HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Penna, G. Articles by Adorini, L. Search for Related Content PubMed PubMed Citation Articles by Penna, G. Articles by Adorini, L. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB 1,25-DIHYDROXYCHOLECALCIFEROL

1,25-Dihydroxyvitamin D₃ Inhibits Differentiation, Maturation, Activation, and Survival of Dendritic Cells Leading to Impaired Alloreactive T Cell Activation

Roche Milano Ricerche, Milan, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃), the active form of vitamin D₃, is a potent immunomodulatory agent. Here we show that dendritic cells (DCs) are major targets of 1,25(OH)₂D₃-induced immunosuppressive activity. 1,25(OH)₂D₃ prevents the differentiation in immature DCs of human monocytes cultured with GM-CSF and IL-4. Addition of 1,25(OH)₂D₃ during LPS-induced maturation maintains the immature DC phenotype characterized by high mannose receptor and low CD83 expression and markedly inhibits up-regulation of the costimulatory molecules CD40, CD80, and CD86 and of class II MHC molecules. This is associated with a reduced capacity of DCs to activate alloreactive T cells, as determined by decreased proliferation and IFN- secretion in mixed leukocyte cultures. 1,25(OH)₂D₃ also affects maturing DCs, leading to inhibition of IL-12p75 and enhanced IL-10 secretion upon activation by CD40 ligation. In addition, 1,25(OH)₂D₃ promotes the spontaneous apoptosis of mature DCs. The modulation of phenotype and function of DCs matured in the presence of 1,25(OH)₂D₃ induces cocultured alloreactive CD4⁺ cells to secrete less IFN- upon restimulation, up-regulate CD152, and down-regulate CD154 molecules. The inhibition of DC differentiation and maturation as well as modulation of their activation and survival leading to T cell hyporesponsiveness may explain the immunosuppressive activity of 1,25(OH)₂D₃.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DC)², a highly specialized APC system critical for the initiation of CD4⁺ T cell responses, are present, in different stages of maturation, in the circulation as well as in lymphoid and nonlymphoid organs (1). Immature DCs, such as Langerhans cells in the skin, are found in nonlymphoid tissues, where they exert a sentinel function. After Ag uptake, they migrate through the afferent lymph to T-dependent areas of secondary lymphoid organs where priming of naive T cells may occur (1, 2). During migration to lymphoid organs, DCs mature into potent APCs by increasing their immunostimulatory properties while decreasing Ag-capturing capacity (3). Recently, it has become clear that DCs can be not only immunogenic but also tolerogenic, both intrathymically (4, 5) and in the periphery (6).

Induction of T cell responses requires T cell receptor activation and costimulatory interactions between DCs and T cells (1); in the absence of costimulation, T cells become anergic (7). The two major costimulatory pathways for T cell activation depend on engagement of CD28 and CD154 on T cells by CD80/CD86 and CD40 on DCs, respectively (8, 9). Once activated, T cells also express CD152, a CD28 homologue that binds to CD80/CD86 with higher affinity than CD28 itself and inhibits IL-2 production, IL-2 receptor expression, and cell cycle progression in activated T cells (10).

Disruption of these costimulatory pathways by biological agents such as CD152-Ig and anti-CD154 mAb has been shown to be beneficial in autoimmune diseases and allograft rejection (8, 9). Interestingly, a short treatment with CD152-Ig and anti-CD154 mAb can induce tolerance to allografts in mice (11) and, possibly, also in nonhuman primates (12, 13). This has stimulated the search for low m.w. compounds able to disrupt costimulatory pathways for T cell activation. The unique capacity of DCs to activate naive T cells correlates with elevated expression of MHC Ags and costimulatory molecules (1), rendering them attractive targets for costimulation blockade.

1,25-Dixydroxyvitamin D₃ (1,25(OH)₂D₃), the biologically active metabolite of vitamin D₃, is a secosteroid hormone that not only regulates bone and calcium/phosphate metabolism but exerts a number of other biological activities, including modulation of the immune response via specific receptors expressed in APC and activated T cells. Immunosuppression by 1,25(OH)₂D₃ and its analogues has been demonstrated in different models of autoimmune diseases and in experimental organ transplantation (14). 1,25(OH)₂D₃ and its analogues can prevent systemic lupus erythematosus in lpr/lpr mice (15), experimental allergic encephalomyelitis (16, 17, 18), and autoimmune diabetes in nonobese diabetic mice (19). In addition, 1,25(OH)₂D₃ and its analogues prolong the survival of heart (20, 21) and small bowel allografts (22) and have been reported to inhibit, in association with cyclosporin A (CsA), not only acute but also chronic allograft rejection (23).

Although 1,25(OH)₂D₃ and its analogues clearly inhibit T cell proliferation and cytokine production (24, 25), it is not yet clear whether these inhibitory effects are exerted directly on T cells (26, 27, 28, 29) or via inhibition of APC activity (30). In the present study, we demonstrate that DCs are primary targets for the immunosuppressive effects of 1,25(OH)₂D₃ on T cell activation. 1,25(OH)₂D₃ inhibits differentiation and maturation of DCs and modulates their activation and survival, leading to a profound modulation of T cell phenotype and function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines and cell culture reagents

Human monocytes were cultured in RPMI 1640 medium supplemented with 10% FCS (HyClone Laboratories, Logan, UT), 2 mM glutamine, 50 µg/ml gentamicin, 1 mM sodium pyruvate, and 1% nonessential amino acids (complete medium). J558L cells expressing CD154 (31) (a gift of Dr. Peter Lane, Basel Institute for Immunology, Basel, Switzerland) were grown in complete medium supplemented with 5 mM L-histidinol dihydrochloride (Sigma, St. Louis, MO).

1,25(OH)₂D₃

Crystalline 1,25(OH)₂D₃ was a gift of Dr. Milan Uskokovic (Hoffmann-La Roche, Nutley, NJ). 1,25(OH)₂D₃ was reconstituted in ethanol and stored in concentrated solutions at -80°C. 1,25(OH)₂D₃ was freshly diluted before each experiment, and the ethanol concentration in the test conditions did not exceed 0.00025%.

Human DC cultures

Immature DCs were prepared as described (32). Briefly, PBMC were isolated from a buffy coat by Ficoll gradient (Pharmacia Biotec AB, Uppsala, Sweden), and monocytes, obtained from PBMC by negative selection with monocyte isolation kit (Milteny Biotec, Bergish Gladblach, Germany), were grown for 6–7 days in medium containing 800 U/ml GM-CSF (Mielogen, Schering-Plough) and 1000 U/ml IL-4 (PharMingen, San Diego, CA). DC maturation was induced by stimulation of immature DCs for 48 h with 200 ng/ml LPS (Escherichia coli 0111:B4; Sigma), 20 ng/ml TNF- (PharMingen), or 1:5000 SAC (Pansorbin cells; Calbiochem, San Diego, CA). For activation, DCs were incubated with CD154-J558L cells at a ratio of 4:1. After 24 h, supernatants were collected, and IL-12 p75 and IL-10 concentrations were measured by ELISA.

Allogeneic and anti-CD3-induced T cell activation

PBMC were separated from buffy coats by Ficoll gradient. For the bidirectional MLR, the same number (3 x 10⁵) of allogeneic PBMC from two different donors were cocultured in 96-well flat-bottom plates. After 5 days, proliferation and cytokine production were measured. For anti-CD3-induced T cell activation, total T cells were purified from PBMC using Pan T Cell isolation kit (Miltenyi Biotec). T cells (10⁵/well) were cultured in 96-well round-bottom plates precoated by overnight incubation with anti-human CD3 mAb (clone TR66) (33) with or without 1 µg/ml soluble anti-human CD28 mAb (CD28.2; PharMingen). After 72 h, proliferation and cytokine production were measured. For the primary T cell response, CD4⁺ cells were purified from PBMC using CD4⁺ T cell isolation kit. CD4⁺ cells (2 x 10⁵/well) were cultured with graded amounts of DCs 300–10(300–10,000) in 96-well flat-bottom plates. After 5 days, proliferation and cytokine production were measured. T cell costimulatory molecules were analyzed 72 h after culture initiation.

Secondary MLR

CD4⁺ T cells (2 x 10⁵/well) were cocultured during the primary stimulation with 10³ DCs. T cells were separated 36 h later by Ficoll gradient, and DCs were removed using FITC anti-human CD1a, anti-human CD14, anti-human CD40, anti-human CD86 (all from PharMingen), followed by anti-FITC microbeads (Miltenyi Biotec). T cells were rested for 2–4 days in complete medium supplemented with 2 U/ml hIL-2 and then restimulated with mature DCs generated from the same donor used for the primary culture. Proliferation and cytokine production were measured 48 h later. T cell costimulatory molecules were analyzed 24 h and 48 h after the beginning of culture.

Flow cytometric analysis

Flow cytometric analysis was performed in the presence of 100 µg/ml mouse IgG using the following mAbs, all from PharMingen except when indicated: anti-CD1a FITC/PE (HI149), anti-CD14 FITC (M5E2), anti-CD25 FITC (M-A251), anti-CD28 PE (CD28.2), anti-CD40 FITC (5C3), anti-CD58 FITC (1C3), anti-CD80 FITC (BB1), anti-CD83 PE (HB15A; Immunotech, Marseille, France), anti-CD86 PE (IT2.2), anti HLA-DR FITC/PE (G46-6), anti-Mannose Receptor PE (19), anti-CD152 PE (BNI3), anti-CD154 PE (TRAP1). For detection of apoptosis, DCs were stained with Annexin-V FITC (PharMingen) and propidium iodide (50 µg/ml, Sigma). Cells were analyzed with a FACScan flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA).

Cytokine analysis

ELISA for IL-12p75 was performed as described (34). Human recombinant IL-12 and anti IL-12 mAbs were a gift from Dr. Maurice Gately (Hoffman-La Roche, Nutley, NJ). ELISA for human IL-10 was performed using commercially available mAbs and standard IL-10 (PharMingen) according to the manufacturer’s instructions. ELISA for human IFN- was performed as described (35). Detection limits were 15 pg/ml for IL-12p75, 10 pg/ml for IL-10, and 50 pg/ml for IFN-.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of APC function by 1,25(OH)₂D₃

1,25(OH)₂D₃ inhibits alloreactive T cell activation, as shown by the inhibition of proliferation and IFN- production in a bidirectional human MLR (Fig. 1A). Compared with other immunosuppressive agents targeting T cells, like mycophenolate mofetil (MMF) or CsA, 1,25(OH)₂D₃ was more potent in the inhibition of IFN- production (IC₅₀ 0.04 nM vs 214 nM for MMF and 30 nM for CsA), but it did not inhibit cell proliferation completely. The inhibitory effect on T cell responses appeared to be indirect because T cell activation by plate-bound anti-CD3, with or without costimulation by anti-CD28, was scarcely affected by 1,25(OH)₂D₃, as determined by cell proliferation or IFN- secretion (Fig. 1B). This indicates that 1,25(OH)₂D₃ inhibits the ability of APCs to induce alloreactive T cell activation, rather than directly inhibiting T cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Inhibition of APC function by 1,25(OH)2D3. A, Allogeneic PBMCs from two different donors (3 x 105 cells/well each) were cocultured in 96-well flat-bottom plates in the presence of the indicated concentrations of MMF, CsA, or 1,25(OH)2D3. After 5 days, proliferation and IFN- secretion were measured. The solid line indicates the control response obtained in the absence of immunosuppressive drugs, and the stippled line 50% of the control response. B, Purified T cells (105/well) were cultured in the presence of 10 nM 1,25(OH)2D3, with or without 1 µg/ml soluble anti-CD28, in 96-well round-bottom plates precoated with the indicated concentrations of anti-CD3 mAb. After 72 h, proliferation and IFN- secretion were measured. The data are from a representative experiment out of two to eight performed.

To determine whether the inhibitory effect of 1,25(OH)₂D₃ on APCs might involve DCs, we have first analyzed its effect on DC differentiation (Fig. 2A). Human peripheral blood monocytes can be differentiated into immature DCs by culture with GM-CSF and IL-4 (36). During this process, they down-regulate the monocyte marker CD14 and express the DC marker CD1a. Addition of 10 nM 1,25(OH)₂D₃ to CD14⁺ human monocytes cultured for 7 days in the presence of GM-CSF and IL-4 completely inhibited the differentiation of CD1a⁺ DCs. IL-10, a cytokine that inhibits APCs at different levels, did not completely prevent DC differentiation when added at 10 ng/ml. Both agents gave rise to cells with down-regulated CD14, compared with freshly isolated monocytes. Monocytes differentiated in the presence of 1,25(OH)₂D₃ or IL-10 yielded APCs with a similarly reduced capacity to stimulate CD4⁺ cell proliferation in a primary MLR assay (Fig. 2B). Thus, 1,25(OH)₂D₃ inhibits phenotypically and functionally the differentiation of peripheral blood monocytes into immature DCs.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Inhibition of DC differentiation by 1,25(OH)2D3. Negatively selected CD14+ monocytes were cultured in medium supplemented with 800 U/ml GM-CSF and 1000 U/ml IL-4 to generate immature DCs. Cells were fed fresh medium every 2–3 days. 1,25(OH)2D3 (10 nM) or IL-10 (10 ng/ml) were added at culture initiation. A, Six days after culture initiation, cells were double stained with anti-CD14 and anti-CD1a mAbs and analyzed by flow cytometry. Values indicate the percentage of positive cells. B, After extensive washing, the APCs described above were cocultured with CD4+ T cells (2 x 105/well) from a different donor. After 5 days, proliferation was measured by [3H]thymidine incorporation. Proliferative responses are shown as mean (±SE) from triplicate cultures. The data are from a representative experiment out of four performed.

Immature DCs obtained by a 7-day culture with GM-CSF and IL-4 can be induced to mature by incubation with LPS (37). DC maturation is accompanied by slight down-regulation of CD1a, decreased expression of the mannose receptor, induction of the maturation marker CD83, and up-regulation of HLA-DR, CD40, and CD86 molecules (Fig. 3A). Addition of 1,25(OH)₂D₃ prevented the LPS-induced maturation of immature DCs, maintaining DCs at the immature stage characterized by high mannose receptor and low CD83 expression (Fig. 3B). Compared with control mature DCs, addition of 1,25(OH)₂D₃ during maturation inhibited by about 50%, as determined by median fluorescence intensity, the expression of MHC class II, CD58, CD40, CD80, and CD86 molecules. A less pronounced effect was observed when the differentiating DCs were exposed to 1,25(OH)₂D₃ or IL-10 for 24 h before LPS-induced maturation (data not shown). The inhibitory effect of 1,25(OH)₂D₃ on DC maturation was comparable to that induced by IL-10, with two exceptions: IL-10 was less efficient in inhibiting CD40 but more efficient in inhibiting CD86 expression. Mature DCs, compared with immature DCs, induced a higher proliferation (Fig. 3C) and IFN- secretion (Fig. 3D) by alloreactive CD4⁺ cells, due to their increased expression of class II and costimulatory molecules. Immature DCs, matured in the presence of either agent, displayed a reduced Ag-presenting capacity in the activation of alloreactive CD4⁺ cells, as determined by the decreased proliferation (Fig. 3C) and IFN- secretion (Fig. 3D) in MLR assays. This reduction was more evident for cells treated with 1,25(OH)₂D₃. Indeed, DCs matured in the presence of 1,25(OH)₂D₃ showed a capacity to activate alloreactive CD4⁺ cell as low as that of immature DCs. Similar results were obtained using negatively selected total T cells, indicating that the presence of CD8⁺ cells in the responding T cell population does not affect the inhibitory capacity of 1,25(OH)₂D₃ (data not shown).

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Inhibition of DC maturation by 1,25(OH)2D3. Maturation was induced by incubation of immature DCs with LPS (200 ng/ml) for 48 h. A, Induction of maturation in control immature DCs, as determined by surface marker expression. Stippled lines refer to isotype controls. The staining profile of immature (solid thin line) and mature (thick line) DCs for the indicated surface molecules is shown. MR, mannose receptor. B, Percentage variation of median fluorescence intensity in DCs matured in the presence of 10 nM 1,25(OH)2D3 or 10 ng/ml IL-10. C and D, After extensive washing, the indicated numbers of DCs, generated as described above, were cocultured with CD4+ T cells (2 x 105/well) from a different donor. After 5 days, proliferation (C) and IFN- secretion (D) were measured. Proliferative responses are shown as mean (±SE) from triplicate cultures. The data are from a representative experiment out of four performed.

Mature DCs can be activated by CD40 cross-linking (38). Activation of mature DCs by incubation with J558L cells transfected with the gene encoding CD154 resulted in IL-12p75 and IL-10 secretion (Fig. 4). Addition of 1,25(OH)₂D₃ during LPS-induced maturation gave rise to DCs unable to secrete IL-12p75 upon CD40 ligation (Fig. 4A) but secreting 7-fold higher levels of IL-10 (Fig. 4B). Conversely, addition of IL-10 during DC maturation up-regulated IL-10 production by only 2-fold and did not significantly inhibit IL-12p75 secretion upon activation by CD154-transduced J558L cells, consistent with the resistance of differentiated DCs to the effects of IL-10 (39, 40). Thus, 1,25(OH)₂D₃ modulates maturing DCs, leading to abrogation of IL-12 but higher IL-10 secretion upon activation.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Abrogation of IL-12 and enhancement of IL-10 secretion upon activation of 1,25(OH)2D3-treated DCs. Maturation of immature DCs was induced by culture for 48h in the presence of 200 ng/ml LPS with or without 10 nM 1,25(OH)2D3 or 10 ng/ml IL-10. After washing, DCs were cultured with (filled bars) or without (open bars) CD154-transfected J558L cells at a ratio of 1:4. After 24 h, IL-12p75 and IL-10 secretion were measured. Bars represent the mean (±SE) from three separate experiments. *, p < 0.05 by Mann-Whitney U test compared with cytokine levels induced by mature DCs.

Apoptosis of mature DCs is strongly enhanced by IL-10 (41). To determine the effect of 1,25(OH)₂D₃ on DC apoptosis, DCs matured in the presence of LPS with or without 1,25(OH)₂D₃ or IL-10 were examined immediately after maturation or following a 24-h culture in complete medium. DC apoptosis was quantified by staining with annexin V, which detects changes in the asymmetry of phosphatidylserine in the cell membrane, an early apoptotic marker. Simultaneous staining of cells with FITC-annexin V and with the non-vital dye propidium iodide (PI) allows the discrimination of intact cells (FITC^-PI^-), early apoptotic (FITC⁺PI^-), and late apoptotic or necrotic cells (FITC⁺PI⁺) (42). As shown in Fig. 5, the apoptosis of DCs immediately after maturation (2% FITC⁺PI^-, 4% FITC⁺PI⁺) was enhanced by addition of 1,25(OH)₂D₃ (8% FITC⁺PI^-, 10% FITC⁺PI⁺) or IL-10 (10% FITC⁺PI^-, 27% FITC⁺PI⁺), confirming the proapoptotic effects of IL-10 and indicating a similar activity of 1,25(OH)₂D₃. The apoptosis of DCs matured in the presence of 1,25(OH)₂D₃ was 6-fold enhanced, compared with controls, following a 24-h incubation in plain culture medium. Thus, 1,25(OH)₂D₃ promotes, when present during DC maturation, DC apoptosis.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. 1,25(OH)2D3 enhances DC apoptosis following maturation. Maturation of immature DCs was induced by culture for 48 h in the presence of 200 ng/ml LPS with or without 10 nM 1,25(OH)2D3 or 10 ng/ml IL-10. DC apoptosis was quantified by double staining with FITC-annexin V and PI immediately after maturation or after a 24-h culture period. The data are from a representative experiment out of three performed.

These results suggest that the ability of 1,25(OH)₂D₃ to prevent differentiation, decrease expression of costimulatory molecules during maturation, and modulate cytokine production upon activation of human DCs might contribute to its inhibitory effect on APC-dependent T cell activation. Thus, we examined whether DCs matured in the presence of 1,25(OH)₂D₃ could modulate T cell responsiveness. Coculture of alloreactive CD4⁺ T cells with 1,25(OH)₂D₃-treated DCs resulted in T cell hyporesponsiveness, as demonstrated by their reduced IFN- secretion upon restimulation by untreated, mature DCs in a secondary MLR assay (Fig. 6). In contrast, T cell proliferation was only slightly inhibited (data not shown). The IFN- secreted, upon restimulation, by CD4⁺ T cells preincubated with immature or 1,25(OH)₂D₃-treated mature DCs was similarly reduced, compared with untreated mature DCs. Induction of T cell hyporesponsiveness by DCs matured in the presence of IL-10 was slightly less pronounced. Addition to cultures of exogenous IL-2 could not reverse the inhibition of IFN- secretion (Fig. 6, right panel).

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Induction of T cell hyporesponsiveness by 1,25(OH)2D3-treated DCs. Allogeneic CD4+ T cells (2 x 105/well) were cocultured with different DC populations (103 cells/well). Immature DCs were generated as described in Fig. 2. Maturation of immature DCs was induced by culture for 48 h in the presence of 200 ng/ml LPS with or without 10 nM 1,25(OH)2D3 or 10 ng/ml IL-10. T cells were recovered 36 h after culture initiation, rested for 2 to 4 days in complete medium supplemented with 2 U/ml IL-2, and restimulated with mature untreated DCs, generated from the same donor used as a DC source for the first culture, with (right panel) or without (left panel) 100 U/ml IL-2. IFN- secretion was measured 48 h after restimulation. Bars represent the mean (±SE) percent variation (n = 3) of IFN- secretion compared with restimulation with untreated mature DCs. IFN- secretion induced by untreated mature DCs in the secondary culture was, in the three individual experiments, 210/2140, 1748/4424, and 345/1038 pg/ml, in the absence or presence of 100 U/ml IL-2, respectively.

To explore possible mechanisms leading to T cell unresponsiveness, we examined whether 1,25(OH)₂D₃-treated DCs could modulate T cell expression of inhibitory molecules such as CD152 or stimulatory ligands like CD154. The percentage of T cells expressing CD152 was reduced by >50% in a primary coculture of alloreactive CD4⁺ cells with mature compared with immature DCs or DCs matured in the presence of 1,25(OH)₂D₃ as well as IL-10. In contrast, CD154 was similarly expressed on T cells stimulated by immature or mature DCs, but CD154-expressing T cells were reduced by >40% in the presence of 1,25(OH)₂D₃ whereas they were unaffected by IL-10 (data not shown). A time course analysis revealed an early up-regulation of CD152, already expressed on 20% of alloreactive CD4⁺ cells following a 24-h interaction with 1,25(OH)₂D₃-treated DCs (Fig. 7). This up-regulation was selective for CD152, because its homologue CD28 was not affected whereas expression of the T cell activation marker CD25 was slightly down-regulated (Fig. 7).

View larger version (26K): [in this window] [in a new window]   FIGURE 7. Interaction with 1,25(OH)2D3-treated DCs up-regulates CD152 on CD4+ alloreactive T cells. Allogeneic CD4+ T cells (2 x 105/well) were cocultured with DCs (103/well) generated as described in Fig. 3. T cells were recovered 36 h after culture initiation, rested for 2 to 4 days in complete medium supplemented with 2 U/ml IL-2, and restimulated with mature untreated DCs, generated from the same donor used as a DC source for the first culture. After 24 h and 48 h, CD152 expression on CD4+ cells was analyzed by flow cytometry. Values refer to the percentage of CD152+ cells. Double staining for CD4 and CD25, CD28, or CD152 expression by T cells analyzed after 48 h of culture with mature DCs is also shown. The data are from a representative experiment out of two performed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our results clearly establish that DCs are a primary target of the immunosuppressive activity of 1,25(OH)₂D₃, which affects all major stages of the DC life cycle: differentiation, maturation, activation, and survival. CD14⁺ peripheral blood monocytes cultured with GM-CSF and IL-4 develop into a homogeneous population of immature DCs, characterized by high capacity for Ag capture but low T cell stimulatory activity (36). Immature DCs can be induced to mature by inflammatory stimuli such as LPS, a process associated with loss of Ag-capturing ability but increased costimulatory capacity for T cell activation (37). T cell-derived signals, such as CD40 ligation by CD154-expressing T cells, can not only induce DC maturation but also activate them to secrete IL-12, a key cytokine for Th1 cell development (43). Finally, mature DCs are programmed to die unless they receive a survival signal from T cells (44). Strikingly, 1,25(OH)₂D₃ profoundly affects all stages of the DC life cycle, inhibiting their differentiation and maturation as well as modulating their activation and survival.

Monocytes (45) and DCs (46) express vitamin D₃ receptors constitutively. Incubation of normal human monocytes with 1,25(OH)₂D₃ has been previously shown to decrease accessory cell function with down-regulation of Ag-presenting capacity (30), but this did not appear to correlate with decreased expression of CD18, CD44, CD54, CD58, or HLA-DR molecules (47), suggesting the involvement of other costimulatory molecules. Decreased CD86 but not CD80 expression was found to be induced by 1,25(OH)₂D₃ on human resting monocytes stimulated with IL-10, IFN-, or TNF-, but not with LPS (48). Indeed, as shown in the present study, CD40, CD58, CD80, and CD86 expression is clearly inhibited by addition of 1,25(OH)₂D₃ during LPS-induced DC maturation.

The inhibition of DC costimulatory molecule expression by 1,25(OH)₂D₃ is intriguing, given the critical role of costimulatory signals delivered by CD40, CD80, and CD86 for optimal T cell activation (8, 9). Administration of anti-CD154 mAb disrupting the CD40-CD154 pathway (9) and/or CD152-Ig blocking the CD80/CD86-CD28 pathway (8, 11) have been shown to be effective in several models of autoimmune diseases and allograft rejection. This suggests that at least part of the immunosuppressive activity exerted by 1,25(OH)₂D₃ in similar models (14) may be due to down-regulation of costimulatory molecule expression. Our results also demonstrate that T cells cultured with 1,25(OH)₂D₃-treated DCs up-regulate CD152, a ligand that inhibits both early and late T cell activation (49), and down-regulate CD154, further contributing to the disruption of costimulatory pathways. The early up-regulation of CD152 coupled with the down-regulation of CD154 may be particularly relevant, considering the capacity of CD152-Ig to synergize with anti-CD154 in costimulation blockade (11). Costimulation blockade has also been shown, in some cases, to induce transplantation tolerance (11). Stimulation by 1,25(OH)₂D₃-treated DCs leads to T cell unresponsiveness, suggesting a similar mechanism of action. Inactivation of host DCs has been recently proposed to prevent graft-vs-host disease in allogeneic bone marrow transplantation (50), and a similar strategy is applicable to solid organ allografts (51). We are currently examining whether a short-term administration of 1,25(OH)₂D₃ to islet allograft recipients may be able, via inhibition of host DCs, to induce long-lasting graft acceptance and transplantation tolerance. If this is the case, a 1,25(OH)₂D₃ analogue with enhanced immunosuppressive activity and reduced effects on the calcium/phosphate metabolism may lead to an effective prevention of human allograft rejection. Efforts to reach this goal are already ongoing (52).

Several agents have been shown to inhibit, at different levels, DCs, including glucocorticoids (53, 54), PGE₂ (55), N-acetyl-L-cysteine (NAC) (56), and IL-10. IL-10 decreases Ag-presenting capacity (57) and IL-12 production (58, 59) by DCs, as well as promoting their apoptosis (41). In addition, IL-10-treated DCs can induce anergy in CD4⁺ cells (39). In the present study, IL-10 has been routinely compared with 1,25(OH)₂D₃, and, overall, their activity is quite similar. However, 1,25(OH)₂D₃ is very effective in inhibiting IL-12 production by mature DCs whereas IL-10 is not, consistent with the resistance of mature DCs to IL-10 effects (39, 40, 60). Moreover, the inhibition of CD40 expression is more evident in DCs matured in the presence of 1,25(OH)₂D₃ rather than IL-10, whereas CD80 and CD86 are similarly inhibited. 1,25(OH)₂D₃ or IL-10-treated DCs induce a similar hyporesponsiveness in alloreactive CD4⁺ cells, as judged by their impairment in IFN- secretion upon restimulation. However, T cells are differentially modulated, as shown by the up-regulation of CD152 expression induced on alloreactive CD4⁺ cells by DCs treated with 1,25(OH)₂D₃ but not IL-10. In addition to glucocorticoids (54), PGE₂ (55), like other cAMP-raising agents (61), and NAC (56) also inhibit IL-12 production by DCs. The inhibition of IL-12 production induced by 1,25(OH)₂D₃ in mature DCs is mediated by inhibition of the transcription factor NF-B (62) and, interestingly, inhibition of IL-12 production by NAC has also been found to be associated with NF-B inhibition (56).

Several TNF family members contribute to DC survival. In addition to TNF- itself (41), and CD154 (38), TNF-related activation-induced cytokine (TRANCE) is a DC-specific survival factor that regulates the expression of the anti-apoptotic molecule Bcl-x_L (63). TRANCE is expressed in activated T cells and promotes, in cooperation with TNF- and CD154, the survival of mature DCs, which selectively express high levels of TRANCE receptor (64). It would be of interest to analyze TRANCE receptor expression in 1,25(OH)₂D₃-treated DCs, in light of our results showing their enhanced apoptosis, given the emerging role of TRANCE and its receptors in the control of DC and bone homeostasis (65). A proapoptotic activity of 1,25(OH)₂D₃ has been described in tumor cells (66) and may also take place in vivo, as suggested by its apoptosis-enhancing effects on diabetogenic Th1 cells (67).

In conclusion, DCs are primary targets for the immunosuppressive activity of 1,25(OH)₂D₃, as indicated by its capacity to inhibit DC differentiation and maturation, leading to marked down-regulation of MHC class II and costimulatory molecules. In addition, 1,25(OH)₂D₃ inhibits IL-12, while enhancing IL-10 production, and promotes DC apoptosis. These effects could contribute substantially to decrease DC-dependent T cell activation and could largely account for the immunosuppressive properties of 1,25(OH)₂D₃. The use of a 1,25(OH)₂D₃ analogue with immunosuppressive activity at nonhypercalcemic doses may permit the exploitation of the modulation of DC activity in the treatment of autoimmune diseases and allograft rejection.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Luciano Adorini, Roche Milano Ricerche, Via Olgettina 58, I-20132 Milan, Italy. E-mail address:

² Abbreviations used in this paper: DC, dendritic cell; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; CsA, cyclosporin A; MMF, mycophenolate mofetil; PI, propidium iodide; NAC, N-acetyl-L-cysteine; TRANCE, TNF-related activation-induced cytokine.

Received for publication September 22, 1999. Accepted for publication December 21, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Banchereau, J., R. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
2. Cyster, J.. 1999. Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs. J. Exp. Med. 189:447.[Free Full Text]
3. Cella, M., F. Sallusto, A. Lanzavecchia. 1997. Origin, maturation and antigen presenting function of dendritic cells. Curr. Opin. Immunol. 9:10.[Medline]
4. Guéry, J.-C., L. Adorini. 1995. Dendritic cells are the most efficient in presenting endogenous naturally processed self epitopes to class II-restricted T cells. J. Immunol. 154:536.[Abstract]
5. Brocker, T., K. Karjalainen. 1997. Targeted expression of MHC class II molecules demonstrates that dendritic cells can induce negative but no positive selection of thymocytes in vivo. J. Exp. Med. 185:541.[Abstract/Free Full Text]
6. Finkelman, F., A. Lees, R. Birnbaum, W. Gause, S. Morris. 1996. Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion. J. Immunol. 157:1406.[Abstract]
7. Schwartz, R. H.. 1990. A cell culture model for T cell anergy. Science 248:1349.[Abstract/Free Full Text]
8. Lenschow, D. J., T. L. Walunas, J. A. Bluestone. 1996. CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14:233.[Medline]
9. Grewal, I., R. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[Medline]
10. Walunas, T. L., C. Y. Bakker, J. A. Bluestone. 1996. CTLA-4 ligation blocks CD28-dependent T cell activation. J. Exp. med. 183:2541.[Abstract/Free Full Text]
11. Larsen, C., E. Elwood, D. Alexander, S. Ritchie, R. Hendrix, C. Tucker-Burden, H. Cho, A. Aruffo, D. Hollenbaugh, P. Linsley, K. Winn, T. Pearson. 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434.[Medline]
12. Kenyon, N., M. Chatzipetrou, M. Masetti, A. Ranuncoli, M. Oliveira, J. Wagner, A. Kirk, D. Harlan, L. Burkly, C. Ricordi. 1999. Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc. Natl. Acad. Sci. USA 96:8132.[Abstract/Free Full Text]
13. Kirk, A., L. Burkly, D. Batty, R. Baumgartner, J. Berning, K. Buchanan, J. J. Fechner, R. Germond, R. Kampen, N. Patterson, S. Swanson, D. Tadaki, C. TenHoor, L. White, S. Knechtle, D. Harlan. 1999. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nat. Med. 6:686.
14. Casteels, K., R. Bouillon, M. Waer, C. Mathieu. 1995. Immunomodulatory effects of 1,25-dihydroxyvitamin D₃. Curr. Opin. Nephrol. Hyperten. 4:313.[Medline]
15. Koizumi, T., Y. Nakao, T. Matsui, T. Nakagawa, S. Matsuda, K. Komoriya, Y. Kawai, T. Fujida. 1985. Effects of corticosteroid and 1,24R-dihydroxy-vitamin D₃ administration on lymphoproliferation and autoimmune disease in MRL/MP lpr/lpr mice. Int. Arch. Allergy Appl. Immunol. 77:396.[Medline]
16. Lemire, J. M., C. Archer. 1991. 1,25-Dihydroxyvitamin D₃ prevents the in vivo induction of murine experimental autoimmune encephalomyelitis. J. Clin. Invest. 87:1103.
17. 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.[Abstract/Free Full Text]
18. Mattner, F., S. Smiroldo, F. Galbiati, M. Muller, P. Di Lucia, P. Poliani, G. Martino, P. Panina-Bordignon, L. Adorini. 2000. Inhibition of Th1 development and treatment of chronic-relapsing experimental allergic encephalomyelitis by a non-hypercalcemic analogue of 1,25-dihydroxyvitamin D₃. Eur. J. Immunol. 30:498.[Medline]
19. Mathieu, C., M. Waer, J. Laureys, O. Rutgeerts, R. Bouillon. 1994. Prevention of autoimmune diabetes in NOD mice by 1,25-dihydroxyvitamin D₃. Diabetologia 37:552.[Medline]
20. Lemire, J., D. Archer, A. Khulkarni, A. Ince, M. Uskokovic, S. Stepkowski. 1992. Prolongation of the survival of murine cardiac allografts by the vitamin D₃ analogue 1,25-dihydroxy-delta16-cholecalciferol. Transplantation 54:762.[Medline]
21. Hullett, D., M. Cantorna, C. Redaelli, J. Humpal-Winter, C. Hayes, H. Sollinger, H. Deluca. 1998. Prolongation of allograft survival by 1,25-dihydroxyvitamin D₃. Transplantation 66:824.[Medline]
22. Johnsson, C., G. Tufveson. 1994. MC 1288: a vitamin D analogue with immunosuppressive effects on heart and small bowel grafts. Transplant. Int. 7:392.[Medline]
23. Raisanen-Sokolowski, A. K., I. S. Pakkala, S. P. Samila, L. Binderup, P. J. Hayry, S. T. Pakkala. 1997. A vitamin D analog, MC1288, inhibits adventitial inflammation and suppresses intimal lesions in rat aortic allografts. Transplantation 63:936.[Medline]
24. Bhalla, A. K., E. P. Amento, B. Serog, L. H. Glimcher. 1984. 1,25-Dihydroxyvitamin D₃ inhibits antigen-induced T cell activation. J. Immunol. 133:1748.[Abstract]
25. Rigby, W. F. C., S. Denome, M. W. Fanger. 1987. Regulation of lymphokine production and human T lymphocyte activation by 1,25-dihydroxyvitamin D₃. J. Clin. Invest. 79:1659.
26. Vanham, G., J. Ceuppens, R. Bouillon. 1989. T lymphocytes and their CD4 subset are direct targets for the inhibitory effect of calcitriol. Cell. Immunol. 124:320.[Medline]
27. Alroy, I., T. Towers, L. Freedman. 1995. Transcriptional repression of the interleukin-2 gene by vitamin D₃: direct inhibition NFATp/AP-1 complex formation by a nuclear hormone receptor. Mol. Cell. Biol. 15:5789.[Abstract]
28. Cippitelli, M., A. Santoni. 1998. Vitamin D₃: a transcriptional modulator of the IFN- gene. Eur. J. Immunol. 28:3017.[Medline]
29. Takeuchi, A., G. Reddy, T. Kobayashi, T. Okano, J. Park, S. Sharma. 1998. Nuclear factor of activated T cells (NFAT) as a molecular target for 1,25-dihydroxyvitamin D₃-mediated effects. J. Immunol. 160:209.[Abstract/Free Full Text]
30. Rigby, W. F. C., M. Waugh, R. F. Graziano. 1990. Regulation of human monocyte HLA-DR and CD4 antigen expression, and antigen presentation by 1,25-dihydroxyvitamin D₃. Blood 76:189.[Abstract/Free Full Text]
31. Lane, P., C. Burdet, F. McConnell, A. Lanzavecchia, E. Padovan. 1995. CD40L-independent B cell activation revealed by CD40L-deficient T cell clones: evidence for distinct activation requirements for antibody formation and B cell proliferation. Eur. J. Immunol. 25:1788.[Medline]
32. Romani, N., D. Reider, M. Heuer, E. E. S., B. Kampgen, D. Eibl, D. Niederwieser, G. Schuler. 1996. Generation of mature dendritic cells from human blood: an improved method with special regard to clinical applicability. J. Immunol. Methods 196:137.[Medline]
33. Lanzavecchia, A., D. Scheidegger. 1987. The use of hybrid hybridomas to target human cytotoxic T lymphocytes. Eur. J. Immunol. 17:105.[Medline]
34. Gately, M. K., R. Chizzonite, D. H. Presky. 1995. Measurement of human and mouse IL-12. Curr. Protocols Immunol. 3, 15:6.16.1.
35. Gallati, H., I. Pracht, J. Schmidt, P. Haering, G. Garotta. 1987. A simple, rapid and large capacity ELISA for biologically active native and recombinant human IFN-. J. Biol. Regul. Homeost. Agents 1:109.[Medline]
36. Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and down-regulated by tumor necrosis factor . J. Exp. Med. 179:1109.[Abstract/Free Full Text]
37. Cella, M., A. Hengering, V. Pinet, J. Pieters, A. Lanzavecchia. 1997. Inflammatory stimuli induce accumulation of MHC class II molecules on dendritic cells. Nature 388:782.[Medline]
38. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263.[Abstract/Free Full Text]
39. Steinbrink, K., M. Wolfl, H. Jonuleit, J. Knop, A. Enk. 1997. Induction of tolerance by IL-10-treated dendritic cells. J. Immunol. 159:4772.[Abstract]
40. Kalinski, P., J. Schiutemaker, C. Hilkens, E. Wierenga, M. Kapsenberg. 1999. Final maturation of dendritic cells is associated with impaired responsiveness to IFN- and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells. J. Immunol. 162:3231.[Abstract/Free Full Text]
41. Ludewig, B., D. Graf, H. Gelderblom, Y. Becker, R. Kroczek, G. Pauli. 1995. Spontaneous apoptosis of dendritic cells is efficiently inhibited by TRAP (CD40-ligand) and TNF-, but strongly enhanced by interleukin-10. Eur. J. Immunol. 25:1943.[Medline]
42. Vermes, I., C. Haanen, H. Steffens-Nakken, C. Reutelingsperger. 1995. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J. Immunol. Methods 184:39.[Medline]
43. Gately, M. K., L. M. Renzetti, J. Magram, A. S. Stern, L. Adorini, U. Gubler, D. H. Presky. 1998. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annu. Rev. Immunol. 16:495.[Medline]
44. De Smedt, T., B. Pajak, G. Klaus, R. Noelle, J. Urbain, O. Leo, M. Moser. 1998. Antigen-specific T lymphocytes regulate lipopolysaccharide-induced apoptosis of dendritic cells in vivo. J. Immunol. 161:4476.[Abstract/Free Full Text]
45. Provvedini, D., S. Manolagas, L. Deftos. 1983. 1,25-dihydroxyvitamin D₃ receptors in human leukocytes. Science 221:1181.[Abstract/Free Full Text]
46. Brennan, A., D. Katz, J. Nunn, S. Barker, M. Hewison, L. Fraher, J. O’Riordan. 1987. Dendritic cells from human tissues express receptors for the immunoregulatory vitamin D₃ metabolite, dihydroxycholecalciferol. Immunology 61:457.[Medline]
47. Rigby, W., M. Waugh. 1992. Decreased accessory cell function and costimulatory activity by 1,25-dihydroxyvitamin D₃-treated monocytes. Arthritis Rheum. 35:110.[Medline]
48. Clavreul, A., C. D’Hellencourt, C. Montero-Menel, G. Potron, D. Couez. 1998. Vitamin D differentially regulates B7.1 and B7.2 expression on human peripheral blood monocytes. Immunology 95:272.[Medline]
49. Bluestone, J. A.. 1997. Is CTLA-4 a master switch for peripheral T cell tolerance?. J. Immunol. 158:1989.[Abstract]
50. Shlomchik, W. D., M. S. Couzens, C. B. Tang, J. McNiff, M. E. Robert, J. Liu, M. J. Shlomchik, S. G. Emerson. 1999. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 285:412.[Abstract/Free Full Text]
51. Thomson, A. W., L. Lu. 1999. Dendritic cells as regulators of immune reactivity: implications for transplantation. Transplantation 68:1.[Medline]
52. Verstuyf, A., S. Segaert, L. Verlinden, K. Casteels, R. Bouillon, C. Mathieu. 1998. Recent developments in the use of vitamin D analogues. Curr. Opin. Nephrol. Hypertens. 7:397.[Medline]
53. Moser, M., T. De Smedt, T. Sornasse, F. Tielemans, A. Chentoufi, E. Muraille, M. Van Mechelen, J. Urbain, O. Leo. 1995. Glucocorticoids down-regulate dendritic cell function in vitro and in vivo. Eur. J. Immunol. 25:2818.[Medline]
54. Piemonti, L., P. Monti, P. Allavena, M. Sironi, L. Soldini, B. Leone, C. Socci, V. Di Carlo. 1999. Glucocorticoids affect human dendritic cell differentiation and maturation. J. Immunol. 162:6473.[Abstract/Free Full Text]
55. Snijdewint, F., P. Kalinski, E. Wierenga, J. Bos, M. Kapsenberg. 1993. Prostaglandin E2 differentially modulates cytokine secretion profiles of human T helper lymphocytes. J. Immunol. 150:5321.[Abstract]
56. Werhasselt, V., W. Vanden Berghe, N. Vanderheyde, F. Willems, G. Haegeman, M. Goldman. 1999. N-acetyl-L-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-B inhibition. J. Immunol. 162:2569.[Abstract/Free Full Text]
57. Caux, C., C. Massacrier, B. Vanbervliet, C. Barthelemy, Y. Liu, J. Banchereau. 1994. Interleukin 10 inhibits T cell alloreaction induced by human dendritic cells. Int. Immunol. 6:1177.[Abstract/Free Full Text]
58. Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kaempgen, N. Romani, G. Schuler. 1996. High level IL-12 production by murine dendritic cells: up-regulation via MHC class II and CD40 molecules and down-regulation by IL-4 and IL-10. J. Exp. Med. 184:741.[Abstract/Free Full Text]
59. Ria, F., G. Penna, L. Adorini. 1998. Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells. Eur. J. Immunol. 28:2003.[Medline]
60. Enk, A., V. Angeloni, M. Udey, S. Katz. 1993. Inhibition of Langerhans cell antigen-presenting function by IL-10: a role for IL-10 in induction of tolerance. J. Immunol. 151:2390.[Abstract]
61. Panina-Bordignon, P., D. Mazzeo, P. Di Lucia, D. D’Ambrosio, R. Lang, C. F. L., C. Self, F. Sinigaglia. 1997. ß₂-Agonists prevent Th1 development by selective inhibition of interleukin 12. J. Clin. Invest. 100:1513.[Medline]
62. D’Ambrosio, D., M. Cippitelli, M. G. Cocciolo, D. Mazzeo, P. Di Lucia, R. Lang, F. Sinigaglia, P. Panina-Bordignon. 1998. Inhibition of IL-12 production by 1,25-dihydroxyvitamin D₃. J. Clin. Invest. 101:252.[Medline]
63. Wong, B., R. Josien, S. Lee, B. Sauter, H. Li, R. Steinman, Y. Choi. 1997. TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J. Exp. Med. 186:2075.[Abstract/Free Full Text]
64. Josien, R., B. Wong, H. Li, R. Steinman, Y. Choi. 1999. TRANCE, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells. J. Immunol. 162:2562.[Abstract/Free Full Text]
65. Wong, B., R. Josien, Y. Choi. 1999. TRANCE is a TNF family member that regulates dendritic cell and osteoclast function. J. Leukocyte Biol. 65:715.[Abstract]
66. Evans, S., V. Soldatenkov, E. Shchepotin, E. Bogrash, I. Shchepotin. 1999. Novel 19-nor-hexafluoride vitamin D₃ analog (Ro 25-6760) inhibits human colon cancer in vitro via apoptosis. Int. J. Oncol. 14:979.[Medline]
67. Casteels, K., M. Waer, R. Bouillon, J. Depovere, D. Valckx, J. Laureys, C. Mathieu. 1998. 1,25-Dihydroxyvitamin D₃ restores sensitivity to cyclophosphamide-induced apoptosis in non-obese diabetic (NOD) mice and protects against diabetes. Cin. Exp. Immunol. 112:181.[Medline]

This article has been cited by other articles:

				R. Bouillon, G. Carmeliet, L. Verlinden, E. van Etten, A. Verstuyf, H. F. Luderer, L. Lieben, C. Mathieu, and M. Demay Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice Endocr. Rev., October 1, 2008; 29(6): 726 - 776. [Abstract] [Full Text] [PDF]

				N. Liu, L. Nguyen, R. F. Chun, V. Lagishetty, S. Ren, S. Wu, B. Hollis, H. F. DeLuca, J. S. Adams, and M. Hewison Altered Endocrine and Autocrine Metabolism of Vitamin D in a Mouse Model of Gastrointestinal Inflammation Endocrinology, October 1, 2008; 149(10): 4799 - 4808. [Abstract] [Full Text] [PDF]

				G. Besin, S. Gaudreau, M. Menard, C. Guindi, G. Dupuis, and A. Amrani Thymic Stromal Lymphopoietin and Thymic Stromal Lymphopoietin-Conditioned Dendritic Cells Induce Regulatory T-Cell Differentiation and Protection of NOD Mice Against Diabetes Diabetes, August 1, 2008; 57(8): 2107 - 2117. [Abstract] [Full Text] [PDF]

				R. Spirig, C. van Kooten, C. Obregon, L. Nicod, M. Daha, and R. Rieben The Complement Inhibitor Low Molecular Weight Dextran Sulfate Prevents TLR4-Induced Phenotypic and Functional Maturation of Human Dendritic Cells J. Immunol., July 15, 2008; 181(2): 878 - 890. [Abstract] [Full Text] [PDF]

				Y. A. Taher, B. C. A. M. van Esch, G. A. Hofman, P. A. J. Henricks, and A. J. M. van Oosterhout 1{alpha},25-Dihydroxyvitamin D3 Potentiates the Beneficial Effects of Allergen Immunotherapy in a Mouse Model of Allergic Asthma: Role for IL-10 and TGF-{beta} J. Immunol., April 15, 2008; 180(8): 5211 - 5221. [Abstract] [Full Text] [PDF]

				M. Cutolo and K. Otsa Review: Vitamin D, immunity and lupus Lupus, January 1, 2008; 17(1): 6 - 10. [Abstract] [PDF]

				C. Daniel, N. A. Sartory, N. Zahn, H. H. Radeke, and J. M. Stein Immune Modulatory Treatment of Trinitrobenzene Sulfonic Acid Colitis with Calcitriol Is Associated with a Change of a T Helper (Th) 1/Th17 to a Th2 and Regulatory T Cell Profile J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 23 - 33. [Abstract] [Full Text] [PDF]

				S. Gorman, L. A. Kuritzky, M. A. Judge, K. M. Dixon, J. P. McGlade, R. S. Mason, J. J. Finlay-Jones, and P. H. Hart Topically Applied 1,25-Dihydroxyvitamin D3 Enhances the Suppressive Activity of CD4+CD25+ Cells in the Draining Lymph Nodes J. Immunol., November 1, 2007; 179(9): 6273 - 6283. [Abstract] [Full Text] [PDF]

				P. Hubert, N. Jacobs, J.-H. Caberg, J. Boniver, and P. Delvenne The cross-talk between dendritic and regulatory T cells: good or evil? J. Leukoc. Biol., October 1, 2007; 82(4): 781 - 794. [Abstract] [Full Text] [PDF]

				J. Ehrchen, L. Helming, G. Varga, B. Pasche, K. Loser, M. Gunzer, C. Sunderkotter, C. Sorg, J. Roth, and A. Lengeling Vitamin D receptor signaling contributes to susceptibility to infection with Leishmania major FASEB J, October 1, 2007; 21(12): 3208 - 3218. [Abstract] [Full Text] [PDF]

				R. Bailey, J. D. Cooper, L. Zeitels, D. J. Smyth, J. H.M. Yang, N. M. Walker, E. Hypponen, D. B. Dunger, E. Ramos-Lopez, K. Badenhoop, et al. Association of the Vitamin D Metabolism Gene CYP27B1 With Type 1 Diabetes Diabetes, October 1, 2007; 56(10): 2616 - 2621. [Abstract] [Full Text] [PDF]