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Topically Applied 1,25-Dihydroxyvitamin D₃ Enhances the Suppressive Activity of CD4⁺CD25⁺ Cells in the Draining Lymph Nodes¹

Shelley Gorman^*, L. Alexandra Kuritzky^*, Melinda A. Judge^*, Katie M. Dixon, Jacqueline P. McGlade^*, Rebecca S. Mason, John J. Finlay-Jones^* and Prue H. Hart^2,*

^* Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Australia; and Bosch Institute and Department of Physiology, University of Sydney, Australia

	Abstract

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The immunomodulatory effects of vitamin D have been described following chronic oral administration to mice or supplementation of cell cultures with 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the active form of vitamin D. In this study, topically applied 1,25(OH)₂D₃, enhanced the suppressive capacity of CD4⁺CD25⁺ cells from the draining lymph nodes. The effects of topical 1,25(OH)₂D₃ were compared with those of UVB irradiation, which is the environmental factor required for 1,25(OH)₂D₃ production in skin. CD4⁺ cells from the skin-draining lymph nodes (SDLN) of either 1,25(OH)₂D₃-treated or UVB-irradiated mice had reduced capacity to proliferate to Ags presented in vitro, and could suppress Ag-specific immune responses upon adoptive transfer into naive mice. This regulation was lost upon removal of CD4⁺CD25⁺ cells. Furthermore, purified CD4⁺CD25⁺ cells from the SDLN of 1,25(OH)₂D₃-treated or UVB-irradiated mice compared with equal numbers of CD4⁺CD25⁺ cells from control mice had increased capacity to suppress immune responses in both in vitro and in vivo assay systems. Following the sensitization of recipient mice with OVA, the proportion of CD4⁺Foxp3⁺ cells of donor origin significantly increased in recipients of CD4⁺CD25⁺ cells from the SDLN of 1,25(OH)₂D₃-treated mice, indicating that these regulatory T cells can expand in vivo with antigenic stimulation. These studies suggest that 1,25(OH)₂D₃ may be an important mediator by which UVB-irradiation exerts some of its immunomodulatory effects.

	Introduction

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As well as being the main initiator of skin neoplasms, the UVB (290–320 nm) component of sunlight is responsible for initiation of the production of the active form of vitamin D (1,25-dihydroxyvitamin D₃; 1,25(OH)₂D₃)³. This lipophilic molecule exerts its actions through the vitamin D receptor (VDR) (1). Upon irradiation with UVB, 7-dehydrocholesterol of skin converts to previtamin D₃, which then isomerises with body heat into vitamin D₃. The bulk of this binds to vitamin D binding protein and is transported to the liver for hydroxylation at the 25 position of vitamin D₃. This metabolite is further hydroxylated in the kidney at the C1 position to form 1,25(OH)₂D₃. In addition, keratinocytes and other skin cells have an autonomous vitamin D₃ pathway and can produce substantial amounts of 1,25(OH)₂D₃ (2). Based on studies in human skin equivalents and in human skin using microdialysis (3, 4), local concentrations of 1,25(OH)₂D₃ in the order of 2–5 nM might be achieved.

The potential immunomodulating effects of vitamin D₃ on the immune system have been largely derived from in vitro observations following the supplementation of DC or T cell cultures with 1,25(OH)₂D₃. This molecule blocks the maturation of myeloid DC by reducing the expression of costimulatory molecules, inhibiting secretion of IL-12 and increasing IL-10 production (5). 1,25(OH)₂D₃ also enhances the secretion of CCL22 by DC in vitro, which is a chemokine that attracts T cells into the skin (5). 1,25(OH)₂D₃ can inhibit the secretion of IFN- by T cells and stimulate Th2 cell development with increased production of IL-4, IL-5, and IL-10 (6). In addition, IL-10-producing regulatory T cells can be induced in vitro by 1,25(OH)₂D₃ in the presence of dexamethasone (7).

Chronic feeding of mice with 1,25(OH)₂D₃ suppressed autoimmune responses in a variety of different mouse models including type 1 diabetes (0.3 µg RI26–2198 (a 1,25(OH)₂D₃ analog)/kg for 16 wk) (8), experimental autoimmune encephalitis (5 µg 1,25(OH)₂D₃/kg thrice weekly for 30 days) (9), arthritis (20 ng 1,25(OH)₂D₃/day, commencing one day before the induction of disease) (10) and asthma (1.6 IU 1,25(OH)₂D₃/g diet) (11). In the model of type 1 diabetes, CD4⁺CD25⁺ regulatory T cell numbers were enhanced in NOD mice chronically fed five times a week for eight weeks with 1,25(OH)₂D₃ (8). Together, these observations indicate that 1,25(OH)₂D₃ induces regulatory-type environments both in vitro and in vivo.

Consequently, the effects of topical application of skin with 1,25(OH)₂D₃ upon subsequent immune responses warrant closer inspection. It is not clear whether topical 1,25(OH)₂D₃ can alter immune responses in models of contact hypersensitivity (CHS). When hapten was applied to the 1,25(OH)₂D₃-treated sites in mice (using a local model of CHS), both no effect (12, 13) and also down-regulation (14) of CHS responses have been reported. In a recent study using human subjects, calcipotriene, a 1,25(OH)₂D₃ analog, significantly suppressed CHS responses by 64% (15). In this paper, increased regulatory activity of CD4⁺CD25⁺Foxp3⁺ cells in the skin-draining lymph nodes (SDLN) following a single topical application of 1,25(OH)₂D₃ is described. The effects of topical 1,25(OH)₂D₃ were compared with those of UVB irradiation as this environmental factor can both induce and activate regulatory T cells in the SDLN of otherwise naive mice (16). These studies indicate that 1,25(OH)₂D₃ may be an important mediator by which UVB-irradiation exerts some of its immunomodulatory effects.

	Materials and Methods

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Mice

Mice transgenic for the OVA_323–339 (ISQAVHAAHAEINEAGR)-specific TCR- (DO11.10) on a BALB/c background were originally purchased from The Jackson Laboratory and bred in-house. Female DO11.10 mice were used between the ages of 8–12 wks. Expression of OVA_323–339-specific TCR- on T cells was confirmed by staining lymph node cells with biotinylated-anti-DO11.10 TCR mAb (KJ1–26; Caltag Laboratories) and then PE-Cy5-streptavidin (BD Biosciences). Female 8-wk-old BALB/c mice were purchased from the Animal Resources Centre (Murdoch, Western Australia). All experiments were performed according to the ethical guidelines of the National Health and Medical Research Council of Australia and with approval from the Telethon Institute for Child Health Research Animal Experimentation Ethics Committee.

Vitamin D application

A 100-µl aliquot containing 125 ng of 1,25-dihyroxyvitamin D₃ (1,25(OH)₂D₃; Sigma-Aldrich) diluted in ethanol, propylene glycol, and water mixed at a 2:1:1 ratio was painted onto a clean-shaven 8 cm² dorsal skin surface of mice. This was equivalent to 37 pmol 1,25(OH)₂D₃ per cm² of skin, or 100 µl of 3 µM 1,25(OH)₂D₃. Alternatively, the vehicle used to dilute 1,25(OH)₂D₃ was applied in a similar manner. In studies of repair of UV-induced thymine dimers, the vehicle or 1,25(OH)₂D₃ was applied immediately after UV irradiation. 1,25(OH)₂D₃ was stored under argon gas at –80°C. The chemical integrity of 1,25(OH)₂D₃ was routinely verified using a scanning spectrophotometer.

UV irradiation

A bank of FS40 sunlamps (Westinghouse) emitting a broad band of UV, 250–360 nm, with 65% of the output in the UVB range (290–320 nm), was used to irradiate mice to deliver a dose of 8 kJ/m² onto clean-shaven 8 cm² dorsal skin as previously described (17, 18). PVC plastic film (0.22 mm thick) was taped to the top of each perspex cage before irradiation to screen out wavelengths <290 nm (UVC radiation). The dose rate of UVB was monitored using a UVX radiometer with a UVX-31 sensor (UV Products) after using the PVC film to remove the UVC wavelengths. Mice were housed in individual compartments of perspex cages during irradiation. UV lamps were held 20 cm above the cages.

Quantification of cyclobutane pyrimidine dimer positive nuclei in UV-irradiated skin

Skin samples were taken from UV-irradiated dorsal skin at 0.5, 3 and 24 h post-UV, fixed in Histochoice (Amresco) and paraffin-embedded. Detection of cyclobutane pyrimidine dimers was by immunohistochemical analysis as previously described (19). Sections were deparaffinised through graded alcohol and endogenous peroxidase blocked by 3% H₂O₂ in H₂O, followed by Ag retrieval with citrate buffer. Nonspecific Ab staining was blocked using 10% normal horse serum in Tris-buffered saline. Sections were incubated with the primary mouse monoclonal IgG1 anti-thymine dimer Ab (Affitech) or isotype control (20 µg/ml) in combination with the Animal Research kit (DakoCytomation).

In vitro proliferation assay

CD4⁺ cells were isolated from the SDLN (inguinal, axillary, and brachial) of treated or control DO11.10 mice. MHC class II⁺ and CD8⁺ cells were removed using supernatant from the TIB-120 hybridoma and rat anti-CD8 mAb (2.5 µg/ml; BD Biosciences) with M-450 sheep anti-rat IgG Dynabeads (Dynal Biotech). A CD4⁺CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec) was used to deplete CD25⁺ cells from CD4⁺ cells. CD4⁺ cells (95% pure, as determined by flow cytometry) were labeled with 5 µM CFSE (Molecular Probes) for 10 min at room temperature. After washing three times, cells were resuspended in RPMI 1640 with 10% FCS and 2 µM 2-ME and aliquoted into round-bottom 96-well plates at 10⁵ cells/0.1 ml/well. OVA_323–339 peptide (Proteomics International) was added at a final concentration of 1 µg/ml. For preparation of APC, lymph nodes of naive BALB/c mice were digested with collagenase type 4 (1 mg/ml; Worthington Biochemical) and DNase (0.1 mg/ml; Sigma-Aldrich) for 30 min at 37°C. CD11c microbeads (Miltenyi Biotec) were used with the autoMACS separator (Miltenyi Biotec) to positively select for CD11c⁺ cells. Cells were washed with RPMI 1640 with 10% FCS and 2 µM 2-ME and added to CD4⁺ cells at varying ratios (1:10 to 1:1000) in replicates of six. This APC population was composed of >90% CD11c⁺ cells, confirmed by staining with PE-anti-CD11c mAb. After incubation for 92 h at 37°C in 5% CO₂, supernatants were harvested and cells washed and stained with PE-Cy5-anti-CD4 mAb. Data were analyzed using the proliferation algorithm of FlowJo software (version 4.6.1; TreeStar) on viable cells, which were gated according to forward and side scatter properties and CD4 expression; 50,000 events were collected on the flow cytometer. The percentage of total cells that had divided was determined by using measurements of the number of viable CD4⁺ CFSE-lo cells. All results shown are of APC cultured at a 1:100 ratio with CD4⁺ cells.

Cytokine detection in supernatants

IL-2, IL-4, IL-5, IL-10, IL-12, and IFN- were detected using rat anti-mouse IL-2, IL-4, IL-5, IL-10, IL-12, or IFN- ELISA capture and detection mAb (BD Biosciences) in a dissociation-enhanced time-resolved fluorescence immunoassay with Europium as the label (sensitivity 25 pg/ml). Recombinant mouse IL-2, IL-4, IL-5, IL-10, IL-12, or IFN- (BD Biosciences) were used as the standards.

Regulation of proliferation by CD4⁺CD25⁺ cells in vitro

CD4⁺CD25⁺ cells (95% pure, as determined by flow cytometry) were isolated from the SDLN of treated or control BALB/c mice using the CD4⁺CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec). CD4⁺CD25^– cells (95% pure) were purified from the lymph nodes of naive DO11.10 mice by also using the CD4⁺CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec) and then labeled with 5 µM CFSE (Molecular Probes). CD4⁺CD25^– cells were resuspended in RPMI 1640 with 10% FCS and 2 µM 2-ME and aliquoted into round-bottom 96-well plates at 10⁵ cells/0.1 ml/well. The CD4⁺CD25⁺ cells were added to the CD4⁺CD25^– cells at ratios of 1:2, 1:5, or 1:10. OVA_323–339 peptide was added at a final concentration of 1 µg/ml. APC were prepared as detailed above, and were added to CD4⁺CD25^– cells at a ratio of 1:100. After incubation for 92 h at 37°C in 5% CO₂, supernatants were harvested and cells washed and stained with PE-Cy5-anti-CD4 mAb. The proliferation of the cells (CD4⁺CD25^–) was determined as described above. Alternatively, methyl-[H³]-thymidine (0.25 mCi; 10 µl/well; Amersham Biosciences) was added to cultures 24 h before the harvest of cells, with [H³]thymidine incorporation used as a measure of cellular proliferation.

Contact hypersensitivity responses

Four days after treatment, 1.5 x 10⁷ CD4⁺ or CD4⁺CD25^– cells or 2.5 x 10⁵ CD4⁺CD25⁺ cells were purified as described above from SDLN of untreated, 1,25(OH)₂D₃-treated or UVB-irradiated BALB/c mice, and adoptively transferred via the tail vein into naive BALB/c mice (five mice per group). Flow cytometry was used to determine that these cells were routinely 95% pure. Twenty-four hours after adoptive transfer, mice were sensitized by painting 25 µl 0.5% 2,4-dinitrofluorobenzene (DNFB; Sigma-Aldrich) diluted in acetone onto the shaved ventral surface. After another 5 days, a CHS response was elicited by painting dorsal and ventral ear surfaces with 10 µl 0.2% DNFB (in acetone). After 24 h, the ear thickness was measured in a blinded manner as previously described (16, 17, 18). The mean ear swelling measured in mice that were challenged but not sensitized with DNFB (0.01 mm) was subtracted from ear measurements in each experiment.

FACS analysis and Abs

The following anti-mouse mAb were obtained from BD Biosciences: FITC- and biotinylated-anti-CD3; FITC-anti-CD62L, PE- and PE-Cy5-anti-CD4; PE-anti-CD44; PE-anti-CD69; biotinylated anti-CD45RB; biotinylated anti-CD54; biotinylated anti-CD103; allyphococyanin-anti-CD25; PE-Cy5-labeled streptavidin; PE-anti-CD11c; and anti-CD16/32. For staining of surface markers, lymph node cells were incubated with Abs and staining buffer (PBS, 0.1% BSA, and 0.01% sodium azide) for 30 min, washed and fixed in 0.3 ml 1% formaldehyde (in PBS). An anti-mouse/rat Foxp3-PE staining kit from eBioscience was used for intracellular staining as described by the manufacturers. A mouse anti-human ki67-FITC kit (BD Biosciences) was used to detect intracellular expression of ki67 in conjunction with the anti-mouse/rat Foxp3-PE staining kit. Data were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo software.

Transfer of CD4⁺CD25⁺ cells and subsequent in vivo OVA sensitization of recipient mice

CD4⁺CD25⁺ cells were purified from the SDLN of 1,25(OH)₂D₃-treated or control DO11.10 mice, as described above. Flow cytometry was used to determine surface expression of CD3, CD4, and CD25 so that CD4⁺CD25⁺ cells were routinely 95% pure. Twenty-four hours following the adoptive transfer of 1 x 10⁵ CD4⁺CD25⁺ cells, naive BALB/c mice were sensitized by i.p. injection of 20 µg OVA protein (Grade V, 98% purity; Sigma-Aldrich) in a suspension with 4 mg of alumium hydroxide (Alu-Gel-S, 2% Al(OH)₃; Serva) in a volume of 200 µl. At 72 h after OVA-sensitization, the lymph nodes that drain the peritoneal cavity (parathymic) were removed from three mice per treatment.

Statistical analyses

Data were compared using the Student’s t test or ANOVA with the Tukey’s posthoc analysis using Microsoft Excel and Prism statistical analysis programs for Macintosh as appropriate.

	Results

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Physiological activity of 1,25(OH)₂D₃

Previous studies have shown that 1,25(OH)₂D₃ can reduce DNA damage induced by UV irradiation (4, 19, 20). To confirm that the 125 ng dose of 1,25(OH)₂D₃ was physiologically active, its capacity to reduce DNA damage was tested by removing skin samples from BALB/c mice at various times after UV-irradiation with either 1,25(OH)₂D₃ or vehicle applied topically immediately after irradiation. 1,25(OH)₂D₃ significantly reduced the number of nuclei positive for cyclobutane pyrimidine dimers as early as 3 h postirradiation (Fig. 1).

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Topical 1,25(OH)2D3 significantly reduced UVB-induced DNA damage in irradiated skin cells. The shaved dorsal skin of BALB/c mice was irradiated with 8 kJ/m2 UVB and then either vehicle () or 125 ng 1,25(OH)2D3 () applied immediately to the irradiated skin. After 0.5, 3, and 24 h, the numbers of cyclobutane pyrimidine dimers were calculated in skin sections as a percentage of total epidermal cell nuclei. Results are shown as mean ± SEM (n = 3 mice/group). *, A significant difference between vehicle and 1,25(OH)2D3 treatments.

To determine whether CD4⁺ cells from the SDLN of 1,25(OH)₂D₃-treated mice had a diminished capacity to proliferate in vitro, these cells were purified from DO11.10 mice at 4 days posttreatment and cultured with OVA peptide and APC (CD11c⁺ cells) derived from the lymph nodes of naive mice. The majority of CD4⁺ cells in the DO11.10 mice express the OVA_323–339 TCR, and will thus proliferate in response to presentation of the OVA_323–339 peptide by APC either in vitro or in vivo. As determined by dilution of the CFSE label on proliferating cells, there was a significant reduction in the ability of CD4⁺ cells purified from the SDLN of 1,25(OH)₂D₃-treated or UVB-irradiated mice to proliferate in vitro (Fig. 2A). For six experiments, the percentage of divided cells (CFSE-lo) was significantly reduced when CD4⁺ cells were derived from the SDLN of 1,25(OH)₂D₃-treated or UVB-irradiated mice (Fig. 2B). However, there was no difference in the extent of proliferation observed between CD4⁺ cell cultures derived from 1,25(OH)₂D₃-treated and UVB-irradiated mice (Fig. 2B).

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Topical 1,25(OH)2D3 significantly reduced the capacity of CD4+ cells from the SDLN of treated mice to proliferate in vitro. In A and B the shaved dorsal skin of DO11.10 mice was topically treated with vehicle, 125 ng 1,25(OH)2D3 (VitD3) or 8 kJ/m2 UVB. Results shown in B have been pooled from six independent experiments (mean + SEM). In C, the shaved dorsal surfaces of DO11.10 mice were topically treated with vehicle (0 ng of 1,25(OH)2D3 or 1, 5, 25, or 125 ng 1,25(OH)2D3. In A–C, four days after treatment, CD4+ cells were purified from the SDLN and cultured with CD11c+ cells purified from the lymph nodes of naive BALB/c mice at a 100:1 ratio, with 1 µg/ml OVA323–339 peptide. In D, mice were administered vehicle (0 days) or 125 ng 1,25(OH)2D3 and after 1, 2, 4, 7, and 21 days, CD4+ cells were purified from the SDLN. In A–D, proliferation of the CD4+ cells was determined by dilution of the intracellular dye CFSE after 92 h of coculture, which are shown as overlays in A. In B, C and D, the percentage of divided live CD4+ (CFSE-lo) cells is shown. In B, C and D, an asterisk (*) denotes a significant suppression following 1,25(OH)2D3 or UVB administration. Results show mean + SEM where cells were pooled from three mice per group and proliferation of cells determined from triplicate wells.

Topical 1,25(OH)₂D₃ did not induce a Th2-bias within cultured CD4⁺ cells

Previous analyses found that lymphocytes cultured with 1,25(OH)₂D₃ added in vitro (21, 22) or derived from 1,25(OH)₂D₃-treated mice (23, 24) secreted increased quantities of Th2-type cytokines such as IL-4 (21, 22), and reduced quantities of the Th1-type cytokine IFN- (22, 23, 24). To determine whether this occurred in our model, cytokine levels were examined in culture supernatants obtained 92 h after the coculture of APC with CD4⁺ cells from the SDLN of treated mice. There was a significant reduction in IL-2 levels in cultures containing CD4⁺ cells from the SDLN of 1,25(OH)₂D₃-treated or UVB-irradiated mice (Fig. 3A). These reductions in IL-2 significantly correlated with the reduced proliferation observed in Fig. 2B (r = 0.99, p < 0.01). Levels of IL-4, IL-5, IL-10, IL-12, or IFN- present in cell culture supernatants of CD4⁺ cells were not altered. IL-4, IL-5, and IFN- levels are shown in Fig. 3B, C, and D, respectively. Thus, with the in vitro conditions used in this study, CD4⁺ cells derived from the SDLN of 1,25(OH)₂D₃-treated mice did not secrete increased quantities of Th2-type cytokines or reduced quantities of Th1-type cytokines.

View larger version (11K): [in this window] [in a new window]   FIGURE 3. CD4+ cells from the SDLN of 1,25(OH)2D3-treated mice secreted less IL-2, but similar quantities of Th1- and Th2-type cytokines as CD4+ cells from vehicle-treated mice. Supernatants were harvested from APC-T cell cocultures after 92 h and three samples were tested per treatment for each of six independent experiments. Levels of IL-2 (A), IL-4 (B), IL-5 (C), and IFN- (D) were detected using a modified ELISA technique. In A, an asterisk (*) denotes a significant difference compared with vehicle treatment only. Results are shown as mean + SEM (n = 18).

Reduced proliferation may reflect control by activated regulatory T cells. To determine whether CD4⁺CD25⁺ cells from the SDLN of 1,25(OH)₂D₃-treated (125 ng) or UVB-irradiated (8 kJ/m²) mice had an increased capacity to suppress the proliferation of cocultured CD4⁺CD25^– cells in vitro, CD4⁺CD25⁺ cells were purified from the SDLN of BALB/c mice at 4 days posttreatment. As controls, CD4⁺CD25⁺ cells were purified from vehicle-treated mice. These cells were cultured with CD4⁺CD25^– cells derived from the lymph nodes of DO11.10 mice at ratios of 1:2, 1:5, or 1:10 with APC from the lymph nodes of naive mice (CD11c⁺ cells) and OVA peptide. As determined by dilution of the CFSE label upon proliferating CD4⁺CD25^– DO11.10 cells, CD4⁺CD25⁺ cells purified from the SDLN of 1,25(OH)₂D₃-treated (Fig. 4A) or UVB-irradiated (Fig. 4B) mice had significantly increased capacity to suppress the proliferation of cocultured CD4⁺CD25^– DO11.10 cells than CD4⁺CD25⁺ cells from control mice. CD4⁺CD25⁺ cells from the SDLN of UVB-irradiated mice suppressed the proliferation of cells at both the 1:5 and 1:10 ratios, while those from 1,25(OH)₂D₃-treated mice were more potent suppressors at only the 1:2 and 1:5 ratios. To confirm these observations with 1,25(OH)₂D₃, a different measure of proliferation, [H³]thymidine incorporation, was used. In this experiment, CD4⁺CD25⁺ cells from BALB/c mice were cultured at a 1:2 ratio with CD4⁺CD25^– DO11.10 cells with APC and OVA peptide. The incorporation of [H³]thymidine was significantly reduced in cocultures containing CD4⁺CD25⁺ cells derived from the SDLN of 1,25(OH)₂D₃-treated compared with those from vehicle-treated mice (Fig. 4C).

View larger version (17K): [in this window] [in a new window]   FIGURE 4. CD4+CD25+ cells from mice topically treated with 1,25(OH)2D3 had enhanced capacity to suppress the proliferation of cocultured CD4+CD25– cells. The shaved dorsal skin of BALB/c mice was topically treated with vehicle, 125 ng 1,25(OH)2D3 (VitD3; A and C) or 8 kJ/m2 UVB (B). CD4+CD25+ cells were purified from the SDLN at four days posttreatment, and then cocultured with CFSE-labeled CD4+CD25– cells from lymph nodes of naive DO11.10 mice at ratio of 1:2, 1:5, or 1:10. OVA323–339 peptide (1 µg/ml) and CD11c+ cells were also added at a 1:100 ratio with the CD4+CD25– cells. Proliferation of the CD4+CD25– cells was determined by dilution of CFSE after 92 h of coculture, which are shown as the percentage of divided live CD4+ cells (CFSE-lo) in A and B. In C, CD4+CD25+ cells were purified from the SDLN of BALB/c mice at four days post 1,25(OH)2D3 treatment, and then cocultured with CD4+CD25– cells from lymph nodes of naive DO11.10 mice at a ratio of 1:2 with CD11c+ cells and OVA peptide. Proliferation of cells in these cultures was determined by the incorporation of [H3]thymidine over the last 24 h of the 92 h culture period. *, A significant difference between treatments. Results show mean + SEM where cells were pooled from at least three mice per group and proliferation of cells determined from triplicate wells.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Depletion of CD25+ cells from CD4+ cells derived from mice treated with 1,25(OH)2D3 ablated the suppression of proliferation observed in T cell cultures. The shaved dorsal skin of DO11.10 mice was topically treated with vehicle, 125 ng 1,25(OH)2D3 (VitD3) or 8 kJ/m2 UVB. Four days after treatment, CD4+ or CD4+CD25– cells were purified from the SDLN of these mice, labeled with CFSE and cultured with CD11c+ cells purified from the lymph nodes of naive BALB/c mice at a 100:1 ratio with 1 µg/ml OVA323–339 peptide. Proliferation of cells was determined by dilution of CFSE after 92 h of coculture, which are shown as overlays in A, and as the percentage of divided live CD4+ cells (CFSE-lo) in B. *, A significant reduction in the percentage of divided cells for CD4+ cells from 1,25(OH)2D3 -treated or UVB-irradiated mice. #, A significant increase in proliferation with the removal of CD4+CD25+ cells by comparing the extent of proliferation observed in cultures containing CD4+ cells with those containing CD4+CD25– cells within treatment groups. ns, Nonsignificant difference in the extent of proliferation was detected for CD4+CD25– cells derived from vehicle and 1,25(OH)2D3-treated or UVB-irradiated mice. Results show mean + SEM where cells were pooled from at least three mice per group and proliferation of cells determined from triplicate wells.

To further confirm that topical 1,25(OH)₂D₃ increases the suppressive activity of CD4⁺CD25⁺ cells in the SDLN, CD4⁺ cells were purified from BALB/c mice treated with either the vehicle, 1,25(OH)₂D₃ or UVB. In a previous study, we showed that transfer of 1.5 x 10⁷ CD4⁺ cells from UVB-irradiated mice was sufficient to suppress contact hypersensitivity (CHS) responses in naive recipients (16). Thus, 1.5 x 10⁷ CD4⁺ cells from the SDLN of either 1,25(OH)₂D₃ or vehicle-treated mice were adoptively transferred into naive BALB/c recipients, which were then sensitized 24 h later with 25 µl 0.5% DNFB. Ear swelling was elicited after another 5 days by applying 10 µl 0.2% DNFB to each ear pinna. Transfer of CD4⁺ cells from the SDLN of mice treated with 1,25(OH)₂D₃ or UVB significantly reduced the ear-swelling response in comparison with that observed in vehicle-treated control mice (Fig. 6).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. CD4+ cells purified from the SDLN of 1,25(OH)2D3-treated or UVB-irradiated mice suppressed contact hypersensitivity responses. The shaved dorsal skin of BALB/c mice was topically treated with vehicle, 125 ng 1,25(OH)2D3 (VitD3) or 8 kJ/m2 UVB. Four days after treatment, CD4+ cells were purified from the SDLN and 1.5 x 107 cells adoptively transferred into recipient BALB/c mice (five mice per group). After 24 h, recipient mice were sensitized on shaved ventral skin by topical application of 25 µl 0.5% DNFB. Ear swelling responses were elicited after a further 5 days by applying 10 µl 0.2% DNFB to each ear pinna. Ear swelling was measured after a further 24 h. *, A significant difference between mice receiving CD4+ cells from vehicle and 1,25(OH)2D3-treated mice (25% reduction) or UVB treatments (36% reduction). Ear swelling responses for mice that did not receive CD4+ cells are also shown. Results are shown as mean + SEM (n = 5).

To determine whether CD4⁺CD25⁺ cells were responsible for reduced CHS responses following the adoptive transfer of CD4⁺ cells from 1,25(OH)₂D₃-treated mice, CD4⁺ cells were depleted of CD25⁺ cells before adoptive transfer. CD4⁺ or CD4⁺CD25^– cells (1.5 x 10⁷) from the SDLN of nonirradiated vehicle-treated, 1,25(OH)₂D₃-treated, or UVB-irradiated BALB/c mice (isolated 4 days posttreatment) were adoptively transferred into naive recipients. CD4⁺ cells from the SDLN of 1,25(OH)₂D₃-treated (Fig. 7A) or UVB-irradiated (Fig. 7B) mice significantly reduced ear-swelling responses in recipient BALB/c mice to a similar extent, in comparison with responses observed in recipients of CD4⁺ cells from vehicle-treated mice. Depletion of CD25⁺ cells before adoptive transfer prevented the suppression observed in recipients of CD4⁺ cells from either 1,25(OH)₂D₃- (Fig. 7A) or UVB-treated (Fig. 7B) mice. In a further experiment, CD4⁺CD25⁺ cells (2.5 x 10⁵ cells) purified from the SDLN of vehicle-treated, 1,25(OH)₂D₃-treated, or UVB-irradiated BALB/c mice (isolated 4 days posttreatment) were adoptively transferred into naive recipients. CD4⁺CD25⁺ cells from vehicle-treated mice did not regulate CHS responses when compared with responses observed in mice that did not receive cells but were sensitized and challenged with DNFB (Fig. 8). CD4⁺CD25⁺ from both 1,25(OH)₂D₃-treated and UVB-irradiated mice significantly suppressed ear swelling responses in recipient mice (Fig. 8). Together these results indicate that the suppressive activity of CD4⁺CD25⁺ regulatory cells is enhanced following topical treatment with 1,25(OH)₂D₃ or irradiation with UVB.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Depletion of CD25+ cells from CD4+ cells before adoptive transfer prevented the suppression of contact hypersensitivity responses in recipients of CD4+ cells from 1,25(OH)2D3-treated mice. The shaved dorsal skin of BALB/c mice was treated with vehicle, 1,25(OH)2D3 (VitD3, A) or 8 kJ/m2 UVB (B). Four days after treatment, CD4+ or CD4+CD25– cells were purified from the SDLN of these mice and 1.5 x 107 cells adoptively transferred into recipient BALB/c mice (five mice per group). After 24 h, recipient mice were sensitized by topical application of 25 µl 0.5% DNFB. Ear swelling responses were elicited after a further 5 days by applying 10 µl 0.2% DNFB to each ear pinna. Ear swelling was measured after a further 24 h. *, A significant difference between treatment pairs. Ear swelling responses for mice that did not receive CD4+ or CD4+CD25– cells are also shown. Results are shown as mean + SEM (n = 5).

View larger version (20K): [in this window] [in a new window]   FIGURE 8. CD4+CD25+ cells purified from the SDLN of 1,25(OH)2D3-treated or UVB-irradiated mice had increased capacity to suppress contact hypersensitivity responses. The shaved dorsal skin of BALB/c mice was topically treated with vehicle, 125 ng 1,25(OH)2D3 (VitD3) or 8 kJ/m2 UVB. Four days after treatment, CD4+CD25+ cells were purified from the SDLN and 2.5 x 105 adoptively transferred into recipient BALB/c mice (five mice per group). After 24 h, recipient mice were sensitized on shaved ventral skin by topical application of 25 µl 0.5% DNFB. Ear swelling responses were elicited after a further 5 days by applying 10 µl 0.2% DNFB to each ear pinna. Ear swelling was measured after a further 24 h. *, A significant difference between mice receiving CD4+CD25+ cells from vehicle and 1,25(OH)2D3-treated (36% reduction) or UVB-irradiated mice (41% reduction). Ear swelling responses for mice that did not receive CD4+CD25+ cells are also shown. Results are shown as mean + SEM (n = 5).

UV-induced regulatory T cells express markers typical of the naturally occurring regulatory T cells, including CD25 (25) and Foxp3 (16, 26). To further examine the phenotype of regulatory T cells in the SDLN of mice topically treated with 1,25(OH)₂D₃, a number of different T cell markers were examined on CD4⁺CD25⁺ or CD4⁺CD25^– cells from the SDLN of vehicle- or 1,25(OH)₂D₃-treated BALB/c mice including the activation markers CD44 and CD69, the lymphocyte/memory markers CD45RB and CD62L, the costimulatory marker CD54, and the regulatory/mucosal cell marker CD103. The expression of these markers was not modified on either CD4⁺CD25⁺ or CD4⁺CD25^– cells from the SDLN of 1,25(OH)₂D₃-treated mice (Table I). The majority of CD4⁺CD25⁺ cells from the SDLN also expressed Foxp3 (>90%). 1,25(OH)₂D₃ had no effect upon the proportion (Fig. 9, A and B) or the number (Fig. 9, E and F) of CD4⁺CD25⁺ Foxp3⁺ cells in the SDLN of either BALB/c or DO11.10 mice. The proportion of CD4⁺ cells expressing Foxp3 and CD25 significantly increased following UVB irradiation in DO11.10 (Fig. 9A) but not BALB/c (Fig. 9B) mice, which have higher baseline levels of Foxp3⁺ cells (Fig. 9B). UVB significantly increased the total number of SDLN cells, and therefore the number of CD4⁺CD25⁺Foxp3⁺ cells in both DO11.10 (Fig. 9, C and E) and BALB/c (Fig. 9, D and F) mice.

View larger version (26K): [in this window] [in a new window]   FIGURE 9. UVB irradiation but not topical 1,25(OH)2D3 induced lymph node hypertrophy and increased the number of CD4+CD25+Foxp3+ cells in the SDLN. The shaved dorsal skin of DO11.10 (A, C, and E) or BALB/c (B, D, and F) mice was topically treated with vehicle, 125 ng 1,25(OH)2D3 (VitD3) or 8 kJ/m2 UVB. After four days, the SDLN were removed and the number of cells counted (C and D). The proportions and numbers of CD3+CD4+ cells expressing CD25 and Foxp3 in the SDLN are shown in (A and B) and (E and F), respectively. Results were derived from six independent experiments where cells from three mice per group were pooled (mean + SEM). *, A significant difference between cells from vehicle and 1,25(OH)2D3-treated or UVB-irradiated mice.

View larger version (14K): [in this window] [in a new window]   FIGURE 10. CD4+CD25+ cells from 1,25(OH)2D3-treated mice proliferated after application of exogenous Ag. In A and B, CD4+CD25+ cells were purified from the lymph nodes of naive DO11.10 mice CFSE-labeled and 1.5 x 105 adoptively transferred into BALB/c recipients. After 18 h, the shaved dorsal skin of these recipient mice (n = 3/treatment) was topically treated with vehicle, 125 ng 1,25(OH)2D3 (VitD3) or 8 kJ/m2 UVB. Four days later, the SDLN were removed and CFSE levels in transferred cells (OVA-TCR+CD4+CD25+) assessed by flow cytometry. Proliferation of the CD4+CD25+ cells was determined by dilution of CFSE, which are shown as the percentage of dividing cells (CFSE-lo) in A and as overlays in B. In A, an asterisk (*) denotes a significant difference in the percentage of dividing cells transferred from UVB-irradiated mice. Results are shown as mean + SEM (n = 3). In B, the numbers 1 and 2 refer to cells, which have undergone 1 or 2 divisions, respectively. In C, the shaved dorsal skin of DO11.10 mice was topically treated with vehicle or 125 ng 1,25(OH)2D3 (VitD3). Four days after treatment, CD4+CD25+ cells were purified from the SDLN and 1 x 105 adoptively transferred into each of five recipient BALB/c mice. After 24 h, recipient mice were sensitized by intraperitoneal injection of 20 µg OVA with 4 mg alum. The PLN were removed 72 h after OVA sensitization and the proportion of OVA-TCR+CD4+Foxp3+ki67+‘donor’ cells in the PLN of recipients of CD4+CD25+ cells investigated. An asterisk (*) denotes a significant increase for cells transferred from 1,25(OH)2D3-treated mice. Results are shown as mean + SEM (n = 3).

As shown above, topical 1,25(OH)₂D₃ alone did not cause the adoptively transferred CD4⁺CD25⁺ cells to proliferate. We then hypothesized that the presentation of Ag to these cells might induce their expansion in vivo. To test this, at four days post 1,25(OH)₂D₃-treatment, CD4⁺CD25⁺ cells were purified from the SDLN of vehicle- or 1,25(OH)₂D₃-treated DO11.10 mice, and 1 x 10⁵ CD4⁺CD25⁺ cells adoptively transferred into naive BALB/c mice. After 24 h, recipient mice were sensitized i.p. with 20 µg OVA with Alum included as an adjuvant. Peritoneal cavity-draining lymph nodes (PLN) were removed at 72 h postsensitization and the expression of the regulatory T cell marker Foxp3 on the donor cells (OVA-TCR⁺) was determined by flow cytometry. In addition, the expression of ki67, a nuclear cell proliferation-associated Ag expressed in all active stages of the cell cycle was determined. At 72 h, the proportion of CD4⁺Foxp3⁺ki67⁺ cells that were derived from the donor mice (OVA-TCR⁺) was significantly increased in the recipients of CD4⁺CD25⁺ cells from 1,25(OH)₂D₃-treated mice (Fig. 10C). The expression of ki67 upon CD4⁺Foxp3⁺ cells indicated that these cells were cycling, and that the increased number in recipients of CD4⁺CD25⁺ cells from 1,25(OH)₂D₃-treated mice was not due to enhanced survival of the transferred population. Instead, the increased proportion of CD4⁺Foxp3⁺ki67⁺ cells of donor origin in recipients of CD4⁺CD25⁺ cells from 1,25(OH)₂D₃-treated mice suggests that OVA sensitization induced expansion of the transferred cells.

	Discussion

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This study demonstrates for the first time that a single topical application of a physiologically relevant amount of 1,25(OH)₂D₃ to the shaved dorsal skin of mice enhances the suppressive capacity of CD4⁺CD25⁺ regulatory T cells in the SDLN. This complements other in vitro studies where 1,25(OH)₂D₃ stimulated regulatory DC and T cells (5, 6, 7), and in vivo models where autoimmunity was reduced by chronic oral administration of 1,25(OH)₂D₃ (8, 9, 10, 11). In this study, CD4⁺CD25⁺ cells activated by topical administration of 1,25(OH)₂D₃ suppressed the proliferation in vitro of cocultured CD4⁺CD25^– cells to a greater extent than equal numbers of CD4⁺CD25⁺ cells from control, vehicle-treated mice. Even though the regulatory activity of the CD4⁺CD25⁺ cells was enhanced by topical 1,25(OH)₂D₃, numbers of CD4⁺CD25⁺Foxp3⁺ cells in the SDLN were not increased. In an in vivo model, when equal numbers of CD4⁺CD25⁺ cells from the SDLN of BALB/c mice topically treated with 1,25(OH)₂D₃ or vehicle were adoptively transferred into BALB/c recipients subsequently sensitized and challenged with hapten, ear swelling responses were significantly reduced in those mice that received cells from 1,25(OH)₂D₃-treated mice.

Transferred CD4⁺CD25⁺ cells were derived from the SDLN, and thus may have been enriched for TCR specific for skin-derived Ags. Upon sensitization with a new Ag, we hypothesize that the transferred cells facilitate proliferation or expansion of new regulatory T cells specific for that Ag. Indeed, following the sensitization of recipient mice with OVA, proportions of CD4⁺Foxp3⁺ cells of donor origin (OVA-TCR⁺) were increased in recipients of CD4⁺CD25⁺ cells from DO11.10 mice treated with 1,25(OH)₂D₃. These cells also expressed ki67, a marker of proliferation, which suggests that transferred CD4⁺CD25⁺ cells expand in recipient mice upon sensitization with Ag. Similar results have been observed in a mouse model of diabetes, where adoptive transfer of regulatory T cells increased the number of Foxp3⁺ cells in pancreatic lymph nodes to block the development of diabetes in recipient NOD mice (27).

In the SDLN of DO11.10 mice, the proportion of cells expressing the OVA-TCR was lower for CD4⁺CD25⁺ (60%) in comparison with CD4⁺CD25^– cells (85%). The levels of expression of the OVA-TCR on CD4⁺CD25⁺ cells from DO11.10 mice were not modified by 1,25(OH)₂D₃ or UVB. The expression of OVA-TCR on the majority of CD4⁺CD25⁺ cells may facilitate the capacity of regulatory T cells from 1,25(OH)₂D₃-treated or UVB-irradiated mice to suppress OVA-specific immune responses. However, experiments using BALB/c mice for the donor cells (e.g., CHS adoptive transfer experiments) demonstrate that the activating effects of 1,25(OH)₂D₃ on CD4⁺CD25⁺ cells could be detected in systems not dependent on the majority of CD4⁺CD25⁺ cells expressing TCR for the experimental Ag. Our experiments suggest that the polyclonal endogenous TCR -chains on the CD4⁺CD25⁺ cells are essential for manifestation of the regulatory function of these cells in different experimental systems.

In the experiments described, topically applied 1,25(OH)₂D₃ failed to modify the balance or quantity of Th1- (IFN-) or Th2-type (IL-4 or IL-5) cytokines secreted by CD4⁺ cells derived from the SDLN when cultured with APC and Ag. This is contrary to previous observations in NOD mice with autoimmune prostatitis, where chronic administration of a VDR agonist (elocalcitol) at 100 µg/kg/day for 10–16 days reduced the capacity of lymph node-derived CD4⁺ T cells to secrete IFN- upon further stimulation in vitro (24). Furthermore, reduced IFN- and increased IL-4 levels were detected in colon protein extracts from mice treated with 0.2 µg of calcitriol/kg/day for 3 days in a model of Th1-mediated colitis (23). In these previous studies, mice were chronically administered significantly greater doses of 1,25(OH)₂D₃ or a VDR agonist than used in the current experiments. In addition, the chronic treatments were previously administered via oral (24) or intraperitoneal (23) routes instead of the single topical treatment with 1,25(OH)₂D₃ given to mice in this study.

The production of 1,25(OH)₂D₃ in the skin of UV-irradiated mice may act as a homeostatic mechanism for controlling some of the inflammatory effects of UVB. 1,25(OH)₂D₃ repaired UVB-induced DNA damage in the epidermis of irradiated BALB/c mice in this study as well as in Skh:HR1 hairless mice (19). UV-induced DNA damage was also reduced by culturing irradiated human skin keratinocytes (4), fibroblasts, or melanocytes (19) with 1,25(OH)₂D₃. In contrast, both 1,25(OH)₂D₃ and UVB increased the regulatory capacity of CD4⁺CD25⁺ cells in the SDLN. Repair of UV-induced DNA damage by 1,25(OH)₂D₃ occurs via a rapid-acting, nongenomic pathway (4, 19). Thus, 1,25(OH)₂D₃ may act via a different pathway to activate regulatory T cells. This alternate pathway may be the genomic pathway, which uses the VDR.

The 125 ng dose of 1,25(OH)₂D₃ applied to the skin of mice in these studies was equivalent to applying 37 pmol 1,25(OH)₂D₃ per cm² of skin or 100 µl of 3 µM 1,25(OH)₂VitD₃. This dose was used because based on the levels measured in human skin equivalents and in human skin using microdialysis (3, 4), local concentration of 1,25(OH)₂D₃ in the order of 2–5 nM might be achieved. The 125 ng dose is similar to that used in other investigations of the immunomodulatory effects of topically applied 1,25(OH)₂D₃ (4, 14, 19, 20). However, lower amounts of 1,25(OH)₂D₃ were also found to be active in our studies. Concentrations of 1,25(OH)₂D₃ of 10–100 nM have been used to treat DC and/or T cells in vitro (5, 6, 7).

UVB irradiation of skin and topical 1,25(OH)₂D₃ were both able to enhance the capacity of CD4⁺CD25⁺ cells to suppress the proliferation of CD4⁺CD25^– cells when cocultured in vitro, and to diminish CHS responses following adoptive transfer in vivo. This study did not identify differences in the mode of activation of CD4⁺CD25⁺ cells after erythemal UVB irradiation or topical 1,25(OH)₂D₃. The phenotype of CD4⁺CD25⁺ cells activated by 1,25(OH)₂D₃ is the subject of further experimentation, as are studies of their mode of action.

The immunomodulatory effects of UVB may be explained by its capacity to promote the secretion of IL-10, prostaglandins, TNF-, and other inflammatory by-products by irradiated keratinocytes (reviewed by Ref. 28). These molecules may further act to expand regulatory T cells in the draining lymph nodes. Indeed, UVB irradiation alone (in the absence of exogenous Ag) caused CD4⁺CD25⁺ cells to proliferate in recipient mice. UVB irradiation also caused hypertrophy of the SDLN, with an increase in the number of CD4⁺CD25⁺ cells as previously described by ourselves (16, 29) and others (30). In contrast, it was only after sensitization with Ag, that CD4⁺CD25⁺ from 1,25(OH)₂D₃-treated mice expanded in vivo.

Although the effects of erythemal UVB are systemic (16), the ability of topical 1,25(OH)₂D₃ to enhance the suppressive capacity of CD4⁺CD25⁺ regulatory cells may be limited to the skin and draining lymph nodes. Topical treatment with 1,25(OH)₂D₃ had no effect upon the ability of CD4⁺ cells from the spleens of DO11.10 mice to proliferate in response to presentation of the OVA peptide by cocultured APC (data not shown). Although the effects of topical 1,25(OH)₂D₃ may not be systemic, suppression of CD4⁺ cell proliferation ex vivo was observed for as long as 21 days after a single treatment, indicating that 1,25(OH)₂D₃ may have long-lasting effects upon regulatory T cells. Together, these differences indicate that UVB irradiation has further proliferative effects upon CD4⁺CD25⁺ regulatory cells that do not occur after application of topical 1,25(OH)₂D₃ and that 1,25(OH)₂D₃ may be one of the molecules generated in skin which is responsible for the activation of the CD4⁺CD25⁺ cells.

In this study, we were not able to determine whether 1,25(OH)₂D₃ applied to skin had direct or indirect effects on CD4⁺CD25⁺ cells. This is the subject of further studies, where we hypothesize that some of the effects of 1,25(OH)₂D₃ and UVB may be due to modified Ag presentation by DC from the skin of treated mice. UVB irradiation also causes skin DC to migrate to the SDLN (31), and once there, the presentation of modified skin Ags by DNA-damaged DC may induce a tolerogenic environment for the activation of regulatory T cells (32). However, the effects of topical 1,25(OH)₂D₃ on skin cells including DC, keratinocytes, and mast cells are not completely understood.

It has been reported that numbers of MHC classII⁺ LC in 1,25(OH)₂D₃-treated ear skin were significantly reduced following topical application of 200 ng 1,25(OH)₂D₃ to the ear skin of mice for 7 days (14). 1,25(OH)₂VitD₃ also inhibited the expression of costimulatory molecules upon LC in vitro, and impaired the ability of these cells to present Ag in a MLR (33). Furthermore, DC can metabolize 1,25(OH)₂D₃ precursors to program T cells to express skin-homing molecules in vitro (34). Additional investigations are required to determine whether LC from 1,25(OH)₂D₃-treated mice have altered capacity to present Ag and whether modified LC are responsible for the activation and increased suppressive capacity of CD4⁺CD25⁺ regulatory T cells in the SDLN. These may focus upon the role of RANK-RANKL (receptor activator of NF-B ligand) interactions in 1,25(OH)₂D₃-treated skin (35, 36), or the role of the inhibitory receptor ILT3 (37). In psoriatic lesions, expression of ILT3 upon DC increased after topical administration of the 1,25(OH)₂D₃ analog calcipotriol, although in vitro studies showed that ILT3 expression was dispensable for the induction of regulatory T cells by 1,25(OH)₂D₃-treated DC (37). Further investigations are required to better understand how 1,25(OH)₂D₃ formed in response to the UVB wavelengths of sunlight may contribute toward curbing over-zealous immune responses.

	Disclosures

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The authors have no financial conflict of interest.

	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 study was supported by the Cancer Council of Western Australia, the National Health and Medical Research Council of Australia, and the University of Western Australia.

² Address correspondence and reprint requests to Dr. Prue Hart, Telethon Institute for Child Health Research, P. O. Box 855, West Perth, Australia. E-mail address: prueh{at}ichr.uwa.edu.au

³ Abbreviations: 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; SDLN, skin-draining lymph nodes; CHS, contact hypersensitivity; PLN, peritoneal cavity-draining lymph nodes; DNFB, 2,4-dinitrofluorobenzene; VDR, vitamin D receptor.

Received for publication April 30, 2007. Accepted for publication August 13, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Norman, A. W., M. T. Mizwicki, D. P. Norman. 2004. Steroid-hormone rapid actions, membrane receptors, and a conformational ensemble model. Nat. Rev. 3: 27-41.
2. Bikle, D. D., M. K. Nemanic, J. O. Whitney, P. W. Elias. 1986. Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3. Biochemistry 25: 1545-1548. [Medline]
3. Lehmann, B., W. Sauter, P. Knuschke, S. Dressler, M. Meurer. 2003. Demonstration of UVB-induced synthesis of 1,25-dihydroxyvitamin D3 (calcitriol) in human skin by microdialysis. Arch. Dermatol. Res. 295: 24-28. [Medline]
4. Gupta, R., K. M. Dixon, S. S. Deo, C. J. Holliday, M. Slater, G. M. Halliday, V. E. Reeve, R. S. Mason. 2007. Photoprotection by 1,25 dihydroxyvitamin D3 is associated with an increase in p53 and a decrease in nitric oxide products. J. Invest. Dermatol. 127: 707-715. [Medline]
5. Penna, G., S. Amuchastegui, N. Giarratana, K. C. Daniel, M. Vulcano, S. Sozzani, L. Adorini. 2007. 1,25-Dihydroxyvitamin D3 selectively modulates tolerogenic properties in myeloid but not plasmacytoid dendritic cells. J. Immunol. 178: 145-153. [Abstract/Free Full Text]
6. Boonstra, A., F. J. Barrat, C. Crain, V. L. Heath, H. F. J. Savelkoul, A. O’Garra. 2001. 1,25-dihydroxyvitamin D3 has a direct effect on naïve CD4⁺ T cells to enhance the development of Th2 cells. J. Immunol. 167: 4974-4980. [Abstract/Free Full Text]
7. Barrat, F. J., D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul, R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, A. O’Garra. 2002. In vitro generation of IL-10-producing regulatory CD4⁺ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 Th1- and Th2-inducing cytokines. J. Exp. Med. 195: 603-616. [Abstract/Free Full Text]
8. Gregori, S., N. Giarratana, S. Smiroldo, M. Uskokovic, L. Adorini. 2002. A 1-,25-dihydroxyvitamin D3 analog enhances regulatory T-cells and arrests autoimmune diabetes in NOD mice. Diabetes 51: 1367-1374. [Abstract/Free Full Text]
9. Cantorna, M. T., C. E. Hayes, H. F. DeLuca. 1996. 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis. Proc. Natl. Acad. Sci. USA 93: 7861-7864. [Abstract/Free Full Text]
10. Cantorna, M. T., C. E. Hayes, H. F. DeLuca. 1998. 1,25-Dihydroxycholecalciferol inhibits the progression of arthritis in murine models of human arthritis. J. Nutr. 128: 68-72. [Abstract/Free Full Text]
11. Matheu, V., O. Back, E. Mondoc, S. Issazadeh-Navikas. 2003. Dual effects of vitamin D-induced alteration of Th1/Th2 cytokine expression: enhancing IgE production and decreasing airway eosinophilia in murine allergic airway disease. J. Allergy Clin. Immunol. 112: 585-592. [Medline]
12. Garrigue, J. L., J. F. Nicolas, A. Demidem, H. Bour, J. Viac, J. Thivolet, D. Schmitt. 1993. Contact sensitivity in mice: differential effect of vitamin D3 derivative (calcipotriol) and corticosteroids. Clin. Immunol. Immunopathol. 67: 137-142. [Medline]
13. Tani, M., Y. Murata, S. Harada, T. Takashima, T. Horikawa. 1989. Increased contact hypersensitivity response in mice by topical application of 1-,25-dihydroxyvitamin D3 to elicitation site. Arch. Dermatol. Res. 281: 355-361. [Medline]
14. Guo, Z., H. Okamoto, S. Imamura. 1992. The effect of 1,25(OH)2-vitamin D3 on Langerhans cells and contact hypersensitivity in mice. Arch. Dermatol. Res. 284: 368-370. [Medline]
15. Hanneman, K. K., H. M. Scull, K. D. Cooper, E. D. Baron. 2006. Effect of topical vitamin D analogue on in vivo contact sensitization. Arch. Dermatol. 142: 1332-1334. [Abstract/Free Full Text]
16. Gorman, S., J. W. Tan, S. T. Yerkovich, J. J. Finlay-Jones, P. H. Hart. 2007. CD4⁺ T cells in lymph nodes of UVB-irradiated mice suppress immune responses to new antigens both in vitro and in vivo. J. Invest. Dermatol. 127: 915-924. [Medline]
17. Hart, P. H., M. A. Grimbaldeston, G. J. Swift, A. Jaksic, F. P. Noonan, J. J. Finlay-Jones. 1998. Dermal mast cells determine susceptibility to ultraviolet B-induced systemic suppression of contact hypersensitivity responses in mice. J. Exp. Med. 187: 2045-2053. [Abstract/Free Full Text]
18. Hart, P. H., A. Jaksic, G. Swift, M. Norval, A. A. el-Ghorr, J. J. Finlay-Jones. 1997. Histamine involvement in UVB- and cis-urocanic acid-induced systemic suppression of contact hypersensitivity responses. Immunology 91: 601-608. [Medline]
19. Dixon, K. M., S. S. Deo, G. Wong, M. Slater, A. W. Norman, J. E. Bishop, G. H. Posner, S. Ishizuka, G. M. Halliday, V. E. Reeve, R. S. Mason. 2005. Skin cancer prevention: a possible role of 1,25dihydroxyvitamin D3 and its analogs. J. Steroid Biochem. Mol. Biol. 97: 137-143. [Medline]
20. Dixon, K. M., S. S. Deo, A. W. Norman, J. E. Bishop, G. M. Halliday, V. E. Reeve, R. S. Mason. 2007. In vivo relevance for photoprotection by the vitamin D rapid response pathway. J. Steroid Biochem. Mol. Biol. 103: 451-456. [Medline]
21. Mahon, B. D., A. Wittke, V. Weaver, M. T. Cantorna. 2003. The targets of vitamin D depend on the differentiation and activation status of CD4-positive T cells. J. Cell. Biochem. 89: 922-932. [Medline]
22. Penna, G., L. Adorini. 2000. 1-,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J. Immunol. 164: 2405-2411. [Abstract/Free Full Text]
23. Daniel, C., H. H. Radeke, N. A. Sartory, N. Zahn, U. Zuegal, A. Steinmeyer, J. Stein. 2006. The new low calcemic vitamin D analog 22-ene-25-oxa-vitamin D prominently ameliorates T helper cell type 1-mediated colitis in mice. J. Pharmacol. Exp. Ther. 319: 622-631. [Abstract/Free Full Text]
24. Penna, G., S. Amuchastegui, N. Giarratana, K. C. Daniel, M. Vulcano, S. Sozzani, L. Adorini. 2006. Treatment of experimental autoimmune prostatitis in nonobese diabetic mice by the vitamin D receptor agonist elocalcitol. J. Immunol. 177: 8504-8511. [Abstract/Free Full Text]
25. Schwarz, A., A. Maeda, M. K. Wild, K. Kernebeck, N. Gross, Y. Aragane, S. Beissert, D. Vestweber, T. Schwarz. 2004. Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity. J. Immunol. 172: 1036-1043. [Abstract/Free Full Text]
26. Ghoreishi, M., J. P. Dutz. 2006. Tolerance induction by transcutaneous immunization through ultraviolet-irradiated skin is transferable through CD4⁺CD25⁺ T regulatory cells and is dependent on host-derived IL-10. J. Immunol. 176: 2635-2644. [Abstract/Free Full Text]
27. Tarbell, K. V., L. Petit, X. Zuo, P. Toy, X. Luo, A. Mqadmi, H. Yang, M. Suthanthiran, S. Mojsov, R. M. Steinman. 2007. Dendritic cell-expanded, islet-specific CD4⁺CD25⁺CD62L⁺ regulatory T cells restore normoglycemia in diabetic NOD mice. J. Exp. Med. 204: 191-201. [Abstract/Free Full Text]
28. Aubin, F.. 2003. Mechanisms involved in ultraviolet light-induced immunosuppression. Eur. J. Dermatol. 13: 515-523. [Medline]
29. Gorman, S., J. W. Tan, J. A. Thomas, S. L. Townley, P. A. Stumbles, J. J. Finlay-Jones, P. H. Hart. 2005. Primary defect in UVB-induced systemic immunomodulation does not relate to immature or functionally impaired APCs in regional lymph nodes. J. Immunol. 174: 6677-6685. [Abstract/Free Full Text]
30. Byrne, S. N., G. M. Halliday. 2005. B cells activated in lymph nodes in response to ultraviolet irradiation or by interleukin-10 inhibit dendritic cell induction of immunity. J. Invest. Dermatol. 124: 570-578. [Medline]
31. Kolgen, W., H. Both, H. van Weelden, K. L. Guikers, C. A. Bruijnzeel-Koomen, E. F. Knol, W. A. van Vloten, F. R. De Gruijl. 2002. Epidermal Langerhans cell depletion after artificial ultraviolet B irradiation of human skin in vivo: apoptosis versus migration. J. Invest. Dermatol. 118: 812-817. [Medline]
32. Schwarz, A., A. Maeda, K. Kernebeck, H. van Steeg, S. Beissert, T. Schwarz. 2006. Prevention of UV radiation-induced immunosuppression by IL-12 is dependent on DNA repair. J. Exp. Med. 201: 173-179.
33. Fujita, H., A. Asahina, M. Komine, K. Tamaki. 2007. The direct action of 1,25(OH)₂-vitamin D₃ on purified mouse Langerhans cells. Cell. Immunol. 245: 70-79. [Medline]
34. Sigmundsdottir, H., J. Pan, G. F. Debes, C. Alt, A. Habtezion, D. Soler, E. C. Butcher. 2007. DCs metabolize sunlight-induced vitamin D3 to ‘program’ T cell attraction to the epidermal chemokine CCL27. Nat. Immunol. 8: 285-293. [Medline]
35. Loser, K., A. Mehling, S. Loeser, J. Apelt, A. Kuhn, S. Grabbe, T. Schwarz, J. M. Penninger, S. Beissert. 2006. Epidermal RANKL controls regulatory T-cell numbers via activation of dendritic cells. Nat. Med. 12: 1372-1379. [Medline]
36. Theill, L. E., W. J. Boyle, J. M. Penninger. 2002. RANK-L and RANK: T cells, bone loss, and mammalian evolution. Annu. Rev. Immunol. 20: 795-823. [Medline]
37. Penna, G., A. Roncari, S. Amuchastegui, K. C. Daniel, E. Berti, M. Colonna, L. Adorini. 2005. 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 106: 3490-3497. [Abstract/Free Full Text]

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