Published online before print July 7, 2004
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* National Institute for Infectious Diseases "Lazzaro Spallanzani," IRCCS, Rome, Italy;
Department of Biology, Laboratory of Immunopathology and Immunochemistry, University of Rome "Tor Vergata," Italy; and
Laboratory of Cell Biology, Istituto Superiore di Sanità, Rome, Italy
1Correspondence: Laboratory of Immunology and UNESCO Center, Istituto Nazionale Malattie Infettive "L.Spallanzani" Hospital, Via Portuense 292, 00149, Rome, Italy. E-mail: martino{at}inmi.it
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, IL-1ß, IL-6) and IL-10 but not IL-12. BCG-DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection. However, BCG-DCs induced IL-4 production when cocultured with human cord-blood mononuclear cells. The induction of IL-4 production by DCs generated by BCG-infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.
Key Words: mycobacteria • differentiation • CD1a • polarization • cytokines
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(TNF-), or phagocytosis have been demonstrated to interfere with DC differentiation [13
14
]. Many pathogens have evolved mechanisms to exploit DC biology in their dissemination within the body and/or to interfere with DC functions to block or delay their elimination by the host. Several pathogens or their components can interact with imDCs or their precursors, influencing DC generation [15
16
]. Recently, it has been demonstrated that a typical intracellular pathogen, Mycobacterium tuberculosis, is able to subvert the differentiation of infected monocytes into DCs, suggesting an escape mechanism, which contributes to mycobacterial, intracellular persistence [17
]. To date, tuberculosis is the sixth largest cause of death, and the death rate is growing with an estimated annual increase of 3% [18 ]. The bacillus Calmette-Guerin (BCG) strain is a live vaccine that has been used in routine vaccination for nearly 80 years. Although it is almost nonpathogenic, it retains the immunogenic properties of M. tuberculosis [19 ]. BCG is effective in protecting severe forms of disseminated tuberculosis, such as meningitis, in childhood [20 ]; however, its efficacy in protecting adult pulmonary tuberculosis has remained stunningly variable [21 22 ]. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses [23 ]. However, the reasons explaining the failure of BCG still need to be fully resolved. DCs are likely to play a key role in the induction of immune response to M. tuberculosis by polarizing T lymphocyte reactivity toward a T helper cell type 1 (Th1) profile, contributing to the generation of protective cellular immunity against mycobacteria [24 ].
In the present work, we investigated the effects of BCG on the differentiation of monocytes into imDCs and mDCs. We isolated peripheral blood monocytes from healthy donors, infected with BCG, and then cultured them with GM-CSF and IL-4. We found that imDCs derived from BCG-infected precursors acquired a mature phenotype, produced IL-10, and directed a Th2-like immune response. These findings could explain one of the mechanisms underlining the failure of the BCG vaccine to prevent pulmonary tuberculosis.
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1 x 106 colony-forming units (CFU) per 100 µl. BCG viability was verified by CFU assay.
Monocyte infection and DC generation
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors by density gradient centrifugation using Lympholyte-H (Cederlane, Canada). Monocytes were positively separated by anti-CD14 magnetic beads [magnetic cell sorter (MACS), Miltenyi Biotec, Germany], according to the manufacturer’s instructions. The cells were then resuspended in RPMI 1640 (Euroclone, UK), supplemented with 10% fetal calf serum (BioWhittaker, Belgium), L-glutamine (2 mM), Hepes buffer (10 mM), sodium piruvate (0.1 M), nonessential amino acids (0.1 M, Euroclone), and gentamycin (10 µg/ml, Sigma-Aldrich, Germany). Monocytes were infected for 3 h with single-cell suspensions of BCG at a multiplicity of infection (MOI) of 1 or were stimulated with lipopolysaccharide (LPS; from Escherichia coli, 1 µg/ml, Sigma-Aldrich). The infection was carried out in the absence of antibiotics. After the treatment, monocytes were washed and cultured with fresh, complete medium for 5 days in the presence of GM-CSF (200 U/ml) and IL-4 (10 ng/ml, Euroclone) to generate imDCs. To induce final maturation of imDCs, LPS (200 ng/ml) was added at the fifth day of culture for 2 days further. Viability of infected cells was determined by trypan blue exclusion.
Fluorescein-activated cell sorter (FACS) analysis
The following fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies—anti-human leukocyte antigen (HLA) class I, HLA-DR, CD1a, CD11c, CD14, CD40, CD64, CD80, CD83, CD86, and CCR7 (Becton Dickinson Bioscience, Mountain View, CA)—were used for direct immunofluorescence staining to characterize the phenotype of DCs generated in different conditions. Briefly, the cells were washed twice in phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), and 0.1% sodium azide and were stained with the monoclonal antibodies (mAb) for 15 min at 4°C. The cells were then washed and acquired using a FACSCalibur instrument running Cellquest software (Becton Dickinson Bioscience).
Transwell experiments
In Transwell experiments, the infected and uninfected monocytes were separated by a membrane (6.5 mm diameter, 0.4 µm pore size) in 24-well plates (Corning-Costar, Cambridge, MA). The lower compartment of the wells contained untreated monocytes (1x106 cells); the upper compartments contained untreated cells or BCG-infected or LPS-treated monocytes (1x106 cells). The cells were cultured in the presence of GM-CSF and IL-4 in 2 ml culture medium. After 5 days, the cells were harvested from the lower and upper compartments, stained with the anti-CD1a mAb, and analyzed for the CD1a expression by FACS.
Confocal laser-scanning microscopy (CLSM) analyses
For CLSM analyses, 5 x 105 DCs were added per well in a U-bottom microtiter plate and were washed once with PBS containing 0.2 mM NaN3 and 1% BSA. Cells were incubated (30 min, +4°C) with appropriate dilution of anti-CD1a–FITC or antiactin–PE mAb. After extensive washes, the cells were fixed with 3% paraformaldehyde, washed once, and mounted on the microscope slide with the Prolong antifade reagent (Molecular Probes, Eugene, OR). The control samples were treated in the same way. For intracellular staining, the cells were fixed and permeabilized with cold methanol (–20°C, 10 min) and then incubated with the antibody for 30 min at 37°C.
CLSM observations were done using a Leica TCS 4D apparatus, equipped with an Argon-Krypton laser, dichroic splitter (488 nm), and 520 nm long-pass filter for observations with FITC-conjugated antibody. Image acquisition and processing were conducted by using the Scanware (Leica Lasertechnik GmbH, Heidelberg, Germany) and Adobe Photoshop software programs.
Cytokine assay
Supernatants of DCs derived from treated or untreated precursors were collected at the fifth day of culture or after stimulation with LPS (200 ng/ml) for 48 h and were stored at –80°C. The levels of IL-10, IL-12, TNF-, IL-1ß, and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA) kits (Pierce, Endogen, Woburn, MA), according to the manufacturer’s instructions. Supernatants of cord-blood mononuclear cells (CBMCs) and purified CD4+CD45RA+ T cells cocultured with DCs generated from untreated, BCG-infected, or LPS-treated monocytes were tested for IL-4 and interferon-
(IFN-) using commercially available ELISA kits (Pierce, Endogen). Results are expressed as pg/ml.
T lymphocyte proliferation and functional polarization
DCs derived from untreated, BCG-infected, or LPS-treated monocytes (0–1x104 cells) were added to 5 x 104 allogeneic PBMCs in 96-well U-bottom microplates (Corning-Costar). The proliferative response was measured after 5 days of culture and 16 h of incubation with [3H]thymidine (Amercharm, Little Chalfont, UK; 1 µCi/well). Incorporated radioactivity was then assessed by a 1450 MicroBeta Trilux (Wallac, Turku, Finland). Results are expressed as means + SD of triplicate wells. T lymphocyte polarization was evaluated incubating DCs with CBMCs and CD4+CD45RA+ T cells at a ratio of 1:5 for 8 days. CD4+CD45RA+ T cells were purified by incubating CBMCs with anti-CD45RA MACS (Miltenyi Biotec). According to the manufacturer’s instruction, after overnight incubation to allow beads degradation, CD4+ T cells were purified further. Supernatants were collected and analyzed for cytokine accumulation.
Statistical analysis
Statistical analysis was determined using a Student’s t-test. Values of P< 0.05 were considered statistically significant.
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Figure 1. BCG-infected monocytes differentiate into mDCs with a failure of CD1a expression. (A) Histogram plots show the phenotype of DCs derived from BCG-infected (BCG-DCs), LPS-treated (LPS-DCs), or untreated monocytes (imDCs) at the fifth day of culture (filled histograms) and after 48 h of LPS stimulation (empty histograms). Purified monocytes were infected with BCG at MOI 1 or were stimulated with LPS (1 µg/ml) for 3 h and then cultured with GM-CSF and IL-4. The cells were stained for the various markers and acquired by FACSCalibur. Numbers indicate mean fluorescence intensity (MFI) values at the fifth day of culture (bold numbers) and after LPS stimulation (normal numbers). One representative result of 10 independent experiments is shown. (B) Histograms show the mean of 10 experiments
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Figure 2. BCG-monocyte interaction is necessary to generate CD1a– DCs. Treated and untreated monocytes were separated by a membrane (6.5 mm diameter, 0.4 µm pore size) in 24-well plates. Untreated monocytes (1x106 cells) were placed in the lower compartments, whereas untreated, BCG-infected, or LPS-treated monocytes were in the upper compartments (1x106 cells). The cells were cultured in the presence of GM-CSF and IL-4 in 2 ml culture medium. After 5 days, imDCs were harvested from the lower compartments (open bars), and imDCs, DCs derived from BCG-infected (BCG-DCs), or LPS-treated (LPS-DCs) monocytes were collected from the upper compartments (solid bars). The cells were stained with an anti-CD1a mAb and analyzed by FACSCalibur. Results of three experiments are expressed as MFI.
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Figure 3. CLSM analyses of localization of CD1a molecules in DCs derived from untreated (imDCs) or BCG-infected (BCG-DCs) monocytes, which were fixed and permeabilized with cold methanol (–20°C, 10 min) and then incubated with the appropriate dilution of the anti-CD1a–FITC (green) and antiactin–PE (red) mAb for 30 min at 37°C. Image acquisition and processing were conducted by using the Scanware and Adobe Photoshop software programs.
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Figure 4. Antigen-presenting capacity of DCs derived from untreated (imDCs), BCG-infected (BCG-DCs), or LPS-treated (LPS-DCs) monocytes. The cells cultured in the presence of GM-CSF and IL-4 for 5 days (open symbols) or induced to maturate in the presence of LPS (solid symbols) were cocultured with allogeneic peripheral blood T lymphocytes. After 5 days, T cell proliferation was assessed by the addition of tritiate thymidine for 16 h. Results of two experiments are expressed as mean counts per minute (CPM) of triplicate cultures.
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, IL-1ß, and IL-6 in imDCs, BCG-DCs, and LPS-DCs was evaluated at the fifth day of culture by ELISA. Uninfected DCs were not able to produce TNF- and IL-1ß cytokines but only low amounts of IL-6 in the absence of maturation stimuli (Fig. 5
). Similar results were obtained by DCs generated from LPS-treated monocytes, although they secreted high levels of IL-6. In contrast, BCG-DCs produced high levels of TNF-, IL-1ß, and IL-6, showing that they have acquired an activated phenotype. Furthermore, the accumulation of IL-10 and IL-12, which are important factors involved in directing immune response, was measured at the fifth day of culture or after LPS stimulation. In the absence of maturation stimuli, imDCs did not produce IL-12 or IL-10, and after LPS stimulation, the production of IL-12 was induced as previously reported (Fig. 6
). Conversely, DCs generated from LPS-treated monocytes secreted high amounts of IL-12 and low levels of IL-10 at the fifth day of culture and after maturation stimuli. In contrast, BCG-DCs produced high levels of IL-10 and were not able to produce IL-12 before or after LPS stimulation. These data indicate that BCG-infected monocytes are able to differentiate into DCs that produce proinflammatory cytokines and IL-10 at 5 days of culture or upon maturation stimuli.
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Figure 5. Proinflammatory cytokine secretion by DCs derived from untreated (imDCs), BCG-infected (BCG-DCs), or LPS-treated (LPS-DCs) monocytes. Cell supernatants were collected at the fifth day of culture and tested for TNF-, IL-1ß, and IL-6 by ELISA. Results are expressed as means (±SD) of six different experiments, and statistical analysis was determined by nonparametric t-test. The significant levels between BCG-DCs and LPS-DCs compared with imDCs were P< 0.05.
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Figure 6. DCs derived from BCG-infected monocytes (BCG-DCs) produce high levels of IL-10. Cell-culture supernatants were collected from untreated DCs (imDCs), BCG-DCs, and DCs derived from LPS-treated monocytes (LPS-DCs) at the fifth day (A) or after a further 48 h of LPS stimulation (B). IL-10 and IL-12 were tested by ELISA. Data are expressed as means (±SD) of six different experiments, and statistical analysis was determined by nonparametric t-test. The significant levels (*) between BCG-DCs compared with imDCs were P< 0.05.
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and significantly less IL-4 (Fig. 7A
). In contrast, CBMCs cultured with BCG-DCs produced high levels of IL-4 and a low amount of IFN-. Conversely, CD4+CD45RA+ T cells cocultured with BCG-DCs are not able to produce IFN- or IL-4 compared with CD4+CD45RA+ T cells stimulated with LPS-DCs, which produced IFN- as expected (Fig. 7B)
. These data show that BCG-infected monocytes differentiate into DCs inducing a Th2-like response and are unable to induce the production of IFN-, which is necessary to elicit a protective, immune response against mycobacteria.
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Figure 7. T cell polarization by DCs derived from untreated (im-DCs), BCG-infected (BCG-DCs), and LPS-treated (LPS-DCs) monocytes, which were cocultured with CBMCs (A) or CD4+CD45RA+ T cells separated from cord blood (B). After 8 days of culture, the supernatants were collected and tested for IFN- and IL-4 by ELISA. Data are expressed as means (±SD) of six different experiments, and statistical analysis was determined by nonparametric t-test. The significant levels (*) between IFN- and IL-4 induced by BCG-DCs and LPS-DCs (A and B) were P< 0.05.
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In the pathology of tuberculosis, the early interaction between DCs, present as a dense network in the airway mucosa, and M. tuberculosis is likely to be critical for mounting a protective, antimycobacterial immune response [31 32 ]. However, M. tuberculosis remains latent much of the time in the lifetime of the host. The unique function of DCs in eliciting and directing the cellular immune response may be modulated by mycobacteria [33 34 35 ]. Recently, it has been demonstrated that M. tuberculosis is able to subvert DC differentiation of infected monocytes to escape immune response [17 ].
The benefits of the only available tuberculosis vaccine, BCG, in the protection against M. tuberculosis have been debated since early in its use, including safety aspects, loss of sensitivity to tuberculin as a diagnostic reagent, and in particular, the failure of BCG in a number of trials in the Third World. There have been many hypotheses to explain the inadequate, protective effect of BCG against tuberculosis as the lack of an effective stimulation of T cell populations [23 ]. However, to develop a novel vaccine strategy superior to BCG, fundamental immunological mechanisms responsible for protection, as well as reasons explaining the failure of BCG, still need to be fully resolved [36 37 ].
We investigated whether the infection of monocytes with BCG could interfere with their differentiation into DCs and analyzed the capacity of BCG-DCs to direct T cell immune response. We found that BCG-infected monocytes differentiate into cells with an acquired, mature phenotype, since they lose CD14, they express CD83 and up-regulate CD80, CD86, and MHC class I and class II molecules in the absence of maturation stimuli. In addition, DCs generated from BCG-infected monocytes express the CCR7 on their surface. The expression of this chemokine receptor in BCG-DCs is particularly relevant, as it allows homing to secondary lymphoid organs, where DCs can present antigens to specific T lymphocytes [38 ]. It is known that during the differentiation of precursors into DCs, the cells acquire CD1a molecules on their surface, allowing them to present lipid and glycolipid antigens to T lymphocytes. However, BCG-DCs do not express CD1a molecules on their surface. In contrast, we found by confocal analyses that CD1a molecules are sequestrated in intracellular compartments. This finding is in accordance to previous investigations revealing that CD1 expression can be down-regulated by mycobacteria in infected DCs [39 40 ]. Conversely, LPS-treated monocytes differentiate into imDCs, showing a phenotype similar to the untreated cells except for the MHC class II up-regulation, suggesting that the treatment of monocytes with inflammatory stimuli does not completely account for the generation of DCs, presenting an early, mature phenotype. Although BCG-DCs show a mature phenotype at the fifth day of culture, they are not more efficient in stimulating T cells compared with imDCs and LPS-DCs. However, upon further maturation stimuli, their antigen-presenting capacity is increased, showing that they are able to acquire the capacity of fully mDCs.
In addition, BCG-DCs acquire an activated phenotype at the fifth day, as they produce proinflammatory cytokines such as TNF-, IL-1ß, and IL-6, showing that they are able to elicit an inflammatory, immune response. It has been reported that the production of proinflammatory cytokines by APCs promotes the recruitment and activation of additional leukocytes, playing a central role to limit the growth of intracellular pathogens [41
].
DCs are known for their capacity to produce immunoregulatory cytokines such as IL-12 or IL-10 upon maturation stimuli. Therefore, the balance of the production of these cytokines plays a pivotal role by orchestrating an innate and acquired immune response and determining the polarization of T cell precursors [42
43
]. It is interesting that we found that BCG-DCs, before or after maturation stimuli, produce high levels of IL-10 and are not able to produce IL-12 levels comparable with imDCs. Conversely, LPS-DCs secrete IL-12 and not IL-10 at the fifth day of culture or after a further LPS-induced maturation. The apparent disagreement of our findings with earlier studies, which showed that BCG is a trigger of IL-12 production of mDCs, can be reconciled on the grounds that cytokine production is modulated differentially, depending on the timing and the maturation stage of target cells. Indeed, in our model, we infected human monocytes and not differentiated DCs, as it was in other experimental studies [44
45
]. The secretion of IL-10 and the inhibition of IL-12 synthesis can account for the inability of BCG-DCs to induce the expansion of effector Th1 lymphocytes. Indeed, BCG-DCs induce a Th2-like immune response when cultured with CBMCs, as shown by high levels of IL-4 and low amounts of IFN- production. However, the production of IL-10 by BCG-DCs cannot completely be taken into account for the IL-4 production, as neutralizing anti-IL-10 antibodies did not reverse the cytokine profile (data not shown). Furthermore, purified CD4+CD45RA+ T cells cocultured with BCG-DCs were not able to produce IL-4 or IFN-. These results could be explained by the presence of multiple cell types in CBMCs able to produce IL-4 such as CD8+ T cells. The loss of IFN--producing T cells could be associated with a nonprotective, immune response and an ineffective role in the killing of intracellular bacteria by macrophages. Several studies indicate that the IFN- is the key cytokine in the control of M. tuberculosis infection, triggering the antimycobacterial mechanism of macrophages in synergism with TNF- [46
47
].
We hypothesize that in the initial steps of exposure, BCG can infect recluted DC precursors and induce them to differentiate into DCs with a mature phenotype producing proinflammatory cytokines that are responsible to amplify the response by promoting the recruitment of additional leukocytes. DCs derived from BCG-infected monocytes migrate to secondary lymphoid organs and produce high levels of IL-10, inducing a Th2-like immune response. These results could account for the inflammatory response induced by BCG vaccination and could explain one mechanism underlining the failure of the BCG vaccine to prevent pulmonary tuberculosis.
Received July 4, 2003; revised April 15, 2004; accepted May 28, 2004.
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switches monocyte differentiation from dendritic cells to macrophages Blood 101,143-150 J. Exp. Med. 179,1109-1118 and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in CD14+ monocytes through distinct pathways that activate NK-
B J. Immunol. 165,3647-3655-independent activation of dendritic cells following treatment with Mycobacterium bovis bacillus Calmette-Guerin Immunology 97,626-633[CrossRef][Medline] B and ß degradation Pediatr. Res. 54,105-112[CrossRef][Medline] in resistance to Mycobacterium tuberculosis infection J. Exp. Med. 178,2249-2254-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals; associations with clinical disease state and effect of treatment J. Immunol. 167,5217-5225This article has been cited by other articles:
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