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Department of Medicine, Division of Infectious Diseases,
Biotechnology Laboratory, Departments of
Medical Genetics,
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Microbiology and Immunology, and
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Zoology,
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Department of Advanced Therapeutics of The British Columbia Cancer Agency, Faculties of Medicine and Science, University of British Columbia; and
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Research Institute of Vancouver Hospital and Health Sciences Center, Vancouver, British Columbia, Canada.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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increased the surface
expression of class II molecules in THP-1 cells and this was markedly
reduced in cells infected with M. tuberculosis. Despite
this effect, steady state levels of HLA-DR
, HLA-DRß, and invariant
(Ii) chains were equivalent in control and infected cells. Metabolic
labeling combined with pulse-chase experiments and biochemical analysis
showed that the majority of class II molecules in infected cells became
resistant to endoglycosidase H, consistent with normal Golgi
processing. However, results of intracellular staining and dual color
confocal microscopy revealed a significant defect in transport of newly
synthesized class II molecules through the endocytic compartment. Thus,
compared with findings in control cells, class II molecules in infected
cells colocalized to a minimal extent with a lysosomal-associated
membrane protein-1+ endosomal compartment. In addition, in
contrast to control cells, class II molecules in infected cells failed
to colocalize with endocytosed BSA under conditions where this marker
is known to label late endosomes, lysosomes, and the MHC class II
compartment. Consistent with defective transport along the endocytic
pathway, the maturation of SDS-stable class II ß dimers—dependent
upon removal of Ii chain and peptide loading of class II dimers in the
MHC class II compartment—was markedly impaired in M.
tuberculosis-infected cells. These findings indicate that
defective transport and processing of class II molecules through the
endosomal/lysosomal system is responsible for diminished cell surface
expression of MHC class II molecules in cells infected with M.
tuberculosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mononuclear phagocytes are the principal host cells for M.
tuberculosis and infected cells have diminished capacity for MHC
class II-restricted Ag presentation and costimulation of Th cells
(7, 8). These defects appear to be related—at least in
part—to reduced expression of class II molecules by infected
macrophages (7, 9, 10). During both the innate and
acquired immune responses, class II expression by APCs is up-regulated
by IFN- (11, 12, 13). However, intracellular infection has
been shown to impair macrophage responses to IFN- (14, 15), and the mechanisms by which M. tuberculosis
interferes with enhanced class II expression in response to IFN- are
not understood.
Recent studies indicate that transcription of MHC class II genes in
response to IFN- is regulated by the MHC class II transactivator
(CIITA)4 protein (16, 17, 18).
CIITA also regulates the expression of Ii chain and HLA-DM. Thus, CIITA
controls expression of multiple gene products involved in Ag
presentation (18). In addition, it has recently been shown
that the intracellular signaling events mediating transcriptional
responses to IFN-—including CIITA—involve the Janus
kinase (Jak)-STAT pathway. When IFN- binds to its receptor, the
nonreceptor tyrosine kinases Jak1 and Jak2 become activated, leading to
tyrosine phosphorylation of STAT1 (19, 20). STAT1
subsequently dimerizes and translocates to the nucleus where it binds
to IFN- activation sequences, leading to transcriptional activation
of IFN--responsive genes including CIITA (for review see
Ref. 21).
Newly synthesized class II molecules and Ii chain exit the endoplasmic
reticulum, traverse the Golgi and trans-Golgi network (TGN),
subsequently localizing to an acidic endosomal compartment. The events
that transpire in this compartment are complex and essential to ensure
appropriate expression of class II dimers. Thus, in this compartment,
Ii undergoes sequential proteolysis (22, 23), and the
final product of Ii degradation, a peptide referred to as CLIP (class
II-associated Ii peptide), is removed and ß dimers are loaded with
antigenic peptide (24). HLA-DM, a nonclassical MHC class
II molecule, stably associates with CLIP-associated ß dimers and
catalyzes the exchange of CLIP with cognate peptide (25, 26). Mature, peptide-loaded, SDS-stable class II dimers are then
transported to the cell surface. Removal of CLIP and peptide loading is
believed to occur in an endosomal/lysosomal compartment referred to as
the MHC class II compartment (MIIC).
This study examined the basis for diminished IFN--induced cell
surface expression of class II molecules in M.
tuberculosis-infected human mononuclear phagocytes. The results
show that infection has no apparent effect on signaling for expression
of CIITA and DRA genes. Rather,
diminished class II expression is related to defective trafficking of
apparently immature class II molecules from the TGN through the
endocytic compartment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 and HBSS were obtained from the Terry Fox Laboratory,
Vancouver, British Columbia, Canada. PMA, PMSF, pepstatin A, aprotinin,
leupeptin, and dextran sulfate were from Sigma Chemical Co. (St. Louis,
MO). Latex particles (1.05 µm diameter) were from Polysciences
(Warrington, PA). Anti-phosphotyrosine mAb 4G10 and rabbit antisera to
Jak1 and Jak2 were from Upstate Biotechnology (Lake Placid, NY). Rabbit
antiserum to STAT1 were kindly provided by Dr. Andrew Larner, Food
and Drug Administration (Bethesda, MD). Human rIFN- was a generous
gift of Genentech (South San Francisco, CA).
Mycobacterium tuberculosis
A virulent strain of M. tuberculosis (Erdman, TMC no. 107, Trudeau Mycobacterial Culture Collection, Saranac Lake, NY) was grown to late log phase in Proskauer and Beck medium supplemented with 0.05% Tween-80. Batch cultures were aliquoted and stored at -70°C. Representative vials were thawed and enumerated for viable CFU on Middlebrook 7H10 agar (Difco Laboratories, Detroit, MI). Prior to infection, bacteria were opsonized as follows: 109 viable organisms were suspended in 1 ml of RPMI 1640 containing 50% AB+ serum and rocked for 30 min at 37°C. Bacteria were then pelleted and resuspended in 1 ml of RPMI 1640 and clumps were disrupted by multiple passages through a 25-gauge needle as described (27). To evaluate the phagocytosis of M. tuberculosis by THP-1 cells, bacteria (109/ml) were labeled by incubation with 0.5 mg of FITC (Sigma) per ml in 0.1 M carbonate buffer (pH 9.0) at 37°C for 2 h. Thereafter, FITC-labeled bacteria were washed twice with PBS to remove unbound FITC, and cells were suspended in fresh Proskauer and Beck medium and kept at -70°C.
Differentiation and infection of THP-1 cells
The monocytic cell line THP-1 (ATCC, Rockville, MD) was cultured in RPMI 1640 supplemented with 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were seeded at a density of 105/cm2 and allowed to adhere and differentiate in the presence of PMA (20 ng/ml) at 37°C in a humidified atmosphere of 5% CO2 for 24 h. Depending on the quantity of cell material needed, either six-well, flat bottom, cell culture plates or 10-cm diameter cell culture dishes (Becton Dickinson, Franklin Lakes, NJ) were used. Cells were then washed three times with HBSS and adherent monolayers were replenished with culture medium without antibiotics. Differentiated THP-1 cells were infected with opsonized M. tuberculosis. After a period of 24 h, noningested bacteria and dead and partially attached cells were removed by extensive washing with HBSS. This resulted in an infection rate of 80 to 90% with an approximate range of 5 to 15 bacteria per cell. The THP-1 monolayers remained stable until the end of the experiments. During the course of the experiments, some cells overloaded with bacteria died and detached from the plastic surface.
Phagocytosis of M. tuberculosis was determined by using the fluorescence quenching technique as described previously (28, 29). Briefly, THP-1 cells were incubated with opsonized FITC-labeled bacteria for 2 h at 37°C. To quench fluorescence of noningested, but membrane-associated bacteria, cells were washed and resuspended in sodium acetate buffer (0.05 M, pH 4.5) containing 0.06% trypan blue for 5 min at 4°C. After two washes with sodium acetate buffer, the number of cells loaded with bacteria was measured by flow cytometry. Appropriate controls were included to validate the quenching of surface fluorescence.
Cell staining and flow cytometry
To measure cell surface expression of HLA-DR, control and M. tuberculosis-infected cells were incubated with anti-HLA-DR mAb (clone HL38, IgG1, Caltag Laboratories, San Francisco, CA) for 30 min, then washed twice and labeled with FITC-conjugated F(ab')2 sheep anti-mouse IgG (Sigma) for 30 min. Cells were also stained with the following mAbs: W6/32 (IgG2a, anti-HLA-class I, ATCC); IB4 (IgG2a, anti-CD18, a gift from Dr. D. Speert, the University of British Columbia, BC, Canada); and 5E9C11 (IgG1, anti-transferrin receptor, ATCC). All staining and washing procedures were performed at 4°C in HBSS containing 0.1% NaN3 and 1% FCS. To control for cell viability, cells were incubated with propidium iodide (0.5 µg/ml in staining buffer) for 10 min. The cells were then washed twice and fixed in 2% paraformaldehyde in staining buffer. The combination 2% paraformaldehyde + 0.1% NaN3 killed both free and cell-associated bacteria. Cell fluorescence was analyzed using a Coulter Elite flow cytometer (Hialeah, FL). Viable cells were identified by exclusion of propidium iodide. Relative fluorescence intensities of 5000 cells were recorded as single-parameter histograms (log scale, 1024 channels, 4 decades) and the mean fluorescence intensity (MFI) was calculated for each histogram. Results are expressed as MFI index, which corresponds to the ratio: MFI of cells + specific Ab/MFI of cells + irrelevant isotype-matched IgG.
Immunoprecipitation and Western blotting
Whole cell lysates were immunoprecipitated with rabbit antisera
to Jak1, Jak2, or STAT1 and analyzed by SDS-PAGE and immunoblotting
with anti-phosphotyrosine mAb (4G10) as previously described
(14). To assess the amount of individual proteins
immunoprecipitated in each sample, after detection of bound
anti-phosphotyrosine Ab, membranes were stripped, probed with
either anti-Jak1, anti-Jak2, or anti-STAT1, and
developed by enhanced chemiluminescence (ECL) as described
(14).
RNA isolation and RT-PCR
RNA preparation, cDNA synthesis, and PCR condition were described previously (30). Sequences of oligonucleotide primers used in PCR amplifications are as follows: DRA sense, CGA GTT CTA TCT GAA TCC TGA CCA; DRA antisense, GTT CTG CTG CAT TGC TTT TGC GCA; CIITA sense, CAA GTC CCT GAA GGA TGT GGA; CIITA antisense, ACG TCC ATC ACC CGG AGG GAC; ß-actin sense, CAC CCC GTG CTG CTG ACC GAG GCC; ß-actin antisense, CCA CAC GGA GTA CTT GCG CTC AGG.
Quantification of mRNA transcripts by photometric enzyme immunoassay
Polyadenylated RNA was isolated from ribosomal RNA and transfer
RNA by base-pairing between the poly(A) residues of the mRNA and a
biotin-labeled oligo(dT)20 probe (Boehringer Mannheim kit,
Montreal, Quebec, Canada) according to the manufacturer’s
instructions. A second kit (Northern-ELISA, Boehringer Mannheim) was
used for direct detection and quantification of DRA transcripts. In
brief, mRNA samples were biotinylated in RNase-free distilled water for
1 h at 65°C and precipitated by incubation in absolute ethanol
for 30 min at -70°C followed by a 30-min centrifugation at
15,000 x g. A digoxigenin (DIG)-labeled DNA probe was
prepared by PCR amplification of cDNA from positive cells (THP-1
cells + IFN-) in the presence of DIG-UTP (Boehringer Mannheim)
(35% DIG-UTP and 65% dTTP) and the sense and anti-sense
oligonucleotide primers for DRA and ß-actin used in the RT-PCR
experiments. A DIG-chloramphenicol acetyl transferase-labeled probe
(Boehringer Mannheim) was used to control for nonspecific
hybridization.
Biotinylated mRNA was diluted in hybridization buffer and prewarmed to 50°C before addition of denatured (5 min, 100°C) DIG-labeled DNA probes. The hybridization mixtures were incubated for 3 h at 50°C and then transferred to streptavidin-coated microtiter plates and incubated for 5 min at 50°C. The microwells were washed and peroxidase-conjugated rabbit anti-DIG-IgG was added for 30 min at 37°C. After washing, the wells were filled with tetramethylbenzidine (TMB) substrate for 20 min and the reaction was quenched by addition of 5% sulfuric acid. Absorbances were measured in an ELISA-reader at 450 nm.
Biosynthetic labeling with [35S]methionine, endoglycosidase H (Endo H) digestion, and SDS stability assay
Control and M. tuberculosis-infected cells were
incubated with IFN- for 36 h and for a further 1 h at
37°C in methionine- and serum-free RPMI 1640. The medium was then
removed and cells were incubated for 30 min in 2 ml of the same medium
supplemented with 5% dialyzed FCS and 200 µCi of
[35S]methionine. After a 30-min incubation, the volume
was brought up to 10 ml with 10% FCS/RPMI 1640, and radiolabeling was
terminated at the end of 2 h. Alternatively, cells were labeled in
presence of 600 to 800 µCi of [35S]methionine for 30
min and chased with medium containing an excess of cold methionine for
the times indicated. At the end of the radiolabeling periods, cells
were extensively rinsed with HBSS and lysed by scraping in
Tris-buffered saline containing 1% Nonidet P-40, 1 mM PMSF, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin, and 0.1%
NaN3 to inactivate live bacteria. Cell debris was removed
by centrifugation in a microfuge for 20 min at 4°C and
immunoprecipitations using the anti-HLA-DR mAb (clone Tu36, Caltag)
were performed 2 h at 4°C using equal amounts of
TCA-precipitable radioactivity from each treatment group. Immune
complexes were collected by adsorption to protein A and released by
boiling agarose beads in 2x SDS sample buffer (31).
Samples were analyzed using 12% SDS-PAGE. To analyze SDS-resistant
class II dimers, radiolabeled immunoprecipitates were incubated for
1 h at room temperature with 2x sample buffer prior to SDS-PAGE
(32, 33). Endo H treatment was performed by dividing DR
immunoprecipitates into two portions. One portion was left untreated,
and the other was incubated for 24 h at 37°C in 25 µl of 0.01
M citric acid, pH 5.5, with 5 mU Endo H (Boehringer Mannheim) and 0.5
mM PMSF. Samples were then analyzed by SDS-PAGE. Fluorography was
performed according to the method of Bonner and Laskey
(34).
Intracellular immunofluorescence
PMA-treated THP-1 cells were adhered to tissue culture-treated
coverslips (Miles Laboratories, Naperville, IL) in 24-well plates and
infected with M. tuberculosis. After IFN- treatment,
intracellular staining was performed essentially as described
(35). Cells were washed with PBS and fixed for 15 min at
37°C with 2.5% paraformaldehyde/PBS, then washed three times for 10
min at 37°C and permeabilized in PBS containing 0.2% saponin and
10% normal goat serum for 5 min. To label the endosomal/lysosomal
compartment, cells were incubated with mouse anti-human Lamp-1 mAb
(H4A3, IgG1, Hybridoma Bank of the University of Iowa, Iowa city, IA)
in PBS/saponin/normal serum for 30 min at room temperature, washed, and
stained with Texas Red-labeled F(ab')2 goat anti-mouse
Ig (Caltag). Coverslips were washed and specific FITC-labeled mAb was
used to stain HLA-DR molecules. Alternatively, cells were stained with
unlabeled anti-HLA-DR mAb and Texas Red-conjugated secondary
Ab.
At the end of the staining procedure, samples were washed three times with PBS and once with distilled water. Coverslips were then mounted in FluorSaveTM (Calbiochem-Novabiochem Corp., La Jolla CA) to minimize photobleaching. To control for nonspecific binding, secondary Ab was used alone, which gave negligible signals in the absence of primary Ab.
Internalization of endocytic tracer
Pulse-chase experiments with FITC-labeled BSA (Sigma) were used
to examine transit through the endocytic pathway. IFN--treated
control and infected cells, on cell culture-treated coverslips, were
pulsed for 30 min in serum-free medium containing FITC-BSA (1 mg/ml),
washed, and chased for either 30 min, 1 h, or 4 h. Cells were
then fixed and stained as described above with unlabeled
anti-HLA-DR mAb and Texas Red-conjugated secondary Ab.
Confocal laser-scanning microscopy
A confocal laser-scanning microscope system (MRC-600; Bio-Rad Laboratories, Hercules, CA) was used to detect intracellular fluochromes. Cell were scanned by dual excitation for FITC (green) and Texas Red (red) fluorescence. A 60x oil objective with numerical aperture of 1.4 was used and the images were captured such that the xyz dimensions were 0.2 µm cubed. NIH Image version 1.60 was used for image analysis, and all images were based on maximal intensity projection. Projections made in NIH Image were saved in TIFF format, then imported to Adobe Photoshop, version 3.0.4, in which the green and red images were assigned to individual RGB channels. To visualize the relative positional distribution of the two fluochromes, the images collected in the red and green channels were merged, and red and green overlapping fluorescence was reflected by a yellow signal.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Flow cytometry and FITC-labeled bacteria were used to measure
phagocytosis of M. tuberculosis by THP-1 cells. THP-1 cells
in suspension (106/ml) were incubated in the presence of
FITC-labeled bacteria (5 x 107/ml, final
concentration) for 2 h and then washed extensively. Fluorescence
of labeled bacteria bound to the outside of the cell was quenched by
trypan blue in sodium acetate buffer. The number of cells loaded with
bacteria was measured by FACS analysis. A signal is obtained only from
bacteria that have been protected from quenching by internalization. As
shown in Figure 1
A, the
proportion of phagocytic cells increased after PMA differentiation (43
vs 14%) and when bacteria were opsonized by fresh, nonimmune
AB+ serum, the frequency of positivity reached 84%. As
determined by FACS analysis, treatment of THP-1 cells with PMA also
resulted in a substantial increase in expression of CD11b and CD14
(data not shown), both of which are considered to be potential
receptors for M. tuberculosis (36, 37).
PMA-differentiated cells were exposed to a range of bacterial
concentrations and maximal loading with opsonized bacteria was obtained
using a bacteria to cell ratio of 50:1. In contrast, undifferentiated
cells exposed to the same range of nonopsonized bacteria showed only
marginal internalization of bacteria (Fig. 1B). This ratio
was used in all subsequent experiments. Alternatively, for each
experiment, routine acid-fast staining was used to confirm the presence
of intracellular bacteria and the experiments were continued when the
proportion of infected cells was >80%.
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-induced HLA-DR
expression in THP-1 cells infected with
M. tuberculosis
Initial experiments examined surface expression of HLA-DR
molecules by PMA-differentiated THP-1 cells in response to incubation
with a range of concentrations of IFN- for 36 h. FACS analysis
indicated that expression of HLA-DR was maximal (MFI index 15 to 25) at
200 U/ml of IFN- (data not shown). This concentration was used in
all subsequent experiments.
When differentiated THP-1 cells were exposed to M.
tuberculosis for 24 h, cell surface expression of HLA-DR in
response to IFN- was nearly completely abrogated (Fig. 2). In contrast, constitutive and
IFN--induced expression of class I molecules were unaltered by
M. tuberculosis infection. The expression of two additional
unrelated surface markers, CD18 and the transferrin receptor, were also
unchanged in infected cells, indicating that the inhibitory effect of
M. tuberculosis on MHC class II expression is selective. As
shown in Table I, reduced fluorescence
intensity for class II expression was first apparent at a bacteria to
cell ratio of 12:1, and the extent of this reduction increased with
greater multiplicities of infection.
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, in contrast to live bacteria,
phagocytosis of killed bacteria led to only a partial attenuation of
HLA-DR expression (42% reduction). To control for potential
nonspecific effects of phagocytosis on HLA-DR expression, cells were
incubated with 0.005% latex particles (uptake of 10 to 15
particles/cell) before treatment with IFN-. Latex particles had no
significant effect on class II induction. In contrast, incubation of
cells with dextran sulfate, which is known to inhibit class II gene
transcription (38), completely abrogated cell surface
class II expression.
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). Human monocytes appeared to be more
sensitive than THP-1 cells to the effects of M. tuberculosis
as bacteria to cell ratios as low as 5:1 markedly inhibited cell
surface expression of class II. In addition, constitutive expression of
class II molecules by cultured monocytes was also diminished by
infection (Table II).
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-dependent activation of the
Jak-STAT pathway
The transcription of class II genes in response to IFN- is
known to be dependent upon signaling through the IFN--activated
Janus tyrosine kinase, Jak1, which phosphorylates the
DNA-binding protein STAT1 (19, 20, 39). To examine the
possibility that attenuation of IFN--induced expression of class II
molecules by M. tuberculosis was related to impaired
signaling through the Jak-STAT pathway, infected (for 24 h) and
non-infected THP-1 cells were incubated with IFN- for 15 min and
cell lysates were analyzed for tyrosine phosphorylation of Jak1, Jak2
and STAT1. As shown in Figure 4,
IFN--induced tyrosine phosphorylation of Jak1, Jak2 and STAT1 was
not influenced by infection with M. tuberculosis. These
results indicate that attenuation of HLA-DR expression in M.
tuberculosis-infected cells is unlikely to be explained by
impaired IFN--activated activation of the Jak-STAT signaling
pathway.
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CIITA is an essential, IFN--inducible, trans-acting
factor required for expression of MHC class II genes
(16, 17, 18). Induction of CIITA is also known to be dependent
on signaling throught the Jak-Stat pathway (39). However,
IFN--induced expression of CIITA and
DRA genes are coordinately attenuated in TGF-ß-treated
cells despite normal function of the Jak-Stat pathway
(30). These suggested the possibility that infection with
M. tuberculosis could affect CIITA expression via
a mechanism independent of Jak-STAT activation. To address this
question, THP-1 cells were exposed to M. tuberculosis for
24 h prior to incubation with IFN- (24 h). Total RNA was
isolated and RT-PCR was performed using primers for CIITA
and DRA. The results shown in Figure 5A demonstrate that mRNA
levels for both CIITA and DRA induced by IFN-
were not influenced by infection with M. tuberculosis. In
contrast, exposure of cells to dextran sulfate, which like M.
tuberculosis inhibits cell surface expression of class II
molecules (38), brought about marked reductions in mRNA
levels for both CIITA and DRA.
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B,
absorbance values, normalized to those of ß-actin, indicated that
there was no significant difference in levels of IFN--induced
DRA transcripts in infected and in control cells. Therefore,
attenuation of IFN--induced expression of MHC class II molecules by
M. tuberculosis is independent of events required for the
transcriptional induction of the CIITA and DRA
genes. Synthesis, assembly and transport of DR polypeptides in M. tuberculosis-infected cells
To determine whether diminished cell surface expression of class
II molecules was related to effects of M. tuberculosis on
either the rate of synthesis or the transport of class II dimers,
radioimmunoprecipitation experiments were done using
[35S]methionine-labeled cells and mAb to class II
molecules. As shown in Figure 6A, three radiolabeled
proteins of approximately 35 kDa (class II -chain), 32 kDa
(invariant chain, Ii) and 30 kDa (class II ß-chain) were
immunoprecipitated from lysates of IFN--treated cells. In contrast,
extracts of untreated cells gave only weak signals for these three
labeled proteins. Importantly, the abundance of these proteins was
equivalent in IFN--treated control cells and in IFN--treated
infected cells. These findings indicate that M. tuberculosis
does not affect the synthesis or steady state levels class II
molecules.
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ß chains to mature Endo
H-resistant molecules that have undergone N-linked glycosylation during
successive exposure to medial Golgi-specific enzymes. IFN--treated
(control and M. tuberculosis-infected) cells were
radiolabeled and chased for various times before Endo H treatment.
After a 1 h of chase, the majority of class II molecules were Endo
H sensitive and control and infected cells displayed similar levels of
immature (indicated by s, ßs, and
Iis) subunits (Fig. 6B). After 8 h of
chase, most class II/Ii molecules were Endo H resistant (indicated by
r, ßr, and Iir) in
IFN--treated control cells and by 20 h there were no apparent
Endo H-sensitive molecules. Substantial Endo H resistance also
developed in M. tuberculosis-infected cells. However a minor
proportion of Endo H-sensitive molecules persisted at 20 h of
chase. Thus, infection appears to have a minimal effect on the assembly
and processing of class II molecules through the Golgi and this is
insufficient to explain the marked decrease in cell surface
expression. Infection with M. tuberculosis inhibits maturation of class II heterodimers during post-Golgi transport to the cell surface
Following the exit from the TGN, Ii chain is partially degraded in
an acidic endosomal/lysosomal compartment. The residual CLIP fragment
in the groove of the ß dimer is exchanged with antigenic peptide
in the MIIC (22). In contrast to their immediate
precursors, mature, peptide-loaded class II dimers are resistant to
dissociation in the presence of SDS at room temperature, thereby
enabling their specific detection (32, 33). To evaluate
the maturity of class II molecules in infected cells,
immunoprecipitates from IFN--treated control and infected cells were
incubated in 1% SDS at room temperature and analyzed by SDS-PAGE (Fig. 7). In control cells, SDS-stable
molecules of the molecular mass expected for the ß dimers (50–55
kDa) were detected at 2 h of chase and steadily increased in
quantity with longer chase times, concomitant with a decrease in
SDS-labile molecules. In contrast, SDS-stable complexes were barely
detectable in both cells infected with M. tuberculosis and
in cells alkalinized by treatment with NH4Cl. These
findings suggest that, if the class II/Ii complex exits the Golgi and
reaches the endosomal compartment in infected cells, either the
enzymatic processing of Ii chain or the loading of ß dimer with
antigenic peptide, or both, are blocked.
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Initially, single-color intracellular staining and epifluorescence
microscopy were used to examine the presence of class II molecules in
large populations of cells. As shown in Table III, for both IFN--treated control and
infected cells, almost 90% of cells stained positively for
intracellular HLA-DR. However, while intracellular staining of control
cells was relatively homogeneous throughout the cytosolic compartment,
peripheral staining was negative in the majority (83 to 89%, n =
3) of infected cells indicating intracellular sequestration of class II
molecules. To examine whether class II molecules are normally targeted
to the endosomal compartment in infected cells, two-color
immunofluorescence staining with specific Abs to HLA-DR and to Lamp-1,
a marker for endosomes and lysosomes was performed. Cells were examined
using a laser, confocal microscope. In IFN--treated control cells,
the staining pattern of HLA-DR (Fig. 8a, green) showed vesicles of
various sizes randomly distributed throughout the cell with a notably
bright peripheral staining. Class II staining overlapped significantly
(yellow) with that of Lamp-1 (red). As Lamp-1 remains associated with
class II vesicles en route to the surface, submembranous localization
of this marker was also noted. The distribution of class II molecules
in infected cells was markedly different. In contrast to control cells,
infected cells showed diffuse, perinuclear staining of the cytosolic
compartment and peripheral staining was negligible (Fig. 8b,
green). Colocalization of class II with Lamp-1 was markedly less than
that observed in IFN--treated control cells. This finding suggests
defective endosomal targeting of class II molecules in infected
cells.
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-treated control
cells, internalized BSA (green) was randomly distributed throughout the
cytoplasm (Fig. 8c, green). Colocalization (yellow) with
HLA-DR (red) was readily observed after a 30-min chase and increased
further at 1 h. After a 4-h chase, internalized BSA completely
colocalized with class II molecules presumably in late endosomes and
lysosomes including the MIIC. This conclusion is based on the findings
that long chase periods (1 h and up) usually allow fluid-phase tracers
to localize in such mature endosomal structures (40, 41).
In M. tuberculosis-infected cells, internalization of
FITC-labeled BSA (Fig. 8d, green) was also observed, but was
much less extensive than in control cells. Notably, in contrast to
control cells, colocalization of HLA-DR with BSA was negligible at both
1 h and 4 h of chase. This finding indicates that the
intersection of vesicles containing MHC class II molecules with late
endosomal structures, including Ag-bearing endosomes, lysosomes, and
the MIIC, is markedly impaired in cells infected with M.
tuberculosis.
The failure of class II molecules to colocalize with endocytosed BSA
may reflect an underlying defect in endocytosis and subsequent
trafficking in the endosomal compartment. To examine endocytic
activity, control and infected cells were incubated with FITC-BSA for
different time periods and were analyzed by FACS. MFI indices (MFI of
labeled cells/MFI of unlabeled cells) of cells internalizing FITC-BSA
were calculated. Compared with control cells, infected cells showed
decreased fluorescence at all time points tested (MFI index at 5 min/15
min/30 min/60 min: control = 17.0/37.3/53.1/59.4 vs infected
= 2.9/4.3/18.2/33.3). These results suggest a defect in endocytosis in
infected cells and may be related to abnormal trafficking of class II
vesicles into the endocytic compartment. Reduced endocytic activity is
consistent with an apparently less extensively developed Lamp-1
positive endosomal compartment in infected cells (compare Fig. 8b with Fig. 8a, red).
Class II molecules intersect with vacuoles containing M. tuberculosis
To examine directly whether class II molecules translocate to
M. tuberculosis-containing vacuoles, cells were incubated
with live or heat-killed (2 h, 60°C) FITC-labeled bacteria before
addition of IFN-. Labeling of mycobacteria with FITC affected
neither cell viability nor replication (data not shown). Moreover, as
determined by FACS analysis, the effect of FITC-labeled bacteria on
surface expression of class II (Table IV)
was similar to that of unlabeled, viable organisms. When intracellular
staining for HLA-DR and confocal laser analysis were performed, the
staining pattern of HLA-DR (red) in cells incubated with killed
bacteria appeared similar to that of control cells (Fig. 9a). Of note, multiple
bacterial fragments (green), resulting from intracellular degradation
of killed bacteria were apparent and these colocalized (yellow) with
HLA-DR. In contrast, in cells infected with viable FITC-labeled
M. tuberculosis (Fig. 9c), bright, predominantly
perinuclear staining for HLA-DR (red) was observed with a periphery
largely devoid of class II molecules. This pattern is qualitatively
similar to that observed with cells infected with unlabeled, viable
organisms (Fig. 8b). In addition, in cells incubated with
FITC-labeled, live bacteria, the organisms (green) appear intact and
are surrounded with substantial yellow color. These findings indicate
that class II molecules colocalize with vacuoles containing either dead
or live M. tuberculosis. However, based upon analysis in
Figure 8, the distribution of class II molecules in the endosomal
compartment is markedly restricted in cells harboring viable
mycobacteria.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced MHC class II
expression in monocytic cells infected with M. tuberculosis.
The human cell line THP-1 (42) was used for this study
because it displays many characteristics of mature monocytes and
macrophages such as Ag processing and presentation to T cells
(43, 44), efficient phagocytosis of M.
tuberculosis (45) (Fig. 1), and substantial
expression of class II molecules in response to IFN- (Fig. 2).
The results obtained show that IFN--induced cell surface expression
of HLA-DR molecules is markedly attenuated in THP-1 cells infected with
viable M. tuberculosis. Inhibition of class II expression is
significantly less when heat-killed bacteria are used (Fig. 3)
indicating that the effects of M. tuberculosis on class II
expression are, at least partially, dependent on metabolically active
organisms. Cell surface HLA-DR expression was also diminished in human
monocytes infected in vitro (Table II), consistent with data previously
reported (7). Compared with THP-1 cells, exposure of
monocytes to a relatively low number of bacteria resulted in high rates
of infection and markedly attenuated class II expression. This relative
increased sensitivity of human monocytes may be related to more
abundant receptors (CD11b and CD14) for M. tuberculosis on
monocyte plasma membranes. In contrast to diminished class II
expression, both constitutive and IFN--induced expression of class I
molecules in the same cells were apparently increased (Fig. 2). This
finding is consistent with a recent report of impaired Ag presentation
through the MHC class II pathway and augmentation of MHC class
I-restricted Ag presentation in M. tuberculosis-infected
monocytes (8).
The possibility that impaired responses to IFN- for induction of
class II expression may be related to defective cell signaling or gene
expression was investigated. IFN- regulates the expression of class
II genes primarily at the level of transcription and this requires
induction of CIITA (18). Moreover, expression
of the CIITA gene itself is dependent on Jak-STAT
signaling (17, 39, 46). The finding that IFN--induced
Jak-STAT activation was not affected by infection with M.
tuberculosis (Fig. 4) suggested the likelihood that
CIITA induction should be observed. In fact, RT-PCR and
Northern analyses (Fig. 5, A B) provided direct evidence
that IFN--induced expression of the CIITA gene occurs
normally in THP-1 cells infected with M. tuberculosis, and
mRNA levels for DRA were also observed to be unaffected.
These results indicated that inhibition of class II expression in
infected cells occurs posttranscriptionally and suggested the
possibilities that M. tuberculosis may be acting either at
the level of translation or upon events involved with processing or
transport of class II proteins. The findings of normal steady state
levels of , ß, and Ii chains (Fig. 6A) were consistent
with effects on either the maturation or transport of class II
molecules, or both.
MHC class II molecules are synthesized as nonameric complexes
consisting of three ß dimers associated with a trimer of Ii chains
[(ß)3/Ii3] (22, 23, 47).
After exiting the TGN, a dileucine-like motif in the cytoplasmic domain
of Ii chain acts as targeting signal and directs
(ß)3/Ii3 complexes to an endosomal
compartment. Here, Ii undergoes sequential proteolysis, giving rise to
an intermediate fragment, CLIP, which remains associated with class II
molecules, occupying the peptide-binding groove. This process requires
an acidic pH and when completed, HLA-DM, a nonclassical MHC class II
molecule, catalyzes CLIP dissociation, allowing class II molecules to
bind antigenic peptide, prior to export to the cell surface. The
cytoplasmic tail of DMß contains a tyrosine-based motif
(Tyr-Thr-Pro-Leu), which directs HLA-DM molecules from the TGN to
endosomes. Studies in a variety of APC types (22) suggest
that removal of CLIP and peptide loading occur in a postendosomal
compartment related to lysosomes, referred to as the MIIC.
Maturation and transport of class II molecules is associated with
characteristic changes in their biochemical properties. Progression of
class II molecules through the Golgi apparatus generates Endo
H-resistant molecules that have undergone N-linked glycan
modification during successive exposure to Golgi-specific enzymes.
Furthermore, peptide loading of class II molecules in the MIIC leads to
the appearance of stable, class II ß dimers that are resistant to
SDS at room temperature (32, 33). Figure 6B
shows that in M. tuberculosis-infected cells, almost all
radiolabeled class II molecules detected after a 20-h pulse-chase are
Endo H resistant. This suggests that ß dimers undergo normal
glycosylation in the medial Golgi. However, pulse-chase analysis also
revealed that M. tuberculosis inhibits the generation of
SDS-resistant ß dimers (Fig. 7). These findings suggested that
M. tuberculosis may interfere with either the delivery of
(ß)3/Ii3 to the endocytic compartment or,
alternatively, with the enzymatic processing of Ii chain and peptide
loading in the MIIC. Confocal laser scanning of 0.2-µm sections
demonstrated that class II molecules colocalized poorly with the
endosomal/lysosomal marker Lamp-1 in infected cells. This provided
direct evidence that the endosomal localization of class II/Ii chain
complexes is defective in infected cells. Fluid-phase tracers of
endocytosis, such as BSA, OVA, or dextran, are commonly used in
pulse-chase experiments to label endosomal structures, such as
lysosomes and the MIIC (40, 41, 48). Cells infected with
M. tuberculosis did endocytose FITC-BSA, albeit at a
significantly reduced level compared with control cells. Nevertheless,
colocalization of class II molecules with internalized BSA was
negligible in infected cells. This was particularly true after longer
periods of chase (compare Fig. 8c to d, 1 h
and 4 h of chase). These findings suggest that class II molecules
exiting the Golgi do not intersect with endosomes containing
internalized Ags, which is an obligatory step before progression to
peptide-loaded, stable molecules in the MIIC. The absence of
SDS-stable, class II dimers in infected cells (Fig. 7) is consistent
with this possibility. Taken together with results from the Endo H
experiments, these findings suggest that, despite normal Golgi
processing, transit of newly synthesized class II molecules into the
endocytic pathway is impaired. Diminished entry or defective transport
of class II molecules in the endocytic pathway may be related to the
finding of an overall reduction in endocytic activity per se.
In contrast to class II molecules, synthesis of heavy and light chains
of class I molecules and peptide loading are endoplasmic
reticulum-associated events. Moreover, class I/peptide complexes travel
to the cell surface along a pathway that does not initially intersect
the endocytic compartment (49). Since this pathway is not
affected by M. tuberculosis, class I expression in infected
cells does not appear to be perturbed (Fig. 2).
Based upon current information about the events involved in the
regulated expression of class II molecules and results of the present
study, Figure 10 provides a model that
may explain the effects of M. tuberculosis on class II
expression. Possible mechanisms accounting for retarded maturation of
class II molecules are depicted. Metabolically active organisms secrete
a variety of products including ammonia (50, 51, 52),
lipoarabinomannan (LAM) (53, 54), and sulfatide (55, 56) that are believed to be virulence factors (57).
Alkalinization of critical intracellular organelles is a potential
mechanism to explain retarded maturation of class II dimers in infected
cells. Ammonia produced by M. tuberculosis, which is known
to inhibit the saltatory movement of lysosomes (50, 58),
may act similarly on endosomes and class II vesicles thereby inhibiting
their fusion. Alternatively, ammonia produced by M.
tuberculosis may diffuse into endosomes containing class II/Ii
complexes and prevent adequate acidification leading to inhibition of
enzymes required for the processing of both Ii chain and internalized
Ags. Consequently, immature class II molecules may be retained in the
endocytic pathway. These hypotheses are supported by the finding that
treatment of cells with NH4Cl, like M.
tuberculosis, also inhibits the generation of SDS-resistant ß
dimers (Fig. 7). Inhibition of at least some proteolytic events is also
consistent with the finding that the M. tuberculosis itself
resists intracellular degradation (Fig. 9c). Defective
maturation of class II dimers could also be related to mycobacterial
sulfatides. These polyanionic glycolipids, which have the ability to
inhibit phagolysosomal fusion (55), could potentially
interfere with the maturation of endosomes to more lysosome-like
organelles (i.e., MIIC).
|
-induced surface expression of class II molecules without
affecting either activation of the Jak-STAT pathway or the accumulation
of class II mRNA (data not shown). Other recent work from this laboratory indicates that incubation of THP-1 cells with live, but not killed, M. tuberculosis promotes both tyrosine phosphorylation and tyrosine dephosphorylation of various proteins (data not shown). This suggests the possibility that infection may lead to changes in the phosphorylation states of tyrosine residues on critical molecules such as the cytoplasmic tail of DMß. Such an effect could influence the endosomal targeting of HLA-DM molecule and impair CLIP removal and peptide loading. It is also possible that dephosphorylation of critical proteins may impair trafficking through the endosomal/lysosomal system.
It is of interest to view the results of the present study in the
context of those of Pancholi et al., who observed sequestration of
mycobacteria growing in macrophages from recognition by immune
CD4+ cells (59). The latter finding was
considered to be independent of effects on class II expression since
these investigators reported that the constitutive expression of class
II molecules was not impaired by infection of human monocytes with
Mycobacterium bovis bacillus Calmette-Guérin. The
present finding of impaired IFN--induced surface expression of class
II molecules suggests an additional mechanism by which mycobacteria may
be able to evade immune recognition.
In summary, the data presented indicate that: 1) M.
tuberculosis markedly attenuates the cell surface expression of
class II molecules in response to IFN-, 2) activation of the
Jak-STAT signaling pathway and expression of CIITA and
DRA in response to IFN- are not inhibited in M.
tuberculosis-infected cells, 3) class II molecules are synthesized
in infected cells and transit the medial Golgi in an apparently normal
manner, but fail to colocalize with late endosomal/lysosomal
organelles, and 4) retardation of intracellular transport and
maturation of HLA class II molecules by M. tuberculosis
prevents their transit to the cell membrane. These findings provide a
basis for investigating products of M. tuberculosis that
interfere with the maturation/processing of MHC class II molecules
leading ultimately to inhibition of class II-restricted Ag
presentation.
|
Acknowledgments |
|---|
, and Genentech Inc. for providing human rIFN-. We also
thank Dr. Q. Gu for help with laser confocal microscopy,
Dr. D. Nandan for assistance with the Jak-STAT analysis, and
Dr. R. Stokes for providing bacterial cultures. |
Footnotes |
|---|
2 Z.H. was a visiting scholar from the University of Fes, Fes, Morocco.
3 Address correspondence and reprint requests to: Dr. Neil E. Reiner, the Division of Infectious Diseases, Department of Medicine, University of British Columbia, Rm 452D, 2733 Heather St., Vancouver, BC, Canada, V5Z 3J5. E-mail address:
4 Abbreviations used in this paper: CIITA, class II transactivator; Ii, invariant; MIIC, MHC class II compartment; Jak, Janus kinase; TGN, trans-Golgi network; CLIP, class II-associated Ii peptide; MFI, mean fluorescence intensity; ECL, enhanced chemiluminescence; DIG, digoxigenin; Endo H, endoglycosidase H; LAM, lipoarabinomannan; Ii chain, invariant chain.
Received for publication November 19, 1997. Accepted for publication June 25, 1998.
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C. Forestier, F. Deleuil, N. Lapaque, E. Moreno, and J.-P. Gorvel Brucella abortus Lipopolysaccharide in Murine Peritoneal Macrophages Acts as a Down-Regulator of T Cell Activation J. Immunol., November 1, 2000; 165(9): 5202 - 5210. [Abstract] [Full Text] [PDF]
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Z. Hmama, D. Nandan, L. Sly, K. L. Knutson, P. Herrera-Velit, and N. E. Reiner 1{alpha},25-Dihydroxyvitamin D3-induced Myeloid Cell Differentiation Is Regulated by a Vitamin D Receptor-Phosphatidylinositol 3-Kinase Signaling Complex J. Exp. Med., December 6, 1999; 190(11): 1583 - 1594. [Abstract] [Full Text] [PDF]
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S. Hussain, B. S. Zwilling, and W. P. Lafuse Mycobacterium avium Infection of Mouse Macrophages Inhibits IFN-{gamma} Janus Kinase-STAT Signaling and Gene Induction by Down-Regulation of the IFN-{gamma} Receptor J. Immunol., August 15, 1999; 163(4): 2041 - 2048. [Abstract] [Full Text] [PDF]
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N. V. Serbina and J. L. Flynn Early Emergence of CD8+ T Cells Primed for Production of Type 1 Cytokines in the Lungs of Mycobacterium tuberculosis-Infected Mice Infect. Immun., August 1, 1999; 67(8): 3980 - 3988. [Abstract] [Full Text] [PDF]
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 L. M. Sly, M. Lopez, W. M. Nauseef, and N. E. Reiner 1alpha ,25-Dihydroxyvitamin D3-induced Monocyte Antimycobacterial Activity Is Regulated by Phosphatidylinositol 3-Kinase and Mediated by the NADPH-dependent Phagocyte Oxidase J. Biol. Chem., September 14, 2001; 276(38): 35482 - 35493. [Abstract] [Full Text] [PDF]
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