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Journal of Virology, February 1999, p. 1411-1418, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Human Parainfluenza Virus Type 3 Up-Regulates Major
Histocompatibility Complex Class I and II Expression on Respiratory
Epithelial Cells: Involvement of a STAT1- and
CIITA-Independent Pathway
Jing
Gao,
Bishnu P.
De, and
Amiya K.
Banerjee*
Department of Virology, The Lerner Research
Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received 20 July 1998/Accepted 2 September 1998
 |
ABSTRACT |
Human parainfluenza virus type 3 (HPIV3) infection causes severe
damage to the lung epithelium, leading to bronchiolitis, pneumonia, and
croup in newborns and infants. Cellular immunity that plays a vital
role in normal antiviral action appears to be involved, possibly
because of inappropriate activation, in the infection-related damage to
the lung epithelium. In this study, we investigated the expression of
major histocompatibility complex (MHC) class I and II molecules on
human lung epithelial (A549) and epithelium-like (HT1080) cells
following HPIV3 infection. MHC class I was induced by HPIV3 in these
cells at levels similar to those observed with natural inducers such as
beta and gamma interferon (IFN-
and -
). MHC class II was also
efficiently induced by HPIV3 in these cells. UV-irradiated culture
supernatants from infected cells were able to induce MHC class I but
not MHC class II, suggesting involvement of released factors for the
induction of MHC class I. Quantitation of IFN types I and II in the
culture supernatant showed the presence of IFN- as the major
cytokine, while IFN- was undetectable. Anti-IFN-, however,
blocked the HPIV3-mediated induction of MHC class I only partially,
indicating that viral antigens, besides IFN-, are directly involved
in the induction process. The induction of MHC class I and class II
directed by the viral antigens was confirmed by using cells lacking
STAT1, an essential intermediate of the IFN signaling pathways. HPIV3 induced both MHC class I and class II molecules in STAT1-null cells.
Furthermore, MHC class II was also induced by HPIV3 in cells defective
in class II transactivator, an important intermediate of the
IFN--mediated MHC class II induction pathway. Together, these data
indicate that the HPIV3 gene product(s) is directly involved in the
induction of MHC class I and II molecules. The induction of MHC class I
and II expression by HPIV3 suggests that it plays a role in the
infection-related immunity and pathogenesis.
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INTRODUCTION |
Human parainfluenza virus type 3 (HPIV3), a paramyxovirus, is an important respiratory pathogen and a
major cause of bronchiolitis and pneumonia in newborns and infants
(12, 19, 31, 38, 42). Reinfection and persistent infection
with the virus have been documented in various clinical situations
(1, 15, 32, 33). These characteristics are different from
those of other paramyxoviruses such as measles and mumps viruses,
because infection with those viruses results in the development of
life-long immunity (37). The HPIV3 infection is often
associated with severe damage to the lung epithelium. A complex
interaction between the virus and the host immune system, the
cell-mediated immune response, and the virus-specific immunoglobulin E
antibody response most likely play a role in the lung epithelial cell
damage (10). The cell-mediated immune response by
up-regulating the expression of major histocompatibility complex (MHC)
molecules has previously been reported for paramyxoviruses, namely,
respiratory syncytial virus and measles virus (11, 30). MHC
class I and II molecules are cell surface glycoproteins that are
involved in the antigen presentation arm of the immune response. MHC
class I molecules are involved in the processing of endogenous antigens
and presenting the antigen-derived peptides to CD8+ T
cells, usually cytotoxic T lymphocytes (CTL). They are ubiquitously expressed, and their basal level of expression can be induced by a
number of cytokines and viral factors (16). In particular, beta and gamma interferon (IFN- and -) are the potent inducers of
MHC class I molecules, and STAT1 (signal transducer and activator of
transcription) plays an essential role in the activation of MHC class I
transcription (14). MHC class II molecules, on the other
hand, are involved in the processing of exogenous antigens and
presenting the processed antigens to the CD4+ T cells,
usually T helper cells. This results in the activation of T cells which
are involved in various functions, including (i) the direct lysis of
virus-infected cells through the activity of CTL and (ii) production of
cytokines that may either activate other cells of the immune system
(macrophage, B cells, and CTL) or interfere with virus replication
(11, 16, 30). Unlike the expression of MHC class I
molecules, the constitutive expression of MHC class II molecules
is restricted primarily to B cells, dendritic cells, thymic epithelium,
and macrophages. The appropriate, constitutive, and inducible
expression of MHC class II is essential for normal immune function,
whereas aberrant expression in various tissues has been implicated in
the pathogenesis of autoimmune diseases such as rheumatoid arthritis,
autoimmune nephritis, insulin-dependent diabetes mellitus, inflammatory
bowel disease, and multiple sclerosis (25, 27). Specific
epithelium-like cells in various tissues can be induced to express MHC
class II molecules upon exposure to IFN-, a potent inducer. The
recently identified class II transactivator (CIITA) has been shown to
be essential for activation of transcription of MHC class II genes in
this process (34). Besides IFN-, other cytokines such as
tumor necrosis factor alpha and interleukin 4 and other agents such as
viral antigens have been shown to induce MHC class II (2, 16, 22,
28, 41). The expression of class II molecules by human bronchial
epithelial cells following IFN- induction or virus infection
suggests that these cells may be involved in mucosal immunity as well
as infection-related immunopathology (16, 40). In this
context, how class II antigen expression on epithelial cells is
modulated by cytokines or directly by the viral antigens remains
unclear. Also, whether epithelial cells bearing class II molecules can
function as antigen-presenting cells (APC) remains unknown.
In this work we have investigated the effect of HPIV3 on the
antigen-presenting macromolecules', i.e., the MHC class I and II
molecules', expression on infected cell surface. Our data clearly demonstrate that cell surface expression of both MHC class I and II
molecules are up-regulated in A549 as well as HT1080 cells following
HPIV3 infection. The induction of MHC class I was mediated through the
production of IFN- and also by the direct interaction of viral
antigens. The MHC class II induction, on the other hand, was directly
mediated by the viral antigens. The induction of MHC class I and II
molecules also occurred on cells lacking the IFN signaling intermediate
STAT1, indicating a direct effect of viral antigens in the induction
process. These results suggest that cell-mediated immune response
involving MHC molecules plays an important role in HPIV3
infection-related immunity and pathogenesis.
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MATERIALS AND METHODS |
Biological reagents.
Human recombinant IFN- was purchased
from Biosource International (Camarillo, Calif.). Human recombinant
IFN- was purchased from Boehringer Mannheim (Indianapolis, Ind.);
poly(I)-poly(C) was purchased from Pharmacia Biotech (Piscataway,
N.J.).
Cell lines and culture conditions.
CV-1 (African green
monkey kidney) cells were used for growing the virus and for plaque
assays. A549 is a lung adenocarcinoma cell line (ATCC CCL 185) which
has been used as a model of type II alveolar epithelial cells. HT1080
(ATCC CCL 121) is a fibrosarcoma cell line. The 2fTGH cell line was
derived from the HT1080 cell line. U3A is a STAT1 mutant cell line, and
G3A is a CIITA-defective cell line; both of them are derived from 2fTGH
(4, 6, 29). 2fTGH, U3A, and G3A cell lines were gifts from
George Stark (Department of Molecular Biology, Lerner Research
Institute, Cleveland Clinic Foundation). All the cell lines mentioned
above were maintained in Dulbecco's modified Eagle medium containing
1% L-glutamine, 1% penicillin-streptomycin, and 10%
fetal bovine serum.
Virus stock and infection.
The HPIV3 viral stock HA-1,
National Institutes of Health catalog no. 47784, was grown in the CV-1
cell line. For the MHC class I assay, the virions released in the
culture medium were purified by centrifugation at 10,000 × g to remove cell debris followed by ultracentrifugation at
100,000 × g for 2 h at 4°C with a SW 50.1 rotor, as described previously (7). The purified virus
pellet was suspended in buffer containing 50 mM HEPES-KOH (pH 7.5) and
50 mM NaCl, and the purity was confirmed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining.
No IFNs were detected in the purified virus pool by antivirus bioassay
and enzyme-linked immunosorbent assay (ELISA). For the MHC class II
assay, infected CV-1 cell supernatants were used as virus stocks.
Titers of both viral sources were determined by plaque assay. The
unpurified stock contained virus at 2 × 106 PFU/ml,
and the purified stock contained virus at 108 PFU/ml. For
some experiments, the virus particles were inactivated with UV light as
previously reported (11).
Experiments were performed by infecting the cells (5 × 105) with HPIV3 at a multiplicity of infection (MOI) of 1 in a total volume of 2 ml in a 12-well plate at 37°C, and the cells
were harvested at 48 and 72 h postinfection for MHC class I and II assays, respectively. The virus infection was conducted in the same
medium used for growing the cells. For the supernatant transfer experiments, the culture supernatants were collected at 48 h
postinfection and transferred (20% volume) to fresh monolayers of
corresponding cell types. The cells were harvested after 48 and 72 h for MHC class I and II assays, respectively.
Transient transfection.
Both A549 and HT1080 cells were
transfected with poly(I)-poly(C) by a FuGENE 6 transfection technique
(Boehringer Mannheim) according to the manufacturer's protocol.
Various doses of poly(I)-poly(C) ranging from 50 to 150 µg/ml and
control medium were added to cells in the absence or presence of FuGENE
6 reagent in 10% fetal bovine serum containing Dulbecco's modified
Eagle medium. Cells were cultured for 48 and 72 h and assayed by
flow cytometry for MHC class I and II molecules, respectively.
Flow cytometry.
The cells were plated at 5 × 105 cells/well into 12-well plates, and after 12 h the
cells were infected with HPIV3 (MOI, 1.0); mock-infected cells served
as the control. After indicated times postinfection, the cells were
removed by short treatment with 0.1 M EDTA, washed twice in
phosphate-buffered saline (PBS), and counted. Viability was determined
by trypan blue dye exclusion. The cells were incubated with specific
antibodies or the same isotype used for the control (1 to 2 µg/sample, according to the manufacturer's protocol) in a 100-µl
reaction mixture containing 1× PBS, 1% bovine serum albumin, and
0.01% sodium azide for 30 min at room temperature. Flow cytometry
analysis of the cells was performed on a FACScan device
(Becton-Dickinson; San Jose, Calif.) with Cyclops software (Cytomation,
Fort Collins, Colo.). Ten thousand cells were analyzed for each sample.
In addition to an ungated analysis, a gate was set (on the basis of the
dot plot for 90° light scatter versus forward-angle light scatter) to
exclude any cell debris or dead cells from the analysis. The antibody
used in the staining for human MHC class I is murine monoclonal
antibody (MAb) to HLA-ABC (W6/32) conjugated directly to fluorescin
isothiocyanate (FITC) obtained from Biodesign, New York, N.Y. The
antibody used in the staining for human MHC class II is murine MAb to
HLA-DR, L243, conjugated directly to FITC (Becton Dickinson). For the
dual color assay for MHC class I and HPIV3, the antibody used in the
staining for human MHC class I antigen is murine MAb to HLA-ABC (W6/32)
conjugated directly to phycoerythrin (Biodesign). The antibody used in
the staining for human MHC class II antigen is murine MAb to HLA-DR,
L243, conjugated directly to phycoerythrin (Becton Dickinson). The
antibody used for HPIV-3 is a MAb specific for HPIV3 surface
glycoproteins conjugated directly to FITC (Biodesign). Nonspecific
background staining was determined with a control FITC-conjugated
isotype-matched Ab (Becton Dickinson).
Cytokine assays.
IFN- was assayed by ELISA (R&D systems,
Minneapolis, Minn.), and IFN- was assayed by ELISA (Biosource
International). The presence of IFN-
/ in the culture supernatant
of HPIV3-infected cells was determined by analyzing their ability to
inhibit vesicular stomatitis virus-induced cytopathic effect on WISH
cells, as described previously (21). Briefly, A549 and
HT1080 cells were infected with HPIV3 at an MOI of 1.0 at 37°C and
the culture supernatant was collected at 48 h postinfection.
Culture supernatant of uninfected cells served as the control. The
culture medium was first exposed to UV light to inactivate the released
virions and then used to measure activity against vesicular stomatitis
virus. Sheep anti-IFN- polyclonal Ab, sheep anti-IFN- polyclonal
Ab (Biosource International), and sheep serum control were used to
quantitate both IFN- and IFN- in the cell supernatants. The assay
was standardized with reference to IFN of known activity. Cell
viability was measured by staining wells with neutral red in PBS and
elution in 50% ethanol in 0.1 M NaH2PO4, and
the absorbance at 540 nm was determined. The results are presented as
percent protection, calculated as (A540 of the
sample 
A540 of the virus
control)/(A540 of the cell control) A540 of the virus control) × 100.
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RESULTS |
Induction of MHC class I and II expression on A549 and HT1080 cells
by HPIV3.
Infection of cells by several paramyxoviruses has been
shown to up-regulate the expression of MHC class I and II molecules, which are important components of the host immune response to these
viruses (11, 26, 30). Up-regulation of the expression of MHC
molecules by these viruses has been suggested to be involved in the
infection-related immunopathology. To understand the molecular mechanism of HPIV3 infection-related immunopathology, manifested by the
damage of respiratory tract epithelium, we examined the effect of HPIV3
on the expression of MHC class I and II molecules on human respiratory
epithelial cells, A549. These are human lung adenocarcinoma cells that
have been used as a model of type II alveolar epithelial cells and are
susceptible to infection with HPIV3 (43). We also used
another human epithelium-like cell line, HT1080, derived from
fibrosarcoma and originally subcultivated under conditions that
eliminate the growth of fibroblasts and favor that of epithelial cells
(17). HT1080 cells were also found to be susceptible to
infection with RNA viruses (17). Furthermore, several mutant
cell lines have been generated from the parental HT1080 cells, and
those have been extensively used for characterizing the components of
the IFN signaling pathway, e.g., JAK (Jenus kinase) and STAT
(6). The use of HT1080 cells would therefore not only help
determine the effect of HPIV3 on MHC class I and II expression, but
also enable us to delineate the molecular mechanism of regulation of
expression of MHC molecules by using various mutant cell lines. The
A549 and HT1080 cells were infected with HPIV3 at an MOI of 1, and the
cells were harvested at 48 h postinfection for MHC class I and
72 h postinfection for MHC class II assays. The cells were viable
by more than 90%, as determined by trypan blue staining. These cells
were processed for flow cytometry by using HLA-ABC and HLA-DR
antibodies, and the fold increase of mean fluorescence intensity (MFI)
was determined. As shown in Fig. 1, cell
surface expression of MHC class I was increased, following HPIV3
infection, by about 3-fold on A549 cells and 4-fold on HT1080 cells,
while class II expression was increased by about 13-fold on A549 cells
and 7-fold on HT1080 cells. These results clearly indicate that HPIV3
upregulates the cell surface expression of both MHC class I and II on
epithelial cells.
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FIG. 1.
Flow cytometric analysis of MHC class I (A) and class II
(B) expression on A549 and HT1080 cells following HPIV3 infection. The
A549 and HT1080 cells (5 × 105 cells) were infected
with HPIV3 (MOI, 1.0) and were harvested at 48 h postinfection for
MHC class I assays and at 72 h postinfection for MHC class II
assays. Mock-treated uninfected cells (Mock) served as the control. In
each panel, the MFI and the percentage of cells staining for MHC class
I or class II are indicated. Results are representative of four
independent assays.
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|
Since, IFNs are the natural inducers of MHC class I and II molecules
during antiviral response, we examined the induction
of MHC molecules
on these cells following IFN-
treatment and
compared this to the
induction observed with HPIV3. Similarly,
induction by IFN-
was
examined because its involvement in the
induction of MHC class I
expression has been previously demonstrated
(
11). As shown
in Fig.
2, MHC class I expression on A549
and
HT1080 cells was induced by about eightfold on A549 and fourfold
on
HT1080 cells following treatment with 100 U of IFN-
/ml. Similarly,
100 U of IFN-
/ml induced MHC class I expression on A549 by about
sevenfold and induced that on HT1080 by about sixfold. MHC class
II
expression, however, was not induced on A549 cells following
IFN-
treatment, as reported previously by other laboratories
(
36), whereas MHC class II expression on HT1080 was induced
by about 16-fold after 72 h. The level of HPIV3-mediated induction
of MHC class I and II expression is therefore comparable to the
level
of induction by the natural inducers, IFN-
and IFN-
.
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FIG. 2.
Flow cytometric analysis of MHC class I (A) and class II
(B) expression on A549 and HT1080 cells induced by IFN- and IFN-.
The A549 and HT1080 cells (5 × 105 cells) were
induced with or without IFN- (100 U/ml) and IFN- (100 U/ml) and
were harvested after 48 h of treatment for MHC class I and after
72 h treatment for MHC class II assays. In each panel, the MFI and
the percentage of cells staining for MHC class I or II are indicated.
Results are representative of four independent assays. NT, no
treatment.
|
|
Production of infectious virions by A549 and HT1080 cells and their
role in MHC class I and II expression.
To determine whether both
A549 and HT1080 cells supported the replication of HPIV3, we examined
the production of infectious virions from these cells. The cells were
infected with HPIV3 at an MOI of 1.0, and at various times (24, 48, and
72 h) postinfection the number of progeny virions released into
the medium was determined by plaque assay. As shown in Table
1, both A549 and HT1080 cells supported
efficient replication of HPIV3.
The roles of viral gene products in the induction of MHC class I and II
were examined by double immunofluorescent labeling
and flow cytometry.
As shown in Fig.
3, MHC class I
expression
occurred on both the cell population containing the viral
antigens
and that without the viral antigens, suggesting a role of
soluble
extracellular factors and possibly also viral gene products.
MHC
class II expression, on the other hand, occurred on the cell
population
containing the viral antigens, suggesting that the viral
gene
products are the major regulator of the induction of MHC class
II
in infected cells.
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FIG. 3.
Dual color staining analysis for MHC class I and HPIV3
(A) and for class II and HPIV3 (B) antigen expression on A549 and
HT1080 cells. A549 and HT1080 cells (5 × 105) were
infected with HPIV3 (MOI, 1.0), and the cells were harvested at 48 h postinfection for dual color staining for MHC class I and HPIV3 and
at 72 h postinfection for dual color staining for MHC class II and
HPIV3. Results are representative of three independent assays. Mock,
mock-treated, uninfected cells.
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|
To determine whether infectious virions were required for the induction
of MHC class I and II, we inactivated the virions
by exposure to UV
light before infection. The absence of infectious
replicating virions
in the UV-treated supernatant was confirmed
by plaque assay. Levels of
expression of MHC class I on A549 and
HT1080 cells were determined at
48 h postinfection, whereas MHC
class II expression was determined
at 72 h postinfection. MHC
class I expression was induced by about
13-fold on A549 cells
and 14-fold on HT1080 cells by UV-inactivated
virus particles
(data not shown). Furthermore, the induction level was
comparable
to that observed with 100 U of IFN-
(Fig.
2). These
results suggest
that the HPIV3-mediated induction of MHC class I
possibly resulted
by the interaction of viral envelope glycoproteins
which can induce
IFN production, as reported previously for other
viruses (
8).
MHC class II induction, on the other hand, was
abrogated by UV
inactivation of the virus particles, indicating a
requirement
of infectious particles in this induction process (data not
shown).
Since nonsegmented negative-strand RNA viruses are known to generate
double-stranded RNA during replication, which in turn
induces IFN, we
examined the effect of various doses (50 to 150
µg) of
poly(I)-poly(C), which is used to mimic intracellular double-stranded
RNA. MHC class I expression occurred on both A549 and HT1080 cells
following poly(I)-poly(C) treatment, indicating that double-stranded
RNAs are possibly involved in MHC class I induction (data not
shown).
By contrast, MHC class II induction was not detected in
either of the
cell lines following poly(I)-poly(C) treatment (data
not shown). These
data suggest that the induction of IFN by the
viral replication
intermediate, double-stranded RNA, is involved,
at least in part, in
the induction of MHC class I. The pattern
of MHC class II induction,
however, suggests that some viral protein(s)
directly induces the MHC
class II, as reported for measles virus
(
30). Alternatively,
viral protein(s) may induce some other
cytokines that in turn induce
MHC class II (
11).
MHC class I, but not MHC class II, can be induced with the culture
supernatant from infected cells.
To investigate the involvement of
soluble factors such as IFN- and IFN- in the induction of MHC
molecules, specifically MHC class I, we transferred the culture
supernatant from infected A549 and HT1080 cells to fresh monolayers of
corresponding cell types. The A549 and HT1080 cells were harvested at
48 and 72 h postinfection, respectively, and MHC class I and II
expression was determined. As shown in Fig.
4, supernatant from infected A549 cells
induced the MFI for MHC class I expression by 3.5-fold on fresh A549
cells, while supernatant from infected HT1080 cells induced this
expression by 3-fold on fresh HT1080 cells. MHC class II expression, on
the other hand, was not induced on fresh monolayers of A549 and HT1080
cell types by the culture supernatants from corresponding infected
cells (data not shown). These data indicate that MHC class I expression
can be transferred with the culture supernatant, perhaps due to the
presence of IFNs, whereas MHC class II expression cannot be
transferred.
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FIG. 4.
Flow cytometric analysis for MHC class I expression on
A549 and HT1080 cells transferred with supernatants from corresponding
HPIV3-infected cell types. The culture supernatants (Sup.) from
HPIV3-infected cells, A549 and HT1080, harvested at 48 postinfection
were inactivated by UV and transferred to fresh monolayers of
corresponding cell types with or without anti-human IFN- Ab (1,000 U/ml) or sheep serum control. The A549 and HT1080 cells were harvested
at 48 and 72 h postinfection, respectively, to determine the
increase in MHC class I and II expression. Results are expressed as
fold MFI increase and are representative of three independent assays.
NT, no treatment.
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Although induction of IFN by some paramyxoviruses in pulmonary
epithelial cells has been reported (
11), such induction of
IFN synthesis by HPIV3 has not been previously investigated. Therefore,
we examined the presence of IFN types I and II in the culture
supernatant from infected A549 and HT1080 cells. We first tested
the
supernatant for the presence of immunoreactive IFN-
by a
sensitive
ELISA and observed, as expected, that IFN-
was not
detected in the
culture supernatant of both A549 and HT1080 cells,
in neither control
nor infected cultures (data not shown). When
IFN-
production was
similarly determined, the culture supernatant
from infected A549 cells
was found to contain about 500 U of IFN-
/ml,
while that from HT1080
cells contained about 300 U of IFN-
/ml
at 48 h postinfection.
By antivirus bioactivity assay, IFN-
was
found to be 80% and
IFN-
was 20% of the total IFN type I in the
culture supernatant.
Consistent with these data, as shown in Fig.
4, neutralizing
anti-IFN-
inhibited a greater part of the culture
supernatant-mediated induction of MHC class I in both cell types,
whereas anti-IFN-
had no effect (data not shown), indicating
the
involvement of IFN-
in the culture supernatant-mediated induction
of
MHC class I. Next, we examined whether the HPIV3-mediated induction
of
MHC class I could also be inhibited by anti-IFN-
, if added
to the
culture medium during infection. The anti-IFN-
inhibited
the
HPIV3-mediated induction of MHC class I only partially (~50%)
(data
not shown). Together, these results indicate that the induction
of MHC
class I in HPIV3-infected cells is mediated partly by IFN-
and
partly by the viral antigens, either directly or through the
production
of some other
cytokines.
HPIV3 induces MHC class I and II expression on STAT1-null and
CIITA-defective cells.
To gain insight into the molecular
mechanism of the induction of MHC class I and class II by HPIV3, we
used STAT1-null cells. STAT1 has been shown to be an essential
component of both IFN type I and type II signal transduction pathways,
being involved in the activation of transcription of IFN-responsive
genes following its phosphorylation by JAK in response to IFNs (6,
35). The STAT1-null cell line (U3A) is therefore defective in
both IFN-/ and IFN- signal transduction and thus provided us a
model for the primary cell response to virus, without endogenous IFN
autocrine effect.
First, the U3A and the parental cells 2fTGH (derived from HT1080) were
treated with IFN-
and IFN-
to confirm the defect
in the signal
transduction pathway. As shown in Fig.
5A, IFN-
and IFN-
treatment of
cells efficiently induced MHC class I on
2fTGH cells but failed to
induce MHC on class I U3A cells. These
cells were then infected with
HPIV3 at an MOI of 1, and the cells
were harvested at 48 h
postinfection and analyzed for MHC class
I expression. As shown in Fig.
5A, MHC class I was induced on
2fTGH and U3A cells, following HPIV3
infection, by about 6-fold
and 3.5-fold, respectively. The culture
supernatant from A549
cells which induced MHC class I on A549 cells by
3.5-fold (Fig.
4) was also tested in these cells and was found to
induce MHC
class I by 3-fold, as expected, only on the 2fTGH cells
(Fig.
5A). These data indicate that in addition to the induction of
MHC
class I by IFN-
, the viral antigens also induce MHC class
I through
a pathway that does not require STAT1.
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FIG. 5.
MHC class I (A) and class II (B) expression on 2fTGH and
U3A cells and comparison with that by IFN-/. The mutant and
parented cells (5 × 105) were induced with IFN-
(100 U/ml) (only for MHC class I expression) or IFN- (100 U/ml) or
were infected with HPIV3 (MOI, 1.0). The cells were harvested after 48 and 72 h to determine the levels of MHC class I and II expression.
In each panel, the MFI and the percentage of cells staining for MHC
class I or II are indicated. Results are representative of four
independent assays. Sup., supernatant.
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Next, the 2fTGH and U3A cells were treated with IFN-
and induction
of MHC class II was determined. As shown in Fig.
5B, MHC
class II was
induced by IFN-
on 2fTGH cells by 2.5-fold, but
no induction was
observed on U3A cells. These results confirm
the defect of U3A cells in
signal transduction. When MHC class
II induction by HPIV3 was examined,
the parental cells, 2fTGH,
as well as the mutant cells, U3A, expressed
MHC class II at the
same level (14-fold in 2fTGH cells and 11-fold in
U3A cells).
The level of induction was comparable to that observed with
infected
A549 and HT1080 cells. These data indicate that IFNs or other
cytokine signaling pathways which require STAT1 are not involved
in the
activation of MHC class II by HPIV3. Thus, it appears that
HPIV3
infection of human epithelial cells induces both MHC class
I and II
directly by viral proteins either interacting with the
promoter or by
activating some other pathway that hitherto remains
uncharacterized.
Finally, to confirm the induction of MHC class II directly by viral
antigens, we also investigated MHC class II induction
on
CIITA-defective cells (G3A). CIITA is the general regulator
of MHC
class II gene expression by IFN-
(
39). In the IFN-
response, STAT1 is phosphorylated and translocates to the nucleus,
where it binds to the GAS element of CIITA promoter IV and activates
transcription of CIITA mRNA, which operates as the essential mediator
of the MHC class II gene and its induction product (
25,
39).
Thus, the CIITA-defective cell line (G3A) is defective in the
production of MHC class II by IFN-
. We infected G3A as well as
parental 2fTGH cells with HPIV3 at an MOI of 1 and examined MHC
class
II expression at 72 h postinfection. As shown in Fig.
6,
MHC class II induction by HPIV3
occurred on both the parental
(2fTGH) and CIITA-defective (G3A) cells
at the similar levels.
MHC class I induction by IFN-
, IFN-
, and
HPIV3 also occurred
on these cells (data not shown), which is
consistent with the
previous findings that CIITA is not involved in the
MHC class
I induction. Together, these data indicate that
HPIV3-mediated
induction of MHC class I and II on pulmonary epithelial
cells
occurs through the direct interaction of viral antigens, which
does not require the involvement of STAT1. Furthermore, MHC class
I and
II induction by HPIV3 can also occur in the absence of CIITA.
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|
FIG. 6.
MHC class II expression on 2fTGH and G3A cells by
comparing with IFN- induction and with HPIV3 infection. These mutant
cells (5 × 105) were induced with IFN- (100 U/ml)
or infected with HPIV3 (MOI, 1.0). At 72 h postinfection, 2fTGH
and G3A cells were harvested to determine the level of MHC class II
expression. In each panel, the MFI and the percentage of cells staining
for MHC class II are indicated. Results are representative of four
independent assays. Mock, mock-treated, uninfected cells; Sup.,
supernatant.
|
|
| |
DISCUSSION |
In this study we investigated the effect of HPIV3 infection of
respiratory epithelial cells on MHC class I and II expression. Although
the importance of MHC molecules in the immune response to HPIV3 in
humans has not been adequately established, studies of Sendai virus
infection in mice demonstrated the importance of both MHC class I and
II in the recovery from pulmonary Sendai virus infection (23,
26). The present studies clearly demonstrate that HPIV3 induces
MHC class I and II expression by the human respiratory epithelial cell
line A549. This suggests that HPIV3-mediated induction of MHC class I
and II accounts, at least in part, for the infection-associated
immunopathology of the respiratory epithelium. MHC class I induction
did not require infectious virions, whereas MHC class II induction
occurred in a virus replication-dependent manner, indicating that viral
proteins, perhaps envelope proteins in the case of MHC class I, are
directly involved in this process. Double labeling of cells with
anti-MHC class I and II and anti-HPIV3 followed by flow cytometric
analysis confirmed that MHC class I expression was mediated by soluble
factors as well as directly by viral antigens, whereas MHC class II
induction occurred primarily by viral antigens. Furthermore, HPIV3
efficiently induced MHC class I and II on STAT1-null and
CIITA-defective cells, indicating that the induction also occurs
without the involvement of cytokine-mediated pathways that require
STAT1 and CIITA, specifically in the case of MHC class II. These
findings are therefore important, particularly in relation to
HPIV3-mediated immunopathology in the airway epithelium.
Similar to those in our studies, several other viruses, including
measles virus, respiratory syncytial virus, and cytomegalovirus, have
been shown to up-regulate MHC class I or II molecules in infected cells
(11, 18, 30). In some cases virus infection was found to
increase the level of cytokine production, which in turn activated the
MHC molecules. For example, in the case of respiratory syncytial virus
infection, MHC class I was induced and the induction was found to be
due to an increased synthesis of IFN- (11). During human
cytomegalovirus infection, on the other hand, the MHC class I molecules
were directly stimulated by the viral antigens (18), whereas
in the case of West Nile virus infection, both IFN- and viral
antigens were involved for the induction of MHC class I (9).
In this respect, the HPIV3-mediated induction of MHC class I molecules
resembles the West Nile virus-mediated MHC class I expression. The MHC
class II molecules, on the other hand, appear to be directly mediated
by the action of viral antigens.
The role of MHC molecules in processing and presentation of viral
antigens to specific T cells for immune response has been well
characterized. MHC class I-restricted T cells (CD8+)
function as CTL which, upon recognition of MHC-antigen complex, kill
the target APC, e.g., virus-infected cells. MHC class II-restricted T
cells (CD4+), on the other hand, play several key roles in
the generation of immune responses; they (i) stimulate
antigen-activated class I-restricted CTL to kill virus-infected cells,
(ii) stimulate antigen-activated B cells to produce antibodies, and
(iii) activate cells of the nonspecific immune responses, e.g.,
macrophages, granulocytes, or eosinophils (13, 16). Some MHC
class II-restricted T cells can also function as CTL and as such may
have an important role in controlling certain virus infections, for
example measles virus and herpes simplex virus infection
(13). MHC class I molecules are ubiquitously expressed, and
their basal level of expression can be induced by a number of cytokines
and viral factors (16). MHC class II molecules are normally
expressed on APC, but a variety of other cells, including epithelial
cells, have been shown to produce MHC class II when activated by
cytokines such as IFN- and other agents (2). In this
respect, our data are the first demonstration that a nonsegmented
negative-strand RNA virus induces MHC class II on epithelial cells in
addition to MHC class I. However, it raises the question as to whether
HPIV3-induced MHC molecules on epithelial cells participate in the
immune response. Although priming of virgin CTL is believed to be
mediated only by specialized APC, nonspecialized APC such as epithelial
cells have also been shown to activate CTL (40). It has been
estimated that an APC has to display a minimum of 2,000 antigenic
complexes to be recognized by activated CTL (2, 13, 16, 41).
The HPIV3-induced expression of MHC molecules seems to be competent for
CTL activation, because the level of MHC class I and II expression is
comparable to that observed with the natural inducers of these
molecules, IFN- and IFN-, respectively. Consistent with this,
other viral infections that enhance the expression of MHC molecules
have been shown to result in increased CTL lysis of infected cells
(11). Further studies are needed to elucidate the mechanism
of MHC class I and II induction by HPIV3 and its possible involvement
in the activation of CTL.
The induction of MHC class I and II molecules on epithelial cells
during HPIV3 infection may play an important role in the protective
immune response of host against the virus by lysis of infected cells.
Alternatively, this may lead to the development of infection-associated
immunopathology by the lysis of both infected and neighboring mucosal
epithelial cells that passively acquire released viral antigens.
Previous findings, however, suggest a role of immunopathologic response
in parainfluenza virus-mediated bronchiolitis (10).
Cell-mediated immune response to parainfluenza viral antigens was found
to be higher among infants who developed bronchiolitis following HPIV3
infection than infants who developed only upper respiratory illness.
Thus, induction of MHC class I and II on airway epithelial cells
following HPIV3 infection possibly results in an increased CTL-mediated
lysis of those cells and thus may contribute to the
infection-associated immunopathology. Alternatively, the HPIV3-induced
expression of MHC molecules may not be able to participate in the
CTL-mediated lysis but rather may inhibit the lysis process. Thus, it
may be a mechanism by which the virus evades the host immune
surveillance. Further studies are, however, needed to gain a deeper
understanding of the molecular mechanism of MHC class I and class II
induction in HPIV3-infected airway epithelial cells and
infection-associated immunopathologic response.
| |
ACKNOWLEDGMENTS |
We thank George R. Stark for providing mutant cell lines
defective in IFN signaling pathways used in our studies. We thank Yoshihiro Ohmori, Xiao Xia Li, and Yu Long Han for discussion and
valuable comments during this work and Amy Raber for FACScan analyses.
This work was supported in part by United States Public Health Services
grant AI32027 (A.K.B.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., NC20, Cleveland, OH 44195. Phone: (216)
444-0625. Fax: (216) 444-0512. E-mail:
banerja{at}cesmtp.ccf.org.
| |
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Abstract
Introduction
