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
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ABSTRACT |
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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)- largely blocked TAP1/LMP2/LMP7 expression, whereas anti-interleukin-1 antibodies were without effect,
and recombinant IFN-
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-
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-
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INTRODUCTION |
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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
(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
-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)-, was largely blocked by
neutralizing IFN-
antibodies, indicating that IFN-
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-
in mediating MHC class I expression and
cell-mediated RSV immunopathology through coordinate expression of the
TAP1/LMP2/LMP7 locus.
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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- (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-
antibody (Chemicon International),
30 µg of anti-human interleukin (IL)-1
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- bioassay.
IFN-
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--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
-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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RESULTS |
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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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RSV-CM contains biologically active
IFN-.
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-
by the ability
of neutralizing antibodies to block its effect. We noted that the RSV-induced increase in IFN-
production within 3 h of RSV
infection was a time preceding TAP1/LMP2/LMP7 expression
(compare with Fig. 1).
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Effect of neutralizing IFN- and
IL-1
in RSV-CM.
To determine the role of IFN-
in RSV-CM-induced TAP1/LMP2/LMP7
expression, RSV-CM treated with excess amounts of neutralizing antibodies to IFN-
or IL-1
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-
was markedly, but not completely, reduced in its ability to induce
TAP1/LMP2/LMP7 expression. By contrast, RSV-CM neutralized
with anti-IL-1
did not inhibit TAP1/LMP2/LMP7 expression. Consistent with the effects observed with recombinant hormones, RSV-CM
neutralized with a combination of anti-IFN-
and anti-IL-1
antibodies showed no effect greater than that of RSV-CM treated with
anti-IFN-
alone. As a control, IFN-
bioactivity in RSV-CM after
antibody neutralization with anti-IFN-
was measured and confirmed to
be <50 U/ml, levels below those needed for its effect (see Fig. 3). To
confirm that IL-1
in RSV-CM was neutralized by anti-IL-1
treatment, we measured IL-1
bioactivity in RSV-CM by a nuclear
factor-
B (NF-
B) activation assay, where RSV-CM neutralized with
anti-IL-1
(but not the preimmune control) failed to activate NF-
B
complex (data not shown). These data indicate that IFN-
, but not
IL-1
in RSV-CM, is predominantly responsible for
TAP1/LMP2/LMP7 expression. However, the inability of
anti-IFN-
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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Effect of IFN- on inducible 26S
proteasome activity.
LMP2 and LMP7 constitute inducible
-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-
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-
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-
,
both rIFN-
and RSV-CM were neutralized with anti-IFN-
antibodies
and used to stimulate cells. Anti-IFN-
completely abrogated
chymotryptic activity in cells treated with rIFN-
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-
in various RSV-CM
preparations). On the other hand, treatment with anti-IL-1
antibodies did not reduce RSV-CM-stimulated chymotryptic activity,
indicating that IFN-
in the RSV-CM largely mediates the increase in
26S proteasome activity.
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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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DISCUSSION |
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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-. 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-.
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.
Secretion of type I IFNs in mucosal pathogen defense.
IFN- and -
, 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
-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-
. This statement is supported
by the following observations: 1) the detection of bioactive
IFN-
in RSV-CM at times preceding TAP1/LMP2/LMP7
expression, activity of which is neutralized specifically by
anti-IFN-
antibodies, 2) the ability of RSV-CM to induce
TAP1/LMP2/LMP7 expression is blocked by 70% following
treatment with anti-IFN-
antibodies, and 3) the ability
of recombinant IFN-
to induce TAP1/LMP2/LMP7 expression
with a similar magnitude as RSV-CM. Although IFN-
appears to be the
predominant activity in RSV-CM, we note that IFN-
stimulation is
transient, whereas RSV-CM stimulation is prolonged, and after IFN-
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
CD4CD8
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-
secretion, MHC class I upregulation is likely to be an important mechanism underlying immunopathological response to RSV.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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