Skip Navigation


Rheumatology Advance Access originally published online on April 4, 2006
Rheumatology 2006 45(10):1230-1237; doi:10.1093/rheumatology/kel106
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
45/10/1230    most recent
kel106v1
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 ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Tzeng, T.-C.
Right arrow Articles by Chiang, B.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tzeng, T.-C.
Right arrow Articles by Chiang, B.-L.
Related Collections
Right arrow Experimental Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dendritic cells pulsed with apoptotic cells activate self-reactive T-cells of lupus mice both in vitro and in vivo

T.-C. Tzeng*, J.-L. Suen1,* and B.-L. Chiang2

Graduate Institute of Immunology, College of Medicine, National Taiwan University, 1Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung and 2Department of Pediatrics, College of Medicine, National Taiwan University, Taiwan, Republic of China.

Correspondence to: B.-L. Chiang, Department of Pediatrics, National Taiwan University Hospital, No. 7, Chung-Shan S. Road, Taipei, Taiwan, ROC. E-mail: gicmbor{at}ha.mc.ntu.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 
Objectives. Systemic lupus erythematosus (SLE) is characterized by the presence of autoantibodies (autoAbs) directed against the nuclear structure. Previous studies have demonstrated that dendritic cells (DCs) can process and present self-antigens (Ags) from apoptotic cells (ACs) in lupus. However, there is no direct evidence demonstrating that ACs provide self-Ags, such as histones, to stimulate autoreactive T-cells in lupus.

Methods. AC-pulsed bone marrow-derived DCs (AC-BMDCs) were used to stimulate autoreactive T-cells in vitro and in vivo.

Results. In our study, we found that AC-BMDCs could induce the proliferation of CD4+ T-cells from unprimed NZB x NZW F1 (BWF1) mice, which spontaneously develop SLE, but not CD4+ T-cells, from non-autoimmune DBA-2 x NZW F1 (DWF1) mice. In addition, AC-BMDCs could induce significant proliferative responses to certain histone peptide-specific T-cells. Furthermore, these AC-BMDCs could induce a considerable anti-DNA Ab response in vivo after adoptive transfer into DWF1 mice, suggesting that AC-BMDCs can break tolerance in normal mice and initiate an autoimmune response.

Conclusion. Our study provides a direct link between self-epitopes from ACs presented by DCs and autoreactive T-cell activation, and demonstrates that ACs are critical for the induction of autoimmunity in vivo.

KEY WORDS: Apoptosis, Autoantigens, CD4+ T-cell, Dendritic cell, SLE


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 
Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease characterized by the production of autoantibodies (autoAbs) directed against ubiquitously expressed self-antigens (Ags). The hallmark of SLE is the production of autoAbs directed against nuclear components, such as double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) and histones as well as nucleosomes. It is known that during the progression of SLE, the serum level of anti-nucleosome and anti-DNA Abs correlates with the severity of the disease [1]. In addition, nucleosomes have been found in the circulation of patients with SLE [2]. Previous studies have shown that such autoAgs can be presented on the surface of apoptotic bodies, while others accumulate inside apoptotic bodies [3]. In addition, the previous work suggested that SLE might be a result of increased apoptotic neutrophils or impaired clearance of apoptotic cells (ACs) by macrophages [4]. Many studies have shown that defects in the clearance of ACs may underlie SLE. For example, characteristic autoAbs and lupus-like pathology arise in mice lacking the complement protein C1q, which binds ACs [5]. Similarly, mice with deficiencies in the serum amyloid P or mutations in the Mer tyrosine kinase develop autoAbs to DNA and exhibit symptoms of autoimmune diseases [6, 7]. Therefore, ACs may be the major reservoir of autoAgs in lupus.

Blanco et al. [8] have demonstrated that serum interferon-alpha (IFN-{alpha}) can induce the differentiation of monocytes to dendritic cells (DCs) in SLE patients. Such DCs might be able to efficiently uptake ACs and nucleosomes present in the blood of SLE patients. Such self-Ag presented by DCs could subsequently activate autoreactive T-cells, which in turn provide help for B-cells in recognizing nuclear autoAgs. A previous study from our laboratory has shown that nucleosome-pulsed DCs can present histone epitopes to stimulate autoreactive CD4+ T-cells from NZB x NZW F1 (BWF1) mice [9]. However, there is no direct evidence demonstrating that these self-Ags derived from ACs could stimulate autoreactive T-cells in lupus. To address this question in this study, we used bone marrow-derived DCs (BMDCs) pulsed with ACs to test for the presence of CD4+ T-cells capable of recognizing ACs in vitro. We also intravenously injected BMDCs pulsed with ACs into non-autoimmune mice to further evaluate the DC-induced immune response directed against ACs.

The results of this study show that BMDCs are capable of processing and presenting ACs, resulting in the stimulation and proliferation of histone peptide-specific CD4+ T-cells from unprimed BWF1 mice in vitro. In addition, AC-pulsed BMDCs (AC-BMDCs) are able to induce persistent Ab responses to ds and ssDNA in normal mice in vivo. Both Ab (IgG) and complement C3 were deposited in the glomeruli of the kidneys in mice immunized with AC-BMDCs. Results from our study will help in understanding how pathogenic autoimmune responses develop in spontaneous SLE.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 
Mice
Female BWF1 mice were purchased from Jackson Laboratories (Bar Harbor, ME). DBA-2 x NZW F1 (DWF1) mice are a non-autoimmune strain with identical major histocompatibility complex (MHC) class II molecules to BWF1 (H-2d/u) mice. At the age of 6–8 weeks, female BWF1 mice and DWF1 mice were used as the source of BMDCs. The young mice were obtained from and maintained by the Animal Center of National Taiwan University in a pathogen-free facility. Studies were performed in accordance with the institutional animal research committee guidelines.

Generation of DCs from bone marrow cells
BMDCs were prepared as described previously [10]. Briefly, DCs were generated from bone marrow cells cultured with murine recombinant granulocyte monoctye-colony-stimulating factor (GM-CSF, 750 U/ml) and interleukin-4 (IL-4, 1000 U/ml) (Pepro Tech Inc. Rocky Hill, NJ) for 4 or 6 days. ACs were added to the BMDC cultures on day 4 or day 6. After 48 h, non-adherent cells were collected and washed extensively to remove the free apoptotic bodies. CD11c+ DCs were positively selected by anti-CD11c-coated magnetic microbeads (Miltenyi Biotec, Auburn, CA). The purity of CD11c+ DCs was (>93%) analysed by flow cytometry, examining the expression of MHC class II, B7-1, B7-2 and CD11c.

Generation of ACs
Thymocytes were retrieved from 6- to 10-week-old mice. ACs were generated by treating single-cell suspensions of thymocytes in a medium with dexamethasone 1.2 x 10–6 M for 12–15 h. We used fluorescein isothiocyanate (FITC)-labelled annexin V (BD PharMingen, San Diego, CA) to evaluate the exposure of phosphatidylserine and DNA-labelled 7-amino-actinomycin D (7-AAD) (BD PharMingen) to assess plasma membrane integrity. Cells were analysed by flow cytometry, and the double-positive cells were >82% after treating with dexamethasone for 12–15 h. For the phagocytosis assay, thymocytes were stained with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular probe, Inc., Eugene, OR) or 7-AAD before treatment with dexamethasone.

Phagocytosis assay
The phagocytosis of ACs was quantified by flow cytometry. CFSE or 7-AAD-labelled ACs (2 x 106 per well) were co-cultured with day-4 or day-6 DCs (2 x 105 per well) for 6 h. In the CSFE-labelled experiment (Fig. 1A), the cells were harvested and stained with 7-AAD to exclude the contamination of dead cells and AC-bound BMDCs. In another experiment, BMDCs were further purified by anti-CD11c-coated magnetic microbeads (Miltenyi Biotec). Purified BMDCs were stained with fluorescence-conjugated anti-MHC class I or anti-MHC class II Ab (BD PharMingen), as indicated in Fig. 1B. Phagocytosis was quantified by flow cytometry as the percentage of double-positive cells.


Figure 1
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1. Phagocytosis of ACs by BMDCs. (A) CFSE-labelled ACs were co-cultured with day-4 or day-6 BMDCs for 6 h at a ratio of 20:1. Cells were harvested and stained with PE-anti-I-Ad monoclonal Ab and 7-AAD. Shown here are 7-AAD negative cells analysed for PE-anti-I-Ad and CFSE staining. (B) Day-4 BMDCs were co-cultured with 7-AAD-labelled ACs for 6 h. CD11c+ BMDCs were further purified and stained with FITC-conjugated anti-MHC class I or class II monoclonal Ab. The MHC class I (left) or MHC class II (right) positive cells gated from AC-BMDCs were used to analyse the ratio of BMDCs engulfing ACs (solid line). The dotted line indicates the BMDCs pulsed with ACs without 7-AAD staining. (C) Confocal microscopy analysis of AC-BMDCs. ACs (arrowhead) were stained with CFSE (green colour), and day-4 BMDCs (arrow) were stained with anti-MHC class II Ab conjugated to PE (red colour).

 
Phagocytosis of ACs was also visualized by confocal microscopy. CFSE-labelled ACs were incubated with BMDCs for 6 h, prepared on a slide with a cytocentrifuge (Cytospin), and fixed in acetone. The slides were stained with 20 µg/ml of phycoerythrin (PE)—anti-I-Ad (BD PharMingen) for 90 min at 37°C, and mounted with 90% glycerol in phosphate-buffered saline (PBS), and observed under a Confocal Spectral Microscope (Leica Microsystems, Wetzlar, Germany).

Cytokine secretion by BMDCs
For cytokine secretion, day-4 or day-6 BMDCs (2 x 105 per well) were co-cultured with an increasing number of syngeneic ACs (1 x 106, 2 x 106, 4 x 106 per well). BMDCs stimulated with lipopolysaccharide (LPS) (1 µg/ml) were used as the positive control. The supernatants were collected after 24 and 48 h and assayed for IL-12p70, IL-12p40, IL-10 and transforming growth factor-beta (TGF-β). The concentration of cytokines in culture supernatant was detected by ELISA (R&D, Minneapoils, MN).

T-cell proliferation assays
All of the BWF1 mice used in this experiment developed anti-dsDNA IgG. CD4+ T-cells were positively selected from splenocytes by anti-CD4-coated magnetic microbeads (Miltenyi Biotec). Day-4 and day-6 BMDCs from young BWF1 mice or DWF1 mice were incubated with syngeneic ACs at different ratios for 48 h. Purified CD4+ T-cells (1–2 x 105 per well) were co-cultured with purified CD11c+ BMDCs (2500–7500 per well) in the presence or absence of anti-I-Ad/Ed (2G9, BD PharMingen) or anti-I-Ak (clone:11-5.2, BD PharMingen) monoclonal Ab for 4–5 days. During the last 4–6 h of culture, 1 µCi of [3H]thymidine was added to each well. The cells were harvested onto glass fibre filters using an automated multisample harvester. [3H]Thymidine incorporation was then measured in a dry scintillation counter (Packard Instrument Co., Meridan, CT). A proliferative response is defined as stimulation index (SI), and SI ≥3 indicates a significant proliferative response. SI was evaluated by dividing the mean counts per minute (cpm) incorporated in cultures of T-cells plus AC-BMDCs by the mean cpm of T-cells plus non-Ag-pulsed BMDCs. The proliferative response of CD4+ T-cells stimulated by LPS-treated BMDCs was used as a positive control.

Immunization of mice
Day-4 BMDCs from DWF1 mice were pulsed with or without ACs for 48 h. BMDCs were further purified using anti-CD11c microbeads. Naïve DWF1 mice (8 weeks old) were intravenously injected with PBS or 1.5 x 105 syngeneic BMDCs, which either had or had not been pulsed with syngeneic ACs. Five days later, for the primary response, the mice that had received PBS or untreated DCs were given an intravenous boost of PBS, whereas mice that had received AC-BMDCs were given an injection of 2 x 106 cells/mice or 1 x 107 cells/mice of ACs. This treatment of BMDC followed by AC was repeated for secondary response after 3 weeks as indicated in Fig. 2A. For the tertiary and quaternary response, the mice received an intravenous injection of PBS or ACs after 3 weeks, respectively. All mice were bled 7 days after treatment with ACs or the PBS boost to evaluate the titre of anti-DNA Abs. In this study, the mice were sacrificed to examine the renal pathology 4 months after their initial treatment.


Figure 2
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2. Induction of anti-DNA Abs in normal mice immunized with AC-BMDCs. (A) Schematic depiction of immunization protocol, as described in ‘Materials and methods' section. Briefly, DWF1 mice were intravenously injected with BMDC alone (group 2), or AC-pulsed day-4 BMDCs (groups 3 and 4) followed by boosts of PBS (group 2) or different doses of AC (group 3 or 4), respectively, at the primary and secondary immunizations. The mice were challenged with ACs (2x106 or 1x107/mice) 7 days after adoptive transfer with AC-BMDCs. At the third and forth immunizations, the mice were only challenged with PBS (group 1 and group 2) or ACs (groups 3 and 4) without BMDCs. Bleeding times are denoted by an asterisk (*). Control mice (group 1) were mice were immunized with PBS only. Each time, 7 days after challenging the mice, anti-dsDNA IgG (B, upper panel) or anti-ssDNA IgG (B, lower panel) in the serum was detected by ELISA. Serum obtained prior to immunization is indicated as pre (pre-immune serum). Results are presented as the mean EU (±SD) in six mice per group at each bleeding time. G, group. *P<0.05; **P<0.01.

 
ELISA for anti-DNA Ab production
Abs specific for dsDNA and ssDNA were evaluated in serum samples by a standard ELISA assay as previously described [11]. Briefly, ELISA plates were coated with 10 µg/ml methylated bovine serum albumin (mBSA; Sigma). dsDNA and ssDNA were coated overnight at 4°C, then washed and blocked with gelatin post-coating solution for 2 h. Serum diluted 100-fold for IgG was applied to each well at 37°C for 45 min. After washing the plates, horseradish peroxidase (HRP)-conjugated goat anti-mouse {gamma}-chain-specific Abs (Sigma) were added. 2,2'-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) solution was used as a substrate, and the optical density (OD) value was evaluated at 405 nm. The levels of anti-IgG are presented as ELISA units (EU/ml) compared with monoclonal Ab 10F10 [11]. This monoclonal Ab is specific to dsDNA or ssDNA. The OD value generated by 37 ng/ml of 10F10 Ab was defined as 1 EU/ml.

Generation of peptide-specific T-cell lines
The generation of peptide-specific T-cell lines was prepared as described previously [11]. In brief, CD4+ T-cells (3 x 106/well) from disease-developing BWF1 mice were cultured with histone peptide- (H2B81–100, H3111–130, H491–110) and U1A201–220 peptide-pulsed syngeneic BMDCs (1 x 105/well) in serum-free medium (AIM-5, Invitrogen, Carlsbad, CA) containing serum replacement TCM (piotide pharmaceuticals, Inc, Paul, MN) for 2–3 days. On day 2, 50 U/ml human recombinant IL-2 was added to each well. After 10–14 days of co-culture, T-cell lines were positively selected by anti-Thy1.2-coated magnetic microbeads (Miltenyi Biotec) and then restimulated with ACs or peptide-pulsed BMDCs (2500 per well) for 5 days. During the last 4–6 h of culture, 1 µCi of [3H]thymidine was added to each well.

Renal deposition of IgG
Histological assessment was performed at 6 months of age. Kidneys were fixed in formalin for haematoxylin and eosin staining was carried out as previously described [12]. Kidney sections for fluorescence microscopy were fixed in an acetone–chloroform (1:1) solution. The slides were stained with 10 µg/ml of fluorescein goat anti-mouse IgG (H+L) (F(ab,)2) (Molecular Probes, Inc., Eugene, OR) or fluorescein-conjugated anti-mouse complement C3(F(ab,)2) (ICN Pharmaceuticals, Inc., Auroua, Ohio), and then counterstained with Evan's Blue, mounted with glycergel (Dako, Japan). Images were generated using fluorescence microscopy (Olympus IX 70).

Statistical analysis
The Mann-Whitney U-test was used to calculate the statistical significance for the difference in the titre of anti-DNA IgG between different groups. A P-value of <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 Acknowledgements
 References
 
Phagocytosis of ACs by BMDCs
To examine the ability of BMDCs to capture ACs, syngeneic thymocytes were treated with dexamethasone for 12–15 h. Staining with annexin V and 7-AAD showed that approximately 82% of the thymocytes were apoptotic after treatment (Supplementary Fig. 1). CFSE-labelled ACs were co-cultured with day-4 or day-6 BMDCs for 6 h at a ratio of 5:1, 10:1 or 20:1. Uptake of ACs by BMDCs increased with the ratio of ACs to BMDCs (data for the 20:1 ratio shown in Fig. 1A). About 23 and 30% of day-4 and day-6 BMDCs (double-positive cells/total I-Ad+ cells) uptook ACs, respectively.

To exclude the contamination of dead cells and non-BMDCs, BMDCs were enriched by anti-CD11c+ microbeads, and the phagocytosis of ACs (7-AAD+) by BMDCs (MHC class I+ or class II+) was determined by flow cytometry. Results showed that more than 75% of MHC I or MHC II positive cells were also 7-AAD positive (Fig. 1B). In addition, the uptake of ACs was also confirmed by confocal microscopy. Effective phagocytosis was indicated by the localization of ACs or bodies (green colour) within the MHC I or MHC II-positive BMDCs (red colour) (Fig. 1C).

ACs can induce the production of IL-12p40 by immature BMDCs
It remains unclear whether ACs can regulate the immune response in lupus mice by promoting or repressing DC maturation. Therefore, in this study, we also investigated the effect of ACs on the maturation of BMDCs. First, we investigated the modulation of surface marker expression known to be upregulated upon DC maturation. The surface expression of molecules such as B7.1, B7.2, MHC class I, MHC class II, CD11c, DEC205 and 33D1 was not altered after either day-4 or day-6 BMDCs from BWF1 or DWF1 mice were pulsed with syngeneic ACs for 48 h (three experiments; Supplementary Fig. 2). We next analysed cytokine production from AC-BMDCs. The IL-12p40 production increased in a dose-dependent manner with the DC AC rate for both day-4 (Fig. 3A), and day-6 (Fig. 3B) BMDCs from BWF1 mice. The amount of IL-12p40 from BWF1 mice was significantly higher than that from DWF1 mice. ACs were not able to induce IL-12p70, IL-10 and TGF-β in these two strains of mice (data not shown). Together, these data suggest that the presence or the phagocytosis of ACs from thymocytes does not induce features of BMDC maturation, with the exception of IL-12p40 production in BWF1 mice.


Figure 3
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 3. IL-12p40 production of BMDCs treated with different numbers of ACs. The day-4 (A) or day-6 (B) BMDCs from BWF1 mice or DWF1 mice were co-cultured with syngeneic ACs at a ratio of 1:5, 1:10 and 1:20. Culture supernatant was collected at 24 and 48 h. As positive controls, the IL-12p40 concentration of LPS (1 µg/ml)-treated BMDCs was 20 ng/ml for day-4 BMDCs and 19 ng/ml for day-6 BMDCs from BWF1 mice, and 6 ng/ml for day-4 BMDCs and 13 ng/ml for day-6 BMDCs from DWF1 mice. There was no obvious difference in IL-12p40 production between 24 and 48 h LPS treatment. The concentration of IL-12p40 was measured using a standard ELISA. The data shown is the result of one of the two repeated experiments.

 
BMDCs can uptake and present autoAgs from ACs to stimulate CD4+ T-cells from BWF1, but not normal mice
To examine whether CD4+ T-cells from BWF1 mice can recognize autoAgs from ACs presented by BMDCs, day-4 and day-6 BMDCs pulsed with syngeneic ACs were used to detect the proliferative response of freshly isolated splenic CD4+ T-cells from disease-developing BWF1 mice or age-matched normal mice (Fig. 4A). A significant response was defined as an SI ≥3.0. CD4+ T-cells from BWF1 mice responded to day-4 BMDCs pulsed with syngeneic ACs in a dose-dependent manner, but not to similarly pulsed day-6 BMDCs. As expected, purified T-cells from DWF1 mice did not exhibit a proliferative response to BMDCs pulsed with syngeneic ACs. In addition, necrotic cell-pulsed BMDCs did not elicit a proliferative response of CD4+ T-cells from BWF1 mice (data not shown).


Figure 4
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 4. CD4+ T-cells from BWF1, but not normal, mice can respond to syngeneic BMDCs pulsed with ACs. (A) Day-4 or day-6 BMDCs (2 x 105 per well) were incubated with syngeneic ACs at a ratio of 1:5, 1:10 and 1:20. CD11c+ BMDCs (5000 cells per well) were co-cultured with syngeneic purified CD4+ T-cells from disease-developing BWF1 mice or normal mice for 4 days. Results are shown as SI (mean ± SD, n = 6–8). Background (T-cells plus non-Ag-pulsed BMDCs) cpm for day-4 or day-6 BMDCs from BWF1 mice were 223 and 845, respectively. Background cpm for day-4 or day-6 BMDCs from DWF1 mice were 2090 and 4581, respectively. The SI of positive control (T-cells stimulated with LPS-treated BMDCs) varied from 12–80 in this experiment. The horizontal line indicates an SI of 3.0 (d4, day 4; d6, day 6), (B) AC-pulsed day-4 BMDCs (the ratio of BMDCs: ACs = 1:20) were pretreated for 30 min with various concentrations of anti-MHC class II (I-Ad or I-Ak) Abs and then co-cultured with CD4+ T-cells from BWF1 mice. The proliferation of CD4+ T-cells without anti-MHC class II Ab treatment was set at 100% (mean cpm = 2127). The data presented are the averages from three mice in each group.

 
To ensure that the proliferative response stimulated by AC-BMDCs was MHC class II-dependent, a blocking Ab (2G9, specific for I-Ad/I-Ed molecules) was used to block the interaction between the TCR and MHC class II molecules. As shown in Fig. 4B, the CD4+ T-cell proliferative response was inhibited by 50% by 2G9 monoclonal Ab, but not by the control monoclonal Ab (anti-I-Ak). The proliferative response cannot be completely inhibited because I-Au/I-Eu molecules may also contribute to the presentation of self-Ags.

To address whether the difference in T-cell proliferative responses between BWF1 and DWF1 mice was due to the abnormality of BMDCs and ACs from BWF1 mice, crossover experiments were performed (Supplementary Fig. 3). Different sources of MHC-matched BMDCs pulsed with syngeneic ACs (Supplementary Fig. 3A) and different sources of ACs pulsed with BMDCs from DWF1 mice (Supplementary Fig. 3B) were used to stimulate CD4+ T-cells from BWF1 mice. The data demonstrates that BMDCs can process and present self-Ags provided by ACs and then stimulate autoreactive CD4+ T-cells from lupus mice, no matter the source of MHC-matched BMDCs and ACs.

BMDCs can uptake ACs and present epitopes of core histones
In our previous study, we identified several potential auto-T-cell epitopes of core histones [9] and U1A protein [11] from BWF1 mice. These include peptides derived from H3 (residue 111–130), H4 (residue 91–110) and U1A (residue 201–220). In order to examine whether ACs can provide such nuclear Ags to BMDCs, CD4+ T-cells from disease-developing BWF1 mice were purified and co-cultured with peptide (H3111–130, H491–110 or U1A201–220)-pulsed BMDCs for 10–14 days to generate peptide-specific T-cell lines as described in the ‘Materials and methods’ section. T-cell lines were then re-stimulated by AC-BMDCs. As a negative control, we used an H2B peptide (residues 81–100), which is not an auto-T-cell epitope in BWF1 mice [9]. As shown in Fig. 5, the specificity of T-cell lines was confirmed by stimulating these T-cells with BMDCs pulsed with the corresponding peptide. Most of the T-cell lines were able to respond to the corresponding peptide-pulsed BMDCs, with the exception of the H2B81–100-specific T-cell line. In addition, H3111–130 and H491–110 but not U1A201–220 or H2B81–100 specific T-cell lines were able to respond to AC-BMDCs. This indicates that BMDCs can process ACs and present certain histone epitopes to autoreactive CD4+ T-cells.


Figure 5
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 5. Histone peptide-specific T-cell lines can respond to AC-BMDCs. T-cell lines were generated by co-culture of CD4+ T-cells from 7-month-old BWF1 mice and histone peptide- or U1A peptide-pulsed syngeneic BMDCs. After 10 days, T-cell lines were harvested and re-stimulated with ACs (the ratio of DCs to ACs was 1:10) or corresponding peptide (10 µg/ml)-pulsed BMDCs. The mean background cpm varied from 293 to 569. The data here is one representative result of two repeated experiments. Results are expressed as the mean SI ± SD from triplicate wells. The dotted line indicates an SI of 3.0.

 
Normal mice injected with AC-BMDCs develop anti-DNA autoAbs and renal deposition of immune complexes
We further investigated whether AC-BMDCs can induce anti-DNA Ab production in normal mice in vivo. In our study, ACs did not alter the phenotype of BMDCs, and the majority of both non-Ag-pulsed and AC-pulsed CD11c+ BMDCs exhibited a mature phenotype (Supplementary Fig. 2). In order to break down the tolerance of CD4+ T- and B-cells, naïve DWF1 mice were intravenously injected AC-BMDCs to activate autoreactive T-cells, followed by a boost of ACs after 5-days to activate autoreactive T-cells, and ACs were subsequent to activate autoreactive B-cells. As shown in Fig. 2, normal mice injected with non-Ag pulsed-BMDCs developed detectable anti-DNA Abs with time compared with mice injected with PBS. However, AC-BMDCs induced statistically significantly higher titres of anti-DNA IgG than did non-Ag-pulsed BMDCs or PBS injected into DWF1 mice.

Due to the high level of autoAb production in mice immunized with ACs, we sought to further characterize their pathogenic effect, since anti-dsDNA IgGs closely correlate with the development of glomerulonephritis. Glomerular samples taken from mice immunized with BMDCs or AC-BMDCs were examined by immunofluorescence. IgG and complement C3 were not detected in the glomeruli of mice immunized with PBS (Fig. 6C and D) or BMDCs (Fig. 6E and F), but IgG and C3 deposition were detected in the glomeruli of mice immunized with AC-BMDCs (Fig. 6G and H) and 8-month-old BWF1 mice (Fig. 6A and B). However, these mice did not develop proteinuria at any point in their lives. This suggests that other susceptibility factors may contribute to the manifestation of renal disease.


Figure 6
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 6. Immune complex and complement C3 deposition in non-autoimmune mice treated with AC-BMDCs. Kidney sections were stained with fluorescein-conjugated anti-mouse IgG (A, C, E, G) or fluorescein-conjugated anti-mouse complement C3 (F(ab,)2) (B,D,F,H). Representative renal glomeruli of 8-month-old BWF1 mice (A, B positive control), and BWF1 mice i.v injected with PBS (C, D), non-Ag-pulsed BMDCs (E, F), or AC-BMDCs (G, H) at 200x magnification. The exposure time for each group was 10 s.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 Acknowledgements
 References
 
Recent studies have indicated that ACs may be a potential reservoir of autoAgs that can initiate and drive systemic autoimmunity. This is the first report demonstrating the existence of histone-specific CD4+ T-cells that can recognize ACs presented by BMDCs in unmanipulated BWF1 mice but not in normal mice. Furthermore, the AC-BMDCs in this study could break tolerance and induce lupus autoAbs in normal mice.

It remains controversial whether ACs can regulate the immune response in SLE by promoting or repressing DC maturation. A previous study [13] suggested that the uptake of ACs, not necrotic cells, allows DCs to induce tolerance to autoAgs. However, it has also been reported that the ingestion of ACs resulted in a reduction of LPS-induced DC maturation, such as by decreasing IL-12 production and CD86 expression [14]. These divergent results might be due to the use of different numbers of ACs to pulse DCs and the different treatment modalities to induce DC maturation. Large amounts of excess ACs can enhance in vitro DC maturation and efficient Ag presentation, whereas fewer numbers of ACs influence the maturation of DCs [15, 16]. Moreover, DC maturation is repressed by the uptake of early ACs but promoted by late ACs [17, 18]. Late ACs have also been shown to be more immunogenic than the early ACs [18]. In our study, immature BMDCs have been cultured with large amounts of late-stage ACs in the in vitro culture, thus allowing autoreactive T-cells from unprimed BWF1 mice to be efficiently stimulated by AC-BMDCs.

Recently, Chang and colleagues [19] demonstrated that oxidants produced during apoptosis can modify protein and lipid moieties on membrane lipid to form neoself-epitopes. Thus, late-stage ACs induced by dexamethasone are immunogenic and pro-inflammatory. However, the surface makers of the CD11c+ BMDCs from BWF1 or control mice, including B7.1, B7.2, MHC II and CD40, did not change significantly after pulsing ACs (Fig. 3). Bioactive IL-12 is a heterodimeric cytokine of 70 kDa comprising of covalently linked p40 and p35 [20]. In our study, ACs could induce IL-12p40 (Fig. 3A), but not p70 secretion of BMDCs in BWF1 mice. Free p40 can form homodimers or be present as free monomers in mice, which have been proposed as natural inhibitors of IL-12 [21]. This suggests that the overproduction of p40 from BMDCs in response to ACs may disturb the function of IL-12 in BWF1 mice. In addition, the role of IL-23, which shares p40 with IL-12 as one of its subunits, needs to be further examined in lupus. It has been found that IL-23 plays an important role in the progression of organ-specific autoimmunity [22, 23].

There have been three histone peptides (H2B10–33, H416–39 and H471–94) that have been reported as epitopes of autoreactive Th cells in (SWR x NZB) lupus mice and have been identified in patients screened with overlapping histone peptides covering the four core histones H2A, H2B, H3 and H4 [24]. These peptides trigger the pathogenic Th cells of SNF1 mice in vivo to induce the development of severe lupus nephritis [25, 26]. In addition, our group recently identified the epitopes of the core nucleosome recognized by autoreactive T-cells from BWF1 mice [9]. The epitopes include the amino acid residues 111–130 of H3 and 91–110 of H4. As shown in Fig. 5, T-cell lines could be generated by non-stimulatory peptide-pulsed BMDCs (residue 81–100 of H2B) [11]. One possible explanation for this result is that the BMDCs could also present self-peptides by MHC molecules by uptaking bystander ACs, even though there were no other Ags or non-stimulatory peptide added during the in vitro culture. Thus, the T-cell line generated by H2B81–100-pulsed BMDCs may contain a broad range of T-cell specificities. This may explain why no significant proliferative response was detected when ‘H2B81–100-specific’ T-cell lines were restimulated by the corresponding peptide-pulsed BMDCs.

Naturally occurring autoreactive T-cells derived from unprimed BWF1 mice encompass a far broader range of specificities against self AGs [27] than do T-cell lines enriched for reactivity to specific self-peptides. Thus, the response from unprimed T-cells represents the sum of the responses from each subset, each with its own specificity. In contrast, T-cell lines contain a few TCR specificities, and the chance of a particular TCR binding to its cognate peptide-bound MHC on AC-BMDCs is much lower. In addition, differences in T-cell response may be due to in vitro culture conditions, as the T-cell lines were cultured in vitro for 2 weeks, vs the unprimed CD4± T-cells, which were stimulated with BMDCs immediately after purification from the spleen. The explanation described above may contribute to the differential proliferative response between unprimed CD4+ T-cells and enriched T-cell lines.

An elegant study by Georgiev and colleagues [28] has demonstrated that mature DCs can break tolerance and induce high titre of autoAbs in normal hosts but do not lead to clinical manifestations of disease. This study showed that mice injected with DCs without feeding dying cells developed autoAbs at levels similar to those in mice receiving DCs that had been pulsed with ACs. The discrepancies between our studies may be explained by differences in several factors, as follows: (1) different MHC haplotype of mice were used (H-2b vs H-2d/u); (2) different DC culture conditions (GM-CSF vs GM-CSF plus IL-4) and different time point of AC-BMDCs were used in the adoptive transfer experiments (day-6 vs day-4 DCs). In our investigation, day-4 and day-6 DCs had a similar ability to engulf ACs (Fig. 1A). However, day-4 DCs could more efficiently stimulate the proliferative responses of autoreactive T-cells than day-6 DCs (Fig. 4A); (3) different numbers of DCs were used for the in vivo experiment (8 x 105 vs 1.5 x 105 cells per mice); and (4) the stage of dying thymocytes fed to DCs was different (early vs late apoptosis). The factors important in DC-induced autoimmunity need to be further elucidated.

Through the phenotypes detected via congenic dissection, disease development of lupus can be organized into three hypothetical pathways [29]. The first pathway requires tolerance to nuclear Ags [30, 31]. Dysregulation of the immune response is also important for pathogenesis to occur. Studies with congenic mice suggest that the break in tolerance to nuclear Ags does not lead to disease unless there is an amplification of the autoimmune response, such as T-cell and B-cell hyperproliferation [32, 33]. The third pathway is the development of end-organ damage [34]. In the present study, IgGs found in the kidneys may likely have been directed against DNA, because a high titre of anti-ssDNA and anti-dsDNA IgG was present in the non-autoimmune mice immunized with AC-BMDCs (Fig. 2). However, no proteinuria was detected in the mice immunized with AC-BMDCs. It has been demonstrated that the pathogenic potential of autoAbs is determined by the IgG subclass [35, 36]. In addition, the receptor involved in complement-fixing also influences the development of severe nephritis [37]. We successfully broke tolerance by inducing anti-DNA Abs in normal mice, although there may be other key factors, such as regulatory T-cells, involved in the mechanism of end-stage disease. It has been reported that the number of regulatory T-cells (CD4+CD25+ cells) significantly decrease in the peripheral blood of SLE patients compared with normal people [38, 39].

The findings in this study suggest an important role for ACs in the initiation and progression of SLE. However, the detailed mechanisms by which ACs regulate DC activity need to be further characterized.

Formula


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
Acknowledgements
 References
 
Supplementary data are available at Rheumatology Online.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
References
 
This work was supported by the grant from National Science Council of Republic of China. We thank Janice Chen for editorial assistance.

The authors have declared no conflict of interest.


   Notes
 
* The first two authors contributed equally to this work. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Acknowledgements
 References
 

  1. Amoura Z, Piette JC, Chabre H, et al. (1997) Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus: correlation with serum antinucleosome antibody titers and absence of clear association with disease activity. Arthritis Rheum 40:2217–25.[ISI][Medline]
  2. Holdenrieder S, Stieber P, Bodenmuller H, et al. (2001) Circulating nucleosomes in serum. Ann N Y Acad Sci 945:93–102.[Abstract/Free Full Text]
  3. Casciola-Rosen LA, Anhalt G, Rosen A. (1994) Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 179:1317–30.[Abstract/Free Full Text]
  4. Ren Y, Tang J, Mok MY, Chan AW, Wu A, Lau CS. (2003) Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus. Arthritis Rheum 48:2888–97.[CrossRef][ISI][Medline]
  5. Botto M, Dell'Agnola C, Bygrave AE, et al. (1998) Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 19:56–9.[CrossRef][ISI][Medline]
  6. Anderson HA, Maylock CA, Williams JA, Paweletz CP, Shu H, Shacter E. (2003) Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells. Nat Immunol 4:87–91.[CrossRef][ISI][Medline]
  7. Cohen PL, Caricchio R, Abraham V, et al. (2002) Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase. J Exp Med 196:135–40.[Abstract/Free Full Text]
  8. Blanco P, Palucka AK, Gill M, Pascual V, Banchereau J. (2001) Induction of dendritic cell differentiation by IFN-{alpha} in systemic lupus erythematosus. Science 294:1540–3.[Abstract/Free Full Text]
  9. Suen JL, Chuang YH, Tsai BY, Yau PM, Chiang BL. (2004) Treatment of murine lupus using nucleosomal T cell epitopes identified by bone marrow-derived dendritic cells. Arthritis Rheum 50:3250–59.[CrossRef][ISI][Medline]
  10. Inaba K, Inaba M, Romani N, et al. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176:1693–702.[Abstract/Free Full Text]
  11. Suen JL, Wu CH, Chen YY, Wu WM, Chiang BL. (2001) Characterization of self-T-cell response and antigenic determinants of U1A protein with bone marrow-derived dendritic cells in NZB x NZW F1 mice. Immunology 103:301–9.[CrossRef][ISI][Medline]
  12. Suen JL, Chuang YH, Chiang BL. (2002) In vivo tolerance breakdown with dendritic cells pulsed with U1A protein in non-autoimmune mice: the induction of a high level of autoantibodies but not renal pathological changes. Immunology 106:326–35.[CrossRef][ISI][Medline]
  13. Sauter B, Albert ML, Francisco L, Larsson M, Somersan S, Bhardwaj N. (2000) Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J Exp Med 191:423–34.[Abstract/Free Full Text]
  14. Stuart LM, Lucas M, Simpson C, Lamb J, Savill J, Lacy-Hulbert A. (2002) Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation. J Immunol 168:1627–35.[Abstract/Free Full Text]
  15. Rovere P, Sabbadini MG, Vallinoto C, et al. (1999) Dendritic cell presentation of antigens from apoptotic cells in a proinflammatory context: role of opsonizing anti-β 2-glycoprotein I antibodies. Arthritis Rheum 42:1412–20.[CrossRef][ISI][Medline]
  16. Rovere P, Vallinoto C, Bondanza A, et al. (1998) Bystander apoptosis triggers dendritic cell maturation and antigen-presenting function. J Immunol 161:4467–71.[Abstract/Free Full Text]
  17. Pietra G, Mortarini R, Parmiani G, Anichini A. (2001) Phases of apoptosis of melanoma cells, but not of normal melanocytes, differently affect maturation of myeloid dendritic cells. Cancer Res 61:8218–26.[Abstract/Free Full Text]
  18. Ip WK and Lau YL. (2004) Distinct maturation of, but not migration between, human monocyte-derived dendritic cells upon ingestion of apoptotic cells of early or late phases. J Immunol 173:189–96.[Abstract/Free Full Text]
  19. Chang MK, Binder CJ, Miller YI, et al. (2004) Apoptotic cells with oxidation-specific epitopes are immunogenic and proinflammatory. J Exp Med 200:1359–70.[Abstract/Free Full Text]
  20. Kobayashi M, Fitz L, Ryan M, et al. (1989) Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 170:827–45.[Abstract/Free Full Text]
  21. Langrish CL, McKenzie BS, Wilson NJ, de Waal Malefyt R, Kastelein RA, Cua DJ. (2004) IL-12 and IL-23: master regulators of innate and adaptive immunity. Immunol Rev 202:96–105.[CrossRef][ISI][Medline]
  22. Langrish CL, Chen Y, Blumenschein WM, et al. (2005) IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 201:233–40.[Abstract/Free Full Text]
  23. Cua DJ, Sherlock J, Chen Y, et al. (2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744–8.[CrossRef][Medline]
  24. Datta SK. (2003) Major peptide autoepitopes for nucleosome-centered T and B cell interaction in human and murine lupus. Ann N Y Acad Sci 987:79–90.[Abstract/Free Full Text]
  25. Kaliyaperumal A, Michaels MA, Datta SK. (1999) Antigen-specific therapy of murine lupus nephritis using nucleosomal peptides: tolerance spreading impairs pathogenic function of autoimmune T and B cells. J Immunol 162:5775–83.[Abstract/Free Full Text]
  26. Kaliyaperumal A, Mohan C, Wu W, Datta SK. (1996) Nucleosomal peptide epitopes for nephritis-inducing T helper cells of murine lupus. J Exp Med 183:2459–69.[Abstract/Free Full Text]
  27. Rozzo SJ, Drake CG, Chiang BL, Gershwin ME, Kotzin BL. (1994) Evidence for polyclonal T cell activation in murine models of systemic lupus erythematosus. J Immunol 153:1340–51.[Abstract]
  28. Georgiev M, Agle LM, Chu JL, Elkon KB, Ashany D. (2005) Mature dendritic cells readily break tolerance in normal mice but do not lead to disease expression. Arthritis Rheum 52:225–38.[CrossRef][ISI][Medline]
  29. Nguyen C, Limaye N, Wakeland EK. (2002) Susceptibility genes in the pathogenesis of murine lupus. Arthritis Res 4:S255–63.[CrossRef][Medline]
  30. Morel L, Croker BP, Blenman KR, et al. (2000) Genetic reconstitution of systemic lupus erythematosus immunopathology with polycongenic murine strains. Proc Natl Acad Sci USA 97:6670–5.[Abstract/Free Full Text]
  31. Mohan C, Alas E, Morel L, Yang P, Wakeland EK. (1998) Genetic dissection of SLE pathogenesis. Sle1 on murine chromosome 1 leads to a selective loss of tolerance to H2A/H2B/DNA subnucleosomes. J Clin Invest 101:1362–72.[ISI][Medline]
  32. Balomenos D, Martin-Caballero J, Garcia MI, et al. (2000) The cell cycle inhibitor p21 controls T-cell proliferation and sex-linked lupus development. Nat Med 6:171–6.[CrossRef][ISI][Medline]
  33. Mohan C, Morel L, Yang P, Wakeland EK. (1997) Genetic dissection of systemic lupus erythematosus pathogenesis: Sle2 on murine chromosome 4 leads to B cell hyperactivity. J Immunol 159:454–65.[Abstract]
  34. Morel L, Tian XH, Croker BP, Wakeland EK. (1999) Epistatic modifiers of autoimmunity in a murine model of lupus nephritis. Immunity 11:131–9.[CrossRef][ISI][Medline]
  35. Karassa FB, Trikalinos TA, Ioannidis JP. (2003) The Fc gamma RIIIA-F158 allele is a risk factor for the development of lupus nephritis: a meta-analysis. Kidney Int 63:1475–82.[CrossRef][ISI][Medline]
  36. Theofilopoulos AN and Dixon FJ. (1985) Murine models of systemic lupus erythematosus. Adv Immunol 37:269–390.[ISI][Medline]
  37. Clynes R, Dumitru C, Ravetch JV. (1998) Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis. Science 279:1052–4.[Abstract/Free Full Text]
  38. Liu MF, Wang CR, Fung LL, Wu CR. (2004) Decreased CD4+CD25+ T cells in peripheral blood of patients with systemic lupus erythematosus. Scand J Immunol 59:198–202.[CrossRef][ISI][Medline]
  39. Crispin JC, Martinez A, Alcocer-Varela J. (2003) Quantification of regulatory T cells in patients with systemic lupus erythematosus. J Autoimmun 21:273–6.[CrossRef][ISI][Medline]
Submitted 7 December 2005; revised version accepted 24 February 2006.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
45/10/1230    most recent
kel106v1
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 ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Tzeng, T.-C.
Right arrow Articles by Chiang, B.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tzeng, T.-C.
Right arrow Articles by Chiang, B.-L.
Related Collections
Right arrow Experimental Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?