CD16+ human monocyte-derived dendritic cells matured with different and unrelated stimuli promote...

CD161 human monocyte-derived dendritic cellsmatured with different and unrelated stimulipromote similar allogeneic Th2 responses:regulation by pro- and anti-inflammatory cytokines

Amaranta Rivas-Carvalho1, Marco A. Meraz-Rıos1, Leopoldo Santos-Argumedo1,Sandra Bajana3, Gloria Soldevila4, Miguel E. Moreno-Garcıa2 and Carmen Sanchez-Torres1

1Department of Molecular Biomedicine and 2Department of Cellular Biology, Centro de Investigacion y de EstudiosAvanzados-IPN, 3Seccion de Estudios de Posgrado e Investigacion, Escuela Superior de Medicina del IPN and4Department of Immunology, Instituto de Investigaciones Biomedicas, UNAM, Mexico City, Mexico

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

Abstract

We previously demonstrated that tumor necrosis factor (TNF)-a-matured CD16� and CD161 humanmonocyte-derived dendritic cells (16�mDC and 161mDC) differentially stimulate naive CD41

lymphocytes by inducing Th1- and Th2-like responses, respectively. Here, we further characterizedthe role of different DC maturation factors on Th polarization. Immature 161mDC and 16�mDC (iDC)obtained by culture of purified monocytes with GM-CSF and IL-4 were maturated with (i) Toll-likereceptor (TLR) ligands [lipopolysaccharide (LPS)], (ii) lymphocyte-derived (soluble CD40 ligand, IFN-c)and (iii) endogenous inflammatory stimuli [TNF-a, prostaglandin (PG)E2]. After activation with thesestimuli, DC secrete IL-12 only in presence of LPS, and 161mDC produced lower amounts of IL-12 andIL-10 than 16�mDC. Allogeneic CD41CD45RO� lymphocytes co-cultured with 161mDC secretedhigher levels of IL-4 and IL-10 than those co-cultured with 16�mDC, regardless of the maturationstimuli. Results were similar when DC were activated with TLR-2 or TLR-3 ligands. The higherinduction of IL-4 by 161mDC was primarily dependent on IL-12, IL-4 and IL-10. IFN-c production byCD41 T cells was similar with all the conditions except with LPS-161mDC, which induced reducedamounts of this cytokine. Those differences were totally eliminated by neutralization of IL-12, IL-4 orIL-10. Finally, 16�mDC could reverse the Th2 phenotype of already committed lymphocytes towarda Th1 pattern in short-term cultures, whereas 161mDC had less ability to skew this phenotype. Theseresults indicate that 161mDC elicit superior Th2 responses independently of the maturation factorsthat they received, and suggest that they could represent an important population of regulatory DC.

Introduction

Circulating blood monocytes are precursors of myeloiddendritic cells (DC) both in vitro (1) and in vivo (2). In vivo,they migrate from blood to inflammatory sites where they seemto differentiate into DC when phagocytic or chemoattractantstimuli are present (2,3). Then, monocyte-derived DC couldreach the draining lymph nodes (2), and they are able topresent antigens to naive lymphocytes (4). CD14+CD16+

human blood monocytes are a cell subset that comprises only1% 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 regularCD14hiCD16� monocytes in a model of transendothelialtrafficking (7). Furthermore, a recent report described thehomologous CD16+ (CD14lowCD16hiCX3CR1hi) and CD16�

(CD14hiCD16low/�CX3CR1low) subsets in mice (8). Afteradoptive transfer, both subpopulations could become DC,but CX3CR1low monocytes differentiate into CD11c+/MHCclass II (I-A)+ DC in inflammatory conditions, whereasCX3CR1hi monocytes might be precursors for resident myeloid

Correspondence to: C. Sanchez-Torres; E-mail: [email protected]

Transmitting editor: K. Inaba Received 18 March 2004, accepted 17 June 2004

International Immunology, Vol. 16, No. 9, pp. 1251–1263 ª 2004 The Japanese Society for Immunologydoi:10.1093/intimm/dxh127 Advance Access publication 26 July 2004

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cells in non-inflamed tissues (8). The biological significance ofthese data is still unknown, but it is conceivable that humanCD16+ monocytes might be relevant precursors of tissue-resident DC in vivo. We have previously demonstrated thathuman CD16+ and CD16� monocytes could differentiate intoDC (subsequently referred as 16+mDC and 16�mDC) whencultured with GM-CSF, IL-4 and tumor necrosis factor (TNF)-aas the maturation stimulus (9). One of the most strikingfeatures of 16+mDC is their preferential activation of allo-geneic Th2 responses, which correlates with low production ofIL-12 p70 and elevated levels of transforming growth factor(TGF)-b mRNA after lipopolysaccharide (LPS) stimulation.Conversely, 16�mDC activate Th1 responses and secreteconsiderable amounts of IL-12 (9).

The Th1/Th2 dichotomy in humans has been useful toexplain certain patterns of immune responses, mainly relatedwith pathological disorders (i.e. allergies, autoimmune dis-eases, responses to infectious agents such as Mycobacteriumleprae) and delayed-type hypersensitivity (10,11). Human DCcould influence the Th1/Th2 phenotype of naive lymphocytesaccording to their cellular lineage (12), their kinetics ofactivation (13) and the effect of qualitatively different signalsfrom their antigenic microenvironment (14,15). The nature ofpathogen- or tissue injured-derived ‘third signal’, as proposedby Kalinski et al. (16), would polarize the DC to establish aninitial Th1/Th2 commitment on T lymphocytes (instructionmodel). However, specialized types of DC could also elicitdifferent 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 appropriateresponses as they encounter Th1- or Th2-promoting factors(16). Alternatively, different sets of receptors for pathogens orinflammatory cytokines could be displayed by subtypes ofDC, thus DC would be intrinsically instructed to skew Th cellresponses (18,19). 16+mDC and 16�mDC behaved accord-ing to this last hypothesis, as they induced differentialresponsiveness on T lymphocytes when they were developedunder the same cytokine environment (9). In the present work,we study in depth this phenomenon by evaluating thefunctional plasticity of 16+mDC and 16�mDC in response toa variety of stimuli, and by identifying the factors involved inDC-induced Th cell polarization. We found that 16+mDCinduced higher levels of IL-4 than those produced by16�mDC after stimulation of allogeneic naive CD4+ lympho-cytes, independently of the stimuli used for maturation. Inaddition, 16+mDC secreted low amounts of IL-12, and thiscytokine, together with IL-10 and IL-4, influenced the inductionof the Th2 pattern.

Methods

Media and reagents

Cells were cultured in RPMI 1640 supplemented with 10%heat-inactivated FCS, 2 mM L-glutamine, 1 mM sodiumpyruvate, 0.1 mM non-essential amino acids (HycloneLaboratories, Logan, UT), 100 U/ml penicillin, 100 lg/mlstreptomycin and 50 lM 2-mercaptoethanol (Gibco BRL,Grand Island, NY), referred to as complete medium (CM). Formonocyte culture, 5% heat-inactivated pooled human serum

was added to the CM. Human recombinant GM-CSF (1000 U/ml), IFN-c (1000 U/ml), IL-12 (1 ng/ml), rat neutralizing mAb toIL-4 (MP4-25D2, 5 lg/ml), to IL-10 (JES3-19F1, 5 lg/ml),murine neutralizing mAb to IL-12 (C8.6, 10 lg/ml), and theirrespective Ig isotype controls, were purchased from BDPharMingen, San Diego, CA. Human IL-2 (20 U/ml) wasobtained from Gibco BRL. Human IL-4 (15 ng/ml) andprostaglandin (PG)E2 (0.1 lg/ml) were provided by Calbio-chem, La Jolla, CA. Human TNF-a (40 ng/ml) and chickenneutralizing polyclonal antibody to TGF-b1 (5 lg/ml) werepurchased from R&D Systems, Minneapolis, MN. Humansoluble CD40 ligand (sCD40L, 1 lg/ml) was provided byPeproTech, Rocky Hill, NJ. Mefenamic acid (10 lg/ml) andLPS (Escherichia coli 0111:B4, 0.5 lg/106 cells/ml) werepurchased from Sigma-Aldrich, St Louis, MO. Peptidoglycan(PGN) from Staphylococcus aureus (10 lg/ml) was obtainedfrom Fluka (Milwaukee, WI), polyinosinic–polycytidylic acid(poly-I:C, 50lg/ml) from Amersham Life Science (Buckingham,UK) and the oligodeoxynucleotide 2006 containing a CpGmotif (2 lM) was purchased from Invitrogen Life Technologies(Carlsbad, CA).

Cell separation and differentiation of peripheral bloodmonocytes

PBMC were isolated from buffy coats of healthy volunteers byFicoll–Hypaque (Gibco BRL) density gradient centrifugation.Subsequently, CD56�CD16+cells and CD56�CD16�CD14+

cells (referred to as CD16+ and CD16� monocytes) wereseparated by magnetic cell sorting, using MACS isolation kits(Miltenyi Biotec, Bergisch Gladbach, Germany), as publishedelsewhere (9). Allogeneic naive CD4+CD45RO� T lympho-cytes from adult blood were separated by negative selectionusing 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+ cellscould be subdivided into two populations according to theirexpression of CD14 (CD14+: 72.8 6 12%, CD14�: 27.1 6

12%) as has been reported (9).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. Thecultures were fed with fresh medium and cytokines every2 days. After 6 days, non-adherent DC were harvested andreplated at 5 3 105 cells/ml in medium containing GM-CSFand IL-4 for two additional days, with or without the followingstimuli: PGE2, TNF-a/PGE2, sCD40L/IFN-c, LPS, LPS/IFN-c,PGN, poly-I:C, or CpG DNA.

Immunofluorescence assays

Monocytes were analyzed immediately after isolation by directstaining 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, Immuno-tech, Marseille, France), followed by FITC-conjugated goatF(ab9)2 anti-mouse Ig polyclonal antibody (Dako, Glostrup,Denmark). Early cell apoptosis was evidenced by staining with

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biotin-labeled annexin V (BD Pharmingen) and PE-conjugatedstreptavidin (Dako). Samples were analyzed on a FACSCaliburusing CellQuest software (Becton Dickinson, MountainView, CA).

Cytokine detection on DC

Production of IL-12 p70, IL-10 and TGF-b1 in the supernatantsof stimulated immature DC (iDC) was quantified by ELISA kitsobtained from BD PharMingen. 105 iDC (day 6 of culture) wereincubated for 24 h with PGE2, TNF-a, TNF-a/PGE2, sCD40L/IFN-c, LPS or LPS/IFN-c, at the concentrations mentioned

above. Controls were established with iDC cultured for 24 hwithout additional stimulus. In some assays, the competitiveinhibitor of cyclooxygenase (COX-1 and -2) mefenamic acid,neutralizing antibody to IL-10, or to TGF-b1, were added to thecultures.

Protein extraction and immunoblotting

Nuclear and cytoplasmic extracts were prepared with theNE-PER� Nuclear and Cytoplasmic Extraction Reagents kit(Pierce Biotechnology, Rockford, IL), according to the manu-facturer’s indications. Protein extracts were separated on 10%

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 isspecified. (C) Analysis of 16�mDC (top panel) and 16+mDC (bottom panel) matured with the different stimuli indicated on the top of thehistograms. Cells were stained with anti-HLA-DR mAb (solid lines), or an isotype-control mAb (dotted lines).

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polyacrylamide gels and transferred to nitrocellulose sheets.Membranes were immunoblotted with rabbit anti-human p50and c-Rel (20) and horseradish peroxidase (HRP)-conjugatedgoat anti-rabbit Ig antibody (Sigma-Aldrich). Protein loadingwas controlled with mouse anti-human b-actin antibody (kindlyprovided by Dr M. Hernandez, CINVESTAV-IPN) for cytoplas-mic extracts, and goat anti-human lamin B antibody (SantaCruz 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, purifiedCD4+CD45RO� Tcells were co-cultured with allogeneic DC at10:1 ratio in CM. Controls were set without addition of DC.When used, neutralizing mAb to IL-4, IL-10, IL-12, or thecytokines IL-4 and IL-12, were added at the beginning of theco-cultures. After 5 days of priming, T cells were expandedwith IL-2 for 6 days. Then, lymphocytes were washedextensively and re-stimulated for 6 h with PMA (20 ng/ml) plusionomycin (500 ng/ml) for intracellular staining of IL-4 and IFN-c,or 24 h with immobilized anti-CD3 mAb (5 lg/ml, UCHT1, BDPharMingen) for quantitation of cytokine secretion. Forintracellular staining of IL-4 and IFN-c, GolgiPlugTM (BDPharMingen) was added to the cultures 4 h before cells wereharvested, to prevent cytokine secretion. Then, cells were fixedand permeabilized with Cytofix/Cytoperm kit (BD PharMingen)and incubated with FITC-labeled anti-IFN-c (4S.B3) and PE-labeled anti-IL-4 (8D4-8) mAb (BD PharMingen). Quantitationof IFN-c, IL-4 and IL-10 secretion in the supernatants wasperformed 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 theabsence of stimulus (naive) or in presence of immobilized anti-CD3 mAb plus IL-12 (4 ng/ml) and anti-IL-4 neutralizing mAb(5 lg/ml) (Th1 conditions) or plus IL-4 (30 ng/ml, Th2conditions). 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 fiveadditional days. Controls were set in the absence of DC. Then,the amount of IFN-c and IL-4 secreted by T lymphocytes wasmeasured after 24 h of re-stimulation with immobilized anti-CD3 mAb, as described above.

Statistical analysis

Data were expressed as mean 6 SD of independent experi-ments. The statistical significance of the data was determinedby the Student’s two-tailed paired t-test, assuming equalvariances.

Results

Phenotype and lymphoproliferative activity of16+mDC and 16�mDC

Phenotype of 16+mDC and 16�mDC is shown in Table 1 andFig. 1(C). DC matured with different stimuli seemed to

generate homogeneous cell populations, as indicated bystaining with anti-HLA-DR antibody (Fig. 1C). Immature DCwere similar in their phenotype, except for the elevatedexpression of CD86 and the low levels of CD1a on 16+mDC.Significant low expression of CD1a by 16+mDC was main-tained after maturation, and it appears to be a feature of thesecells (9). Enhanced expression of CD86 was also conservedafter maturation, with the exception of DC matured with LPSand lymphocyte-derived stimuli, where its expression becamesimilar. CD40 tended to be augmented on 16�mDC, whereasCD80 expression was significantly increased on 16+mDCmatured with endogenous inflammatory stimuli. RegardingCD83 expression, PGE2 alone and sCD40L/IFN-c were weakermaturation factors compared to LPS/IFN-c, LPS or TNF-a/PGE2. For sCD40L/IFN-c, these data differ from previousreports (21). It is possible that the CD40L used here, which issoluble and monomeric, might deliver a low activating signalcompared with membrane or soluble CD40L trimers used inother works. Taking all markers together, LPS/IFN-c was thebest maturation stimulus for both DC, followed by TNF-a/PGE2

for 16+mDC and LPS for 16�mDC. The increment of totalnumbers of CD4+ lymphocytes after stimulation with alloge-neic DC showed that mature DC induced higher proliferativeresponses than iDC (Table 1). TNF-a/PGE2 and LPS/IFN-cwere the strongest stimuli of maturation for 16�mDC, whereasresults were similar for 16+mDC with all stimuli. Immature andLPS-16+mDC showed significant higher lymphoproliferativeactivity than 16�mDC.

Production of IL-12 and IL-10 by DC activatedwith different stimuli

Secretion of IL-12 and IL-10 produced by iDC was consistentlyobserved in presence of LPS (Fig. 2A) and not with the otherstimuli, although in a few experiments sCD40L/IFN-c- andTNF-a/PGE2-16�mDC also produced low but detectableamounts of IL-12 (not shown). Combination of LPS with IFN-cwas a powerful stimulator of IL-12 secretion, while it decreasedthe production of IL-10 (Fig. 2A). Secretion of IL-12 and IL-10by 16+mDC was significantly lower than produced by16�mDC. However, 16+mDC have the potential to secretehigh levels of IL-12 when the appropriate stimulus is given (i.e.LPS/IFN-c; Fig. 2A). The differences observed in cytokineexpression were not due to early apoptosis, since <7% of cellsstained positively for annexin V after stimulation (not shown).

We also performed analysis of TGF-b1 production by DC, aswe have previously reported the augmented expression ofTGF-b1 mRNA on 16+mDC after LPS stimulation (9). However,we detected production of this cytokine only in one out of fourexperiments, and values were superior on 16�mDC (notshown).

Modulation of IL-12 production by DC

In an attempt to assess the factors involved in the differentialproduction of IL-12 by 16+mDC and 16�mDC, we performeda blockade of IL-10, TGF-b1 or COX during LPS or LPS/IFN-cstimulation (Fig. 2B). IL-10 had potent suppressor effects onIL-12 production by DC, while TGF-b1 and PGE2 had onlymarginal effects, and no synergy with IL-10 was observed inany experiment (not shown). The dependence on IL-10 was


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obviously related to the ability of DC to produce IL-10 afterLPS or LPS/IFN-c activation. Then, as 16�mDC alwayssecreted IL-10 with both stimuli, blockade of IL-10 alwaysresulted in increased production of IL-12. On the other hand,16+mDC secreted IL-10 only in some experiments in responseto LPS, and we detected IL-10 after LPS/IFN-c stimulation onlyin one out of four experiments (Fig. 2A). Therefore, IL-10blockade led to an augmented expression of IL-12 by16+mDC in some but not all assays. However, in experimentsT

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Fig. 2. Cytokine production by 16�mDC and 16+mDC. (A) IL-12 andIL-10 production by immature 16�mDC (black bars) and 16+mDC(gray bars) stimulated for 24 h with LPS or LPS/IFN-c. Results are themean 6 SD of six (IL-12) or four (IL-10) independent experiments.Statistical analysis: *P < 0.05. (B) Regulation of IL-12 expression byimmature 16�mDC (black bars) and 16+mDC (gray bars) stimulatedfor 24 h with LPS (left) or LPS/IFN-c (right). DC were activated with LPSor LPS/IFN-c in absence (control) or presence of neutralizing antibodyagainst IL-10, TGF-b1 and the COX inhibitor mefenamic acid. Data areexpressed as the mean 6 SD of four (LPS) and three (LPS/IFN-c)independent experiments. (C) Visualization of NF-jB subunits c-Reland p50 in nuclear and cytoplasmic extracts of 16�mDC and16+mDC after 4 h of LPS stimulation. Proteins were separated ona 10% SDS–polyacrylamide gel and electrophoretically transferred toa nitrocellulose membrane. Lamin B and b-actin were used as proteinloading control for nuclear and cytoplasmic extracts, respectively.


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where LPS-stimulated 16+mDC produced IL-10, blockade ofthis cytokine did not lead to a production of IL-12 as high as16�mDC. Consequently, IL-10 production by 16+mDC is notprimarily responsible for their low secretion of IL-12.

Neutralization of these factors when DC were activated withPGE2, TNF-a/PGE2, or sCD40L/IFN-c did not result in detect-able production of IL-12 (not shown).

Specific NF-jB subunits such as p50 and c-Rel areresponsible for IL-12 transcription on DC (22,23). Thus, wehypothesized that low production of IL-12 by 16+mDC couldbe the consequence of a lower rate of nuclear translocation ofp50 or c-Rel, or lower levels of these proteins, compared to16�mDC. However, Fig. 2(C) shows that after 4 h ofstimulation with LPS, p50 and c-Rel subunits are detectedon nuclear and cytoplasmic extracts of both DC subtypes, andsimilar or even higher levels (c-Rel) were found on 16+mDC.Therefore, p50 and c-Rel do not seem to be directly involved indifferential production of IL-12 by these DC.

16+mDC stimulate alloantigen-specific T cells to producehigh levels of IL-4 and IL-10

Given the differences on IL-12 production by 16�mDC and16+mDC, we asked if this event would be reflected in theextent of naive CD4+ T cell polarization. As shown in Fig. 3(A),both DC induced secretion of IFN-c, IL-4 and IL-10 inallogeneic assays. Similar responses were observed withoutIL-2 expansion (not shown). Primed T cells secreted similaramounts of IFN-c 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+mDCthan with 16�mDC (2–6-fold higher for IL-4, 1.4–2-fold higherfor IL-10), regardless of the stimuli used for maturation. Valuesof IFN-c/IL-4 ratios were superior with 16�mDC than with16+mDC, mainly due to the low levels of IL-4 produced duringco-cultures of 16�mDC and T cells (Fig. 3A). The highestdifferences (4–5-fold) were observed with LPS- and LPS/IFN-c-DC, and the lowest differences corresponded to TNF-a/PGE2-DC (2-fold). Secretion of IL-4 and IL-10 by lymphocytesprimed with 16+mDC was highly correlated, and the correla-tion was less prominent with 16�mDC (Fig. 3B). At the singlecell level, the number of IL-4+ cells showed fewer divergencesbetween 16�mDC and 16+mDC (19.4 6 8.6% and 24.9 6

3.6%, respectively), but they were significant for iDC, PGE2-and TNF-a/PGE2-DC (Fig. 3C).

DC matured with other TLR ligands induce similar responseson T lymphocytes

In order to further characterize the DC1 and DC2 nature of16�mDC and 16+mDC, respectively, we matured both DCwith additional stimuli known to generate Th1-inducing DC. Forthis purpose, DC were stimulated with PGN [Toll-like receptor(TLR)-2 ligand], poly-I:C (TLR-3 ligand) and CpG DNA (TLR-9ligand). Although is has been reported that monocyte-derivedDC do not express TLR-9 (24), we employed CpG DNA to seektheir effects on 16+mDC, since it has not been previouslyinvestigated. Both PGN and poly-I:C induce CD83 expressionon DC (Fig. 4A), but their effects were less prominent on16+mDC. According to CD83 expression, CpG DNA did notappear to be a maturation stimulus for any DC subtype.

Importantly, the IFN-c/IL-4 ratio observed after co-cultures ofthese DC with naive CD4+ T lymphocytes (Fig. 4B) showed thesame pattern previously seen with other maturation stimuli(3–6-fold higher with 16�mDC), which was fundamentally dueto the elevated levels of IL-4 secreted by 16+mDC-stimulated

Fig. 3. Production of IFN-c, IL-4 and IL-10 after co-cultures ofallogeneic CD4+CD45RO� T lymphocytes with 16�mDC (black bars)or 16+mDC (gray bars). (A) Lymphocytes were cultured with eitheriDC or DC matured with PGE2, TNF-a/PGE2, sCD40L/IFN-c, LPS orLPS/IFN-c. After 5 days of priming, lymphocytes were expanded withIL-2 for six additional days. Then, T cells were re-stimulated for 24 hwith immobilized anti-CD3 mAb. Analysis of IFN-c, IL-4 and IL-10secretion on culture supernatants was performed by ELISA. Pro-duction of cytokines by lymphocytes cultured in absence DC wassubtracted. Results are the mean 6 SD of 12 independent experi-ments. The bottom right graph corresponds to the mean 6 SD ofIFN-c/IL-4 ratios. (B) Analysis of the correlation between IL-4 andIL-10 secretion by lymphocytes primed with 16�mDC or 16+mDC:(open diamonds) iDC, (closed squares) PGE2-, (open triangles)TNF-a/PGE2-, (closed diamonds) sCD40L/IFN-c-, (closed triangles)LPS-, (open squares) LPS/IFN-c-DC. Each point represents thecorrelation index between IL-4 and IL-10 values obtained in 12individual experiments. (C) Percentage of IL-4+ lymphocytes afterDC:T cells co-cultures, as described above. After a 6 h re-stimulationwith PMA plus ionomycin, IL-4 detection was performed by cyto-fluorometry. Data shown are the mean 6 SD of 10 independentexperiments. Statistical analysis: *P < 0.05.


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T cells (Fig. 4B), whereas the amount of IFN-c was much thesame (data not shown).

The Th2 profile of primed T cells is dependent onendogenous IL-4

Neutralization of IL-4 during T cell stimulation by DCcompletely abrogated the production of this cytokine (Fig.5A and B). Therefore, endogenous IL-4 is essential to achieveits own secretion. Addition of exogenous IL-4 to 16�mDCduring co-cultures showed that 16�mDC tended to increasethe secretion of IL-4 to the levels reached by 16+mDC, exceptwith iDC and LPS/IFN-c-DC (Fig. 5C). Overall, we concludethat endogenously-produced IL-4 by 16+mDC-activatedlymphocytes plays a critical role in their higher secretion ofIL-4. However, the low levels of IL-4 induced by iDC and LPS/

IFN-c-16�mDC are independent of the amount of IL-4 atpriming.

High secretion of IL-4 by 16+mDC-stimulated T cells relieson IL-12 and IL-10 levels

Differences on IL-4 secretion induced by 16+mDC and16�mDC disappeared by blockade or addition of IL-12 whenlymphocytes were stimulated with PGE2-, TNF-a/PGE2-,sCD40L/IFN-c- and LPS-DC (Fig. 6). The same effect wasfound when IL-10 was neutralized (Fig. 6). Thus, differenceson IL-4 secretion by 16�mDC- and 16+mDC-stimulated Tlymphocytes depended on distinct production or responsive-ness to IL-12 and IL-10. Notably, adding IL-12 led to a similarproduction of IL-10 when using 16+mDC or 16�mDC asantigen-presenting cells (APC) on those co-cultures (Fig. 6).

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 (fullgray histograms) and 16+mDC (empty black histograms) activated for 2 days with PGN, poly-I:C, LPS or CpG DNA. Dotted histogramscorrespond to the fluorescence of an isotype-matched control antibody. (B) Co-cultures of these DC and naive CD4+ T lymphocytes wereestablished as in Fig. 3. Then, IFN-c and IL-4 in the supernatants were quantified by ELISA. Data are presented as the IFN-c/IL-4 ratio (lefthistogram) and the IL-4 secretion (right histogram) for each condition, and are representative of one out of two independent experiments.


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Furthermore, differential secretion of IL-4 by T cells activatedwith iDC or LPS/IFN-c-DC was independent of IL-12 or IL-10,and addition of IL-12 did not remarkably alter their differentialsecretion of IL-10, although differences were partially abol-ished with LPS/IFN-c-DC (Fig. 6).

Strikingly, we could not detect IL-12 at any time during co-cultures of DC:Tcells (not shown). However, blockade of IL-12enhanced IL-4 production by CD4+ lymphocytes cultured witheither 16�mDC or 16+mDC (Fig. 6B), which indicates thatthey secreted enough IL-12 along with their interaction withlymphocytes to influence the Th pattern. Moreover, IL-12neutralization led to a decrement of IFN-c production, althoughit was not abolished (Table 2). Blockade of IL-10 alwayscarried a decrease of IL-4 levels on 16+mDC-activatedlymphocytes, and in 3/6 conditions with 16�mDC (iDC,PGE2- and sCD40L/IFN-c-DC) (Fig. 6B). Overall, neutralizationof IL-10 did not enhance the priming of IFN-c production, with

the exception of scarce conditions (Table 2), and it abrogatedits own production (data not shown).

Differences on IFN-c induction by LPS-matured DC dependon IL-4, IL-10 and IL-12

Despite the differences on IL-12 production, 16+mDC and16�mDC induced similar amounts of IFN-c on allogeneicnaive lymphocytes. However, LPS-16�mDC induced signifi-cantly greater levels of this cytokine compared to 16+mDC(Fig. 7A), although as for IL-4, differences in the number ofIFN-c+ cells were less pronounced than the amount ofsecreted cytokine (Fig. 7B). IL-12, as well as IL-4 and IL-10,were involved in the differential secretion of IFN-c, andneutralization of each cytokine separately equals the levelsof IFN-c on the supernatants (Fig. 7A). As expected, blockadeof IL-4 increased, while blockade of IL-12 decreased thesecretion of IFN-c with both DC. IL-10 neutralization only

Fig. 5. Effect of IL-4 on Th phenotype development. IL-4 secretion byallogeneic naive CD4+ T lymphocytes stimulated with 16�mDC (A) or16+mDC (B) in presence of isotype-matched control mAb (16�mDC:black bars, 16+mDC: gray bars) or anti-IL-4 mAb (white bars). Notethat the values of the white bars are nearly imperceptible. Data arerepresentative of one out of four experiments. (C) Effect of the additionof IL-4 on the secretion of IL-4 by T lymphocytes cultured with16�mDC. Co-cultures were set as described in Fig. 3, in absence ofany additional stimulus (16�mDC, black bars; 16+mDC, gray bars) orin presence of exogenous IL-4 for 16�mDC (white bars). Analysis ofIL-4 on culture supernatants was performed by ELISA after stimulationwith anti-CD3 mAb. Data are representative of one out of twoindependent experiments.

Fig. 6. Effect of IL-12 and IL-10 on the secretion of IL-4 and IL-10 by Tlymphocytes cultured with 16�mDC (black and dotted bars) or16+mDC (gray and white bars). Cultures were established asdescribed in Fig. 3, and they were treated with (A) isotype controlantibody or (B) neutralizing antibody to IL-10, to IL-12, or with thecytokine IL-12. Analysis of IL-4 and IL-10 on culture supernatants wasperformed by ELISA after stimulation with anti-CD3 mAb. Data are themean 6 SD of three (antibodies: anti-IL-10 and IL-12) and five(antibody: anti-IL-12) independent experiments.


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augmented the amount of IFN-c with 16+mDC, to a point thatreached the levels observed for 16�mDC (Fig. 7A). Neutral-ization of those cytokines affects similarly the percentage ofIFN-c+ cells with both subtypes of DC, and Fig. 7(C) showsa representative experiment with 16�mDC. Likewise, block-ade of IL-4 augmented, and blockade of IL-12 diminished, thepercentage of IFN-c+ lymphocytes. Surprisingly, neutralizationof IL-10 led to the highest increment on IFN-c+ cells for bothDC, although it does not carry a similar increase of its secretion(Fig. 7A).

Reversibility of Th phenotype by DC

Once naive CD4+ T cells have been developed in vitro underTh1 or Th2 conditions during a short-term culture, we asked ifthe two types of DC could reverse this initial commitment. Toaddress this issue, we cultured naive T cells for a 7 day periodin absence of stimuli (referred to as naive Tcells) or under Th1or Th2 conditions. Then, lymphocytes were stimulated withPGE2- or LPS-DC. Values of IFN-c and IL-4 secretion for LPS-DC are presented in Fig. 8, and results were essentially similarfor PGE2-DC. 16�mDC induced higher amounts of IFN-c than16+mDC in all conditions, the greatest differences being withTh2 lymphocytes (up to 3-fold). Both 16+mDC and 16�mDCwere capable of maintaining the IFN-c/IL-4 ratio of Th1 cellswith respect to Th1 lymphocytes in the absence of DC.Nevertheless, there were some differences between both DCsubtypes (1.3–2-fold) on the IFN-c/IL-4 ratio values for Th1cells. Importantly, 16�mDC augmented up to 9-fold the ratioof 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 towarda Th1 phenotype. On the other hand, 16+mDC could maintainthe Th1 phenotype of already committed lymphocytes, butthey had little influence on the reversion of the Th2 phenotype.

Discussion

The present study evaluates the influence of differentmaturation factors on two subsets of human DC, 16�mDCand 16+mDC. Distinct signaling pathways are induced withthe different stimuli tested (25–27) and the resulting DCshowed variations not only in their phenotype, but also in theirlymphoproliferative abilities. The best maturation stimulus

tested for 16+mDC and 16�mDC was LPS/IFN-c. However,some discrepancies were observed comparing phenotypiccharacteristics and induction of lymphocyte proliferation bymature DC. For instance, PGE2 alone did not produce fullymature DC at the phenotypic level, but its stimulationgenerated DC that induce a high rate of lymphocyte pro-liferation, in agreement with other reports (28). On the otherhand, LPS-16�mDC were phenotypically better APC andmore mature DC than 16+mDC, but their lymphoproliferativeactivity was significantly diminished compared with 16+mDC.This could be explained by a differential expression ofcostimulatory molecules, or by a distinct pattern of cytokinesecretion by DC (i.e. IL-10) (29), and requires further analysis.

According to previous data (9), 16+mDC induced a Th2-likeresponse on allogeneic cultures but, surprisingly, we show inthis work that they behave as DC2 after maturation witha variety of unrelated stimuli. The greatest differences on Thpolarization between 16�mDC and 16+mDC were observedwith DC matured in the presence of TLR ligands. Therefore, itmight have important consequences in vivo during infectionswith bacteria or viruses, and perhaps 16+mDC couldcounterbalance the effect of exacerbated Th1 responses bystimulating naive Tcells to differentiate toward the Th2 pattern.Interestingly, we found similar amounts of IFN-c induced by16�mDC and 16+mDC (except for LPS-DC), but higher levelsof IL-4 in all conditions. The number of IL-4+ Tcells and IFN-c+

(for LPS-DC) T cells showed little differences between16+mDC and 16�mDC, as well as the amount of cytokineproduced by individual cells (see Fig. 7). Those discrepancieshave been reported by others (30), and may be due either toa differential release of both cytokines by T lymphocytes or tomethodological approaches, based on the use of immobilizedanti-CD3 mAb to evaluate cytokine secretion on culturesupernatants, and PMA plus ionomycin to evaluate intra-cellular cytokine production (31).

The influence of different DC lineages on Th polarization isstill inconclusive. Despite early reports indicating the exist-ence of DC subtypes with fixed characteristics to promoteTh1 or Th2 responses (12,17,32), recent work rules out thenotion of pre-determined ‘DC1’ and ‘DC2’ subsets, and showsthat the flexibility of DC in directing Th development is basedon 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

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

Maturation stimulus 16�mDC 16+mDC

Isotype controla Anti-IL-12b Anti-IL-10c Isotype control Anti-IL-12 Anti-IL-10

iDC 35.8 6 15 13.6 6 8.7*,d 15.9 6 12* 26.8 6 9 17.4 6 6.1* 33.6 6 12PGE2 37.5 6 7 21.7 6 8.9* 39.1 6 7.6 33.1 6 10 21.7 6 10.8* 19.5 6 1TNF-a/PGE2 33.6 6 4 21.6 6 8.4 37.2 6 5 31.2 6 8 18.8 6 9.3* 36.1 6 14sCD40L/IFN-c 22.8 6 7 16.4 6 12.3 26.6 6 9 27.9 6 10 18.1 6 16.8* 29.6 6 7LPS 36.9 6 12 10.9 6 5.2* 29.7 6 12 13.6 6 4 7.3 6 3.6 27.6 6 6.2*LPS/IFN-c 48.7 6 12 16.7 6 5.1* 53.1 6 12 44.5 6 6 11.2 6 4.2* 48.6 6 12

IFN-c secretion (mean 6 SD of five independent experiments in ng/ml) by allogeneic naive CD4+ T lymphocytes stimulated with immature ormature 16�mDC and 16+mDC. Co-cultures of T cells and DC were established as described in Methods.aIsotype-matched control, banti-IL-12 or canti-IL-10 mAb were added at the beginning of the culture.dStatistical analysis (16�mDC or 16+mDC, treated vs control): *P < 0.05.


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immune response against a particular pathogen. We have notyet defined the pattern of expression of TLR on 16+mDC or16�mDC, but a recent report showed that CD16+ monocytesexpress higher levels of TLR-2 compared with CD16� cells(35), suggesting that they might not share the same type ofTLR once they become DC. In fact, CD83 expression inresponse to PGN and poly-I:C was low on 16+mDC comparedto 16�mDC, while it was similar after LPS stimulation (Fig. 4A).These data support a model where either the number ofsurface TLR-2 and TLR-3 molecules is diminished on 16+mDC

or the intracellular signaling mediators for both receptorspresent some differences.

The Th1/Th2 pattern induced by 16�mDC and 16+mDC wasmediated essentially by IL-4, IL-12 and IL-10, except for iDCand LPS/IFN-c-DC, which appeared to be independent of anytested factor. The differential levels of IL-12, IL-4 and IL-10produced during DC:T cells co-cultures, or the distinct re-sponsiveness of lymphocytes to these cytokines, had a deeperglobal effect on IL-4 production than on IFN-c. The mechanismsthat elicit high levels of IL-4 by iDC and LPS/IFN-c-16+mDC arecurrently under study, but it is possible that for immature16+mDC, their higher CD86 expression could influence theobserved phenotype, since CD86 costimulation has beeninvolved in the generation of IL-4-producing T cells (36).

We confirm in this work that endogenous IL-4 present duringT cell stimulation was essential to attain its own expression, ashas been reported (37). Furthermore, addition of IL-4 at thebeginning of the co-cultures notably augmented the expres-sion of this cytokine by 16�mDC-stimulated T cells, at levelscomparable to those induced by 16+mDC. It is possible that16�mDC are unable to induce enough IL-4 on T lymphocytesat priming, perhaps influenced by the presence of IL-12, andthis in turn results in low expression of IL-4 at the end of thecultures. Addition of exogenous IL-4 will promote the expres-sion of high levels of this cytokine (38) through up-regulationof 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 abolishIFN-c production (34), and other cytokines could partiallyreplace the IL-12 deficiency (40,41). Herein, we demonstratethat 16+mDC secrete lower amounts of IL-12 than 16�mDC.The reason is currently undetermined, but it appears to beindependent of several factors known to reduce IL-12 pro-duction, such as IL-10, TGF-b1, PGE2, or the differentialactivation of the NF-jB subunits p50 and c-Rel. Subsets of DCproducing differential levels of IL-12 have been described inhumans and rodents (12,17), where higher IL-12 producersare associated with Th1- and lower producers with Th2-typeresponses. Nevertheless, the DC subsets showed someflexibility, since both can promote Th1 responses in thepresence of stimuli that elicit IL-12 production (i.e. CpGDNA) or Th2 responses upon exposure to IL-10-inducingagents (i.e. zymosan) (33). In our system, signals that inducehigh IL-12 secretion (LPS, poly-I:C) (42) promoted Th1

Fig. 7. Analysis of IFN-c production by T lymphocytes stimulated withLPS-matured 16�mDC (black bars) or 16+mDC (gray bars). (A)Secretion of IFN-c 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:Tcell co-cultures were set as in Fig. 3. Data wereobtained by ELISA after stimulation with anti-CD3 mAb, and representthe mean 6 SD of three independent experiments. Statistical analysis:*P < 0.05. (B) Two-color immunofluorescence analysis of IL-4 and IFN-c expression on lymphocytes stimulated by LPS-matured 16�mDC(left) or 16+mDC (right). CD4+ Tcells were cultured with allogeneic DCas described in Fig. 3, followed by re-stimulation with PMA plusionomycin. Percentage of positive cells is indicated in each quadrant.(C) Percentage of IFN-c+ T lymphocytes after co-culture with 16�mDCas described in (A). Markers were set according to the profiles of anisotype-matched irrelevant antibody. Percentage of IFN-c+ lympho-cytes is indicated on the top of the histograms. Results are from oneout of three experiments.


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responses, and stimuli that enhanced IL-10 production withoutinducing secretion of IL-12 such as PGE2 (28,43), elicitedlower IFN-c/IL-4 ratios on regular 16�mDC, but this is not thecase on 16+mDC. This result challenges the notion of flexibilityon this particular subpopulation of DC. Although there areseveral maturation factors that we have not yet tested, wecover a wide range of such stimuli, and we suggest that16+mDC might act as regulatory DC with a fixed phenotype.We wanted to further address this phenomenon by analyzingthe ability of both subpopulations of DC to reverse the patternof short-term cultured Th1 and Th2 populations. The resultssuggest that 16�mDC and 16+mDC had little influence inmodifying the Th1 pattern, although 16+mDC lowered theIFN-c/IL-4 ratio of Th1 cells compared with 16�mDC, which isin agreement with a putative role of 16+mDC in counter-balancing Th1 responses. Furthermore, 16�mDC would havean important effect in the redirection of primed Th2 lymphocytestoward a Th1 phenotype, while 16+mDC would perpetuate theTh2 pattern. The low capacity of 16+mDC to induce IFN-c

production on Th2 cells substantially accounted for thisactivity, which might be related to their low production ofIL-12 (44).

Involvement of IL-12 in differential Th1/Th2 responses mightbe paradoxical since 16+mDC and 16�mDC induced similaramounts of IFN-c after interaction with T lymphocytes, anddifferences in IL-4 production are the bases of the Th2responses promoted by 16+mDC. We may argue that the lowlevels of IL-12 secreted by 16+mDC are sufficient to inducea certain level of IFN-c on T lymphocytes, but these amountsare insufficient to block the secretion of IL-4, while 16�mDCprobably produce a higher concentration of IL-12, and theseamounts are enough to block partially the production of IL-4without increasing the secretion of IFN-c with respect to16+mDC.

The effect of IL-12 on IL-4 production is still uncertain. Theinhibition of IL-4 production by IL-12 on bulk Tcell cultures mayinvolve preferential expansion of Th1 cells (40), or selectivegrowth inhibition of Th2 cells by IFN-c. That explanation mayfunction for LPS-16�mDC, since they induced higher levels ofIFN-c than 16+mDC, but it is unlikely for the other factors.Additional data could clarify that question. The conditionswhere higher production of IL-4 are dependent upon IL-12(PGE2-, TNF-a/PGE2-, sCD40L/IFN-c- and LPS-DC) inducedsimilar levels of IL-10 during DC:Tcells co-cultures when IL-12was added to the cultures, and this in turn results in similarproduction of IL-4 by both DC subtypes. By contrast, theconditions independent of IL-12 (iDC and LPS/IFN-c-DC) didnot equal the levels of IL-10 after addition of IL-12, which wasfollowed by a divergent production of IL-4. These data suggestthat the synthesis of IL-10 and IL-4 are intimately linked, andthat IL-10 might have a role in inducing the secretion of IL-4.This hypothesis is further supported by the decrease of IL-4secretion after blockade of IL-10. Therefore, IL-12 could takepart in the regulation of IL-4 production indirectly by regulatingthe levels of IL-10 (40). Importantly, differences in IL-4 secretiondisappeared when IL-10 was neutralized (except for iDC andLPS/IFN-c-DC). As a result, IL-10 is also responsible for the Th2-like phenotype induced by 16+mDC.

The differential production of IFN-c with LPS-DC was alsodependent on IL-4, IL-12 or IL-10. This network of interrelatedcytokines might act as a cascade. First, LPS-16�mDC secretehigher levels of IL-12 than 16+mDC, and IL-12 is clearlyimplicated in the high rate of transcription of the ifn-c locus(39,40). Second, blockade of IL-10 increases the secretion ofIFN-c only by 16+mDC-stimulated lymphocytes. This might beassociated with the high production of IL-10 during 16+mDC-Tcell co-cultures, which could in turn diminish their productionof IL-12. Finally, high levels of IL-4 induced by 16+mDC couldbe the consequence of a default mechanism due to their lowlevels of IL-12 production (45).

Our data support a model where myeloid 16+mDC elicitTh2-like responses when they are activated with either Th1- orTh2-promoting factors. These results suggest that 16+mDCdisplay a pre-determined DC2 phenotype, and they show littlechanges in the presence of stimuli that elicit Th1 responses(i.e. LPS). By contrast, regular 16�mDC tended to displaya DC1 phenotype, but they can modify the magnitude of theresponse according to the ability of the maturation stimulus topromote IL-12 secretion. Our results indicated that both DC

Fig. 8. Reversibility of Th1 and Th2 phenotypes by 16�mDC and16+mDC. Allogeneic naive CD4+ T lymphocytes, or lymphocytesdeveloped 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-cand IL-4 on the supernatants were analyzed by ELISA. The IFN-c/IL-4ratios are represented on the bottom panel. Results are representativeof one out of two independent experiments.


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secreted enough levels of IL-12 after interaction with naiveCD4+ T lymphocytes to induce similar amounts of IFN-c(except for LPS-DC), but IL-4 secretion is higher with16+mDC, and it is greatly influenced by their low productionof IL-12. Lower levels of IL-12 at the initial priming of T cellsmight account for higher production of IL-4 by a defaultmechanism, 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 byincreasing the secretion of IL-4, either directly or indirectlyby suppressing IL-12 production.

Acknowledgements

The support of the Juarez Hospital Blood Bank staff for providingadult blood samples is gratefully acknowledged. We also thank VictorH. Rosales for help with flow cytometry, Juana Narvaez for experttechnical assistance and Ms Ninfa Arreola for aid in the preparation ofthe manuscript. This work was supported by a grant to C.S.T. fromCONACYT (33075-N). A.R.C., S.B. and M.E.M.G. are recipients ofCONACYT pre-doctoral scholarships (nos 149529, 144142 and119307, respectively).

Abbreviations

16�mDC CD16� monocyte-derived DC16+mDC CD16+ monocyte-derived DCAPC antigen-presenting cellsCM complete mediumCOX cyclooxygenaseDC dendritic celliDC immature DCPBMC peripheral blood mononuclear cellssCD40L soluble CD40 ligandTLR Toll-like receptor

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