas Medical Branch) for advice and discussion.


    FOOTNOTES

This project was supported by National Institute of Allergy and Infectious Diseases Grants R01-AI-40218 to A. R. Brasier and R01-AI-15939 to R. P. Garofalo, National Institute of Child Health and Human Development Grant R30-HD-27841 and National Institute of Environmental Health Sciences Grant P30-ES-06676 to R. S. Lloyd (University of Texas Medical Branch). A. R. Brasier is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: A. R. Brasier, Div. of Endocrinology, MRB 8.138, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-1060 (E-mail: arbrasie{at}utmb.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 25 April 2000; accepted in final form 7 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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