Diabetes
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Halteren, A. G.S.
Right arrow Articles by Mathieu, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Halteren, A. G.S.
Right arrow Articles by Mathieu, C.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Diabetes 51:2119-2125, 2002
© 2002 by the American Diabetes Association, Inc.

Redirection of Human Autoreactive T-Cells Upon Interaction With Dendritic Cells Modulated by TX527, an Analog of 1,25 Dihydroxyvitamin D3

Astrid G.S. van Halteren1, Evelyne van Etten2, Esther C. de Jong3, Roger Bouillon2, Bart O. Roep1, and Chantal Mathieu2

1 Department of Immunohematology and Blood Transfusion, Leids Universitair Medisch Centrum, Leiden, the Netherlands
2 Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Katholieke Universiteit Leuven, Leuven, Belgium
3 Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The active form of vitamin D3, 1{alpha},25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent immunomodulator known to affect T-cells through targeting antigen-presenting cells such as dendritic cells (DCs). We studied the effects of a novel nonhypercalcemic 1,25(OH)2D3 analog, TX527, on DC differentiation, maturation, and function with respect to stimulation of a committed human GAD65-specific autoreactive T-cell clone. Continuous addition of TX527 impaired interleukin (IL)-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF)-driven DC differentiation as well as lipopolysaccharide (LPS) and interferon-{gamma} (IFN-{gamma})-induced maturation into Th1-promoting DC (DC1), as characterized by marked changes in DC morphology and abrogation of IL-12p70 release upon CD40 ligation. Addition of TX527 during maturation did not affect DC morphology but significantly changed DC cytokine profiles. The potential of treated DCs to alter the response pattern of committed autoreactive T-cells was found to depend on the timing of TX527 exposure. Continuously TX527-treated DCs significantly inhibited T-cell proliferation and blocked IFN-{gamma}, IL-10, but not IL-13 production, whereas DCs treated during maturation failed to inhibit T-cell proliferation but affected IL-10 and IFN-{gamma} production. Collectively, we provide evidence that nonhypercalcemic TX527 is a potent in vitro DC modulator, yielding DCs with the potential to change cytokine responses of committed autoreactive T-cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because preventing and curing type 1 diabetes are priorities in health care, much effort is put into gaining insight into the pathogenesis of the disease. In the NOD mouse model, it is clear that primary prevention of type 1 diabetes, e.g., autoimmunity itself, is easily achievable (1). Secondary prevention and especially prevention of recurrence of autoimmune diabetes after islet transplantation are much harder to realize. In the latter situation, an already activated immune system needs to be silenced or preferentially redirected (2). One of the protocols that are able to prevent diabetes in NOD mice, even when started after insulitis is present, includes the use of 1{alpha},25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D3 (3,4). Although intervention in humans is not yet possible, epidemiologic data support the hypothesis that vitamin D status is an important risk factor for the development of type 1 diabetes, because dietary vitamin D supplementation during infancy was shown to be associated with a reduced risk of type 1 diabetes later in life (5).

1,25(OH)2D3 is a secosteroid hormone that has several distinct functions in both bone and calcium/phosphate metabolism as well as in the regulation of immune responses (6). A variety of cells within the immune system are known to express vitamin D receptors, including antigen-presenting cells (APCs) and activated T-cells (7). 1,25(OH)2D3 directly affects proliferation and polarization of naive murine T-cells by preventing the development of Th1 cells (8). However, inhibition of Th1 development by 1,25(OH)2D3 is thought to be mediated primarily via its action on APCs, leading to reduced IL-12 production after 1,25(OH)2D3 treatment (9). In addition, 1,25(OH)2D3 was recently shown to affect the maturation, activation, and survival of human DCs, leading to impaired priming of alloreactive T-cell responses in vitro (10–12). These results demonstrate that APCs, in particular DCs, are primary targets for the immunomodulatory activity of 1,25(OH)2D3.

Because hypercalcemia seriously hampers clinical application of 1,25(OH)2D3, however, much effort has been put into the development of 1,25(OH)2D3 analogs with reduced hypercalcemic capacity (13). Protective effects of 1,25(OH)2D3 analogs have been studied extensively in the NOD mouse, demonstrating prevention of both insulitis and diabetes (14). Moreover, progression to clinically overt diabetes and recurrence of diabetes after syngeneic islet transplantation has been prevented by analogs of 1,25(OH)2D3 (15). Both studies point out that 1,25(OH)2D3 analogs interfere with progression of autoimmune diabetes, i.e., changing an ongoing autoimmune reaction, which suggests an effect on committed autoreactive T-cells. Immunomodulatory agents, such as 1,25(OH)2D3, that are able to shift dominant pathogenic (Th1 associated) T-helper responses (16) and possibly induce regulatory T-cells (17) are extremely interesting.

The aim of the present study was to analyze whether incubation of DCs with TX527, a nonhypercalcemic analog of 1,25(OH)2D3, could alter DCs in such a manner that a change in the response pattern of an already committed GAD65-autoreactive T-cell clone is induced. The T-cell clone used in this study was derived from a prediabetic type 1 diabetes patient and recognizes naturally processed GAD65 as well as a defined peptide epitope in the context of HLA-DR3 (18). First we studied phenotypic and functional changes in DCs induced by TX527 or 1,25(OH)2D3 treatment during various stages of their development from monocytes. In addition, we determined proliferative and cytokine responses of T-cells cocultured with nontreated TX527 or 1,25(OH)2D3-treated immature and mature DCs.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of human DCs.
Human HLA-DR3 homozygous peripheral blood mononuclear cells were isolated by Ficoll gradient from HLA-typed buffy coats, obtained from healthy blood donors. Monocytes were obtained via positive selection on a MACS column after staining peripheral blood mononuclear cells with CD14-MicroBeads according to the supplier’s protocol (Milteny Biotec via CLB, Amsterdam, the Netherlands). Isolated monocytes (0.5 x 106/ml, routinely >90% pure) were subsequently cultured for 6 days in RPMI 1640 medium (Gibco Life Technologies, Breda, The Netherlands) supplemented with 10% heat-inactivated FCS, 2 mmol/l glutamine, 100 IU/ml penicillin, 100 IU/ml streptomycin, 1,000 units/ml recombinant human IL-4 (Strathmann Biotec AG, Hannover, Germany), and 800 units/ml recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) (Leucomax, Novartis Pharma, Arnhem, the Netherlands) in 24-well tissue culture plates (Costar, Cambridge, MA). Medium and cytokines were refreshed every 3 days. At day 6, when DCs had obtained an immature phenotype, DC1 maturation was induced by addition of 100 ng/ml Escherichia coli-derived lipopolysaccharide (LPS) (Sigma Aldrich Chemie, Zwijndrecht, The Netherlands), 1,000 units/ml recombinant human IFN-{gamma} (Peprotech, Rocky Hill, NJ), and 800 units/ml GM-CSF. DCs were harvested after 48 h for further analysis.

In some experiments, macrophages were generated in parallel to DCs by culturing monocytes in RPMI 1640 medium supplemented with 10% FCS, 2 mmol/l glutamine, 100 IU/ml penicillin, 100 IU/ml streptomycin, and 50 ng/ml recombinant human M-CSF (Peprotech) in 24-well tissue culture plates. Medium and cytokines were refreshed at days 3 and 6.

1,25(OH)2D3 or TX527 treatment of DCs.
1,25(OH)2D3 was obtained from J.P. Vandevelde (Duphar, Weesp, the Netherlands). The analog TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (19,20) was synthesized by M. Vandewalle and P. De Clercq (University of Ghent, Belgium) and further obtained from Théramex S.A. (Monaco). Both 1,25(OH)2D3 and TX527 were used at a final concentration of 10-8 mol/l and refreshed at days 3 and 6, respectively. They were added to the culture medium either during the complete culture period from monocyte to immature DCs to mature DCs or only during the maturation phase.

Analysis of DC surface markers, cytokine production, and phagocytosis capacity.
For evaluating DCs’ surface expression, DCs were incubated for 30 min at 4°C with the following FITC- or PE-conjugated murine antibodies (all from Becton Dickinson, San Jose, CA): CD1a, CD14, CD83, CD86, CD80, CD54, HLA-DR, CD40, and control mIgG, all diluted in PBS containing 0.1% BSA. After extensive washing, DCs were subsequently analyzed on a FACS Calibur (Becton Dickinson).

Because mature DC1 cells are unresponsive to bacterial or viral compounds (21), 4 x 105 DCs were incubated with 4 x 105 CD154 expressing L cells (provided by Dr. C. van Kooten, Department of Nephrology, LUMC, the Netherlands, [22]) in the presence of 1,000 units/ml recombinant human IFN-{gamma} to trigger cytokine production by untreated 1,25(OH)2D3 or TX527 modulated DCs. After 24 h, supernatants were collected and cytokine levels were determined by enzyme-linked immunosorbent assay (ELISA) by using commercial kits specific for IL-12p70 and IL-10 (R&D Systems, Minneapolis, MN).

Fluorescein-labeled zymosan particles (Saccharomyces cerevisiae; Molecular Probes Europe, Leiden, the Netherlands) were used to evaluate the phagocytosis capacity of variously treated DCs and macrophages. Briefly, the various cell suspensions were incubated for 2 h with fluoresceinated particles (1–100 particles/cell), after which the cells were washed and dissolved in 100 µl of Trypan Blue. After extensive washing, the cells were resuspended in 2% paraformaldehyde and analyzed by FACS. Phagocytosis capacity is expressed as percentage of fluorescein-positive cells.

T-cell assays.
To test the T-cell stimulatory capacities of nontreated-1,25(OH)2D3 or TX527-treated DCs, we used a GAD65-specific T-cell clones derived from a prediabetic individual, which recognizes naturally processed GAD65 antigen (Diamyd, Stockholm, Sweden) and the minimal GAD65 epitope p (339–352) in the context of HLA-DR3, as described previously (18). For proliferation or cytokine induction, 1 x 104 T-cells were incubated with an equal number of variously treated DCs, diluted in Iscove’s modified Dulbecco’s medium (IMDM), supplemented with 2 mmol/l glutamine and 10% pooled human AB serum. T-cells were cultured for, respectively, 3 days (proliferation) or 24 h (ELIspot) in 96-well flat-bottom plates (Costar, Cambridge, MA). After 3 days, cultures were pulsed with [3H]thymidine (0.5 µCi/well) for another 18 h, after which 3H incorporation was measured by liquid scintillation counting. Results are expressed as means ± SD of triplicate wells. For detection of the T-cell cytokines IL-2, IFN-{gamma}, IL-10, and IL-13, T-cells were harvested by gently rinsing the wells and washing the collected T-cells in a large volume of IMDM (23). T-cells were subsequently plated on anti-cytokine antibody-precoated ELISA plates and cultured for 2 h (IL-2), 5 h (IFN{gamma}, IL-13), or overnight (IL-10) in IMDM supplemented with 2% pooled human AB serum at 37°C 5% CO2. After lysis of the cells with ice-cold deionized water, the plates were washed with PBS/0.05% Tween-20 and incubated with biotinylated detector antibody for 1 h at 37°C, followed by a second incubation with gold-labeled anti-biotin antibody (GABA) for 1 h at 37°C. All antibodies were diluted in PBS/1% BSA. After extensive washing with PBS/0.05% Tween-20, the plates were developed according to the manufacturer’s protocol (U-CyTech, Utrecht, the Netherlands). Spots were counted on an Olympus microscope and analyzed with Olympus Micro Image 4.0 software (Paes Nederland, Zoeterwoude, the Netherlands). Results are expressed as means ± SD of triplicate wells.

Statistical analysis.
The Mann-Whitney U test was used to compare results obtained from untreated DCs with either TX527- or 1,25(OH)2D3-treated DCs in various experiments. Significance was defined at the 0.05 level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of TX527 or 1,25(OH)2D3 on morphological features and functional profile of human dendritic cells.
To compare the effects of TX527 and its parent compound 1,25(OH)2D3 on in vitro DC generation, we added TX527 or 1,25(OH)2D3 to monocytes that were cultured with GM-CSF and IL-4 for 6 days to generate immature DCs. On the basis of earlier findings (10,24), the concentration chosen in our study was 10-8 mol/l for both drugs, although TX527 was found to be equally potent at 10- to 100-fold lower concentrations (data not shown).

Addition of 1,25(OH)2D3 or TX527 impaired downregulation of the monocyte marker CD14 and interfered with upregulation of the skin DC marker CD1a when compared with untreated immature DCs (Fig. 1, left). Levels of MHC class II (HLA-DR) as well as costimulatory molecules CD86, CD80, CD54, and CD40 were unchanged compared with nontreated immature DCs (data not shown for CD80, CD54, and CD40). The DC maturation marker CD83 was obviously not expressed on nontreated TX527 or 1,25(OH)2D3-treated immature DCs.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 1. Characterization of cell surface markers on human immature (day 6) and mature DCs (day 8) generated from isolated monocytes in the presence of GM-CSF and IL-4. DC cultures were either nontreated (dotted line) or treated with 10-8 mol/l 1,25(OH)2D3 (solid thin line) or TX527 (solid thick line). DC maturation was induced by addition of LPS and IFN-{gamma}.

 
After exposure to LPS and IFN-{gamma} to induce DC maturation into DC1 (25), TX527- or 1,25(OH)2D3-treated DCs continued to express CD14 but failed to upregulate CD1a or CD83 (Fig. 1, right). In addition, TX527 or 1,25(OH)2D3 treatment resulted in a 10-fold lower expression of HLA-DR and CD86, whereas expression of CD80, CD40, or CD54 was not different from control mature DCs (data not shown).

This TX527- or 1,25(OH)2D3-induced altered DC phenotype was accompanied by typical morphological features (Fig. 2). Upon culture for 6 days with GM-CSF and IL-4, DCs became nonadherent and expressed protruding veils (data not shown). After the addition of LPS and IFN-{gamma}, maturing DCs formed large clusters of nonadherent cells with even more pronounced cytoplasmic projections, whereas continuous addition of TX527 and, to a lesser extent, 1,25(OH)2D3 yielded small clusters of mostly adherent growing spindle-shaped cells.



View larger version (82K):
[in this window]
[in a new window]
  		FIG. 2. Morphology of variously treated mature DCs. Cultures were either nontreated (NT) or continuously treated with 10-8 mol/l 1,25(OH)2D3 or TX527.

 
In addition to the induction of phenotypic differences, prolonged TX527 or 1,25(OH)2D3 treatment was found to affect DC cytokine production induced by CD40 triggering via CD40L-expressing cells (Fig. 3). Untreated DCs express high levels of IL-12p70, whereas TX527 or 1,25(OH)2D3 treatment almost completely abrogated IL-12p70 production. The production of IL-10 was not different from untreated DC.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 3. 1,25(OH)2D3 or TX527 treatment affects DC cytokine production induced by CD40 triggering. Variously treated DCs were cultured for 24 h with CD40L-transfected L cells after which supernatant was harvested for the determination of IL-12p70 () or IL-10 ({blacksquare}) by ELISA.

 
Because TX527 treatment in particular yielded adherent growing cells, which lack the expression of typical DC markers, e.g., CD1a and CD83, and more closely resemble macrophages, we compared the phagocytosis capacity of variously treated DCs and macrophages generated in parallel (Table 1). The phagocytosis capacity of TX527- or 1,25(OH)2D3-treated DCs did not differ from untreated mature DCs. All three types of DCs showed a lower capacity to take up fluorescent particles when compared with macrophages or immature DCs.


View this table:
[in this window]
[in a new window]
  		TABLE 1 Capacity of macrophages and variously treated dendritic cells to take up fluorescein-labeled zymosan particles by phagocytosis

 
Taken together, TX527 and its parent compound 1,25(OH)2D3 affect the differentiation of DCs from monocytes and their subsequent maturation, yielding morphologically altered cells that lack DC-specific features and express lower levels of cell surface molecules involved in antigen presentation. Moreover, TX527- or 1,25(OH)2D3-modulated immature DCs were unable to convert into classical mature DC1 cells upon withdrawal of TX527 or 1,25(OH)2D3 and addition of LPS + IFN-{gamma}, as determined by FACS analysis and analysis of IL-12p70 levels induced by CD40 ligation (data not shown), demonstrating that the effects of both drugs on DC differentiation are persistent.

Stimulation of autoreactive T-cells by TX527- or 1,25(OH)2D3-treated DCs.
Because lower levels of MHC class II and CD86 expression as well as reduced cytokine production by TX527- or 1,25(OH)2D3-treated DCs could affect the immune-stimulatory potential of these DCs, we tested proliferative and cytokine responses of a GAD65-specific T-cell clone upon coculture with variously treated DCs. Typically, this T-cell clone displays a Th0 cytokine profile when stimulated with peptides that contain the 339–352 sequence, i.e., IFN-{gamma}, IL-10, and IL-13 (18). To exclude any (selective) effects of TX527 or 1,25(OH)2D3 on T-cells (8,24), we washed DCs extensively before addition to T-cell cultures.

Because immature DCs are most efficient in processing complete protein into peptides (26), we cocultured variously treated immature DCs with T-cells in the presence of GAD65 protein or the minimal peptide epitope 339–352 (Fig. 4). TX527 or 1,25(OH)2D3 pretreatment of DCs had no significant effect on GAD65-induced proliferative responses of the T-cells, whereas cytokine responses were completely blocked (P < 0.001). When the minimal peptide epitope was directly loaded onto DCs, proliferation of the T-cell clone was again not influenced by TX527 pretreatment of DCs. However, peptide-induced cytokine responses were differently affected by TX527 pretreatment, varying from complete abrogation of IL-10 (P < 0.001), significantly reduced IFN-{gamma} (P = 0.04), and unchanged IL-13 production, whereas 1,25(OH)2D3 pretreatment of DCs was found to block IL-10 levels (P = 0.002) without significantly affecting IFN-{gamma} or IL-13.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4. Modulation of autoreactive mol/l-cell responses by 1,25(OH)2D3- or TX527-treated immature DCs. GAD65-specific T-cells were cocultured with either nontreated ({square}) or 10-8 mol/l 1,25(OH)2D3-treated () or TX527-treated ({blacksquare}) immature DCs in the presence of 10 µg/ml GAD65 protein (top) or 0.2 µg/ml GAD (339–352) peptide (bottom) for the induction of T-cell proliferation and cytokine production. A representative experiment of two is shown.

 
Mature DCs, however, do not have the capacity to process proteins into peptides efficiently. Therefore, the T-cell clone was incubated only with its minimal peptide epitope in the presence of variously treated DCs (Fig. 5). APCs that were generated from monocytes in the continuous presence of TX527 or 1,25(OH)2D3 induced significantly lower proliferation (P = 0.002 for TX527 vs. P = 0.009 for 1,25(OH)2D3) and remarkably affected the cytokine profile of the T-cell clone. Both IFN-{gamma} and IL-10 were almost completely inhibited (P = 0.002), whereas IL-13 was not affected. Of both drugs, TX527 pretreatment induced the most pronounced inhibitory effects on the response pattern of the committed T-cell.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 5. Effect of 1,25(OH)2D3 or TX527 treatment on T-cell stimulatory capacity of mature DCs. Mature DCs were generated from isolated monocytes in the absence ({square}) or presence of 10-8 mol/l 1,25(OH)2D3 () or TX527 ({blacksquare}), after which GAD65-specific T-cells were added. T-cells were cultured in the presence of 0.2 µg/ml GAD (339–352) peptide for the induction of T-cell proliferation and cytokine production. A representative experiment of five is shown.

 
DC phenotype and function upon addition of TX527 or 1,25(OH)2D3 only during the maturation phase.
To evaluate the effects of TX527 or 1,25(OH)2D3 on the process of LPS- and IFN-{gamma}-induced DC maturation (phase II), we added TX527 or 1,25(OH)2D3 during the last 2 days of culture of immature DCs generated in the presence of GM-CSF and IL-4 only. Introduction of TX527 or 1,25(OH)2D3 resulted in upregulation of CD14 on a low percentage of mature DCs (mean fluorescence intensity [MFI] 225.73 in TX527- and MFI 129.76 in 1,25(OH)2D3-treated DCs vs. MFI 49.38 in control untreated DCs). Addition of TX527 or 1,25(OH)2D3 did not affect expression of CD1a, CD83, CD80, CD86, CD40, CD54, or HLA-DR, because levels of these markers expressed on TX527- or 1,25(OH)2D3-modulated DCs were identical to levels expressed by nontreated mature DCs (data not shown). Nevertheless, DC cytokine profiles released upon CD40 triggering were significantly altered (Fig. 6). In contrast to untreated mature DCs, which express high levels of IL-12p70 and low levels of IL-10, TX527 or 1,25(OH)2D3 treatment during maturation resulted in DCs that were unable to secrete IL-12p70 but secreted four- to eightfold increased levels of IL-10 upon CD40 ligation. Thus, TX527, as well as the parent compound, redirects mature DCs, resulting in functionally different DCs compared with nontreated mature DCs.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 6. Abrogation of IL-12p70 and enhancement of IL-10 secretion upon CD40 ligation of DCs treated with 1,25(OH)2D3 or TX527 during maturation only. Immature DCs were generated from isolated monocytes, after which maturation was induced in the absence or presence of 10-8 mol/l 1,25(OH)2D3 or TX527. Variously treated mature DCs were subsequently cocultured with CD40L-expressing cells, after which supernatant was collected for determination of IL-12p70 () or IL-10 ({blacksquare}) by ELISA.

 
Profile of autoreactive T-cells incubated with DCs exposed to TX527 or 1,25(OH)2D3 during the maturation phase only.
Despite that TX527 or 1,25(OH)2D3 treatment did not affect peptide-induced proliferative responses of the T-cell clone, cytokine responses were affected to various extents (Fig. 7). Again, TX527-modulated DCs were more effective than 1,25(OH)2D3-treated DCs in redirecting cytokine responses, as demonstrated by a complete reduction in IL-10 (P < 0.001) and significant reduction in IFN-{gamma} (P = 0.002) release. 1,25(OH)2D3-pretreated DCs significantly affected IL-10 and IL-13 (P = 0.002) but not IFN-{gamma} production.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 7. 1,25(OH)2D3 or TX527 treatment during maturation yields DCs that marginally affect GAD65-committed T-cells. Immature DCs were matured in the absence ({square}) or presence of 10-8 mol/l 1,25(OH)2D3 () or TX527 ({blacksquare}). After 48 h, DCs were cocultured with GAD65-specific T-cells in the absence or presence of 0.2 µg/ml GAD (339–352) peptide for the induction of proliferation and cytokine production. A representative experiment of three is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that TX527 is a potent 1,25(OH)2D3 analog that is capable of targeting DCs at various stages throughout their in vitro generation from monocytes. TX527 was found to be biologically active in a broader range (10-7 to 10-10 mol/l) when compared with its parent compound 1,25(OH)2D3 (10-7 to 10-8 mol/l), yielding DCs with the highest potential to alter the cytokine profile of a committed autoreactive T-cell clone. This modulating effect was mediated via the antigen-presenting DCs, because TX527 or 1,25(OH)2D3 was not added during T-cell cultures.

Most classical immunomodulators and immune suppressants modulate T-cells through direct interaction with T-cell proliferation or cytokine production. Very few strategies also affect antigen-presenting DCs, the central cell of the immune system able to polarize the immune system into different directions, e.g., Th1 versus Th2 (27). The active form of vitamin D3, 1,25(OH)2D3, is one of the few products available (10,12), together with glucocorticoids (28–31), that is able to change DC differentiation and function in vitro.

In the present study, we demonstrated that a novel nonhypercalcemic 1,25(OH)2D3 analog, TX527, is able to modify significantly the phenotype of DCs as characterized by major changes in cell surface marker expression, morphology, and cytokine profile. In contrast to another study (12), we demonstrated that TX527 or 1,25(OH)2D3 treatment does not simply abrogate differentiation and maturation but leads to the emergence of a different cell that lacks typical DC features. Moreover, these modulated DCs were not able to equalize the high phagocytosis capacity that is observed for untreated macrophages or immature DCs. This is a strong argument against the postulated 1,25(OH)2D3-mediated arrest in the immature stage of DC development (11). It also confirms that 1,25(OH)2D3 or TX527 does not simply mediate differentiation toward macrophages (12). In the past, however, a clear, positive effect of 1,25(OH)2D3 on the differentiation of macrophages was shown under different culture conditions (32). Monocytes may be considered as relative immature precursors with multiple differentiation potentials, depending on the microenvironment during differentiation (33). In vitro, cytokines and inflammatory substances play an important role in the final determination of whether monocytes will acquire DC, macrophage, osteoclastic, or other characteristics and functions (34,35). Additional analysis of phenotype and functionality will be necessary to characterize fully the cell type generated by continuous presence of TX527 or 1,25(OH)2D3.

In vivo, 1,25(OH)2D3 analogs induce an immune shift that is characterized by downregulation of Th1 cytokines and upregulation of Th2 cytokines in response to pancreatic autoantigens such as GAD65 (16). Our in vitro data on committed human autoreactive T-cells indicate that TX527 treatment of DCs indeed inhibited IFN-{gamma} release but did not increase Th2-linked IL-13 production. It is conceivable that this partial discordance with the in vivo data results from a direct action of 1,25(OH)2D3 on T-cells during in vivo treatment, because 1,25(OH)2D3 was shown to promote directly Th2-associated cytokine production in the absence of APCs (8).

Another important finding in our study is the redirection by TX527-modulated DCs of committed T-cells toward a different cytokine profile without completely blocking their proliferative capacity. The persistence of the proliferative capacity suggests that the autocrine stimulation of IL-2 was unaffected. Indeed, IL-2 production was not changed from that of autoreactive T-cells stimulated with untreated DCs (data not shown). This finding indicates that for complete blockade of committed autoreactive T-cells, a combined immunosuppressive therapy that targets both IL-2-dependent (e.g., cyclosporine, tacrolimus, or dacliximab [36]) and -independent pathways (TX527 or 1,25(OH)2D3) of T-cell activation would be preferred. This is in accordance with data on prevention of recurrent autoimmune disease after syngeneic islet transplantation in diabetic NOD mice treated with a combination of a vitamin D3 analog and cyclosporine, whereas the individual drugs were less effective (15).

In addition, treatment with 1,25(OH)2D3 in combination with the IL-2-independent drug mycophenolate mofetil was recently shown to induce transplantation tolerance associated with an increased frequency of regulatory T-cells that bear the IL-2 receptor (CD152+CD4+CD25+ [17]). Mycophenolate mofetil is a selective lymphocyte inhibitor that blocks the progression of T-cells into the S phase without affecting the IL-2 pathway (24). The IL-2 pathway is known to be critical in the development of CD25-expressing regulatory T-cells (37) that mediate transplantation tolerance (17) and control autoimmunity in NOD mice (38).

1,25(OH)2D3- or analog-induced changes in cytokine profile of committed T-cells may in vivo result in impaired autoimmune effector cells. In addition, naive T-cells were shown to become hyporesponsive through priming by 1,25(OH)2D3-modulated DCs (10,12). When extrapolating these in vitro data to the in vivo situation, where the generation of suppressor cells by 1,25(OH)2D3 and its analogs has been confirmed in different settings (4,17), these studies collectively define 1,25(OH)2D3, especially its nonhypercalcemic analogs, as ideal immunosuppressants in autoimmunity and as candidate drugs to be combined with typical anti-T-cell immunosuppressants to intervene in hyperactivated immune systems, e.g., in islet transplantation. We conclude that a new structurally modified analog of vitamin D3, TX527, modulates morphological features and cytokine production of human DCs, yielding altered DCs that redirect an already committed T-cell clone toward a cell with a reduced pathogenic potential.


   ACKNOWLEDGMENTS
 
This work was supported by Grant 2000.00.056 and 1998.01.001 from the Diabetes Fonds Nederland, Grant 99/10 from the Geconcerteerde OnderzoeksActie, and Grant 3.0332.98 from the Flemish Research Foundation (Fonds voor Wetenschappelijk Onderzoek).

Erica F.M. Meulenberg is greatly acknowledged for excellent technical assistance.


   FOOTNOTES
 
Address correspondence and reprint requests to Dr. B.O. Roep, Department of Immunohematology and Blood Transfusion, Leids Universitair Medisch Centrum, E3Q, LUMC, P.O. Box 9600, 2300 RC Leiden, the Netherlands. E-mail: A.G.S.van_Halteren{at}lumc.nl.

Received for publication 16 November 2001 and accepted in revised form 21 February 2002.

1,25(OH)2D3, 1{alpha},25-dihydroxyvitamin D3; APC, antigen-presenting cells; DC, dendritic cell; DC1, Th1-promoting DC; ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte/macrophage colony-stimulating factor; IFN-{gamma}, interferon-{gamma}; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium; LPS, lipopolysaccharide; MFI, mean fluorescence intensity.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Atkinson MA, Leiter EH: The NOD mouse model of type 1 diabetes: as good as it gets? Nat Med 5:601–604, 1999[Medline]
  2. Roep BO, Stobbe I, Duinkerken G, van Rood JJ, Lernmark A, Keymeulen B, Pipeleers D, Claas FH, de Vries RR: Auto- and alloimmune reactivity to human islet allografts transplanted into type 1 diabetic patients. Diabetes 48:484–490, 1999[Abstract]
  3. Mathieu C, Laureys J, Sobis H, Vandeputte M, Waer M, Bouillon R: 1,25-Dihydroxyvitamin D3 prevents insulitis in NOD mice. Diabetes 41:1491–1495, 1992[Abstract]
  4. Mathieu C, Waer M, Laureys J, Rutgeerts O, Bouillon R: Prevention of autoimmune diabetes in NOD mice by 1,25 dihydroxyvitamin D3. Diabetologia 37:552–558, 1994[Medline]
  5. Hyppönen E, Läärä E, Reunanen A, Järvelin M-J, Virtanen SM: Intake of vitamin D and risk of type 1 diabetes: a birth control study. Lancet 358:1500–1503, 2001[Medline]
  6. Lemire JM: Immunomodulatory actions of 1,25-dihydroxyvitamin D3. J Steroid Biochem Mol Biol 53:599–602, 1995[Medline]
  7. Verstuyf A, Segaert S, Verlinden L, Bouillon R, Mathieu C: Recent developments in the use of vitamin D analogs. Expert Opin Investig Drugs 9:443–455, 2000[Medline]
  8. Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HF, O’Garra A: 1{alpha},25-Dihydroxyvitamin D3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells. J Immunol 167:4974–4980, 2001[Abstract/Free Full Text]
  9. D’Ambrosio D, Cippitelli M, Cocciolo MG, Mazzeo D, Di Lucia P, Lang R, Sinigaglia F, Panina-Bordignon P: Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-{kappa}B downregulation in transcriptional repression of the p40 gene. J Clin Invest 101:252–262, 1998[Medline]
  10. Penna G, Adorini L: 1{alpha},25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 164:2405–2411, 2000[Abstract/Free Full Text]
  11. Griffin MD, Lutz W, Phan VA, Bachman LA, McKean DJ, Kumar R: Dendritic cell modulation by 1{alpha},25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo. Proc Natl Acad Sci U S A 98:6800–6805, 2001[Abstract/Free Full Text]
  12. Piemonti L, Monti P, Sironi M, Fraticelli P, Leone BE, Dal Cin E, Allavena P, Di Carlo V: Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells. J Immunol 164:4443–4451, 2000[Abstract/Free Full Text]
  13. Bouillon R, Okamura WH, Norman AW: Structure-function relationships in the vitamin D endocrine system. Endocr Rev 16:200–257, 1995[Medline]
  14. Casteels KM, Mathieu C, Waer M, Valckx D, Overbergh L, Laureys JM, Bouillon R: Prevention of type I diabetes in nonobese diabetic mice by late intervention with nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 in combination with a short induction course of cyclosporin A. Endocrinology 139:95–102, 1998[Abstract/Free Full Text]
  15. Casteels K, Waer M, Laureys J, Valckx D, Depovere J, Bouillon R, Mathieu C: Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine. Transplantation 65:1225–1232, 1998[Medline]
  16. Overbergh L, Decallonne B, Waer M, Rutgeerts O, Valckx D, Casteels KM, Laureys J, Bouillon R, Mathieu C: 1{alpha},25-dihydroxyvitamin D3 induces an autoantigen-specific T-helper 1/T-helper 2 immune shift in NOD mice immunized with GAD65 (p524–543). Diabetes 49:1301–1307, 2000[Abstract]
  17. Gregori S, Casorati M, Amuchastegui S, Smiroldo S, Davalli AM, Adorini L: Regulatory T cells induced by 1{alpha},25-dihydroxyvitamin D(3) and mycophenolate mofetil treatment mediate transplantation tolerance. J Immunol 167:1945–1953, 2001[Abstract/Free Full Text]
  18. Schloot NC, Batstra MC, Duinkerken G, de Vries RR, Dyrberg T, Chaudhuri A, Behan PO, Roep BO: GAD65-reactive T cells in a non-diabetic stiff-man syndrome patient. J Autoimmun 12:289–296, 1999[Medline]
  19. Verlinden L, Verstuyf A, Van Camp M, Marcelis S, Sabbe K, Zhao XY, De Clercq P, Vandewalle M, Bouillon R: Two novel 14-Epi-analogues of 1,25-dihydroxyvitamin D3 inhibit the growth of human breast cancer cells in vitro and in vivo. Cancer Res 60:2673–2679, 2000[Abstract/Free Full Text]
  20. Verlinden L, Verstuyf A, Quack M, Van Camp M, van Etten E, De Clercq P, Vandewalle M, Carlberg C, Bouillon R: Interaction of two novel 14-epivitamin D3 analogs with vitamin D3 receptor-retinoid X receptor heterodimers on vitamin D3 responsive elements. J Bone Miner Res 16:625–638, 2001[Medline]
  21. Kalinski P, Schuitemaker JH, Hilkens CM, Wierenga EA, Kapsenberg ML: Final maturation of dendritic cells is associated with impaired responsiveness to IFN-gamma and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells. J Immunol 162:3231–3236, 1999[Abstract/Free Full Text]
  22. Caux C, Massacrier C, Vanbervliet B, Dubois B, van Kooten C, Durand I, Banchereau J: Activation of human dendritic cells through CD40 cross-linking. J Exp Med 180:1263–1272, 1994[Abstract/Free Full Text]
  23. Hiemstra HS, Schloot NC, van Veelen PA, Willemen SJ, Franken KL, van Rood JJ, de Vries RR, Chaudhuri A, Behan PO, Drijfhout JW, Roep BO: Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase. Proc Natl Acad Sci U S A 98:3988–3991, 2001[Abstract/Free Full Text]
  24. van Etten E, Branisteanu DD, Verstuyf A, Waer M, Bouillon R, Mathieu C: Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for classical immunosuppressants. Transplantation 69:1932–1942, 2000[Medline]
  25. Vieira PL, de Jong EC, Wierenga EA, Kapsenberg ML, Kalinski P: Development of Th1-inducing capacity in myeloid dendritic cells requires environmental instruction. J Immunol 164:4507–4512, 2000[Abstract/Free Full Text]
  26. Sallusto F, Lanzavecchia A: Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179:1109–1118, 1994[Abstract/Free Full Text]
  27. Kalinski P, Hilkens CM, Wierenga EA, Kapsenberg ML: T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol Today 20:561–567, 1999[Medline]
  28. de Jong EC, Vieira PL, Kalinski P, Kapsenberg ML: Corticosteroids inhibit the production of inflammatory mediators in immature monocyte-derived DC and induce the development of tolerogenic DC3. J Leukoc Biol 66:201–204, 1999[Abstract]
  29. Vieira PL, Kalinski P, Wierenga EA, Kapsenberg ML, de Jong EC: Glucocorticoids inhibit bioactive IL-12p70 production by in vitro-generated human dendritic cells without affecting their T cell stimulatory potential. J Immunol 161:5245–5251, 1998[Abstract/Free Full Text]
  30. Vanderheyde N, Verhasselt V, Goldman M, Willems F: Inhibition of human dendritic cell functions by methylprednisolone. Transplantation 67:1342–1347, 1999[Medline]
  31. Rea D, van Kooten C, van Meijgaarden KE, Ottenhoff TH, Melief CJ, Offringa R: Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10. Blood 95:3162–3167, 2000[Abstract/Free Full Text]
  32. Fritsche J, Moser M, Faust S, Peuker A, Buttner R, Andreesen R, Kreutz M: Molecular cloning and characterization of a human metalloprotease disintegrin: a novel marker for dendritic cell differentiation. Blood 96:732–739, 2000[Abstract/Free Full Text]
  33. Palucka KA, Taquet N, Sanchez-Chapuis F, Gluckman JC: Dendritic cells as the terminal stage of monocyte differentiation. J Immunol 160:4587–4595, 1998[Abstract/Free Full Text]
  34. Duddy ME, Dickson G, Hawkins SA, Armstrong MA: Monocyte-derived dendritic cells: a potential target for therapy in multiple sclerosis (MS). Clin Exp Immunol 123:280–287, 2001[Medline]
  35. Chomarat P, Banchereau J, Davoust J, Palucka AK: IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat Immunol 1:510–514, 2000[Medline]
  36. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV: Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238, 2000[Abstract/Free Full Text]
  37. Papiernik M, de Moraes ML, Pontoux C, Vasseur F, Penit C: Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency. Int Immunol 10:371–378, 1998[Abstract/Free Full Text]
  38. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA: B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12:431–440, 2000[Medline]

Add to CiteULike CiteULike   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Chen, G. P. Sims, X. X. Chen, Y. Y. Gu, S. Chen, and P. E. Lipsky
Modulatory Effects of 1,25-Dihydroxyvitamin D3 on Human B Cell Differentiation
J. Immunol., August 1, 2007; 179(3): 1634 - 1647.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Penna, S. Amuchastegui, N. Giarratana, K. C. Daniel, M. Vulcano, S. Sozzani, and L. Adorini
1,25-Dihydroxyvitamin D3 Selectively Modulates Tolerogenic Properties in Myeloid but Not Plasmacytoid Dendritic Cells
J. Immunol., January 1, 2007; 178(1): 145 - 153.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
I. Durinovic-Bello, S. Rosinger, J. A. Olson, M. Congia, R. C. Ahmad, M. Rickert, J. Hampl, H. Kalbacher, J. W. Drijfhout, E. D. Mellins, et al.
DRB1*0401-restricted human T cell clone specific for the major proinsulin73-90 epitope expresses a down-regulatory T helper 2 phenotype
PNAS, August 1, 2006; 103(31): 11683 - 11688.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Delgado, E. Gonzalez-Rey, and D. Ganea
The Neuropeptide Vasoactive Intestinal Peptide Generates Tolerogenic Dendritic Cells
J. Immunol., December 1, 2005; 175(11): 7311 - 7324.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
G. Penna, A. Roncari, S. Amuchastegui, K. C. Daniel, E. Berti, M. Colonna, and L. Adorini
Expression of the inhibitory receptor ILT3 on dendritic cells is dispensable for induction of CD4+Foxp3+ regulatory T cells by 1,25-dihydroxyvitamin D3
Blood, November 15, 2005; 106(10): 3490 - 3497.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. C. Gauzzi, C. Purificato, K. Donato, Y. Jin, L. Wang, K. C. Daniel, A. A. Maghazachi, F. Belardelli, L. Adorini, and S. Gessani
Suppressive Effect of 1{alpha},25-Dihydroxyvitamin D3 on Type I IFN-Mediated Monocyte Differentiation into Dendritic Cells: Impairment of Functional Activities and Chemotaxis
J. Immunol., January 1, 2005; 174(1): 270 - 276.
[Abstract] [Full Text] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
E. VAN ETTEN, O. DARDENNE, C. GYSEMANS, L. OVERBERGH, and C. MATHIEU
1,25-Dihydroxyvitamin D3 Alters the Profile of Bone Marrow-Derived Dendritic Cells of NOD Mice
Ann. N.Y. Acad. Sci., December 1, 2004; 1037(1): 186 - 192.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
M T Abreu, V Kantorovich, E A Vasiliauskas, U Gruntmanis, R Matuk, K Daigle, S Chen, D Zehnder, Y-C Lin, H Yang, et al.
Measurement of vitamin D levels in inflammatory bowel disease patients reveals a subset of Crohn's disease patients with elevated 1,25-dihydroxyvitamin D and low bone mineral density
Gut, August 1, 2004; 53(8): 1129 - 1136.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. M. Woltman and C. van Kooten
Functional modulation of dendritic cells to suppress adaptive immune responses
J. Leukoc. Biol., April 1, 2003; 73(4): 428 - 441.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Halteren, A. G.S.
Right arrow Articles by Mathieu, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Halteren, A. G.S.
Right arrow Articles by Mathieu, C.
Social Bookmarking
Add to CiteULike