Published online before print November 21, 2003
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,,1
,¶,2
* Graduate Institute of Life Sciences, Departments of
Microbiology and Immunology,
Medicine, and
|| Surgery, National Defense Medical Center, Taipei, Taiwan;
Department of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan;
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan;
¶ Department of Chemical Engineering, National Taipei University of Technology, Taiwan; and
** Division of General Surgery, Armed Force Taoyuan General Hospital, Taiwan
1 Correspondence: Department of Microbiology and Immunology, National Defense Medical Center, 161, Section 6, MinChuan East Road, Neihu, Taipei, Taiwan 114. E-mail: sytwu{at}ndmctsgh.edu.tw
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from T cells cocultured with DcR3.Fc-treated DCs was profoundly suppressed, indicating that DcR3 exerts a Th1-suppressing effect on differentiating DCs. Furthermore, adoptive transfer experiments revealed that NOD/severe combined immunodeficiency mice received DcR3.Fc-treated DCs, and subsequently, autoreactive T cells showed delayed onset of diabetes and a decrease in diabetic severity compared with mice that received normal DCs and T cells, suggesting a future therapeutic potential in autoimmune diabetes. Data from two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight analysis show an up-regulation of some proteins—such as mitogen-activated protein kinase p38 ß, cyclin-dependent kinase 6, and signal-induced proliferation-associated gene 1—and a down-regulation of the IL-17 precursor; tumor necrosis factor-related apoptosis-inducing ligand family member-associated nuclear factor-
B activator-binding kinase 1; and Golgi S-nitroso-N-acetylpenicillamine in cells treated with DcR3, further demonstrating its effect on DC differentiation and function.
Key Words: CD80 • CD86 • IFN- • proteomics
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DcR3, also known as TR6 or M68, belongs to the tumor necrosis factor (TNF) receptor superfamily and is a decoy receptor for Fas ligand (FasL) [18 ], a receptor homologous to lymphotoxins (LTs), exhibits inducible expression and competes with Herpes simplex virus glycoprotein D for the Herpesvirus entry mediator (HVEM) and is expressed by T lymphocytes (LIGHT) [19 ], and TNF-like molecule 1A (TL1A) [20 ]. Like osteoprotegerin [21 ], DcR3 lacks a transmembrane domain and is regarded as a secreted rather than a membrane-bound protein, with a molecular weight (Mw) of 35 kDa. DcR3 binds to FasL and LIGHT, thereby neutralizing the proapoptotic actions induced by the Fas-FasL interaction or inhibiting LIGHT-mediated biological effects by blocking the interaction between LIGHT and the LT-ß receptor (LTßR) or HVEM. As DcR3 is genetically amplified in colon and lung carcinomas [18 ] and is frequently overexpressed by malignant tumors arising from the pulmonary or gastrointestinal tracts [22 ], it has been postulated that DcR3 promotes tumor growth by escaping FasL- and LIGHT-mediated immunosurveillance. As in cancer patients, the DcR3 gene is also overexpressed in patients with silicosis or systemic lupus erythematosus [23 ]. As LIGHT is also expressed in DCs and acts as a costimulatory factor in priming T cell responses [13 , 24 , 25 ], we examined whether DcR3 modulates T cell-mediated immunity by interfering with the differentiation and function of DCs.
We recently demonstrated that soluble DcR3.Fc binds to CD14+ monocytes and modulates their differentiation and maturation into DCs [14 ]. The expression of human leukocyte antigen-DR, CD1a, CD40, CD54, and CD80/B7.1 was reduced in DcR3.Fc-treated DCs. However, the expression of CD86/B7.2 was up-regulated under the same conditions. It is interesting that DcR3.Fc-treated DCs caused T cell differentiation to be biased toward the T helper 2 (Th2) phenotype in allogenic mixed lymphocyte reactions (MLR) [14 ]. DCs are not only critical for the induction of primary immune responses but are also important for the induction of immunological tolerance, as well as for the regulation of some types of T cell-mediated autoimmune diseases. Therefore, in this study, we further tested whether DcR3 modulates the differentiation and function of bone marrow-derived DCs (BM-DCs) from nonobese diabetic (NOD) mice, which have been well characterized as a model of Th1-mediated autoimmune disease that resembles human insulin-dependent diabetes mellitus (IDDM).
IDDM is caused by progressive autoimmune destruction of the insulin-producing ß cells in the pancreatic islets of Langerhans. Genetic predisposition and environmental factors contribute to its pathogenesis. Extensive studies of the immunodiabetogenic mechanisms of this disease indicate that Th1 lymphocytes play an important role in the initiation and propagation of the diabetogenic process in NOD mice. Therefore, we characterized the modulatory effects of DcR3 on NOD BM-DCs, seeking to take advantage of the biased Th2 phenotype induced by DcR3.Fc-treated DCs as a potential therapy in this disease model.
Our results reveal that the expression of CD11c, CD40, CD54, and major histocompatibility complex (MHC) I-Ag7 was reduced in cells cultured with additional DcR3.Fc, compared with DCs incubated with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4, indicating that DcR3 interferes with the differentiation and maturation of BM-DCs. It is interesting that CD86 was up-regulated, and CD80 was down-regulated in DcR3.Fc-treated DCs, suggesting a modulatory potential to skew the T cell response toward the Th2 phenotype. Moreover, the proliferation of CD4+ T cells cocultured with DcR3.Fc-treated DCs was significantly reduced compared with that of T cells stimulated by normal DCs. Furthermore, the secretion of interferon (IFN)- from T cells cocultured with DcR3.Fc-treated DCs was profoundly suppressed, indicating that DcR3 has a Th1-suppressing effect on differentiating DCs. To further investigate quantitative changes in proteins differentially expressed in normal and DcR3.Fc-treated BM-DCs, we performed two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analysis. Our data show an up-regulation of some proteins, such as mitogen-activated protein kinase (MAPK) p38 ß, cyclin-dependent kinase 6, and signal-induced proliferation-associated gene 1, and a down-regulation of the IL-17 precursor, TNF-related apoptosis-inducing ligand family member-associated nuclear factor-B (NF-B) activator (TANK)-binding kinase 1, and Golgi S-nitroso-N-acetylpenicillamine (SNAP) in cells incubated with DcR3.Fc. This confirms the modulatory effects of DcR3 on DC differentiation and function. In summary, our results indicate that DcR3 may act not only as a decoy receptor for its known ligands but also as an effector molecule that modulates the differentiation and function of DCs, thus down-regulating the Th1 immune response. This modulatory effect of DcR3 in restoring or enhancing the Th2 immune response in NOD mice may provide the basis for a future therapeutic application in autoimmune diabetes.
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Purification of recombinant DcR3.Fc (rDcR3.FC) protein
DcR3.Fc protein was produced as described previously [14
]. Briefly, the open-reading frame of the human DcR3 gene was isolated by reverse transcriptase-polymerase chain reaction using the forward primer, 5'-GGAATTCAAGGACCATGAGGGCGCTG-3', and the reverse primer, 5'-GGAATTCGTGCACAGGGAGGAAGCGC-3'. The amplified product was ligated in-frame into the EcoRI-cut pUC19-immunoglobulin G (IgG)1-Fc vector containing the cDNA for human (h)IgG1 Fc. The fusion gene was then subcloned into the pBacPAK9 vector (Clontech Laboratories, Palo Alto, CA) and was cotransfected with linearized BacPAK6 DNA (Clontech Laboratories) into Sf21 cells. The supernatant from recombinant virus-infected Sf21 cells was filtered and purified on protein A-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). Bound DcR3.Fc protein was then eluted with 0.1 M citric acid buffer (pH 3.0; Sigma Chemical Co., St. Louis, MO) followed by dialysis against phosphate-buffered saline (PBS). Purified, recombinant protein was desalted on NAPTM 10 columns (Amersham Pharmacia Biotech) before use.
Western blot analysis
Purified DcR3.Fc protein (10 µg/lane) was separated on 12% polyacrylamide gels and blotted onto nitrocellulose membranes by standard procedures. The membranes were washed, incubated with anti-DcR3 (Anawrahta Biotech, Taipei, Taiwan) or anti-hIgG1 (Chemicon, El Segundo, CA) primary antibodies (Ab), washed, and then incubated with horseradish peroxidase-conjugated goat anti-mouse Ig secondary Ab (Chemicon). Enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) were used to detect positive signals.
Assessment of DcR3-mediated inhibition of activation-induced cell death
Jurkat cells were seeded in 96-well plates at a density of 105/well, stimulated with phorbol 12-myristate-13-acetate (PMA; 30 ng/ml) plus ionomycin (2 µM) overnight at 37°C, and then incubated with DcR3.Fc (10 µg/ml), Fas.Fc (10 µg/ml), or hIgG1 (10 µg/ml) for 20 h. CD4+ T cells were purified from spleen cells by magnetic cell sorting. Briefly, single-cell suspensions of NOD splenocytes were incubated with magnetic cell sorter (MACS) CD8
and B220 microbeads (Miltenyi Biotec, Germany) for 15 min at 6°C. Non-Ab-bound cells were sorted by negative selection using autoMACS (Miltenyi Biotec) according to the manufacturer’s instructions. The sorted CD4+ T cells were checked by flow cytometric analysis, and the purity was 90–95%. These freshly prepared CD4+ T cells were then added to anti-CD3 (0.3 µg/ml) Ab-coated plates and were supplied with 5 µg/ml IL-2 for 4 days. CD4+ T cells were incubated for 24 h without anti-CD3 stimulation and at a density of 5 x 105/well, were restimulated with anti-CD3 Ab (1 µg/ml). They were then incubated with DcR3.Fc (10 µg/ml), Fas.Fc (10 µg/ml), or hIgG1 (10 µg/ml) in 96-well plates for 20 h. To quantitate FasL-induced cell apoptosis and DcR3-mediated inhibition, cells were stained with propidium iodide (PI) or annexin V (PharMingen, San Diego, CA) for 15 min at room temperature. Apoptotic cells were then measured by flow cytometry using a Becton Dickinson (San Jose, CA) FACSCalibur cytometer and CellQuest software.
BM cell isolation and culture
DCs were obtained in vitro by growing BM stem cells from tibias and femurs in minimum essential medium (Gibco-BRL, UK), supplemented with 5% fetal calf serum (Gibco-BRL), 5 x 10-5 M 2-mercaptoethanol (Gibco-BRL), 5 mg/ml L-glutamine (Sigma, UK), 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco-BRL), and NaHCO3 (Sigma, UK), and containing 1000 U/ml GM-CSF and 1000 U/ml IL-4. Myeloid progenitor cells were cultured in 24-well plates at 106 cells/well. Cultures were fed every 2 days by aspirating off 75% of the medium and adding fresh medium containing cytokines. On day 4 of culture, nonadherent and loosely adherent clusters were transferred to new plates for subsequent culture, and IL-4 was added to a concentration of 3000 U/ml from this time point. In addition to this normal culture of BM-DCs from NOD mice, some BM cells were originally incubated with additional rDcR3.Fc protein at a concentration of 5 µg/ml. GM-CSF and IL-4 used in these experiments were kindly provided by Dr. Tao Mi-Hua of the Institute of Biomedical Science, Academic Sinica (Taipei, Taiwan).
Flow cytometric analysis of cell-surface markers
After 4, 6, or 8 days of culture under different conditions, NOD or BALB/c BM-DCs were harvested, washed, and resuspended at 1 x 106 cells/ml. DCs (2x105) were incubated in 100 µl buffer with fluorescein-5-isothiocyanate (FITC)-conjugated monoclonal Ab (mAb) specific for B220, DEC205, CD40, CD80, or CD86 (PharMingen), phycoerythrin-conjugated mAb specific for CD11c or CD54, or allophycocyanin-conjugated mAb for CD86 and the appropriate isotype-matched controls for 30 min on ice. The NOD-specific MHC class II Ab used in the study was anti-I-Ag7 clone 10-2.16 (purchased from American Type Culture Collection, Manassas, VA), and subsequent staining was performed using FITC-conjugated goat anti-mouse IgG Ab (PharMingen).
T cell proliferation assay
CD4+ T cells were purified as described above. Enriched CD4+ T cells (1x105) were seeded in triplicate in a 96-well flat-bottom plate (Falcon, Becton Dickinson) together with titrated numbers of normal or DcR3.Fc-treated BM-DCs. In a glutamate decarboxylase 65 (GAD65)-specific T cell proliferation assay, the synthetic GAD peptides (p247–266) were added in culture wells at the concentration of 15 µg/ml. After 3 days, cell cultures were pulsed with 1 µCi/well 3H-methyl thymidine (Amersham Pharmacia Biotech) overnight for 16 h. The plates were then harvested onto glass fiber, and the incorporated 3H-methyl thymidine was detected with TopCount (Packard, PerkinElmer, Boston, MA).
Cytokine assay
The concentrations of cytokines IL-4, IL-10, IL-13, and IFN- secreted by CD4+ T cells cocultured with normal BM-DCs or DcR3.Fc-treated BM-DCs were determined by enzyme-linked immunosorbent assay (ELISA). Briefly, the cultured supernatants were harvested after 48 h, and cytokine ELISAs were performed using purified mAb-coated plates. All procedures followed the standard protocols provided by R&D Systems (Minneapolis, MN). Cytokine concentrations were read with an MRX microplate reader (Dynex Technologies, Chantilly, VA) at 450 nm.
Flow cytometric detection of DcR3.Fc-binding on BM-DC
To detect DcR3.Fc-binding on NOD BM-DC, cells cultured for 6 days after IL-4/GM-CSF treatment were harvested and washed twice in PBS (pH 7.4) containing 1% bovine serum albumin and 0.01% sodium azide. The samples were then incubated with different biotin-conjugated proteins including DcR3.Fc-biotin, LTßR.Fc-biotin, control hIgG1-biotin, or anti-FasL Ab (PharMingen) for 30 min. After two additional washes with PBS buffer, the samples were incubated with streptavidin conjugated with allophycocyanin (PharMingen) for 25 min at 4°C. After a final wash, the cells were analyzed by flow cytometer (Becton Dickinson FACSCalibur with CellQuest software).
Adoptive transfer of DCs and T cells
To investigate the therapeutic potential of DcR3.Fc-treated DCs in autoimmune diabetes, three groups—regular, hIgG-treated, or DcR3.Fc-treated BM-DCs (2x105 cells/group)—on day 8 of culture were injected intravenously (i.v.) into 8-week-old NOD/SCID. One week after DC transfer, mice subsequently received 2 x 107 splenocytes isolated from 12-week-old NOD mice. After T cell transfer, mice were then checked for blood sugar levels and glycosuria every week using Optium detection kit (Abbott Laboratories, MediSense Products, Abbott Park, IL) and Chemstrip UG (Boehringer Mannheim, Indianapolis, IN), respectively. When animals were positive (
250 mg/dl) in two consecutive blood tests, they were scored as diabetic.
2D gel electrophoresis and image analysis
BM-DC cell pellets (5x106 cells) were solubilized in lysis buffer containing 7 M urea (Boehringer Mannheim, Germany), 2 M thiourea (Aldrich, Milwaukee, WI), and 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS; J. T. Baker, Phillipsburg, NJ). After sonication, 1 mg total protein was loaded onto immobilized pH gradient (IPG) gel strips (pH 3–10, 18 cm long; Amersham Pharmacia Biotech) that had been rehydrated overnight before use in a solution of 7 M urea, 2 M thiourea, 4% CHAPS, 4 mM Tris base, 2% IPG ampholyte, 65 mM dithioerythritol (DTE), and 0.0002% bromophenol blue. For separation in the first dimension, isoelectric focusing was performed using the IPGphor system (Amersham Pharmacia Biotech) at 20°C and 8000 V for a total of 75 kVh. After isoelectric focusing, the IPG strips were equilibrated for 15 min in equilibration solution [50 mM Tris-HCl, pH 8.8, 6 M urea, 2% sodium dodecyl sulfate (SDS), 30% glycerol, 2% DTE] and were then attached with 0.5% agarose to the top of a vertical 10–15% linear gradient SDS-polyacrylamide gel. Second-dimensional electrophoresis was performed with a Protean II multicell (Bio-Rad, Hercules, CA) at 45 mA per gel for 5 h until the bromophenol blue reached the bottom of the gel. The gels were fixed in 10% methanol and 7% acetic acid for 30 min, stained in 350 ml Sypro Ruby Protein gel-stain solution overnight, and then soaked in deionized water for 20 min to wash residual dye from the polyacrylamide matrix. The developed gels were digitally scanned as 2D images using a Typhoon 9200 fluorescence image scanner (Amersham Pharmacia Biotech) and were then analyzed using ImageMaster software (Amersham Pharmacia Biotech) to detect and quantify protein spots automatically. Intensity levels were normalized between gels as a proportion of the total protein intensity detected for the entire gel. Normalized protein intensity data for the matched spots with a twofold difference were exported to Gel Report (Amersham Pharmacia Biotech).
In-gel digestion and MALDI-TOF-mass spectrometry (MS)
From the 2D gel analysis of control and induced samples, MS selected differentially expressed proteins for further identification. These spots were cut from the 2D gels, sliced into 1–2 mm2 pieces, and then destained three times with 23 mM ammonium bicarbonate buffer (pH 8.0) in 50% acetonitrile for 15 min. The gel pieces were dehydrated in 100% acetonitrile for 5 min and then dried for 20 min in a vacuum centrifuge. Enzymatic digestion was achieved by adding 15 µl trypsin in 25 mM ammonium bicarbonate to a final concentration of 0.0225 µg per sample at 37°C for 16 h. Peptide fragments were extracted twice with 50 µl 50% acetonitrile-0.1% trifluoroacetic acid. Acetonitrile was removed by centrifugation under vacuum, and the peptides were concentrated using C18 Zip-Tip, eluted with 2 µl 100% acetonitrile, and directly spotted onto the sample plate of a MALDI-TOF-MS. Finally, 0.5 µl -cyano-4-hydroxycinnamic acid (10 mg/ml) was applied to each spot, and the spots were air-dried at room temperature before mass spectra were acquired (M@LDITM, Micromass, Manchester, UK). The resultant peptide masses were matched with the theoretical peptide masses of all proteins from all species in the SWISS-PROT database using Masslynx 3.4 software. Peptide mass mapping is particularly successful for the identification of proteins, as described in the literature [26
27
28
], so we chose this method to identify proteins. Our protein selection criteria were a match of at least four fragments from a single 2D gel spot against a single protein sequence entry in the database; a high coverage value; and a sequence of mouse origin. This protein was then considered as a candidate [29
].
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Figure 1. Characterization of DcR3.Fc protein. (A) Western blot analysis of DcR3.Fc protein. rDcR3.Fc fusion protein was produced from a baculoviral expression system and analyzed by a Western blot probed with anti-DcR3 Ab (lane 2) or anti-hIgG Ab (lane 3). Lane 1 is a negative control probed with anti-LTßR Ab. (B and C) Inhibition of activation-induced apoptosis in Jurkat cells and CD4+ T cells by DcR3.Fc. Jurkat cells (105/well) were stimulated with PMA (30 ng/ml) and ionomycin (2 µM) for 20 h (B), and CD4+ T cells (5x105/well) were activated with immobilized anti-CD3 (0.3 µg/ml) for 5 days (C) in 96-well microtiter plates, followed by restimulation for 20 h in the presence of Fas.Fc (10 µg/ml), DcR3.Fc (10 µg/ml), or hIgG1 (10 µg/ml). PI staining and flow cytometric analysis determined percentage cell death.
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Figure 2. Microscopic examination of BM-DCs from NOD mice. BM cells from 8-week-old NOD mice were cultured with IL-4 and GM-CSF (A and C) or incubated with additional DcR3.Fc (B and D). On day 8, cells were observed microscopically (original magnification, 40x, A and B; original magnification, 200x, C and D). Expression of DEC205 on cultured cells was further characterized by flow cytometic analysis (E, cells cultured with IL-4/GM-CSF; F, cells cultured with additional DcR3.Fc).
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Table 1. Identification by MS of the Protein Spots During DcR3.Fc-Treated DCs
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Figure 3. Flow cytometric analysis of BM-DCs generated in the presence or absence of DcR3.Fc protein. Flow cytometric analysis was used to characterize the expression of CD11c (A), CD80 (B), CD86 (C), MHC class II I-Ag7 (D), CD40 (E), and CD54 (F) by DCs treated with (open histograms) or without (solid histograms) DcR3.Fc at day 8 of culture. Composite data are from two of four representative experiments. Compared with DCs incubated only with GM-CSF and IL-4, the expression of CD11c (A), CD40 (E), CD54 (F), CD80 (B), and MHC I-Ag7 (D) was reduced in cells cultured with additional DcR3.Fc, but the expression of CD86 (C) in these cells was up-regulated.
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Table 2. MFI of Surface Markers on Regular and DcR3.Fc-Treated DCs*
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, and anti-FasL Ab do not bind these cells. This suggests that DcR3.Fc interacts with a surface molecule on CD14+ monocytes that is distinct from LIGHT, the membrane form of LT, and FasL. To address this finding further, we used biotin-conjugated DcR3.Fc, LTßR.Fc, or Fas.Fc fusion proteins as well as anti-FasL and anti-LIGHT Ab to stain BM-DCs from NOD mice. As shown in Figure 4A
, strong fluorescence was detected in cultured BM-DCs stained with DcR3.Fc-biotin and subsequently with streptavidin-allophycocyanin, whereas LTßR.Fc-biotin only detected a weak signal on those cells. No signal was detected on cells stained with Fas.Fc-biotin (data not shown) or anti-FasL (Fig. 4B)
, suggesting that FasL plays no role in the DcR3-driven differentiation and maturation of BM-DCs. In previous studies, we demonstrated that LIGHT is expressed on the surfaces of immature human DCs but is lost from mature DCs and is barely detectable by LTßR.Fc [14
]. Whereas the strong signal did not change in cells stained with DcR3.Fc-biotin when those cells were treated with LPS, the weak signal detected in cultured cells stained with LTßR.Fc-biotin disappeared (data not shown). From these results, we conclude that the molecule detected by DcR3.Fc on mature DCs is distinct from LIGHT, indicating that DcR3 binds to LIGHT and an unidentified novel ligand in immature DCs.
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Figure 4. DcR3.Fc-binding on BM-DCs from NOD mice. Flow cytometric analysis of DcR3.Fc-binding on BM-DCs cultured with GM-CSF and IL-4 for 6 days. (A) The primary staining reagents were DcR3.Fc-biotin (solid line), LTßR.Fc-biotin (dashed line), or hIgG1-biotin (solid histograms), and the secondary reagent was streptavidin conjugated with allophycocyanin. (B) DCs were cultured for 6 days and stained with anti-FasL Ab (open histogram) or Ig-matched control Ab (solid histogram).
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from naïve CD4+ T cells cocultured with DcR3.Fc-treated BM-DCs. The secretion of IFN- from T cells stimulated with BM-DCs that had been pretreated with DcR3.Fc is much lower than that from T cells incubated with normally cultured DCs (Fig. 6A
). This is also consistent with our previous finding that CD14+ monocyte-derived DCs treated with DcR3.Fc down-regulated the secretion of IFN- from CD4+ T cells in allogenic MLR. Despite the presence of IFN-, the secretion of IL-10 (Fig. 6B)
, IL-13 (Fig. 6C)
, and IL-4 (Fig. 6D) from T cells incubated with the two types of DCs did not differ. Unexpectedly, the secretion of IL-4 from T cells stimulated with DcR3.Fc-treated DCs did not increase, suggesting an intrinsic defect in IL-4 production in NOD mice. To investigate this point further, we performed the similar experiment in a non-NOD mouse strain. Results shown in Figure 7D
indicated that the secretion of IL-4 from CD4+ T cells incubated with DcR3.Fc-treated DCs in a BALB/c background is significantly increased (P<0.01), demonstrating that the failure of IL-4 up-regulation in NOD mice is an intrinsic feature of this strain.
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Figure 5. Modulatory effect of DcR3.Fc-treated DCs on T cell proliferation. Splenic CD4+ T cells were stimulated with irradiated syngenic splenocytes (iSpl + T; A, lane 2), normal cultured DCs (iDC + T; A, lane 3), or DcR3.Fc-treated DCs [iDC(DcR3) + T; A, lane 4] for 3 days. Similar cultures were set up in the presence of GAD65 peptide at the concentration of 15 µg/ml (B). [3H]Thymidine was added to the cultures during the last 16 h before the cells were harvested on day 3. Results are from one of three representative experiments conducted in triplicate (mean±SD).
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Figure 6. Modulatory effects of DcR3.Fc-treated DCs on cytokine production in a NOD strain. The secretion of IFN- (A), IL-10 (B), IL-13 (C), and IL-4 (D) by NOD CD4+ T cells stimulated with concanavalin A (conA; lane 1) or cocultured with syngenic, irradiated, normal DCs (iDC; lane 2), DcR3-Fc-treated DCs [iDC(DcR3); lane 3], or splenocytes (isplenocyte; lane 4) was detected by ELISA on day 3. Data represent one of three independent experiments conducted in triplicate (mean±SD).
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Figure 7. Modulatory effects of DcR3.Fc-treated DCs on cytokine production in BALB/c strain. The secretion of IFN- (A), IL-10 (B), IL-13 (C), and IL-4 (D) by BALB/c CD4+ T cells cocultured with syngenic, irradiated, normal DCs (iDC; lane 1), DcR3-Fc-treated DCs [iDC(DcR3); lane 2], or splenocytes (isplen; lane 3) were detected by ELISA on day 3. Data represent one of three independent experiments conducted in triplicate (mean±SD).
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secretion by NOD T cells may provide a potential basis for clinical application, we performed DC and T cell transfer experiments. NOD/SCID mice that received DcR3.Fc-treated DCs and subsequently, autoreactive T cells showed delayed onset of diabetes and a decrease in diabetic frequency compared with NOD/SCID mice that received normal DCs and T cells (Fig. 8
, P<0.05). Forty-one days after T cell transfer, 60% of mice that originally received regular DCs developed diabetes. However, none of the mice that originally received DcR3.Fc-treated DCs and subsequently, T cells developed diabetes at this time. All mice that received regular DCs developed diabetes 61 days after T cell transfer, and less than 40% of mice that received DcR3.Fc-treated DCs developed diabetes. This result suggests a future therapeutic potential of DcR3-modulated DCs in autoimmune diabetes.
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Figure 8. Adoptive transfer of DCs and T cells. Three groups of BM-DCs—regular (DC), hIgG-treated (IgG), or DcR3.Fc-treated (DcR3; 2x105 cells/group)—on day 8 of culture were injected i.v. into 8-week-old NOD/SCID (number of mice for each group: 10, 10, and 12, respectively). One week after DC transfer, mice subsequently received 2 x 107 splenocytes isolated from 12-week-old NOD mice. After T cell transfer, mice were then checked for glycosuria every week. When animals were positive (250 mg/dl) in two consecutive urine tests, they were scored as diabetic.
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Figure 9. 2D profiles of NOD BM-DCs. DcR3.Fc-treated (left panel) and normal BM-DCs (right panel) were harvested on day 8 of culture and analyzed by 2D gel electrophoresis. Twenty-five spots, indicated by black arrows (left panel), represent the proteins up-regulated during DcR3.Fc treatment. Ten spots, indicated by white arrows (right panel), represent the proteins down-regulated in DcR3.Fc-treated DCs. These results are representative of three independent experiments.
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The data presented in Figure 4
suggest that a novel ligand is expressed on DCs other than LIGHT and FasL. Based on the following reasons, we could rule out the possibility that DcR3.Fc-biotin may just be a better reagent than the LTßR.Fc-biotin and further deduce that a novel ligand for DcR3 is expressed on DCs: (1) In this study, we found that DcR3.Fc but not LTßR.Fc or hIgG1 modulates the expression of several surface molecules on BM-DCs. As DcR3.Fc and LTßR.Fc can bind LIGHT, however, only DcR3.Fc modulates DC differentiation, suggesting a biological function-distinct, and LIGHT-independent receptor for DcR3. (2) We previously demonstrated that DcR3.Fc but not LTßR.Fc binds freshly isolated CD14+ monocytes and that anti-LIGHT, anti-LT, and anti-FasL Ab do not bind these cells. This suggests that DcR3.Fc interacts with a surface molecule on CD14+ monocytes that is distinct from LIGHT, the membrane form of LT, and FasL. (3) We also demonstrated that LIGHT is expressed on the surfaces of immature human DCs but is lost from mature DCs and is barely detectable by LTßR.Fc [14
]. Whereas the strong signal did not change in cells stained with DcR3.Fc-biotin when those cells were treated with LPS, the weak signal detected in cultured cells stained with LTßR.Fc-biotin disappeared, indicating that a LIGHT-independent, novel molecule is expressed on culture DCs. (4) It is also unlikely that the DcR3.Fc-biotin may just be a better reagent than the LTßR.Fc-biotin (data presented in Fig. 4
), as the data presented in other groups demonstrated that LTßR.Fc and DcR3.Fc bind LIGHT with almost the same fluorescence intensity [19
].
The effects of DcR3.Fc on DC differentiation and function in human CD14+ monocytes and NOD BM cells are almost identical in terms of cell-surface marker expression and T cell-stimulating ability, demonstrating the general modulatory potential of this protein on DC differentiation and function. One of the most striking effects of DcR3.Fc on DC differentiation is the up-regulation of CD86/B7.2 and the down-regulation of CD80/B7.1. Cytokines such as IFN- up-regulate CD80/B7.1 and CD86/B7.2 expression, whereas IL-10 down-regulates both proteins [33
, 34
]. Therefore, the unique feature of DcR3-mediated up-regulation of CD86/B7.2 with simultaneous down-regulation of CD80/B7.1 might provide an invaluable tool with which to dissect the mechanisms underlying the differential regulation of CD80/B7.1 and CD86/B7.2 expression. Moreover, it has been demonstrated that although CD80/B7.1 and CD86/B7.2 equivalently costimulate IL-2 and IFN- production, CD86/B7.2 preferentially induces greater IL-4 production than CD80/B7.1 [35
, 36
]. However, in this study, the secretion of IL-4 from T cells incubated with DcR3.Fc-treated DCs in the NOD system was much lower than that from T cells stimulated with DcR3.Fc-treated DCs in the human monocyte system. We previously demonstrated that CD14+ monocyte-derived DCs treated with DcR3.Fc suppressed CD4+ T cell proliferation in allogenic MLR and profoundly up-regulated IL-4 secretion from CD4+CD45RA+ T cells. NOD mice spontaneously develop autoimmune diabetes, which is characterized by a progressive insulitis followed by selective destruction of ß cells in pancreatic islets. Extensive evidence suggests that effector CD4+ T cells, which preferentially secrete IFN- and TNF- (Th1 cells), mediate ß cell destruction, at least predominantly [37
]. Therefore, the inability of NOD DCs to trigger Th2 effector cells, which secrete IL-4, may contribute to the immunopathogenic process and may have an impact on the breakdown of self-tolerance in autoimmune diabetes. Nevertheless, the strong effects of DcR3.Fc-treated DCs on the down-regulation of IFN- secretion by NOD T cells provide a potential basis for clinical application. To test this idea further, we performed DC transfer experiments. NOD/SCID mice that received DcR3.Fc-treated DCs and subsequently, autoreactive T cells showed delayed onset of diabetes and a decrease in diabetic severity compared with NOD/SCID mice that received normal DCs and T cells (Fig. 8)
. These data are generally consistent with the hypothesis that immature DCs or cytokine (e.g., IL-10)-treated DCs, which express lower levels of MHC proteins or costimulatory molecules and higher levels of the programmed cell death-1 ligand (PD-L1), have a balance of stimulatory versus inhibitory molecules that favors inhibition. They are, therefore, more responsible for the induction of tolerance [38
, 39
]. The potential role of PD-L1 and its ligands in DcR3-treated, DC-mediated immunomodulation in NOD mice is currently under investigation in our laboratory.
DC differentiation from naïve BM precursor cells is a tightly regulated and highly complicated process influenced by many environmental cytokines. To systematically investigate how DcR3 modulates the differentiation of BM-DCs and their subsequent T cell-stimulatory potential, we took advantage of recently developed proteomic technology to profile changes in gene expression in DcR3.Fc-treated BM-DCs. DCs acquire their function during differentiation, which occurs through the programmed expression of specific proteins. To better identify specific genes involved in the DcR3-mediated modulation of DCs, we performed 2D gel electrophoresis and MALDI-TOF-MS analysis. This approach, which relies on the quantitative analysis and identification of proteins by proteomic techniques, has additional advantages over the quantitative analysis of mRNA by oligonucleotide microarrays: Proteins represent the most functional compartment of a cell, and the information obtained at the protein level cannot be predicted simply by examining expression at the RNA level; and the proteomics approach can identify post-translational modifications, which may regulate important functions of these proteins. In this study, we identified almost 7% of the proteins shown to be differentially regulated in the two DC groups. These differentially regulated genes were predominantly related to cell-growth control, signaling, the regulation of the immune response, or lipid metabolism. They include some genes previously reported to be modulated during DC differentiation. A large number of additional genes not previously reported in DCs were also expressed during DcR3 modulation.
Immature DCs move from the circulating blood to inflammatory tissues where they take up and process Ag. These cells then migrate to the draining lymphoid organs and become mature with the up-regulation of costimulatory and MHC molecules, which subsequently prime naïve T cells. We previously identified a large number of genes encoding proteins involved in cell adhesion and motility and Ag uptake, processing, and presentation, which are regulated during DC differentiation [40
]. For example, the expression of CD11a/lymphocyte function-associated antigen-1a, syndecan 2, CD44E, and presenilin 1, which are involved in cell adhesion and motility, is down-regulated in mature DCs. In contrast, expression of the Fc receptor, MHC class II proteins, and heat shock protein 73, which represent Ag-presentation capacity, is up-regulated in mature DCs, as expected. It is interesting that the expression of most genes involved in cell adhesion and motility or Ag uptake, processing, and presentation, which are regulated during DC differentiation and maturation, did not differ between normal and DcR3.Fc-treated DCs, emphasizing the phenotypic similarity of these two DC groups. However, some genes encoding proteins involved in cell growth, signaling, and metabolism were differentially regulated in DCs after DcR3.Fc treatment. MAPK p38 ß is a kinase-phosphorylated protein, which is induced in response to inflammation, stress, and environmental cytokines. Data from kinase assays in our laboratory also demonstrated a significant increase in MAPK p38 activity in DcR3.Fc-incubated DCs treated with LPS (data not shown), suggesting a critical role for this kinase in DC differentiation during DcR3 modulation. As MAPK p38 affects cell-cycle progression and differentiation, and expression of this protein is up-regulated in DCs treated with LPS or other stimuli [41
], the up-regulation of this protein in DcR3-modulated DCs may indicate that DcR3 plays a regulatory role in DC differentiation. In addition to MAPK p38, vimentin, GAPDH, cyclin-dependent kinase 6, signal-induced proliferation-associated gene 1, and Ras-GTPase-activating protein SH3 domain-binding protein, which is up-regulated in DcR3.Fc-treated DCs, are all involved in the cell cycle, differentiation, or growth control, indicating a significant effect of DcR3 on DC differentiation and maturation. mIL-17, one of the 10 down-regulated proteins detected in DcR3.Fc-treated DCs, was initially cloned by subtractive hybridization from an activated T cell hybridoma and designated mCTL-associated Ag 8 [42
]. IL-17 induces epithelial, endothelial, and fibroblastic cells to activate NF-B and to produce proinflammatory cytokines such as IL-6, IL-8, GM-CSF, and prostaglandin E2 [43
, 44
]. It can also induce fibroblasts to support better the growth and differentiation of CD34+ progenitor cells. Therefore, IL-17 is considered to be an inducer of stromal cell proinflammatory and hematopoietic cytokines. Down-regulation of this protein in DcR3.Fc-treated DCs further supports the proposition that DcR3 is negatively modulated in the DC-mediated immune response.
Extensive evidence defines DCs as principal, professional APCs involved in T cell priming. This evidence includes several experiments: On one hand, DCs were compared directly with other APCs, such as B cells and monocytes, in terms of their strong ability to prime alloreactive, naïve TCR transgenic T cells in vitro or to significantly activate and expand Ag-specific, naïve precursors from polyclonal populations [45 ]. Consistent with these results, our study reveals that NOD BM-DCs have a much stronger stimulatory effect on naïve syngenic CD4+ T cells than on conventional APCs. Conversely, injection of Ag-loaded DCs also induced potent, primary CD4+ and CD8+ T cell responses in vivo [46 , 47 ]. Furthermore, DCs destroy neonatal T cell tolerance [48 ] and peripheral tolerance against soluble Ag [49 ], transplantation Ag [50 ], peripheral tissue Ag [51 , 52 ], and tumor Ag [53 , 54 ]. However, accumulating data also indicate that DCs are involved in central and peripheral tolerance. The presence of DCs in the thymic medulla suggests that they may participate in the establishment of central tolerance. This point was supported by Brocker et al. [55], who demonstrated that the exclusive expression of MHC class II proteins in DCs was sufficient to delete Vß-specific T cells through a retrovirally encoded super-Ag. Moreover, several studies reported that thymic [56 ] and splenic DCs [57 58 59 ], in addition to B cells, were involved in Ag presentation after i.v. injections of high doses of tolerogenic Ag. Furthermore, mature DCs from the T cell areas of lymph nodes present self-Ag at the periphery [60 , 61 ], indicating an important role for these cells in the maintenance of tolerance. Finally, DCs also participate in the induction of tolerance against liver allografts [62 ]. Therefore, the newly identified role of DcR3 in the differentiation and function of BM-DCs in NOD mice further confirms the action of cytokine-modulated DCs in the induction and maintenance of tolerance. This effect of DcR3 in modulating the suppression of Th1 and the restoration or enhancement of the Th2-immune response in NOD mice may provide the basis for future therapeutic applications in autoimmune diabetes.
Received March 25, 2003; revised October 6, 2003; accepted October 21, 2003.
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-stimulated monocytes: a potential mechanism for amplification of T cell activation through the CD28 pathway Cell. Immunol. 137,429-437[CrossRef][Medline]B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells Blood 96,1039-1046This article has been cited by other articles:
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