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,
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Department of Pathology,
Division of Infectious Diseases, and
Tuberculosis Research Unit, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, 44106;
Center for Microbial Pathogenesis, University of Connecticut Health Center, Farmington, CT 06030;
¶ Department of Microbiology, Colorado State University, Fort Collins, CO 80523; and
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Maxwell Finland Laboratory for Infectious Diseases, Boston Medical Center, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibition of MHC-II expression or Ag processing does not require viable bacilli and can be achieved by exposure of macrophages to MTB lysate (4, 5, 6, 11, 12, 13). In this study, we biochemically fractionated MTB lysate to identify MTB 19-kDa lipoprotein as an inhibitor of MHC-II Ag processing. MTB 19-kDa lipoprotein inhibited macrophage expression of MHC-II molecules, Ag processing, and presentation of MHC-II-restricted Ag. Studies with cells from receptor-knockout or -deficient mice showed that the effects of MTB 19-kDa lipoprotein were dependent on Toll-like receptor (TLR) 2 and independent of TLR 4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unless otherwise specified, all experiments were performed at 37°C in 5% CO2 atmosphere and standard medium composed of DMEM (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FCS (HyClone Laboratories, Logan, UT), 50 µM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer, nonessential amino acids, and antibiotics. CBA/J (H-2k), C3H/HeJ (H-2k), C3H/HeOuJ (H-2k), and C57BL/6 (H-2b) female retired breeder mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions. H2-DM knockout mice (14) were generously provided by L. Van Kaer (Vanderbilt University, Nashville, TN) and bred at Case Western Reserve University under specific pathogen-free conditions. TLR 2 knockout mice (15) were generously provided by O. Takeuchi and S. Akira (Osaka University, Osaka, Japan) and bred onto C57BL/6 background. Unless otherwise specified, macrophages were derived from bone marrow precursors that were harvested from femur marrow and differentiated in bacterial grade dishes for 7 days in standard medium supplemented with 10% L929 cell-conditioned medium. The resultant macrophages were used during the following week. Con A-elicited peritoneal macrophages (16) were used for initial characterization of BB7 T hybridoma cells. Bovine ribonuclease A (RNase; Sigma, St. Louis, MO) or hen egg lysozyme (HEL; Sigma) peptide-MHC-II complexes were detected with T hybridomas obtained from P. Allen and E. Unanue (Washington University, St. Louis, MO): TS12, specific for RNase42–56:I-Ak; A6.A2, specific for HEL34–45:I-Ak; and 3A9, specific for HEL48–61:I-Ak. DOBW T hybridoma cells (17) were used to detect OVA323–339:I-Ab complexes. BB7, a T hybridoma cell line specific for Ag85B241–256:I-Ab complexes, was generated by fusion of BW5147 cells with lymph node cells from C57BL/6 mice immunized with MTB Ag 85B (18).
Abs and reagents
The World Health Organization mAb bank at Colorado State
University (Fort Collins, CO) provided supernatants containing IgG1
anti-19-kDa lipoprotein mAbs IT-12 (19) and IT-19
(19, 20), and the IT-19 cell line (with permission of J.
Ivanyi, King’s College, London, U.K.). B8-24-3 cells, producing murine
IgG1 anti-Kb mAb, were obtained from the
American Type Culture Collection (Manassas, VA). IT-19 and B8-24-3 Abs
were purified from culture supernatants by protein G affinity
chromatography. Dako (Carpinteria, CA) was the source of a rabbit
anti-M. bovis bacillus Calmette-Guérin (BCG) serum
that recognizes 
100 BCG Ags, many of which are common to other
mycobacteria. MTB 85B Ag was obtained through the Tuberculosis Research
Materials and Vaccine Testing Contract at Colorado State University.
LPS from Escherichia coli 055:B5 was obtained from Sigma.
The synthetic Treponema pallidum lipopeptide 47L and its
nonacylated control peptide 47 were synthesized as described
(21).
Culture and biochemical fractionation of bacteria
MTB H37Ra (American Type Culture Collection) was grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, MI) with albumin, dextrose, and catalase enrichments (Difco), harvested, and frozen at -70°C as described (22). Bacterial titer was determined by counting CFU on 7H10 medium (Difco). Unless otherwise specified, MTB lysate refers to a MTB cytosol preparation. This preparation was obtained by resuspending MTB H37Ra in deionized water containing 7.5 mM EDTA, 0.7 µg/ml leupeptin (Sigma), 0.2 mM PMSF (Sigma), 0.7 µg/ml pepstatin A (Sigma), 10 U/ml DNase (Sigma), and 25 U/ml RNase A (Boehringer Mannheim, Indianapolis, IN), passing the suspension through a French press 2–3 times, centrifuging the suspension for 1–2 h at 100,000 x g, and harvesting the supernatant. Protein concentration was estimated by an assay (Bio-Rad, Richmond, CA) with BSA standards. For Triton X-114 (TX114) extraction (23), MTB lysate was rotated at 4°C for 1 h with ice-cold 2% TX114 (Sigma) in 50 mM sodium phosphate, pH 7.4 (total protein concentration, 0.5–1 mg/ml), warmed to 37°C for 15 min, and centrifuged at 37°C for 10–15 min at 2400 x g to separate aqueous and TX114 (detergent) layers. The aqueous layer was removed, cold 50 mM phosphate buffer was added to the detergent layer, and the tube was incubated on ice until the phases merged and then warmed and centrifuged as described above. The TX114 layer was washed 3–5 times in this manner and then precipitated by overnight incubation at -20°C with 10 volumes cold acetone (Fisher Chemicals, Fair Lawn, NJ). The pellet was washed once with cold 80% acetone and then resuspended in PBS, DMSO (Sigma), or 6–8 M urea (Sigma) to produce a detergent-free "TX114 fraction" that could be added to cell cultures (with prior dialysis in some cases). For purification of lipoproteins by electroelution, the acetone precipitate was solubilized in reducing SDS-PAGE sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 700 mM 2-ME, 0.01 µg/ml bromophenol blue), boiled for 5 min, loaded onto a 12% SDS polyacrylamide preparative gel, and electroeluted using a Bio-Rad model 491 Prep Cell. Fractions were collected every 8 min for 20 h in elution buffer (25 mM Tris, 192 mM glycine, pH 8.3, flow rate 0.1 ml/min), and the fraction containing the most bromophenol blue dye marker was designated fraction 0. For immunoaffinity purification of MTB 19-kDa lipoprotein, the TX114 phase from extraction of 1.5 mg MTB lysate was passed over either IT-19 (anti-19-kDa lipoprotein) or B8-24-3 (control Ab) linked to AminoLink gel matrix (Pierce, Rockford, IL). The columns were washed with 0.1% TX114 in PBS, and bound material was eluted with 0.1% TX114 in 0.1 M glycine, pH 2.5. Each eluted fraction was neutralized, and the TX114 phase was acetone precipitated and solubilized in water.
SDS-PAGE and Western blot analysis
Samples were boiled in SDS-PAGE sample buffer under reducing conditions, electrophoresed on 12% SDS polyacrylamide gels, and stained using the Bio-Rad Silver Stain Plus kit. For Western blotting, proteins were transferred onto 0.2 µm nitrocellulose membranes (Bio-Rad), incubated for 1 h at room temperature in blocking buffer (5% Carnation nonfat dry milk (Nestle, Solon, OH) in PBS with 0.1% Tween 20), and probed with primary Ab in blocking buffer for 1 h at room temperature or overnight at 4°C. Blots were washed extensively, incubated with HRP-labeled secondary Ab (Amersham Pharmacia, Piscataway, NJ) for 1 h at room temperature, and developed with a Supersignal West Pico chemiluminescence kit (Pierce). Blots were stripped with Re-blot Western Blot Recycling kit (Chemicon, Temecula, CA) and reprobed.
Ag processing and presentation assays
Macrophages were removed from bacteriologic grade dishes with
trypsin-versene (BioWhittaker), plated in 96-well flat-bottom plates
(BD Biosciences, Franklin Lakes, NJ) at 5 x
104 macrophages/well, and incubated with 2 ng/ml
rIFN-
(Genzyme, Cambridge, MA) for 20–24 h. Cells were incubated
for an additional 22–26 h (or in some cases 48 h) with 2 ng/ml
rIFN- with or without bacterial preparations, washed, incubated with
RNase, OVA, or HEL for 1–3 h, fixed in 1% paraformaldehyde, washed
extensively, and incubated with T hybridoma cells
(105/well for 20–24 h). In assays using MTB
bacilli as Ag, macrophages were infected in medium without antibiotics
for 2 h as previously described (6), washed, fixed,
and processed as described for soluble Ag. To monitor infection,
parallel cultures of macrophages in Lab-Tek chamber slide systems
(Nalge Nunc International, Naperville, IL) were infected, fixed with
100% methanol, and stained for acid-fast bacilli by the cold Kinyoun
method (24); percentage of infection was determined from
the mean of three individual slides, counting 100 cells/slide.
Supernatants from the T hybridoma assay were assessed for IL-2 using a
CTLL-2 cell bioassay with a colorimetric determination (25, 26) using Alamar Blue (Alamar Biosciences, Sacramento, CA) and a
Bio-Rad 550 microplate reader (Bio-Rad). Unless otherwise noted, all
data points represent the mean response from triplicate wells ±
SD (when error bars are not visible they are smaller than the symbol
width).
Detection of I-Ak and I-Ab by flow cytometry
Macrophages were plated at 2 x 106
cells/100-mm petri dish, cultured for 24 h with IFN- (R&D
Systems, Minneapolis, MN), and then cultured for 24 or 48 h with
IFN- with or without MTB 19-kDa lipoprotein. Macrophages were
removed with trypsin plus 0.02% EDTA (Life Technologies, Grand Island,
NY) and placed in V-bottom 96-well plates at 2 x
105/well. Cells were incubated with 10% normal
mouse serum (Sigma) and 1% FBS (HyClone Laboratories) in PBS
and then stained with biotinylated 10.3.6-2
anti-I-Ak, biotinylated Y3P
anti-I-Ab, or biotinylated IgG2a isotype
control Ab (BD PharMingen, San Diego, CA) at 5 µg/ml. Cells were
washed, incubated with streptavidin-cychrome (1:100; PharMingen), and
resuspended in 1% paraformaldehyde. Analysis was performed with a
FACScan flow cytometer (BD Immunocytometry Systems, San Jose,
CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate inhibition of MHC-II Ag processing by MTB, murine
macrophages were infected with MTB H37Ra for 2 h, washed, cultured
(generally for 22–26 h), pulsed with soluble Ag, and fixed with
paraformaldehyde to prevent further processing (6).
Presentation of peptide-MHC-II complexes was determined with T
hybridoma cells. Infection of macrophages for 12 h or more
inhibited subsequent processing of model Ags, including RNase (Fig. 1
A) and HEL (data not shown). Inhibition of Ag processing was
observed after prolonged (>12 h) MTB infection of macrophages
(detected first after 12 h of infection and maximal after 20–24 h
of infection). Inhibition did not result from loss of cell viability,
decreased Ag endocytosis, or decreased Ag degradation (6).
Similar inhibition was produced by exposure of macrophages to MTB H37Ra
lysate (soluble fraction of MTB homogenate) (6) for
24 h (Fig. 1A), indicating that the inhibitor was a
constitutive component of MTB. Inhibitory activity was found in
different fractions of MTB bacilli, including preparations of MTB
cytosol, cell wall, cell membranes, and culture filtrate (data not
shown). Lysates from virulent MTB H37Rv, avirulent MTB H37Ra, and
M. avium had similar inhibitory activity (data not shown and
Ref. 6).
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B). The sample was incubated at
37°C, above the cloud point of TX114, to separate aqueous and TX114
(detergent) phases. The TX114 phase was precipitated with acetone and
solubilized to produce a detergent-free TX114 fraction. Both the
aqueous and TX114 fractions then were added to macrophages to determine
their ability to inhibit the processing of RNase to
RNase42–56:I-Ak as
described above. The TX114 fraction contained substantial inhibitory
activity, whereas the aqueous fraction had no inhibitory activity (Fig. 1C). The TX114 fraction also inhibited processing of
additional epitopes derived from HEL and MTB Ag85B (data not shown). A
TX114 extract of lysate buffer had no effect on Ag processing,
confirming that inhibition was not due to residual detergent.
The inhibitor was substantially enriched by TX114 extraction
because the TX114 fraction contained only 1.5% of total MTB lysate
protein but at least 10% of the starting inhibitory activity in MTB
lysate. The degree of purification was visualized by Western analysis
of whole MTB lysate, aqueous fraction, and TX114 fraction using a
polyclonal anti-BCG rabbit serum (Fig. 1D). Whole MTB
lysate and aqueous fraction contained numerous bands, whereas the TX114
fraction contained only 8–10 dominant bands, at least two of which
were enriched in this fraction relative to MTB lysate. We conclude that
a small number of species, including an MTB-derived inhibitor of Ag
processing, were enriched in the TX114 fraction.
Purification of the inhibitory activity and identification of MTB 19-kDa lipoprotein as an inhibitor of MHC-II expression and Ag processing
For further purification, the TX114 fraction was eluted
electrophoretically from a preparative 12% SDS-polyacrylamide gel, and
fractions were tested for inhibition of Ag processing (Fig. 2A). At 1/2000 dilution, peak inhibitory activity was found in
fractions 10–18 (Fig. 2B). No inhibition was produced by
elution buffer (1/20 dilution) or fractions eluted before the dye front
(fraction -15) or after fraction 110. Analytical SDS-PAGE
and silver staining showed that peak inhibitory activity correlated
with a prominent band of 20 kDa (Fig. 2C, peak level in
fractions 12 and 16) that reacted with anti-BCG polyclonal serum
(Fig. 2D, peak level in fractions 12 and 16).
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E) and IT-19 (data not shown). Much lower
levels of MTB 19-kDa lipoprotein were detected in some other fractions,
consistent with the distribution of the 20-kDa band seen in
silver-stained gel (Fig. 2C) and Western blot with
anti-BCG polyclonal serum (Fig. 2D). Peak levels of both
MTB 19-kDa lipoprotein and inhibitory activity coincided in fractions
10–18. Based on total protein determination, MTB 19-kDa lipoprotein
from fraction 14 produced inhibition at a final concentration of 2.4 nM
(Fig. 2B).
Earlier studies indicated that exposure to MTB or MTB lysate for 24–48
h inhibited expression of MHC-II molecules by macrophages
(6). To determine the effect of purified MTB 19-kDa
lipoprotein on MHC-II expression, IFN--stimulated macrophages were
treated with or without 7.5 nM electroeluted MTB 19-kDa lipoprotein for
24–48 h, and I-Ak expression was assessed by
flow cytometry (Fig. 3A). After 24 h, the mean fluorescence value (MFV) of
I-Ak staining was 747 without MTB 19-kDa
lipoprotein vs 408 with MTB 19-kDa lipoprotein. After 48 h, MFV
was 835 without MTB 19-kDa lipoprotein vs 154 with MTB 19-kDa
lipoprotein. Thus, MTB 19-kDa lipoprotein produced substantial
inhibition of MHC-II expression by macrophages.
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B). Therefore, both electroelution and
immunoaffinity purification identified the MTB 19-kDa lipoprotein as an
inhibitor of MHC-II expression and Ag processing by macrophages. MTB 19-kDa lipoprotein is a potent inhibitor of processing of multiple antigenic epitopes
The ability of MTB 19-kDa lipoprotein to produce generalized
inhibition of processing of multiple antigenic epitopes was determined
by incubating macrophages with or without 19-kDa lipoprotein for
24 h before the addition of soluble Ags. MTB 19-kDa lipoprotein
inhibited processing of all Ag epitopes studied, including an epitope
from RNase (Fig. 4A), two different epitopes from HEL (Fig. 4B), and
an epitope from OVA. The potency of the inhibitor was assessed using
different dilutions of MTB 19-kDa lipoprotein. Processing was inhibited
at a 1/2000 dilution of a fraction containing 90 µg/ml protein,
indicating that 2.4 nM MTB 19-kDa lipoprotein was sufficient to
inhibit Ag processing.
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To test the impact of MTB 19-kDa lipoprotein on processing of an
Ag expressed by intact MTB bacilli, we generated MTB-specific murine T
hybridomas after immunization of C57BL/6 mice with MTB Ag 85B
(18, 28). Of the resulting Ag 85B-reactive hybridomas, BB7
was characterized and showed specific, dose-dependent secretion of IL-2
in response to Ag 85B presented by macrophages (Fig. 5A), responding to as little as 10 ng/ml of purified Ag. To
map the epitope recognized by BB7, we used overlapping 16-mer synthetic
peptides that spanned 60% of the MTB Ag85B sequence and included
previously reported T cell epitopes in M. bovis BCG
(29). BB7 responded to two overlapping peptides, Ag
85B236–251 and Ag
85B241–256 (Fig. 5A, inset),
suggesting that the core epitope contained residues 241–251 of MTB Ag
85B. Because C57BL/6 mice do not express I-E,
I-Ab was the only potential MHC-II restriction
element, and BB7 recognition of Ag 85B241–256
was blocked by two anti-I-Ab Abs, Y-3P and
AF6-120.1.2, confirming that this peptide was presented by
I-Ab (data not shown). BB7 also responded to
macrophages that were infected with viable MTB bacilli for 1–2 h (Fig. 5B), indicating that the BB7 epitope was processed and
presented in the course of infection with MTB. Processing of this
epitope from MTB bacilli was inhibited by chloroquine, ammonium
chloride, or the use of macrophages from H2-DM knockout mice (data not
shown). These studies established that BB7 recognizes an MTB Ag 85B
epitope processed from both soluble Ag and intact bacilli.
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B) for 24 h. Macrophages were then infected with MTB
for 2 h, fixed, and assessed for presentation of MTB Ag 85B to BB7
T hybridoma cells. TX114 fraction and purified 19-kDa lipoprotein both
inhibited processing of this epitope from intact bacilli. The inhibitor
did not alter the number of MTB bacilli internalized per macrophage
(data not shown). TX114 fraction also inhibited processing of MTB
bacilli by a human macrophage cell line (THP-1) for presentation of Ag
85B to HLA-DR1-restricted T cells, confirming this effect for human
macrophages (data not shown). Thus, 19-kDa lipoprotein inhibits
processing of Ag expressed by intact MTB bacilli in infected cells and
may inhibit processing of MTB Ags during chronic infection. MTB 19-kDa lipoprotein and other microbial lipopeptides share a common ability to inhibit Ag processing via a TLR 4-independent mechanism
Because MTB 19-kDa lipoprotein and other bacterial lipoproteins
may represent a pathogen-associated molecular pattern (PAMP)
(30) that is associated with many bacterial species, we
investigated the ability of synthetic analogs of lipopeptides from a
different bacterium, T. pallidum, to inhibit Ag processing.
Synthetic lipopeptide Tp47L, a hexapeptide derived from T.
pallidum 47-kDa lipoprotein, inhibited Ag processing (Fig. 6, A and B) at concentrations similar to those that
induce other responses (e.g., NF-
B-dependent gene transcription and
cytokine secretion) (21). Nonacylated control peptide Tp47
had no inhibitory activity (Fig. 6, A and B),
demonstrating that acylation is required for this activity. Tp17L, a
similar T. pallidum lipopeptide with the hexapeptide
sequence CGSSHH, gave similar results but was less potent than Tp47L
(data not shown).
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). Thus, inhibition of Ag processing by MTB
19-kDa lipoprotein, MTB lysate, and Tp47L was not due to LPS
contamination and was independent of TLR 4. Expression of TLR 2 is required for inhibition of MHC-II expression and Ag processing by MTB 19-kDa lipoprotein
Because lipoproteins require TLR 2 to induce NF-B-dependent
gene transcription and cytokine secretion (36, 37), we
investigated whether lipoprotein-induced inhibition of MHC-II
expression and Ag processing also depends on signaling via TLR 2.
Macrophages were prepared from TLR 2 knockout (TLR
2-/-) mice (on C57BL/6 background), their
heterozygous (TLR 2+/-) littermates, and C57BL/6
mice. Macrophages were incubated for 48 h with or without
electroelution-purified MTB 19-kDa lipoprotein, and
I-Ab expression was evaluated by flow cytometry.
MTB 19-kDa lipoprotein decreased expression of MHC-II by C57BL/6 and
TLR 2+/- mice but not TLR
2-/- mice (Fig. 7A). We also assessed the role of TLR 2 in the effects of
lipoprotein on Ag processing with a T cell readout (in a
H-2b system with DOBW T hybridoma cells due to
the background of the TLR 2 knockout mice). C57BL/6, TLR
2+/-, and TLR 2-/-
macrophages were incubated for 24 h with or without MTB 19-kDa
lipoprotein, exposed to OVA for 1 h, and fixed. Presentation of
OVA was assessed with DOBW T hybridoma cells. MTB 19-kDa lipoprotein
inhibited processing and presentation of OVA by C57BL/6 and TLR
2+/- macrophages but not TLR
2-/- macrophages (Fig. 7B). MTB
19-kDa lipoprotein also failed to inhibit processing of MTB bacilli by
TLR 2-/- macrophages for presentation to BB7 T
hybridoma cells (processing tested as in Fig. 5B, data not
shown). Thus, TLR 2 was required for MTB 19-kDa lipoprotein to inhibit
expression of MHC-II molecules and their use in the processing of Ag
for presentation to T cells.
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MTB has been shown to contain agonists for both TLR 2 and TLR 4
(40, 41). We also observed that a TLR 4 agonist, e.g.,
LPS, can inhibit MHC-II Ag processing (Fig. 6 and Ref. 16)
similar to a TLR 2 agonist, e.g., MTB 19-kDa lipoprotein. Thus, it was
important to determine the relative importance of TLR 4 and TLR 2 in
MTB-mediated inhibition of MHC-II Ag processing. TLR 4-defective
C3H/HeJ and wild-type C3H/HeOuJ macrophages were infected with MTB
(multiplicity of infection (MOI) of 10:1) for 24 h and then tested
for the ability to process exogenous model Ag (as in Fig. 1A). Infection with MTB produced similar inhibition of
MHC-II Ag processing by macrophages from both mouse strains (Fig. 8A), indicating that TLR 4 is not essential for inhibition of
MHC-II Ag processing by MTB. In contrast, macrophages from TLR
2-/- mice were substantially more resistant to
MTB inhibition of MHC-II Ag processing than macrophages from C57BL/6
mice (Fig. 8B), although a slight TLR 2-independent decrease
in MHC-II Ag processing was observed with TLR
2-/- macrophages (mediated through unknown
receptors potentially including other TLRs or non-TLRs). In addition,
MHC-II Ag processing by TLR 2-/- macrophages
was not inhibited by a lysate of MTB that inhibited MHC-II Ag
processing by C57BL/6 macrophages (Fig. 8C). This
observation confirms the dominant effect of TLR 2 in the inhibition and
indicates that TLR 4 does not contribute substantially to this effect
(TLR 4 agonists may still be present in MTB and the lysate preparation
without impacting on these mechanisms). Macrophages from TLR
2-/- and C57BL/6 macrophages responded
similarly to LPS (Fig. 8D), indicating that TLR
2-/- macrophages are capable of responding to a
TLR 4 agonist. In summary, the experiments described in this section
compare the effects of purified MTB 19-kDa lipoprotein to the effects
of infection with viable MTB bacilli, confirming that the effects and
TLR signaling mechanisms of the two agents are similar. Taken
together, these data also establish that MTB inhibits MHC-II Ag
processing by TLR 2-dependent signaling that does not require TLR 4,
although these observations do not exclude roles for TLR 4 or other
TLRs in different responses to MTB.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). One interesting question is whether MTB 19-kDa lipoprotein, shown here to inhibit MHC-II expression and MHC-II Ag processing, has more general inhibitory effects on Ag processing and presentation, e.g., by MHC-I molecules. Other studies from our laboratory (N. S. Potter, R. K. Pai, W. H. Boom, and C. V. Harding, manuscript in preparation) show that the inhibitory effect of MTB 19-kDa lipoprotein is specific to MHC-II. MTB 19-kDa lipoprotein did not inhibit MHC-I expression (by flow cytometry) or function in alternate MHC-I Ag processing of exogenous particulate Ags, e.g., E. coli HB101.Crl-OVA (expressing a fusion protein containing OVA sequence and the SIINFEKL Kb epitope) for presentation to T cells. Because alternate MHC-I Ag processing in this system (45, 46) depends on MHC-I molecules, phagocytosis, phagolysosomal function, and lysosomal proteases, this finding implies that MTB 19-kDa lipoprotein does not generally inhibit Ag uptake, proteolytic processing, or the expression of MHC-I molecules. In addition, we have previously demonstrated that Ag uptake and catabolism are not affected by MTB (6). These observations suggest that the inhibitory effects of MTB and 19-kDa lipoprotein are largely attributable to decreased MHC-II expression.
Lipoproteins belong to a larger group of microbial molecules
categorized as PAMPs that contain molecular motifs characteristic of
microbes and absent from mammalian hosts (30). Recognition
of PAMPs, such as LPS and lipoproteins, can initiate or enhance
microbicidal mechanisms (including production of toxic intermediates
such as NO), cytokine secretion, and other functions that enhance both
innate and acquired immunity. PAMPs trigger these responses by binding
to pattern recognition receptors, e.g., TLR 2 and TLR 4 (30, 47). Inhibition of macrophage MHC-II expression and Ag
processing appears to be another consequence of the recognition of
multiple diverse PAMPs, because such inhibition is produced by LPS and
bacterial CpG DNA as well as lipoproteins (16). Like other
immunomodulatory activities of LPS, the inhibition of MHC-II expression
and Ag processing by LPS is abrogated in TLR 4 mutant macrophages (Fig. 6F). However, the lipoprotein-induced inhibition of Ag
processing is independent of TLR 4 (Fig. 6, B and
D). Lipoproteins signal via TLR 2 to induce
NF-B-dependent gene transcription and cytokine secretion (36, 37). Our studies with macrophages from TLR 2 knockout mice show
that lipoprotein-induced inhibition of MHC-II Ag processing also
involves signaling via TLR 2. In addition, macrophages that lack TLR 2
were largely resistant to inhibition of MHC-II Ag processing following
infection with MTB bacilli (Fig. 8). These studies indicate that
MTB-mediated inhibition of MHC-II Ag processing is dependent on TLR 2
and does not require TLR 4. Because inhibitory activity was largely
limited to fractions containing MTB 19-kDa lipoprotein (data not shown
and Figs. 1C and 2), we conclude that MTB-mediated
inhibition of MHC-II Ag processing was mediated primarily by MTB 19-kDa
lipoprotein signaling through TLR 2 (although other PAMPs and receptors
may be involved in different responses to MTB).
Although inhibition of Ag processing after chronic microbial stimulation may benefit the host under some conditions, MTB may exploit this mechanism to evade T cell effector responses and maintain chronic infection inside host cells. MTB peptide-MHC-II complexes are presented by macrophages shortly after infection, leading to CD4+ T cell responses and control of infection. However, small numbers of bacilli survive and persist inside macrophages. Inhibition of MHC-II Ag processing by MTB 19-kDa lipoprotein may allow MTB residing in chronically infected macrophages to avoid detection by effector CD4+ T cells, promoting persistence of MTB infection. Recent evidence suggests that TLR 2 can sample material present in phagosomal compartments (35, 48). Thus, phagosomal MTB 19-kDa lipoprotein may chronically activate TLR 2 in cells that are infected with MTB, leading to decreased MHC-II Ag processing. Inhibition of MHC-II Ag processing by 19-kDa lipoprotein may also explain why vaccination with recombinant M. vaccae or M. smegmatis expressing 19-kDa lipoprotein resulted in less protection against MTB than vaccination with nonrecombinant strains of these bacteria (49, 50). We speculate that the efficacy of live mycobacterial vaccines, e.g., BCG or attenuated MTB, may be improved by the development of vaccine vectors that lack molecules that inhibit Ag processing.
Inhibition of macrophage MHC-II Ag processing by PAMPs, including LPS
(16), bacterial CpG DNA (16), and
lipoproteins (
Figs. 3–6), seems discordant with the ability of PAMPs
to enhance microbicidal functions and stimulate innate and acquired
immunity, but the delayed kinetics of inhibition suggest a resolution
to this paradox. Upon immediate exposure of macrophages to microbes,
microbial Ags will be processed before inhibition. Subsequent
inhibition of Ag processing may be accompanied by stabilization of
existing peptide-MHC-II complexes, enhancing the half-life of a cohort
of peptide-MHC-II complexes containing microbial peptides. This would
freeze the selection of Ags presented and prolong the presentation of a
frame of microbial Ags, a model we term the "freeze-frame
hypothesis". The ability of PAMPs to induce dendritic cell maturation
(51, 52, 53) is consistent with this hypothesis. In addition,
inhibition of Ag processing may limit unnecessary enhancement of Ag
presentation beyond the initial period of microbial exposure,
contributing to down-regulation of immune responses when
infection has been controlled. Thus, our results are consistent with a
model wherein PAMPs, e.g., MTB 19-kDa lipoprotein, initially activate
microbicidal mechanisms and innate immunity (also impacting on specific
immunity) but later cause inhibition of certain immune processes, e.g.,
MHC-II Ag processing. In the context of tuberculosis, we propose that
inhibition of MHC-II Ag processing by MTB 19-kDa lipoprotein decreases
presentation of MTB Ag to CD4 T cells, allowing intracellular bacilli
to evade immune surveillance and promoting chronic infection.
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Acknowledgments |
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Footnotes |
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2 E.H.N. and R.K.P. shared first authorship (these authors contributed equally to this work).
3 W.H.B. and C.V.H. shared senior authorship.
4 Address correspondence and reprint requests to Dr. Clifford V. Harding, Department of Pathology, BRB 925, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4943. E-mail address: cvh3{at}po.cwru.edu; or Dr. W. Henry Boom, Division of Infectious Diseases, BRB 1031, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4984. E-mail address: whb{at}po.cwru.edu
5 Abbreviations used in this paper: MHC-II, class II MHC; MTB, Mycobacterium tuberculosis; TLR, Toll-like receptor; HEL, hen egg lysozyme; RNase, bovine ribonuclease A; TX114, Triton X-114; PAMP, pathogen-associated molecular pattern; MFV, mean fluorescence value; BCG, bacillus Calmette-Guérin; MOI, multiplicity of infection.
Received for publication February 8, 2001. Accepted for publication May 3, 2001.
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References |
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Janus kinase-STAT signaling and gene induction by down-regulation of the IFN- receptor. J. Immunol. 163:2041. transcriptional responses without inhibiting activation of STAT1. J. Immunol. 163:3898.
2-microglobulin in the processing of bacterial or particulate antigens via an alternate class I MHC processing pathway. J. Immunol. 156:4182.[Abstract]
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L.-Y. Gao, R. Groger, J. S. Cox, S. M. Beverley, E. H. Lawson, and E. J. Brown Transposon Mutagenesis of Mycobacterium marinum Identifies a Locus Linking Pigmentation and Intracellular Survival Infect. Immun., February 1, 2003; 71(2): 922 - 929. [Abstract] [Full Text] [PDF]
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D. van der Kleij, E. Latz, J. F. H. M. Brouwers, Y. C. M. Kruize, M. Schmitz, E. A. Kurt-Jones, T. Espevik, E. C. de Jong, M. L. Kapsenberg, D. T. Golenbock, et al. A Novel Host-Parasite Lipid Cross-talk. SCHISTOSOMAL LYSO-PHOSPHATIDYLSERINE ACTIVATES TOLL-LIKE RECEPTOR 2 AND AFFECTS IMMUNE POLARIZATION J. Biol. Chem., December 6, 2002; 277(50): 48122 - 48129. [Abstract] [Full Text] [PDF]
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L. E. DesJardin, T. M. Kaufman, B. Potts, B. Kutzbach, H. Yi, and L. S. Schlesinger Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, Fc{gamma}RII and the mannose receptor Microbiology, October 1, 2002; 148(10): 3161 - 3171. [Abstract] [Full Text] [PDF]
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K. Hashimoto, Y. Maeda, H. Kimura, K. Suzuki, A. Masuda, M. Matsuoka, and M. Makino Mycobacterium leprae Infection in Monocyte-Derived Dendritic Cells and Its Influence on Antigen-Presenting Function Infect. Immun., September 1, 2002; 70(9): 5167 - 5176. [Abstract] [Full Text] [PDF]
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 D. G. Russell, H. C. Mwandumba, and E. E. Rhoades Mycobacterium and the coat of many lipids J. Cell Biol., August 5, 2002; 158(3): 421 - 426. [Abstract] [Full Text] [PDF]
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R. K. Pai, D. Askew, W. H. Boom, and C. V. Harding Regulation of Class II MHC Expression in APCs: Roles of Types I, III, and IV Class II Transactivator J. Immunol., August 1, 2002; 169(3): 1326 - 1333. [Abstract] [Full Text] [PDF]
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J. Caamano and C. A. Hunter NF-{kappa}B Family of Transcription Factors: Central Regulators of Innate and Adaptive Immune Functions Clin. Microbiol. Rev., July 1, 2002; 15(3): 414 - 429. [Abstract] [Full Text] [PDF]
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R. van Crevel, T. H. M. Ottenhoff, and J. W. M. van der Meer Innate Immunity to Mycobacterium tuberculosis Clin. Microbiol. Rev., April 1, 2002; 15(2): 294 - 309. [Abstract] [Full Text] [PDF]
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L. Grode, M. Kursar, J. Fensterle, S. H. E. Kaufmann, and J. Hess Cell-Mediated Immunity Induced by Recombinant Mycobacterium bovis Bacille Calmette-Guerin Strains Against an Intracellular Bacterial Pathogen: Importance of Antigen Secretion or Membrane-Targeted Antigen Display as Lipoprotein for Vaccine Efficacy J. Immunol., February 15, 2002; 168(4): 1869 - 1876. [Abstract] [Full Text] [PDF]
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L. Ramachandra, E. Noss, W. H. Boom, and C. V. Harding Processing of Mycobacterium tuberculosis Antigen 85B Involves Intraphagosomal Formation of Peptide-Major Histocompatibility Complex II Complexes and Is Inhibited by Live Bacilli that Decrease Phagosome Maturation J. Exp. Med., November 12, 2001; 194(10): 1421 - 1432. [Abstract] [Full Text] [PDF]
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C. Neufert, R. K. Pai, E. H. Noss, M. Berger, W. H. Boom, and C. V. Harding Mycobacterium tuberculosis 19-kDa Lipoprotein Promotes Neutrophil Activation J. Immunol., August 1, 2001; 167(3): 1542 - 1549. [Abstract] [Full Text] [PDF]
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