CD16+ human monocyte-derived dendritic cells matured with different and unrelated stimuli promote similar allogeneic Th2 responses: regulation by pro- and anti-inflammatory cytokines

Amaranta Rivas-Carvalho1, Marco A. Meraz-Ríos1, Leopoldo Santos-Argumedo1, Sandra Bajaña3, Gloria Soldevila4, Miguel E. Moreno-García2 and Carmen Sánchez-Torres1

1 Department of Molecular Biomedicine and 2 Department of Cellular Biology, Centro de Investigación y de Estudios Avanzados-IPN, 3 Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina del IPN and 4 Department of Immunology, Instituto de Investigaciones Biomédicas, UNAM, Mexico City, Mexico

Correspondence to: C. Sánchez-Torres; E-mail: csanchez{at}mail.cinvestav.mx


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We previously demonstrated that tumor necrosis factor (TNF)-{alpha}-matured CD16 and CD16+ human monocyte-derived dendritic cells (16–mDC and 16+mDC) differentially stimulate naive CD4+ lymphocytes by inducing Th1- and Th2-like responses, respectively. Here, we further characterized the role of different DC maturation factors on Th polarization. Immature 16+mDC and 16–mDC (iDC) obtained by culture of purified monocytes with GM-CSF and IL-4 were maturated with (i) Toll-like receptor (TLR) ligands [lipopolysaccharide (LPS)], (ii) lymphocyte-derived (soluble CD40 ligand, IFN-{gamma}) and (iii) endogenous inflammatory stimuli [TNF-{alpha}, prostaglandin (PG)E2]. After activation with these stimuli, DC secrete IL-12 only in presence of LPS, and 16+mDC produced lower amounts of IL-12 and IL-10 than 16–mDC. Allogeneic CD4+CD45RO lymphocytes co-cultured with 16+mDC secreted higher levels of IL-4 and IL-10 than those co-cultured with 16–mDC, regardless of the maturation stimuli. Results were similar when DC were activated with TLR-2 or TLR-3 ligands. The higher induction of IL-4 by 16+mDC was primarily dependent on IL-12, IL-4 and IL-10. IFN-{gamma} production by CD4+ T cells was similar with all the conditions except with LPS-16+mDC, which induced reduced amounts of this cytokine. Those differences were totally eliminated by neutralization of IL-12, IL-4 or IL-10. Finally, 16–mDC could reverse the Th2 phenotype of already committed lymphocytes toward a Th1 pattern in short-term cultures, whereas 16+mDC had less ability to skew this phenotype. These results indicate that 16+mDC elicit superior Th2 responses independently of the maturation factors that they received, and suggest that they could represent an important population of regulatory DC.

Keywords: allogeneic CD4+ T cell differentiation, antigen presenting cell subsets


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Circulating blood monocytes are precursors of myeloid dendritic cells (DC) both in vitro (1) and in vivo (2). In vivo, they migrate from blood to inflammatory sites where they seem to differentiate into DC when phagocytic or chemoattractant stimuli are present (2,3). Then, monocyte-derived DC could reach the draining lymph nodes (2), and they are able to present antigens to naive lymphocytes (4). CD14+CD16+ human blood monocytes are a cell subset that comprises only 1% of circulating peripheral blood mononuclear cells (PBMC) (5). However, important evidence for their accumulation in the marginal intravascular pool has been provided (6). Notably, they showed a higher potential to become DC than regular CD14hiCD16 monocytes in a model of transendothelial trafficking (7). Furthermore, a recent report described the homologous CD16+ (CD14lowCD16hiCX3CR1hi) and CD16 (CD14hiCD16low/–CX3CR1low) subsets in mice (8). After adoptive transfer, both subpopulations could become DC, but CX3CR1low monocytes differentiate into CD11c+/MHC class II (I-A)+ DC in inflammatory conditions, whereas CX3CR1hi monocytes might be precursors for resident myeloid cells in non-inflamed tissues (8). The biological significance of these data is still unknown, but it is conceivable that human CD16+ monocytes might be relevant precursors of tissue-resident DC in vivo. We have previously demonstrated that human CD16+ and CD16 monocytes could differentiate into DC (subsequently referred as 16+mDC and 16–mDC) when cultured with GM-CSF, IL-4 and tumor necrosis factor (TNF)-{alpha} as the maturation stimulus (9). One of the most striking features of 16+mDC is their preferential activation of allogeneic Th2 responses, which correlates with low production of IL-12 p70 and elevated levels of transforming growth factor (TGF)-ß mRNA after lipopolysaccharide (LPS) stimulation. Conversely, 16–mDC activate Th1 responses and secrete considerable amounts of IL-12 (9).

The Th1/Th2 dichotomy in humans has been useful to explain certain patterns of immune responses, mainly related with pathological disorders (i.e. allergies, autoimmune diseases, responses to infectious agents such as Mycobacterium leprae) and delayed-type hypersensitivity (10,11). Human DC could influence the Th1/Th2 phenotype of naive lymphocytes according to their cellular lineage (12), their kinetics of activation (13) and the effect of qualitatively different signals from their antigenic microenvironment (14,15). The nature of pathogen- or tissue injured-derived ‘third signal’, as proposed by Kalinski et al. (16), would polarize the DC to establish an initial Th1/Th2 commitment on T lymphocytes (instruction model). However, specialized types of DC could also elicit different Th cell responses when the same stimulus is given (lineage model) (12,17). The latter system might be inefficient, because DC need some flexibility to induce the appropriate responses as they encounter Th1- or Th2-promoting factors (16). Alternatively, different sets of receptors for pathogens or inflammatory cytokines could be displayed by subtypes of DC, thus DC would be intrinsically instructed to skew Th cell responses (18,19). 16+mDC and 16–mDC behaved according to this last hypothesis, as they induced differential responsiveness on T lymphocytes when they were developed under the same cytokine environment (9). In the present work, we study in depth this phenomenon by evaluating the functional plasticity of 16+mDC and 16–mDC in response to a variety of stimuli, and by identifying the factors involved in DC-induced Th cell polarization. We found that 16+mDC induced higher levels of IL-4 than those produced by 16–mDC after stimulation of allogeneic naive CD4+ lymphocytes, independently of the stimuli used for maturation. In addition, 16+mDC secreted low amounts of IL-12, and this cytokine, together with IL-10 and IL-4, influenced the induction of the Th2 pattern.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Media and reagents
Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (Hyclone Laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µM 2-mercaptoethanol (Gibco BRL, Grand Island, NY), referred to as complete medium (CM). For monocyte culture, 5% heat-inactivated pooled human serum was added to the CM. Human recombinant GM-CSF (1000 U/ml), IFN-{gamma} (1000 U/ml), IL-12 (1 ng/ml), rat neutralizing mAb to IL-4 (MP4-25D2, 5 µg/ml), to IL-10 (JES3-19F1, 5 µg/ml), murine neutralizing mAb to IL-12 (C8.6, 10 µg/ml), and their respective Ig isotype controls, were purchased from BD PharMingen, San Diego, CA. Human IL-2 (20 U/ml) was obtained from Gibco BRL. Human IL-4 (15 ng/ml) and prostaglandin (PG)E2 (0.1 µg/ml) were provided by Calbiochem, La Jolla, CA. Human TNF-{alpha} (40 ng/ml) and chicken neutralizing polyclonal antibody to TGF-ß1 (5 µg/ml) were purchased from R&D Systems, Minneapolis, MN. Human soluble CD40 ligand (sCD40L, 1 µg/ml) was provided by PeproTech, Rocky Hill, NJ. Mefenamic acid (10 µg/ml) and LPS (Escherichia coli 0111:B4, 0.5 µg/106 cells/ml) were purchased from Sigma-Aldrich, St Louis, MO. Peptidoglycan (PGN) from Staphylococcus aureus (10 µg/ml) was obtained from Fluka (Milwaukee, WI), polyinosinic–polycytidylic acid (poly-I:C, 50 µg/ml) from Amersham Life Science (Buckingham, UK) and the oligodeoxynucleotide 2006 containing a CpG motif (2 µM) was purchased from Invitrogen Life Technologies (Carlsbad, CA).

Cell separation and differentiation of peripheral blood monocytes
PBMC were isolated from buffy coats of healthy volunteers by Ficoll–Hypaque (Gibco BRL) density gradient centrifugation. Subsequently, CD56CD16+cells and CD56CD16CD14+ cells (referred to as CD16+ and CD16 monocytes) were separated by magnetic cell sorting, using MACS isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany), as published elsewhere (9). Allogeneic naive CD4+CD45RO T lymphocytes from adult blood were separated by negative selection using the MACS CD4+ T cell isolation kit (Miltenyi Biotec), followed by incubation with MACS anti-CD45RO antibody. These procedures routinely provided 90–95% pure monocytes (Fig. 1A and B) and 85–95% pure naive T cells. CD16+ cells could be subdivided into two populations according to their expression of CD14 (CD14+: 72.8 ± 12%, CD14: 27.1 ± 12%) as has been reported (9).



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Fig. 1. Purity of CD16 and CD16+ monocytes after isolation from PBMC. (A) Forward and side scatter of total PBMC, CD16 and CD16+ monocytes. (B) Staining of monocyte subpopulations with FITC-labeled anti-CD16 and PE-labeled anti-CD14 mAb. Percentage of positive cells is specified. (C) Analysis of 16–mDC (top panel) and 16+mDC (bottom panel) matured with the different stimuli indicated on the top of the histograms. Cells were stained with anti-HLA-DR mAb (solid lines), or an isotype-control mAb (dotted lines).

 
Isolated CD16+ and CD16 monocytes were cultured at 106 cells/ml in polystyrene six-well plates (Costar Corp., Cambridge, MA), supplemented with GM-CSF and IL-4. The cultures were fed with fresh medium and cytokines every 2 days. After 6 days, non-adherent DC were harvested and replated at 5 x 105 cells/ml in medium containing GM-CSF and IL-4 for two additional days, with or without the following stimuli: PGE2, TNF-{alpha}/PGE2, sCD40L/IFN-{gamma}, LPS, LPS/IFN-{gamma}, PGN, poly-I:C, or CpG DNA.

Immunofluorescence assays
Monocytes were analyzed immediately after isolation by direct staining with FITC-labeled anti-CD16 (3G8) and phycoerythrin (PE)-labeled anti-CD14 (M5E2) mAb. At the end of the culture (day 8), DC were stained with anti-HLA–DR (TU36), -CD1a (HJ149), -CD40 (5C3), -CD80 (BB1), -CD83 (HB15a) and -CD86 (IT2.2) mAb (BD PharMingen except CD83, Immunotech, Marseille, France), followed by FITC-conjugated goat F(ab')2 anti-mouse Ig polyclonal antibody (Dako, Glostrup, Denmark). Early cell apoptosis was evidenced by staining with biotin-labeled annexin V (BD Pharmingen) and PE-conjugated streptavidin (Dako). Samples were analyzed on a FACSCalibur using CellQuest software (Becton Dickinson, Mountain View, CA).

Cytokine detection on DC
Production of IL-12 p70, IL-10 and TGF-ß1 in the supernatants of stimulated immature DC (iDC) was quantified by ELISA kits obtained from BD PharMingen. 105 iDC (day 6 of culture) were incubated for 24 h with PGE2, TNF-{alpha}, TNF-{alpha}/PGE2, sCD40L/IFN-{gamma}, LPS or LPS/IFN-{gamma}, at the concentrations mentioned above. Controls were established with iDC cultured for 24 h without additional stimulus. In some assays, the competitive inhibitor of cyclooxygenase (COX-1 and -2) mefenamic acid, neutralizing antibody to IL-10, or to TGF-ß1, were added to the cultures.

Protein extraction and immunoblotting
Nuclear and cytoplasmic extracts were prepared with the NE-PER® Nuclear and Cytoplasmic Extraction Reagents kit (Pierce Biotechnology, Rockford, IL), according to the manufacturer's indications. Protein extracts were separated on 10% polyacrylamide gels and transferred to nitrocellulose sheets. Membranes were immunoblotted with rabbit anti-human p50 and c-Rel (20) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig antibody (Sigma-Aldrich). Protein loading was controlled with mouse anti-human ß-actin antibody (kindly provided by Dr M. Hernández, CINVESTAV-IPN) for cytoplasmic extracts, and goat anti-human lamin B antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for nuclear extracts, followed by HRP-conjugated goat anti-mouse or rabbit anti-goat Ig antibody (Zymed Laboratories, South San Francisco, CA), respectively.

T cell activation
To assess cytokine production by T lymphocytes, purified CD4+CD45RO T cells were co-cultured with allogeneic DC at 10:1 ratio in CM. Controls were set without addition of DC. When used, neutralizing mAb to IL-4, IL-10, IL-12, or the cytokines IL-4 and IL-12, were added at the beginning of the co-cultures. After 5 days of priming, T cells were expanded with IL-2 for 6 days. Then, lymphocytes were washed extensively and re-stimulated for 6 h with PMA (20 ng/ml) plus ionomycin (500 ng/ml) for intracellular staining of IL-4 and IFN-{gamma}, or 24 h with immobilized anti-CD3 mAb (5 µg/ml, UCHT1, BD PharMingen) for quantitation of cytokine secretion. For intracellular staining of IL-4 and IFN-{gamma}, GolgiPlugTM (BD PharMingen) was added to the cultures 4 h before cells were harvested, to prevent cytokine secretion. Then, cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD PharMingen) and incubated with FITC-labeled anti-IFN-{gamma} (4S.B3) and PE-labeled anti-IL-4 (8D4-8) mAb (BD PharMingen). Quantitation of IFN-{gamma}, IL-4 and IL-10 secretion in the supernatants was performed by ELISA kits from BD PharMingen.

Analysis of the reversibility of Th1 and Th2 phenotype by DC
CD4+CD45RO T lymphocytes were cultured for 7 days in the absence of stimulus (naive) or in presence of immobilized anti-CD3 mAb plus IL-12 (4 ng/ml) and anti-IL-4 neutralizing mAb (5 µg/ml) (Th1 conditions) or plus IL-4 (30 ng/ml, Th2 conditions). After that time, lymphocytes were harvested, washed and co-cultured with allogeneic PGE2- or LPS-matured 16+mDC or 16–mDC (1:10 DC:T cell ratio) for five additional days. Controls were set in the absence of DC. Then, the amount of IFN-{gamma} and IL-4 secreted by T lymphocytes was measured after 24 h of re-stimulation with immobilized anti-CD3 mAb, as described above.

Statistical analysis
Data were expressed as mean ± SD of independent experiments. The statistical significance of the data was determined by the Student's two-tailed paired t-test, assuming equal variances.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Phenotype and lymphoproliferative activity of 16+mDC and 16–mDC
Phenotype of 16+mDC and 16–mDC is shown in Table 1 and Fig. 1(C). DC matured with different stimuli seemed to generate homogeneous cell populations, as indicated by staining with anti-HLA-DR antibody (Fig. 1C). Immature DC were similar in their phenotype, except for the elevated expression of CD86 and the low levels of CD1a on 16+mDC. Significant low expression of CD1a by 16+mDC was maintained after maturation, and it appears to be a feature of these cells (9). Enhanced expression of CD86 was also conserved after maturation, with the exception of DC matured with LPS and lymphocyte-derived stimuli, where its expression became similar. CD40 tended to be augmented on 16–mDC, whereas CD80 expression was significantly increased on 16+mDC matured with endogenous inflammatory stimuli. Regarding CD83 expression, PGE2 alone and sCD40L/IFN-{gamma} were weaker maturation factors compared to LPS/IFN-{gamma}, LPS or TNF-{alpha}/PGE2. For sCD40L/IFN-{gamma}, these data differ from previous reports (21). It is possible that the CD40L used here, which is soluble and monomeric, might deliver a low activating signal compared with membrane or soluble CD40L trimers used in other works. Taking all markers together, LPS/IFN-{gamma} was the best maturation stimulus for both DC, followed by TNF-{alpha}/PGE2 for 16+mDC and LPS for 16–mDC. The increment of total numbers of CD4+ lymphocytes after stimulation with allogeneic DC showed that mature DC induced higher proliferative responses than iDC (Table 1). TNF-{alpha}/PGE2 and LPS/IFN-{gamma} were the strongest stimuli of maturation for 16–mDC, whereas results were similar for 16+mDC with all stimuli. Immature and LPS-16+mDC showed significant higher lymphoproliferative activity than 16–mDC.


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Table 1. Phenotype and lymphoproliferative activity of 16–mDC and 16+mDC

 
Production of IL-12 and IL-10 by DC activated with different stimuli
Secretion of IL-12 and IL-10 produced by iDC was consistently observed in presence of LPS (Fig. 2A) and not with the other stimuli, although in a few experiments sCD40L/IFN-{gamma}- and TNF-{alpha}/PGE2-16–mDC also produced low but detectable amounts of IL-12 (not shown). Combination of LPS with IFN-{gamma} was a powerful stimulator of IL-12 secretion, while it decreased the production of IL-10 (Fig. 2A). Secretion of IL-12 and IL-10 by 16+mDC was significantly lower than produced by 16–mDC. However, 16+mDC have the potential to secrete high levels of IL-12 when the appropriate stimulus is given (i.e. LPS/IFN-{gamma}; Fig. 2A). The differences observed in cytokine expression were not due to early apoptosis, since <7% of cells stained positively for annexin V after stimulation (not shown).



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Fig. 2. Cytokine production by 16–mDC and 16+mDC. (A) IL-12 and IL-10 production by immature 16–mDC (black bars) and 16+mDC (gray bars) stimulated for 24 h with LPS or LPS/IFN-{gamma}. Results are the mean ± SD of six (IL-12) or four (IL-10) independent experiments. Statistical analysis: *P < 0.05. (B) Regulation of IL-12 expression by immature 16–mDC (black bars) and 16+mDC (gray bars) stimulated for 24 h with LPS (left) or LPS/IFN-{gamma} (right). DC were activated with LPS or LPS/IFN-{gamma} in absence (control) or presence of neutralizing antibody against IL-10, TGF-ß1 and the COX inhibitor mefenamic acid. Data are expressed as the mean ± SD of four (LPS) and three (LPS/IFN-{gamma}) independent experiments. (C) Visualization of NF-{kappa}B subunits c-Rel and p50 in nuclear and cytoplasmic extracts of 16–mDC and 16+mDC after 4 h of LPS stimulation. Proteins were separated on a 10% SDS–polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane. Lamin B and ß-actin were used as protein loading control for nuclear and cytoplasmic extracts, respectively.

 
We also performed analysis of TGF-ß1 production by DC, as we have previously reported the augmented expression of TGF-ß1 mRNA on 16+mDC after LPS stimulation (9). However, we detected production of this cytokine only in one out of four experiments, and values were superior on 16–mDC (not shown).

Modulation of IL-12 production by DC
In an attempt to assess the factors involved in the differential production of IL-12 by 16+mDC and 16–mDC, we performed a blockade of IL-10, TGF-ß1 or COX during LPS or LPS/IFN-{gamma} stimulation (Fig. 2B). IL-10 had potent suppressor effects on IL-12 production by DC, while TGF-ß1 and PGE2 had only marginal effects, and no synergy with IL-10 was observed in any experiment (not shown). The dependence on IL-10 was obviously related to the ability of DC to produce IL-10 after LPS or LPS/IFN-{gamma} activation. Then, as 16–mDC always secreted IL-10 with both stimuli, blockade of IL-10 always resulted in increased production of IL-12. On the other hand, 16+mDC secreted IL-10 only in some experiments in response to LPS, and we detected IL-10 after LPS/IFN-{gamma} stimulation only in one out of four experiments (Fig. 2A). Therefore, IL-10 blockade led to an augmented expression of IL-12 by 16+mDC in some but not all assays. However, in experiments where LPS-stimulated 16+mDC produced IL-10, blockade of this cytokine did not lead to a production of IL-12 as high as 16–mDC. Consequently, IL-10 production by 16+mDC is not primarily responsible for their low secretion of IL-12.

Neutralization of these factors when DC were activated with PGE2, TNF-{alpha}/PGE2, or sCD40L/IFN-{gamma} did not result in detectable production of IL-12 (not shown).

Specific NF-{kappa}B subunits such as p50 and c-Rel are responsible for IL-12 transcription on DC (22,23). Thus, we hypothesized that low production of IL-12 by 16+mDC could be the consequence of a lower rate of nuclear translocation of p50 or c-Rel, or lower levels of these proteins, compared to 16–mDC. However, Fig. 2(C) shows that after 4 h of stimulation with LPS, p50 and c-Rel subunits are detected on nuclear and cytoplasmic extracts of both DC subtypes, and similar or even higher levels (c-Rel) were found on 16+mDC. Therefore, p50 and c-Rel do not seem to be directly involved in differential production of IL-12 by these DC.

16+mDC stimulate alloantigen-specific T cells to produce high levels of IL-4 and IL-10
Given the differences on IL-12 production by 16–mDC and 16+mDC, we asked if this event would be reflected in the extent of naive CD4+ T cell polarization. As shown in Fig. 3(A), both DC induced secretion of IFN-{gamma}, IL-4 and IL-10 in allogeneic assays. Similar responses were observed without IL-2 expansion (not shown). Primed T cells secreted similar amounts of IFN-{gamma} with the exception of LPS-matured cells, where 16+mDC induced lower levels of this cytokine (P = 0.01). Production of IL-4 and IL-10 was greater with 16+mDC than with 16–mDC (2–6-fold higher for IL-4, 1.4–2-fold higher for IL-10), regardless of the stimuli used for maturation. Values of IFN-{gamma}/IL-4 ratios were superior with 16–mDC than with 16+mDC, mainly due to the low levels of IL-4 produced during co-cultures of 16–mDC and T cells (Fig. 3A). The highest differences (4–5-fold) were observed with LPS- and LPS/IFN-{gamma}-DC, and the lowest differences corresponded to TNF-{alpha}/PGE2-DC (2-fold). Secretion of IL-4 and IL-10 by lymphocytes primed with 16+mDC was highly correlated, and the correlation was less prominent with 16–mDC (Fig. 3B). At the single cell level, the number of IL-4+ cells showed fewer divergences between 16–mDC and 16+mDC (19.4 ± 8.6% and 24.9 ± 3.6%, respectively), but they were significant for iDC, PGE2- and TNF-{alpha}/PGE2-DC (Fig. 3C).



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Fig. 3. Production of IFN-{gamma}, IL-4 and IL-10 after co-cultures of allogeneic CD4+CD45RO T lymphocytes with 16–mDC (black bars) or 16+mDC (gray bars). (A) Lymphocytes were cultured with either iDC or DC matured with PGE2, TNF-{alpha}/PGE2, sCD40L/IFN-{gamma}, LPS or LPS/IFN-{gamma}. After 5 days of priming, lymphocytes were expanded with IL-2 for six additional days. Then, T cells were re-stimulated for 24 h with immobilized anti-CD3 mAb. Analysis of IFN-{gamma}, IL-4 and IL-10 secretion on culture supernatants was performed by ELISA. Production of cytokines by lymphocytes cultured in absence DC was subtracted. Results are the mean ± SD of 12 independent experiments. The bottom right graph corresponds to the mean ± SD of IFN-{gamma}/IL-4 ratios. (B) Analysis of the correlation between IL-4 and IL-10 secretion by lymphocytes primed with 16–mDC or 16+mDC: (open diamonds) iDC, (closed squares) PGE2-, (open triangles) TNF-{alpha}/PGE2-, (closed diamonds) sCD40L/IFN-{gamma}-, (closed triangles) LPS-, (open squares) LPS/IFN-{gamma}-DC. Each point represents the correlation index between IL-4 and IL-10 values obtained in 12 individual experiments. (C) Percentage of IL-4+ lymphocytes after DC:T cells co-cultures, as described above. After a 6 h re-stimulation with PMA plus ionomycin, IL-4 detection was performed by cytofluorometry. Data shown are the mean ± SD of 10 independent experiments. Statistical analysis: *P < 0.05.

 
DC matured with other TLR ligands induce similar responses on T lymphocytes
In order to further characterize the DC1 and DC2 nature of 16–mDC and 16+mDC, respectively, we matured both DC with additional stimuli known to generate Th1-inducing DC. For this purpose, DC were stimulated with PGN [Toll-like receptor (TLR)-2 ligand], poly-I:C (TLR-3 ligand) and CpG DNA (TLR-9 ligand). Although is has been reported that monocyte-derived DC do not express TLR-9 (24), we employed CpG DNA to seek their effects on 16+mDC, since it has not been previously investigated. Both PGN and poly-I:C induce CD83 expression on DC (Fig. 4A), but their effects were less prominent on 16+mDC. According to CD83 expression, CpG DNA did not appear to be a maturation stimulus for any DC subtype. Importantly, the IFN-{gamma}/IL-4 ratio observed after co-cultures of these DC with naive CD4+ T lymphocytes (Fig. 4B) showed the same pattern previously seen with other maturation stimuli (3–6-fold higher with 16–mDC), which was fundamentally due to the elevated levels of IL-4 secreted by 16+mDC-stimulated T cells (Fig. 4B), whereas the amount of IFN-{gamma} was much the same (data not shown).



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Fig. 4. Effect of different TLR ligands on the Th polarization pattern induced by 16–mDC and 16+mDC. (A) Expression of CD83 on 16–mDC (full gray histograms) and 16+mDC (empty black histograms) activated for 2 days with PGN, poly-I:C, LPS or CpG DNA. Dotted histograms correspond to the fluorescence of an isotype-matched control antibody. (B) Co-cultures of these DC and naive CD4+ T lymphocytes were established as in Fig. 3. Then, IFN-{gamma} and IL-4 in the supernatants were quantified by ELISA. Data are presented as the IFN-{gamma}/IL-4 ratio (left histogram) and the IL-4 secretion (right histogram) for each condition, and are representative of one out of two independent experiments.

 
The Th2 profile of primed T cells is dependent on endogenous IL-4
Neutralization of IL-4 during T cell stimulation by DC completely abrogated the production of this cytokine (Fig. 5A and B). Therefore, endogenous IL-4 is essential to achieve its own secretion. Addition of exogenous IL-4 to 16–mDC during co-cultures showed that 16–mDC tended to increase the secretion of IL-4 to the levels reached by 16+mDC, except with iDC and LPS/IFN-{gamma}-DC (Fig. 5C). Overall, we conclude that endogenously-produced IL-4 by 16+mDC-activated lymphocytes plays a critical role in their higher secretion of IL-4. However, the low levels of IL-4 induced by iDC and LPS/IFN-{gamma}-16–mDC are independent of the amount of IL-4 at priming.



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Fig. 5. Effect of IL-4 on Th phenotype development. IL-4 secretion by allogeneic naive CD4+ T lymphocytes stimulated with 16–mDC (A) or 16+mDC (B) in presence of isotype-matched control mAb (16–mDC: black bars, 16+mDC: gray bars) or anti-IL-4 mAb (white bars). Note that the values of the white bars are nearly imperceptible. Data are representative of one out of four experiments. (C) Effect of the addition of IL-4 on the secretion of IL-4 by T lymphocytes cultured with 16–mDC. Co-cultures were set as described in Fig. 3, in absence of any additional stimulus (16–mDC, black bars; 16+mDC, gray bars) or in presence of exogenous IL-4 for 16–mDC (white bars). Analysis of IL-4 on culture supernatants was performed by ELISA after stimulation with anti-CD3 mAb. Data are representative of one out of two independent experiments.

 
High secretion of IL-4 by 16+mDC-stimulated T cells relies on IL-12 and IL-10 levels
Differences on IL-4 secretion induced by 16+mDC and 16–mDC disappeared by blockade or addition of IL-12 when lymphocytes were stimulated with PGE2-, TNF-{alpha}/PGE2-, sCD40L/IFN-{gamma}- and LPS-DC (Fig. 6). The same effect was found when IL-10 was neutralized (Fig. 6). Thus, differences on IL-4 secretion by 16–mDC- and 16+mDC-stimulated T lymphocytes depended on distinct production or responsiveness to IL-12 and IL-10. Notably, adding IL-12 led to a similar production of IL-10 when using 16+mDC or 16–mDC as antigen-presenting cells (APC) on those co-cultures (Fig. 6). Furthermore, differential secretion of IL-4 by T cells activated with iDC or LPS/IFN-{gamma}-DC was independent of IL-12 or IL-10, and addition of IL-12 did not remarkably alter their differential secretion of IL-10, although differences were partially abolished with LPS/IFN-{gamma}-DC (Fig. 6).



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Fig. 6. Effect of IL-12 and IL-10 on the secretion of IL-4 and IL-10 by T lymphocytes cultured with 16–mDC (black and dotted bars) or 16+mDC (gray and white bars). Cultures were established as described in Fig. 3, and they were treated with (A) isotype control antibody or (B) neutralizing antibody to IL-10, to IL-12, or with the cytokine IL-12. Analysis of IL-4 and IL-10 on culture supernatants was performed by ELISA after stimulation with anti-CD3 mAb. Data are the mean ± SD of three (antibodies: anti-IL-10 and IL-12) and five (antibody: anti-IL-12) independent experiments.

 
Strikingly, we could not detect IL-12 at any time during co-cultures of DC:T cells (not shown). However, blockade of IL-12 enhanced IL-4 production by CD4+ lymphocytes cultured with either 16–mDC or 16+mDC (Fig. 6B), which indicates that they secreted enough IL-12 along with their interaction with lymphocytes to influence the Th pattern. Moreover, IL-12 neutralization led to a decrement of IFN-{gamma} production, although it was not abolished (Table 2). Blockade of IL-10 always carried a decrease of IL-4 levels on 16+mDC-activated lymphocytes, and in 3/6 conditions with 16–mDC (iDC, PGE2- and sCD40L/IFN-{gamma}-DC) (Fig. 6B). Overall, neutralization of IL-10 did not enhance the priming of IFN-{gamma} production, with the exception of scarce conditions (Table 2), and it abrogated its own production (data not shown).


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Table 2. Effect of IL-12 and IL-10 blockade on Th1 phenotype development

 
Differences on IFN-{gamma} induction by LPS-matured DC depend on IL-4, IL-10 and IL-12
Despite the differences on IL-12 production, 16+mDC and 16–mDC induced similar amounts of IFN-{gamma} on allogeneic naive lymphocytes. However, LPS-16–mDC induced significantly greater levels of this cytokine compared to 16+mDC (Fig. 7A), although as for IL-4, differences in the number of IFN-{gamma}+ cells were less pronounced than the amount of secreted cytokine (Fig. 7B). IL-12, as well as IL-4 and IL-10, were involved in the differential secretion of IFN-{gamma}, and neutralization of each cytokine separately equals the levels of IFN-{gamma} on the supernatants (Fig. 7A). As expected, blockade of IL-4 increased, while blockade of IL-12 decreased the secretion of IFN-{gamma} with both DC. IL-10 neutralization only augmented the amount of IFN-{gamma} with 16+mDC, to a point that reached the levels observed for 16–mDC (Fig. 7A). Neutralization of those cytokines affects similarly the percentage of IFN-{gamma}+ cells with both subtypes of DC, and Fig. 7(C) shows a representative experiment with 16–mDC. Likewise, blockade of IL-4 augmented, and blockade of IL-12 diminished, the percentage of IFN-{gamma}+ lymphocytes. Surprisingly, neutralization of IL-10 led to the highest increment on IFN-{gamma}+ cells for both DC, although it does not carry a similar increase of its secretion (Fig. 7A).



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Fig. 7. Analysis of IFN-{gamma} production by T lymphocytes stimulated with LPS-matured 16–mDC (black bars) or 16+mDC (gray bars). (A) Secretion of IFN-{gamma} on culture supernatants in presence of isotype-matched irrelevant antibody (control), or neutralizing antibody to IL-10, to IL-4 or to IL-12. DC:T cell co-cultures were set as in Fig. 3. Data were obtained by ELISA after stimulation with anti-CD3 mAb, and represent the mean ± SD of three independent experiments. Statistical analysis: *P < 0.05. (B) Two-color immunofluorescence analysis of IL-4 and IFN-{gamma} expression on lymphocytes stimulated by LPS-matured 16–mDC (left) or 16+mDC (right). CD4+ T cells were cultured with allogeneic DC as described in Fig. 3, followed by re-stimulation with PMA plus ionomycin. Percentage of positive cells is indicated in each quadrant. (C) Percentage of IFN-{gamma}+ T lymphocytes after co-culture with 16–mDC as described in (A). Markers were set according to the profiles of an isotype-matched irrelevant antibody. Percentage of IFN-{gamma}+ lymphocytes is indicated on the top of the histograms. Results are from one out of three experiments.

 
Reversibility of Th phenotype by DC
Once naive CD4+ T cells have been developed in vitro under Th1 or Th2 conditions during a short-term culture, we asked if the two types of DC could reverse this initial commitment. To address this issue, we cultured naive T cells for a 7 day period in absence of stimuli (referred to as naive T cells) or under Th1 or Th2 conditions. Then, lymphocytes were stimulated with PGE2- or LPS-DC. Values of IFN-{gamma} and IL-4 secretion for LPS-DC are presented in Fig. 8, and results were essentially similar for PGE2-DC. 16–mDC induced higher amounts of IFN-{gamma} than 16+mDC in all conditions, the greatest differences being with Th2 lymphocytes (up to 3-fold). Both 16+mDC and 16–mDC were capable of maintaining the IFN-{gamma}/IL-4 ratio of Th1 cells with respect to Th1 lymphocytes in the absence of DC. Nevertheless, there were some differences between both DC subtypes (1.3–2-fold) on the IFN-{gamma}/IL-4 ratio values for Th1 cells. Importantly, 16–mDC augmented up to 9-fold the ratio of Th2 lymphocytes, whereas 16+mDC increased this ratio <3-fold. Therefore, 16–mDC could preserve the Th1 phenotype, while they had the potential to modify the Th2 pattern toward a Th1 phenotype. On the other hand, 16+mDC could maintain the Th1 phenotype of already committed lymphocytes, but they had little influence on the reversion of the Th2 phenotype.



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Fig. 8. Reversibility of Th1 and Th2 phenotypes by 16–mDC and 16+mDC. Allogeneic naive CD4+ T lymphocytes, or lymphocytes developed under Th1 and Th2 conditions (as described in Methods) were stimulated for 5 days with LPS-matured 16–mDC (black bars), 16+mDC (gray bars) or were left in the absence of DC (white bars). Then, lymphocytes were re-stimulated with anti-CD3 mAb, and IFN-{gamma} and IL-4 on the supernatants were analyzed by ELISA. The IFN-{gamma}/IL-4 ratios are represented on the bottom panel. Results are representative of one out of two independent experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present study evaluates the influence of different maturation factors on two subsets of human DC, 16–mDC and 16+mDC. Distinct signaling pathways are induced with the different stimuli tested (2527) and the resulting DC showed variations not only in their phenotype, but also in their lymphoproliferative abilities. The best maturation stimulus tested for 16+mDC and 16–mDC was LPS/IFN-{gamma}. However, some discrepancies were observed comparing phenotypic characteristics and induction of lymphocyte proliferation by mature DC. For instance, PGE2 alone did not produce fully mature DC at the phenotypic level, but its stimulation generated DC that induce a high rate of lymphocyte proliferation, in agreement with other reports (28). On the other hand, LPS-16–mDC were phenotypically better APC and more mature DC than 16+mDC, but their lymphoproliferative activity was significantly diminished compared with 16+mDC. This could be explained by a differential expression of costimulatory molecules, or by a distinct pattern of cytokine secretion by DC (i.e. IL-10) (29), and requires further analysis.

According to previous data (9), 16+mDC induced a Th2-like response on allogeneic cultures but, surprisingly, we show in this work that they behave as DC2 after maturation with a variety of unrelated stimuli. The greatest differences on Th polarization between 16–mDC and 16+mDC were observed with DC matured in the presence of TLR ligands. Therefore, it might have important consequences in vivo during infections with bacteria or viruses, and perhaps 16+mDC could counterbalance the effect of exacerbated Th1 responses by stimulating naive T cells to differentiate toward the Th2 pattern. Interestingly, we found similar amounts of IFN-{gamma} induced by 16–mDC and 16+mDC (except for LPS-DC), but higher levels of IL-4 in all conditions. The number of IL-4+ T cells and IFN-{gamma}+ (for LPS-DC) T cells showed little differences between 16+mDC and 16–mDC, as well as the amount of cytokine produced by individual cells (see Fig. 7). Those discrepancies have been reported by others (30), and may be due either to a differential release of both cytokines by T lymphocytes or to methodological approaches, based on the use of immobilized anti-CD3 mAb to evaluate cytokine secretion on culture supernatants, and PMA plus ionomycin to evaluate intracellular cytokine production (31).

The influence of different DC lineages on Th polarization is still inconclusive. Despite early reports indicating the existence of DC subtypes with fixed characteristics to promote Th1 or Th2 responses (12,17,32), recent work rules out the notion of pre-determined ‘DC1’ and ‘DC2’ subsets, and shows that the flexibility of DC in directing Th development is based on microbial signals and antigen dose (33,34). Nevertheless, different subpopulations of DC display distinct sets of TLR (18,19) which could program the type of DC that elicits the immune response against a particular pathogen. We have not yet defined the pattern of expression of TLR on 16+mDC or 16–mDC, but a recent report showed that CD16+ monocytes express higher levels of TLR-2 compared with CD16– cells (35), suggesting that they might not share the same type of TLR once they become DC. In fact, CD83 expression in response to PGN and poly-I:C was low on 16+mDC compared to 16–mDC, while it was similar after LPS stimulation (Fig. 4A). These data support a model where either the number of surface TLR-2 and TLR-3 molecules is diminished on 16+mDC or the intracellular signaling mediators for both receptors present some differences.

The Th1/Th2 pattern induced by 16–mDC and 16+mDC was mediated essentially by IL-4, IL-12 and IL-10, except for iDC and LPS/IFN-{gamma}-DC, which appeared to be independent of any tested factor. The differential levels of IL-12, IL-4 and IL-10 produced during DC:T cells co-cultures, or the distinct responsiveness of lymphocytes to these cytokines, had a deeper global effect on IL-4 production than on IFN-{gamma}. The mechanisms that elicit high levels of IL-4 by iDC and LPS/IFN-{gamma}-16+mDC are currently under study, but it is possible that for immature 16+mDC, their higher CD86 expression could influence the observed phenotype, since CD86 costimulation has been involved in the generation of IL-4-producing T cells (36).

We confirm in this work that endogenous IL-4 present during T cell stimulation was essential to attain its own expression, as has been reported (37). Furthermore, addition of IL-4 at the beginning of the co-cultures notably augmented the expression of this cytokine by 16–mDC-stimulated T cells, at levels comparable to those induced by 16+mDC. It is possible that 16–mDC are unable to induce enough IL-4 on T lymphocytes at priming, perhaps influenced by the presence of IL-12, and this in turn results in low expression of IL-4 at the end of the cultures. Addition of exogenous IL-4 will promote the expression of high levels of this cytokine (38) through up-regulation of the Th2-specific transcription factor GATA-3 (39).

IL-12 is a crucial cytokine in Th1 phenotype development (12,32). Nevertheless, its absence did not completely abolish IFN-{gamma} production (34), and other cytokines could partially replace the IL-12 deficiency (40,41). Herein, we demonstrate that 16+mDC secrete lower amounts of IL-12 than 16–mDC. The reason is currently undetermined, but it appears to be independent of several factors known to reduce IL-12 production, such as IL-10, TGF-ß1, PGE2, or the differential activation of the NF-{kappa}B subunits p50 and c-Rel. Subsets of DC producing differential levels of IL-12 have been described in humans and rodents (12,17), where higher IL-12 producers are associated with Th1- and lower producers with Th2-type responses. Nevertheless, the DC subsets showed some flexibility, since both can promote Th1 responses in the presence of stimuli that elicit IL-12 production (i.e. CpG DNA) or Th2 responses upon exposure to IL-10-inducing agents (i.e. zymosan) (33). In our system, signals that induce high IL-12 secretion (LPS, poly-I:C) (42) promoted Th1 responses, and stimuli that enhanced IL-10 production without inducing secretion of IL-12 such as PGE2 (28,43), elicited lower IFN-{gamma}/IL-4 ratios on regular 16–mDC, but this is not the case on 16+mDC. This result challenges the notion of flexibility on this particular subpopulation of DC. Although there are several maturation factors that we have not yet tested, we cover a wide range of such stimuli, and we suggest that 16+mDC might act as regulatory DC with a fixed phenotype. We wanted to further address this phenomenon by analyzing the ability of both subpopulations of DC to reverse the pattern of short-term cultured Th1 and Th2 populations. The results suggest that 16–mDC and 16+mDC had little influence in modifying the Th1 pattern, although 16+mDC lowered the IFN-{gamma}/IL-4 ratio of Th1 cells compared with 16–mDC, which is in agreement with a putative role of 16+mDC in counterbalancing Th1 responses. Furthermore, 16–mDC would have an important effect in the redirection of primed Th2 lymphocytes toward a Th1 phenotype, while 16+mDC would perpetuate the Th2 pattern. The low capacity of 16+mDC to induce IFN-{gamma} production on Th2 cells substantially accounted for this activity, which might be related to their low production of IL-12 (44).

Involvement of IL-12 in differential Th1/Th2 responses might be paradoxical since 16+mDC and 16–mDC induced similar amounts of IFN-{gamma} after interaction with T lymphocytes, and differences in IL-4 production are the bases of the Th2 responses promoted by 16+mDC. We may argue that the low levels of IL-12 secreted by 16+mDC are sufficient to induce a certain level of IFN-{gamma} on T lymphocytes, but these amounts are insufficient to block the secretion of IL-4, while 16–mDC probably produce a higher concentration of IL-12, and these amounts are enough to block partially the production of IL-4 without increasing the secretion of IFN-{gamma} with respect to 16+mDC.

The effect of IL-12 on IL-4 production is still uncertain. The inhibition of IL-4 production by IL-12 on bulk T cell cultures may involve preferential expansion of Th1 cells (40), or selective growth inhibition of Th2 cells by IFN-{gamma}. That explanation may function for LPS-16–mDC, since they induced higher levels of IFN-{gamma} than 16+mDC, but it is unlikely for the other factors. Additional data could clarify that question. The conditions where higher production of IL-4 are dependent upon IL-12 (PGE2-, TNF-{alpha}/PGE2-, sCD40L/IFN-{gamma}- and LPS-DC) induced similar levels of IL-10 during DC:T cells co-cultures when IL-12 was added to the cultures, and this in turn results in similar production of IL-4 by both DC subtypes. By contrast, the conditions independent of IL-12 (iDC and LPS/IFN-{gamma}-DC) did not equal the levels of IL-10 after addition of IL-12, which was followed by a divergent production of IL-4. These data suggest that the synthesis of IL-10 and IL-4 are intimately linked, and that IL-10 might have a role in inducing the secretion of IL-4. This hypothesis is further supported by the decrease of IL-4 secretion after blockade of IL-10. Therefore, IL-12 could take part in the regulation of IL-4 production indirectly by regulating the levels of IL-10 (40). Importantly, differences in IL-4 secretion disappeared when IL-10 was neutralized (except for iDC and LPS/IFN-{gamma}-DC). As a result, IL-10 is also responsible for the Th2-like phenotype induced by 16+mDC.

The differential production of IFN-{gamma} with LPS-DC was also dependent on IL-4, IL-12 or IL-10. This network of interrelated cytokines might act as a cascade. First, LPS-16–mDC secrete higher levels of IL-12 than 16+mDC, and IL-12 is clearly implicated in the high rate of transcription of the ifn-{gamma} locus (39,40). Second, blockade of IL-10 increases the secretion of IFN-{gamma} only by 16+mDC-stimulated lymphocytes. This might be associated with the high production of IL-10 during 16+mDC-T cell co-cultures, which could in turn diminish their production of IL-12. Finally, high levels of IL-4 induced by 16+mDC could be the consequence of a default mechanism due to their low levels of IL-12 production (45).

Our data support a model where myeloid 16+mDC elicit Th2-like responses when they are activated with either Th1- or Th2-promoting factors. These results suggest that 16+mDC display a pre-determined DC2 phenotype, and they show little changes in the presence of stimuli that elicit Th1 responses (i.e. LPS). By contrast, regular 16–mDC tended to display a DC1 phenotype, but they can modify the magnitude of the response according to the ability of the maturation stimulus to promote IL-12 secretion. Our results indicated that both DC secreted enough levels of IL-12 after interaction with naive CD4+ T lymphocytes to induce similar amounts of IFN-{gamma} (except for LPS-DC), but IL-4 secretion is higher with 16+mDC, and it is greatly influenced by their low production of IL-12. Lower levels of IL-12 at the initial priming of T cells might account for higher production of IL-4 by a default mechanism, and IL-4 will in turn up-regulate its own secretion. On the other hand, 16+mDC also induce higher levels of IL-10, which may act in a positive feedback mechanism by increasing the secretion of IL-4, either directly or indirectly by suppressing IL-12 production.


    Acknowledgements
 
The support of the Juarez Hospital Blood Bank staff for providing adult blood samples is gratefully acknowledged. We also thank Victor H. Rosales for help with flow cytometry, Juana Narváez for expert technical assistance and Ms Ninfa Arreola for aid in the preparation of the manuscript. This work was supported by a grant to C.S.T. from CONACYT (33075-N). A.R.C., S.B. and M.E.M.G. are recipients of CONACYT pre-doctoral scholarships (nos 149529, 144142 and 119307, respectively).


    Abbreviations
 
16–mDC   CD16 monocyte-derived DC
16+mDC   CD16+ monocyte-derived DC
APC   antigen-presenting cells
CM   complete medium
COX   cyclooxygenase
DC   dendritic cell
iDC   immature DC
PBMC   peripheral blood mononuclear cells
sCD40L   soluble CD40 ligand
TLR   Toll-like receptor

    Notes
 
Transmitting editor: K. Inaba

Received 18 March 2004, accepted 17 June 2004.


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 Introduction
 Methods
 Results
 Discussion
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