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Differential Regulation of Vitamin D Receptor and Its Ligand in Human Monocyte-Derived Dendritic Cells¹

Martin Hewison^*, Lisa Freeman, Susan V. Hughes^*, Katie N. Evans^*, Rosemary Bland^*, Aristides G. Eliopoulos, Mark D. Kilby, Paul A. H. Moss and Ronjon Chakraverty^2,

Departments of ^* Medical Sciences, Hematology, Cancer Studies, and Reproductive and Child Health, University of Birmingham, Edgbaston, Birmingham, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The functions of dendritic cells (DCs) are tightly regulated such that protective immune responses are elicited and unwanted immune responses are prevented. 125-dihydroxyvitamin D₃ (125(OH)₂D₃) has been identified as a major factor that inhibits the differentiation and maturation of DCs, an effect dependent upon its binding to the nuclear vitamin D receptor (VDR). Physiological control of 125(OH)₂D₃ levels is critically dependent upon 25-hydroxyvitamin D₃-1-hydroxylase (1OHase), a mitochondrial cytochrome P450 enzyme that catalyzes the conversion of inactive precursor 25-hydroxyvitamin D₃ (25(OH)D₃) to the active metabolite 125(OH)₂D₃. Using a human monocyte-derived DC (moDC) model, we have examined the relationship between DC VDR expression and the impact of exposure to its ligand, 125(OH)₂D₃. We show for the first time that moDCs are able to synthesize 125(OH)₂D₃ in vitro as a consequence of increased 1OHase expression. Following terminal differentiation induced by a diverse set of maturation stimuli, there is marked transcriptional up-regulation of 1OHase leading to increased 1OHase enzyme activity. Consistent with this finding is the observation that the development and function of moDCs is inhibited at physiological concentrations of the inactive metabolite 25(OH)D₃. In contrast to 1OHase, VDR expression is down-regulated as monocytes differentiate into immature DCs. Addition of 125(OH)₂D₃ to moDC cultures at different time points indicates that its inhibitory effects are greater in monocyte precursors than in immature DCs. In conclusion, differential regulation of endogenous 125(OH)₂D₃ ligand and its nuclear receptor appear to be important regulators of DC biology and represent potential targets for the manipulation of DC function.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Changes in dendritic cell (DC)³ function represent the net cellular response to a large repertoire of microenvironmental positive and negative stimuli (1). An understanding of how developing or terminally differentiated DCs integrate these signals is of crucial importance in terms of defining regulatory pathways that contribute to the homeostatic control of their functions.

One important regulatory mechanism involves the actions of the secosteroid hormone, 125-dihydroxyvitamin D₃ (125(OH)₂D₃), the biologically active metabolite of vitamin D₃, which influences the immune response at multiple levels (2), including at the level of the APC. For example, 125(OH)₂D₃ inhibits the release of inflammatory cytokines such as IL-12 (3) or TNF- by macrophages (4). Furthermore, in vitro 125(OH)₂D₃ inhibits the differentiation and maturation of human monocyte-derived DCs (moDCs), leading to reduced cell surface expression of CD40, CD80, and CD86 costimulatory molecules, reduced allostimulatory function, and decreased survival (5, 6). Similar findings are observed in murine bone marrow-derived DCs which have been propagated in the presence of 125(OH)₂D₃ analogs (7). Indeed, infusion of small numbers of these altered DCs is sufficient to induce tolerance to male Ags in an HY mismatch model (7).

The major 125(OH)₂D₃-mediated effects upon DCs are mediated through a nuclear vitamin D receptor (VDR) (8). The ligand-VDR complex heterodimerizes with the 9-cis-retinoic acid liganded retinoid X receptor, before binding as a transregulatory complex to vitamin D₃-responsive cis elements in target gene promoters (9). These genes include IL-12p35 and p40 (3), that are expressed upon CD40-mediated licensing of DCs and consequently the repressive effect of 125(OH)₂D₃ upon IL-12 secretion is lost in DCs derived from VDR knockout mice (7). Indeed, the lymph nodes in VDR knockout mice are increased in size as compared with wild type and the proportion of mature DCs within them is greater, suggesting that 125(OH)₂D₃ exerts an essential negative regulatory function in vivo that limits the degree to which DCs undergo spontaneous maturation (7).

The observed effects of 125(OH)₂D₃ upon APCs in vitro are at concentrations greater than that readily measured in vivo, suggesting that its influence upon DC function occurs across microenvironmental gradients where 125(OH)₂D₃ is generated locally. Thus, the impact of 125(OH)₂D₃ upon DC function is likely to be dependent not only upon cellular expression of VDR but also its local synthesis and or breakdown. 125(OH)₂D₃ is generated upon the 1- hydroxylation of an inactive precursor metabolite, 25-hydroxyvitamin D₃ (25(OH)D₃), which exists at a 2–3 log higher concentration than the daughter molecule (10). The level of 125(OH)₂D₃ is, thus, critically dependent upon cellular expression of the enzyme, 25-hydroxyvitamin D₃ (25(OH)D₃) 1--hydroxylase (1OHase) which catalyzes this reaction. 1OHase is expressed predominantly in proximal renal tubular cells (11) where it is subject to tight regulation, but is also expressed in cells from extrarenal sites including skin, endothelium, lymphoid organs, and decidua (12, 13, 14). Macrophages constitute one of the primary sources of extrarenal 125(OH)₂D₃ production by virtue of their ability to up-regulate 1OHase expression (15). Indeed, in the context of aberrant immune responses such as those associated with systemic sarcoidosis, unregulated 125(OH)₂D₃ synthesis can lead to defective calcium homeostasis (16).

Myeloid DCs can be differentiated from monocyte/macrophage precursors in vitro and share much of the cellular apparatus required to react to microbial pathogens and other proinflammatory signals (17). This suggests the possibility that the capacity to synthesize 125(OH)₂D₃ is conserved within DCs. Therefore, the aim of this study was to determine whether moDCs also generate 125(OH)₂D₃ and if so, under what conditions. Further, we aimed to establish the relationship between the synthesis of 125(OH)₂D₃ and the expression of the VDR in DCs. We confirm that differentiation and maturation of human moDCs is associated with significant 125(OH)₂D₃ synthesis, as a result of transcriptional up-regulation of 1OHase. Furthermore, we demonstrate that as moDCs differentiate, there is a significant down-regulation of VDR expression. This differential regulation of inhibitory ligand and its nuclear receptor are indicative of potential autocrine/paracrine regulation of DC differentiation and function, and suggest possible targets for manipulation of DC function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of moDCs

PBMCs were derived from buffy coats of healthy donors by density centrifugation over Lymphoprep (Robbins Scientific, Sunnyvale, CA), resuspended in RPMI 1640/2% human AB serum, and allowed to adhere to plastic at 37°C for 2 h. Nonadherent cells were then removed and monocytes were cultured in IMDM (Life Technologies, Rockville, MD) plus 5% human AB serum (HD Supplies, Buckinghamshire, U.K.) plus 2 mM L-glutamine plus 100 µg/ml streptomycin plus 100 µg/ml penicillin for 7 days in the presence of 800 U/ml GM-CSF (Novartis Pharmaceuticals, East Hanover, NJ) and 1000 U/ml IL-4 (R&D Systems, Abingdon, U.K.) in 75-cm² flasks (Iwaki, Tokyo, Japan). In parallel experiments, moDCs were propagated under serum-free conditions in macrophage serum-free media (Life Technologies). 125(OH)₂D₃ (10^-8 M), 25(OH)D₃ (10^-8–10^-7 M), or vehicle (Sigma-Aldrich, St. Louis, MO) were added in some experiments either at the beginning of culture or at time points indicated in the text. Media were replenished at 3-day intervals. Immature DC were resuspended in the above media ± vitamin D₃ metabolites (on day 7) and incubated in 12-well plates at a concentration of 5 x 10⁵/ml and matured for 48 h in the presence of one of the following maturation stimuli: 5 µg/ml G28.5 CD40 Ab (kindly provided by Prof. J. Gordon, Department of Immunology, University of Birmingham, Birmingham, U.K.) plus 5 µg/ml goat anti-mouse Ig Fc cross-linker (Pierce, Rockford, IL), 50 ng/ml TNF- (Peprotech, London, U.K.), 1000 U/ml IFN-, 1 µg/ml LPS Escherichia coli, or 20 µg/ml poly I:C (all obtained from Sigma-Aldrich). In control experiments, 5 µg/ml nonspecific mouse IgG1 MOPC21 (Sigma-Aldrich) plus 5 µg/ml goat anti-mouse Ig Fc cross-linker (Sigma-Aldrich) or PBS were substituted for the various maturation stimuli. In experiments to test the regulation of 1OHase, 20 µM p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (Sigma-Aldrich) or 50 µM of the NF-B inhibitor N--tosyl-L-phenylalanine chloromethyl ketone (TLCK; Sigma-Aldrich) were added 60 min before the maturation stimulus.

Flow cytometric analysis

The cell surface phenotype of moDCs or DCs propagated in the presence of vitamin D₃ metabolites was analyzed using the following mAbs: anti-CD1a-fluorescecin isothiocyanate (HI149;IgG1), anti-CD14-PE (M5E2. IgG2a), anti-CD83-PE (HB15E, IgG1), anti-CD86-PE (IT2.2, IgG2b), anti-HLA-DR-Cychrome (G46.6, IgG2a; San Diego, CA), and the isotype controls labeled with the appropriate fluorochrome (MOPC21, IgG1; G155–178, IgG2a; and 27–35, IgG2b) were obtained from BD PharMingen (San Diego, CA). The staining of the samples was measured on a FACS Coulter Epics XL (Beckman Coulter, Fullerton, CA), using System II software (Beckman Coulter), and was analyzed with WinMDI software (from Prof. J. Trotter, The Scripps Research Institute, San Diego, CA).

Cytokines

In experiments to test the effect of vitamin D₃ metabolites upon IL-12 production by moDCs, day 7 DCs were incubated for 48 h in 48-well plates at a concentration of 5 x 10⁵/ml with CD40 ligand (CD40L)-transfected or control nontransfected L cells at a ratio of 2:1. IL-12p70 and IL-12p40 levels were evaluated by ELISA kits according to the manufacturer’s instructions (BD PharMingen). The IL-12p40 kit detects the IL-12p40 homodimer or monomer but not the bioactive IL-12p70 heterodimer.

Cytochemistry staining

Cytospins were prepared from monocytes, immature moDC, and mature moDC, washed with PBS and stained for 1 h at room temperature with sheep anti-1OHase Ab (12), which had been diluted 1/200. After washing, the slides were incubated with secondary biotinylated donkey anti-sheep Ab (Binding Site, Birmingham, U.K.) for 30 min, followed by a second 30-min incubation with a streptavidin-biotin complex (strepABComplex; DAKO, Cambridgeshire, U.K.). The staining was visualized using 3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Finally, the cells were counterstained with Mayer’s hematoxylin. The percentage of stained cells and intensity of staining were measured by counting a minimum of 200 cells per slide. All analyses were performed single blind.

EMSAs

These were performed as previously described (18). For the detection of NF-B binding activity, an oligonucleotide containing the HIV-long terminal repeat NF-B consensus binding sequence (5'-GAT CAG GGA CTT TCC GCT GGG GAC TTT CC-3') was used.

Phospho-p38-immunoblots

Phospho-p38 immunoblots were performed as described previously (18) using a phosphospecific p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) Ab (New England Biolabs, Beverly, MA) and were compared with total p38 MAPK.

Quantitative RT-PCR analysis of 1-OHase, VDR, and 25(OH)D₃ 24-hydroxylase (24(OH)ase) mRNA expression

1OHase, VDR, and 24(OH)ase mRNA levels were analyzed using an ABI 7700 sequence detection system (PE Biosystems, Warring, U.K.) using previously reported protocols (12). RNA was extracted from cell pellets using the StrataPrep total RNA miniprep kit (Stratagene, Amsterdam, The Netherlands) and was reverse-transcribed using AMV reverse transcriptase (Promega, Madison, WI) and random hexamers in 40-µl reaction volumes according to manufacturer’s instructions. Amplification of cDNAs was performed in 25-µl volumes on 96-well plates, in a reaction buffer containing TaqMan Universal PCR Master Mix, 3 mM Mn(Oac)₂, 200 µM dNTPs, 1.25 U AmpliTaq Gold polymerase, 1.25 U AmpErase UNG, 150 nmol TaqMan probe, 900 nmol primers, and 50 ng of cDNA. All reactions were multiplexed with the housekeeping gene 18S rRNA, provided as a preoptimized control probe (PE Biosystems) enabling data to be expressed in relation to an internal reference to allow for differences in sampling and RT efficiency. Data were obtained as cycle threshold (Ct) values (the cycle number at which logarithmic PCR plots cross a calculated threshold line) according to the manufacturer’s guidelines and were used to determine Ct values (Ct = Ct of the target gene - Ct of the housekeeping gene). Measurements were conducted in triplicate for each individual experiment. All PCRs were conducted with the primers and probes outlined below using the following reaction conditions: 50°C for 2 min, 95°C for 10 min; 44 cycles of 95°C for 15 s; 60°C for 1 min.

Primers and probes for PCRs

PCR primer and probe sequences for 1OHase were as follows: forward primer, 5'-CACCCGACACGGAGACCTT-3'; reverse primer, 5'-TCAACAGCGTGGACACAAACA-3' and TaqMan probe, 5'-TCCGCGCTGTGGGCTCGG-3'. For 24(OH)ase: forward primer, 5'-CAAACCGTGGAAGGCCTATC-3'; reverse primer, 5'-AGTCTTCCCCTTCCAGGATCA-3' and Taqman probe, 5'-ACTACCGCAAAGAAGGCTACGGGCT-3'. For VDR: forward primer, 5'-CTTCAGGCGAAGCATGAAGC-3'; reverse primer, 5'-CCTTCATCATGCCGATGTCC-3' and TaqMan probe, 5'-AAGGCACTATTCACCTGCCCCTTCAA-3'. In each case, the housekeeping gene used to derive Ct values was 18S rRNA which was analyzed using primers and probes provided by the manufacturer (PE Biosystems).

1OHase activity

Cells were washed in serum-free RPMI 1640 and were then resuspended at 1 x 10⁶ cells/ml. Aliquots of this suspension (400 µl) were then incubated with 10 nM [³H]25(OH)D₃ (specific activity 180 Ci/mmol; Amersham, London, U.K.) for 4 h at 37°C. The reaction was terminated by freezing at -20°C. Cell extracts and medium were combined and vitamin D metabolites were extracted in 2.5 ml of chloroform:methanol (4:1 vol:vol). Following evaporation of the organic phase, steroids were resuspended in 50 µl of dichloromethane:isopropanol (9:1 vol:vol). Standard lanes were included which contained only [³H]25(OH)D₃ or [³H]125(OH)₂D₃. Production of [³H]125(OH)₂D₃ was measured on a Bioscan System 200 imaging TLC plate scanner (Bioscan, Edmonds, WA). Data were reported as fmol of 125(OH)₂D₃ produced per hour per 10⁶ cells.

Analysis of nuclear binding of 125(OH)₂D₃

Numbers of intracellular receptors for 125(OH)₂D₃ were assessed by whole cell nuclear association assays. Briefly, cells were resuspended in serum-free RPMI 1640 to give 0.5 x 10⁷ cells/ml. Aliquots (100 µl) of these suspensions were then added to glass tubes containing a single saturating dose (5 nM) of [³H]125(OH)₂D₃ (specific activity 180 Ci/mmol; Amersham) in the presence or absence of a 200-fold excess of unlabeled 125(OH)₂D₃ (to determine nonspecific binding). Cells were then incubated for 1 h to allow association of ligand-VDR complexes with nuclei and then washed twice with 1 ml of PBS at 4°C to remove unincorporated [³H]125(OH)₂D₃. The final cell pellets were incubated with 0.5 ml of lysis buffer (0.25 M sucrose, 20 mM HCl, 1.1 mM magnesium chloride, and 0.5% Triton X-100, pH 7.4) and centrifuged at 1000 x g for 5 min. This step was repeated and the resulting crude nuclear pellet was resuspended in 100 µl of PBS and 100 µl of ethanol, and transferred to 4 ml of scintillation fluid before scintillation counting. Maximal binding was determined as fmol 125(OH)₂D₃ bound per 10⁶ cells and then converted to VDR per cell using Avogadro’s constant.

Data analysis

Statistical analysis was performed using one-way or repeated measure ANOVA, with posthoc testing using the Student Newmans Keuls multiple comparison posttest (Instat version 2.04a; GraphPad Software, San Diego, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human moDCs synthesize 125(OH)₂D₃

To determine whether human DCs are able to synthesize 125(OH)₂D₃, monocytes were incubated in the presence of IL-4 and GM-CSF for 7 days and were then matured by CD40 ligation for 48 h. At various time points, RNA was prepared and real time RT-PCR was performed to determine 1OHase mRNA expression. Differentiation and maturation of moDCs was accompanied by a marked increase in 1OHase mRNA expression as compared to the starting population, increasing >10-fold in day 7 immature moDCs and >60-fold in moDC matured by CD40 ligation (Fig. 1A). The amplified product corresponded to the expected size and sequence indicating that, in common with macrophages, the transcript is identical to that detected in proximal renal tubular cells (data not shown). We also examined the effect of other maturation stimuli representative of both T cell and microbial origin for their ability to induce the transcriptional up-regulation of 1OHase. As shown in Fig. 1B, under the conditions tested, CD40 ligation, TNF-, IFN-, poly I:C, and LPS treatments of day 7 immature moDCs were associated with 5- to 12-fold induction of 1OHase as compared with nontreated controls. Immunocytochemical staining of monocytes, immature and mature moDCs for the 1OHase enzyme demonstrated a similar increase in protein expression, with dense staining, in particular of mature moDCs (Figs. 1C and 3B). In parallel, 125(OH)₂D₃ synthesis, as estimated by thin layer chromatography of cellular extracts incubated in the presence of the [³H]25(OH)D₃ precursor, was absent in monocytes and increased dramatically following differentiation into immature moDC (Fig. 1, D and E). CD40 ligation-induced moDC maturation further boosted 125(OH)₂D₃ synthesis by >3-fold as compared with immature moDCs or mock-treated cells (Fig. 1, D and E, and data not shown). moDC 1OHase mRNA expression and 125(OH)₂D₃ synthesis was similar to that of GM-CSF-propagated macrophages before and following activation (Fig. 1E and data not shown). In additional experiments, we confirmed that moDC generated under serum-free conditions also up-regulated 1OHase gene transcription and activity, indicating that this phenomenon is reproducible under alternate culture conditions (data not shown). Thus, moDCs develop the capacity to synthesize 125(OH)₂D₃ in vitro to an extent which is comparable to macrophages and which is enhanced upon terminal differentiation induced by a diverse set of stimuli.

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Human moDCs synthesize 125(OH)2D3. A, Monocytes were cultured in 1000 U/ml IL-4 and 800 U/ml GM-CSF for 7 days and then immature moDCs were matured by CD40 ligation for 48 h. RNA was extracted from cells at the indicated time points and real time RT-PCR was performed to evaluate 1OHase expression. Mean fold change (±SEM) in 1OHase mRNA expression (y-axis) compared with monocytes vs day of culture (x-axis) is shown. Data shown are for eight independent experiments. Significant differences were evident between day 1 and both days 7 and 9 (p < 0.001 both comparisons) and between days 7 and 9 (p < 0.01). B, moDCs were propagated as above and on day 7 were left untreated or matured with CD40 mAb, 50 ng/ml TNF-, 1000 U/ml IFN-, 1 µg/ml LPS E. coli, or 20 µg/ml poly I:C for 48 h. Maturation with each stimulus was confirmed by flow cytometric staining for CD83 (not shown). Mean fold increases (±SEM) in 1OHase mRNA expression compared with nontreated cells are shown. Significant increases were observed between nontreated cells and all maturation stimuli tested (CD40 mAb, TNF-, LPS (p < 0.001), IFN-, poly I:C (p < 0.01)). C, Photomicrographs (x400) of immunocytochemical staining of cytospins prepared from monocytes, day 7 immature moDCs, and TNF--matured moDC using polyclonal antisera directed against 1OHase. Negative staining of cytospins incubated without primary Ab or with an excess of the peptide to which the primary was directed confirmed the specificity of staining (not shown). Data are representative of four independent experiments performed. D, Thin layer chromatographic analysis of products following incubation of 1 x 106/ml CD40 ligation-matured moDCs with 10-8 M [3H]25(OH)D3 for 4 h at 37°C. Graph shows disintegrations per minute (y-axis, dpm) vs migration (x-axis, mm). Positions of 125(OH)2D3 (a) and 25(OH)D3 (b) as evaluated by running standards in parallel assays are shown. Data shown are representative of three independent experiments performed. E, Monocytes were cultured in 1000 U/ml IL-4 and 800 U/ml GM-CSF or 800 U/ml GM-CSF alone for 7 days to generate immature DCs or macrophages, respectively, and then were activated by CD40 ligation. The percentages of cell staining for CD14 were 3.3 and 69.5% on day 7 immature moDCs and macrophages, respectively. Evaluation of 1OHase was performed as above and the mean (±SEM triplicate values) of 1OHase activity in fmol 125(OH)2D3 synthesis per 106 cells per hour is shown. The data shown is representative of three independent experiments.

Loss of the 1-hydroxyl group from 125(OH)₂D₃ is associated with an 500-fold reduction in the binding affinity of ligand to VDR (19) and, thus, no VDR-mediated biological activity is demonstrable at physiological concentrations of 25(OH)D₃ (20). The ability of human moDCs to generate 125(OH)₂D₃ from the inactive precursor 25(OH)D₃ suggested that the latter could have inhibitory effects upon the differentiation or function of human moDCs. To test this hypothesis we exposed moDC cultures from the first day of culture to 10^-8–10^-7 M (10–100 nM) 25(OH)D₃, representative of concentrations below or within the normal physiological range (19) and evaluated the effect upon DC differentiation, maturation, allostimulatory potential, and survival. In the same experiments, as a positive control, DCs were propagated in the presence of 10^-8 M (10 nM) 125(OH)₂D₃, the concentration at which maximal inhibitory effects are observed (5, 6). By day 7, immature DCs cultured in the presence of GM-CSF and IL-4 were CD14-negative and had acquired surface CD1a expression (Fig. 2, A and B). As in previous studies, DCs differentiated in the presence of 10^-8 M 125(OH)₂D₃ showed a marked decrease in CD1a expression, while CD14 remained elevated. Addition of 10^-8–10^-7 M 25(OH)D₃ at the initiation of culture also resulted in marked inhibition of CD1a expression at all concentrations tested. Furthermore, we observed a concentration-dependent failure to down-regulate CD14 with relatively minor effects observed at 10^-8 25(OH)D₃ but significant inhibition at 10^-7 M 25(OH)D₃, equivalent to that observed in the presence of 10^-8 M 125(OH)₂D₃. Consistent with this, day 7 immature moDCs that had been cultured in the presence of 10^-8–10^-7 M 25(OH)D₃ had impaired allostimulatory function as compared with controls (data not shown). As shown in Fig. 2, C and D, and in accordance with other studies (5, 6), DCs generated in the presence of 10^-8 M 125(OH)₂D₃ failed to up-regulate CD83 expression in response to CD40 ligation to the same degree as controls. Again, similar concentration-dependent effects were observed following the addition of 10^-8–10^-7 M 25(OH)D₃, with greater inhibition at 10^-7 M 25(OH)D₃, equivalent to that observed in the presence of10^-8 M 125(OH)₂D₃. In addition, DCs cultured in the presence of 10^-8 M 125(OH)₂D₃ and higher concentrations of 25(OH)D₃ both failed to up-regulate other molecules involved in the development of accessory functions, including CD80, CD86, and HLA-DR (Fig. 2, C and D). Similar results were observed when DCs were matured in the presence of TNF- (data not shown). Furthermore, as compared to controls, DCs differentiated in the presence of both 10^-8 M 125(OH)₂D₃ and 10^-7 M 25(OH)D₃ demonstrated an almost complete failure in their capacity to produce IL-12p70 and IL-12p40 in response to CD40 ligation (Fig. 2E) and accordingly had weak APC activity in an allogeneic mixed leukocyte reaction (Fig. 2F). At the concentrations used and under the specific conditions described above, we observed no effects of 25(OH)D₃ upon day 9 DC viability as evaluated by annexin V-FITC (FITC)-propidium iodide (PI) staining (control-FITC^-PI^- 82.4 ± 3.4%; 10^-8 M 125(OH)₂D₃-FITC^-PI^- 80.0 ± 3.0%; and 10^-7 M 25(OH)D₃- FITC^-PI^- 84.0 ± 1.6%, n = 6). In conclusion, these results suggest that the active synthesis of 125(OH)₂D₃ from its inactive precursor restricts the differentiation of immature moDCs in vitro and their subsequent sensitivity to maturation stimuli. This phenomenon appears to operate at physiological concentrations of 25(OH)D₃.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. 25(OH)D3 inhibits DC differentiation, maturation, and allostimulatory potential. A, Monocytes were cultured in 1000 U/ml IL-4 and 800 U/ml GM-CSF for 7 days. 125(OH)2D3 (10-8 M, 10 nM), 25(OH)D3 (10-8–10-7 M, 10–100 nM), or vehicle was added at the start of culture. On day 7, the percentage of cells staining positively for cell surface CD1a (top graph) and CD14 (bottom graph) was evaluated. Results shown are representative of six independent experiments. For CD1a, significant differences were evident between controls and 125(OH)2D3 (p < 0.001) and all concentrations of 25(OH)D3 tested (p < 0.001). For CD14, significant differences were evident between controls and 125(OH)2D3 (p < 0.05) and at 10-7 M 25(OH)D3 (p < 0.05). B, Representative flow cytometric dot plots for cell surface CD1a and CD14 staining of day 7 DCs cultured in the presence of vehicle, 125(OH)2D3 (10-8 M) or 25(OH)D3 (10-7 M). Percentage of positive cells, set according to isotype controls, are shown in each quadrant. C, Histogram to show inhibitory effects of differing concentrations of 25(OH)D3 (10-8–10-7 M, 10–100 nM) as compared with 125(OH)2D3 (10-8 M, 10 nM) upon membrane phenotype of day 9 moDCs/vitamin D3 metabolite-treated moDCs matured by CD40 ligation. Percentage inhibition of mean cellular fluorescence (MCF) = (1 - (MCF test/MCF control)) x 100. D, Flow cytometric histograms showing staining for CD83, CD80, CD86, and HLA-DR for day 9 moDCs/vitamin D3 metabolite-treated moDCs matured by CD40 ligation (isotype control, open histograms; day 9 DC, filled histograms). Figures in the upper right corner of each histogram represent MCF of day 9 DCs stained with specific fluorochrome-labeled Ab minus that of fluorochrome-labeled isotype controls. Results shown are representative of at least six independent experiments, except for CD80 which was evaluated in three independent experiments with similar results. E, moDCs were cultured in the presence of vehicle, 125(OH)2D3 (10-8 M), or 25(OH)D3 (10-7 M) and on day 7 were cultured in the presence of CD40L-transfected cells or nontransfected control cells. At 48 h, culture supernatants were removed and IL-12p70 and IL-12p40 concentrations were measured using an ELISA method. Histogram shows mean ± SEM IL-12p70 (, n = 4) or IL-12p40 (, n = 3) for each condition. Concentration of IL-12 (picograms per milliliter) = IL-12 concentration in the presence of CD40L transfectants - IL-12 concentration in the presence of nontransfected controls. Significant differences were evident between control DCs and 125(OH)2D3 (IL-12p70, p < 0.001; IL-12p40, p < 0.05) and 25(OH)D3 (IL-12p70, p < 0.001; IL-12p40, p < 0.05). F, Day 9 moDCs/vitamin D3 metabolite-treated moDCs matured by CD40 ligation were extensively washed, irradiated 3000 rad, and 1.5 x 104 cells and doubling dilution thereof were added to 96-well round bottom plates. Purified allogeneic 105 CD4+CD45RA+ T cells were added to each well and the cells were incubated for 5 days. Cell proliferation was assessed by [3H]thymidine incorporation after cells were pulsed with [3H]thymidine for the last 16 h of culture. All assays were performed in triplicate. Graph shows proliferation (cpm, y-axis) vs number of moDCs per well (x-axis). Result is representative of six independent experiments. Similar results were obtained when moDCs or vitamin D3 metabolite-treated moDCs were matured in the presence of TNF-.

Regulation of 1OHase

The above data are indicative of a link between immature moDC maturation and the up-regulation of 1OHase expression. Integral to the maturation program of DCs in response to divergent inflammatory stimuli is the activation and cooperative interaction of the p38 MAPK and NF-B pathways (21, 22). Therefore, we reasoned that specific inhibition of each of these pathways would also inhibit the up-regulation of 1OHase expression. As shown in Fig. 3, A and B, inhibition of the p38 MAPK pathway using the pyridinyl imidazole compound SB203580 resulted in a major reduction in the expression of the 1OHase induced by TNF- as demonstrated by immunocytochemical staining. Western blots of total cell lysates were performed in parallel to confirm that SB203580 inhibited p38 MAPK phosphorylation (Fig. 3C). Furthermore, inhibition of nuclear NF-B translocation using the proteosomal inhibitor TLCK was also associated with a failure to up-regulate 1OHase expression induced by TNF- (Fig. 3, A and B). We confirmed in these experiments that TLCK effectively inhibited TNF--induced increases in nuclear NF-B binding (Fig. 3D). In contrast, SB203580 had no effect upon nuclear NF-B binding, confirming that under the conditions used in these experiments, its effects were limited to inhibition of the p38 MAPK pathway alone. We confirmed, as reported previously (23), that inhibition of both these pathways inhibited up-regulation of cell surface CD83, but had no effect on moDC viability (data not shown). In additional experiments (Fig. 3E), we confirmed that disabling either the p38 MAPK or NF-B pathways led to almost complete inhibition of the transcriptional up-regulation of 1OHase upon maturation.

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Regulation of 1(OH)ase. A, Photomicrographs (x400) of immunocytochemical staining of cytospins prepared from TNF--matured moDC cultured in the presence or absence of TLCK (50 µM) or SB203580 (20 µM) using polyclonal antisera directed against 1OHase. Data are representative of three independent experiments performed. B, Stained cytospins were scored for the number of densely staining cells (defined as cytoplasmic staining that obscures the nuclear outline). All analyses were performed single blind and a minimum of 200 cells were counted. Mean percentage (±SEM) of densely staining monocytes, day 7 immature moDCs, and day 9 moDCs matured with TNF- (50 ng/ml) in the presence or absence of TLCK or SB203580 are shown for three independent experiments. C, Immature moDCs were treated for 60 min with SB203580 (20 µM) or were mock-treated before the addition of 50 ng/ml TNF-. After 30 min, cell lysates were prepared and subjected to immunoblotting using mAbs specific for total (p38) or phospho-p38 MAPK (p-p38) as indicated by the arrows. D, Immature moDCs were treated for 60 min with TLCK (50 µM) or SB203580 (20 µM) or mock-treated, before the addition of TNF- (50 ng/ml). After 30 min, nuclear extracts were prepared and nuclear NF-B activity was determined (arrow). E, Immature moDCs were left untreated, or were matured by CD40 ligation with or without the prior addition 60 min before of TLCK (50 µM), SB203580 (20 µM), or 125(OH)2D3 (10-8 M). RNA was extracted and real time RT-PCR was performed to evaluate 1OHase expression. Mean fold change (±SEM) in 1OHase mRNA expression compared with untreated cells is shown; three independent experiments were performed. F, moDCs were prepared as above, with or without the addition separately of 125(OH)2D3 (10-8 M) or 25(OH)D3 (10-7 M). RNA was extracted at the indicated time points and real time RT-PCR was performed to evaluate 24(OH)ase mRNA expression. Similar results were obtained in a second experiment performed.

In summary, 1OHase up-regulation in moDCs is associated with the p38 MAPK-and NF-B-dependent maturation of DCs, but its transcription is resistant to the negative regulatory effects of 125(OH)₂D₃. A degree of control for 125(OH)₂D₃ synthesis may be imparted by the induction of an inactivating enzyme, 24(OH)ase, particularly at early time points during differentiation.

VDR expression is down-regulated during DC differentiation

A major arbiter of the dendritic cellular response to either exogenously or endogenously derived 125(OH)₂D₃ ligand will be the level of expression of VDR and/or the presence of coactivators that are recruited to the nuclear receptor complex. One indirect measure of the integrity of this pathway within cells is the 125(OH)₂D₃-mediated induction of 24(OH)ase transcription (8). Our finding that the induction of 24(OH)ase transcription by 125(OH)₂D₃ in moDCs was greatest at early time points during DC differentiation, then followed by a significant decline, suggested the possibility that VDR levels change during DC differentiation. To evaluate VDR expression we performed real time RT-PCR from RNA extracts of moDC cultures at a number of time points. The DC-derived amplified product was identical in size to the product from the HL-60 cell line that is known to express VDR (not shown). Immature and mature moDCs demonstrated about a 4-fold reduction in VDR gene transcription as compared with the starting population (Fig. 4A). These changes were mirrored by similar reductions in the level of VDR protein expression as evaluated by nuclear binding of the [³H]125(OH)₂D₃ ligand which showed a >2-fold reduction in VDR expression in immature and mature moDCs (Fig. 4B). In contrast to 1OHase, there was no significant difference between VDR expression (either at the transcript or protein level) in immature and mature DCs. Thus, the down-regulation of VDR was not clearly linked to the terminal differentiation of DCs. Consistent with this interpretation, the decline in VDR transcription was not arrested by specific inhibition of p38 MAPK or NF-B pathways (data not shown). We also confirmed that neither 125(OH)₂D₃ nor 25(OH)D₃ treatment of moDCs was associated with significant differences in VDR expression as compared with controls (data not shown).

View larger version (24K): [in this window] [in a new window]   FIGURE 4. VDR expression is down-regulated during DC differentiation. A, Immature moDCs were propagated as stated previously and matured by CD40 ligation. RNA was extracted at the indicated time points and real time RT-PCR was performed to evaluate VDR expression. Mean fold change (±SEM) in VDR mRNA expression (y-axis) compared with monocytes vs day of culture (x-axis) is shown. Data shown are for eight independent experiments, except for day 3 which represents two independent experiments. Significant differences were evident between day 1 and both days 7 and 9 (p < 0.05 both comparisons). B, VDR expression in moDC cultures was assessed by whole cell nuclear association assays, where monocytes (mo), immature moDCs (imDC), and CD40 ligation-matured moDCs (mDC) were incubated at 1 x 106/ml for 1 h with saturating concentrations of [3H]125(OH)2D3, washed, and then lysed to form nuclear pellets before scintillation counting. Mean (±SEM) of VDR per cell are shown. Significant differences were evident between day 1 and both days 7 and 9 (p < 0.001 both comparisons). Similar results were obtained when moDCs were matured in the presence of TNF-. C, moDCs were cultured and immature moDCs were matured by CD40 ligation as before in the presence of vehicle or 125(OH)2D3 (10-8 M) added for 72 h from days 1–3, 4–6, or 7–9. Every 72 h cultured cells in all groups were washed extensively and resuspended in fresh media. On day 9 output cells were used a stimulators in an allogeneic mixed leukocyte reaction as outlined in Fig. 2F. The graph shows proliferation (cpm, y-axis) vs number of moDCs per well (x-axis). The result is representative of three independent experiments. Similar results were obtained when moDCs or vitamin D3 metabolite-treated moDCs were matured in the presence of TNF-. D, Flow cytometric histograms of CD86 staining in day 9 moDCs/vitamin D3 metabolite-treated moDCs cultured under the above conditions (isotype control, thin line; day 9 DC, filled histograms). Figures in the upper right corner of each histogram represent mean cellular fluorescence of day 9 DCs stained with specific fluorochrome-labeled Ab minus that of fluorochrome-labeled isotype controls. Results shown are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this work, we have demonstrated that human moDCs can synthesize the immunosuppressive steroid hormone 125(OH)₂D₃, suggesting autocrine and/or paracrine regulation of DC function. Synthesis of 125(OH)₂D₃ increases markedly as DC differentiate from monocyte precursors. However, and in direct contrast to monocytes, VDR expression is reduced in immature DCs. These data are consistent with a model in which differential regulation of ligand and nuclear receptor contributes to a relative resistance of immature and mature DCs to the inhibitory effect of ligand as compared with their monocyte precursors.

Recent evidence supports the existence of an autocrine element to the homeostatic control of DC development and function (29, 30, 31). For example, certain maturation stimuli induce the autocrine synthesis of IL-10, which appears to bind selectively to its receptor on the surface of immature but not mature DCs, the latter having lost cell surface expression of the receptor (30). Consistent with this, blocking IL-10 enhances sustained NF-B activation and maturation of DCs (32). In another example, exposure of immature moDCs to LPS or TNF- in the presence of serum leads to activation of the extracellular signal-regulated protein kinase-signaling pathway which is a negative regulator of DC maturation (33). These potential autoregulatory phenomena and the work in this study suggest that as DCs integrate positive proinflammatory signals inducing maturation, they simultaneously begin to generate negative signals that limit the extent to which they or other developing DCs can propagate an immune response. Our data suggests that active 125(OH)₂D₃ synthesis by DCs is able to limit the full acquisition of APC function by inhibiting costimulatory molecule expression and the secretion of bioactive IL-12p70. The lack of specific in vitro inhibitors of 1OHase activity means that it has not been possible to exclude the possibility that the effects upon DC function observed in this study result, in part, from a direct effect of 25(OH)D₃. However, loss of the 1-hydroxyl group from 125(OH)₂D₃ is associated with an 500-fold reduction in the binding affinity of ligand to VDR (19) and no biological activity is demonstrable at physiological concentrations of 25(OH)D₃ (20), suggesting that this is unlikely.

The potential role of such inhibitory signals may be to reduce the possibility of bystander activation of the immune response. By maintaining DCs in an immature state or modulating their survival (5, 6), negative feedback mediated by 125(OH)₂D₃ could conceivably contribute to the maintenance of peripheral tolerance to self-Ags. The phenotype of VDR knockout mice which have enlarged lymph nodes containing increased numbers of mature DCs is consistent with a nonredundant physiological role for this ligand in limiting DC activation and/or survival (7). Furthermore, transfer of vitamin D₃ analog-treated donor-derived murine bone marrow-derived DCs can induce tolerance to alloantigens (7). Indeed, systemic administration of 125(OH)₂D₃ prolongs allograft survival, an effect associated with greatly reduced IL-12 production and costimulatory molecule expression by graft-associated DCs, and the induction of a CD4⁺CD25⁺ T cell population with regulatory properties (34). The generation of high local concentrations of 125(OH)₂D₃ upon ligation of the CD40 receptor, as shown in this study, suggests that one potential target is the interacting CD4⁺ T cell that up-regulates CD40L following signaling via its TCR. By limiting the capacity of DCs to become activated by CD40 ligation, 125(OH)₂D₃ could conceivably increase the threshold at which licensing occurs, such that low affinity T cells are less likely to generate unwanted immune responses. Alternatively, the actions of 125(OH)₂D₃ may directly affect CD4⁺ T cells, perhaps by skewing them toward a Th2 phenotype (35), or modulating their sensitivity to Fas-mediated activation-induced cell death (36), or, in conjunction with glucocorticoids, enhancing the development of IL-10-producing regulatory T cells (37).

The ability of any particular cell type to generate 125(OH)₂D₃ will depend upon regulation of 1OHase expression/activity, access to the substrate (25(OH)D₃) and the competing effects of inhibitory enzymes, such as 24(OH)ase. This study reaffirms important differences between renal and extrarenal sites of 125(OH)₂D₃ production. First, DCs and macrophages (38), but not proximal renal tubular cells, (39) respond to a number of proinflammatory stimuli by increasing synthesis of 125(OH)₂D₃. Consistent with this, inhibition of intracellular p38 MAPK and NF-B pathways that promote terminal differentiation of DCs (23) prevents the transcriptional up-regulation of 1OHase. Definition of the minimal promoter region of the 1OHase gene in DCs or macrophages has yet to be determined. Thus, while the 1OHase gene has a putative NF-B binding sequence in its promoter region (40), it is quite possible that the stimulatory effects of proinflammatory signals upon its expression are indirect rather than direct. The failure of inflammatory mediators to enhance proximal renal tubular cell expression of 1OHase, may reflect a relative lack of the cellular apparatus required to respond to such signals. In this context it is noteworthy that differential expression of Toll-like receptors in cell lines isolated from the proximal and distal nephron has been implicated in the variable transcriptional up-regulation of 1OHase in response to LPS (39). A second difference between renal and extrarenal 1OHase regulation is that in contrast to proximal renal tubular cells (24), both DCs and macrophages (26) demonstrate relative insensitivity to the negative regulatory effects of 125(OH)₂D₃. Variations in the absolute levels of VDR between the cell types or the ability of VDR ligand to recruit coactivators/partner receptors may explain this difference. Indeed, it is noteworthy that IFN--activated macrophages can recruit complexes containing 125(OH)₂D₃ into other pathways that directly antagonize the binding of VDR-ligand complexes to vitamin D₃-responsive cis elements (41). Whether such mechanisms are operative in human DCs and interfere with VDR-mediated repression of 1OHase expression remains to be determined. Third, whereas 25(OH)D₃ complexed to vitamin D binding protein is taken up by receptor-mediated endocytosis in proximal renal tubular cells (42), this means of controlling access of enzyme to substrate does not appear to be operative in moDCs which do not express the transcript for megalin, one of the key endocytic receptors involved in this process (M. Hewison and R. Chakraverty, unpublished observations). Despite this, during only relatively short incubations in vitro, 5–10% of the input 25(OH)D₃ is converted to 125(OH)₂D₃, suggesting that that the process whereby vitamin D₃ metabolites enter DCs is highly efficient. Whether this is entirely dependent upon passive diffusion (as with other steroid hormones) or is regulated/facilitated by other means is not known.

Both macrophages (26) and DCs (the latter at the level of transcription) demonstrate the capacity to express the enzyme 24(OH)ase which inactivates 125(OH)₂D₃. The role this enzyme plays in limiting the activity of 125(OH)₂D₃ is unclear at present, because 125(OH)₂D₃ (added directly to the culture or generated by differentiating DCs from the precursor molecule 25(OH)D₃) is clearly able to inhibit DC development. Indeed, the inhibitory effects of 125(OH)₂D₃ was at its greatest during the early culture period, during which the capacity of DCs to up-regulate 24(OH)ase transcript levels in response to ligand is at its highest. Although we have not formally evaluated DC 24(OH)ase enzyme activity, this finding suggests that inactivation of 125(OH)₂D₃ by 24(OH)ase is not significant under the specific conditions studied here. One possibility is that 24(OH)ase activity is more relevant to lower concentrations of 125(OH)₂D₃ that may exist, for example, at the peripheries of focal areas of local synthesis.

The capacity of cells to respond to 125(OH)₂D₃ will be related to their expression of VDR, the presence or absence of intracellular pathways that transduce VDR-mediated responses or, indeed, their ability to invoke other nongenomic signaling pathways (43). In common with macrophages (38), differentiation of moDCs is associated with down-regulation of the VDR. This may explain in part the relative resistance of immature DCs as compared with monocyte precursors to the inhibitory effects of the ligand. Although immature DCs still retain some sensitivity to 125(OH)₂D₃ (5, 6, 44), mature DCs are almost completely resistant to its inhibitory effects (44) despite similar expression of VDR. Thus, other factors, including expression of partner receptors/coactivators or the integrity of downstream signaling pathways, are likely to contribute to this resistance. The precise factors that regulate VDR cellular levels are not yet defined in detail, although the ligand appears to have an important role in increasing its expression via a posttranscriptional mechanism in nonhemopoietic cells (45). In this regard, tissue-specific major N-terminal differences in human VDR mRNAs have been described that could conceivably contribute to variations in renal cell and myeloid hemopoietic cell responsiveness to ligand (46). We did not identify any significant changes in moDC VDR expression in response to exogenous 125(OH)₂D₃ (data not shown), although we did not determine VDR activity in this particular context. Although data have yet to establish a link between any particular VDR gene polymorphisms and absolute levels of VDR mRNA expression (47) or protein synthesis (48) this potential relationship has not been explored in hemopoietic cells. The findings in this study that VDR is down-regulated as DCs differentiate from monocytes and that transcription and activity fall in parallel provide a potentially useful model system in which regulation of VDR expression could be dissected in detail.

In summary, we have demonstrated that differentiation of human moDCs is associated with the active synthesis of 125(OH)₂D₃. In contrast, differentiation of moDCs is associated with significant reductions in the expression of VDR as compared with monocyte precursors. Thus, generation of 125(OH)₂D₃ appears to exert an autoregulatory function by partially prohibiting the differentiation of monocyte precursors into immature DCs and their subsequent ability to undergo terminal differentiation in response to maturation stimuli. These findings suggest a model in which DC synthesis of 125(OH)₂D₃ acts to limit unwanted immune reactivity. In this regard, we are currently investigating the regulation of 125(OH)₂D₃ synthesis and VDR expression in other human DC subsets. How this phenomenon impacts upon the induction of adaptive immunity in vivo will require the detailed analysis of how VDR or 1OHase knockout mice (49) generate immune responses to infectious agents, model, or transplanted Ags.

	Acknowledgments

 
We gratefully acknowledge Moray Campbell and Jasbir Moore (Medical Sciences, Birmingham, U.K.) for advice with real-time RT-PCR analyses.

	Footnotes

 
¹ R.C. was supported by a Senior Fellowship in Experimental Hematology from the Leukemia Research Fund U.K. A.E. was supported by a Medical Research Council Career Development Award and by Cancer Research U.K. Grant SP2584. This work was also supported by grants from the National Kidney Research Fund U. K. (to R.B.) and Biotechnology and Biological Sciences Research Council Research Grant 6/S14523 (to M.H. and M.K.).

² Address correspondence and reprint requests to Dr. Ronjon Chakraverty at the current address: Transplant Biology Research Center/Bone Marrow Transplantation Section, Massachusetts General Hospital, Harvard Medical School, Massachusetts General Hospital East, Building 149-5102, 13th Street, Boston, MA 02129. E-mail address: Ronjon.Chakraverty{at}tbrc.mgh.harvard.edu

³ Abbreviations used in this paper: DC, dendritic cell; 125(OH)₂D₃, 125-dihydroxyvitamin D₃; moDC, monocyte-derived DC; VDR, vitamin D receptor; 25(OH)D₃, 25-hydroxyvitamin D₃; 1OHase, 25(OH)D₃ 1-hydroxylase; 24(OH)ase, 25(OH)D₃ 24-hydroxylase; TLCK, N--tosyl-L-phenylalanine chloromethyl ketone; CD40L, CD40 ligand; MAPK, mitogen-activated protein kinase; Ct, cycle threshold; PI, propidium iodide.

Received for publication October 11, 2002. Accepted for publication March 20, 2003.

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