| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institut für Klinische Mikrobiologie, Immunologie und Hygiene der Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IFN-
, TGF-ß, IL-4), only IL-10
modulated the growth in human DC. This effect was specific for immature
dendritic cells, as IL-10 did not induce growth inhibition in human
macrophages. In searching for the mechanism of growth inhibition, we
found that IL-10 induces the down-regulation of the DC marker CD1,
while the macrophage marker CD14 was up-regulated. Functionally,
IL-10-treated cells had a reduced capacity to induce an alloresponse,
but phagocytic uptake of M. tuberculosis was more
efficient. We also show that DC are inferior to macrophages in
containing mycobacterial growth. These findings show that IL-10
converts DC into macrophage-like cells, thereby inducing the growth
inhibition of an intracellular pathogen. At the site of a local immune
response, such as a tuberculous granuloma, IL-10 might therefore
participate in the composition of the cellular microenvironment by
affecting the maturity and function of DC. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, as mice or humans that lack components of the
IFN- signaling pathway are highly susceptible to mycobacterial
infection (3). Nevertheless, in vitro studies that involve
human macrophages fail to demonstrate the activation of
antimycobacterial effects by IFN- (4, 5). Thus, the
activation of macrophages by IFN- is necessary, but not sufficient,
for eradication of intracellular microorganisms. Therefore, additional
mechanisms must be involved in the clearance of these organisms,
particularly at the site of primary infection. M. tuberculosis infects humans primarily via the respiratory route. Inhaled particles that are small enough to gain access to the terminal alveoli (<5 µm) are phagocytosed, processed, and presented mainly by alveolar macrophages (6). Another cell type present in the bronchoalveolar space, the airway epithelium, and lung parenchyma are dendritic cells (DC)3 (7, 8). Immature pulmonary DC are strategically located in pulmonary airways and distal alveoli, where they may function as sentinels for inhaled pathogens. Recent reports indicate an increased trafficking of DC into mucosal tissues in response to local bacterial stimuli such as Moraxella, Bordatella or Mycobacterium bovis bacillus Calmette-Guérin (BCG) (9, 10, 11). This property is likely to be central to the role of mucosal DC in surveillance of these front-line tissues for incoming microbial pathogens.
More recently, it has been discovered that DC can internalize live pathogens including BCG (12), M. tuberculosis (13, 14), Bordatella bronchosepticum (15), Chlamydia trachomatis (16, 17), Borrelia burgdorferi (18), Trypanosoma cruzi (19), and Leishmania major (20). Although the in vivo interaction of intracellular microorganisms and lung DC is essential for priming naive T cells, the ability of bacteria to survive in DC has not been elucidated. Human DC infected in vitro with M. tuberculosis or BCG undergo activation and maturation (13, 21, 22) that presumably enhance their potential to stimulate T cells. Also, murine DC infected with BCG and injected into the foot pad of mice can induce a specific T cell response and provide protection against a subsequent aerosol challenge with M. tuberculosis (23). These findings support the hypothesis that DC strengthen the cellular immune response against mycobacteria.
We designed experiments to investigate whether DC also participate in protective immunity by mediating direct antimycobacterial activity. Growth of intracellular bacteria was investigated in the absence or presence of cytokines, which had previously been shown to modulate mycobacterial growth in murine macrophages. Among a variety of cytokines tested, only IL-10, a product of macrophages with normally immunosuppressive effects, decreased the intracellular growth of M. tuberculosis. In parallel, IL-10-treatment reduced the ability of DC to present mycobacterial lipid Ags to CD1-restricted T cells. This differential activity of IL-10 on DC may provide a mechanism to maintain the balance between a protective immune response and excessive cellular activation in the microenvironment of a tuberculous granuloma.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cells were cultured in RPMI 1640 (Biochrom KG, Berlin, Germany) supplemented with 10% heat-inactivated FCS (Sigma, St. Louis, MO), glutamine (2 mM, Sigma), 10 mM HEPES, 13 mM NaHCO3, 100 µg/ml streptomycin, and 60 µg/ml penicillin (all purchased from Biochrom). Experiments involving the infection of cells with M. tuberculosis were performed in the absence of antibiotics, and FCS was replaced by pooled human serum (generated from the blood of healthy volunteers) to optimize the phagocytosis of the bacteria.
Cytokines and Abs
The following cytokines and Abs were used:
recombinant human (rhu) IL-2 (Proleukin; Chiron, Ratingen, Germany),
rhu IL-4 (Strathmann Biotech, Hannover, Germany), rhu TNF-, rhu
IL-10 (both purchased from Endogen, Woburn, MA), purified TGF-ß1,
anti IL-10 (R&D Systems, Wiesbaden, Germany), and rhu GM-CSF (kindly
supplied by Novartis, Vienna, Austria).
The following Abs were used for flow cytometry and immunostaining: CD83-PE, CD56 (both from Immunotech, Marseilles, France), CD19, MHC class II-FITC, CD14-PE, CD86-PE, CD40-FITC (all from Cymbus Biotechnology, Chandlers Ford, U.K.), CD3 (clone OKT3, obtained from American Type Culture Collection, Manassas, VA), CD1a-FITC (Serotec, Oxford, U.K.), goat-anti mouse-FITC (Jackson ImmunoResearch, West Grove, PA), Texas Red-conjugated goat-anti-mouse Ab (Caltag, Burlingame, CA), CD1b (clone bCD1b3.1, kindly provided by S. Porcelli, Boston, MA), CD1c (clone 10C3, kindly provided by W. Knapp, Vienna, Austria), lipoarabinomannan (kindly provided by J. Belisle, Fort Collins, CO). Isotype controls were all purchased from Cymbus Biotechnology.
Growth of M. tuberculosis
M. tuberculosis (virulent strain H37Rv) was grown in suspension with constant, gentle rotation in roller bottles containing Middlebrook 7H9 broth (Becton Dickinson, Heidelberg, Germany) supplemented with 1% glycerol (Roth, Karlsruhe, Germany), 0.05% Tween 80 (Sigma), and 10% Middlebrook oleic acid albumin dextrose catalase (OADC) enrichment (Becton Dickinson). Aliquots from logarithmically growing cultures were frozen in PBS containing 10% glycerol, and representative vials were thawed and enumerated for viable CFU on Middlebrook 7H11 plates. Staining of bacterial suspensions with fluorochromic substrates differentiating between live and dead bacteria (BacLight, Molecular Probes, Leiden, The Netherlands) revealed a viability of the bacteria above 90%. Because clumping of mycobacteria is a common problem that can influence the validity and reproducibility of the experiments, we undertook several precautions to minimize clumps. First, culture conditions (rotation, Tween) were chosen to support the growth of single cell suspensions. Second, before in vitro infection M. tuberculosis bacilli were sonicated to disrupt small aggregates of bacteria. Third, the multiplicity of infection (MOI) was selected such that there were only two to three bacilli per DC.
Generation of DC and macrophages
PBMCs from healthy donors were isolated from buffy coats
obtained from the Institute for Transfusion Medicine (University of
Erlangen, Erlangen, Germany) by Ficoll-Hypaque (Pharmacia, Freiburg,
Germany) density gradient centrifugation. Cells were allowed to adhere
to Nunclon culture flasks (Nunc, Roskilde, Denmark) in RPMI 1640 plus
10% FCS. After 2 h at 37°C, the nonadherent cells were removed
by vigorous washing with PBS. In control experiments, cells were
detached by incubation with Mg2+- and
Ca2+-free PBS containing 1 mM EDTA at 37°C for
10 min and harvested for flow cytometry. Cell-surface staining showed
that the adherent population contained >95% monocytes. Adherent cells
were incubated in culture medium plus 10% FCS supplemented with GM-CSF
(1000 U/ml) and IL-4 (1000 U/ml). Cytokines and 50% of the culture
medium were replaced after 3 days of culture. After 7 days, nonadherent
(>85% of the total population) cells were harvested and used as the
starting population for the following experiments. This method is
widely used becasue it yields substantial and pure populations of
immature DC (24, 25, 26). To induce maturation of immature DC,
cultures were supplemented with TNF- (10 ng/ml) and incubated for an
additional 48 h. Macrophages were generated by incubating PBMC in
a culture flask for 1–2 h. Nonadherent cells were removed by three
thorough washing steps. The adherent cells were then cultured in
culture medium for 7 days before harvesting. The purity of the
macrophage population was confirmed by FACS staining
(CD14+, CD11b+,
CD19-, CD56-, CD3 <
2%) and was above 98% in all experiments.
Infection of DC
DC were infected with single-cell suspensions of M. tuberculosis. After 4 h of incubation at 37°C, DC were harvested (slightly adherent cells were detached by vigorous pipetting) and centrifuged at 800 rpm for 8 min. This low-speed centrifugation selectively spins down DC while extracellular bacteria remain in the supernatant. After three cycles of centrifugation, the majority of extracellular bacteria were removed as determined by auramine-rhodamine stain (TB-fluor, Merck, Darmstadt, Germany). Infected cells were then plated at a concentration of 1 x 106 cells/ml in a 24-well plate in culture medium without antibiotics plus 10% human serum. The efficiency of infection, as quantitated by staining of control cultures on Permanox chamber slides (Nunc) in every experiment was dependent on the MOI. The microscopic evaluation of infected macrophages under the fluorescence microscope confirmed the absence of any mycobacterial aggregates. Cell viability of infected DC was determined by trypan blue exclusion and was >99% in all experiments.
Cytokine treatment of cell cultures
Dendritic cells or macrophages were harvested, washed, and plated into tissue cultures plates. After the pulse infection with mycobacteria, cytokines or anti IL-10 were added. The cytokines were not renewed (with the exception of one experimental series) and were present throughout the incubation period.
Confocal laser microscopy
Mycobacteria were incubated with an Ab directed against lipoarabinomannan (1:1 dilution of pure hybridoma supernatant) diluted in PBS/1% BSA/20%FCS for 30 min at room temperature. Lipoarabinomannan was labeled by incubation of a 1:50 dilution (PBS/1% BSA/20%FCS) of a Texas Red-conjugated goat-anti-mouse Ab for 30 min. After an additional washing step, stained mycobacteria were resuspended and used for infection of DC, which were cultured on Permanox chamber slides. After 4 h, nonphagocytosed bacteria were removed by washing with PBS, and infected cells were stained with a FITC-conjugated Ab recognizing CD1a (1:20 dilution, 30 min, room temperature). Finally, cells were fixed with 4% paraformaldehyde (Sigma), mounted (Aquatex, Merck), and analyzed using a confocal microscope (Leica, Solms, Germany).
Quantification of mycobacterial growth
To ensure the reliable quantification of intracellular M. tuberculosis we employed three independent methods. The first method used acid-fast stain (auramine-rhodamine). The second method employed colony forming units (CFU). After various time points of incubation, cells were lysed with 0.3% saponin (Sigma) to release intracellular bacteria. At all time points, an aliquot of unlysed, infected cells was harvested and counted. This allowed an exact quantification of cells as well as the determination of cellular viability by trypan blue exclusion. Recovery of cells was >80% in all experiments, with cell viability regularly exceeding 90% of total cells. Lysates of infected cells were resuspended vigorously, transferred into screw caps, and sonicated in a preheated (37°C) waterbath sonicator (Elma, Singen, Germany) for 5 min. Aliquots of the sonicate were diluted 10-fold in 7H9 medium. Four dilutions of each sample were plated in duplicates on 7H11 agar plates and incubated at 37°C and 5% CO2 for 21 days. The third method involved [3H]uracil incorporation. Incorporation of [3H]-labeled uracil into the mycobacterial RNA was determined following the method published by Rook and Rainbow (27) with several modifications. First, 1 x 106 infected DC were cultured in duplicates as described above. At the end of the incubation period, cells were lysed using 0.3% saponin, resuspended vigorously, and transferred into screw caps. Lysates were centrifuged in an aerosol-tight microfuge (3000 rpm, 20 min) and resuspended in 100 µl of 7H9 to allow optimal growth of the released mycobacteria. Lysates were then transferred into 96-well round-bottom plates (Nunc) and incubated in the presence of 3 µCi [3H]uracil (Amersham-Pharmacia, Freiburg, Germany). After 24 h, mycobacteria were killed by treatment with paraformaldehyde (final concentration, 4%) for 30 min. The mycobacteria were harvested onto glass fiber filters (Inotech, Dottikon, Switzerland), and [3H]uracil incorporation was measured in a beta counter (Berthold, München, Germany). Background radioactivity in uninfected cells was below 500 cpm in all experiments. To document the suitability of our quantification methods to detect antimycobacterial activity, we performed some experiments in the presence of the mycobactericidal drug rifampicin (Sigma).
Flow cytometry
A total of 3 x 105 cells were resuspended in 100 µl staining buffer (2% FCS, 1% NaN3, PBS without Mg2+/Ca2+) and incubated with unconjugated or conjugated Abs for 30 min on ice. Samples were washed twice in staining buffer and if necessary incubated for an additional 30 min on ice with goat anti mouse-FITC Abs (1:500). Cells were then fixed in 2% paraformaldehyde and stored at 4°C until analysis in a FACScan flow cytometer. Data were analyzed using CellQuest software (Becton Dickinson).
Mixed lymphocyte reaction
DC were irradiated with 30 Gy (137Cs source). Subsequently, graded numbers of these stimulator cells were mixed with a fixed amount of purified peripheral blood CD4+ cells (5 x 104) and seeded into 96-well round-bottom tissue culture plates in a final volume of 200 µl. CD4+ cells were enriched by indirect immunomagnetic depletion of cells expressing CD8, CD14, CD19, and CD56 (sheep anti mouse Dynabeads, Dynal, Oslo, Norway) from PBMC prepared by Ficoll-Hypaque density centrifugation. Purity, as determined by flow cytometry, proved to be >95%. Controls included DC alone, responder cells alone, and responder cells cultured in the presence of 100 U/ml IL-2. All cultures were set up as triplicates. After 5 days of culture, 0.5 µCi [3H]thymidine (Amersham-Pharmacia) was added to each well, and thymidine incorporation was measured 18 h later using a cell harvester and a betaplate counter.
CD1-restricted cell lines
CD1-restricted cell lines were generated as described earlier
(28). DN7 was derived from a healthy, purified protein
derivative-positive donor. DN7 is TCRß+,
CD4-, CD8-, and
specifically recognizes purified lipoarabinomannan from M.
tuberculosis in the context of the nonclassical MHC molecule CD1b.
To measure the Ag-specific response, DN7 (1 x
104) was incubated in the presence of
IL-10-treated or control DC (1 x 104) and
lipoarabinomannan (1 µg/ml) for 3 days. The last 4 h of
incubation were performed in the presence of 0.5 µCi
[3H]thymidine. Incorporated radioactivity was
measured after harvesting cells onto glass fiber filters in a beta
counter.
Statistical analysis
Data are presented as mean value ± SEM except where stated otherwise. Student’s t test was used to determine statistical significance between two differentially treated cultures. Differences were considered significant if p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To monitor the growth of M. tuberculosis in DC, it was
necessary to determine the MOI, which allowed us to culture infected
cells for at least 7 days. Therefore, we infected DC with an increasing
MOI. Four hours later, extracellular bacteria were removed by
differential centrifugation, and the number of infected cells was
determined by acid-fast stain (Fig. 1
A). MOIs of 2.5 or 12.5
resulted in a high infection efficiency of 62% and 93%, respectively.
However, the high bacterial burden of the cells (Fig. 1B)
resulted in cell death within 48 h (data not shown). A MOI of 0.5
resulted in infection of 24% ± 0.4 of the cells, whereby each cell
harbored an average of 2.2 ± 0.3 bacteria. This infection rate
did not result in rapid cell death and was chosen for studying the
intracellular growth of M. tuberculosis in DC for 7 days. To
ensure that we were studying the growth of intracellular, but not
extracellular, mycobacteria, we double-stained infected DC with Abs
directed against mycobacteria and DC. Bacteria were labeled with an
anti-lipoarabinomannan Ab and a Texas Red-conjugated secondary Ab
before infection of DC. Four hours after infection, DC were stained
with an Ab recognizing the CD1a-FITC glycoprotein on the cell surface
of DC. Confocal microscopy revealed that over 90% of the bacteria were
localized intracellularly (Fig. 1C). To confirm the
intracellular localization of M. tuberculosis, DC were
harvested and centrifuged at 800 rpm. At this low speed, cells are
preferentially spun down, while the majority of bacteria will remain in
the supernatant. Plating of these supernatants revealed that <0.1% of
the initial inoculum remained in the extracellular compartment (not
shown).
|
Using this model of low-dose infection (MOI 0.5), we found that
after an initial quiescent phase of 24 h the number of M.
tuberculosis increased 4-fold within 1 day and continued to
multiply throughout the observation period (Fig. 2). Total growth of the bacteria was two
orders of magnitude within 7 days, which is equivalent to an average
generation time of M. tuberculosis in human DC of 26 h.
The replication of bacteria resulted in an increased bacterial burden
of individual cells, as well as a higher number of infected cells
(Table I). Even though this finding
indicates that some cells must have been lysed to allow spreading of
the mycobacteria, the number of viable cells after 7 days of infection
was above 90% of the initial inoculum (data not shown). To verify that
we were not studying the growth of extracellular bacteria that had been
released into the culture medium by dying cells, we centrifuged
representative lysates at 800 rpm. These supernatants contained <0.5%
of the total bacteria in the culture plate. Even though there was a
considerable variabilty in the ability of the DC of individual donors
to phagocytose M. tuberculosis (21–32%), the generation
time remained similar. An extension of the observation period beyond 7
days was not informative as cell viability declined drastically.
|
|
and TNF- on the growth of M.
tuberculosis
Next, we designed experiments to investigate whether IFN- and
TNF-, alone or in combination, would be able to induce
antimycobacterial activity in DC. While it is well established that
IFN- activates antimycobacterial effector mechanisms in mice
(29), evidence for comparable action on human macrophages
is lacking (4, 5). Similarly, the role of TNF- in the
activation of human phagocytes is poorly defined, despite one study
clearly showing an antimycobacterial effect of TNF- on alveolar
macrophages (30). Neither IFN- nor TNF- (both at 10
ng/ml) reduced the metabolic activity of virulent M.
tuberculosis in DC, as determined by incorporation of
[3H]uracil (Fig. 3A). Higher cytokine
concentrations up to 50 ng/ml also had no effect on bacterial growth
(data not shown). TNF--treated cells showed a tendency to increase
the bacterial burden. Even though this increase did not reach a
significant level, it was reproducible in four of four independent
experiments. Similarly, incubation of DC in the presence of a
combination of IFN- and TNF- had no effect on mycobacterial
growth. As a control, we treated infected cells with the
mycobactericidal drug rifampicin, which reduced the uracil
incorporation to background levels (<500 cpm) (Fig. 3A).
This documents the suitability of our evaluation system to detect
antimycobacterial activity.
|
Prototypic cytokines involved in the down-regulation of the
protective immune response against mycobacterial disease are TGF-ß
(31, 32, 33), IL-4 (34), and IL-10
(35, 36, 37, 38). However, neither exogenous TGF-ß nor IL-4
inhibited the metabolic activity of M. tuberculosis after an
incubation period of 7 days in DC (Fig. 3
B). To exclude that
we had missed an early antimycobacterial effect of these cytokines, we
also quantitated the bacterial load of cytokine-treated DC after
72 h of infection. No difference could be detected as determined
by auramine-rhodamine stain (data not shown). Moreover, representative
experiments revealed that replenishment of cytokines or Abs at day 3
did not alter the course of infection (data not shown). In contrast,
IL-10 reduced the uracil uptake of M. tuberculosis from
16,265 cpm to 6,187 cpm by 62.5% (Fig. 3B). This unexpected
finding was confirmed by determining the number of CFU of IL-10-treated
and control cultures (Fig. 4A). A total of 1 ng/ml of
IL-10 was already sufficient to decrease the mycobacterial growth by
43.5%. Increasing concentrations of IL-10 (10 ng/ml) enhanced the
growth inhibition up to 65%. At 10 ng/ml, the activity of IL-10
reached a plateau and could not be augmented further (Fig. 4A). The reduced growth of M. tuberculosis in DC
induced by IL-10 was not detectable 24 h after infection, ruling
out an early antibactericidal effect associated with the uptake of the
microorganism (Fig. 4B). Despite significant reduction of
mycobacterial growth after 3 days of incubation (32%), the maximum
inhibition was observed on day 7. To obtain a phenotypical correlate of
the decreased bacterial burden in IL-10-treated DC, we stained infected
cultures with acid-fast stain and visualized mycobacteria by confocal
microscopy (Fig. 4C). Decreases in metabolic activity and
limited growth on agar plates correlated with a drastically reduced
number of mycobacteria residing within DC. The bacterial burden per
infected cell was also substantially lower (Table II). These data show by three independent
methods of quantification that IL-10 reduces the growth of virulent
M. tuberculosis in human DC. Because IL-10 is also secreted
by DC infected with live M. tuberculosis (13),
we considered the possibility that neutralization of endogenous IL-10
would increase mycobacterial survival. However, no difference in
mycobacterial growth was observed as compared with the uninfected
controls (Fig. 4B). This result is in agreement with our
failure to detect IL-10 in the supernatant of infected DC (<20 pg/ml,
data not shown). To our knowledge, only one study has shown the
secretion of IL-10 by M. tuberculosis-infected immature DC
(13). Using a MOI of 5–10 (as opposed to the MOI of 0.5
used in our experiments), they found 100 pg/ml of IL-10 in the
supernatant. These values are one order of magnitude lower than the
amounts needed to observe an antimycobacterial effect in our study.
|
|
To unveil the mechanism of the IL-10-induced growth inhibition, we
considered the possibility that IL-10 influences the phenotypic and
functional maturity of monocyte-derived DC. We incubated uninfected DC,
generated as described above, for an additional 48 h in the
presence of IL-10 or medium alone (Fig. 5A). This resulted in a marked
up-regulation of the monocyte marker CD14 (mean fluorescence intensity
345 vs 1987 after IL-10 treatment). The CD1a molecule, which is highly
expressed on the cell surface of immature DC, almost disappeared from
the cell surface (Fig. 5A). The functional impact of the
down-regulation of CD1 was demonstrated by the failure of these cells
to present lipoarabinomannan to a CD1b-restricted T cell line (Fig. 5B). To further characterize the functional impact of IL-10
treatment on immature DC, we measured the ability to induce a mixed
lymphocyte reaction. Incorporation of
[3H]thymidine by heterologous,
CD4+ T cells was significantly lower when
IL-10-treated cells were used as APC (Fig. 5C). Because DC
lose the ability to phagocytose soluble and particulate Ags during
their maturation from monocytes (39, 40), we asked whether
IL-10 treatment of immature DC would reverse this effect and increase
the ability to phagocytose virulent M. tuberculosis. IL-10
treatment increased the phagocytic activity of DC in a dose-dependent
manner from 32 ± 2% in untreated cells up to nearly 80 ±
4% by treatment with 10 ng/ml of IL-10 (Fig. 5D). In
summary, these experiments demonstrate that IL-10 converts immature DC
into macrophage-like cells according to phenotypical and functional
criteria. Therefore, the reduction of mycobacterial growth in
IL-10-treated immature DC may be the result of the modulation of the
maturity of the host cell, rather than a consequence of
antimycobacterial effector mechanisms in DC.
|
To investigate whether the described effects of IL-10 are unique
to immature DC or represent a general phenomenon, we examined the
growth of M. tuberculosis in human macrophages. Macrophages
were derived from peripheral blood monocytes and cultured for 7 days
before infection with M. tuberculosis with a MOI of 0.5.
IL-10 was added after the end of the pulse infection at various
concentrations (ranging from 1 ng/ml to 50 ng/ml), and mycobacterial
growth was determined after 7 days of culture. We found no influence on
mycobacterial growth at all concentrations tested (Fig. 6A). Similarly, treatment of
macrophages with IL-10 (10 ng/ml) for 48 h did not modulate the
expression of CD14 and CD1a on the cell surface (Fig. 6B) as
opposed to its effect on DC (Fig. 5A). To verify the
biologic activity of IL-10 on macrophages, we also measured MHC class
II expression, which is known to be down-regulated by IL-10
(41). As expected, expression of MHC class II molecules on
the cell surface was down-regulated by IL-10 (mean fluorescence
intensity of 199 vs 49 after treatment), whereas it had no effect on
MHC class II expression on immature DC (Fig. 5A). Taken
together, these data demonstrate that the induction of
antimycobacterial activity and regulation of CD14/CD1a by IL-10 is
specific for immature DC and is not a general phenomenon of host cells
for intracellular pathogens.
|
Our previous experiments had demonstrated that IL-10
converts immature DC into cells resembling macrophages. These
macrophage-like cells might be more competent in combating an infection
with intracellular pathogens. To test this hypothesis, we compared the
intracellular growth of M. tuberculosis in macrophages and
DC derived from the same donor. In four independent experiments using
different donors, we found that mycobacterial growth was significantly
lower in macrophages than in DC after 7 days of incubation. Using an
identical MOI (0.5), we found that the initial inoculum was higher in
macrophages than in DC (data not shown), consistent with our previous
data (Fig. 5D). After 3 days of infection, and even more
significantly after 7 days of infection, mycobacterial multiplication
was lower in macrophages as compared with DC. Within the 7-day
observation period, the bacterial burden in DC increased 72-fold in DC
and only 24-fold in macrophages. This results in a doubling time of
M. tuberculosis in DC of 24 h and for bacteria residing
within macrophages of 42 h (Fig. 7).
To rule out that the difference in the initial inoculum influences the
generation time of mycobacteria, we adjusted the MOI in macrophages
such that the infection rate was similar to DC (23%). This had no
significant impact on the intracellular growth of M.
tuberculosis in macrophages (data not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Intracellular bacteria that can invade DC comprise relevant human pathogens (12, 13, 14, 15, 16, 17, 18, 19, 20). Therefore, it is becoming increasingly important to learn more about the intracellular fate of live micoorganisms residing within DC. Human DC infected in vitro with M. tuberculosis undergo a direct activation and maturation that presumably enhance their efficacy at stimulating T cells (13). In contrast, M. tuberculosis has evolved evasion mechanisms to prevent Ag presentation of lipid Ags to cytolytic T cells by down-regulating one of the Ag-presenting molecules, i.e., CD1 (42). We extend these findings by showing that invasion of DC by virulent bacteria may be beneficial for the pathogen as it can multiply liberally. Therefore, invasion of DC may be advantageous for intracellular organisms allowing their unlimited multiplication and spreading. In contrast to the phagocytosis of soluble microbial Ag, which leads to the maturation of DC and subsequent activation of the specific immune system, uptake of viable pathogens might be harmful for the host. Therefore, the role of DC in the local immune response might be both harmful or beneficial to the host, depending on whether soluble Ags or live microbes are taken up.
M. tuberculosis infects humans primarily by the respiratory
route. The majority of cells present in the alveolar spaces are
alveolar macrophages that phagocytose the microbial invader
(6). However, alveolar macrophages are not efficient in
inhibiting the growth of intracellular pathogens and have even been
shown to depress the protective immune response of Ag-specific T cells
(43). Another cell type present in the airway epthilium
are DC (7). They presumably acquire Ag and then migrate to
the lymph node. In contrast to alveolar macrophages, pulmonary DC are
very efficient in initiating a protective immune reponse
(8). Therefore, they might be crucial for the initiation
of the early immune response, which inhibits clinically overt
tuberculosis in >95% of infected humans. The experiments presented in
this study do not support the hypothesis that DC are involved in
elimination of intracellular bacteria by exerting immediate
antibacterial activity. More likely, the innate immune system
consisting of alveolar macrophages and freshly recruited monocytes is
responsible for the early containment of infection. Mycobacterial Ags
released into the extracellular space by macrophages could gain access
to the Ag-presenting network of DC by macropinocytosis. Alternatively,
a fraction of macrophages might undergo apoptotic cell death induced by
an overwhelming bacterial burden. These apoptotic bodies could then be
taken up by DC, which present mycobacterial Ags to T cells. While
direct evidence for the uptake of regurgitated mycobacterial products
by DC is lacking (44), DC that have taken up Ags derived
from apoptotic bodies have been shown to activate
CD8+ cytolytic T cells (45).
CD8+ T cells have been suggested to play a
special role in the human immune response to M. tuberculosis
by injecting anti-mycobacterial effector molecules such as
granulysin into the target cell (46). The special role of
DC in Ag presentation is underscored by their unique ability to present
nonprotein Ags to T cells via the nonclassical MHC molecules CD1a, -b,
and -c (47). Because our experiments demonstrate that DC
are poor antibacterial effector cells (Figs. 2 and 7) and are clearly
inferior to macrophages in this regard, their prominent function is
more likely to link the innate and acquired immune response by
recruiting and activating Ag-specific T cells.
IL-10 was first detected based on its cytokine synthesis inhibitory activity mainly on macrophages (48, 49, 50). Recently, evidence is accumulating that DC are another major target for the action of this immunosuppressive cytokine. IL-10 was reported to inhibit the Ag-presenting capacity of DC (51, 52, 53, 54, 55), to reduce the expression of Ag-presenting and costimulatory molecules (56, 57, 58, 59), and to interfere with the maturation of monocytes to DC (60, 61, 62). Taken together, these studies suggest an overall picture in which IL-10 prevents the differentiation of monocytes to mature DC but promotes their maturation to macrophages (63). Recent findings demonstrate that maturation of monocytes to DC also occurs in vivo (64, 65). These findings point out that the development of monocytic cells into either mature tissue-macrophages or DC depends on the local microenvironment. The opposite development of immature DC into macrophages as shown here in an in vitro culture system has not been formally proven in vivo. However, the studies discussed above demonstrate the potential of immature cells to develop differentially in vivo. This may serve to pave the way for the maturation of cells optimally equipped to meet the functional requirements of the local immune response. Specifically, in the case of a tuberculous granuloma, the local cytokine microenvironment may be dominated by IL-10 in certain instances (36), thereby supporting the development of macrophage-like cells. These will then complement effector mechanisms of the protective cellular immune response and contribute to the eradication of M. tuberculosis.
One mechanism of infected cells to eliminate intracellular pathogens is to undergo apoptosis, thereby exposing the microbes to the extracellular environment. IL-10 has been shown to inhibit apoptosis of cells infected with intracellular bacteria including mycobacteria (66, 67). However, we did not observe apoptosis in untreated or IL-10-treated immature DC as determined by annexin V staining (data not shown). The failure to detect apoptotic cells was most likely a consequence of the low MOI, which was chosen to allow a 7-day observation period of mycobacterial growth. Also, if IL-10 would inhibit apoptosis of infected DC, bacterial growth would more likely be increased, rather than inhibited as observed in this study. Therefore, growth inhibition of mycobacteria in immature DC by IL-10 is unlikely to be mediated by the modulation of apoptosis.
In vivo studies revealed that IL-10-expressing cells tend to accumulate in patients suffering from an unfavorable outcome of disease (35, 68). However, the data from murine studies argue against a critical, nonredundant role of IL-10 in immunity to tuberculosis because IL-10-deficient mice are not or are only partially protected from tuberculosis (38, 69).
This study exemplifies the versatility of the immune system with an armamentarium of effector cells, each having specific and specialized functions in immunity to microorganisms. In the setting of a local cellular immune response, as is typical for a tuberculous granuloma, the differential effects of IL-10 on macrophages and DC may contribute to the fine-tuned balance that must provide protection from the pathogen, as well as limitation of excessive tissue destruction.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address of correspondence and reprint requests to Dr. Steffen Stenger, Friedrich-Alexander Universität Erlangen-Nürnberg, Institut fuer Klinische Mikrobiologie, Immunologie und Hygiene, Wasserturmstrasse 3, D-91054 Erlangen, Germany.
3 Abbreviations used in this paper: DC, dendritic cell(s); MOI, multiplicity of infection; BCG, bacillus Calmette-Guérin; rhu, recombinant human.
Received for publication December 7, 1999. Accepted for publication May 2, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in host defense against mycobacteria and salmonella in mice and men. Curr. Opin. Immunol. 3:346.
interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunology 57:159.[Medline]
independent activation of dendritic cells following treatment with Mycobacterium bovis bacillus Calmette-Guerin. Immunology 97:626.[Medline]
interferon, IL-4 and tumor necrosis factor. Infect. Immun. 58:2675.-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar macrophages. J. Immunol. 152:743.[Abstract]
production. J. Immunol. 150:3755.[Abstract]
or by interleukin-10. Eur. J. Immunol. 25:394.[Medline]
. J. Immunol. 161:2636. and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages. J. Immunol. 162:6122.This article has been cited by other articles:
|
|
 |
|
  S. Kumar and R. Jack Invited review: Origin of monocytes and their differentiation to macrophages and dendritic cells Innate Immunity, October 1, 2006; 12(5): 278 - 284. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Ben Nasr, J. Haithcoat, J. E. Masterson, J. S. Gunn, T. Eaves-Pyles, and G. R. Klimpel Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of immature DC and intracellular survival of the bacteria J. Leukoc. Biol., October 1, 2006; 80(4): 774 - 786. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. H. Tan, C. Meinken, M. Bastian, H. Bruns, A. Legaspi, M. T. Ochoa, S. R. Krutzik, B. R. Bloom, T. Ganz, R. L. Modlin, et al. Macrophages Acquire Neutrophil Granules for Antimicrobial Activity against Intracellular Pathogens J. Immunol., August 1, 2006; 177(3): 1864 - 1871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sinha, A. Singh, V. Satchidanandam, and K. Natarajan Impaired Generation of Reactive Oxygen Species during Differentiation of Dendritic Cells (DCs) by Mycobacterium tuberculosis Secretory Antigen (MTSA) and Subsequent Activation of MTSA-DCs by Mycobacteria Results in Increased Intracellular Survival J. Immunol., July 1, 2006; 177(1): 468 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. de Gruijl, C. C. Sombroek, S. M. Lougheed, D. Oosterhoff, J. Buter, A. J. M. van den Eertwegh, R. J. Scheper, and H. M. Pinedo A Postmigrational Switch among Skin-Derived Dendritic Cells to a Macrophage-Like Phenotype Is Predetermined by the Intracutaneous Cytokine Balance. J. Immunol., June 15, 2006; 176(12): 7232 - 7242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Giacomini, A. Sotolongo, E. Iona, M. Severa, M. E. Remoli, V. Gafa, R. Lande, L. Fattorini, I. Smith, R. Manganelli, et al. Infection of Human Dendritic Cells with a Mycobacterium tuberculosis sigE Mutant Stimulates Production of High Levels of Interleukin-10 but Low Levels of CXCL10: Impact on the T-Cell Response Infect. Immun., June 1, 2006; 74(6): 3296 - 3304. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Vakkila, R. Jaffe, M. Michelow, and M. T. Lotze Pediatric cancers are infiltrated predominantly by macrophages and contain a paucity of dendritic cells: a major nosologic difference with adult tumors. Clin. Cancer Res., April 1, 2006; 12(7): 2049 - 2054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Stegelmann, M. Bastian, K. Swoboda, R. Bhat, V. Kiessler, A. M. Krensky, M. Roellinghoff, R. L. Modlin, and S. Stenger Coordinate Expression of CC Chemokine Ligand 5, Granulysin, and Perforin in CD8+ T Cells Provides a Host Defense Mechanism against Mycobacterium tuberculosis J. Immunol., December 1, 2005; 175(11): 7474 - 7483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Mu, X. Ouyang, A. Agarwal, L. Zhang, D. A. Long, P. E. Cruz, C. A. Roncal, O. Y. Glushakova, V. A. Chiodo, M. A. Atkinson, et al. IL-10 Suppresses Chemokines, Inflammation, and Fibrosis in a Model of Chronic Renal Disease J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3651 - 3660. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mocellin, F. M. Marincola, and H. A. Young Interleukin-10 and the immune response against cancer: a counterpoint J. Leukoc. Biol., November 1, 2005; 78(5): 1043 - 1051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Stenger Immunological control of tuberculosis: role of tumour necrosis factor and more Ann Rheum Dis, November 1, 2005; 64(suppl_4): iv24 - iv28. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Tanaka, Y. Hoshino, J. Gold, S. Hoshino, F. Martiniuk, T. Kurata, R. Pine, D. Levy, W. N. Rom, and M. Weiden Interleukin-10 Induces Inhibitory C/EBP{beta} through STAT-3 and Represses HIV-1 Transcription in Macrophages Am. J. Respir. Cell Mol. Biol., October 1, 2005; 33(4): 406 - 411. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Buettner, C. Meinken, M. Bastian, R. Bhat, E. Stossel, G. Faller, G. Cianciolo, J. Ficker, M. Wagner, M. Rollinghoff, et al. Inverse Correlation of Maturity and Antibacterial Activity in Human Dendritic Cells J. Immunol., April 1, 2005; 174(7): 4203 - 4209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Berger, R. Tigelaar, J. Cohen, K. Mariwalla, J. Trinh, N. Wang, and R. L. Edelson Cutaneous T-cell lymphoma: malignant proliferation of T-regulatory cells Blood, February 15, 2005; 105(4): 1640 - 1647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Gerlini, A. Tun-Kyi, C. Dudli, G. Burg, N. Pimpinelli, and F. O. Nestle Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions Am. J. Pathol., December 1, 2004; 165(6): 1853 - 1863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mariotti, R. Teloni, E. Iona, L. Fattorini, G. Romagnoli, M. C. Gagliardi, G. Orefici, and R. Nisini Mycobacterium tuberculosis Diverts Alpha Interferon-Induced Monocyte Differentiation from Dendritic Cells into Immunoprivileged Macrophage-Like Host Cells Infect. Immun., August 1, 2004; 72(8): 4385 - 4392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Marino, S. Pawar, C. L. Fuller, T. A. Reinhart, J. L. Flynn, and D. E. Kirschner Dendritic Cell Trafficking and Antigen Presentation in the Human Immune Response to Mycobacterium tuberculosis J. Immunol., July 1, 2004; 173(1): 494 - 506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Mailaender, N. Reiling, H. Engelhardt, S. Bossmann, S. Ehlers, and M. Niederweis The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis Microbiology, April 1, 2004; 150(4): 853 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Alaniz, S. Sandall, E. K. Thomas, and C. B. Wilson Increased Dendritic Cell Numbers Impair Protective Immunity to Intracellular Bacteria Despite Augmenting Antigen-Specific CD8+ T Lymphocyte Responses J. Immunol., March 15, 2004; 172(6): 3725 - 3735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Rotta, E. W. Edwards, S. Sangaletti, C. Bennett, S. Ronzoni, M. P. Colombo, R. M. Steinman, G. J. Randolph, and M. Rescigno Lipopolysaccharide or Whole Bacteria Block the Conversion of Inflammatory Monocytes into Dendritic Cells In Vivo J. Exp. Med., October 20, 2003; 198(8): 1253 - 1263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Kumar, R. R. Amara, V. K. Challu, V. K. Chadda, and V. Satchidanandam The Apa Protein of Mycobacterium tuberculosis Stimulates Gamma Interferon-Secreting CD4+ and CD8+ T Cells from Purified Protein Derivative-Positive Individuals and Affords Protection in a Guinea Pig Model Infect. Immun., April 1, 2003; 71(4): 1929 - 1937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Bleharski, V. Kiessler, C. Buonsanti, P. A. Sieling, S. Stenger, M. Colonna, and R. L. Modlin A Role for Triggering Receptor Expressed on Myeloid Cells-1 in Host Defense During the Early-Induced and Adaptive Phases of the Immune Response J. Immunol., April 1, 2003; 170(7): 3812 - 3818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Lopez-Maderuelo, F. Arnalich, R. Serantes, A. Gonzalez, R. Codoceo, R. Madero, J. J. Vazquez, and C. Montiel Interferon-{gamma} and Interleukin-10 Gene Polymorphisms in Pulmonary Tuberculosis Am. J. Respir. Crit. Care Med., April 1, 2003; 167(7): 970 - 975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Tailleux, O. Neyrolles, S. Honore-Bouakline, E. Perret, F. Sanchez, J.-P. Abastado, P. H. Lagrange, J. C. Gluckman, M. Rosenzwajg, and J.-L. Herrmann Constrained Intracellular Survival of Mycobacterium tuberculosis in Human Dendritic Cells J. Immunol., February 15, 2003; 170(4): 1939 - 1948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Brown, D. M. Dorfman, F.-R. Ma, E. L. Sullivan, O. Munoz, C. R. Wood, E. A. Greenfield, and G. J. Freeman Blockade of Programmed Death-1 Ligands on Dendritic Cells Enhances T Cell Activation and Cytokine Production J. Immunol., February 1, 2003; 170(3): 1257 - 1266. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H.E. Kaufmann and U. E. Schaible A Dangerous Liaison between Two Major Killers: Mycobacterium tuberculosis and HIV Target Dendritic Cells through DC-SIGN J. Exp. Med., January 6, 2003; 197(1): 1 - 5. [Full Text] [PDF]
|
|
|
|
|
|
L. Tailleux, O. Schwartz, J.-L. Herrmann, E. Pivert, M. Jackson, A. Amara, L. Legres, D. Dreher, L. P. Nicod, J. C. Gluckman, et al. DC-SIGN Is the Major Mycobacterium tuberculosis Receptor on Human Dendritic Cells J. Exp. Med., January 6, 2003; 197(1): 121 - 127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 D. P. Portales-Perez, L. Baranda, E. Layseca, N. A. Fierro, H. de la Fuente, Y. Rosenstein, and R. Gonzalez-Amaro Comparative and Prospective Study of Different Immune Parameters in Healthy Subjects at Risk for Tuberculosis and in Tuberculosis Patients Clin. Vaccine Immunol., March 1, 2002; 9(2): 299 - 307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Jiao, R. Lo-Man, P. Guermonprez, L. Fiette, E. Deriaud, S. Burgaud, B. Gicquel, N. Winter, and C. Leclerc Dendritic Cells Are Host Cells for Mycobacteria In Vivo That Trigger Innate and Acquired Immunity J. Immunol., February 1, 2002; 168(3): 1294 - 1301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Engele, K. Castiglione, N. Schwerdtner, M. Wagner, P. Bolcskei, M. Rollinghoff, and S. Stenger Induction of TNF in Human Alveolar Macrophages As a Potential Evasion Mechanism of Virulent Mycobacterium tuberculosis J. Immunol., February 1, 2002; 168(3): 1328 - 1337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wille, A. Szekeres, O. Majdic, E. Prager, G. Staffler, J. Stockl, D. Kunthalert, E. E. Prieschl, T. Baumruker, H. Burtscher, et al. Characterization of CDw92 as a Member of the Choline Transporter-Like Protein Family Regulated Specifically on Dendritic Cells J. Immunol., November 15, 2001; 167(10): 5795 - 5804. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Newman and A. Holly Candida albicans Is Phagocytosed, Killed, and Processed for Antigen Presentation by Human Dendritic Cells Infect. Immun., November 1, 2001; 69(11): 6813 - 6822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Jotwani, A. K. Palucka, M. Al-Quotub, M. Nouri-Shirazi, J. Kim, D. Bell, J. Banchereau, and C. W. Cutler Mature Dendritic Cells Infiltrate the T Cell-Rich Region of Oral Mucosa in Chronic Periodontitis: In Situ, In Vivo, and In Vitro Studies J. Immunol., October 15, 2001; 167(8): 4693 - 4700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. W. Cutler, R. Jotwani, and B. Pulendran Dendritic Cells: Immune Saviors or Achilles' Heel? Infect. Immun., August 1, 2001; 69(8): 4703 - 4708. [Full Text] [PDF]
|
|
|
|
|
|
E. Giacomini, E. Iona, L. Ferroni, M. Miettinen, L. Fattorini, G. Orefici, I. Julkunen, and E. M. Coccia Infection of Human Macrophages and Dendritic Cells with Mycobacterium tuberculosis Induces a Differential Cytokine Gene Expression That Modulates T Cell Response J. Immunol., June 15, 2001; 166(12): 7033 - 7041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ameixa and J. S. Friedland Down-Regulation of Interleukin-8 Secretion from Mycobacterium tuberculosis-Infected Monocytes by Interleukin-4 and -10 but Not by Interleukin-13 Infect. Immun., April 1, 2001; 69(4): 2470 - 2476. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Ancuta, Y. Bakri, N. Chomont, H. Hocini, D. Gabuzda, and N. Haeffner-Cavaillon Opposite Effects of IL-10 on the Ability of Dendritic Cells and Macrophages to Replicate Primary CXCR4-Dependent HIV-1 Strains J. Immunol., March 15, 2001; 166(6): 4244 - 4253. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
