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Infection and Immunity, December 2001, p. 7461-7470, Vol. 69, No. 12
Department of Neuroscience, University of
Rome "Tor Vergata,"1 Institute of
Neurobiology and Molecular Medicine, National Council of Research
(CNR),2 Laboratory of Bacteriology and
Medical Mycology, "Istituto Superiore di
Sanità,"4 and
"Istituto Dermopatico dell'Immacolata"
(IDI-IRCCS),6 Rome, Italy; Division of
Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital
and Harvard Medical School, Boston, Massachusetts
021153; and Department of Microbiology
and Immunology, Albert Einstein College of Medicine, Bronx, NY
104615
Received 30 May 2001/Returned for modification 13 July
2001/Accepted 11 September 2001
Nonpeptide antigens (including glycolipids of microbial origin) can
be presented to T cells by CD1 molecules expressed on monocyte-derived
dendritic cells. These HLA unrestricted responses appear to play a role
in host immunity against Mycobacterium tuberculosis and
other pathogenic bacteria. It is known that vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG)
has limited efficacy in many clinical settings, although the reasons
for its inadequacy remain unclear. Here we have investigated the
influence of BCG on the induction of CD1b on human monocytes by
granulocyte-macrophage colony-stimulating factor (GM-CSF), which is
believed to be the principal inducer of this antigen-presenting
molecule. Although BCG alone led to a slight induction of CD1b
expression, this agent reduced markedly the ability of GM-CSF to induce
high levels of CD1b that were typically observed in uninfected cells.
Inhibition of CD1b expression in BCG-infected monocytes was apparent at
both the mRNA transcript and CD1b protein levels. Down-regulation of CD1b expression by BCG was mediated, at least in part, by
one or more soluble factors and could not be reversed with high
concentrations of GM-CSF or a variety of other cytokines. The present
results suggest that BCG could diminish the efficiency of
CD1-restricted T-cell responses against nonpeptide mycobacterial
antigens by reducing CD1 expression on antigen-presenting cells. These
findings have potential implications for understanding the nature of
the immune response elicited by BCG in humans and suggest potential strategies that could be important for the development of better vaccines for the prevention of tuberculosis.
Data from the world literature show
that morbidity and mortality from mycobacterial infections are
continuously increasing (3, 9, 17). This appears to be due
not only to a higher transmission rate of the disease, especially in
immunocompromised human immunodeficiency virus (HIV)-infected patients
(2, 5), but also to the emergence of multidrug-resistant
strains of Mycobacterium tuberculosis (17, 21,
25). Therefore, effective early vaccination of individuals at
high risk for developing active tuberculosis has been targeted as an
important approach for tuberculosis control. Vaccination against
M. tuberculosis has been attempted on a large scale using
Mycobacterium bovis bacillus Calmette-Guérin (BCG), a
live attenuated strain. However, the results of clinical trials that
enrolled an extraordinary number of cases immunized with BCG were not
consistently appealing (24, 29). A recent meta-analysis of
the literature showed that the vaccine significantly reduces the risk
of tuberculosis by an average of only 50% (4). The reasons why BCG does not provide optimal protection are not clear, since the organism is known to share a number of major
histocompatibility complex (MHC)-restricted antigens with virulent
M. tuberculosis and also activates At present, the most important cell-mediated mechanism involved in
protective sensitization against mycobacteria appears to rely on the
classical HLA-restricted responses against bacterial peptides
(35) mediated mainly by gamma interferon
(IFN- A fraction of the T cells that respond to mycobacterial lipids and
glycolipids presented by CD1 molecules comes from the
CD4 There is a general consensus that the known CD1b presented antigens,
such as ManLAM(s) and mycolic acids, isolated from BCG (43) and M. tuberculosis (10, 15)
share the same basic structures. It follows that sensitization with BCG
is expected to generate CD1-restricted T cells that could be cytotoxic
for M. tuberculosis and possibly for other pathogenic
mycobacteria and for dendritic cells and macrophages containing viable
bacilli. However, Stenger et al. found that infection with virulent
M. tuberculosis severely depresses the expression of CD1b in
GM-CSF-activated monocytes (36). These observations
prompted us to explore whether BCG itself could play a negative role in
the CD1-dependent immune system similar to that described for M. tuberculosis. In particular, we focused on the question of whether
the process of CD1b induction by GM-CSF could be inhibited by BCG
infection rather than the expression of CD1b protein already present in
adherent mononuclear cells preactivated by the cytokine. Our results
indicated that BCG affects adversely the GM-CSF-dependent induction of
all group I CD1 molecules. In contrast, we found that the microorganism does not seem to produce similar effects in preactivated,
CD1b+ adherent cells, in which the vaccine
produced only a modest downregulation of this molecule. These findings
raise the question of whether BCG vaccination could produce
self-limiting cellular and/or humoral signals with respect to
CD1-dependent immunity, which could be a potential factor contributing
to its limited efficacy in tuberculosis prevention.
Reagents and antibodies.
All chemical reagents, if not
otherwise specified, were obtained from Sigma Chemical Co. (St. Louis,
Mo.). Recombinant human GM-CSF and IL-4 were obtained from Sandoz
(Milan, Italy) and Genzyme (Cambridge, Mass.), respectively.
Recombinant human IL-10 was obtained from R&D Systems (Minneapolis,
Minn.).
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.12.7461-7470.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Influence of Mycobacterium bovis
Bacillus Calmette Guérin on In Vitro Induction of CD1 Molecules
in Human Adherent Mononuclear Cells
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
T cells that may
facilitate the responses of CD4+ and
CD8+ responder T cells that are important in
maintaining immunity to M. tuberculosis (14).
)-producing CD4 T cells (6, 8). However, in recent
years growing interest has been elicited in an arm of the immune system
involving T-cell reactivity directed against lipid or glycolipid
antigens presented by CD1 molecules (27). The CD1
molecules are expressed most prominently on antigen-presenting cells of
the myeloid lineage, including dendritic cells derived from
circulating monocytes. Adherent blood mononuclear cells can be
activated by granulocyte-macrophage colony-stimulating factor
(GM-CSF) to express group I CD1 (i.e., CD1a, CD1b, CD1c) proteins
(16, 27, 38). These molecules are the products of the
CD1A, -B, and -C genes and are known
to be involved in the presentation of nonpeptide microbial antigens (27, 33). Among the CD1-restricted antigens, of primary
interest are lipoarabinomannan (ManLAM), phosphatidylinositol
mannosides, mycolic acids, and glycosylated mycolates, all of which are
abundant constituents of the cell wall of the mycobacterial species
(33).
CD8 phenotypic
subset of CD3+ 
T-cell receptor (TCR) T
cells. These cells, sometimes referred to as double negative T
lymphocytes (26), proliferate and generate cytotoxic
clones following interaction with mycobacterial glycolipids, presented
by CD1+ monocyte-derived dendritic cells that
develop from blood monocytes preactivated with GM-CSF, alone or in
combination with interleukin-4 (IL-4) (26). More recently,
CD8+ TCR T-cell clones with similar
properties have also been demonstrated (37), and a recent
report has described CD4+ TCR T cells that
react with mycobacterial antigens presented by CD1 molecules
(34). Thus, responder cells that potentially play a role
in CD1-restricted responses to nonpeptide antigens have been
demonstrated among all of the major phenotypic subsets of T cells, as
presently defined (27)
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
, K5-1B8 clone; Ancell, Bayport, Minn.).
FITC-conjugated MAb recognizing CD14 (IgG2a, UCHM1 clone) was obtained
from Ancell. FITC-conjugated mouse IgG1 or IgG2a (Becton Dickinson,
Oxnard, Calif.) was used as a negative control. Purified anti-HLA-ABC
MAb (IgG2a, W6/32 hybridoma) was obtained from Dako (Dakopatts,
Copenhagen, Denmark), and FITC-conjugated anti-HLA-DR MAb (IgG2a, L243
hybridoma) was obtained from Becton Dickinson.
Cell lines and culture conditions.
The CD1b-transfected
C1R.b6 lymphoblastoid cell line was generated by stably transfecting
human lymphoblastoid C1R cells with pSR-NEO vector containing CD1b
cDNA, as previously described (1). The control,
CD1b-negative C1R/MOCK subline was obtained following transfection of
C1R cells with a mock plasmid. Lymphoblastoid cells were grown in
suspension in RPMI 1640 medium (GIBCO, Paisley, Scotland) supplemented
with 10% fetal calf serum (GIBCO), 10 mM HEPES (Flow Laboratories,
McLean, Va.), and 2 mM L-glutamine (Flow) and subcultured
two or three times weekly.
Preparation and in vitro culture of human AMNC. Peripheral blood mononuclear cells were separated from heparinized whole blood obtained from healthy donors on a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient, washed twice in RPMI 1640 medium, and resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mM HEPES, 2 mM L-glutamine, 0.8 mM nonessential amino acids (GIBCO), 0.4 mM essential amino acids, and 50 µM 2-mercaptoethanol (hereafter referred to as complete medium [CM]). Adherent mononuclear cells (AMNC) were isolated by plastic adherence at 37°C for 2 h, followed by extensive washing with RPMI 1640 at 37°C. In the majority of cases, more than 70% of AMNC were positive for CD14 on day 0, whereas marked down-regulation of this antigen occurred progressively in the following days of culture (data not shown). On the contrary, CD1b was essentially absent in all AMNC preparations on day 0. To activate AMNC, when not otherwise stated, GM-CSF was used at a concentration of 200 IU/ml and left in the medium for the entire period of cell culture.
Cultivation of BCG and infection of AMNC.
BCG (ATCC 27291)
was grown in Middlebrook 7H10 agar (Difco Laboratories, Detroit, Mich.)
at 37°C under a humidified 5% CO2 atmosphere
for 2 weeks. Bacterial suspensions were prepared by dispersing colonies
with glass beads in RPMI 1640. The tubes were vortexed for 1 min and
allowed to stand for 30 min to allow larger particles to settle. The
upper supernatant was stored at 
40°C until use. CFU were counted by
the standard viable count technique on Middlebrook 7H10 agar plates.
Flow cytometry analysis. Cultured cells were washed twice in phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin and 0.02% sodium azide (PBS-A). One million cells were resuspended in 50 µl of the same medium containing the appropriate mouse immunoglobulin. Samples were incubated at 4°C for 30 min and then washed in PBS-A. Pellets were resuspended in 1% formaldehyde (Merck KGaA, Darmstadt, Germany) to inactivate BCG organisms and left at 4°C for 30 min. The labeled samples were then washed with PBS-A, resuspended in the same medium, and analyzed with a FACScan flow cytometer (Becton Dickinson). Data were collected on 104 viable cells as determined by forward and side angle light scatter. Data analysis was performed by using Lysis II software (Becton Dickinson).
Northern blot analysis.
Total cellular RNA was extracted
using the TriPure isolation reagent (Boehringer Mannheim, Indianapolis,
Ind.). RNAs were fractionated by electrophoresis on a
formaldehyde-containing 1.2% agarose gel, transferred to a nylon
membrane (Gene Screen Plus; NEN Research Products, Boston, Mass.), and
hybridized at 68°C for 1 h with a
32P-labeled probe using the QuickHyb
hybridization solution (Stratagene, Cambridge, United Kingdom).
CD1b-specific transcripts were detected as previously described using
as a probe a 266-bp cDNA fragment corresponding to the second exon of
the CD1B gene, which encodes the first extracellular domain
of the mature CD1b protein, designated 1 (11, 19, 26).
The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe, a 0.9-kb
EcoRI fragment of the human GAPDH gene, was a generous gift
from R. Dalla Favera (Department of Pathology, Columbia University, New
York, N.Y.). Washing of the blots was performed according to the
manufacturer's instructions. The blotted membrane was exposed to X-ray
film at 80°C (Kodak, Rochester, N.Y.).
RT-PCR analysis. cDNA was synthesized by incubating 1.5 µg of total RNA with 0.5 U of avian myeloblastosis virus reverse transcriptase (RT) and 0.2 µg of oligo(dT) primer at 42°C for 1 h using the cDNA Cycle kit from Invitrogen (Carlsbad, Calif.). The PCR was performed by adding cDNA samples to a solution (total volume, 50 µl) containing 1× PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin) and 200 µM (each) dCTP, dATP, dGTP, and dTTP. Ten picomoles each of two synthetic oligonucleotide primers (Biogen, Rome, Italy) was added to the mixture, and amplification was performed using Taq DNA polymerase (1.25 U) (Boehringer Mannheim) for 28 cycles in a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, Conn.). Each cycle consisted of denaturation at 95°C for 45 s, annealing at 57°C (CD1b) or 53°C (GAPDH) for 1.30 min, and extension at 72°C for 2 min.
The oligonucleotide primer pairs used for CD1b amplification were 5'-CCTTCCAGGGGCCGACCTCCTTT-3' and 5'-CATGGGATATTCTGATATGACCG-3'. These primers allow amplification of a 940-bp DNA fragment, spanning from exon 2 to the exon 5-6 boundary of the CD1b sequence (19). The primers used for GAPDH (5'-TGGTATCGTGGAAGGACTCATGAC-3' and 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3') amplify a 190-bp product.Nucleotide sequence analysis. For nucleotide sequence analysis, the amplified products derived from RT-PCR were subjected to electrophoresis and purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). The nucleotide sequence of the purified PCR products was determined using the dye terminator cycle sequencing kit with Amplitaq DNA polymerase (Perkin Elmer, Foster City, Calif.) and run on an Applied Biosystem PRISM 377 DNA sequencer.
Western blot analysis. Cells were washed extensively with PBS. The cell pellet was suspended in 5 volumes of lysis buffer (25 mM HEPES [pH 7.5], 2.5 mM MgCl2, 2.5 mM EGTA, 50 mM 2-mercaptoethanol, 200 µg of leupeptin per ml, 5 µg of aprotinin per ml, 1 mM phenylmethylsulfonyl fluoride, 400 µg of soybean trypsin inhibitor per ml), sonicated at 4°C for 5 s, and centrifuged at 100,000 × g at 4°C for 1 h. The supernatant was collected and designated the cytosol fraction. The pellet was resuspended in lysis buffer containing 1% Triton X-100, sonicated for 5 s, and centrifuged at 15,000 × g at 4°C in a microcentrifuge for 10 min. The supernatant was collected and defined as the membrane fraction.
Membrane and cytosol fractions were separated in sodium dodecyl sulfate-12% (wt/vol) polyacrylamide gels as described by Laemmli (18) and transferred to nitrocellulose filters, according to the method described by Towbin et al. (42), using a Bio-Rad (Hercules, Calif.) electrophoretic mini blotting apparatus. Filters were incubated with 3% (wt/vol) nonfat dry milk (Bio-Rad) in Tris-HCl (20 mM; pH 7.5) plus 0.9% NaCl (TBS) for 1 h and then with rabbit anti-CD1b serum diluted 1:2,000 in TBS containing 0.05% Tween 20 (TBST) for 30 min. Thereafter, the membranes were washed twice with TBST and incubated with alkaline phosphatase-coupled secondary antibody diluted 1:7,500 in TBST for 1 h. The bands were visualized using the Protoblot (Promega Biotec, Madison, Wis.) reagents according to the procedures provided by the manufacturer.| |
RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Influence of BCG on the induction of group I CD1 molecules by GM-CSF on AMNC. A series of preliminary experiments was conducted to obtain quantitative information on the effect of different levels of BCG exposure of AMNC on GM-CSF-induced CD1b expression. AMNC were infected with BCG at MOIs ranging from 0.25 to 10 and incubated with GM-CSF alone. On day 3, flow cytometry analysis of CD1b expression showed that BCG at all concentrations used antagonized the effect of GM-CSF on CD1b induction. When BCG was used at a high MOI (i.e., greater than 5), the expression of CD1b declined markedly and reached almost undetectable values at an MOI of 10 (data not shown). Although a certain degree of donor-dependent variability was apparent, induction of CD1b (in terms of percentage of CD1b-positive cells) by GM-CSF was found to be consistently inhibited (by 35 to 75%) by infection of AMNC at an MOI of 1 in more than 10 different AMNC samples studied.
Time-course studies were performed by exposing AMNC to graded amounts of BCG (from 0.25 to 1 MOI), followed by GM-CSF. Flow cytometry analysis of CD1b antigen was performed on days 3, 5, and 7 of culture. The results of one of two comparable experiments (Fig. 1) show a reasonably good concentration-inhibition relationship between BCG amount and CD1b expression on day 3, as confirmed by the regression line analysis illustrated in the legend to Fig. 1. A similar inhibitory effect of BCG was maintained up to 7 days in culture, as evidenced by the 50% inhibitory concentration (IC50) values relative to MOI calculated on days 5 and 7 (see legend of Fig. 1). It must be pointed out that persistence of intracellular bacilli was found in approximately 50% of infected AMNC in all experiments up to 7 days after BCG infection (data not shown).
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Influence of BCG on the level of CD1b-specific transcripts.
To
investigate whether reduced expression of CD1b protein might be the
consequence of decreased mRNA levels, Northern blot analysis was
performed on total RNA extracted from AMNC that were either
unstimulated or stimulated with GM-CSF for 72 h with or without
prior BCG infection. Hybridization with the CD1b probe revealed CD1b
transcripts only in GM-CSF-activated AMNC, either uninfected or BCG
infected (Fig. 3A), whereas hybridization
of the same blot with the GAPDH probe allowed detection of the
corresponding transcript in all samples (Fig. 3B). The hybridization
signals were quantified by densitometric scanning of the autoradiograms and CD1b expression was normalized in relation to GAPDH. This demonstrated that BCG-infected AMNC had lower levels of CD1b
transcripts with respect to uninfected and GM-CSF-activated AMNC
(approximately 35% reduction).
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3 domain sequence (nucleotides 115 and 116 of exon
4) is joined to the splice acceptor site of the
transmembrane-cytoplasmic domain (exon 5) (data not shown). This
alternative mRNA splicing was previously predicted to result in a
transcript encoding a truncated and likely inactive form of the protein
(44).
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Western blot analysis of the influence of BCG on induction of the
CD1b protein.
Western blot analysis of cytosol and cell membrane
preparations was performed using the rabbit polyclonal antiserum that
recognizes the denatured form of CD1b protein (11). The
results of one of two similar independent experiments (Fig.
5A) showed two bands that were detectable
in the membrane extract of either C1R.b6 cells and AMNC activated by
GM-CSF. The upper band showed a molecular weight of about 45 kDa,
corresponding to the expected size of the mature form of CD1b (i.e.,
with multiple sialic acid additions). The lower band (an approximately
40-kDa molecule) most likely corresponded to a less glycosylated (i.e.,
immature) form of CD1b. These bands were almost undetectable in the
membrane extract of nonstimulated AMNC and were absent in that of
C1R/MOCK cells (data not shown). Exposure of AMNC to BCG prior to
treatment with GM-CSF reduced significantly the GM-CSF-dependent
induction of CD1b expression, as confirmed by densitometric analysis
illustrated in Fig. 5B. No bands corresponding to the expected
molecular weights of CD1b were found in the cytosolic fraction of AMNC.
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A soluble factor(s) is involved in the inhibition of CD1b induction
by BCG.
A transwell system consisting of an upper and lower well
separated by a permeable 0.40-µm-pore-size membrane was used
to investigate whether soluble inhibitory factors could be released
from BCG-infected cells. The experimental design adopted in these
experiments, illustrated in Table 4, was
validated by the observation that GM-CSF added to the upper well was
able to activate AMNC of the lower well (group 1 versus group 11), as
expected for a soluble, diffusible factor. BCG infection of AMNC in
either the upper or lower wells antagonized the induction of CD1b by
GM-CSF on AMNC cultured in the lower wells (groups 4 and 10). This
indicated that a soluble factor produced by BCG-infected AMNC was able
to pass through the membrane separating the upper and lower wells and
mediate the inhibition of CD1b induction on uninfected AMNC (group 10). Interestingly, when BCG was placed in the upper well without AMNC, no
inhibition but rather an increase of GM-CSF-induced CD1b expression was
detected in AMNC of the lower well (group 2 versus group 9). This
indicated that the inhibitory factor was most likely not produced by
live extracellular BCG organisms but was released following infection
of AMNC.
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Attempts to revert BCG-mediated impairment of CD1b induction using
high concentrations of GM-CSF or GM-CSF combined with various
cytokines.
Previous studies performed in our laboratory showed
that induction of CD1b expression by GM-CSF in AMNC reaches a plateau at a concentration of 200 IU/ml (unpublished data). Although higher concentrations of the cytokine did not substantially increase antigen
levels, attempts were made in the present study to revert BCG-induced
down-regulation of CD1b expression using high amounts of GM-CSF. This
approach stemmed from the consideration that the effect of BCG might be
to make infected cells partially refractory to this cytokine.
Therefore, AMNC were exposed to BCG (MOI of 1) and activated with
"standard" (200 IU/ml), high (2,000 IU/ml), or extremely high
(20,000 IU/ml) concentrations of GM-CSF. The results of two comparable
experiments (Fig. 6 and data not shown) showed that these treatments failed to significantly overcome the
negative effect of BCG on CD1b expression. It was also demonstrated that treatment with GM-CSF plus IL-4 can induce CD1b expression higher
than that obtainable with GM-CSF alone (references 26 and
38 and the present report). However, exposure of AMNC to GM-CSF associated with IL-4 or with other cytokines (i.e., IL-6, IL-15,
IFN-, IFN-) did not revert the inhibitory effects of BCG on CD1b
expression (Table 1; Fig. 2; data not shown).
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DISCUSSION |
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Abstract
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Materials and Methods
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Discussion
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The worldwide spread of tubercular infections, especially in malnourished and/or immunocompromised subjects, and the rapid increase of drug-resistant mycobacteria strains (2, 25) encourage investigation on immunological approaches to control these diseases. Several lines of evidence are in favor of a relationship between CD1-dependent immune reactivity and tuberculosis in humans (7, 30, 32, 33, 37, 39). Therefore, biological and pharmacological agents able to influence the expression of CD1 molecules on AMNC could have a significant impact on resistance against tuberculosis and related diseases. In the present study, we have shown that pretreatment of AMNC with BCG inhibited the induction of high levels of CD1 molecules by GM-CSF, which is believed to be a key inducer of the expression of this family of antigen-presenting molecules on myeloid lineage cells. This effect, which is detectable at both the protein and mRNA levels, was mediated at least in part by a soluble factor(s) released by target cells following interaction with either live or heat-killed BCG.
Circulating monocytes give rise to CD1-positive dendritic cells upon
activation with selected cytokines, i.e., GM-CSF (26) or
IL-3 in combination with IL-4 (41). These cytokines can be released during inflammatory processes and sensitization procedures, including exposure to BCG, which activates sequential production of Th1
and Th2 cytokines (31). Group I CD1 (i.e., CD1a,
CD1b, and CD1c [26]) molecules present glycolipids
(33), and in particular ManLAM of mycobacterial origin,
through the hydrophobic interactions between ManLAM fatty acids
and nonpolar amino acids of the CD1 groove to the antigen-binding site
of the TCR of T cells (23). Although M. tuberculosis-reactive CD1-restricted T cells have been isolated
from healthy donors (30), in other cases CD1-dependent
anti-M. tuberculosis activity was detected in patients
preexposed to mycobacterial infections, including leprosy patients
(34), or in HIV-positive patients who may have been
sensitized by increased exposure to opportunistic mycobacteria (13). Moreover, CD1c-restricted T cells recognizing
mannosyl-1-phosphodolichols of M. tuberculosis origin
have been found in the peripheral blood of human subjects infected with
M. tuberculosis, but not in the blood of healthy controls
(22).
Previous investigations revealed that CD1 expression induced by GM-CSF in AMNC can be up-regulated by rifampin, an antitubercular antibiotic (12, 38), or down-regulated by the Th2 cytokine IL-10 (40) and by infection with virulent M. tuberculosis (36). The present study showed that BCG negatively influences the GM-CSF-mediated induction phase of CD1 expression in AMNC at the relatively low MOI of 1. It is reasonable to suggest that a similar biological situation could occur in the initial steps of BCG vaccination in vivo. Actually, no data are available on the precise role played by the CD1 system in the long-term protection against mycobacterial infections in vaccinated subjects. It can be hypothesized that CD1-restricted responses could act in concert with classical MHC-restricted T-cell responses directed against peptides of mycobacterial origin (35). If this is the case, the present results could offer novel insights into the overall pattern of host-BCG interaction involved in the immune responses against M. tuberculosis. Moreover, the observation that the inhibitory effects mediated by BCG show measurable differences among the healthy donors tested could account, at least in part, for the well-known variability of the protective action of this vaccine.
Stenger et al. (36) reported that virulent M. tuberculosis is able to severely down-regulate transcription of CD1 genes and expression of the corresponding products on the membranes of GM-CSF-activated AMNC. Their experimental model was designed to analyze the effect of mycobacteria on AMNC preactivated by GM-CSF plus IL-4 and already expressing CD1 molecules. In this case the model was properly tailored to recreate events occurring in an established infection, in which cytokine-mediated activation of AMNC would be likely to have already occurred during the initial stages of development of the infection. In contrast, in the experimental design of our study, BCG has been applied in a manner more consistent with its role as a vaccine rather than as an infectious agent causing a systemic disease. Therefore, the analysis of the influence of the bacterium on the CD1 system was performed during the induction phase of cytokine-mediated CD1 generation. No experiments with virulent M. tuberculosis have been conducted using this model. It follows that the results obtained with BCG cannot be compared directly with those previously described for virulent M. tuberculosis. However, when CD1-positive AMNC preactivated with GM-CSF were exposed to BCG, only limited down-regulation of the antigen-presenting molecule was detected (Table 2), suggesting a significant difference between the effects of BCG and virulent M. tuberculosis in this regard.
The present study showed that BCG, like M. tuberculosis, reduces CD1b mRNA levels, suggesting that it interferes with CD1B gene transcription or accelerates degradation of its transcripts. The results illustrated in Fig. 4B demonstrate that preexposure of AMNC to BCG consistently reduces the levels of two mRNA transcripts of CD1B origin, namely the main final product and an alternatively spliced mRNA. Alternative splicing of mRNA has already been described for CD1A and CD1C gene transcripts, revealing rather complex and tissue-specific mRNA splicing patterns (44). However, no data are available from the literature concerning splicing pattern of mRNA transcribed from CD1B gene in GM-CSF-activated human AMNC. In any case, it has been suggested that the less abundant alternatively spliced form of mRNA, shown in Fig. 4B, would encode a truncated and likely inactive CD1b protein (S. Porcelli, unpublished data).
BCG-mediated reduction of CD1B gene transcript levels was followed by a consistent decrease of CD1b on the cell membrane, as evidenced by flow cytometry and Western blot analysis. It seems likely that the molecular mechanism involved in the prevention of GM-CSF-dependent up-regulation of the protein is at least in part based on the reduced availability of CD1B gene transcripts, although it remains possible that additional posttranscriptional mechanisms could also be involved. Moreover, since both principal and alternatively spliced mRNA transcripts are lower in BCG-infected GM-CSF-activated AMNC, it is not likely that alternative splicing mechanisms would play a significant role in the effect of BCG on CD1 expression.
The results of the present investigation point out that one or more soluble factors are involved in the effect of BCG on CD1b expression. Previous studies showed that pretreatment of AMNC with IL-10 markedly down-regulated CD1 expression in human adherent cells exposed to GM-CSF (40). Since BCG is able to induce production of IL-10 in adherent cells (31), this cytokine appeared to be a good candidate to play a role in the mechanism of action of BCG. However, treatment of AMNC with neutralizing MAbs directed against IL-10 or against IL-10 receptor prior to (30 min) and during (4 h) exposure of target cells to BCG did not prevent the adverse effects of the microorganism on the induction of GM-CSF-dependent CD1b expression. On the contrary, both MAbs consistently abrogated the inhibition produced by IL-10 on the induction of CD1b by GM-CSF. In a representative experiment, treatment with GM-CSF alone induced 60% of CD1b-positive AMNC on day 3 of culture. Addition of IL-10 (10 ng/ml) reduced the percentage of CD1b+ cells to 43%, whereas treatment with IL-10 plus both types of MAbs restored the full response of AMNC to GM-CSF. Moreover, it must be pointed out that a concentration of IL-10 of 10 ng/ml is usually higher than that obtainable after a 3-day coculture of AMNC and BCG under our experimental conditions (data not shown). Nevertheless, it cannot be ruled out that IL-10 would contribute to the overall effect of the mycobacterium, sharing this role with other unrecognized factors.
Several attempts have been made to overcome the detrimental influence of BCG on CD1 induction by GM-CSF. Actually, a limited increment of AMNC-associated CD1b expression can be provoked by the organism itself in the absence of added GM-CSF, as shown in Table 1. This could be explained by direct influence of the organism on monocytes or by BCG-induced release of cytokines (e.g., GM-CSF or IL-3 [31]) able to increase CD1 expression (26, 41). Thus, infection with this microorganism appears to be followed by induction of a threshold CD1 level that cannot be increased even by extremely high amounts of GM-CSF exogenously added to infected cultures (see Fig. 6). In addition, the synergistic effect of IL-4 on CD1b induction was also blocked by BCG infection.
In conclusion, the present study reveals for the first time that BCG is a potent negative modulator of GM-CSF-induced expression of group I CD1 molecules. This suggests that BCG could adversely influence nonpeptide antigen presentation to lymphocyte subsets involved in resistance against tuberculosis during the induction phase of the sensitization process. Our results call attention to one potential limitation of live attenuated mycobacteria such as BCG as vaccine strains. Given that these organisms have coevolved with the vertebrate immune system, it appears likely that they have incorporated numerous mechanisms to subvert the immune response of the hosts that they infect and parasitize. However, identification of specific immune modulating activities of mycobacterial vaccine strains will potentially allow their genetic manipulation to inactivate the genes that control their ability to prevent effective immunization. In the case of BCG, modification of the bacillus to abrogate its ability to down-regulate group I CD1 molecules represents a potential strategy by which the efficacy of this vaccine for prevention of tuberculosis might be significantly improved.
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant of "Progetto AIDS" of the National Institutes of Health, Rome, Italy (Istituto Superiore di Sanità; contract # 50C.1, 2000), and in part by a grant from the Ministry of the University and Research of the Italian Government (MURST; Research Unit Graziani, 1999). S.A.P. was supported by grants from the NIH (AI45888) and from the Irene Diamond Foundation.
We thank G. Girolomoni for fruitful discussion and suggestions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461. Phone: (718) 430-3228, -3227, or -3226. Fax: (718) 430-8711. E-mail: porcelli{at}aecom.yu.edu.
Editor: S. H. E. Kaufmann
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, December 2001, p. 7461-7470, Vol. 69, No. 12
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.12.7461-7470.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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