Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;72:953-961.)
© 2002 by Society for Leukocyte Biology

Cyclosporin A inhibits dendritic cell maturation promoted by TNF-{alpha} or LPS but not by double-stranded RNA or CD40L

K. Duperrier, A. Farre, J. Bienvenu, N. Bleyzac, J. Bernaud, L. Gebuhrer, D. Rigal and A. Eljaafari

Departments of Cell Therapy, Immunology, HLA Clinical Pharmacology, Etablissement Français du Sang région Rhone-Alpes, site de Lyon, Centre Hospitalier Lyon-Sud, and Hopital Debrousse, France, Jeune equipe universitaire, 2267, UCLB, France

Correspondence: Dr. Assia Eljaafari, M.D., Ph.D., Banque de Tissus et Cellules, Pavillon I, Hôpital Edouard Herriot, 5 Place d’Arsonval, 69003 Lyon, France. E-mail: assia.eljaafari{at}efs.sante.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Here, we investigated the influence of cyclosporin A (CsA) on dendritic cell (DC) generation. With this aim, human DC were propagated from monocytes in serum-free medium with granulocyte macrophage-colony stimulating factor and interleukin-4. DC were then exposed to tumor necrosis factor {alpha} (TNF-{alpha}) for maturation. Our results show that CsA does not impair commitment of monocytes into DC, as assessed by loss of CD14 and increase of CD40 and CD1a. However, TNF-{alpha}-induced DC maturation was affected, as CsA-treated DC expressed lower levels of human leukocyte antigen and costimulatory molecules but sustained levels of CD1a, and less DC expressed DC-lysosomal-associated-membrane-protein (LAMP) and CD83. Accordingly, CsA inhibited the allostimulatory and accessory cell functions of DC. Surprisingly, when other maturation stimuli were used, we observed that CsA significantly inhibited maturation induced by lipopolysaccharides but not by polyribocytidylic acid or CD40 ligand, as assessed by DC phenotype and functions. Therefore, our results indicate that CsA may differentially affect DC maturation.

Key Words: differential effect • immunosuppressive drug • antigen presenting cell function • accessory cell function • environmental stimulus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclosporin A (CsA) is a potent immunosuppressive agent, which is classically used in organ transplantation to prevent allograft rejection [1 ]. Indeed, CsA is known to reversibly block the early events in T cell as well as in B cell and natural killer cell responses, leading to an alteration of the immune response [2 , 3 ]. In particular, CsA is demonstrated to inhibit T cell-mediated alloimmune responses and the activation cascade, resulting in the secretion of cytokines through inhibition of calcium-related cytoplasmic processes and calcineurin inhibition [4 , 5 ]. Furthermore, CsA has been shown to impair the antigen (Ag)-presenting cell (APC) functions of several cell types, including dendritic cells (DC), Langerhans cells, and monocytes [6 7 8 9 ]. Thus, Whisler et al. [6 ] have shown that CsA affects human leukocyte Ag (HLA)-DR expression on monocytes and impairs their ability to stimulate allogeneic T cells, whereas Dupuy et al. [7 ] have reported that CsA alters the APC functions of freshly isolated human Langerhans cells. Finally, different studies have shown that CsA affects the accessory cell function of DC [8 , 9 ].

DC are potent APC that are involved in the initiation of the immune response [10 , 11 ]. They play a critical role in the stimulation of T cells, and in a context of organ transplantation, in which numerous HLA mismatches exist among the donor/recipient pairs, DC are known to potentiate graft rejection [12 , 13 ]. However, it has also been shown that infusion of bone marrow-derived DC (Bm-DC) together with the organ transplant rather increases tolerance of the donor organ by anergyzing the allogeneic recipient T cells as a result of a deficiency in the cell surface expression of B7.1/B7.2 [14 15 16 ]. Therefore, the decreased expression of costimulatory molecules that are required to induce optimal T cell activation could account for the deficiency of CsA-treated DC to stimulate T cells. Such a possibility has been addressed in different studies in murine and human models. However, too many discrepant results have been reported that did not allow a conclusive idea about the mechanisms involved by CsA to inhibit DC functions. Thus, in mouse, Lee et al. [17 ] have clearly shown that CsA inhibits Bm-DC functions through down-regulation of CD40, CD80, and CD86 expression, whereas three different studies in human led to discordant results [18 19 20 ]. Indeed, Woltman et al. [18 ] have reported that treatment of DC with CsA results only in a partial reduction in tumor necrosis factor {alpha} (TNF-{alpha}) production, without modulation of costimulatory molecules, whereas Manome et al. [19 ] have observed a modulation of CD86 expression in CsA-treated DC that have been induced to mature by superantigens but not by other stimuli, such as lipopolysaccharides (LPS), cytokines, or anti-CD40 activation. Finally, Szabo et al. [20 ] have not observed any inhibitory effect of CsA on major histocompatibility complex (MHC), CD80, and CD86 molecule expression in DC but have shown an inhibition of their allostimulatory capacities.

CsA is an agent that binds to lipoproteins, which buffer its effects through limitation of the active, unbound fraction [21 , 22 ]. Because of the presence of lipoproteins in the serum, the mode of culture of DC is an important bias that could participate in these discrepant results. Thus, in Manome et al. [19 ], as little as 1 µg/ml was sufficient to observe an effect, whereas in Woltman et al. [18 ], 10 µg/ml was required. To address whether CsA could modulate DC costimulatory molecule expression, in support of its effect on APC and accessory cell functions, we therefore cultured DC in serum-free medium to avoid CsA absorption by serum lipoproteins. Immature DC were propagated from human monocytes in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) plus interleukin (IL)-4 and were driven into maturation by subsequent exposure with various stimuli, i.e., TNF-{alpha}, an inflammatory cytokine [23 ]; Escherichia coli LPS, a gram-negative bacterial toxin that triggers innate immunity [23 ]; polyriboinosinic polyribocytidylic acid [poly(I:C)], a synthetic, double-stranded RNA that mimics viral RNA [24 , 25 ]; and CD40L, which is expressed by activated lymphocytes and participates in the triggering of adaptative immunity [26 27 28 ]. The effects of CsA on monocyte-derived (Mo)-DC at the various stages of their differentiation were analyzed with regards to their phenotype, their ability to capture Ag, and their immunostimulatory properties. It is interesting that we observed that at as little as 0.5 µg/ml CsA was sufficient to inhibit DC-costimulatory phenotype and functions. However, such inhibition was dependent on the stimulus that was used to trigger DC maturation and on the stage of DC; i.e., inhibition by CsA was observed in mature but not immature DC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and reagents
Culture medium consisted of X-VIVOTM 20 serum-free medium (BioWhittaker, Walkersville, MD). Recombinant human (rh)GM-CSF, IL-4, and TNF-{alpha} were purchased from R&D Systems (Abingdon, UK). CsA, LPS, and poly(I:C) were obtained from Sigma Chemical Co. (St. Quentin Fallavier, France). CD40L-transfected cell lines were kindly provided by S. Lebecque, Schering-Plough, Lyon, France).

Generation of DC from monocytes
Generation of DC from monocytes was performed using a modified protocol of Sallusto and Lanzavecchia [29 ], as previously reported [30 , 31 ]. Briefly, peripheral blood was harvested from normal, healthy donors and depleted from platelets by gentle centrifugation. Peripheral blood mononuclear cells were then isolated by centrifugation in lymphocyte-separation medium (Eurobio, Les Ulis, France). After two washes, a two-step, discontinuous density gradient was performed to isolate monocytes. With this aim, 50% (6 ml) and 40% (3 ml) dilutions of stock isoosmotic solution of Percoll (1.130 g/ml; Pharmacia LKB, Uppsala, Sweden) in Dulbecco’s calcium- and magnesium-free phosphate-buffered saline (PBS) containing 5% human serum (HS) were layered sequentially. Low-density cells, mostly monocytes, were collected from the interface of the two Percoll solutions. Monocytes were finally isolated by negative selection, using a cocktail of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, anti-immunoglobulin E (IgE) antibodies, and magnetic cell sorter (MACS) microbeads coupled to the antihapten monoclonal antibody (mAb; Miltenyi Biotec, Paris, France). These magnetically labeled cells were depleted by retaining them on a MACS column in the magnetic field of the MidiMACS, whereas monocytes were eluted from the column by several washes.

The monocytes were plated on tissue-culture wells at 37°C in humidified 5% CO2 in air. Medium was supplemented with 200 U/ml rhGM-CSF and 500 U/ml rhIL-4 in a 6-ml final volume. On day 6, cells were transferred into Teflon wells and cultured at a density of 5 x 105 cells per well in a 3-ml final volume for 2 additional days in the presence of rhTNF-{alpha} (200 U/ml), LPS (1 µg/ml), or poly(I:C; 12.5 µg/ml) or were transferred into six-well plates where 106 Mo-DC per ml were cultured in the presence of 105-irradiated (7000 rad) fibroblastic CD40L-transfected L cells per ml. When required, CsA was added at 0.5 or 1 µg/ml from the beginning of cultures and during the entire period of cultures or during the maturation step only.

In some experiments, DC were generated in RPMI-1640 culture medium (Life Technologies, Eggenstein, Germany) supplemented with 10% HS. In those experiments, CsA was added at 1 or 5 µg/ml from the beginning of cultures.

Flow cytometry labeling
Cell staining was performed using fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)- conjugated and affinity-purified mouse mAb or unconjugated antibodies in combination with FITC-conjugated goat F(ab')2 anti-mouse Ig (FITC control) for indirect staining. Analyses were performed using a FACSCalibur flow cytometer and the CELL QUEST® software (Becton Dickinson, Erembodegem-Aalst, Belgium). The mAb anti-CD14 (IgG2), anti-CD54 (IgG1), anti-CD80 (IgG1), anti-CD40 (IgG1), anti-CD86 (IgG1), anti-CD1a (IgG2), and anti-HLA-DR (IgG2) were purchased from Becton Dickinson. The anti-CD83 mAb (IgG2) was purchased from Immunotech (Marseille, France).

For intracellular staining of DC-LAMP (Immunotech), cells were fixed in 4% paraformaldehyde for 10 min and were then permeabilized with 0.25% saponin (Sigma Chemical Co., St. Louis, MO) and 4% bovine serum albumin for 1 h. After washes, cells were incubated for 30 min with anti-DC-LAMP and were then washed twice with 0.1% saponin. Staining was revealed with FITC-conjugated F(ab')2 fragment anti-mouse Ig.

FITC-dextran internalization
DC (105) were resuspended in PBS/5% HS and cultured at 37°C (or 0°C for control) for 15 min. They were then incubated with 1 mg/ml FITC-dextran at 37°C (or 0°C for control) for 30 min. The reaction was stopped with cold PBS containing 5% HS and 0.1% sodium azide. Cells were washed three times with cold PBS/HS/azide and analyzed on a FACScan flow cytometer.

Allogeneic and autologous mixed lymphocyte reaction (MLR)
DC stimulatory function was assessed by incubating 105 allogeneic T cells with graded numbers of irradiated DC (30 Gy) in U-bottom microwells or by stimulating 105 autologous CD45RA or CD45RO T cells with phytohemagglutinin (PHA; 0.1 µg/ml) in the absence or presence of irradiated DC. Cell proliferation was measured by [3H]-thymidine incorporation at day 3 of culture for PHA-induced, autologous MLR or at day 4 for allogeneic MLR.

Supernatants (SN) from CsA-pretreated Mo-DC
Day 8-cultured DC generated from monocytes in serum-free medium, which had been treated or not by CsA (0.5 µg/ml or 1 µg/ml) were washed and maintained at 37°C for 2 additional days in fresh-culture medium. SN were then discarded and used as a culture medium for MLRs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of CsA on immature DC properties
We first tested the effect of CsA on DC differentiation. With this aim, we generated DC from monocytes by culturing them in serum-free medium and the relevant cytokines. CsA was added or not at various concentrations, i.e., 0.5 or 1 µg/ml, during the entire period of culture. As shown in Figure 1 , culture of monocytes with GM-CSF plus IL-4 for 6 days in the absence of CsA resulted in the generation of immature DC, as assessed by down-regulation of CD14—a monocyte surface marker—up-regulation of CD40 molecule, and induction of CD1a expression. Addition of CsA to these cultures at 0.5 or 1 µg/ml did not markedly influence the cell-surface profile, which suggests that CsA does not prevent commitment of monocytes into immature DC.



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  		Figure 1. Effect of CsA on monocyte differentiation into immature DC. Monocytes were cultured in the absence or presence of 0.5 µg/ml or 1 µg/ml CsA, with GM-CSF plus IL-4 for 6 days in serum-free medium. Cells were stained with conjugated mAb and analyzed by flow cytometry fluorescence value on all viable cells represented. As control, dotted histograms represent the fluorescence values of monocytes at day 0 of culture.

 
CsA inhibits TNF-{alpha}-induced DC maturation
Immature DC, pretreated or not with CsA, were subsequently exposed to TNF-{alpha} for 2 additional days to induce their maturation. During this step, CsA was added at 0.5 or 1 µg/ml to CsA-pretreated DC. As shown in Table 1 (row A), day 8-cultured DC expressed high levels of HLA-DR, CD80, CD86, and CD54. Except for CD40, CsA treatment resulted in a dose-dependent decrease of the MFI of these molecules and the percent of cells expressing CD80 and CD86. CD40 expression, which is classicaly up-regulated during DC maturation, appeared not very sensitive, however, to CsA treatment. Moreover, CD1a, a molecule that is known to disappear upon DC maturation, was sustained at relatively high levels in CsA-treated DC. In accordance with these results, the expression of CD83 and DC-LAMP, two markers that are neo-expressed upon DC maturation, was markedly reduced, as compared with untreated DC (Fig. 2A ). Thus, our results show that CsA treatment inhibits TNF-{alpha}-induced Mo-DC maturation. It is interesting that when DC were cultured in the presence of 10% HS, as much as 5 µg/ml CsA did not result in such a drastic inhibition, as assessed by the sustained ratio of cells expressing CD86, albeit with a lower MFI; the weaker decrease of HLA-DR MFI; the only two-fold increase of cells expressing CD1a instead of threefold in serum-free medium; and the lower inhibition of CD83 and DC-LAMP-expressing cells (Table 1 , rows D–F and Fig. 2B ).


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  		Table 1. Influence of CsA on DC Maturation

 


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  		Figure 2. Expression of specific DC maturation markers is altered by CsA. DC were harvested at day 8 of culture and tested for the expression of DC-specific maturation markers. Cells were cultured from the beginning of cultures in the presence of 0.5, 1, or 5 µg/ml CsA or not and were then stained with CD83 and DC-LAMP mAb, directly conjugated to PE or indirectly conjugated to FITC, respectively, and analyzed by flow cytometry. Dotted histograms represent the fluorescence values of isotypic controls. (A) Cells were cultured in serum-free medium. (B) Cells were cultured in the presence of 10% HS.

 
CsA alters the capacity of DC to mature, starting from the initial period of culture
To analyze in more details the effect of CsA on DC maturation, we added CsA at different periods of culture. As shown above, treatment of DC with 1 µg/ml CsA during the entire period of culture resulted in a decrease of CD83, CD80, and CD86 expression (Fig. 3B ), as compared with untreated DC (Fig. 3A) . However, in cells treated with CsA during the first period of culture only, i.e., during commitment of monocytes into immature DC (Fig. 3C) or during the maturation phase induced by TNF-{alpha} exposure (Fig. 3D) , CD83, CD80, and CD86 expression was also decreased, albeit less, as assessed by the percent of positive cells or MFI. Thus, these results suggest that CsA may alter the responsiveness of immature DC to subsequent TNF-{alpha} exposure.



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  		Figure 3. CsA alters the capacity of DC to mature from the early step of monocyte commitment into DC. Monocytes were cultured in the presence of GM-CSF plus IL-4 during 6 days and for an additional 2 days with TNF-{alpha} in the absence of CSA; (A) DC were cultured without CSA. (B–D) DC were treated with 1 µg/ml CsA at different periods of culture; i.e., CsA was added during the entire period of culture (B), during the first 6 days of culture only (C), or during the last 2 days of culture (D). CD83, CD80, and CD86 staining at day 8 of culture was analyzed by flow cytometry. Dotted histograms represent the fluorescence values of isotypic controls.

 
Effects of CsA on DC functional properties
To get further insight into the influence of CsA on DC maturation, we then analyzed its effect at the functional level, paying particular attention to dextran-FITC capture, immunostimulatory capacity, and accessory cell function. Cells were thus treated with CsA during the entire period of culture or not. As in the case of untreated DC, we observed that capture of dextran-FITC by CsA-treated, immature DC was intense but was completely abrogated when DC became mature (Fig. 4A ). However, CsA-treated DC lost part of their ability to stimulate allogeneic T cells, as compared with untreated DC (Fig. 4B) .



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  		Figure 4. Influence of CsA on DC functional properties. (A) DC were tested for their ability to internalize FITC-dextran, as described in Materials and Methods. CsA was added during the entire period of cultures. As control, dotted histograms represent the fluorescence values of FITC-dextran uptake at 0°C. The immunostimulatory (B and D) and accessory cell functions (C) of untreated DC and of CsA-treated DC were measured following exposure to TNF-{alpha} for 2 days. In MLR experiments (B, D), graded numbers of DC were used to stimulate 105 T cells, whereas to measure the accessory cell function (C), 3 x 103 DC were added to autologous T cells preactivated with 0.1 µg/ml PHA. (D) SN were discarded and added as a culture medium to MLR cultures with untreated DC. Proliferative response was measured by [3H]-thymidine incorporation, as described in Materials and Methods. Results are representative of three different experiments.

 
The accessory cell function can be measured by the capacity of APC to support autologous T cell proliferation following stimulation by a suboptimal signal. As opposed to the allostimulatory function, this capacity only belongs to highly competent APC, such as mature DC. To evaluate the CsA effect on accessory cell function, autologous T cells were activated with a suboptimal level of PHA, which by itself was unable to trigger significant T cell proliferation, and DC, previously treated with CsA or not, were added. As shown in Figure 4C , a strong T cell proliferation occurred in the presence of untreated DC. However, T cell proliferation was markedly decreased in the presence of 1 µg/ml CsA-treated DC, thus indicating that CsA may alter the accessory cell function of mature DC. That inhibition of Mo-DC-allostimulatory function by CsA could be an result of drug carryover by DC and therefore to a direct, immunosuppressive effect of CsA on T cells, according to Péguet-Navarro et al. [32 ], was ruled out in this experiment. Indeed, Mo-DC that were generated in the presence of 0.5 or 1 µg/ml CsA were washed out extensively at day 8 of culture and maintained for 2 additional days at 37°C in fresh culture medium. SN were discarded and used as culture medium for fresh MLR in which untreated DC were added as stimulating cells. As shown in Figure 4D , no inhibition of T cell proliferation was induced when washout CsA SN was added to MLR cultures, as compared with Figure 4B and 4C . Therefore, these results indicate that CsA may alter DC functional properties independently of drug carryover by DC.

CsA differentially modulates DC maturation, depending on the stimulus used, i.e., TNF-{alpha} and LPS but not poly(I:C) or CD40L
As DC maturation could be triggered by different environmental signals such as TNF-{alpha}, LPS, poly(I:C), or CD40L, we then analyzed the effect of CsA on those different DC. However, in those experiments, CsA was added to DC cultures during the maturation phase only. In TNF-{alpha}- and LPS-induced, mature DC, CsA treatment altered expression of CD83 and CD86 costimulatory molecules. In accordance with these results, CsA treatment resulted in an increased expression of CD1a. However, no significant effect of CsA was observed in poly(I:C)- or CD40L-treated DC (Fig. 5 ). Accordingly, the immunostimulatory and accessory cell functions of poly(I:C) and CD40L mature DC were not significantly inhibited by CsA, as opposed to the drastic effect observed in TNF-{alpha}- and LPS-induced, mature DC (Fig. 6 ). Thus, this suggests that DC maturation is regulated by CsA, depending on the environmental stimulus.



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  		Figure 5. CsA differentially modulates DC differentiation at the phenotypical level depending on the stimulus that was used for maturation, i.e., TNF-{alpha}, LPS, poly(I:C), or CD40L. Monocytes were cultured in the presence of GM-CSF plus IL-4 during 6 days and an additional 2 days with TNF-{alpha}, LPS, poly(I:C), or CD40L. Influence of CsA on CD83, CD80, CD86, and CD1a molecule expression at day 8 of culture was analyzed by flow cytometry. DC were untreated (solid thin lines) or treated with 1 µg/ml CsA (thick lines) at day 6 of culture. Dotted histograms represent the fluorescence values of isotypic controls.

 


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  		Figure 6. CsA differentially modulates DC differentiation at the functional level depending on the stimulus that was used for maturation, i.e., TNF-{alpha}, LPS, poly(I:C), or CD40L. The immunostimulatory (A and B) and accessory cell functions (C and D) of untreated DC (open bars) and of CsA-treated DC (solid bars) were measured. With this aim, immature DC were induced to mature in the presence of TNF-{alpha}, LPS, poly(I:C), or CD40L. When required, CsA was added at 1 µg/ml during the maturation step only. In MLR experiments (A and B), graded numbers of DC were used to stimulate 105 T cells, whereas to measure the accessory cell function, 3 x 103 DC were added to autologous T cells preactivated with 0.1 µg/ml PHA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have investigated the effect of CsA on the phenotype and functions of DC propagated in vitro from human monocytes. The present study was realized under serum-free conditions to avoid the possible buffering of CsA by lipoproteins present in the serum. We found that at the phenotypical level, commitment of monocytes into immature DC was not altered by CsA (Fig. 1) . However, TNF-{alpha}-induced DC maturation was affected, as CsA treatment of cells cultured in serum-free medium resulted in a marked reduction of the expression levels of several molecules that increase or appear upon maturation of DC, i.e., costimulatory, adhesion, and MHC molecules or DC-LAMP and CD83, respectively (Table 1 , rows A–C and Fig. 2A ). An inhibitory effect of CsA on DC maturation was also observed in cells cultured with 10% HS, but the concentration of CsA that was required was too high to allow a clear evaluation. Indeed, as shown in Table 1 (rows D–F) and Figure 2B , some degree of inhibition of DC maturation by CsA was observed at 1 µg/ml and upgraded at 5 µg/ml. However, this inhibition did not reach the levels observed in serum-free medium. In accordance with our results, previous studies reported by Woltman et al. [18 ] and Manome et al. [19 ] have also shown some but not a clear inhibitory effect of CsA on human DC cultured with 10% fetal calf serum. However, Szabo et al. [20 ] did not find any inhibitory effect of CsA on DC phenotype while using almost 10 µg/ml CsA. Nevertheless, it is can be pointed out that these authors investigated the effect of CsA on immature DC but not on mature DC. Thus, according to our results shown in Figure 1 , this could explain their negative results.

The effect of CsA on mature DC cultured in serum-free medium was then analyzed at the functional level. We observed that the ability of Mo-DC to stimulate allogeneic T cells was impaired by CsA together with the accessory cell function, independently of drug carryover by DC (Fig. 4) . Thus, these results suggest that CsA may inhibit recognition of alloantigens by decreasing the accessory cell function as well as the stimulatory capacity of Mo-DC. Another property of DC is their ability to capture pathogens by phagocytosis or macropinocytosis at the immature stage. This property is lost when DC become mature. Unexpectedly, the prevention of the loss of this function was not observed in CsA-treated DC, as assessed by down-regulation of dextran-FITC internalization (Fig. 4A) . Thus, this suggests that CsA might differentially act on the various aspects of DC maturation by inhibiting the capacity of DC to stimulate T cells without impairing the ability of DC to capture Ag. Thus, a type of alternative DC maturation state could be induced by CsA. It is interesting that this differential impairment of DC maturation by CsA is likely to be specific for CsA, as IL-10, transforming growth factor-ß (TGF-ß), or glucocorticoid have been reported to affect mature DC phenotype and stimulatory functions but also the ability of DC to internalize dextran-FITC [33 34 35 36 ]. However, the possibility that the various aspects of DC maturation can be triggered by different biochemical pathways is supported by Ardeshna et al. [37 ] who have reported that inhibitors of p38SAPK, a kinase that plays an important role in DC maturation, significantly reduce LPS-mediated up-regulation of CD80, CD83, and CD86 on Mo-DC but do not affect macropinocytosis.

To get further insight into the stages of CsA action, washout experiments were performed on CsA-treated, immature DC. Washed cells were then exposed to TNF-{alpha} for 2 days. Results showed that even in that case, cells still expressed an altered, mature phenotype, thus suggesting that CsA acts during the first steps of Mo-DC differentiation and that TNF-{alpha} does not reverse the CsA-inhibitory effect. One possible mechanism that could explain the early effect of CsA on DC differentiation is that CsA affects DC maturation by impairing the transcription of genes involved in the driving of immature DC on the way to DC maturation. Nuclear factor (NF)-{kappa}B is a good candidate, as this factor and more particularly, the RelB factor appear to play a predominant role in the regulation of DC maturation. Indeed, mice deficient in RelB have been shown to express immature Langerhans cells but not mature DC [38 ]. Moreover, Lee et al. [17 ] have shown that CsA inhibits NF-{kappa}B activation in mouse Bm-DC. Alternatively and as suggested by a recent study showing that CsA may activate TGF-ß production in malignant cells [39 ], another hypothesis could be that CsA stimulates TGF-ß production by immature DC. Indeed, this cytokine has been shown to prevent DC maturation [34 ]. However, this cytokine has also been shown to bias the differentiation of DC toward Langherans cells [40 41 42 ], which should result in an increased number of cells expressing CD1a. Whereas Figure 1 could favor this hypothesis, the complete abrogation of dextran-FITC capture following TNF-{alpha} treatment (Fig. 4A) is in discordance, as reported by Geissmann et al. [34 ].

As maturation of DC could be induced by different stimuli, including those provided by innate immunity or adaptative immunity, we then analyzed the effect of CsA on DC activated by LPS, a gram-negative bacterial toxin; poly(I:C), a synthetic double-stranded RNA that mimicks viral RNA stimulus; and CD40L, a molecule that is expressed by activated T cells and that serves to activate DC through CD40. We observed that CsA drastically inhibited maturation of DC induced by LPS but had only little or no effect on DC activated by poly(I:C) or CD40 ligation. It must be pointed out that the absence of the CsA effect on the allostimulatory capacity of poly(I:C)- or CD40L-treated DC allowed us to exclude the possibility that a nonspecific CsA release by DC, through binding of CsA to their membrane lipids, could account for the inhibition of T cell alloresponse to TNF-{alpha}- or LPS-mature DC.

These differential effects of CsA on LPS- and CD40-dependent pathways of DC maturation are highly reminiscent with those of Vanderheyde et al. [36 ]. Indeed, these authors have shown that methylprednisolone inhibits LPS- but not CD40 ligation-induced DC maturation, as assessed by costimulatory molecule expression.

Moreover, such a differential effect was also observed with IL-10, which inhibited LPS- but not CD40L-induced DC differentiation [43 ]. Therefore, altogether, these data suggest that LPS and CD40 ligation may trigger distinct activation pathways in DC. Moreover, TNF-{alpha} and CD40L might also use distinct pathways, as assessed by a recent study showing a differential state of activation of NF-{kappa}B by these two stimuli [44 ].

That poly(I:C) may trigger a pathway distinct from that mediated by LPS is very likely, as poly(I:C) has been shown to induce DC maturation through the Toll-like receptor 3 (TLR3) signaling pathway, whereas gram-negative LPS is known to use TLR4 (see ref. [45 ] for review). Human TLRs have been described to use a common pathway that involves MyD88, an adapator molecule, and that results in NF-{kappa}B activation [45 ]. However, as shown by Alexopoulou et al. [46 ] and Kawai et al. [47 ], in addition to this pathway, TLR3 and TLR4 can activate other pathways that also lead to NF-{kappa}B activation and result in DC maturation. Therefore, the differential effects of CsA, depending on the mode of stimulation, suggest that different signaling pathways, which depend on calcineurin or not, may lead to DC maturation. From our results, one could suggest that the MyD88-independent pathway triggered by TLR4 is dependent on calcineurin. Whereas this hypothesis remains to be analyzed, the differential effects of CsA on mature DC, which we report herein, could become a useful tool for dissecting the pathways triggered by TLRs. It would therefore be interesting to examine the effects of CsA on maturation of DC induced by other stimuli that activate other TLRs.

In conclusion, we show herein that depending on the maturation stimulus, CsA differentially inhibits the functions of DC generated from human monocytes. Indeed, TNF-{alpha}- and LPS- but not poly(I:C)- or CD40L-induced, mature DC were markedly affected by CsA treatment. Thus, this suggests that in addition to its direct immunosuppressive effect on T cells, CsA may alter the immune response through inhibition of APC functions. However, this latter effect of CsA is likely to be influenced by the microenvironment.


   ACKNOWLEDGEMENTS
 
We thank Dr. Tuna Mutis for helpful comments on the manuscript, as well as Pr. J. P. Revillard, Pr. D. Schmitt, and Pr. J. C. Bensa.

Received May 13, 2002; revised July 11, 2002; accepted July 29, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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