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Laboratories of
*
Immunology and
Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome, Italy; and
Department of Virology, National Public Health Institute, Helsinki, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and IL-6 were
secreted mainly by MDM. As regards the production of IFN-
-inducing
cytokines, IL-12 and IFN-, was seen almost exclusively from infected
MDDC, while IL-18 was secreted preferentially by macrophages. Moreover,
Mtb-infected MDM also produce the immunosuppressive cytokine IL-10.
Because IL-10 is a potent inhibitor of IL-12 synthesis from activated
human mononuclear cells, we assessed the inhibitory potential of this
cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12
secretion from Mtb-infected MDM. In line with these findings,
supernatants from Mtb-infected MDDC induced IFN- production by T
cells and enhanced IL-18R expression, whereas supernatants from MDM
failed to do that. Neutralization of IFN-, IL-12, and IL-18 activity
in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12
and, to a lesser extent, IFN- and IL-18 play a significant role in
enhancing IFN- synthesis by T cells. During Mtb infection,
macrophages and DC may have different roles: macrophages secrete
proinflammatory cytokines and induce granulomatous inflammatory
response, whereas DC are primarily involved in inducing
antimycobacterial T cell immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Once inhaled, Mtb particles are readily phagocytosed, processed, and presented by alveolar macrophages (1). Initially, the establishment of a productive infection depends on the ability of the mycobacteria to invade the alveolar space and to survive within the macrophages. In contrast, infection of the macrophages by Mtb leads to the activation of multiple microbicidal mechanisms, including phagolysosome fusion and respiratory burst, and the production of proinflammatory cytokines, which limit the growth of ingested organisms and the recruitment and activation of additional leukocytes (2, 3).
While innate immune responses initially predominate, the subsequent
recruitment of T lymphocytes to the lung is necessary to the
containment of Mtb within granulomas, which consist of activated
macrophages surrounded by T lymphocytes, fibroblasts, and epitheloid
cells (4). The kinetics of production and the balance
between proinflammatory (IL-1, IL-6, IL-12, and TNF-) and inhibitory
(IL-10 and TGF-
) cytokines secreted by mononuclear phagocytes after
the exposure to microbial Ags regulate subsequent T cell responses and
are also critical for the formation and maintenance of the granuloma.
In turn, cytokines produced by T cells, such as IFN-, can activate
monocyte and macrophages to become microbicidal. Therefore, the
cytokine cross-talk between T cells and mononuclear phagocytes is
essential for the final result of Mtb infection. Four potential
outcomes of Mtb infection can occur according to the fate of the
microorganism inside the macrophages. In fact, Mtb can be immediately
eliminated, becomes dormant indefinitely inside the host, causes a
primary tuberculosis, or reactivates many years after the primary
infection.
Recent studies support the hypothesis that dendritic cells (DC) also strengthen the cellular immune response against mycobacterium infection (5, 6, 7, 8, 9). Even if the critical role of DC in the initiation of immune response has been established (10), their involvement in Mtb infection is poorly defined. DC are highly represented in sites of Mtb infection at the onset of the inflammatory response (11, 12, 13). Immature DC present in the lung mucosa are specialized for Ag up-take and processing. After interacting with pathogens, they mature and migrate in lymphoid organs where they prime T cells through the cell surface expression of MHC and costimulatory molecules and the secretion of immunoregulatory cytokines such as IL-12 (7, 10).
In this study, we have investigated the interactions of virulent Mtb
H37Rv strain with human peripheral blood monocyte-derived macrophages
(MDM) and monocyte-derived dendritic cells (MDDC). We have examined the
modulation of host cell activation markers during the infection and
analyzed the kinetics of cytokine gene expression from
Mtb-infected cells focusing on their ability to stimulate
IFN- production or enhance IL-18R expression on T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAbs specific for CD1a, CD1b, CD14, CD11b, CD64, CD86, CD83,
CD40, CD54, HLA-DR, HLA-DQ, CD58, CD80, mannose receptor, IgG1, IgG2a
(BD PharMingen, San Diego, CA), and IL-18R (R&D Systems, Abingdom, U.K)
were used as pure Abs or as direct conjugates to FITC or PE. Goat
anti-mouse IgG F(ab')2 FITC was used as
secondary Ab where necessary. Anti-CD3 Ab (OKT3, 1 mg/ml; Ortho
Diagnostics, Raritan, NJ) was used for precoating the plate wells for
1 h at 37°C. Following removal of unbound Ab, T cells were
added. Neutralizing anti-IL-12 Ab (R&D Systems) and control IgG (BD
PharMingen) were used at 20 µg/ml after preincubation for 1 h at
37°C with supernatants to neutralize the IL-12 production.
Neutralizing mouse monoclonal anti-IL-18 Ab was used at the
concentration of 40 µg/ml (Euroclone, Devon, U.K.) and rabbit
polyclonal anti-IFN- was used at 20 µg/ml (PBL Biomedical
Laboratories, New Brunswick, NJ). Recombinant human soluble IL-10R was
purchased from R&D Systems and used at 5 µg/ml after preincubation
for 1 h at 37°C with the supernatants. IL-12 was obtained from
R&D Systems. A concentration of 1 µg/ml LPS from Escherichia
coli 0111:B4 (Sigma, St. Louis, MO) was used to induce cytokine
gene expression.
Monocytes, macrophages, DC, and T cells
PBMCs were isolated from freshly collected buffy coats obtained from healthy voluntary blood donors (Blood Bank of University "La Sapienza", Rome, Italy) by density gradient centrifugation using Lympholyte-H (Cederlane, Hornby, Ontario, Canada). Monocytes were purified by positive sorting using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladsbach, Germany). The recovered cells were >99% CD14+ as determined by flow cytometry with anti-CD14 Ab. Macrophages were obtained by culturing adherent monocytes in six-well tissue cultures plates (Costar, Cambridge, MA) with 0.1 ng/ml GM-CSF (Schering-Plough, Innishannon, Ireland) for 5 days at 0.5 x 106 cells/ml in RPMI 1640 (BioWhittaker Europe, Verviers, Belgium) supplemented with 2 mM L-glutamine and 15% FCS (BioWhittaker Europe). DC were generated by culturing adherent monocytes in six-well tissue cultures plates (Costar) with 25 ng/ml GM-CSF and 1000 U/ml IL-4 (R&D Systems) for 5 days at 0.5 x 106 cells/ml in RPMI 1640 with supplements as above. No antibiotics were ever added to the cultures. After day 5 of culture, the cells were analyzed for the expression of surface markers associated with DC as well as macrophage differentiation. The resulting DC were 70–80% CD1a+ and 95% CD14-, while the macrophages were 80–90% CD14+.
T cells were purified by negative sorting using magnetic microbeads (Miltenyi Biotec). The recovered cells were >96% CD3+ as determined by cytometry with anti-CD3 Ab. Purified T cells were primarily stimulated with plate-bound anti-CD3 mAb and cultured for 5 days in the presence of 100 U/ml IL-2 (BD PharMingen) in RPMI 1640 supplemented with 10% FCS. IL-2-containing medium was removed from T cells 16 h before stimulation with MDM or MDDC supernatants.
Mtb and infection of MDM and MDDC
Mtb H37Rv (ATCC 27294; American Type Culture Collection, Manassas, VA) was grown with gentle agitation (80 rpm) in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with 0.05% Tween 80 (Sigma) and 10% Middlebrook oleic acid albumine dextrose catalase enrichment (Becton Dickinson, Sparks, MD). Logarithmically growing cultures were centrifuged at 800 rpm for 10 min to eliminate clumped mycobacteria and then washed three times in RPMI 1640. Mycobacteria were resuspended in RPMI 1640 containing 10% FCS and 10% glycerol and then stored at -80°C. Vials were thawed and bacterial viability was 90% as enumerated by CFU on Middlebrook 7H10 agar plates. All Mtb preparations were analyzed for LPS contamination by the Limulus lysate assay (BioWhittaker Europe) and contained <10 pg/ml LPS.
Bacterial suspensions, at a multiplicity of infection (MOI) from 0.1 to 10 Mtb/cell, were added on macrophages and DC, and, after 16 h of infection at 37°C, the cultures were gently washed (three times) with medium. Macrophages and DC were centrifuged at 800 rpm for 10 min to selectively spin down cells while extracellular bacteria remain in the supernatants. Cells were resuspended in RPMI 1640 supplemented with 2% FCS and cultured for the times indicated in each experiment.
CFU assay
Triplicate samples were assayed for CFU. Culture medium was removed and cells were lysed with water containing 0.06% SDS. Serial dilutions of the bacterial suspensions were plated (six replicates for each dilution) on Middlebrook 7H10 agar plates.
Acid-fast staining
The medium overlying the infected cells attached on coverslips (Nunc, Roskilde, Denmark) was gently aspirated. The monolayers were fixed in 2% formalin for 10 min, dried, and stained with the Kinyoun method (14). After drying and mounting, bacteria were observed by light microscopy. Duplicate monolayers were prepared for each experimental condition.
FACS analysis
Approximately 1–2 x 105 cells were aliquoted into tubes and washed once in PBS containing 2% FCS. The cells were incubated with purified mAbs at 4°C for 45 min. The cells were then washed and fixed overnight with 2% paraformaldeyde before analysis on a FACSCalibur using CellQuest software (Becton Dickinson, Mountain View, CA).
Cytokine determinations
Supernatants from control and Mtb-infected macrophage and DC
cultures were harvested at different times after infection, filtered
(0.2-µm filters) and stored at -80°C. Ab pairs used in ELISA for
IL-1, IL-6, IL-10, and TNF- cytokine levels were obtained from
R&D Systems. IL-12- and IFN--specific ELISA kits were obtained from
R&D Systems, IL-18 ELISA was obtained from Hayashibara Biochemical
Laboratories (Fujisaki Institute, Okayama, Japan), and IFN- ELISA
was obtained from PBL Biomedical Laboratories. Supernatants from
6 to 10 separate experiments were considered. All ELISA were conducted
according to manufacturers’ instructions.
RNase protection assay (RPA)
RNA was extracted from MDM and MDDC with RNeasy kit (Qiagen,
Valencia, CA) according to the manufacturer’s instructions. A
phenol/chloroform extraction was performed to inactivate residual
mycobacterial particles. Then 5 µg of each target RNA was analyzed by
RPA using the hCK-2 multiprobe template set (Riboquant; BD PharMingen).
Linearized DNA templates were used for T7-directed synthesis of
32P-labeled riboprobes using
[-32P]UTP (3000 Ci/mmol, 10 mCi/ml; Amersham
Life Science, Amersham, U.K.). The probes were hybridized overnight and
then digested with RNase T1 and RNase A to remove unhybridized probes
and mRNAs. The protected probes were purified and electrophoresed on a
6% denaturing polyacrylamide gels. Bands were visualized by
autoradiography (XAR film; Eastman Kodak, Rochester, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial studies were designed to determine the infectivity of Mtb
in macrophages and DC. MDM and immature MDDC were generated from the
same blood donors and were allowed to differentiate in culture for 5
days. Once their differentiated phenotypes were acquired, the cells
were infected with increasing MOI, starting at 0.1, 1, and 10 per cell
(Fig. 1
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A). A clear difference was
observed in the number of internalized bacteria, which was 2-fold
higher in DC compared with macrophages (at MOI = 1 the DC harbored
3.1 ± 0.4 bacteria vs 1.4 ± 0.3 bacteria present in
macrophages Fig. 1B).
Cell viability was evaluated both by phase-contrast light microscopic
examination and trypan blue dye exclusion method. Infections of MDM and
MDDC with a MOI of 0.1 and 1 apparently had no effect on cell viability
during a 6-day follow-up period, whereas high cell mortality was seen
in MDDC cultures infected with a MOI of 10 after 3 days. Moreover, the
morphology attained by infected vs uninfected cultures was dependent on
the bacterial doses used. In fact, the morphology acquired by the
infected macrophages with MOIs of 1 or 10 clearly showed cells firmly
attached to the plastic surface (Fig. 1C). Likewise, the
percentage of DC showing the typical morphology with extended fine
dendrites increases when MOIs of 1 or 10 were used to infect the cells
(Fig. 1D). We choose the infectious dose of MOI = 1 to
perform the experiments, because it resulted in cellular activation and
maturation without considerably inducing cell death.
To investigate the differential ability of DC vs macrophages to
internalize mycobacteria, the cell surface expression of two markers
involved in the receptor-mediated uptake (15), i.e.,
mannose receptor and CD11b, was analyzed (Fig. 1E). No
differences were detected in the expression of mannose receptor, while
the levels CD11b were higher in DC compared those present on the
surface of macrophages.
Up-regulation of markers peculiar of activated macrophages and mature DC by Mtb infection
To analyze whether the effect of Mtb infection alters
cell surface expression of markers involved in Ag presentation and T
cell interaction, MDM and MDDC were infected with Mtb and the cell
surface expression of MHC class II DR and DQ, ICAM-1 (CD54), B7.1
(CD80) and B7.2 (CD86), CD40, FcRI (CD64), LFA-3 (CD58), and CD83
was examined (Fig. 2).
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A). No changes in
CD80, CD86, CD64, and MHC class II DR were observed. Conversely, a
strong increase in CD83 expression as well as in costimulatory
molecules CD40, CD80, CD86 and adhesion molecules CD58 and CD54 was
observed in Mtb-infected MDDC (Fig. 2B). Contrary to MDM,
MDDC showed a significant up-regulation of class II DR and DQ
molecules, suggesting that Mtb-infected DC are likely to function as
efficient APCs compared with Mtb-infected macrophages. These
observations are in line with previous reports describing a diminished
capacity for MHC class II-restricted Ag presentation of macrophages
following Mtb infection (16, 17). Also, the low expression
of CD1a and CD1b molecules on macrophages, as compared with the level
present on DC, did not comply with CD1-restricted Mtb Ag presentation
to T cells (data not shown). Cytokine secretion from Mtb-infected macrophages and DC
Next we analyzed the kinetics and the profile of cytokine
secretion from MDM and MDDC during Mtb infection. Cell culture
supernatants were collected at different time points after the
infection and cytokine levels were determined by ELISA (Fig. 3, A and B). MDM
infected with Mtb showed enhanced production of IL-1, IL-6, IL-10,
TNF-, and IL-18. Some differences in the kinetics were seen. In
fact, IL-1 and IL-18 production was fast and evident already at
6–16 h after infection, while IL-6, IL-10 and TNF- steadily
increased up to 24 or 48 h after infection. A clearly different
situation was observed in Mtb-infected MDDC, which produced
low, but reproducible, levels of IL-12 p70 and significant amount of
IFN-, up to 400 pg/ml (equivalent to 
40 IU/ml) (Fig. 3B). In MOI = 1-infected MDDC, some IL-1, TNF-,
and IL-18 production was seen, whereas IL-6 and IL-10 secretion was not
clearly detectable.
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), but the relative
cytokine expression profile remained similar to the one seen at a lower
MOI value. Similarly, in macrophages the cytokine expression pattern
was not markedly altered by a higher infectious dose of Mtb (MOI of 10)
and no production of IL-12 and IFN- was detected (Fig. 4). Thus, the
inhibitory role of IL-10 on IL-12 production was evaluated. IL-12
production was restored in Mtb-infected macrophages when the effect of
IL-10 was blocked by the addition of soluble IL-10R (Fig. 5). A 1.5-fold increase in IL-12 was also
found in Mtb-infected MDDC, pointing to a low production of IL-10,
probably undetectable by conventional ELISA (Fig. 5).
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). In particular, there was a
clear correlation between the IL-12 p70 secretion from DC and the
up-regulation of IL-12 p35 at the mRNA level, which was observed at
16 h after infection. IL-12 p40 mRNA expression, instead, was
strongly induced as early as 8 h after exposure of DC to
Mtb. Conversely, LPS-stimulated IL-12 p35 gene transcription
both in MDM and MDDC. High-level expression of IL-1 mRNA was
detected in DC and especially in macrophages infected with Mtb, whereas
IL-10 gene expression starting at 3 h after infection was only
seen in macrophages. The expression of IL-1, IL-1Ra (receptor
antagonist) and IL-6 was observed in higher levels in
Mtb-infected MDM compared with MDDC (Fig. 6).
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production and IL-18R
expression by cytokines secreted from macrophage and DC infected with
Mtb
Next we investigated whether the cytokines produced by
Mtb-infected macrophages and DC were able to induce IFN- production
from T cells. For these experiments, MDM, MDDC, and T cells were
obtained from the same blood donor. Purified T cells were initially
stimulated with plate-bound anti-CD3 mAbs and cultured in presence
of 100 IU/ml IL-2 for 5 days. IL-2 was removed from T cells 16 h
before stimulation with supernatants from infected MDM and MDDC
cultures collected at 24 h after infection. T cells were incubated
for 16, 24, or 48 h and secreted IFN- levels were analyzed by
ELISA (Fig. 7A). As a control,
T cells were also stimulated with IL-12 (20 ng/ml). Supernatants
obtained from MDM culture were unable to stimulate IFN- production,
whereas MDDC supernatants readily induced IFN- synthesis in T cells
(Fig. 7A).
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gene expression, neutralization and
immunodepletion experiments were conducted. Because IL-10 may
down-regulate IFN- production (18), we tested whether
soluble IL-10R affected the capacity of Mtb-infected MDM supernatants
to stimulate IFN- production (Fig. 7B). Pretreatment of
MDM supernatants with soluble IL-10R leads to a clearly detectable
increase in T cell IFN- secretion (Fig. 7B). To determine
the role of IL-12, IFN-, and IL-18 in the induction of IFN- gene
expression, neutralizing anti-IFN-, anti-IL-12, or
anti-IL-18 Abs were used. Pretreatment of infected MDDC
supernatants with anti-IL-12 Abs significantly down-regulated T
cell IFN- production, while anti-IFN- and anti-IL-18 Abs
exerted a less pronounced, but clearly detectable reduction of IFN-
secretion (Fig. 7C).
In addition of enhancing IFN- production, IFN- and IL-12 may
stimulate a Th1-type response by inducing the expression of IL-18R
(19). Therefore, we tested whether supernatants from
Mtb-infected MDM and MDDC would also stimulate T cell IL-18R
expression. Infected DC supernatants as well as IL-12 were able to
enhance the expression IL-18R on the T cell surface as examined by FACS
analysis (Fig. 8). Treatment of
Mtb-infected MDDC supernatant with anti-IL-12 and anti-IFN-
Abs reduced, at different extent, the number of T cells expressing
IL-18R. Supernatant from infected MDM was a poor inducer of IL-18R, but
after pretreatment with soluble IL-10R the supernatant gained some
ability to stimulate expression of IL-18R on the surface of T
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T
lymphocytes, and T lymphocytes of CD4 and CD8 phenotype are
recruited in a sequential order after Mtb infection. All these cells
share potential cytolytic activity and are able to produce IFN-,
which plays a central role in the host defense against the Mtb.
Activation of these cell subsets is primarily regulated by cytokine
production and presentation of Mtb Ags by infected macrophages
(3, 21). Characterization of some human severe
immunodeficiencies has highlighted an essential role of IL-12 and
IFN- in the control of Mtb (22, 23, 24, 25, 26). Therefore, studies
committed to investigate the balance existing in the granulomatous
response between mononuclear phagocytes and T cells are essential to
understand the changes leading to the dissemination of mycobacteria and
disease or to the reactivation of latent infection.
To investigate the effects of the initial interactions between Mtb and
macrophages or DC on the profile of secreted cytokines, we used in
vitro-cultured human immature MDDC and MDM. Both cell types took up the
Mtb although MDDC appeared to be more active than MDM to internalize
bacteria probably through CD11b-mediated uptake (Fig. 1). The invasion
of DC may be advantageous for intracellular mycobacteria because it may
allow their multiplication and spreading into draining lymph nodes and
lungs.
Moreover, we extended our analysis on the expression of cell surface
markers (Fig. 2). DC infected with Mtb expressed high levels of
costimulatory and adhesion molecules, while macrophages exhibited only
a considerable induction of CD40 and CD54 following Mtb infection.
Moreover, a significant increase of MHC class II DR and DQ was observed
in MDDC while the constitutive expression of MHC class II molecules was
slightly down-modulated in infected MDM as previously reported
(16). Conversely, the up-regulation of these surface
markers in infected DC underlines the capacity of DC to mature
following Mtb infection, which correlates with the acquired ability to
present Ag to T lymphocytes. Thus, our results suggest that while Mtb
infection results in the direct activation and maturation of DC
followed by enhanced presentation of Ag and capacity to stimulate T
cells (7), it impairs the ability of macrophages to
process and/or present soluble Ag and in turn, to serve as accessory
cells in T cell activation.
The production of proinflammatory cytokines is essential for host
resistance against Mtb infection. TNF- production is an important
early event that leads to granuloma formation and a protective host
immune response (27, 28). Macrophage-derived IL-1 enhances
IL-2 production, IL-2R expression, and subsequent clonal expansion of
the CD4+ T cells (3). IL-6 has also
been suggested to be a pivotal proinflammatory cytokine during acute
infection (29). It has been recently found that IL-18,
another proinflammatory cytokine that enhances innate and specific Th1
immune response (30), is important for the generation of
protective immunity to mycobacteria (31, 32). The
differentiation process of T cells is generally initiated by triggering
the Ag receptor and is directed by cytokines present at the time of
priming (33). The expression of inflammatory and
immunoregulatory cytokines was therefore analyzed in supernatants
obtained from MDM and MDDC infected with Mtb. Proinflammatory cytokines
TNF-, IL-1, IL-6, and IL-18 were secreted rapidly at high levels and
in a sustained fashion, preferentially by Mtb-infected MDM, whereas
MDDC produced low or undetectable levels of these cytokines (Fig. 3).
However, when MOIs of 10 were used to infect MDDC, low, but
reproducible, levels of TNF-, IL-1, IL-6 were secreted, suggesting
that a stronger stimulus is required to induce the expression of these
inflammatory cytokines (Fig. 4). This suggests that macrophages and DC
respond to Mtb in a different fashion. A different mechanism is instead
responsible for the absence of IL-18 production from infected DC. It is
likely that caspase activation, which is a prerequisite for the
processing and secretion of IL-18 (30), was not taking
place in DC. In fact, it has been recently described that the precursor
form of IL-18 is constitutively produced by DC although the secretion
of the biologically active form requires CD40 engagement of DC
(34). Similarly, we observed a clear induction of IL-1
mRNA in Mtb-infected MDDC at all examined time points,
whereas a modest secretion of mature IL-1 protein was seen. Whether
this is due to differential ability of Mtb to induce caspase activation
in macrophages and DC is presently not known.
Mtb-infected macrophages produce the immunosuppressive cytokine IL-10.
IL-10 has been shown to inhibit the activation of macrophages
(18, 35, 36) and more recently the differentiation of DC
(37, 38). It is thus likely that high IL-10 production
levels in Mtb-infected macrophages plays an antiinflammatory role
through the inhibition of IL-12 expression (39, 40, 41) as
well as the inhibition of the MHC class II transport to the cell
membrane (42). In line with this concept is that the
neutralization of IL-10 rescued IL-12 production from
Mtb-infected macrophages (Fig. 6); however, we cannot exclude that
other mechanisms could suppress IL-12 synthesis in Mtb-infected
macrophages. Some increase of IL-12 synthesis was also observed in
Mtb-infected MDDC (Fig. 6), suggesting a low level of IL-10 synthesis
in DC as well. However, despite low IL-10 production, MDDC were able to
produce high levels of IL-12 compared with MDM.
IL-10 also inhibits IFN- production and Ag-specific proliferation of
Th1 (43). The hypothesis that IL-10 secretion from
infected macrophages may down-modulate the T cell responses was
confirmed by adding soluble IL-10R to supernatants obtained from
Mtb-infected-MDM (Fig. 8). The presence of IL-18 in the supernatants of
infected macrophages was not sufficient to stimulate IFN- production
from T cells despite the neutralization of IL-10. In fact, it has been
described that IL-18 is a weak inducer of IFN- synthesis from T
cells without the cooperation of IL-12 and IFN- (44).
Therefore, the present findings of impaired ability of Mtb-infected MDM
to stimulate T cells suggests a possible mechanism by which
mycobacteria may evade immune recognition through the reduced
expression of MHC class II molecules and the increased IL-10 production
by infected macrophages.
A typical pattern of Th1/IFN--inducing cytokine production was
produced by DC after Mtb infection. The contact between Mtb and MDDC
resulted in an elevated expression of IL-12 and IFN-. This
observation is in line with the results obtained in vivo with human
subjects demonstrating enhanced IL-12 expression in skin lesions of
patients with tuberculoid leprosy and in tuberculous pleuritis
(45). Moreover, our findings in DC are in agreement with
the recent observations by Mohagheghpour et al. who showed that
Mycobacterium avium-infected DC secreted larger amounts of
IL-12 than infected macrophages (9). Few in vitro studies
have examined the ability and mechanisms of Mtb to directly stimulate
the production of the bioactive p70 IL-12 in human monocytes (46, 47) or in DC (7). In fact, the presence of
activated T lymphocytes that produce IFN- or express CD40 ligand is
generally required to obtain the expression of p70 IL-12 from
macrophages or DC.
IFN- production following Mtb infection of DC is a novel finding. In
fact, IFNs were originally identified as cytokines that mediate
antiviral immunity, but were also found to mediate a protective role
against bacterial infections (48, 49). Our results about
IFN- production from Mtb-infected MDDC are consistent with the data
of Cella et al., who found plasmacytoid DC produce type I IFN in
mycobacteria-infected lymph nodes (50). It has been also
shown that Mtb infection leads to secretion of type I IFN from THP-1
cells (48). All together these observations indicate that
the production of IFN type I could play a dual role in Mtb infection by
promoting both Th1 and DC differentiation (51, 52, 53, 54).
Next, we examined the effects of the cytokines produced by infected MDM
and MDDC on T cell stimulation measured by IFN- release, a parameter
that is indicative of a favorable outcome of tuberculosis (Fig. 7).
Supernatants obtained from MDM culture were unable to stimulate IFN-
production, whereas MDDC supernatants readily induced IFN- synthesis
in T cells (Fig. 7A). Similar effects were observed when MDM
and MDDC supernatants were used to study the expression of IL-18R (Fig. 8). Immunodepletion of IL-12 produced in infected DC significantly
down-regulated IFN- synthesis and IL-18R expression in T cells,
whereas the anti-IFN- and anti-IL-18 Abs exerted less
pronounced effects, but consistent reduction of both IFN- secretion
and IL-18R expression, indicating that both cytokines are inducers
of Th1 cell response. These observations are in line with recent
data indicating that IL-12, IL-18, and IFN- have a significant role
in enhancing Th1 immune response by inducing T cell IFN- production
and the expression of Th1-type cytokine receptors (19, 44, 51, 55).
In the present study, we have demonstrated that human macrophages and
DC are infected by Mtb and these cell types have a unique way to
respond to the infection. Both cell types showed a differential
expression of some cellular adhesion molecules and activation markers
following Mtb infection, in particular MHC class II gene expression
that resulted up-regulated in DC but down-modulated in macrophages.
Moreover, macrophages readily produced proinflammatory cytokines and
IL-10 in response to mycobacteria infection, whereas DC failed to
produce these cytokines in significant amounts and instead released
Th1/IFN--inducing cytokines IL-12 and IFN-. These features also
correlate with the different localizations of activated cells, in
particular the infected DC migrate to lymphoid organs where they liase
with and activate Ag-specific T cells while macrophages are inside the
granuloma and are involved in the establishment of inflammation. Thus,
the results suggest that macrophages and DC clearly have a different
role in Mtb infection. DC are engaged in inducing T cells in virtue of
their production of Th1/IFN--inducing cytokines and expression of
costimulatory molecules while macrophages are primarily involved in the
formation of the granuloma where tissue macrophages harboring tubercle
bacilli are surrounded by and interact with effector T
lymphocytes. Thus, the development of a new generation of vaccine
against tuberculosis has to elicit a strong activation of DC to
stimulate the maximal Ag presentation, the production of IFN- and
IL-12 cytokines, and consequently a protective T cell response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eliana Coccia, Laboratory of Immunology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail address: e.coccia{at}iss.it
3 Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; DC, dendritic cells; MDM, monocyte-derived macrophages; MDDC, monocyte-derived dendritic cells; MOI, multiplicity of infection; RPA, RNase protection assay.
Received for publication November 27, 2000. Accepted for publication April 2, 2001.
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