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(Journal of Leukocyte Biology. 2002;72:1180-1189.)
© 2002 by Society for Leukocyte Biology

CD40 ligation and phagocytosis differently affect the differentiation of monocytes into dendritic cells

Michelle Rosenzwajg, Frédéric Jourquin, Ludovic Tailleux and Jean Claude Gluckman

Institut National de la Recherche Scientifique EMI-0013 and Laboratoire d’Immunologie Cellulaire et Immunopathologie de l’Ecole Pratique des Hautes Etudes, Institut Universitaire d’Hématologie, Hôpital Saint-Louis, Paris, France

Correspondence: Michelle Rosenzwajg, M.D., Ph.D., INSERM EMI-0013, Institut Universitaire d’Hématologie, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France. E-mail: michelle.rosenzwajg{at}sat.ap-hop-paris.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
That monocytes can differentiate into macrophages or dendritic cells (DCs) makes them an essential link between innate and adaptive immunity. However, little is known about how interactions with pathogens or T cells influence monocyte engagement toward DCs. We approached this point in cultures where granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 induced monocytes to differentiate into immature DCs. Activating monocytes with soluble CD40 ligand (CD40L) led to accelerated differentiation toward mature CD83+ DCs with up-regulated human leukocyte antigen-DR, costimulatory molecules and CD116 (GM-CSF receptor), and down-regulation of molecules involved in antigen capture. Monocytes primed by phagocytosis of antibody-opsonized, killed Escherichia coli differentiated into DCs with an immature phenotype, whereas Zymosan priming yielded active DCs with an intermediate phenotype. Accordingly, DCs obtained from cultures with CD40L or after Zymosan priming had a decreased capacity to endocytose dextran, but only DCs cultured with CD40L had increased capacity to stimulate allogeneic T cells. DCs obtained after E. coli or Zymosan priming of monocytes produced high levels of proinflammatory tumor necrosis factor {alpha} and IL-6 as well as of regulatory IL-10, but they produced IL-12p70 only after secondary CD40 ligation. Thus, CD40 ligation on monocytes accelerates the maturation of DCs in the presence of GM-CSF/IL-4, whereas phagocytosis of different microorganisms does not alter and even facilitates their potential to differentiate into immature or active DCs, the maturation of which can be completed upon CD40 ligation. In vivo, such differences may correspond to DCs with different trafficking and T helper cell-stimulating capacities that could differently affect induction of adaptive immune responses to infections.

Key Words: cell differentiation • cell activation • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) are essential antigen-presenting cells (APCs) that initiate and regulate adaptive immune responses [1 ]. In fact, distinct DC populations develop from different hematopoietic progenitors already committed to the lymphoid or the myeloid lineages [2 ] and in the latter case, even from monocytes [3 4 5 ]. Thus, the latter cells appear as versatile precursors that differentiate into macrophages or DCs depending on the microenvironment [6 , 7 ], e.g., "danger" signals provided by microorganisms [7 , 8 ] and/or the "cytokine field" into which they are immersed [9 , 10 ]. Macrophages are essential players of innate immunity by their scavenger function [11 ] mostly based on high phagocytosis and endocytic capacity and by their ability to signal infection by releasing proinflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor {alpha} (TNF-{alpha}), and IL-12 [12 13 14 ]. DCs, which also can phagocytose particles and capture soluble antigens by macropynocytosis or receptor-mediated endocytosis [15 ], are the only cells capable to prime naive T cells upon antigen presentation and the most potent inducers of secondary T cell responses [1 ]; therefore, they are considered as the "true" professional APCs for adaptive immune responses. Thus, as a result of this dual differentiation capacity, monocytes should represent an essential link between the two types of immunity.

Monocytes differentiate into immature DCs upon culture with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 or after transmigration through vascular endothelial cells and phagocytosis [3 4 5 , 16 , 17 ]. These DCs react to a wide range of stimuli (including inflammatory cytokines and microbial products) and upon the activation/maturation process that ensues, undergo coordinated phenotypic and functional switches resulting in antigen capture, down-modulation of their processing capacity, and up-regulation of their presentation capacity. This process is reflected by increased major histocompatibility complex (MHC) class II [18 ], costimulatory and adhesion molecule expression, decreased expression of receptors involved in antigen capture [19 ], modified chemokine receptor expression, and change in cytokine production profile [1 , 20 ]. The process is completed when membrane CD40 on these active DCs is ligated by CD40 ligand (CD40L), expressed by the T cells with which they interact and activate, leading to terminal differentiation into fully mature DCs [21 ].

The effect of danger signals or interactions with T cells on already differentiated DCs or macrophages has been extensively investigated [5 ], but the differentiation potential of monocytes under such circumstances is poorly known. Monocytes express a variety of surface proteins that play an important role in antigen uptake, presentation, and contact interactions with lymphocytes [12 , 14 , 22 ]. Engagement of different membrane receptors by pathogens could then direct the subsequent differentiation of monocytes as well as the functions of the resulting cells. For example, bacterial lipopolysaccharide (LPS), known to induce macrophage as well as DC maturation, may block differentiation of monocytes into DCs, except if signal triggering DC differentiation is delivered concomitantly [7 ]. Here, we investigated whether activation via phagocytosis of opsonized, killed bacteria (Escherichia coli) or yeast-wall particles (Zymosan), as representatives of fungi or via CD40 ligation as a schematic model of the basic interaction with T cells, could affect the potential of monocytes to differentiate into DCs when cultured with GM-CSF and IL-4.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation and culture of monocytes
Culture medium was RPMI 1640, 1% L-glutamine, 1% penicillin/streptomycin (Life Technologies, Grand Island, NY), 10% heat-inactivated fetal calf serum (FCS; Dutscher, Brumath, France). Endotoxin level in complete culture medium was <0.5 EU/ml (LAL kit, BioWhittaker, Walkersville, MD).

Cytaphereses of normal blood donors (Etablissement Français du Sang, Paris, France) were obtained according to institutional guidelines. Peripheral blood mononuclear cells (PBMC) were enriched by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) centrifugation. The PBMC in phosphate-buffered saline (PBS), 2% FCS, were depleted of T and B cells using sheep erythrocyte rosetting (Mérieux, Marcy l’Etoile, France) and M-450 Pan B/CD19 Dynabeads (Dynal, Oslo, Norway), resulting in 91 ± 6% CD14+ cells (n=12), a population referred to thereafter as monocytes. T cells (>90% CD3+ cells) obtained after rosetting were cryopreserved for use as responders in subsequent allogeneic mixed leukocyte reactions (MLR).

DCs were generated in Costar plates (Cambridge, MA) by 7-day culture of 2 x 106 monocytes/3 ml in medium supplemented with 5 ng/ml human recombinant GM-CSF and IL-4 (R&D Systems, Minneapolis, MN). Cultures were fed every 2 days by removing one-third of the supernatant and adding fresh medium (half of culture volume) with full doses of cytokines. [5 ]. Human soluble trimeric CD40L (CD40LT; 250 ng/ml; gift of Immunex, Seattle, WA) was added on days 0, 3, and 5. In some experiments, CD40LT was discontinued after 2 days culture, it was added only from day 5, or it was used without cytokines. Monocytes were stimulated for 3 h with antibody-opsonized E. coli(10 bacteria/cell; Orpegen Pharma, Heidelberg, Germany) or with 18 µg/ml Zymosan (yeast-wall particles from Saccharomyces cerevisiae; Sigma-Aldrich), washed, and cultured as above or without added cytokines in some cases. E. coli and Zymosan concentrations had been determined in preliminary experiments as resulting in phagocytosis by nearly 100% of monocytes (data not shown). In some experiments, day 5 immature DCs were stimulated for 3 h with antibody-opsonized, killed E. coli or with Zymosan, washed, and cultured as above for 2 additional days.

Multiparameter flow cytometry analysis
For immunophenotyping, cells were washed in PBS, 2% FCS, incubated with antibodies for 30 min at 4°C, washed, and fixed in PBS, 1% paraformaldehyde, before analysis with a FACScalibur (Becton Dickinson, San Jose, CA). The following monoclonal antibodies (mAb), directly labeled with fluorescein isothiocyanate (FITC), PtdEtn, or PyC5, were used: CD3, CD14, CD20, CD54, and anti-human leukocyte antigen (HLA)-DR (Becton Dickinson); CD58, CD80, CD83, and CD116 (Beckman Coulter France, Villepinte, France); mannose receptor (MR), CD1a, CD36, CD40, CD86, CXCR4, and CCR5 (PharMingen, San Diego, CA); and CXCR1 and CCR6 (R&D Systems).

Endocytic activity
Endocytic activity of DCs was measured by the uptake of FITC-dextran (Sigma-Aldrich). DCs were incubated in PBS, 2% FCS, with 1 mg/ml FITC-dextran at 4°C to measure nonspecific binding or at 37°C to measure specific uptake. After 1 h incubation, cells were washed in cold PBS-FCS and analyzed by flow cytometry.

Allogeneic MLR assay
The MLR was performed using culture medium with 10% heat-inactivated normal human AB serum. Allogeneic T cells (1x105/well) were cultured for 6 days in 96-well culture microplates (Costar) as responders to 0.1 - 10 x 103 20 Gray-irradiated DCs. [3H]Thymidine ([3H]dThd) incorporation (specific activity, 1 Ci/mM; Amersham, Little Chalfont, UK) was measured after 8–16 h pulse with 1 µCi/well. Results are shown as mean cpm of triplicates; incorporation in negative control wells, with responder T cells alone, was always <=50 cpm.

Cytokine determination in culture supernatants
For determining cytokine levels, supernatants were recovered on culture days 2, 3, 5, and 7. Enzyme-linked immunosorbent assay (ELISA) kits for quantification of IL-6, IL-10, IL-12p40, IL12p70, and TNF-{alpha} were from R&D Systems. When CD40LT was added from day 5 to day 7, culture medium and cytokines were totally renewed, and supernatants were recovered after 2-day stimulation with CD40LT.

Statistics
Statistical analyses were performed with the paired Student’s t-test or when stated, by analysis of covariance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Monocytes were cultured in GM-CSF and IL-4 to induce differentiation into immature DCs (control cultures). Alternatively, CD40LT was added from the start, or monocytes were first allowed to phagocytose antibody-opsonized, killed E. coli or Zymosan before being cultured under the same condition as in control cultures. DC differentiation, maturation, and functions under the different conditions were assessed on day 7 unless otherwise stated. When we attempted to use the same stimuli alone in monocyte cultures without added cytokines, cell viability was severely affected, and <10% of cells survived on day 7, which did not allow us to investigate whether any of the stimuli affected monocyte differentiation into DCs or maturation of the latter cells. This is at variance with a report showing that CD40 ligation induced monocytes to differentiate into DCs in the absence of exogenous cytokines [23 ], but the CD40L-transfected murine fibroblasts used in this case may presumably produce unaccounted for growth factors [24 ].

Culture of monocytes with CD40LT elicits DCs with a mature phenotype
Culture day 7 DCs were first examined using CD1a and CD83 expression as markers of immature and mature DCs, respectively [1 ]. As already known, CD14 had by then disappeared (data not shown), and 78 ± 4% of cells had become CD1a+ (n=18) in control cultures (Fig. 1A ). Most CD1a+ DCs exhibited an immature phenotype, and only 11 ± 3% were CD83+; they expressed MHC class II, costimulatory molecules CD40, CD54, CD58, CD80, and CD86, and molecules involved in antigen capture: MR, CD11b (CR3), CD11c (CR4), CD32 [Fc receptor for immunoglobulin G (Fc{gamma}R)II], and CD36, whereas CD16 (Fc{gamma}RIII) and CD64 (Fc{gamma}RI) were undetectable (Fig. 2A ; and data not shown). As to chemokine receptors, CXCR1, CXCR4, and CCR5 were readily detected, CXCR4 expression was relatively low, and CCR6 was undetectable, as reported [25 ] (Fig. 2B) .



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  		Figure 1. CD1a and CD83 expression on cells derived from monocytes activated by different stimuli and cultured with GM-CSF/IL-4. (A) Day 7 CD1a+ and CD83+ cell percentages from cultures without monocyte priming (GI), in the presence of CD40LT (GI+CD40LT), or after priming with E. coli (GI+E.coli) or Zymosan (GI+Zymosan); results are presented as means + SEM (n=18). (B) Sequential analysis of CD1a+ and CD83+cell percentages (means + SEM; n=2) in monocyte cultures in which, when CD40LT was added, it was used for the first 2 days or for the entire 7 days.

 


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  		Figure 2. Characterization of DCs from 7-day monocyte cultures with GM-CSF/IL-4 without monocyte priming (GI), in the presence of CD40LT (GI+CD40LT), or after priming with E. coli (GI+E.coli) or Zymosan (GI+Zym). (A) Membrane markers: Dotted histograms indicate control labeling with an irrelevant mAb; bold histograms indicate staining by the relevant mAb as indicated; specific mean fluorescence intensities are indicated; the results shown are a composite of data from two experiments and are representative of 11 experiments. (B) Expression of chemokine receptors: the data are from one representative experiment out of seven.

 
In cultures where CD40LT was added to GM-CSF/IL-4 from the start, most day 7 cells had a mature DC phenotype: CD1a+ cell percentages were lower than in control cultures (65±6%; P=0.02; n=18), whereas 85 ± 4% of cells (P<0.001) were CD83+ (Fig. 1A) . Adhesion and costimulatory molecules were up-regulated, whereas molecules involved in antigen capture such as MR, CD32, and CD36 were down-regulated, and in accordance with terminal maturation, CD115 (M-CSF receptor) expression was abolished, and CD116 (GM-CSF receptor) expression increased (Fig. 2A ; and data not shown). As expected, CXCR1 and CCR5 were down-regulated, whereas CXCR4 expression increased (Fig. 2B) . Day 7 DC yields were comparable whether or not cultures were conducted with CD40LT; starting from 2 x 106 monocytes, 0.74 ± 0.11 and 0.74 ± 0.11 x 106 (n=10) cells, respectively, were noted then, which is in line with previous findings in this model [5 ].

It is interesting that after just 2-day culture with CD40LT, there were already ~60% CD1a+ and CD83+ cells versus 30% and none, respectively, under the control condition; then, discontinuing CD40LT resulted in CD1a+ cell percentages that leveled off and CD83+ cell percentages that decreased to ~35% on days 5–7 (Fig. 1B) , presumably as a result of subsequent dilution of mature DCs among newly generated, immature DCs, possibly through attrition of CD83+ DCs, as their life expectancy is limited in the absence of further stimuli [1 ]. These findings suggest that under the conditions used, CD40LT has an early effect on the differentiation of monocytes into mature DCs rather than merely inducing already differentiated, immature DCs to become mature.

Phagocytosis of opsonized E. coli or Zymosan by monocytes differently affects their subsequent differentiation and maturation into DCs
Upon stimulation with bacterial products, DCs undergo phenotypic and functional changes in a process known as DC activation, which ultimately leads to development of fully mature and competent APCs [20 ]. We previously found that LPS, which binds to CD14, partially blocked DC differentiation from monocytes [7 ]. It was therefore of interest to investigate whether other bacterial stimuli could also affect DC differentiation and function. Accordingly, before culture with GM-CSF/IL-4 was initiated, monocytes were first exposed to antibody-opsonized, killed E. coli, which was phagocytosed by the great majority of monocytes. Upon 7-day culture, the resulting DCs differed from control culture DCs by lower CD1a+ cell percentages (49±7%; P=0.01; n=10), but CD83+ cell percentages (14±3%) did not change (Fig. 1A) . Expression of the other markers investigated was homogeneous and for most of them comparable to that of control DCs, corresponding to an immature DC phenotype (Fig. 2A and 2B) .

We next examined the effect of the phagocytosis by monocytes of Zymosan, which is mainly taken up by the MR [26 ]. Zymosan priming resulted in culture day 7 DCs with decreased CD1a+ cell percentages (17±5%) and increased CD83+ cell percentages (48±6%) relative to the control condition (P<0.001; n=13 in both cases; Fig. 1A ). MR, CD36, and CD115 expression was decreased, and that of MHC class II and costimulatory molecules was up-regulated, but to a lesser extent than in cultures with CD40LT; CD116 expression was unchanged (Fig. 2A) . The chemokine receptor expression pattern also corresponded to a mature phenotype, with down-regulated CXCR1 and CCR5 and up-regulated CXCR4 (Fig. 2B) . Thus, these cells could be considered as active DCs.

Although the effect of the stimuli on monocyte differentiation was different, day 7 DC yields from cultures of monocytes stimulated by E. coli or Zymosan were lower than in control cultures: 0.39 ± 0.07 and 0.36 ± 0.07, respectively, versus 0.71 ± 0.11 x 106 cells (P<0.001; n=9) were then recovered from 2 x 106 initial monocytes.

We also examined how these same stimuli did affect already differentiated day 5 immature DCs. Therefore, such DCs were stimulated for 3 h with antibody-opsonized, killed E. coli or with Zymosan, washed, and cultured as above for 2 additional days; as control, CD40LT was added at that time and maintained for the next 2 days in the cultures. Relative to control DCs, expression of CD83, MHC class II, and costimulatory molecules (CD40, DC80, and CD86) was up-regulated to the same extent as in cultures with CD40LT. Thus, in a different manner than for monocytes, these stimuli then elicited full maturation of the DCs (Fig. 3 ).



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  		Figure 3. Activation and maturation markers of DCs from monocytes cultured for 7 days with GM-CSF/IL-4 (GI) or 2 days after being stimulated by CD40LT (GI+CD40LT), E. coli (GI+E.coli), or Zymosan (GI+Zym) on day 5. Dotted histograms: Control labeling with an irrelevant mAb; bold histograms: staining by the relevant mAb, as indicated. The results shown are representative of two experiments.

 
The macropinocytosis capacity of DCs
Immature DCs efficiently capture antigens by endocytosis, a capacity that decreases upon maturation concomitantly with the appearance of increased antigen-presenting function [20 ]. Thus, we monitored FITC-dextran uptake to assess the capacity of DCs at macropinocytosis [15 ]. As expected, the mature DCs from cultures of monocytes with CD40LT had a lower capacity to take up FITC-dextran than the immature DCs of control cultures. Also, in line with their respective immature and active phenotype, respectively, DCs derived from E. coli-primed monocytes retained high dextran-FITC uptake ability, whereas it was reduced in DCs derived from Zymosan-primed monocytes (Fig. 4A ).



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  		Figure 4. Functional capacities of DCs from 7-day monocyte cultures with GM-CSF/IL-4 without monocyte priming (GI), in the presence of CD40LT (GI+CD40LT), or after priming with E. coli (GI+E.coli) or Zymosan (GI+Zym). (A) FITC-dextran uptake: cells were incubated with FITC-dextran for 1 h at 37°C (bold histograms) or at 4°C (dotted histograms) to comparatively assess specific uptake and nonspecific binding, respectively; percentages of cells with a fluorescence intensity distinct from that of the nonspecific binding at 4°C are indicated; representative data of two different experiments. (B) MLR-stimulating capacity: the proliferative response of 1 x 105 allogeneic T lymphocytes, cultured for 6 days as responder cells to different amounts of irradiated DCs from monocytes cultured under the indicated conditions, was assessed by [3H]dThd incorporation (shown as kcpm); means ± SEM of four different paired experiments.

 
Capacity of DCs to stimulate allogeneic T cells
The ability of the DCs obtained under each condition to stimulate T cells was then assayed in the allogeneic MLR (Fig. 4B) . As expected, the mature phenotype of DCs from cultures with CD40LT was associated with the greatest stimulatory capacity (P=0.003; covariance analysis; n=9), whereas DCs obtained from E. coli-primed monocytes simulated the MLR to the same levels as controls. Surprisingly, despite an activated phenotype and reduced endocytic capacity, DCs derived from Zymosan-primed monocytes did not stimulate allogeneic T cells to a significantly greater extent than controls.

Cytokine production by DCs
The outcome of DC encounters with T cells depends in part on the nature of the cytokines released by the DCs, especially in regard to T cell response polarization [27 ]. Therefore, we assessed the kinetics of cytokine production in supernatants of monocyte cultures under the different conditions assayed (Fig. 5 and Table 1 ). In control cultures, IL-6 secretion decreased with time, TNF-{alpha} and IL-10 levels were consistently low, and almost no IL-12 (p40 or p70) was found. In the presence of CD40LT, IL-6, and IL-10, levels were comparable to those in control cultures, TNF-{alpha} production increased but not significantly, whereas IL-12p40 and bioactive IL-12p70 levels increased as early as day 2, in line with the mature phenotype of ~60% of DCs already noted at that time. When E. coli was used to prime monocytes, IL-6 production was higher than in control cultures from day 2 onward and remained elevated thereafter, whereas TNF-{alpha}- as well as IL-10-level increases peaked on day 2 and subsequently declined; IL-12 production did not significantly increase even if interferon-{gamma} was added [27 ] (data not shown). Almost the same pattern was noted in cultures of Zymosan-primed monocytes but for about twofold higher amounts of TNF-{alpha} than in the former cultures.



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  		Figure 5. Kinetics of IL-6, TNF-{alpha}, IL-12p40, IL-12p70, and IL-10 production by DCs differentiating from monocytes cultured with GM-CSF/IL-4 without priming (GI), in the presence of CD40LT (+CD40LT), or after priming with E. coli or Zymosan. Supernatants were collected at days 2, 5, and 7 and were assayed by ELISA. Data are shown as means + SEM of different experiments (IL-6, TNF-{alpha}, and IL-12p40: n=6; IL-12p70: n=9; IL-10: n=5); statistically significant differences (P<0.05) are indicated: *, Day 5 versus day 2; **, day 7 versus day 2 and day 5.

 

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  		Table 1. Statistical Significances of Differences in Cytokine Production Among the Different Conditions Depicted in Fig. 5

 
Inasmuch as IL-10 promotes monocyte differentiation into macrophages and inhibits DC differentiation as well as production of proinflammatory cytokines such as IL-12 [28 ], high IL-10 levels in cultures of E. coli- or Zymosan-primed monocytes could negatively affect maturation of DCs and account for their lack of increased allogeneic MLR-stimulating capacity. However, culturing E. coli- or Zymosan-primed monocytes in the presence of a neutralizing anti-IL-10 mAb did not affect the phenotype or the function of the resulting DCs (data not shown), which indicates that the effect of endogenously produced IL-10 should differ from that noted with exogenous IL-10 [29 , 30 ].

DCs derived from E. coli- or Zymosan-primed monocytes mature differently after activation by CD40LT
As DCs derived from E. coli-primed monocytes were phenotypically and functionally rather immature and did not produce IL-12, we examined if nonetheless these cells could undergo terminal maturation and produce the cytokine when subsequently activated by CD40LT. Thus, culture day 5 DCs from E. coli-primed monocytes were cultured with CD40LT for 2 more days, before their phenotype and IL-12 production were examined. As expected, 58 ± 11% of the cells (P=0.01; n=6) then became CD83+ and acquired a mature DC phenotype, with up-regulated HLA-DR, CD80, and CD86 (Fig. 6 ). This also resulted in increased IL-12p40/p70 production relative to control cultures, which was nevertheless associated with persistently high IL-10 levels (Fig. 7 ).



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  		Figure 6. Expression of maturation surface markers on DCs from 5-day culture with GM-CSF/IL-4 of monocytes primed with E. coli or Zymosan and then activated or not by CD40LT for 2 additional days. Dotted histograms: No CD40LT added; bold histograms: CD40LT activation. The results shown are a composite of data from two experiments and are representative of six different experiments.

 


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  		Figure 7. Production of IL-12p40, IL-12p70, and IL-10 by DCs from 5-day cultures with GM-CSF/IL-4 of monocytes, nonprimed (GI), or primed with E. coli or Zymosan, followed by activation by CD40LT (+CD40LT) or not for 2 additional days. Supernatants collected on day 7 were assayed by ELISA. Data are from two experiments with different donors (#1 and #2).

 
The same was found in regards to IL-12 and IL-10 production when DCs derived from Zymosan-primed monocytes were treated with CD40LT in the same manner (Fig. 7) , but no change of phenotype was observed as a result of an already active phenotype (Fig. 6) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is well established that monocytes differentiate into DCs or macrophages depending on micro-environmental conditions [6 ]. How pathophysiological events affect the capacity of monocytes that migrate from the blood to tissues to differentiate into DCs and to what extent this could influence the function of the resulting DCs, has not been yet well characterized. Indeed, to fully exert their antigen-presenting function, DCs need to be activated and eventually become mature under the influence of stimuli such as live bacteria, double-stranded RNA, inflammatory cytokines, and ultimately T cell signals such as CD40 ligation [1 ]. Here, we examined how some of these stimuli influenced monocyte differentiation into DCs. Our key observation is that under conditions leading to DC differentiation (i.e., in culture with GM-CSF and IL-4), phagocytosis of antibody-opsonized, killed bacteria does not alter, and that of yeast cell-wall extracts facilitates the potential of monocytes to differentiate into immature or active DCs with distinct phenotypes and functions, respectively, whereas when monocytes are activated by CD40 ligation, this results in accelerated maturation of the DCs.

We previously showed that priming monocytes with LPS from E. coli, which binds to CD14 and Toll-like receptor 4 [7 , 31 ], altered their early and late differentiation into DC, except if signal-triggering DC differentiation was delivered concomitantly [6 , 7 ]. Therefore, we investigated here whether priming monocytes with the bacteria themselves had the same effect. Actually, percentages of CD1a+ DCs differentiating from such primed monocytes were lower than in control cultures, but markers known to play a role in antigen uptake, MHC class II, adhesion, and costimulatory molecules, were homogeneously expressed by the cells in the same manner as in controls. Functionally, the DCs took up dextran-FITC and stimulated allogeneic T cells to the same level as control DCs. These findings indicate that despite lower CD1a expression, DCs obtained from monocytes primed with antibody-opsonized, killed E. coli phenotypically and functionally correspond to immature DCs.

Priming monocytes with Zymosan gave somewhat different results. Most DCs were CD1a-, and ~50% were CD83+ and displayed an overall more mature phenotype than control DCs. They lost the capacity to take up dextran-FITC but did not stimulate allogeneic T cells to a greater extent than control DCs. These data indicate that DCs obtained from Zymosan-primed monocytes should correspond to active rather than fully mature DCs [32 ].

Of note, when added to already differentiated DCs, E. coli and Zymosan stimuli elicited their full maturation, which may be reminiscent of the different effect that LPS exerts on DC maturation on the one hand and on monocyte differentiation into DCs on the other [7 ].

Despite different phenotypes, DCs from cultures of antibody-opsonized E. coli- and Zymosan-primed monocytes displayed a similar pattern of cytokine production, releasing high amounts of IL-6 and TNF-{alpha} that can mediate DC maturation in an autocrine manner [1 ]. DCs can produce IL-12 at different stages of maturation: Immature DCs may produce IL-12 for natural killer cells, macrophages, and memory T cells to home at the site of infection, whereas mature DCs would release IL-12 to regulate development of T cells specific for the antigen they present [21 , 33 , 34 ]. However, unlike DCs obtained from cultures to which CD40LT was added from the start, DCs obtained from E. coli- and Zymosan-primed monocytes were ineffective at producing bioactive IL-12p70. In contrast, they released high levels of IL-10, which may serve as a counter-regulatory signal for blocking DC activation [35 36 37 38 ]. Because of its potentially deleterious effect, the inflammatory response must be coordinated via anti-inflammatory mechanisms such as IL-10 production, which will allow the return to the steady state by down-regulating IL-12 production and/or inhibiting maturation of DCs into potent T cell stimulators [27 ]. After systemic triggering in vivo, DCs have been shown to undergo a refractory state during which they cannot produce IL-12 upon new stimulation [39 ], a paralysis that may represent a feedback aimed at limiting the immunopathology associated with prolonged exposure to this cytokine [27 , 40 ]. Here, we found that the capacity of DCs obtained from E. coli- or Zymosan-primed monocytes to produce IL-12p70 upon secondary CD40 ligation was not suppressed and that the inability to produce this cytokine in the absence of CD40LT was not a result of a refractory state, comparable with that noted in endotoxin tolerance [37 ], but rather of a lack of or an incomplete maturation process [41 ].

Altogether, our data suggest that phagocytosis of different microorganisms does not alter or even may facilitate the potential of monocytes to differentiate into immature or active DCs, the final maturation of which can be completed upon CD40 ligation. In vivo, such differences may correspond to DCs with different trafficking and T helper cell-stimulating capacities that could differently affect induction of adaptive, immune responses to infections.


   ACKNOWLEDGEMENTS
 
This work was supported by the Agence Nationale de Recherche contre le SIDA, the Association de Recherche contre le Cancer, the Comité de Paris de la Ligue Nationale contre le Cancer, and the Association pour la Recherche sur les Déficits Immunitaires Viro-Induits (Paris, France). We thank Dr. A. K. Palucka and N. Taquet for their help at the initiation of this work. We gratefully acknowledge the help of Dr. E. Thomas (Immunex, Seattle, WA) for the gift of CD40LT.

Received February 26, 2002; revised September 10, 2002; accepted September 17, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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