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Infection and Immunity, October 1998, p. 4624-4632, Vol. 66, No. 10
Departments of Medicine and Medical
Microbiology and Immunology, University of Alberta, Edmonton,
Canada,1 and
Department of Medicine,
Received 10 November 1997/Returned for modification 26 January
1998/Accepted 23 June 1998
We have analyzed proteasomal adaptation and associated changes in
the B27-bound peptide repertoire in response to cellular invasion with
Salmonella. The peptide repertoire of HLA-B27 complexes was
analyzed by two different methods: (i) high-pressure liquid chromatography (HPLC) profiles of newly synthesized peptides eluted from B27 following metabolic labeling with arginine and (ii)
reactivities with two B27 monoclonal antibodies, Ye-2 and B27.M2,
sensitive to peptide-induced conformational changes. LMP, MECL, and
PA28 expression was analyzed by reverse transcription-PCR (RT-PCR) of
mRNA and by Western blot analysis for LMP2. Invasion of
HLA-B27-transfected HeLa cells by Salmonella typhimurium
induced significant changes in the reactivities of HLA-B27 with these
two antibodies, which was accompanied by significant quantitative and
qualitative changes in the HPLC profile of peptides eluted from
HLA-B27. We also observed increases in the RT-PCR values for the LMP2,
LMP7, and MECL proteasome subunit genes, as well as the proteasomal
activator PA28 The class I major histocompatibility
complex (MHC) consists of the polymorphic heavy chain,
Studies of the immune response towards bacterium-infected cells have so
far been limited to the recognition of peptides generated from
bacterial proteins. One study has shown that class I presentation of
bacterium-derived peptides is decreased by inhibitors of the proteasome
(17). It is known that the proteolytic specificity of an
uninfected cell can change by modulation of the relative contributions
of the various components to the entire proteasome complex. The genes
of two components, the LMP2 and LMP7 genes, reside in the MHC class II
region (11, 16). Their transcription is modulated by
activators such as gamma interferon (IFN- In this work, we have studied the effect of Salmonella
invasion on the peptide-sensitive antibody reactivity of HLA-B27 which has been transfected into HeLa cells and have compared the results to
the effects of IFN- Cell lines and culture conditions.
The HeLa cell line was
purchased from the American Type Culture Collection, Rockville, Md. We
obtained the cDNA for the B*2705 gene, which was already inserted into
the RSV5.neo vector, from Beatrice Carreno (University of Washington,
Seattle). The vector was transfected into HeLa cells with Lipofectin by
a procedure provided by the manufacturer (Gibco BRL, Gaithersburg,
Md.). G418-resistant clones were screened by immunofluorescence for the
expression of HLA-B27. This transfectant was designated HLA-B27-HeLa.
It was cultured in medium supplemented with 0.5 mg of G418 per ml.
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Invasion by Salmonella typhimurium Induces Increased
Expression of the LMP, MECL, and PA28 Proteasome Genes and Changes
in the Peptide Repertoire of HLA-B27
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and -
genes, and increased expression of the LMP2
protein by Western blotting. Upregulation of LMP2, but not LMP7, gene
expression showed a close correlation with the changes in antibody
reactivities observed upon bacterial invasion. We observed similar
changes in reactivity with the Ye-2 or the B27.M2 antibody of
lymphoblastoid cells upon gamma interferon treatment, which
significantly correlated with the increased RT-PCR values for the LMP2
gene. This was accompanied by consistent HPLC profile changes for
eluted peptides. Thus, Salmonella invasion leads to
serologically recognizable changes in the B27-bound peptide repertoire,
which may include peptides of host origin potentially through
modulation of proteasome LMP2 subunit expression and, as a consequence,
proteasomal activities.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
2-microglobulin, and antigenic peptides. Antigenic
peptides are derived from endogenous proteins as well as from the
proteins of intracellular pathogens such as viruses and invasive
bacteria. To provide antigenic peptides, these proteins are first
degraded by the proteolytic multicatalytic proteasome complex
(24) and then transported into the endoplasmic reticulum,
where they associate with the class I molecule to form the class I MHC.
One of the physiological functions of class I MHCs is recognition by
peptide-specific T-cell receptors of cytolytic T lymphocytes (CTL). In
the case of virally or bacterially infected cells, cytolysis serves to
eliminate cells harboring pathogens. It has been demonstrated that for
some intracellular bacteria, CTL activity against bacterium-infected
cells not only is induced (29) but also plays a role in host
defense (5). It has also been postulated that similar CTL
activities play a role in autoimmune disease, such as Salmonella
typhimurium-induced HLA-B27-related arthritis (12).
) (31). A change
in the LMP2-to-LMP7 ratio in the complex has been shown to induce
changes in the carboxyl termini of the peptides which are generated
(10). We have reported previously that in the case of
HLA-B27, these IFN--induced changes can be detected by a monoclonal
antibody, Ye-2 (32), which demonstrates specificity for
peptide C-terminal residues and is sensitive to peptide-induced conformational changes of the heavy chain (9). We later
reported on the reactivity of another monoclonal antibody, B27.M2,
which is also sensitive to peptide-induced conformational change
(15). Proteasomal activity and proteolytic specificity are
also influenced by the binding of an IFN--inducible activator
protein, PA28 and - (18), which generates peptides
with C termini tailored for binding to class I molecules
(7). A further IFN--inducible, though non-MHC-encoded,
proteasome subunit, MECL, has recently been identified (21)
and may be functional in MHC class I-restricted antigen presentation.
. We found that Salmonella invasion
could induce changes in peptide-sensitive antibody reactivities
parallel to those induced by IFN- and changes in expression of LMP2,
LMP7, MECL, and PA28 and - genes, as evaluated by reverse
transcription-PCR (RT-PCR). Using regression analysis, we were able to
pinpoint the LMP2 gene as the LMP gene whose expression correlated with the observed changes in peptide-sensitive antibody reactivities. In
addition, we have taken advantage of the fact that nearly all peptides
that bind HLA-B27 contain an arginine at position 2 (20), and we therefore labeled cells with a [3H]arginine amino
acid precursor for incorporation into proteins. Following degradation,
these proteins provide labeled peptides which are eventually loaded
into class I molecules, such as HLA-B27, and can then be analyzed by
high-pressure liquid chromatography (HPLC). This approach increases the
sensitivity of the assay and greatly diminishes the quantity of cells
necessary for these analyses. HPLC analysis of newly synthesized
peptides eluted from B27 revealed significant qualitative and
quantitative differences following either bacterial invasion or
incubation with IFN-. Hence, invasion by Salmonella
can lead to recognizable changes in the B27-bound peptide
repertoire, possibly through modulation of LMP2 subunit expression.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, cells were cultured at 1 ml per well in
24-well plates at 37°C in a humidified 5% CO2 incubator. In preliminary experiments, we determined that the optimum conditions for inducing changes in expression of HLA-B27 were 105
cells per ml, 600 U of IFN- (Biosource International, Camarillo, Calif.) per ml, and a total culture period of 72 h.
Bacterial strain and invasion procedure. The Salmonella wild-type invasive strain used was S. typhimurium SL1344, provided by Brett Finlay, University of British Columbia, Vancouver, Canada. A noninvasive strain (SB111) was also provided. An inoculum of each bacterial strain in a 10-ml aliquot of Luria broth was cultured overnight at 37°C without shaking the culture flask. HeLa or HLA-B27-HeLa cells were seeded at a density of 2 × 106 cells per 100-mm-diameter culture dish. The next day, salmonellae were added to achieve a bacterium/human cell ratio of 25:1. The preparations were then cultured at 37°C for 1 h. Afterwards, they were washed four times with phosphate-buffered saline and incubated in the same volume of RPMI-10% fetal calf serum containing 5 µg of gentamicin sulfate per ml. The culture was continued for another 24 h before the cells were harvested for assay. Control mock-invaded samples were tested in parallel and were the same except that no bacteria were added.
Monoclonal antibodies and immunofluorescence assay. The anti-HLA-B27 antibodies were ME1, B27.M2, and Ye2. The first was immunoglobulin G (IgG), and the latter two were IgM. The method for indirect immunofluorescence analysis with flow cytometry (fluorescence-activated cell sorting [FACS]; Becton Dickinson, San Jose, Calif.) followed previously described procedures (9). Briefly, cells were incubated first with saturating amounts of first antibodies and then with phycoerythrin-conjugated goat anti-mouse IgG or IgM (Jackson ImmunoResearch Laboratories, West Grove, Pa.). The degree of reactivity was computed as the mean intensity of fluorescence expressed on an arithmetic scale.
Total RNA extraction, reverse transcription, and adjustment of
sample dilutions.
Total RNA was extracted by the guanidium
thiocyanate procedure according to the protocol provided by the
manufacturer (Micro RNA isolation kit; Stratagene, La Jolla, Calif.).
The concentration of RNA was assessed by spectrophotometry with
GenQuant II (Pharmacia, Piscataway, N.J.). Reverse transcription was
carried out in 20-µl reaction volumes. Besides total RNA, each sample
contained the following: 200 U of Moloney murine leukemia virus reverse
transcriptase (Gibco BRL), 2 U of RNase inhibitor (Promega, Madison,
Wis.), 2 µl of 10× PCR buffer II, 5 mM MgCl2
(Perkin-Elmer, Foster City, Calif.), 5 pmol of oligo(dT) (Promega), and
2 mM each dATP, dCTP, dGTP, and dTTP. This mixture was incubated at
42°C for 40 min. The reaction was terminated by incubation at 99°C
for 5 min. In preliminary experiments, we tested 10-fold-increasing
amounts of total RNA from 0.001 to 1.0 µg per sample. The
single-stranded cDNA generated by each sample was then subjected to PCR
with primers for -actin by a PCR method described below. The optimum
amount of total RNA for each reverse transcription experiment was
determined to be 1.0 µg. This was the amount used in all experiments
reported in this paper.
PCR amplification.
PCR amplification of a targeted gene
sequence was carried out with an automated thermocycler (RoboCycler 40;
Stratagene). Each 20-µl reaction volume consisted of water, 2 µl of
10× PCR buffer, 0.2 µl of 10 mM deoxynucleoside triphosphate mix
(Boehringer, Indianopolis, Ind.), 0.2 µl of Taq polymerase
(Perkin-Elmer), 2 µl of first-strand cDNA, and 20 pmol of each
primer. The reaction mixture was subjected to 30 amplification cycles,
each consisting of 96°C for 1 min, 57°C for 30 s, and 72°C
for 1 min. The PCR products were separated by electrophoresis in a 2%
agarose gel. To assess the amount of amplified product, the DNA was
stained with ethidium bromide and photographed with Polaroid
(Cambridge, Mass.) type 55 films. The negatives of the films were then
subjected to densitometry measurements (UltroScan XL; Pharmacia). All
of the densitometry measurements obtained in this study fell in a linear relationship with the amount of DNA. Primer sequences were as
follows: for -actin, sense 5'-AAC TGG GAC GAC ATG GAG AA-3' and antisense 5'-CCACGT CGC AGCCAT ACA TAT-3'; for
HLA-B27, sense 5'-GAC GAC ACG CTG TTC GTG-3' and antisense
5'-CCA CGT CGC AGC CAT ACA TAT-3'; for LMP2, sense
5'-GGC GTT GTG ATG GGT TCT GAT TCC-3' and antisense
5'-AAG ATG ACT CGA TGG TCC ACA CCG-3'; for LMP7, sense
5'-CCC TGT TTC CAG CGG ATG C-3' and antisense 5'-GCA GCA GGT CAC TGA CAT CTG-3'; for PA28, sense 5'-GCA AAC
AGG TGG AGG TCT TCAGG-3' and antisense 5'-CAT TACATG AGT
CTCCTT GGAGG-3'; for PA28, sense 5'-GTG GAT GTG TTT CGT
GAA GACCT-3' and antisense 5'-GCT GCT TGG CTG CTT TAG
TCACT-3'; and for MECL, sense 5'-CGA ACATGACGCTGGAGG CTG-3'
and antisense 5'-CTG GGTCAGGACAGC TGT GGT-3'.
-actin were those previously
published by other investigators (8). The primers for LMP2 and LMP7 were minor modifications of those published by other investigators (22). These two pairs of primers were designed to generate PCR products of 536 and 690 bp, respectively. The validity
of these primers was verified by sequencing the PCR products (data not
shown). The PA28 and PA28 primers were designed from published
sequence data (1, 23) and were verified by sequencing the
PCR products (data not shown). These primers generated PCR products of
535 and 515 bp, respectively. The primers to amplify human MECL were
based on the published genomic DNA sequence (19). The
antisense primer for MECL spanned two intron-exon junctions and
generated a PCR product of 212 bp; the validity of the primers was
verified by sequencing the PCR products (data not shown). The primers
to amplify HLA-B27 were based on published sequence data. The antisense
primer in exon 3 is specific for HLA-B27, while the sense primer is in
exon 2, generating a PCR product of 266 bp, which was verified by
sequencing (data not shown).
Semiquantitative analysis of proteasome subunit gene expression
following Salmonella invasion or IFN- treatment.
To
allow comparison of RT-PCR values for LMP2, LMP7, MECL, HLA-B27, and
PA28 and - genes derived from different samples, the
concentration of cDNA in each sample was adjusted so that they would
all yield similar amounts of PCR product when amplified by primers for
-actin. In our experiments, the variation of -actin PCR products
between adjusted samples of cDNA did not exceed 5%. Quantitation was
computed as a ratio of the densitometry reading for the test gene to
that for -actin. In addition, cDNAs were serially diluted and PCR
amplified with specific primers and -actin primers; test
gene/-actin ratios were calculated by determining the linear range
for each sample, plotting the best-fit line, and determining the test
gene/-actin ratio at the x intercept. Samples from
control cells and cells invaded by Salmonella or incubated
with IFN- were analyzed on the same day and run on adjacent lanes.
The changes induced by Salmonella or IFN- were expressed
as a percentage of the values for the control samples. In preliminary
experiments, we assayed gene expression at various time points (4, 8, 16, and 24 h) following bacterial invasion (data not shown).
Maximal expression was observed at 16 h after invasion, and these
data are presented below.
Preparation of LMP2 and anti-human MHC class I antisera. Rabbit antiserum was generated against the C-terminal peptide of LMP2 (HRVILLNELPKFYDE) (2). Rabbits were primed and challenged repeatedly with peptide conjugated to keyhole limpet hemocyanin emulsified in complete Freund's adjuvant and incomplete Freund's adjuvant. Prebleed sera lacked reactivity to proteasome components. The murine H-2Db molecule was purified from EL4 tumor cells grown as ascites fluid in 6- to 8-month old mice as previously described (28). Detergent lysates from 1010 EL4 cells were passaged over a Sepharose 4B precolumn followed by a B22.249 monoclonal antibody column (4). Columns were washed with 0.1% deoxycholate (DOC)-40 mM NaCl-10 mM Tris (pH 8.2) and 0.5% DOC-0.65 M NaCl-10 mM Tris (pH 8.5). The bound H-2Db was eluted with 0.5% DOC-0.15 M NaCl-15 mM Na2CO3 (pH 10.5). Solid-phase enzyme-linked immunosorbent assay with the B22.249 monoclonal antibody was performed to identify fractions containing H-2Db. Purity of the fractions was assessed by silver staining. Protein quantitation was determined by a micro-bicinchoninic acid assay (Pierce Chemical Co., Rockford, Ill.). Peak fractions, determined by enzyme-linked immunosorbent assay, from each preparation were pooled. Approximately 0.8 ml containing 50 to 150 µg of H-2Db was mixed with an equal volume of complete Freund's adjuvant and injected into a rabbit. Three additional injections were performed at 1-month intervals with 0.8 ml of the H-2Db samples mixed with incomplete Freund's adjuvant. The rabbit was bled, and the serum was found to immunoblot several murine class I heavy-chain molecules, including H-2Kb, Db, Kk, Dk, and Dd, and to cross-react with human class I MHC molecules, including HLA-B27, but it did not immunoblot control proteins such as murine class II molecules or antibodies. No reactivity to the class I MHC molecules was observed with the serum from the rabbit prebleed.
Western blot analysis. Twenty million cells were lysed at 4°C for 30 min in 0.5% Nonidet P-40-150 mM NaCl-50 mM Tris HCl-1 mM phenylmethylsulfonyl fluoride-10 mM iodoacetamide-5 mM EDTA-10 µg of aprotinin per ml. Equivalents of 5 × 105 cells were boiled for 3 min in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer, and the proteins were separated by SDS-10.5% polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane. The membrane was incubated with rabbit anti-LMP2 serum at a 1:100 dilution. Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, Calif.) by using the supersignal CL-HRP substrate system (Pierce, Rockford, Ill.).
In other experiments, cell lysates from 20 × 106 cells were immunoprecipitated with 250 µl (500 µg) of ME1 (HLA-B27-specific) antibody on beads for 24 h. The beads were washed three times in 20 mM Tris (pH 7.5)-150 mM NaCl and then once in 50 mM ammonium acetate (pH 7). Beads were resuspended in reducing buffer, and serial dilutions from salmonella-invaded and control cells were run on an SDS-12.5% acrylamide gel. Proteins were transferred onto an Immobilon-P membrane (Millipore Corporation) over 3 h at 400 mA and blocked overnight with 4% bovine serum albumin. The membrane was incubated with the rabbit anti-human MHC class I serum at a 1:500 dilution. Bound antibodies were detected with HRP-conjugated goat anti-rabbit IgG by using enzyme chemiluminescence (New England Life Science Products).Isolation and analysis of metabolically labeled B27-bound peptides. Following incubation with the two S. typhimurium strains, HeLa cells were incubated for 16 h at 37°C at 2 × 106 cells per ml in medium containing 90 µCi of [3H]arginine (Amersham, Oakville, Ontario, Canada) per ml. Following metabolic labeling, the cells were solubilized in lysis buffer composed of 0.5% Triton X-100, 10 mM Tris, and 50 mM NaCl (pH 8.0). The HLA-B27 molecules were then immunoprecipitated with ME1 monoclonal antibody-coupled Sepharose beads by rotation for 4 h at 4°C in the presence of protease inhibitors, aprotinin, and phenylmethylsulfonyl fluoride (Sigma, St. Louis, Mo.). The beads were acid eluted with 10% (vol/vol) acetic acid in water, and low-molecular-weight material corresponding to eluted peptides was recovered after Centricon 10 (Amicon, Beverly, Mass.) filtration by previously described procedures (27). Eluted peptides were washed twice with distilled water and dried with a speed vacuum device. Isolated peptides were resuspended in 0.1% trifluoracetic acid (TFA) in water and separated on a narrow-bore C18 (2 mm by 25 cm) column set up on a Beckman System Gold HPLC with solution A consisting of 0.1% TFA in water and solution B consisting of 0.1%TFA, 80% acetonitrile, and 20% H2O. The flow rate (0.15 to 0.2 ml/min) and rate of increase of the acetonitrile gradient (15 to 50%) were adjusted to maximize resolution of the labeled peptides. Collected fractions (200 µl each) were dried with the speed vacuum device, and radioactivity was determined by scintillation counting.
Statistics. Statistical comparisons were made only for the percent changes in values for antibody fluorescence intensity and RT-PCR values for gene expression as the methods used were semiquantitative. To compare two parameters, the percent changes in each parameter were entered into separate columns in the Instat software (Graph Pad, San Diego, Calif.). The linear regression figures and the r2 values were generated by the software.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The IFN--induced increase in reactivity with the
peptide-sensitive anti-HLA-B27 Ye-2 and B27.M2 antibodies correlates
with LMP2 gene expression.
The parallel use of antibodies
sensitive to peptide-induced conformational change of the B27 heavy
chain and RT-PCR assays of LMP mRNA constitutes a novel approach. To
assess the possibility of data correlations with these assays, we
tested the effect of IFN- on 11 HLA-B27-positive lymphoblastoid cell
lines. Depending on the particular cell line, there was considerable
variation in changes in RT-PCR values of LMP2 or LMP7, as well as the
reactivities with each of the three antibodies B27.M2, Ye-2, and ME1.
Strikingly, the variations were not random. The increases in reactivity
with either the B27.M2 or the Ye-2 peptide-sensitive antibody
correlated remarkably well with increases in LMP2 RT-PCR values
(r2 = 0.71 and 0.68, respectively). In
contrast, the correlation with increases in LMP7 RT-PCR values was poor
(r2 = 0.21 and 0.03, respectively). The
results of these experiments are shown in Fig.
1. That these antibody
assays were reliable was demonstrated by the finding that none of the
HLA-B27-negative lymphoblastoid cell lines reacted positively with
B27.M2 or Ye-2, and the IFN--induced increases in reactivities with
these two particular peptide-sensitive antibodies in the
HLA-B27-positive cells correlated with one another
(r2 = 0.77) (Fig. 1). Changes in
reactivities with B27.M2 and Ye-2 were not secondary to changes in
expression of HLA-B27, because there was no correlation between these
changes in B27.M2 and Ye-2 reactivities and changes in reactivities
with the non-peptide-sensitive anti-HLA-B27 ME1 antibody (Fig. 1). An
increase in the LMP2 protein was also documented by Western blot
analysis following incubation with IFN- (Fig.
2).
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Salmonella invasion into HLA-B27 HeLa cells induces
increased reactivities with the peptide-sensitive Ye-2 and B27.M2
antibodies.
HLA-B27 HeLa cells were subjected to
Salmonella invasion, and the antibody reactivities were
compared to those for a sample which was mock invaded and a sample
which was cultured with the noninvasive mutant SB111. Compared to the
mock-invaded sample, significant increases in reactivities were
observed with the peptide-sensitive antibodies Ye-2 and B27.M2
(Fig. 2), which was consistently observed in three separate
assays (Fig. 2). There was no increase in reactivity with the
non-peptide-sensitive ME1 antibody (data not shown). With the parent
HeLa cell line, which carries the HLA-A3 and -B5 alleles, no reactivity
was observed with anti-HLA-B27 antibodies regardless of invasion. Data
from the sample cultured with the noninvasive mutant were similar to
those for uninvaded controls (data not shown). As a positive control,
increases were also observed with IFN- incubation compared to
control uninfected HeLa cells (Fig. 2).
Salmonella invasion into HLA-B27-HeLa cells induces
increased LMP, MECL, and PA28, but not HLA-B27, expression.
In
parallel with increases in reactivities with the peptide-sensitive Ye-2
and B27.M2 antibodies, an increase in RT-PCR values was observed for
the LMP2 gene in HLA-B27-HeLa cells following Salmonella
invasion (Fig. 3). Increased RT-PCR
values for LMP7, MECL, and PA28 and -, but not HLA-B27, were also
observed in cDNA samples where the concentration was adjusted to yield
similar amounts of -actin after PCR amplification (Fig. 3).
Incubation with IFN- served as a positive control. Test
gene/-actin ratios following PCR amplification of serially diluted
template were consistent with the data summarized in Fig. 3 (data not
shown). An increase in the LMP2 protein was also documented by Western blot analysis following Salmonella invasion as well as
incubation with IFN- (Fig. 2). That the increased reactivity of the
Ye-2 and B27.M2 antibodies was not due to upregulation of B27
expression was confirmed in immunoprecipitation experiments
demonstrating no change in the amount of the HLA-B27 protein following
Salmonella invasion (Fig. 4).
Incubation of HeLa cells with supernatant from cell cultures with
invasive bacteria did not induce LMP2 gene expression, indicating that
such expression was unlikely to be secondary to autocrine stimulation
with IFN- following bacterial invasion (data not shown).
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HPLC profiles of newly synthesized HLA-B27-bound peptides. The preceding results indicate that Salmonella invasion of HeLa cells results in the induction of LMP2 expression, a change associated with alteration of the binding characteristics of monoclonal antibodies that recognize peptide-induced conformational change of the B27 heavy chain, suggesting that invasion may alter the peptides bound by HLA-B27 in the invaded cell. To address this possibility more directly, we analyzed the repertoire of B27-bound peptides of B27-transfected HeLa cells following bacterial invasion and compared this with the peptide profile of B27-bound peptides from the same HeLa cells in the absence of invasion. The HPLC profile of [3H]arginine-labeled peptides extracted from HLA-B27 molecules from noninvaded cells shows a diverse profile of peptides that distinguishes at least 40 peptide peaks, including approximately 20 major peptide peaks (Fig. 5). Salmonella invasion substantially alters the profile of bound peptides found in HLA-B27. Qualitative changes include both the appearance of prominent new peptide peaks and the reduction in yield or disappearance of others, particularly in the late elution fractions. Quantitative changes include an overall diminution in [3H]arginine-labeled peptides eluted from B27. These conclusions are based on reproducible changes in HPLC peptide profiles observed in four separate experiments. In particular, repeated analyses demonstrated that the prominent new peaks following bacterial invasion highlighted in Fig. 5 were consistently observed (data not shown). Thus, despite the fact that overall B27 expression as measured by ME1 FACS and immunoprecipitation of HLA-B27 is unchanged following Salmonella invasion (Fig. 4), significant changes are occurring in the repertoire of arginine-containing peptides bound by B27.
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. Again, significant HPLC
profile differences were observed (Fig.
6), and certain prominent new peaks after
stimulation with IFN- (Fig. 6) were consistently detected in
repeated analyses (data not shown). Despite some differences in the
overall yield of [3H]arginine-labeled peptides between
experiments, superimposition of HPLC profiles following either
Salmonella invasion or incubation with IFN- revealed that
two induced peptide peaks appeared to correspond in retention time
(Fig. 5 and 6). The radioactivity in HPLC profiles of acid-eluted
peptides from antibody isotype control beads incubated with lysates
from S. typhimurium-invaded HeLa cells and HeLa cells
incubated with and without IFN- was less than 25 cpm per fraction
(data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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While the effectiveness of CTL in defense against viral infection has been known and manipulated for decades, study of their role in bacterial infection is more recent. Nevertheless, multiple investigators have succeeded in generating MHC class I-restricted CTL against target cells infected in vitro with, for example, S. typhimurium, Yersinia eneterocolitica, and Chlamydia trachomatis (12, 29, 30). Only a small number of bacterial proteins which provide peptides for T-cell recognition have been identified (29, 30). Mounting evidence suggests that pathogen-derived proteins are processed by the same cytosolic pathway utilized by host proteins (17). Not surprisingly, certain viruses have developed subversive strategies to evade class I-restricted antigen processing and presentation (13). Strategies by which intracellular bacteria might attempt to evade or modify antigen processing have not yet been reported. Understanding the interplay between host cells and invading gram-negative bacteria with respect to class I-restricted antigen processing and presentation may yield insights toward defining the molecular basis of HLA-B27-associated arthritides induced by disease-associated bacteria.
The major contribution of the present study is that it shows that
invasion by S. typhimurium induces transcription of the LMP2, LMP7, MECL, and PA28 and - genes and expression of the LMP2
protein, a subunit of the cytosolic proteasome complex responsible for
class I-restricted antigen processing. These changes parallel those
reported with IFN-. In the case of IFN-, the LMP2, LMP7, and
recently described MECL1 (21) subunits become integrated into the 20S complex of the proteasome, displacing the respective constituents Y, X, and Z (3, 14). As a result, there is
modification of the terminal cleavage specificity of the catalytic
core. The results of our experiments with IFN- and
HLA-B27-transfected HeLa cells are consistent with the above-described
general principle and indicate that cellular stimuli resulting in
modification of proteasome subunit expression can be extended to
include encounters with invasive bacteria (26). Furthermore,
we were able to identify LMP2 expression in particular as
correlating with changes to the B27-bound peptide repertoire as
detected by peptide-sensitive antibodies. However, this type of
correlation analysis alone offers only indirect evidence for the role
of LMP2 in inducing changes in peptide repertoire. Direct evidence
requires the analysis of the peptide repertoire.
A previous study has demonstrated alterations in the HPLC profiles of
HLA-B27-bound peptides following Salmonella infection of CIR
B27-transfected cells, although the changes observed were primarily
quantitative rather than qualitative as they appear in our study
(25). A distinction from the previous study with respect to
B27-bound peptide analysis is that we performed metabolic labeling of
peptides with radiolabeled arginine, a nearly canonical B27 peptide
anchor residue for the B27 peptide binding groove. This approach can
provide greater sensitivity for peptide repertoire changes
subsequent to or as a consequence of bacterial invasion or
IFN- treatment, since it identifies newly generated peptides that
are not displayed against a potentially large background of
previously processed B27-bound peptides.
A recent study has shown altered expression of serologic HLA-B27 epitopes on human monocytes following exposure to Yersinia or invasion by Salmonella enteritidis, although in contrast with our data, ME1 epitope expression was decreased (33). This could reflect differences in signal transduction and/or antigen processing associated with bacterial invasion. In addition, similar Salmonella invasion assays with the U937 cell line in our lab resulted in toxicity, consistent with other data indicating that Salmonella invasion of phagocytic cell lines results in cell death (15a), suggesting that decreased ME1 epitope expression may reflect a toxicity phenomenon.
Based on previous findings with IFN- (10), it is likely
that activation of the LMP2 gene induced by S. typhimurium
invasion will alter antigen processing and consequently the HLA-B27
peptide repertoire. Indeed, our results from experiments using
peptide-sensitive antibodies as well as HPLC analysis of newly
synthesized peptides are consistent with this hypothesis. It seems
unlikely that LMP2 gene activation following bacterial invasion is
secondary to autocrine stimulation with IFN-. Incubation of HeLa
cells with supernatant from cell cultures with invasive bacteria did
not induce LMP2 gene expression. Furthermore, we did not observe
increased expression of the IFN--inducible HLA-B27 gene following
bacterial invasion.
An intriguing consequence of the changes in LMP2 expression induced by Salmonella invasion is that they may result in changes in the processing of self proteins to yield autoantigens. We have reported previously that activation of synovial T lymphocytes by bacterium-invaded stimulator cells can lead to generation of "autoreactive" CD8+ CTL (12). The findings in this paper provide a potential mechanism for generation of immunogenic self peptides.
Finally, the present findings are also important in clearly emphasizing
that invasion of bacteria into host cells is associated with complex
events. The view that they merely provide target peptides is likely an
oversimplification. A major clue as to how Salmonella might
induce multiple cellular events is provided by recent findings that
bacterial invasion activates signal transduction systems of host cells
(6). This activation probably leads to generation of
transcription factors such as AP1 and NF-
B. Since a particular
transcription factor is utilized by enhancer regions of multiple genes,
several unrelated events will be triggered. In addition, if
cytokine genes are triggered, the cytokines can in turn exert an
autocrine effect and recruit additional gene activation. These events
might hold the key to defense or generation of autoimmunity.
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ACKNOWLEDGMENTS |
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We thank Elizabeth Hermann for review of the project.
Walter P. Maksymowych and Takashi Ikawa contributed equally to this work.
This work was supported by the Medical Research Council (MRC) of Canada, the Nora Eccles Treadwell Foundation, and the U.S. Department of Agriculture. W.P.M. is a Scholar with the Alberta Heritage Foundation for Medical Research (AHFMR), and K.P.K. is an AHFMR Senior Scholar and MRC Scholar.
| |
FOOTNOTES |
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* Corresponding author. Mailing address: 562 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. Phone: (403) 492-1964. Fax: (403) 492-6055. E-mail: maksymow{at}gpu.srv.ualberta.ca.
Editor: R. N. Moore
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Infection and Immunity, October 1998, p. 4624-4632, Vol. 66, No. 10
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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