Suppressive Effect of 1,25-Dihydroxyvitamin D3 on Type I IFN-Mediated Monocyte Differentiation into Dendritic Cells: Impairment of Functiona
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免疫学杂志 2005年第1期
Abstract
Dendritic cells (DCs) generated by a single-step exposure of human monocytes to type I IFN and GM-CSF (IFN-DCs) are endowed with potent immunostimulatory activities and a distinctive migratory response to specific chemokines. In this study, we evaluated the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active metabolite of vitamin D3, on the DC differentiation/activation induced by type I IFN. We found that 1,25(OH)2D3 prevented the generation of IFN-DCs when added to freshly isolated monocytes, and was capable of redirecting already differentiated IFN-DCs toward a more immature stage, as revealed by their immunophenotype, reduced allostimulatory activity, and impaired LPS-induced production of Th1-polarizing cytokines. Control and 1,25(OH)2D3-treated IFN-DCs exhibited a similar expression of vitamin D receptor, as well as comparable cell death rates. Furthermore, the chemotactic response of IFN-DCs to CCL4 and CCL19 was markedly reduced or completely abrogated by 1,25(OH)2D3. Despite these changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. These findings indicate that, in this innovative single-step DC generation model from monocytes, the suppressive effect of 1,25(OH)2D3 is associated with a potent impairment of DC migration in response to inflammatory and lymph node-homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)2D3-mediated immunomodulation.
Introduction
Dendritic cells (DCs)3 are potent APCs that play a pivotal role in the immune response, by linking innate and adaptive immune responses ( 1). These cells are well-recognized inducers and modulators of T cell immunity and increasingly viewed as mediators of T cell tolerance ( 2, 3). Integral to their APC function is the DC migratory behavior, as these cells continuously traffic between peripheral tissues and secondary lymphoid organs, performing specific functions at each site ( 4). In peripheral tissues, resting immature DCs exert a sentinel function, taking up and processing available Ags. Upon activation, they migrate to draining lymph nodes and home to T cell-rich areas, where they interact with T cells to initiate immune responses. A spontaneous migration of DCs is also observed under healthy steady state conditions, and increasing evidence demonstrates that these steady state migrating DCs, loaded with tissue self Ags, contribute to the maintenance of peripheral tolerance ( 2). Each step of DC trafficking is likely to be controlled, at least in part, by chemokines. Immature, tissue resident DCs respond to many inflammatory/inducible chemokines (CCL3, CCL4, CCL5, and CCL20), while maturing DCs lose responsiveness to most of these chemokines, as a result of down-regulation of cognate receptor expression or function. In a coordinated process, maturing DCs up-regulate CCR7 and gain responsiveness to lymph node-homing chemokines (CCL19 and CCL21). Increasing evidence indicates that soluble factors present in the DC microenvironment are instrumental in determining the outcome of DC differentiation (immunostimulatory vs tolerogenic) and, as a direct consequence, the quality of the T cell response generated, in particular tolerance, memory, and polarized Th1/Th2 differentiation ( 1, 2, 3).
Type I IFNs are cytokines spontaneously expressed at low levels under physiologic conditions whose expression is highly enhanced soon after cell exposure to viruses or other stimuli. Although first characterized as potent antiviral molecules, type I IFNs are also endowed with immunoregulatory activities and serve as important links between innate and adaptive immune responses ( 5, 6). DCs and their precursors are important cellular targets of type I IFN immunomodulatory activities, as demonstrated by the profound effects of these cytokines on DC activation, maturation, and survival ( 7). Although apparently contrasting results have been reported on the effects of type I IFNs on monocyte differentiation into DCs in different experimental models ( 8, 9, 10, 11, 12), we have previously shown that a single-step treatment of freshly isolated monocytes with type I IFN, together with GM-CSF added as survival factor, results in the rapid generation of DCs (IFN-DCs) endowed with important immune priming activities ( 13). IFN-DCs potently stimulate the in vitro proliferative response of allogeneic T cells, and, after viral priming, of autologous PBL, inducing a strongly Th1-polarized response. In vivo studies have shown that virus-pulsed IFN-DCs are potent inducers of both humoral and CD8-mediated immune responses in the human PBL-SCID mouse model ( 13, 14). Of note, IFN-DCs show a unique pattern of expression of chemokine receptors and migratory response to proinflammatory and lymph node-homing chemokines. Compared with GM-CSF/IL-4-generated monocyte-derived DCs (IL-4-DCs), IFN-DCs express higher levels of CCR5 associated with a stronger chemotactic response to -chemokines, express CCR7, and are able to migrate in response to its ligand CCL19 without the need for any further maturation stimuli ( 15).
The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a secosteroid hormone with pleiotropic actions, including maintenance of bone and calcium/phosphate homeostasis, regulation of growth and differentiation of cancer cells, and modulation of the immune response ( 16, 17). The immunoregulatory properties of 1,25(OH)2D3 and its analogues have been demonstrated in different models of autoimmune diseases and in experimental organ transplantation ( 18, 19, 20). DCs are both primary cellular targets and mediators of the immunomodulatory effects of 1,25(OH)2D3. Studies performed either on human monocyte-derived DCs or on bone marrow mouse DCs have clearly demonstrated that 1,25(OH)2D3 inhibits their in vitro differentiation and maturation ( 21, 22, 23). Moreover, vitamin D receptor (VDR)-deficient mice have increased numbers of DCs in lymph nodes, suggesting a physiologically relevant inhibition of DC differentiation by 1,25(OH)2D3 in vivo ( 24). By modulating DC activation and survival, 1,25(OH)2D3 also profoundly affects the phenotype and function of interacting T cells, ultimately leading to the induction of T cell hyporesponsiveness in vitro ( 21) and of regulatory T cells mediating transplantation tolerance ( 25) and arrest of autoimmune diseases in vivo ( 26).
The aim of this study was to investigate the effects of 1,25(OH)2D3 on the type I IFN-mediated pathway for the differentiation of monocytes into DCs. We found that 1,25(OH)2D3 prevents the generation of IFN-DCs when added to freshly isolated monocytes, and is capable of redirecting already differentiated IFN-DCs toward a more immature stage. The suppression of IFN-DC differentiation/activation by 1,25(OH)2D3 is associated with disruption of their capability to migrate in response to inflammatory and lymph node-homing chemokines, although the expression of the cognate receptors and the calcium response are unaffected.
Materials and Methods
Cell separation and culture
Peripheral blood monocytes from healthy donors were isolated by counterflow centrifugal elutriation and further purified by depleting the nonmonocytic population by immunomagnetic beads selection (MACS monocyte isolation kit from Miltenyi Biotec) using the manufacturer’s instructions. To obtain IFN-DCs, monocytes were seeded at 1 x 106 cells/ml and cultured for 3–5 days in RPMI 1640 (Invitrogen Life Technologies) medium containing 10% FBS, supplemented with 50 ng/ml human rGM-CSF (a generous gift from Schering-Plough) and 1000 U/ml human rIFN- (a generous gift from Serono). Depending on the experimental setting, 10 nM 1,25(OH)2D3 (BioXell) was added at seeding or at day 3.
Real-time quantitative RT-PCR
RNA was extracted using TRIzol (Invitrogen Life Technologies), according to the manifacturer’s instructions. Reverse transcription was performed, and real-time quantitative RT-PCR of total cDNA using specific primers was conducted using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) and either Taqman or SIBRgreen chemistry. The primer sequences used are available on request. Relative quantification of target cDNA was determined by calculating the difference in cross-threshold (CT) values after normalization to hypoxanthine phosphoribosyltransferase or GAPDH signals, according to the CT 2–CT. To exclude amplification of genomic DNA, RNA samples were treated with DNase (Sigma-Aldrich).
Flow cytometric analysis
For the analysis of the surface immunophenotype and of CCR5, cells were stained with FITC-conjugated mAbs (BD Pharmingen). The following mAbs were used: CD14 (M5E2), CD1a (HI149), CD80 (L307.4), CD83 (HB15e), HLA-DR (G46-6), CD40 (5C3), CCR5 (2D7), and CD54. Abs directed against and catenins (BD Transduction Laboratories), VCAM (Chemicon International) E-cadherin, and CD44 (Santa Cruz Biotechnology) were used for indirect fluorescence studies by using as secondary Ab the R-PE F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories). Briefly, 1–2 x 105 cells were preincubated for 30 min on ice with PBS 10% human AB serum to block nonspecific Ig binding, and then incubated for an additional 30 min with the appropriate mAb. Cells were washed in PBS 10% human serum and fixed in 1% formaldehyde. For detection of apoptosis/cell death, IFN-DCs were stained with Annexin VFITC and propidium iodide (BD Pharmingen). Cells were analyzed with a FACS flow cytometer (BD Biosciences) using the CellQuest software.
Cytokine analysis
IL-12 and IL-10 secreted in the culture supernatant were quantified using commercial ELISA kits (R&D Systems and Pierce; detection limit of 7.8 and 32.5 pg/ml, respectively). Type I IFNs secreted in culture supernatants were measured by using a cytopathic effect reduction assay with HeLa cells (1 x 104 cells/well in 96-well microplates) and vesicular stomatitis virus, as challenge virus, at the multiplicity of infection of 0.1 PFU/cell. Human IFN- reference standard (National Institutes of Health; Ga23-902-530) was used at the dilution of 500 IU/ml. The sensitivity of the assay was in the range of 2–8 IU/ml.
Mixed leukocyte reaction
The MLR was performed in RPMI 1640 culture medium supplemented with 5% heat-inactivated normal human AB serum. Allogeneic T cells (1–2 x 105/well) were cultured for 5 days in 96-well culture microplates as responder cells with 1–20 x 103 stimulatory cells (DCs). [3H]Thymidine incorporation (sp. act., 5 Ci/mmol; Amersham Bioscience Europe) was measured after a 16-h pulse with 0.5 μCi/well. Results are shown as mean cpm of triplicate samples. [3H]Thymidine incorporation in negative control wells, with responder T cells or stimulator DCs alone, was always <800 cpm.
Chemotaxis assay
Blind well chemotaxis chambers (NeuroProbe) with a lower well volume of 200 μl were used. A maximum volume of 200 μl of RPMI 1640 containing 1% BSA was placed in the lower well in the presence or absence of chemokines. Cells (2 x 105) were placed in the upper compartments of Boyden chambers above the filters. The chambers were incubated for 3 h at 37°C in a 5% CO2 incubator. The filters were then removed, dehydrated, stained with 15% Giemsa for 7 min, and then mounted on glass slides. Cells in 10 high power fields from four to six filters were counted and averaged for each sample. Migration index was calculated as the number of cells migrating toward the concentration gradient of chemokines divided by the number of cells migrating toward medium only.
Measurement of intracellular calcium
Cells (1 x 107/ml) were resuspended in a buffer containing 0.25% BSA, 145 mM NaCl, 5 mM KCl, 10 mM sodium/MOPS, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4, and incubated with 2 μM fura 2-AM (Molecular Probes) for 40 min at room temperature. The cells were washed once, resuspended in the buffer containing 0.25% BSA, and kept at room temperature in the dark. Just before use, cell aliquots (1 x 106) were washed and resuspended in 2 ml of buffer containing 0.005% BSA in a stirred cuvette at 37°C. Measurement of Ca2+ was performed, as previously described ( 27).
Results
The 1,25(OH)2D3 strongly impairs differentiation, maturation, and function of IFN-DCs
In a first set of experiments, we investigated the effect of 1,25(OH)2D3 on the in vitro generation of IFN-DCs. To this aim, freshly isolated monocytes were cultured with GM-CSF and IFN- in the continuous presence of 1,25(OH)2D3, and their immunophenotype and functional activities were compared with control IFN-DCs. IFN-DCs generated in the presence of 1,25(OH)2D3 (D3 IFN-DCs) failed to up-regulate the CD1a differentiation marker as well as the CD83 maturation marker, while retaining high levels of CD14 (Fig. 1A). Moreover, expression of CD40, CD80, and MHC class II molecules was significantly reduced in D3 IFN-DCs, compared with untreated IFN-DCs. In addition, the expression of a panel of adhesion molecules, including CD54, CD44, VCAM, E-cadherin, and and catenins, has been analyzed. None of these molecules, with the only exception of CD54, was found to be modulated by 1,25(OH)2D3 treatment, with respect to control cells. CD54 was abundantly expressed by 99% of both control and 1,25(OH)2D3-treated cells.
However, a 50% reduction in the mean fluorescence intensity was consistently observed in these latter (2611 ± 256 SE in control cells vs 1154 ± 84 SE in 1,25(OH)2D3-treated cells). Consistently with their altered immunophenotype, D3 IFN-DCs also displayed strongly impaired functional activities. IFN-DCs exhibited a potent allostimulatory capacity, while cells generated in the presence of 1,25(OH)2D3 were almost unable to stimulate T cell proliferation (Fig. 1B). In addition, while IFN-DCs produced high levels of IL-12p70 upon LPS stimulation, no detectable LPS-induced IL-12 secretion was found in D3 IFN-DC cultures (Fig. 1C). Thus, similar inhibitory effects are induced by 1,25(OH)2D3 on the differentiation and maturation of IFN-DCs and IL-4-DCs.
FIGURE 1. Effect of 1,25(OH)2D3 on the generation of IFN-DCs. Negatively selected peripheral blood monocytes were cultured for 4 days in the presence of GM-CSF (50 ng/ml) and IFN- (1000 IU/ml). The 1,25(OH)2D3 (10 nM) was added soon after cell seeding. A, Immunophenotype. Cytofluorimetric analysis was performed at day 4. Cells were stained with FITC-conjugated mAbs specific for the indicated markers. Filled histograms represent the background staining of isotype-matched control Ab. A representative experiment of eight performed is shown. B, MLR assay. Purified lymphocytes were seeded into 96-well microplates at the concentration of 1 x 105 cells/well. Allogeneic IFN-DCs were added at graded doses to lymphocytes. After 5 days, [3H]thymidine incorporation was measured. A representative experiment of three performed is shown. C, LPS-induced IL-12 secretion. IFN-DCs or D3 IFN-DCs were treated with LPS (200 ng/ml) at day 3, and culture supernatants were collected 24 h later. IL-12p70 secreted in culture supernatants was quantified by ELISA. Each bar represents the value obtained with cells from a different donor.
1,25(OH)2D3 partially reverts IFN-DC differentiation/activation
Next, we evaluated the effect of 1,25(OH)2D3 on already differentiated IFN-DCs. As previously described ( 13), after a 3-day culture, IFN-DCs are irreversibly committed to DC differentiation/maturation and retain a DC phenotype upon cytokine removal, while IL-4-DCs readily reacquire macrophage features. Thus, 3-day IFN-DCs were exposed to 1,25(OH)2D3 or left untreated, upon removal of GM-CSF and IFN-, and their immunophenotype and functional properties were analyzed 48 h later. As shown in Fig. 2A, in the presence of 1,25(OH)2D3, IFN-DCs regained high levels of CD14 expression, while the expression of CD83 was markedly down-modulated. No significant differences were observed for CD80, CD86, CD40, and HLA-DR (data not shown). Likewise, the extent of apoptosis/cell death in control and 1,25(OH)2D3-treated IFN-DCs was comparable (Fig. 2B). The allostimulatory activity (Fig. 2C) and LPS-induced IL-12 production (Fig. 2D) were also significantly reduced, although not completely abolished, as observed when the immunomodulatory agent was added from the beginning of the culture (Fig. 1). These results indicate that 1,25(OH)2D3 can also revert differentiated IFN-DCs toward a more immature phenotype and function.
FIGURE 2. Effect of 1,25(OH)2D3 on differentiated IFN-DCs. After 3 days of culture, IFN-DCs were washed and reseeded without GM-CSF and IFN-, in the presence or in the absence of 1,25(OH)2D3. Phenotype and functional activities were analyzed 48 h later. A, Immunophenotype. Cytofluorimetric analysis was performed, as described in Fig. 1. Filled histograms represent the background staining of isotype-matched control Ab. A representative experiment of four performed is shown. B, Cell death rates. IFN-DC apoptosis/necrosis was quantified by double staining with Annexin VFITC and propidium iodide 48 h upon removal of GM-CSF/IFN- in the presence or in the absence of 1,25(OH)2D3. A representative experiment of three performed is shown. C, MLR assay. The assay was performed, as described in the legend to Fig. 1. A representative experiment of three performed is shown. D, LPS-induced IL-12 secretion. LPS (100 ng/ml) was added at day 3, 1 h after reseeding in the presence or in the absence of 1,25 (OH)2D3. Supernatants were collected 48 h later, and secreted IL-12p70 was quantified by ELISA, as described in the legend to Fig. 1. Each bar represents IL-12 production from a different donor.
Effect of 1,25(OH)2D3 on LPS-induced type I IFN production in response to activating stimuli
It is well recognized that, upon exposure to a variety of maturation-inducing stimuli, IL-4-DCs secrete type I IFNs ( 28, 29, 30). Thus, we investigated whether production of type I IFNs in response to LPS (a prototypic type I IFN inducer in DCs) also occurred in IFN-DC cultures and whether it was affected by 1,25(OH)2D3. As shown in Fig. 3, IFN-DCs produced significant amounts of biologically active type I IFNs following LPS stimulation (ranging between 100 and 1000 IU/ml, with the majority of donors producing 250–500 IU/ml). This response to LPS stimulation was 10-fold reduced when cells were generated in the continuous presence of 1,25(OH)2D3. On the contrary, exposure of already differentiated IFN-DCs to 1,25(OH)2D3 did not affect their ability to produce type I IFNs in response to LPS. Likewise, the production of LPS-induced IL-10 by IFN-DCs (238.9 pg/ml ± 160.7) was not significantly modified when these cells were cultured in the continuous presence of 1,25(OH)2D3 (210.6 pg/ml ± 161.9) or treated at day 3 for 48 h (384.7 pg/ml ± 81.5).
FIGURE 3. Effect of 1,25(OH)2D3 on type I IFN production in response to LPS. LPS stimulation (100 ng/ml; 48 h) was performed at day 3, after extensive washing and reseeding without GM-CSF and IFN-, to ensure removal of the exogenous IFN. The 1,25(OH)2D3 has been added at culture initiation (D3 IFN-DCs) or at day 3, 1 h before LPS addition (IFN-DCs D3 48 h). The presence of biologically active type I IFN in culture supernatants collected 48 h after LPS stimulation was assessed in a vesicular stomatitis virus-cytopathic reduction assay. Each bar represents type I IFN production from a different donor.
VDR and 25-hydroxyvitamin D-24-hydoxylase (CYP24) mRNA expression during in vitro differentiation/maturation of IFN-DCs
The biological effects of 1,25(OH)2D3 are mediated by the VDR, a member of the superfamily of nuclear hormone receptors, and the enzyme CYP24 has been described as a primary vitamin D-responsive gene ( 31). Therefore, we investigated, by real-time RT-PCR analysis, whether expression of these genes could be modulated during the course of the type I IFN-driven in vitro differentiation of monocytes into DCs. As shown in Fig. 4, time course experiments indicated that VDR mRNA expression did not change significantly, neither during the in vitro differentiation process, nor after a short (2-h) or prolonged treatment with 1,25(OH)2D3 of both monocytes and IFN-DCs. Conversely, the basal levels of CYP24 transcripts observed in monocytes as well as in 3- and 5-day cultured IFN-DCs, were strongly up-regulated upon 1,25(OH)2D3 exposure. In particular, a 2-h treatment with 1,25(OH)2D3 resulted in a 1,000-fold increase of the expression level of CYP24 mRNA in monocytes, and in a 100-fold in IFN-DCs. Upon longer treatments, as routinely performed in this study, the increase in the levels of CYP24 was even stronger, reaching 100,000-fold in D3 IFN-DCs and 10,000-fold in IFN-DCs D3 48 h, compared with control IFN-DCs.
FIGURE 4. Effect of 1,25(OH)2D3 on VDR and CYP24 mRNA expression. RNA samples were obtained at the indicated time points by freshly isolated monocytes and fully differentiated IFN-DCs treated with 1,25(OH)2D3 or left untreated. Quantification of VDR and CYP24 transcripts by real-time RT-PCR was performed, as described in Materials and Methods. The levels of mRNA are shown as arbitrary units normalized to GAPDH expression. Data are from one representative experiment of three performed.
Migration of IFN-DCs in response to CCL4 and CCL19 is abolished in the presence of 1,25(OH)2D3
Because IFN-DCs display a distinctive pattern of chemokine receptor expression and migratory response to specific chemokines ( 15), we investigated the effect of 1,25(OH)2D3 on the expression and function of CCR5 and CCR7 in IFN-DCs. As shown in Fig. 5, CCR5 was expressed at the surface of control IFN-DCs (Fig. 5A), and its triggering by CCL4 induced a significant elevation in intracellular Ca2+ (Fig. 5B) as well as a marked chemotactic response (Fig. 5C). The 1,25(OH)2D3 neither decreased receptor expression nor impaired CCL4-induced raise in intracellular Ca2+. In contrast, the chemotactic response to CCL4 (Fig. 5C) was nearly abolished in the presence of 1,25(OH)2D3. Although CCR7 expression was barely detectable at the surface of control IFN-DCs, as assessed by using the currently available Abs to CCR7 (data not shown), CCL19 addition to these cultures induced a rapid rise in intracellular Ca2+, which was not significantly modified in the presence of 1,25(OH)2D3 (Fig. 6A). In contrast, the migratory response to CCL19 was almost completely abolished by the addition of 1,25(OH)2D3 (Fig. 6B). Thus, in 1,25(OH)2D3-treated IFN-DCs, CCR5 and CCR7 expression and CCL4- and CCL19-triggered calcium mobilization were uncoupled from chemotaxis.
FIGURE 5. Effect of 1,25(OH)2D3 on CCR5 expression and response to CCL4. A, CCR5 expression. Cytofluorimetric profiles of CCR5 surface staining on IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3. Filled histograms represent the background staining of isotype-matched control Ab. A representative experiment of three performed is shown. B, Intracellular calcium levels. IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3 were labeled with fura 2-AM and stimulated with 1 μg/ml CCL4. Arrows indicate the time of chemokine addition. Similar results have been obtained with 100 ng/ml (data not shown). C, Migratory response. Chemotaxis assay was performed on IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3, in the presence of 50 ng/ml CCL4. Similar results were obtained at the CCL4 concentration of 5 ng/ml (data not shown).
FIGURE 6. Effect of 1,25(OH)2D3 on the response to CCL19. A, Intracellular calcium levels. IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3 were stimulated with 40 ng/ml CCL4. Similar results were obtained with 100 ng/ml (data not shown). B, Migratory response. Chemotaxis assay was performed on IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3 in the presence of 50 pg/ml CCL19. Similar results were obtained with 500 pg/ml CCL19 (data not shown).
Discussion
In this study, we demonstrate that 1,25(OH)2D3 potently inhibits the type I IFN-mediated pathway of monocyte differentiation into DCs. These results are in agreement with previous reports on the inhibitory effect of 1,25(OH)2D3 on differentiation and maturation of IL-4-DCs ( 21, 22), further highlighting the role of 1,25(OH)2D3 as a DC modulator. Even though the IL-4/GM-CSF culture method has allowed the performance of many important studies on DC biology, this differentiation pathway may not truly reflect the physiologic process by which monocytes differentiate in vivo into DCs, as high levels of IL-4 are unlikely to be present, for example, during the immune response to infections. Conversely, concentrations of type I IFNs similar to those used in our in vitro model are likely to be transiently present in the microenvironment of circulating or tissue monocytes during viral infections or in other pathological conditions. Indeed, it has been reported that IFN- in sera from patients with systemic lupus erythematosus induced normal monocytes to differentiate into DCs ( 32). Thus, it is reasonable to hypothesize that exposure of monocytes to type I IFNs can represent at least one of the early mechanisms involved in the in vivo generation of immunostimulatory DCs in response to pathogen invasion or in the pathogenesis of autoimmune diseases. The observation that 1,25(OH)2D3 potently inhibits this DC differentiation pathway could be important both in light of DC manipulation for therapeutic intervention, and in consideration of recent evidence pointing to 1,25(OH)2D3 as a physiological regulator of DC homeostasis ( 33). VDR-deficient mice display hypertrophy of s.c. lymph nodes and an increase in mature DCs in lymph nodes, indicating that the 1,25(OH)2D3/VDR complex can mediate a physiologically relevant inhibition of DC maturation in vivo ( 24). Furthermore, two very recent studies have demonstrated that monocyte-derived DCs are able to synthesize 1,25(OH)2D3. This capability is up-regulated upon induction of terminal maturation, while a concomitant down-modulation of the receptor is observed, suggesting a potential autocrine/paracrine loop in the regulation of DC differentiation and function by 1,25(OH)2D3 ( 34, 35).
In our in vitro model of DC differentiation/activation, the addition of the immunomodulatory agent to freshly isolated monocytes completely abrogated the generation of IFN-DCs. Cells cultured in the continuous presence of 1,25(OH)2D3 retained high levels of CD14 and did not gain expression of CD1a and CD83. Moreover, the expression of costimulatory molecules was significantly lower than in control IFN-DCs. Conversely, 1,25(OH)2D3 treatment did not modify the expression of the mannose receptor, thus suggesting that the immunomodulatory agent does not affect the phagocytic activity of IFN-DCs. Similarly, we observed that both subunits of the type I IFN receptor were expressed on IFN-DCs, and were not modulated in the presence of 1,25(OH)2D3 (data not shown). Notably, D3 IFN-DCs were unable to stimulate allogeneic T cell proliferation and to produce IL-12 and type I IFNs in response to further stimulation with LPS. When the immunomodulatory agent was added to already differentiated IFN-DCs, a qualitatively similar effect was observed, although less dramatic. Interestingly, although no significant changes were observed in the expression of VDR under all experimental conditions tested, a clear-cut up-modulation of CYP24 was detected in IFN-DCs treated with 1,25(OH)2D3 for 48 h, and to a higher extent in IFN-DCs generated in the continuous presence of 1,25(OH)2D3. Similarly, we observed a higher up-modulation of CYP24 upon a 2-h treatment with 1,25(OH)2D3 in monocytes than in IFN-DCs. These results indicate that freshly harvested monocytes exhibit a higher responsiveness to 1,25(OH)2D3 compared with IFN-DCs, as assessed by CYP24 mRNA induction. This higher responsiveness is most likely correlated with the stronger inhibitory activity of 1,25(OH)2D3 on the IFN-mediated DC differentiation/maturation pathway observed when it is added to monocytes at seeding, compared with its activity on already differentiated IFN-DCs.
Despite the fact that 1,25(OH)2D3 induces overall similar effects on the differentiation/maturation and functional activities of DCs generated under classical conditions or in the presence of IFN, some differences were observed. We have previously reported that 1,25(OH)2D3 promotes the spontaneous apoptosis of mature IL-4 DCs ( 21). In the present study, the already high levels of spontaneous apoptosis detected in DC generated in the presence of IFN, in agreement with previous data ( 13), were not significantly modified in IFN-DCs treated with 1,25(OH)2D3 for 48 h. This excludes that the inhibitory effect of 1,25(OH)2D3 is due to a selective loss of mature DCs. In addition, while a significant increase in IL-10 production was observed in 1,25(OH)2D3-treated IL-4-DCs in response to maturation stimuli ( 21, 36, 37), the secretion of this cytokine was not significantly modified in IFN-DCs exposed to 1,25(OH)2D3 as compared with control cultures.
In contrast with the results on IL-12 production, the type I IFN response appears to depend on the timing of 1,25(OH)2D3 addition. Although IFN production was strongly reduced when 1,25(OH)2D3 was added to monocytes at seeding, it was not affected when 1,25(OH)2D3 treatment was performed on already differentiated IFN-DCs. With the exception of a study reporting an inhibitory effect of 1,25(OH)2D3 on IFN- production by PBMCs of psoriatic patients ( 38), no data are available on the effects of this immunomodulatory agent on the type I IFN induction pathway. Although the main type I IFN-producing cell is the plasmacytoid DC ( 39), both mouse myeloid DCs ( 40) and human monocyte-derived DCs (28, 29, 30) can also produce high amounts of type I IFNs when appropriately activated. This ability of nonplasmacytoid DCs to produce type I IFNs may be of biological importance because, at variance with plasmacytoid DCs ( 41), they are present at sites of pathogen entry. The IFNs produced by nonplasmacytoid DCs in response to danger signals, besides their direct antiviral effect, could contribute to the differentiation/activation of DCs and then promote protective immune responses. In addition, once DCs have migrated into the lymph node, type I IFNs could induce, in synergy with IL-12, Th1 responses ( 42).
Our data show that chemotaxis of IFN-DCs in response to both CCL4 and CCL19 is strongly impaired in the presence of 1,25(OH)2D3. To the best of our knowledge, this is the first report showing an inhibitory effect of 1,25(OH)2D3 on DC migration. However, despite the dramatic changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. Uncoupling of inflammatory chemokine receptor expression and migratory responses has been described in human DCs and monocytes exposed to IL-10 ( 43) as well as in blood plasmacytoid DCs ( 44), most likely representing a strategy to block excessive DC recruitment and activation upon inflammation. Although the immunological consequences of DC migration impairment by IL-10 in IL-4-DCs and by 1,25(OH)2D3 in IFN-DCs could be similar, the molecular mechanisms involved appear to be different. Indeed, no downstream signaling event was activated after receptor engagement in IL-4-DCs treated with IL-10 ( 43), whereas in IFN-DCs treated with 1,25(OH)2D3 the calcium mobilization in response to both CCL4 and CCL19 was unaffected. The results that we obtained in IFN-DCs are in keeping with the previously observed dissociation between chemotaxis and calcium mobilization ( 45) and suggest that other intracellular pathways required for migration, downstream or parallel to calcium mobilization, could be affected by 1,25(OH)2D3. Although adhesion molecules play a key role in regulating cell migration, they do not seem to be the main targets of the 1,25(OH)2D3 inhibitory activity on IFN-DC migration. Indeed, FACS analysis of a panel of these molecules, including CD44, VCAM, E-cadherin, and and catenins, did not reveal differences in their expression in control and 1,25(OH)2D3-treated IFN-DCs, while a reduction in the amount of CD54 expressed per cell was observed in the presence of 1,25(OH)2D3. It remains to be determined whether integrin expression is affected under these experimental conditions.
Engagement of chemokine receptors by their cognate ligands induces an elaborate biochemical program, ultimately leading to the induction of a variety of functions, including cell migration and proliferation ( 46). The biochemical events include G protein-coupled receptor-dependent calcium flux, as well as changes in intracellular cAMP levels and phosphoinositol lipid metabolism ( 46). Chemokine receptors and their downstream signaling cascades, including activation of c-Src, p125FAK, PyK2, paxillin, and MAPK, are known to be involved in the regulation of chemotactic migratory properties of DCs ( 47). Interestingly, some of these signaling pathway components (i.e., c-Src family proteins and MAPK) have been reported to be modulated by 1,25(OH)2D3 in other cell types ( 48, 49, 50).
Localization in tissues and migration to lymphoid organs are essential steps in DC immunobiology that are linked to the differentiation and T cell stimulatory function of these cells. Thus, our finding that 1,25(OH)2D3 markedly impairs chemotactic responses of IFN-DCs to both proinflammatory and lymph node-homing chemokines unravels a novel mechanism potentially contributing to the multiple immunomodulatory properties of this hormone.
Acknowledgments
We thank A. M. Fattapposta for excellent editorial assistance, and Giuseppe Penna for helpful discussion.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the European Community Contract QLK2-CT-2001-02103 to S.G., A.A.M., and L.A.
2 Address correspondence and reprint requests to Dr. Sandra Gessani, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail address: gessani@iss.it
3 Abbreviations used in this paper: DC, dendritic cell; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; CYP24, 25-hydroxyvitamin D-24-hydoxylase; D3 IFN-DC, IFN-DC generated in the continuous presence of 1,25(OH)2D3; VDR, vitamin D receptor.
Received for publication April 2, 2004. Accepted for publication October 22, 2004.
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Dendritic cells (DCs) generated by a single-step exposure of human monocytes to type I IFN and GM-CSF (IFN-DCs) are endowed with potent immunostimulatory activities and a distinctive migratory response to specific chemokines. In this study, we evaluated the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active metabolite of vitamin D3, on the DC differentiation/activation induced by type I IFN. We found that 1,25(OH)2D3 prevented the generation of IFN-DCs when added to freshly isolated monocytes, and was capable of redirecting already differentiated IFN-DCs toward a more immature stage, as revealed by their immunophenotype, reduced allostimulatory activity, and impaired LPS-induced production of Th1-polarizing cytokines. Control and 1,25(OH)2D3-treated IFN-DCs exhibited a similar expression of vitamin D receptor, as well as comparable cell death rates. Furthermore, the chemotactic response of IFN-DCs to CCL4 and CCL19 was markedly reduced or completely abrogated by 1,25(OH)2D3. Despite these changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. These findings indicate that, in this innovative single-step DC generation model from monocytes, the suppressive effect of 1,25(OH)2D3 is associated with a potent impairment of DC migration in response to inflammatory and lymph node-homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)2D3-mediated immunomodulation.
Introduction
Dendritic cells (DCs)3 are potent APCs that play a pivotal role in the immune response, by linking innate and adaptive immune responses ( 1). These cells are well-recognized inducers and modulators of T cell immunity and increasingly viewed as mediators of T cell tolerance ( 2, 3). Integral to their APC function is the DC migratory behavior, as these cells continuously traffic between peripheral tissues and secondary lymphoid organs, performing specific functions at each site ( 4). In peripheral tissues, resting immature DCs exert a sentinel function, taking up and processing available Ags. Upon activation, they migrate to draining lymph nodes and home to T cell-rich areas, where they interact with T cells to initiate immune responses. A spontaneous migration of DCs is also observed under healthy steady state conditions, and increasing evidence demonstrates that these steady state migrating DCs, loaded with tissue self Ags, contribute to the maintenance of peripheral tolerance ( 2). Each step of DC trafficking is likely to be controlled, at least in part, by chemokines. Immature, tissue resident DCs respond to many inflammatory/inducible chemokines (CCL3, CCL4, CCL5, and CCL20), while maturing DCs lose responsiveness to most of these chemokines, as a result of down-regulation of cognate receptor expression or function. In a coordinated process, maturing DCs up-regulate CCR7 and gain responsiveness to lymph node-homing chemokines (CCL19 and CCL21). Increasing evidence indicates that soluble factors present in the DC microenvironment are instrumental in determining the outcome of DC differentiation (immunostimulatory vs tolerogenic) and, as a direct consequence, the quality of the T cell response generated, in particular tolerance, memory, and polarized Th1/Th2 differentiation ( 1, 2, 3).
Type I IFNs are cytokines spontaneously expressed at low levels under physiologic conditions whose expression is highly enhanced soon after cell exposure to viruses or other stimuli. Although first characterized as potent antiviral molecules, type I IFNs are also endowed with immunoregulatory activities and serve as important links between innate and adaptive immune responses ( 5, 6). DCs and their precursors are important cellular targets of type I IFN immunomodulatory activities, as demonstrated by the profound effects of these cytokines on DC activation, maturation, and survival ( 7). Although apparently contrasting results have been reported on the effects of type I IFNs on monocyte differentiation into DCs in different experimental models ( 8, 9, 10, 11, 12), we have previously shown that a single-step treatment of freshly isolated monocytes with type I IFN, together with GM-CSF added as survival factor, results in the rapid generation of DCs (IFN-DCs) endowed with important immune priming activities ( 13). IFN-DCs potently stimulate the in vitro proliferative response of allogeneic T cells, and, after viral priming, of autologous PBL, inducing a strongly Th1-polarized response. In vivo studies have shown that virus-pulsed IFN-DCs are potent inducers of both humoral and CD8-mediated immune responses in the human PBL-SCID mouse model ( 13, 14). Of note, IFN-DCs show a unique pattern of expression of chemokine receptors and migratory response to proinflammatory and lymph node-homing chemokines. Compared with GM-CSF/IL-4-generated monocyte-derived DCs (IL-4-DCs), IFN-DCs express higher levels of CCR5 associated with a stronger chemotactic response to -chemokines, express CCR7, and are able to migrate in response to its ligand CCL19 without the need for any further maturation stimuli ( 15).
The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a secosteroid hormone with pleiotropic actions, including maintenance of bone and calcium/phosphate homeostasis, regulation of growth and differentiation of cancer cells, and modulation of the immune response ( 16, 17). The immunoregulatory properties of 1,25(OH)2D3 and its analogues have been demonstrated in different models of autoimmune diseases and in experimental organ transplantation ( 18, 19, 20). DCs are both primary cellular targets and mediators of the immunomodulatory effects of 1,25(OH)2D3. Studies performed either on human monocyte-derived DCs or on bone marrow mouse DCs have clearly demonstrated that 1,25(OH)2D3 inhibits their in vitro differentiation and maturation ( 21, 22, 23). Moreover, vitamin D receptor (VDR)-deficient mice have increased numbers of DCs in lymph nodes, suggesting a physiologically relevant inhibition of DC differentiation by 1,25(OH)2D3 in vivo ( 24). By modulating DC activation and survival, 1,25(OH)2D3 also profoundly affects the phenotype and function of interacting T cells, ultimately leading to the induction of T cell hyporesponsiveness in vitro ( 21) and of regulatory T cells mediating transplantation tolerance ( 25) and arrest of autoimmune diseases in vivo ( 26).
The aim of this study was to investigate the effects of 1,25(OH)2D3 on the type I IFN-mediated pathway for the differentiation of monocytes into DCs. We found that 1,25(OH)2D3 prevents the generation of IFN-DCs when added to freshly isolated monocytes, and is capable of redirecting already differentiated IFN-DCs toward a more immature stage. The suppression of IFN-DC differentiation/activation by 1,25(OH)2D3 is associated with disruption of their capability to migrate in response to inflammatory and lymph node-homing chemokines, although the expression of the cognate receptors and the calcium response are unaffected.
Materials and Methods
Cell separation and culture
Peripheral blood monocytes from healthy donors were isolated by counterflow centrifugal elutriation and further purified by depleting the nonmonocytic population by immunomagnetic beads selection (MACS monocyte isolation kit from Miltenyi Biotec) using the manufacturer’s instructions. To obtain IFN-DCs, monocytes were seeded at 1 x 106 cells/ml and cultured for 3–5 days in RPMI 1640 (Invitrogen Life Technologies) medium containing 10% FBS, supplemented with 50 ng/ml human rGM-CSF (a generous gift from Schering-Plough) and 1000 U/ml human rIFN- (a generous gift from Serono). Depending on the experimental setting, 10 nM 1,25(OH)2D3 (BioXell) was added at seeding or at day 3.
Real-time quantitative RT-PCR
RNA was extracted using TRIzol (Invitrogen Life Technologies), according to the manifacturer’s instructions. Reverse transcription was performed, and real-time quantitative RT-PCR of total cDNA using specific primers was conducted using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) and either Taqman or SIBRgreen chemistry. The primer sequences used are available on request. Relative quantification of target cDNA was determined by calculating the difference in cross-threshold (CT) values after normalization to hypoxanthine phosphoribosyltransferase or GAPDH signals, according to the CT 2–CT. To exclude amplification of genomic DNA, RNA samples were treated with DNase (Sigma-Aldrich).
Flow cytometric analysis
For the analysis of the surface immunophenotype and of CCR5, cells were stained with FITC-conjugated mAbs (BD Pharmingen). The following mAbs were used: CD14 (M5E2), CD1a (HI149), CD80 (L307.4), CD83 (HB15e), HLA-DR (G46-6), CD40 (5C3), CCR5 (2D7), and CD54. Abs directed against and catenins (BD Transduction Laboratories), VCAM (Chemicon International) E-cadherin, and CD44 (Santa Cruz Biotechnology) were used for indirect fluorescence studies by using as secondary Ab the R-PE F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories). Briefly, 1–2 x 105 cells were preincubated for 30 min on ice with PBS 10% human AB serum to block nonspecific Ig binding, and then incubated for an additional 30 min with the appropriate mAb. Cells were washed in PBS 10% human serum and fixed in 1% formaldehyde. For detection of apoptosis/cell death, IFN-DCs were stained with Annexin VFITC and propidium iodide (BD Pharmingen). Cells were analyzed with a FACS flow cytometer (BD Biosciences) using the CellQuest software.
Cytokine analysis
IL-12 and IL-10 secreted in the culture supernatant were quantified using commercial ELISA kits (R&D Systems and Pierce; detection limit of 7.8 and 32.5 pg/ml, respectively). Type I IFNs secreted in culture supernatants were measured by using a cytopathic effect reduction assay with HeLa cells (1 x 104 cells/well in 96-well microplates) and vesicular stomatitis virus, as challenge virus, at the multiplicity of infection of 0.1 PFU/cell. Human IFN- reference standard (National Institutes of Health; Ga23-902-530) was used at the dilution of 500 IU/ml. The sensitivity of the assay was in the range of 2–8 IU/ml.
Mixed leukocyte reaction
The MLR was performed in RPMI 1640 culture medium supplemented with 5% heat-inactivated normal human AB serum. Allogeneic T cells (1–2 x 105/well) were cultured for 5 days in 96-well culture microplates as responder cells with 1–20 x 103 stimulatory cells (DCs). [3H]Thymidine incorporation (sp. act., 5 Ci/mmol; Amersham Bioscience Europe) was measured after a 16-h pulse with 0.5 μCi/well. Results are shown as mean cpm of triplicate samples. [3H]Thymidine incorporation in negative control wells, with responder T cells or stimulator DCs alone, was always <800 cpm.
Chemotaxis assay
Blind well chemotaxis chambers (NeuroProbe) with a lower well volume of 200 μl were used. A maximum volume of 200 μl of RPMI 1640 containing 1% BSA was placed in the lower well in the presence or absence of chemokines. Cells (2 x 105) were placed in the upper compartments of Boyden chambers above the filters. The chambers were incubated for 3 h at 37°C in a 5% CO2 incubator. The filters were then removed, dehydrated, stained with 15% Giemsa for 7 min, and then mounted on glass slides. Cells in 10 high power fields from four to six filters were counted and averaged for each sample. Migration index was calculated as the number of cells migrating toward the concentration gradient of chemokines divided by the number of cells migrating toward medium only.
Measurement of intracellular calcium
Cells (1 x 107/ml) were resuspended in a buffer containing 0.25% BSA, 145 mM NaCl, 5 mM KCl, 10 mM sodium/MOPS, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4, and incubated with 2 μM fura 2-AM (Molecular Probes) for 40 min at room temperature. The cells were washed once, resuspended in the buffer containing 0.25% BSA, and kept at room temperature in the dark. Just before use, cell aliquots (1 x 106) were washed and resuspended in 2 ml of buffer containing 0.005% BSA in a stirred cuvette at 37°C. Measurement of Ca2+ was performed, as previously described ( 27).
Results
The 1,25(OH)2D3 strongly impairs differentiation, maturation, and function of IFN-DCs
In a first set of experiments, we investigated the effect of 1,25(OH)2D3 on the in vitro generation of IFN-DCs. To this aim, freshly isolated monocytes were cultured with GM-CSF and IFN- in the continuous presence of 1,25(OH)2D3, and their immunophenotype and functional activities were compared with control IFN-DCs. IFN-DCs generated in the presence of 1,25(OH)2D3 (D3 IFN-DCs) failed to up-regulate the CD1a differentiation marker as well as the CD83 maturation marker, while retaining high levels of CD14 (Fig. 1A). Moreover, expression of CD40, CD80, and MHC class II molecules was significantly reduced in D3 IFN-DCs, compared with untreated IFN-DCs. In addition, the expression of a panel of adhesion molecules, including CD54, CD44, VCAM, E-cadherin, and and catenins, has been analyzed. None of these molecules, with the only exception of CD54, was found to be modulated by 1,25(OH)2D3 treatment, with respect to control cells. CD54 was abundantly expressed by 99% of both control and 1,25(OH)2D3-treated cells.
However, a 50% reduction in the mean fluorescence intensity was consistently observed in these latter (2611 ± 256 SE in control cells vs 1154 ± 84 SE in 1,25(OH)2D3-treated cells). Consistently with their altered immunophenotype, D3 IFN-DCs also displayed strongly impaired functional activities. IFN-DCs exhibited a potent allostimulatory capacity, while cells generated in the presence of 1,25(OH)2D3 were almost unable to stimulate T cell proliferation (Fig. 1B). In addition, while IFN-DCs produced high levels of IL-12p70 upon LPS stimulation, no detectable LPS-induced IL-12 secretion was found in D3 IFN-DC cultures (Fig. 1C). Thus, similar inhibitory effects are induced by 1,25(OH)2D3 on the differentiation and maturation of IFN-DCs and IL-4-DCs.
FIGURE 1. Effect of 1,25(OH)2D3 on the generation of IFN-DCs. Negatively selected peripheral blood monocytes were cultured for 4 days in the presence of GM-CSF (50 ng/ml) and IFN- (1000 IU/ml). The 1,25(OH)2D3 (10 nM) was added soon after cell seeding. A, Immunophenotype. Cytofluorimetric analysis was performed at day 4. Cells were stained with FITC-conjugated mAbs specific for the indicated markers. Filled histograms represent the background staining of isotype-matched control Ab. A representative experiment of eight performed is shown. B, MLR assay. Purified lymphocytes were seeded into 96-well microplates at the concentration of 1 x 105 cells/well. Allogeneic IFN-DCs were added at graded doses to lymphocytes. After 5 days, [3H]thymidine incorporation was measured. A representative experiment of three performed is shown. C, LPS-induced IL-12 secretion. IFN-DCs or D3 IFN-DCs were treated with LPS (200 ng/ml) at day 3, and culture supernatants were collected 24 h later. IL-12p70 secreted in culture supernatants was quantified by ELISA. Each bar represents the value obtained with cells from a different donor.
1,25(OH)2D3 partially reverts IFN-DC differentiation/activation
Next, we evaluated the effect of 1,25(OH)2D3 on already differentiated IFN-DCs. As previously described ( 13), after a 3-day culture, IFN-DCs are irreversibly committed to DC differentiation/maturation and retain a DC phenotype upon cytokine removal, while IL-4-DCs readily reacquire macrophage features. Thus, 3-day IFN-DCs were exposed to 1,25(OH)2D3 or left untreated, upon removal of GM-CSF and IFN-, and their immunophenotype and functional properties were analyzed 48 h later. As shown in Fig. 2A, in the presence of 1,25(OH)2D3, IFN-DCs regained high levels of CD14 expression, while the expression of CD83 was markedly down-modulated. No significant differences were observed for CD80, CD86, CD40, and HLA-DR (data not shown). Likewise, the extent of apoptosis/cell death in control and 1,25(OH)2D3-treated IFN-DCs was comparable (Fig. 2B). The allostimulatory activity (Fig. 2C) and LPS-induced IL-12 production (Fig. 2D) were also significantly reduced, although not completely abolished, as observed when the immunomodulatory agent was added from the beginning of the culture (Fig. 1). These results indicate that 1,25(OH)2D3 can also revert differentiated IFN-DCs toward a more immature phenotype and function.
FIGURE 2. Effect of 1,25(OH)2D3 on differentiated IFN-DCs. After 3 days of culture, IFN-DCs were washed and reseeded without GM-CSF and IFN-, in the presence or in the absence of 1,25(OH)2D3. Phenotype and functional activities were analyzed 48 h later. A, Immunophenotype. Cytofluorimetric analysis was performed, as described in Fig. 1. Filled histograms represent the background staining of isotype-matched control Ab. A representative experiment of four performed is shown. B, Cell death rates. IFN-DC apoptosis/necrosis was quantified by double staining with Annexin VFITC and propidium iodide 48 h upon removal of GM-CSF/IFN- in the presence or in the absence of 1,25(OH)2D3. A representative experiment of three performed is shown. C, MLR assay. The assay was performed, as described in the legend to Fig. 1. A representative experiment of three performed is shown. D, LPS-induced IL-12 secretion. LPS (100 ng/ml) was added at day 3, 1 h after reseeding in the presence or in the absence of 1,25 (OH)2D3. Supernatants were collected 48 h later, and secreted IL-12p70 was quantified by ELISA, as described in the legend to Fig. 1. Each bar represents IL-12 production from a different donor.
Effect of 1,25(OH)2D3 on LPS-induced type I IFN production in response to activating stimuli
It is well recognized that, upon exposure to a variety of maturation-inducing stimuli, IL-4-DCs secrete type I IFNs ( 28, 29, 30). Thus, we investigated whether production of type I IFNs in response to LPS (a prototypic type I IFN inducer in DCs) also occurred in IFN-DC cultures and whether it was affected by 1,25(OH)2D3. As shown in Fig. 3, IFN-DCs produced significant amounts of biologically active type I IFNs following LPS stimulation (ranging between 100 and 1000 IU/ml, with the majority of donors producing 250–500 IU/ml). This response to LPS stimulation was 10-fold reduced when cells were generated in the continuous presence of 1,25(OH)2D3. On the contrary, exposure of already differentiated IFN-DCs to 1,25(OH)2D3 did not affect their ability to produce type I IFNs in response to LPS. Likewise, the production of LPS-induced IL-10 by IFN-DCs (238.9 pg/ml ± 160.7) was not significantly modified when these cells were cultured in the continuous presence of 1,25(OH)2D3 (210.6 pg/ml ± 161.9) or treated at day 3 for 48 h (384.7 pg/ml ± 81.5).
FIGURE 3. Effect of 1,25(OH)2D3 on type I IFN production in response to LPS. LPS stimulation (100 ng/ml; 48 h) was performed at day 3, after extensive washing and reseeding without GM-CSF and IFN-, to ensure removal of the exogenous IFN. The 1,25(OH)2D3 has been added at culture initiation (D3 IFN-DCs) or at day 3, 1 h before LPS addition (IFN-DCs D3 48 h). The presence of biologically active type I IFN in culture supernatants collected 48 h after LPS stimulation was assessed in a vesicular stomatitis virus-cytopathic reduction assay. Each bar represents type I IFN production from a different donor.
VDR and 25-hydroxyvitamin D-24-hydoxylase (CYP24) mRNA expression during in vitro differentiation/maturation of IFN-DCs
The biological effects of 1,25(OH)2D3 are mediated by the VDR, a member of the superfamily of nuclear hormone receptors, and the enzyme CYP24 has been described as a primary vitamin D-responsive gene ( 31). Therefore, we investigated, by real-time RT-PCR analysis, whether expression of these genes could be modulated during the course of the type I IFN-driven in vitro differentiation of monocytes into DCs. As shown in Fig. 4, time course experiments indicated that VDR mRNA expression did not change significantly, neither during the in vitro differentiation process, nor after a short (2-h) or prolonged treatment with 1,25(OH)2D3 of both monocytes and IFN-DCs. Conversely, the basal levels of CYP24 transcripts observed in monocytes as well as in 3- and 5-day cultured IFN-DCs, were strongly up-regulated upon 1,25(OH)2D3 exposure. In particular, a 2-h treatment with 1,25(OH)2D3 resulted in a 1,000-fold increase of the expression level of CYP24 mRNA in monocytes, and in a 100-fold in IFN-DCs. Upon longer treatments, as routinely performed in this study, the increase in the levels of CYP24 was even stronger, reaching 100,000-fold in D3 IFN-DCs and 10,000-fold in IFN-DCs D3 48 h, compared with control IFN-DCs.
FIGURE 4. Effect of 1,25(OH)2D3 on VDR and CYP24 mRNA expression. RNA samples were obtained at the indicated time points by freshly isolated monocytes and fully differentiated IFN-DCs treated with 1,25(OH)2D3 or left untreated. Quantification of VDR and CYP24 transcripts by real-time RT-PCR was performed, as described in Materials and Methods. The levels of mRNA are shown as arbitrary units normalized to GAPDH expression. Data are from one representative experiment of three performed.
Migration of IFN-DCs in response to CCL4 and CCL19 is abolished in the presence of 1,25(OH)2D3
Because IFN-DCs display a distinctive pattern of chemokine receptor expression and migratory response to specific chemokines ( 15), we investigated the effect of 1,25(OH)2D3 on the expression and function of CCR5 and CCR7 in IFN-DCs. As shown in Fig. 5, CCR5 was expressed at the surface of control IFN-DCs (Fig. 5A), and its triggering by CCL4 induced a significant elevation in intracellular Ca2+ (Fig. 5B) as well as a marked chemotactic response (Fig. 5C). The 1,25(OH)2D3 neither decreased receptor expression nor impaired CCL4-induced raise in intracellular Ca2+. In contrast, the chemotactic response to CCL4 (Fig. 5C) was nearly abolished in the presence of 1,25(OH)2D3. Although CCR7 expression was barely detectable at the surface of control IFN-DCs, as assessed by using the currently available Abs to CCR7 (data not shown), CCL19 addition to these cultures induced a rapid rise in intracellular Ca2+, which was not significantly modified in the presence of 1,25(OH)2D3 (Fig. 6A). In contrast, the migratory response to CCL19 was almost completely abolished by the addition of 1,25(OH)2D3 (Fig. 6B). Thus, in 1,25(OH)2D3-treated IFN-DCs, CCR5 and CCR7 expression and CCL4- and CCL19-triggered calcium mobilization were uncoupled from chemotaxis.
FIGURE 5. Effect of 1,25(OH)2D3 on CCR5 expression and response to CCL4. A, CCR5 expression. Cytofluorimetric profiles of CCR5 surface staining on IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3. Filled histograms represent the background staining of isotype-matched control Ab. A representative experiment of three performed is shown. B, Intracellular calcium levels. IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3 were labeled with fura 2-AM and stimulated with 1 μg/ml CCL4. Arrows indicate the time of chemokine addition. Similar results have been obtained with 100 ng/ml (data not shown). C, Migratory response. Chemotaxis assay was performed on IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3, in the presence of 50 ng/ml CCL4. Similar results were obtained at the CCL4 concentration of 5 ng/ml (data not shown).
FIGURE 6. Effect of 1,25(OH)2D3 on the response to CCL19. A, Intracellular calcium levels. IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3 were stimulated with 40 ng/ml CCL4. Similar results were obtained with 100 ng/ml (data not shown). B, Migratory response. Chemotaxis assay was performed on IFN-DCs, D3 IFN-DCs, or IFN-DCs treated for 24 h with 1,25(OH)2D3 in the presence of 50 pg/ml CCL19. Similar results were obtained with 500 pg/ml CCL19 (data not shown).
Discussion
In this study, we demonstrate that 1,25(OH)2D3 potently inhibits the type I IFN-mediated pathway of monocyte differentiation into DCs. These results are in agreement with previous reports on the inhibitory effect of 1,25(OH)2D3 on differentiation and maturation of IL-4-DCs ( 21, 22), further highlighting the role of 1,25(OH)2D3 as a DC modulator. Even though the IL-4/GM-CSF culture method has allowed the performance of many important studies on DC biology, this differentiation pathway may not truly reflect the physiologic process by which monocytes differentiate in vivo into DCs, as high levels of IL-4 are unlikely to be present, for example, during the immune response to infections. Conversely, concentrations of type I IFNs similar to those used in our in vitro model are likely to be transiently present in the microenvironment of circulating or tissue monocytes during viral infections or in other pathological conditions. Indeed, it has been reported that IFN- in sera from patients with systemic lupus erythematosus induced normal monocytes to differentiate into DCs ( 32). Thus, it is reasonable to hypothesize that exposure of monocytes to type I IFNs can represent at least one of the early mechanisms involved in the in vivo generation of immunostimulatory DCs in response to pathogen invasion or in the pathogenesis of autoimmune diseases. The observation that 1,25(OH)2D3 potently inhibits this DC differentiation pathway could be important both in light of DC manipulation for therapeutic intervention, and in consideration of recent evidence pointing to 1,25(OH)2D3 as a physiological regulator of DC homeostasis ( 33). VDR-deficient mice display hypertrophy of s.c. lymph nodes and an increase in mature DCs in lymph nodes, indicating that the 1,25(OH)2D3/VDR complex can mediate a physiologically relevant inhibition of DC maturation in vivo ( 24). Furthermore, two very recent studies have demonstrated that monocyte-derived DCs are able to synthesize 1,25(OH)2D3. This capability is up-regulated upon induction of terminal maturation, while a concomitant down-modulation of the receptor is observed, suggesting a potential autocrine/paracrine loop in the regulation of DC differentiation and function by 1,25(OH)2D3 ( 34, 35).
In our in vitro model of DC differentiation/activation, the addition of the immunomodulatory agent to freshly isolated monocytes completely abrogated the generation of IFN-DCs. Cells cultured in the continuous presence of 1,25(OH)2D3 retained high levels of CD14 and did not gain expression of CD1a and CD83. Moreover, the expression of costimulatory molecules was significantly lower than in control IFN-DCs. Conversely, 1,25(OH)2D3 treatment did not modify the expression of the mannose receptor, thus suggesting that the immunomodulatory agent does not affect the phagocytic activity of IFN-DCs. Similarly, we observed that both subunits of the type I IFN receptor were expressed on IFN-DCs, and were not modulated in the presence of 1,25(OH)2D3 (data not shown). Notably, D3 IFN-DCs were unable to stimulate allogeneic T cell proliferation and to produce IL-12 and type I IFNs in response to further stimulation with LPS. When the immunomodulatory agent was added to already differentiated IFN-DCs, a qualitatively similar effect was observed, although less dramatic. Interestingly, although no significant changes were observed in the expression of VDR under all experimental conditions tested, a clear-cut up-modulation of CYP24 was detected in IFN-DCs treated with 1,25(OH)2D3 for 48 h, and to a higher extent in IFN-DCs generated in the continuous presence of 1,25(OH)2D3. Similarly, we observed a higher up-modulation of CYP24 upon a 2-h treatment with 1,25(OH)2D3 in monocytes than in IFN-DCs. These results indicate that freshly harvested monocytes exhibit a higher responsiveness to 1,25(OH)2D3 compared with IFN-DCs, as assessed by CYP24 mRNA induction. This higher responsiveness is most likely correlated with the stronger inhibitory activity of 1,25(OH)2D3 on the IFN-mediated DC differentiation/maturation pathway observed when it is added to monocytes at seeding, compared with its activity on already differentiated IFN-DCs.
Despite the fact that 1,25(OH)2D3 induces overall similar effects on the differentiation/maturation and functional activities of DCs generated under classical conditions or in the presence of IFN, some differences were observed. We have previously reported that 1,25(OH)2D3 promotes the spontaneous apoptosis of mature IL-4 DCs ( 21). In the present study, the already high levels of spontaneous apoptosis detected in DC generated in the presence of IFN, in agreement with previous data ( 13), were not significantly modified in IFN-DCs treated with 1,25(OH)2D3 for 48 h. This excludes that the inhibitory effect of 1,25(OH)2D3 is due to a selective loss of mature DCs. In addition, while a significant increase in IL-10 production was observed in 1,25(OH)2D3-treated IL-4-DCs in response to maturation stimuli ( 21, 36, 37), the secretion of this cytokine was not significantly modified in IFN-DCs exposed to 1,25(OH)2D3 as compared with control cultures.
In contrast with the results on IL-12 production, the type I IFN response appears to depend on the timing of 1,25(OH)2D3 addition. Although IFN production was strongly reduced when 1,25(OH)2D3 was added to monocytes at seeding, it was not affected when 1,25(OH)2D3 treatment was performed on already differentiated IFN-DCs. With the exception of a study reporting an inhibitory effect of 1,25(OH)2D3 on IFN- production by PBMCs of psoriatic patients ( 38), no data are available on the effects of this immunomodulatory agent on the type I IFN induction pathway. Although the main type I IFN-producing cell is the plasmacytoid DC ( 39), both mouse myeloid DCs ( 40) and human monocyte-derived DCs (28, 29, 30) can also produce high amounts of type I IFNs when appropriately activated. This ability of nonplasmacytoid DCs to produce type I IFNs may be of biological importance because, at variance with plasmacytoid DCs ( 41), they are present at sites of pathogen entry. The IFNs produced by nonplasmacytoid DCs in response to danger signals, besides their direct antiviral effect, could contribute to the differentiation/activation of DCs and then promote protective immune responses. In addition, once DCs have migrated into the lymph node, type I IFNs could induce, in synergy with IL-12, Th1 responses ( 42).
Our data show that chemotaxis of IFN-DCs in response to both CCL4 and CCL19 is strongly impaired in the presence of 1,25(OH)2D3. To the best of our knowledge, this is the first report showing an inhibitory effect of 1,25(OH)2D3 on DC migration. However, despite the dramatic changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. Uncoupling of inflammatory chemokine receptor expression and migratory responses has been described in human DCs and monocytes exposed to IL-10 ( 43) as well as in blood plasmacytoid DCs ( 44), most likely representing a strategy to block excessive DC recruitment and activation upon inflammation. Although the immunological consequences of DC migration impairment by IL-10 in IL-4-DCs and by 1,25(OH)2D3 in IFN-DCs could be similar, the molecular mechanisms involved appear to be different. Indeed, no downstream signaling event was activated after receptor engagement in IL-4-DCs treated with IL-10 ( 43), whereas in IFN-DCs treated with 1,25(OH)2D3 the calcium mobilization in response to both CCL4 and CCL19 was unaffected. The results that we obtained in IFN-DCs are in keeping with the previously observed dissociation between chemotaxis and calcium mobilization ( 45) and suggest that other intracellular pathways required for migration, downstream or parallel to calcium mobilization, could be affected by 1,25(OH)2D3. Although adhesion molecules play a key role in regulating cell migration, they do not seem to be the main targets of the 1,25(OH)2D3 inhibitory activity on IFN-DC migration. Indeed, FACS analysis of a panel of these molecules, including CD44, VCAM, E-cadherin, and and catenins, did not reveal differences in their expression in control and 1,25(OH)2D3-treated IFN-DCs, while a reduction in the amount of CD54 expressed per cell was observed in the presence of 1,25(OH)2D3. It remains to be determined whether integrin expression is affected under these experimental conditions.
Engagement of chemokine receptors by their cognate ligands induces an elaborate biochemical program, ultimately leading to the induction of a variety of functions, including cell migration and proliferation ( 46). The biochemical events include G protein-coupled receptor-dependent calcium flux, as well as changes in intracellular cAMP levels and phosphoinositol lipid metabolism ( 46). Chemokine receptors and their downstream signaling cascades, including activation of c-Src, p125FAK, PyK2, paxillin, and MAPK, are known to be involved in the regulation of chemotactic migratory properties of DCs ( 47). Interestingly, some of these signaling pathway components (i.e., c-Src family proteins and MAPK) have been reported to be modulated by 1,25(OH)2D3 in other cell types ( 48, 49, 50).
Localization in tissues and migration to lymphoid organs are essential steps in DC immunobiology that are linked to the differentiation and T cell stimulatory function of these cells. Thus, our finding that 1,25(OH)2D3 markedly impairs chemotactic responses of IFN-DCs to both proinflammatory and lymph node-homing chemokines unravels a novel mechanism potentially contributing to the multiple immunomodulatory properties of this hormone.
Acknowledgments
We thank A. M. Fattapposta for excellent editorial assistance, and Giuseppe Penna for helpful discussion.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the European Community Contract QLK2-CT-2001-02103 to S.G., A.A.M., and L.A.
2 Address correspondence and reprint requests to Dr. Sandra Gessani, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail address: gessani@iss.it
3 Abbreviations used in this paper: DC, dendritic cell; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; CYP24, 25-hydroxyvitamin D-24-hydoxylase; D3 IFN-DC, IFN-DC generated in the continuous presence of 1,25(OH)2D3; VDR, vitamin D receptor.
Received for publication April 2, 2004. Accepted for publication October 22, 2004.
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