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,25-Dihydroxyvitamin D3 on Type I IFN-Mediated Monocyte Differentiation into Dendritic Cells: Impairment of Functional Activities and Chemotaxis1
* Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy;
BioXell, Milan, Italy; and
Department of Anatomy, University of Oslo, Oslo, Norway
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Abstract |
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,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 |
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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.
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Materials and Methods |
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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).
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Results |
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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.
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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.
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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).
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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.
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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.
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Discussion |
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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.
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Acknowledgments |
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Footnotes |
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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{at}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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D. Braun, L. Galibert, T. Nakajima, H. Saito, V. V. Quang, M. Rubio, and M. Sarfati Semimature Stage: A Checkpoint in a Dendritic Cell Maturation Program That Allows for Functional Reversion after Signal-Regulatory Protein-{alpha} Ligation and Maturation Signals J. Immunol., December 15, 2006; 177(12): 8550 - 8559. [Abstract] [Full Text] [PDF]
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 S. S. Barros, M. P. Soares, and E. J. Gimeno Macrophages and Giant Cell Proliferation Associated with Bone Protein Synthesis and Calcification in the Trachea and Bronchi of Rabbits Intoxicated with Solanum glaucophyllum. Vet. Pathol., July 1, 2006; 43(4): 494 - 499. [Abstract] [Full Text] [PDF]
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G. Penna, A. Roncari, S. Amuchastegui, K. C. Daniel, E. Berti, M. Colonna, and L. Adorini Expression of the inhibitory receptor ILT3 on dendritic cells is dispensable for induction of CD4+Foxp3+ regulatory T cells by 1,25-dihydroxyvitamin D3 Blood, November 15, 2005; 106(10): 3490 - 3497. [Abstract] [Full Text] [PDF]
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J A Hardin Dendritic cells: potential triggers of autoimmunity and targets for therapy Ann Rheum Dis, November 1, 2005; 64(suppl_4): iv86 - iv90. [Full Text] [PDF]
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 M. Del Corno, M. C. Gauzzi, G. Penna, F. Belardelli, L. Adorini, and S. Gessani Human Immunodeficiency Virus Type 1 gp120 and Other Activation Stimuli Are Highly Effective in Triggering Alpha Interferon and CC Chemokine Production in Circulating Plasmacytoid but Not Myeloid Dendritic Cells J. Virol., October 1, 2005; 79(19): 12597 - 12601. [Abstract] [Full Text] [PDF]
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P. H. Tan, P. Sagoo, C. Chan, J. B. Yates, J. Campbell, S. C. Beutelspacher, B. M. J. Foxwell, G. Lombardi, and A. J. T. George Inhibition of NF-{kappa}B and Oxidative Pathways in Human Dendritic Cells by Antioxidative Vitamins Generates Regulatory T Cells J. Immunol., June 15, 2005; 174(12): 7633 - 7644. [Abstract] [Full Text] [PDF]
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 M. C. Gauzzi, C. Purificato, L. Conti, L. Adorini, F. Belardelli, and S. Gessani IRF-4 expression in the human myeloid lineage: up-regulation during dendritic cell differentiation and inhibition by 1{alpha},25-dihydroxyvitamin D3 J. Leukoc. Biol., June 1, 2005; 77(6): 944 - 947. [Abstract] [Full Text] [PDF]
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