- Email: [email protected]
Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus
Immunology Letters 138 (2011) 179–186
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus Mariann H. Jørgensen a , Ole Petter Rekvig a , Rasmus S. Jacobsen b , Søren Jacobsen b , Kristin A. Fenton a,∗ a b
Molecular Pathology Research Group, Institute of Medical Biology, Faculty of Health Sciences, University of Tromsø, N-9037 Tromsø, Norway Department of Rheumatology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
a r t i c l e
i n f o
Article history: Received 20 December 2010 Received in revised form 5 April 2011 Accepted 11 April 2011 Available online 17 April 2011 Keywords: Anti-dsDNA antibodies Lupus nephritis Chromatin Systemic lupus erythematosus (NZBxNZW)F1 mice
a b s t r a c t Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known. Serum samples from autoimmune (NZBxNZW)F1 mice, healthy BALB/c mice, patients with SLE, RA and normal healthy individuals were analyzed for presence and amount of circulating anti-dsDNA antibodies and nucleosomal DNA. Here we use a quantitative PCR to measure circulating DNA in sera. We demonstrate an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli. The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect the development of lupus nephritis at an early stage. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Systemic lupus erythematosus (SLE) is an autoimmune syndrome that is characterized by the production of anti-DNA antibodies and appearances of immune complex mediated inflammation in various organs [1]. These autoantibodies form complexes with nucleosomes that deposit within the kidney and the skin [2], and the affection of the kidney causes a most serious and potentially fatal complication in human SLE; lupus nephritis [3]. Other nuclear autoantibodies like anti-nucleosome, anti-nucleolin and anti-centromere protein antibodies has been implicated in the development of lupus nephritis [4–6]. However, there is currently no definitive distinction that allows us to separate non-pathogenic from pathogenic anti-dsDNA antibodies. It is therefore of great importance to identify cellular processes and/or available molecular structures that determine anti-dsDNA antibody-binding in the kidney contributing to the induction of nephritis. The presence of anti-chromatin antibodies have been linked to the development of glomerulonephritis and disease activity in SLE [7]. In recent studies we have shown that anti-dsDNA and anti-nucleosome antibodies recognize selectively ectopic, intraglomerular chromatin structures in vivo [8,9]. Autoantibody
∗ Corresponding author. Tel.: +47 77646834; fax: +47 77645350. E-mail address: [email protected] (K.A. Fenton). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.04.006
deposits in vivo are strictly localized to electron-dense structures (EDS) associated with mesangial matrix (MM) and glomerular basement membranes (GBM), and they co-localize with experimental antibodies to chromatin structures and nicked DNA [8–12]. This demonstrates that the antibodies bind to exposed chromatin fragments. Recently, on the basis of a longitudinal study in (NZBxNZW)F1 (B/W) mice we have shown that autoantibodies deposit first in the MM of the glomeruli and, as the disease develops into a full blown nephritis, they deposit in the GBM [13]. Further, we demonstrated that serial injection of anti-dsDNA antibodies into healthy mice induced the formation of immune complex deposits within the MM [14]. These data confirmed that anti-dsDNA antibodies form complexes with nucleosomes that deposit in glomerular membranes [11,12,15]. In SLE, defective clearance of apoptotic nucleosomes has been described [16–18] which is believed to lead to chromatin fragment deposition within glomerular membranes, where they have been shown to bind at high affinity [10]. The origin of the nucleosomes and when they appear is not known. Since anti-DNA antibodies and nucleosomes most likely form complexes in the circulation [19–21], serum is the most relevant biological compartment to perform the present investigations. Plasma DNA has been difficult to study in SLE due to low concentrations present in most SLE specimens [22,23], Amoura et al. measured the circulating plasma levels of nucleosomes by ELISA [24]. They found that the nucleosome levels in plasma of SLE patients were significantly higher than in the
180
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
Table 1 Demographic and clinical characteristics of 99 patients with systemic lupus erythematosus, 57 RA patients and 44 NHC.
Female gender, n (%) Age, yrs Disease duration, yrs SLEDAI score ACR criteria fulfilled – Malar rash, n (%) – Discoid rash, n (%) – Photosensitivity, n (%) – Oral ulcers, n (%) – Arthritis, n (%) – Serositis, n (%) – Renal disease, n (%) – CNS disease, n (%) – Hematological disorder, n (%) – Immunological disorder, n (%) – Antinuclear antibodies, n (%)
Table 2 The primers and probe sequences used for real-time PCR of murine (B1-qPCR) and human (Alu-qPCR) sera.
SLE
RA
NHC
Primer/probe
Sequence
90 (91) 46 (18–83) 12 (0.02–36) 4 (0–20)
47 (82) 53 (20–82) – –
32 (73) 49 (21–72) – –
Mouse B1 forward Mouse B1 reverse Mouse B1 probe Human Alu forward Human Alu reverse Human Alu probe
5 -GCACACCTTTAATCCCAGCA-3 5 -CTCTGTGTAGCCCTGGCTGT-3 5 -GATTTCTGAGTTCGAGGCCA-3 5 -CCTGAGGTCAGGAGTTCGAG-3 5 -CCCGAGTAGCTGGGATTACA-3 5 -TGGTGAAACCCCGTCTCTAC-3
45 (45) 20 (20) 33 (33) 12 (12) 55 (56) 34 (34) 51 (52) 3 (3.0) 72 (73) 86 (87) 96 (97)
Continuous data presented as mean (range).
control group. Chen et al. purified DNA from sera and plasma and quantified the amount using the Picogreen assay and found significantly elevated levels of DNA in SLE patients compared with normal healthy donors [25]. Circulating DNA has been shown to be of low molecular weight, corresponding to mono- and di-nucleosomes [22,23,26,27]. Quantification of nucleosomes as DNA in the circulation by real-time PCR is a widely accepted method. By using Alu or B1 specific primers and probe it is possible to detect all kinds of free and protein bound circulating DNA [28,29]. Alu or B1 elements are short interspersed elements (SINEs). Alu sequences occupy 10.7% of the human genome [30], while B1 sequences in mice comprise 2.7% of the mouse genome [31]. In this study we have investigated if deposition of nucleosomes in the MM and GBM may reflect increased or decreased levels of circulating chromatin. DNA concentrations, Dnase1 activity, anti-dsDNA antibodies, and immune complex deposition in the glomeruli have been analyzed in sera and kidneys from normal and lupus prone mice in addition to sera from SLE patients, RA patients and normal healthy controls (NHC). The data demonstrate an inverse correlation between levels of circulating chromatin and anti-dsDNA antibodies that may reflect glomerular deposition of chromatin-containing immune complexes. 2. Materials and methods 2.1. Human serum samples Serum samples from SLE or rheumatoid arthritis (RA) patients were obtained from Rigshospitalet, The Copenhagen University Hospital. Forty-four normal sera from age and sex matched healthy controls (NHC) of the same ethnic group with no previous history of any significant clinical manifestation were included in this study. This study was approved by the Ethical committee in Copenhagen. Demographic and clinical characteristics of the 99 patients with SLE, 57 RA and NHC 44 are listed in Table 1. 2.2. Animals BALB/c mice were obtained from Harlan (Oxon, England) and (NZBxNZW)F1 (B/W) were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). Treatment and care of animals were conducted in accordance with guidelines of the Norwegian Ethical and Welfare Board for Animal Research, and the institutional review board approved the study.
Amplicon size (bp)
107
115
2.3. Longitudinal study of B/W mice For a longitudinal study, animals were sacrificed approximately every second week (in groups of 3 mice) from the age of 4 weeks old (w.o.) until development of severe proteinuria (≥20 g/l) [13]. Proteinuria was measured using Uristix from Bayer Diagnostics (Leverkusen, Germany) measuring values ranging from +1, ≤0.3 g/l protein in urine, +2, ≤1 g/l; +3, ≤3 g/l and +4, ≤20 g/l; +3 and +4 are regarded as indication of severe nephritis. Serum samples were collected at 2 week intervals and stored at −80 ◦ C. Kidneys were collected at endpoint, fixed in 8% paraformaldehyde and embedded in sucrose for immune electron microscopy (IEM) according to the Tokuyasu method [32]. 2.4. Murine serum samples Serum samples were obtained every week for a time study by making a small incision on the mouse leg and collecting the blood sample using a capillary tube. The blood samples were centrifuged at 4500 × g for 20 min at 4 ◦ C, and sera were stored at −20 ◦ C. When the mice were sacrificed, blood samples were drained from the heart and put directly on ice. The blood samples were centrifuged as described above and sera were stored at −20 ◦ C. 2.5. ELISA and antibodies Serum anti-dsDNA antibodies were detected and controlled by ELISA as described [33,34], using microtiter plates (Nunc MaxiSorp; Nunc, Copenhagen, Denmark) coated with calf thymus dsDNA (10 g/ml in PBS, Sigma–Aldrich). Monoclonal anti-DNA antibody 163p.87 was obtained from Dr. Tony Marion (Memphis TE, USA) [35] and was used as a positive control for primary antibody in the enzyme-linked immunosorbent assay (ELISA) experiments. Antimouse IgG and anti-human IgG peroxidase conjugated antibody was obtained from Sigma–Aldrich (Saint Louis, USA). 2.6. Quantitative real-time PCR (qPCR) The consensus sequences for B1 in mouse and Alu sequences in human were taken from Detter et al. and Umetani et al. [36,37], and the appropriate primers and probes were designed by using the primer design tool from GenScript (http://www.genscript.com). The probes were labeled with a reporter dye (FAM) at its 5 -end and a non-fluorescence quencher and MGB at its 3 -end. Primers and probe were purchased from Applied Biosystems (Foster City, CA, USA) and are listed in Table 2. Sera were diluted 1:2 in a buffer composed of 0.25% Tween 20, 50 mM Tris, and 1 mM EDTA and digested with 0.4 g/l proteinase K at 50 ◦ C for 1 h, followed by 5 min heat inactivation at 95 ◦ C. The samples were centrifuged at 10,000 × g for 20 min and 1 l of the supernatant was used as template for B1-specific-qPCR and 0.2 l was used for the Alu-specific qPCR. The reaction mixture consisted of a template, 0.2 M each of forward, reverse primers and probe, 1× TaqMan FastUniversal PCR Master Mix (2×, Applied
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
181
hydrolyzed DNA was calculated by using the program cellFˆ (Olympus, Oslo, Norway). The values for standards and samples were plotted and the concentrations of Dnase1 in serum samples was calculated using cellFˆ (Olympus). 2.9. Statistics
Fig. 1. B1 qPCR on proteinase K treated sera from 20 w.o. B/W and BALB/c. The serum samples were tested five times by qPCR. Five parallels of each serum sample were tested each time. For each qPCR the mean value and SEM for each mouse are plotted.
Biosystems) in a total reaction volume of 10 l. The qPCR amplification was performed using the ABI 7900 HT Fast Real Time PCR system and was initiated with 95 ◦ C for 20 s, followed by 40 cycles of denaturation at 95 ◦ C for 1 s and annealing at 60 ◦ C for 20 s. The amount of DNA in each sample was determined using a standard curve with serial dilutions (1 ng–0.01 pg) of genomic DNA purified from murine fibroblast cells (A31) and from human full blood. Both DNA preparations were purified by the BioRobot® EZ1 (Qiagen). The results were analyzed by using the SDS 2.3 software program; threshold was set to 0.13 for all runs. Graphs were made in GraphPad Prism 5. The B1 specific qPCR method was validated by testing sera from four B/W mice and three BALB/c mice. These sera were tested five different times with five parallels for each mouse for each run, to determine intra- and inter-assay variations. In general, both intra- and inter-assay variation were regarded as acceptable (see Fig. 1, were sera from 20 w.o. B/W and BALB/c were analyzed). Similar results were obtained using human serum samples (data not shown). 2.7. Dnase I gel zymography DNA degrading activity by Dnase1 was determined after separation of serum proteins in a 10% SDS–polyacrylamide gel containing 100 g/ml heat-denatured salmon sperm DNA (Invitrogen Corp., Carlsbad, CA) as described [38], Eight l of 10× diluted serum sample were mixed with 2 l of 5× sample buffer, and 10 l of each sample was loaded on the gel. Dnase1 (10 U/l) isolated from bovine pancreas (Amersham Biosciences) was diluted to 0.0004 U/l and used as a positive control. Eight l of this dilution was mixed with 2 l of 5× sample buffer to a final concentration of 0.00032 U/l. The running conditions were set to 150 V/25 mA/10 W. Electrophoresis was run for 70 min at 4 ◦ C. The gel was soaked in washing buffer for 1 h at room temperature under constant agitation, and then in reaction buffer at 37 ◦ C for 18 h, and photographed in the UV illuminator Gel Doc 2000 (BioRad). 2.8. Radial enzyme diffusion – total nuclease activity A 1% agarose gel in 1× Dnase reaction buffer was prepared and 150 l heat-denatured salmon sperm DNA (Invitrogen Corp., Carlsbad, CA, 10 g/ml) and 50 l EtBr (1 g/ml) was added. A standard curve was prepared by Dnase1 starting at 0.1 U/l and five steps of five fold serial dilutions. Serum protein concentrations were measured by bicinchoninic acid (BCA) protein assay (Pierce Biochemicals, Rockford, IL, USA) and normalized for total protein content. Three l standard dilutions or normalized serum samples were added to the wells. The gels were incubated in darkness at 37 ◦ C for 18 h. The gels were then photographed in the UV illuminator Gel Doc 2000 (BioRad) and the area of cleared circles of
Unpaired two tailed t-test was performed to compare the means of two sets of measurements. One-way Anova test was performed to compare three or more groups. Spearman correlation analyses were done to see if there could be any connection between chromatin levels and anti-dsDNA antibodies production. 3. Results 3.1. Anti-dsDNA antibody titers and chromatin levels are inversely correlated in circulation of human SLE patients, RA patients and normal healthy controls (NHC) First, we analyzed if serum levels of circulating DNA and antidsDNA antibody correlated with each other in sera from 99 SLE patients, 57 RA patents and 44 NHC (Fig. 2A–C, respectively). The results revealed a significant variance in the anti-dsDNA antibody levels and in the chromatin concentrations (Fig. 2D, for chromatin concentrations). A biplot of the two parameters revealed a negative correlation (R = −0.219, p = 0.0018) demonstrating that high anti-dsDNA antibody titer correlated with low levels of DNA in the circulation (Fig. 2E). 3.2. Chromatin levels in circulation of (NZBxNZW)F1 mice are negatively correlated with anti-dsDNA antibody titers as determined in a longitudinal study Chromatin concentrations estimated by qPCR in sera of BALB/c mice and B/W mice at different ages revealed substantial individual variances (Fig. 3A and B, for BALB/c and B/W mice, respectively). Chromatin levels in sera from BALB/c mice were significantly higher compared with sera from B/W mice producing anti-dsDNA antibodies (Fig. 3C, p = 0.0428). Mice with antibody positive sera were divided into groups based on immune complex deposits in MM only or deposition also in GBM. This group distinction caused an increase in the significantly inverse correlation between circulating DNA levels and anti-dsDNA antibody positive sera taken from mice with MM deposits compared to BALB/c (Fig. 3D, p = 0.0308). Circulating DNA levels were not correlated with anti-dsDNA antibody titers when analyzing all sera from BALB/c and B/W mice irrespective whether producing anti-dsDNA antibodies or not (Fig. 3E). However, in anti-dsDNA antibody positive sera, levels of circulating chromatin were inversely correlated with amount of anti-dsDNA antibodies produced (Fig. 3F). 3.3. Chromatin levels correlate negatively with anti-dsDNA antibodies production, but not with total serum nuclease activity during progression of murine lupus nephritis Considerable individual differences of serum DNA levels were observed between the BALB/c and B/W mice in the different age groups (Fig. 3B). Therefore, sera from five (NZBxNZW)F1 mice taken at different time points from age of 12 w.o. to the development of end-stage renal disease were analyzed by (i) anti-dsDNA antibodies as detected by ELISA, (ii) real-time PCR to measure chromatin levels, and (iii) total serum nuclease activity as determined by radial diffusion nuclease assay. Anti-dsDNA antibody production profile for each mouse is presented in Fig. 4A. Serum chromatin concentrations for each individual mouse demonstrated a cyclical pattern
182
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
Fig. 2. Quantification of anti-dsDNA antibodies and chromatin levels in sera from SLE, RA and NHC. Anti-dsDNA antibodies and chromatin concentrations were measured in native sera and in proteinase K treated sera from SLE patients (A), RA patients (B) and normal healthy donors (NHC) (C) respectively. A comparison of chromatin levels in the three different groups demonstrated a significant lower amount in sera from SLE patients than in RA patients and NHC (unpaired t-test p = 0.0018 and p < 0.0001, respectively, D). A Spearman correlation analysis on all subjects demonstrated a negative correlation of R = −0.219 (p = 0.018) were high amounts of anti-dsDNA antibodies correlated to lower concentrations of chromatin (E).
with decreasing levels at the end point (Fig. 4B). The activity of circulating total nucleases showed the same cyclical pattern as chromatin levels (Fig. 4C). Chromatin levels plotted against antidsDNA antibody levels revealed an inverse correlation between the 2 parameters (Fig. 4D). The chromatin levels showed no correlation with total serum nuclease activity (Fig. 4E). Dnase1 zymography corresponded to the observed total nuclease activity (data not shown). DNA levels measured in the circulation of BALB/c mice from 22 to 36 w.o. was significantly higher than DNA levels in the circulation of B/W mice of the same age, in which presence of anti-dsDNA antibodies correlated inversely with DNA concentration (Fig. 5A and B). There were no significant differences in the levels of total nuclease activity in sera from B/W compared to sera from BALB/c (Fig. 5C). Interestingly, the levels of total nuclease activity in sera were more stable in BALB/c than in sera of B/W mice (Fig. 5C). 4. Discussion In this study, we determined the impact of three parameters that may in an interdependent manner have impact on devel-
opment of SLE and lupus nephritis. These parameters are levels of circulating DNA, anti-dsDNA antibodies and serum nucleases. One perception from this study is reduced DNA concentration in sera from SLE patients compared to RA patients and NHC, and an inverse correlation between DNA concentration and anti-dsDNA antibody levels. This is in contrast to results published by other groups [22,24,25,27,39–42]. For example, Amoura et al. measured significantly higher levels of nucleosomes in plasma of 13 of 58 SLE patients compared to the healthy individuals [24]. Similar results were obtained using the Picogreen assay to quantify DNA in sera and plasma of SLE patients [25]. However, none of the 13 patients with elevated DNA concentration had detectable antinucleosome or anti-dsDNA antibodies. Interestingly, Amoura et al. demonstrated that there was an inverse correlation between DNA concentration, anti-nucleosome antibodies and anti-dsDNA antibodies when the entire population of 58 SLE patients was compared [24]. Bengtsson et al. measured DNA levels in circulating immune complexes by a quantitative immunochemical assay and found a decrease in DNA concentration at severe SLE flares [43]. Notably, they demonstrated an inverse correlation between immunecomplexed DNA levels and anti-dsDNA antibody concentrations
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
183
Fig. 3. Circulating chromatin levels in groups of BALB/c control mice and B/W mice are presented. Sera taken consecutively every second week in groups of 3 were analyzed for levels of circulating chromatin (A and B for BALB/c and B/W mice respectively). There was a significant difference in serum chromatin concentrations in sera from BALB/c mice compared to anti-dsDNA positive B/W sera (C, unpaired t-test p = 0.0428). Dividing the anti-dsDNA positive sera into sera from mice with MM deposits or GBM deposits the significant difference between BALB/c and B/W with MM deposits increased (D, p = 0.0308). The chromatin levels in all BALB/c and B/W sera were not correlated with anti-dsDNA antibody levels (E).
measured by the following methods: Critidiae Luciliae assay, ELISA, plasmid DNA-based assay and Farr assay. The inverse relationship between DNA and anti-dsDNA antibodies in plasma from lupus patients was also observed by Coubrey-Hoyer et al. [22]. However many patients had high levels of plasma DNA and anti-dsDNA antibodies without clinical nephritis. This suggests that other factors than the presence of chromatin and anti-dsDNA antibodies is important in the initiation of glomerular damage in SLE. Analyzing serum levels of circulating chromatin over time in B/W mice similarly revealed a weak inverse correlation with the production of anti-dsDNA antibodies. Due to the design of the clinical part of our study, we have not been able to address if similar time dependent dynamics are present in patients with SLE. However, since there seem to be a cyclical increase and decrease in the amount of circulating chromatin measured in mice, this may be important to investigate as this may explain the flares of the disease often seen in SLE.
Our findings in SLE patients demonstrating an inverse correlation between anti-dsDNA antibody levels and circulating concentrations of DNA were confirmed in lupus prone B/W mice. High levels of anti-dsDNA antibodies corresponded to reduced levels of DNA in the circulation. Since this was limited to mice producing anti-dsDNA antibodies, reduced levels of DNA in circulation also correlated with progression of lupus and lupus nephritis. However, there was no significant differences in levels of circulating DNA between anti-dsDNA antibody negative B/W and anti-dsDNA positive B/W, in contrast to the inverse correlation between anti-dsDNA antibodies and circulating DNA between BALB/c and B/W mice. This discrepancy may be explained by masking the production of anti-dsDNA antibodies by the formation of immune complexes and deposition in the kidney [13]. A masked increase of circulating DNA may be explained by the deposition of the immune complexes containing chromatin. It is reasonable
184
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
Fig. 4. Anti-dsDNA antibody levels, chromatin concentrations and total nuclease activity in sera of five B/W mice taken every week from 12 w.o. until development of proteinuria. Anti-dsDNA antibodies were measured by ELISA and individual levels for each mouse (T1–5) are shown in (A). The individual mean ± SEM of circulating chromatin in sera of the five mice taken at different time points (age) demonstrated a cyclical pattern with decreasing amount at end point (B). The total nuclease activity demonstrated the same cyclical pattern over time (C). A Spearman correlation analysis demonstrated a negative correlation R = −0.306, p = 0.003, were high amounts of anti-dsDNA antibodies correlated to lower concentrations of chromatin (D). There was no correlation between chromatin concentration and total nuclease activity, R = 0.125, p = 0.267 (E).
to assume that immune complex deposition in the glomeruli stimulates an inflammatory cascade characterized by complement activation and neutrophil granulocytes and macrophage infiltration [44]. Progressive inflammation can cause release of chromatin from the tissue and cells [45], and may explain the higher chromatin concentration in sera from mice with GBM deposits in contrast to levels in sera from B/W mice with immune complex deposits only located in MM. In line with this, Williams et al. demonstrated that the levels of circulating nucleosomes were raised in SLE patients with very active central nervous system and renal involvement [46]. However, this is in contrast to our overall results and to previously results from Bengtsson et al. showing decreased levels of circulating nucleosomes during flares of the disease [43]. The inverse correlation between DNA concentration and antidsDNA antibodies in the circulation demonstrated in this study, as
Fig. 5. Chromatin levels and total nuclease activity measured consecutively in the circulation of BALB/c and B/W mice. Serum levels of chromatin in sera from BALB/c mice taken from the age of 22–36 weeks were compared with levels in sera from age-matched B/W mice (A). There was a significantly higher amount of circulating chromatin fragments in sera from BALB/c mice compared to sera from B/W mice (B) (unpaired t-test p < 0.0001). There was no significant difference between the total nuclease activity in sera taken over time from BALB/c and B/W mice (C).
well as by others [22,43,47–49] suggest that anti-dsDNA antibodies influence the levels of circulating nucleosomes. Anti-dsDNA antibodies may form immune complexes with chromatin fragments in the circulation which are possible cleared by a combination of phagocytosis and by deposition in e.g. the kidneys. Such a process indicates that initial events in progressive lupus nephritis involve production of anti-dsDNA/nucleosome antibodies that bind available nucleosomes in circulation, and capture of the formed immune complexes by mesangial cells through binding to Fc␥R [50] with subsequent release to the mesangial matrix. In this microenvironment the immune complexes may trigger inflammatory pathways having detrimental effects on glomeruli with renal failure as the
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
end result. Subsequent shut down of renal nucleases may further facilitate this process [13,51]. Thus, progressive lupus nephritis may have the origin in the fatal meeting of 2, by themselves nonpathogenic factors, anti-dsDNA antibodies and circulating DNA. As single factors, they are epiphenomenons with no pathogenic impact; together they start the processes that threaten the function of the kidneys. This hypothesis has its origin in a recent longitudinal study of progressive lupus nephritis in B/W mice [13]. We have recently published data on the effect of heparin treatment leading to enhanced nucleosome degradation and reduced chromatin deposition within the MM and the GBM of glomeruli of (NZBXNZW)F1 mice [52]. Heparin binds to chromatin and makes it more susceptible for degradation by both proteases and nucleases. This treatment led to delayed production of anti-dsDNA antibodies and reduced immune complex deposition within the mesangial matrix and glomerular basement membrane in heparin treated B/W mice compared to untreated animals. The important observation in this study was that increased variation of circulating chromatin levels in heparin treated mice were observed compared to non-treated control mice. A prevention of the development of lupus nephritis could thus hypothetically be achieved by hindering the process of immune complex formation by anti-dsDNA antibodies and chromatin in the circulation or the deposition of them to the mesangial matrix and GBM can be hindered. This makes the immune complexes or the deposition of them a possible target for treatment of lupus nephritis.
Acknowledgments We are grateful for the anti-dsDNA mAb from Tony N Marion. Berit Tømmerås, Natalya Seredkina and Annica Hedberg are appreciated for their technical help. This work was funded by the Foundation for Health and Rehabilitation through the Norwegian Rheumatology Organization (project 2008/2/0229) (KF), Northern Norway Regional Health Authoroty medical Research Progema (OPR, Grant # SFP-100-04, SFP 100-04) and the Novo Nordisk Foundation (SJ). This study was approved by the Ethical committee in Copenhagen.
References [1] Lahita R. The clinical presentation of systemic lupus erythematosus. In: Lahitha R, editor. Systemic lupus erythematosus. San Diego: Academic Press; 1999. p. 325–36. [2] Fismen S, Hedberg A, Fenton KA, Jacobsen S, Krarup E, Kamper AL, et al. Circulating chromatin–anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis. Lupus 2009;18:597–607. [3] Balow JE, Boumpas DT, Austin III HA. Systemic lupus erythematosus and the kidney. In: Lahita RG, editor. Systemic lupus erythematosus. New York: Elsevier Academic Press; 2004. p. 877–912. [4] Manson JJ, Ma A, Rogers P, Mason LJ, Berden JH, d. van V, et al. Relationship between anti-dsDNA, anti-nucleosome and anti-alpha-actinin antibodies and markers of renal disease in patients with lupus nephritis: a prospective longitudinal study. Arthritis Res Ther 2009;11:R154. [5] Fritzler MJ, Rattner JB, Luft LM, Edworthy SM, Casiano CA, Peebles C, et al. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F. Autoimmun Rev 2011;10:194–200. [6] Minota S, Jarjour WN, Suzuki N, Nojima Y, Roubey RA, Mimura T, et al. Autoantibodies to nucleolin in systemic lupus erythematosus and other diseases. J Immunol 1991;146:2249–52. [7] Gomez-Puerta JA, Burlingame RW, Cervera R. Anti-chromatin (antinucleosome) antibodies: diagnostic and clinical value. Autoimmun Rev 2008;7:606–11. [8] Kalaaji M, Mortensen E, Jorgensen L, Olsen R, Rekvig OP. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells. Am J Pathol 2006;168:1779–92. [9] Kalaaji M, Fenton KA, Mortensen ES, Olsen R, Sturfelt G, Alm P, et al. Glomerular apoptotic nucleosomes are central target structures for nephritogenic antibodies in human SLE nephritis. Kidney Int 2007;71:664–72.
185
[10] Mjelle JE, Rekvig OP, Fenton KA. Nucleosomes possess a high affinity for glomerular laminin and collagen IV and bind nephritogenic antibodies in murine lupus-like nephritis. Ann Rheum Dis 2007;66:1661–8. [11] Kramers C, van Bruggen MCJ, Walgreen B, Elema JD, Kallenberg CGM, van den Born J, et al. Nucleosomes and histones are present in glomerulardeposits in human lupus nephritis. J Am Soc Nephrol 1995;6:425. [12] Malide D, Londono I, Russo P, Bendayan M. Ultrastructural localization of DNA in immune deposits of human lupus nephritis. Am J Pathol 1993;143:304– 11. [13] Fenton K, Fismen S, Hedberg A, Seredkina N, Fenton C, Mortensen ES, et al. Anti-dsDNA antibodies promote initiation, and acquired loss of renal Dnase1 promotes progression of lupus nephritis in autoimmune (NZBxNZW)F1 mice. PLoS One 2009;4:e8474. [14] Fenton KA, Tommeras B, Marion TN, Rekvig OP. Pure anti-dsDNA mAbs need chromatin structures to promote glomerular mesangial deposits in BALB/c mice. Autoimmunity 2010;43:179–88. [15] van Bruggen MC, Kramers C, Walgreen B, Elema JD, Kallenberg CG, van den Born J, et al. Nucleosomes and histones are present in glomerular deposits in human lupus nephritis. Nephrol Dial Transplant 1997;12:57–66. [16] Herrmann M, Voll RE, Zoller OM, Hagenhofer M, Ponner BB, Kalden JR. Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. Arthritis Rheum 1998;41:1241–50. [17] Baumann I, Kolowos W, Voll RE, Manger B, Gaipl U, Neuhuber WL, et al. Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus. Arthritis Rheum 2002;46:191–201. [18] Gaipl US, Munoz LE, Grossmayer G, Lauber K, Franz S, Sarter K, et al. Clearance deficiency and systemic lupus erythematosus (SLE). J Autoimmun 2007;28:114–21. [19] Liou LB, Chao WJ. Changing cytokine patterns in systemic lupus: A prospective longitudinal study. J Microbiol Immunol Infect 2010;43:18–25. [20] Emlen W, Mannik M. Clearance of circulating DNA–anti-DNA immune complexes in mice. J Exp Med 1982;155:1210–5. [21] Mannik M. Mechanisms of tissue deposition of immune complexes. J Rheumatol Suppl 1987;14(Suppl. 13):35–42. [22] Coubrey-Hoyer A, Okarma TB, Holman HR. Partial purification and characterization of plasma DNA and its relation to disease activity in systemic lupus erythematosus. Am J Med 1984;77:23–34. [23] Steinman CR. Circulating DNA in systemic lupus erythematosus. Isolation and characterization. J Clin Invest 1984;73:832–41. [24] Amoura Z, Piette JC, Chabre H, Cacoub P, Papo T, Wechsler B, et al. 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 1997;40:2217–25. [25] Chen JA, Meister S, Urbonaviciute V, Rodel F, Wilhelm S, Kalden JR, et al. Sensitive detection of plasma/serum DNA in patients with systemic lupus erythematosus. Autoimmunity 2007;40:307–10. [26] Sano H, Morimoto C. DNA isolated from DNA/anti-DNA antibody immune complexes in systemic lupus erythematosus is rich in guanine-cytosine content. J Immunol 1982;128:1341–5. [27] Raptis L, Menard HA. Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus. J Clin Invest 1980;66:1391–9. [28] Holdenrieder S, Stieber P, Chan LY, Geiger S, Kremer A, Nagel D, et al. Cellfree DNA in serum and plasma: comparison of ELISA and quantitative PCR. Clin Chem 2005;51:1544–6. [29] Umetani N, Kim J, Hiramatsu S, Reber HA, Hines OJ, Bilchik AJ, et al. Increased integrity of free circulating DNA in sera of patients with colorectal or periampullary cancer: direct quantitative PCR for ALU repeats. Clin Chem 2006;52:1062–9. [30] Umylny B, Presting G, Efird JT, Klimovitsky BI, Ward WS. Most human Alu and murine B1 repeats are unique. J Cell Biochem 2007;102:110–21. [31] Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, et al. Initial sequencing and comparative analysis of the mouse genome. Nature 2002;420:520–62. [32] Tokuyasu KT, Singer SJ. Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol 1976;71:894–906. [33] Haugbro K, Nossent JC, Winkler T, Figenschau Y, Rekvig OP. Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity. Ann Rheum Dis 2004;63:386–94. [34] Rekvig OP, Moens U, Sundsfjord A, Bredholt G, Osei A, Haaheim H, et al. Experimental expression in mice and spontaneous expression in human SLE of polyomavirus T-antigen. A molecular basis for induction of antibodies to DNA and eukaryotic transcription factors. J Clin Invest 1997;99:2045–54. [35] Krishnan MR, Jou NT, Marion TN. Correlation between the amino acid position of arginine in VH-CDR3 and specificity for native DNA among autoimmune antibodies. J Immunol 1996;157:2430–9. [36] Detter JC, Nguyen QA, Kingsmore SF. Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR. Nucleic Acids Res 1998;26:4091–2. [37] Umetani N, Hiramatsu S, Hoon DS. Higher amount of free circulating DNA in serum than in plasma is not mainly caused by contaminated extraneous DNA during separation. Ann NY Acad Sci 2006;1075:299–307. [38] Rosenthal AL, Lacks SA. Nuclease detection in SDS–polyacrylamide gel electrophoresis. Anal Biochem 1977;80:76–90.
186
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
[39] Morimoto C, Sano H, Abe T, Homma M, Steinberg AD. Correlation between clinical activity of systemic lupus erythematosus and the amounts of DNA in DNA/anti-DNA antibody immune complexes. J Immunol 1982;129:1960–5. [40] Licht R, van Bruggen MCJ, Oppers-Walgreen B, Rijke TPM, Berden JHM. Plasma levels of nucleosomes and nucleosome-autoantibody complexes in murine lupus – effects of disease progression and lipopolysaccharide administration. Arthritis Rheum 2001;44:1320–30. [41] Tsai CY, Yu CL, Huang DF, Hsieh SC, Tsai YY, Tsai ST. The elevation of plasma DNA in patients with systemic lupus erythematosus is attributable to increased DNA release and defective DNA binding of mononuclear cells. Zhonghua Yi Xue Za Zhi (Taipei) 1994;54:291–9. [42] Atamaniuk J, Hsiao YY, Mustak M, Bernhard D, Erlacher L, Fodinger M, et al. Analysing cell-free plasma DNA and SLE disease activity. Eur J Clin Invest 2010. [43] Bengtsson A, Nezlin R, Shoenfeld Y, Sturfelt G. DNA levels in circulating immune complexes decrease at severe SLE flares-correlation with complement component C1q. J Autoimmun 1999;13:111–9. [44] Albrightson CR. Role of eicosanoids in renal disease. In: Goldstein RS, editor. Mechanisms of injury in renal disease and toxicity. CRC Press; 1994. p. 63–102. [45] Jiang N, Pisetsky DS. The effect of inflammation on the generation of plasma DNA from dead and dying cells in the peritoneum. J Leukoc Biol 2005;77:296–302.
[46] Williams Jr RC, Malone CC, Meyers C, Decker P, Muller S. Detection of nucleosome particles in serum and plasma from patients with systemic lupus erythematosus using monoclonal antibody 4H7. J Rheumatol 2001;28:81–94. [47] Steinman CR. Circulating DNA in systemic lupus erythematosus. Association with central nervous system involvement and systemic vasculitis. Am J Med 1979;67:429–35. [48] Nezlin R, Dayan M, Zinger H, Mozes E. DNA levels in immune complexes circulating in mice with induced systemic lupus erythematosus. Immunol Lett 1999;67:85–90. [49] Nezlin R. A quantitative approach to the determination of antigen in immune complexes. J Immunol Methods 2000;237:1–17. [50] Bergtold A, Gavhane A, D’Agati V, Madaio M, Clynes R. FcR-bearing myeloid cells are responsible for triggering murine lupus nephritis. J Immunol 2006;177:7287–95. [51] Seredkina N, Zykova SN, Rekvig OP. Progression of murine lupus nephritis is linked to acquired renal Dnase1 deficiency and not to up-regulated apoptosis. Am J Pathol 2009;175:97–106. [52] Hedberg A, Fismen S, Fenton KA, Fenton C, Osterud B, Mortensen ES, et al. Heparin exerts a dual effect on lupus nephritis by enhancing enzymatic chromatin degradation and preventing chromatin-binding in glomerular membranes. Arthritis Rheum 2010.
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus Mariann H. Jørgensen a , Ole Petter Rekvig a , Rasmus S. Jacobsen b , Søren Jacobsen b , Kristin A. Fenton a,∗ a b
Molecular Pathology Research Group, Institute of Medical Biology, Faculty of Health Sciences, University of Tromsø, N-9037 Tromsø, Norway Department of Rheumatology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
a r t i c l e
i n f o
Article history: Received 20 December 2010 Received in revised form 5 April 2011 Accepted 11 April 2011 Available online 17 April 2011 Keywords: Anti-dsDNA antibodies Lupus nephritis Chromatin Systemic lupus erythematosus (NZBxNZW)F1 mice
a b s t r a c t Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known. Serum samples from autoimmune (NZBxNZW)F1 mice, healthy BALB/c mice, patients with SLE, RA and normal healthy individuals were analyzed for presence and amount of circulating anti-dsDNA antibodies and nucleosomal DNA. Here we use a quantitative PCR to measure circulating DNA in sera. We demonstrate an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli. The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect the development of lupus nephritis at an early stage. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Systemic lupus erythematosus (SLE) is an autoimmune syndrome that is characterized by the production of anti-DNA antibodies and appearances of immune complex mediated inflammation in various organs [1]. These autoantibodies form complexes with nucleosomes that deposit within the kidney and the skin [2], and the affection of the kidney causes a most serious and potentially fatal complication in human SLE; lupus nephritis [3]. Other nuclear autoantibodies like anti-nucleosome, anti-nucleolin and anti-centromere protein antibodies has been implicated in the development of lupus nephritis [4–6]. However, there is currently no definitive distinction that allows us to separate non-pathogenic from pathogenic anti-dsDNA antibodies. It is therefore of great importance to identify cellular processes and/or available molecular structures that determine anti-dsDNA antibody-binding in the kidney contributing to the induction of nephritis. The presence of anti-chromatin antibodies have been linked to the development of glomerulonephritis and disease activity in SLE [7]. In recent studies we have shown that anti-dsDNA and anti-nucleosome antibodies recognize selectively ectopic, intraglomerular chromatin structures in vivo [8,9]. Autoantibody
∗ Corresponding author. Tel.: +47 77646834; fax: +47 77645350. E-mail address: [email protected] (K.A. Fenton). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.04.006
deposits in vivo are strictly localized to electron-dense structures (EDS) associated with mesangial matrix (MM) and glomerular basement membranes (GBM), and they co-localize with experimental antibodies to chromatin structures and nicked DNA [8–12]. This demonstrates that the antibodies bind to exposed chromatin fragments. Recently, on the basis of a longitudinal study in (NZBxNZW)F1 (B/W) mice we have shown that autoantibodies deposit first in the MM of the glomeruli and, as the disease develops into a full blown nephritis, they deposit in the GBM [13]. Further, we demonstrated that serial injection of anti-dsDNA antibodies into healthy mice induced the formation of immune complex deposits within the MM [14]. These data confirmed that anti-dsDNA antibodies form complexes with nucleosomes that deposit in glomerular membranes [11,12,15]. In SLE, defective clearance of apoptotic nucleosomes has been described [16–18] which is believed to lead to chromatin fragment deposition within glomerular membranes, where they have been shown to bind at high affinity [10]. The origin of the nucleosomes and when they appear is not known. Since anti-DNA antibodies and nucleosomes most likely form complexes in the circulation [19–21], serum is the most relevant biological compartment to perform the present investigations. Plasma DNA has been difficult to study in SLE due to low concentrations present in most SLE specimens [22,23], Amoura et al. measured the circulating plasma levels of nucleosomes by ELISA [24]. They found that the nucleosome levels in plasma of SLE patients were significantly higher than in the
180
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
Table 1 Demographic and clinical characteristics of 99 patients with systemic lupus erythematosus, 57 RA patients and 44 NHC.
Female gender, n (%) Age, yrs Disease duration, yrs SLEDAI score ACR criteria fulfilled – Malar rash, n (%) – Discoid rash, n (%) – Photosensitivity, n (%) – Oral ulcers, n (%) – Arthritis, n (%) – Serositis, n (%) – Renal disease, n (%) – CNS disease, n (%) – Hematological disorder, n (%) – Immunological disorder, n (%) – Antinuclear antibodies, n (%)
Table 2 The primers and probe sequences used for real-time PCR of murine (B1-qPCR) and human (Alu-qPCR) sera.
SLE
RA
NHC
Primer/probe
Sequence
90 (91) 46 (18–83) 12 (0.02–36) 4 (0–20)
47 (82) 53 (20–82) – –
32 (73) 49 (21–72) – –
Mouse B1 forward Mouse B1 reverse Mouse B1 probe Human Alu forward Human Alu reverse Human Alu probe
5 -GCACACCTTTAATCCCAGCA-3 5 -CTCTGTGTAGCCCTGGCTGT-3 5 -GATTTCTGAGTTCGAGGCCA-3 5 -CCTGAGGTCAGGAGTTCGAG-3 5 -CCCGAGTAGCTGGGATTACA-3 5 -TGGTGAAACCCCGTCTCTAC-3
45 (45) 20 (20) 33 (33) 12 (12) 55 (56) 34 (34) 51 (52) 3 (3.0) 72 (73) 86 (87) 96 (97)
Continuous data presented as mean (range).
control group. Chen et al. purified DNA from sera and plasma and quantified the amount using the Picogreen assay and found significantly elevated levels of DNA in SLE patients compared with normal healthy donors [25]. Circulating DNA has been shown to be of low molecular weight, corresponding to mono- and di-nucleosomes [22,23,26,27]. Quantification of nucleosomes as DNA in the circulation by real-time PCR is a widely accepted method. By using Alu or B1 specific primers and probe it is possible to detect all kinds of free and protein bound circulating DNA [28,29]. Alu or B1 elements are short interspersed elements (SINEs). Alu sequences occupy 10.7% of the human genome [30], while B1 sequences in mice comprise 2.7% of the mouse genome [31]. In this study we have investigated if deposition of nucleosomes in the MM and GBM may reflect increased or decreased levels of circulating chromatin. DNA concentrations, Dnase1 activity, anti-dsDNA antibodies, and immune complex deposition in the glomeruli have been analyzed in sera and kidneys from normal and lupus prone mice in addition to sera from SLE patients, RA patients and normal healthy controls (NHC). The data demonstrate an inverse correlation between levels of circulating chromatin and anti-dsDNA antibodies that may reflect glomerular deposition of chromatin-containing immune complexes. 2. Materials and methods 2.1. Human serum samples Serum samples from SLE or rheumatoid arthritis (RA) patients were obtained from Rigshospitalet, The Copenhagen University Hospital. Forty-four normal sera from age and sex matched healthy controls (NHC) of the same ethnic group with no previous history of any significant clinical manifestation were included in this study. This study was approved by the Ethical committee in Copenhagen. Demographic and clinical characteristics of the 99 patients with SLE, 57 RA and NHC 44 are listed in Table 1. 2.2. Animals BALB/c mice were obtained from Harlan (Oxon, England) and (NZBxNZW)F1 (B/W) were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). Treatment and care of animals were conducted in accordance with guidelines of the Norwegian Ethical and Welfare Board for Animal Research, and the institutional review board approved the study.
Amplicon size (bp)
107
115
2.3. Longitudinal study of B/W mice For a longitudinal study, animals were sacrificed approximately every second week (in groups of 3 mice) from the age of 4 weeks old (w.o.) until development of severe proteinuria (≥20 g/l) [13]. Proteinuria was measured using Uristix from Bayer Diagnostics (Leverkusen, Germany) measuring values ranging from +1, ≤0.3 g/l protein in urine, +2, ≤1 g/l; +3, ≤3 g/l and +4, ≤20 g/l; +3 and +4 are regarded as indication of severe nephritis. Serum samples were collected at 2 week intervals and stored at −80 ◦ C. Kidneys were collected at endpoint, fixed in 8% paraformaldehyde and embedded in sucrose for immune electron microscopy (IEM) according to the Tokuyasu method [32]. 2.4. Murine serum samples Serum samples were obtained every week for a time study by making a small incision on the mouse leg and collecting the blood sample using a capillary tube. The blood samples were centrifuged at 4500 × g for 20 min at 4 ◦ C, and sera were stored at −20 ◦ C. When the mice were sacrificed, blood samples were drained from the heart and put directly on ice. The blood samples were centrifuged as described above and sera were stored at −20 ◦ C. 2.5. ELISA and antibodies Serum anti-dsDNA antibodies were detected and controlled by ELISA as described [33,34], using microtiter plates (Nunc MaxiSorp; Nunc, Copenhagen, Denmark) coated with calf thymus dsDNA (10 g/ml in PBS, Sigma–Aldrich). Monoclonal anti-DNA antibody 163p.87 was obtained from Dr. Tony Marion (Memphis TE, USA) [35] and was used as a positive control for primary antibody in the enzyme-linked immunosorbent assay (ELISA) experiments. Antimouse IgG and anti-human IgG peroxidase conjugated antibody was obtained from Sigma–Aldrich (Saint Louis, USA). 2.6. Quantitative real-time PCR (qPCR) The consensus sequences for B1 in mouse and Alu sequences in human were taken from Detter et al. and Umetani et al. [36,37], and the appropriate primers and probes were designed by using the primer design tool from GenScript (http://www.genscript.com). The probes were labeled with a reporter dye (FAM) at its 5 -end and a non-fluorescence quencher and MGB at its 3 -end. Primers and probe were purchased from Applied Biosystems (Foster City, CA, USA) and are listed in Table 2. Sera were diluted 1:2 in a buffer composed of 0.25% Tween 20, 50 mM Tris, and 1 mM EDTA and digested with 0.4 g/l proteinase K at 50 ◦ C for 1 h, followed by 5 min heat inactivation at 95 ◦ C. The samples were centrifuged at 10,000 × g for 20 min and 1 l of the supernatant was used as template for B1-specific-qPCR and 0.2 l was used for the Alu-specific qPCR. The reaction mixture consisted of a template, 0.2 M each of forward, reverse primers and probe, 1× TaqMan FastUniversal PCR Master Mix (2×, Applied
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
181
hydrolyzed DNA was calculated by using the program cellFˆ (Olympus, Oslo, Norway). The values for standards and samples were plotted and the concentrations of Dnase1 in serum samples was calculated using cellFˆ (Olympus). 2.9. Statistics
Fig. 1. B1 qPCR on proteinase K treated sera from 20 w.o. B/W and BALB/c. The serum samples were tested five times by qPCR. Five parallels of each serum sample were tested each time. For each qPCR the mean value and SEM for each mouse are plotted.
Biosystems) in a total reaction volume of 10 l. The qPCR amplification was performed using the ABI 7900 HT Fast Real Time PCR system and was initiated with 95 ◦ C for 20 s, followed by 40 cycles of denaturation at 95 ◦ C for 1 s and annealing at 60 ◦ C for 20 s. The amount of DNA in each sample was determined using a standard curve with serial dilutions (1 ng–0.01 pg) of genomic DNA purified from murine fibroblast cells (A31) and from human full blood. Both DNA preparations were purified by the BioRobot® EZ1 (Qiagen). The results were analyzed by using the SDS 2.3 software program; threshold was set to 0.13 for all runs. Graphs were made in GraphPad Prism 5. The B1 specific qPCR method was validated by testing sera from four B/W mice and three BALB/c mice. These sera were tested five different times with five parallels for each mouse for each run, to determine intra- and inter-assay variations. In general, both intra- and inter-assay variation were regarded as acceptable (see Fig. 1, were sera from 20 w.o. B/W and BALB/c were analyzed). Similar results were obtained using human serum samples (data not shown). 2.7. Dnase I gel zymography DNA degrading activity by Dnase1 was determined after separation of serum proteins in a 10% SDS–polyacrylamide gel containing 100 g/ml heat-denatured salmon sperm DNA (Invitrogen Corp., Carlsbad, CA) as described [38], Eight l of 10× diluted serum sample were mixed with 2 l of 5× sample buffer, and 10 l of each sample was loaded on the gel. Dnase1 (10 U/l) isolated from bovine pancreas (Amersham Biosciences) was diluted to 0.0004 U/l and used as a positive control. Eight l of this dilution was mixed with 2 l of 5× sample buffer to a final concentration of 0.00032 U/l. The running conditions were set to 150 V/25 mA/10 W. Electrophoresis was run for 70 min at 4 ◦ C. The gel was soaked in washing buffer for 1 h at room temperature under constant agitation, and then in reaction buffer at 37 ◦ C for 18 h, and photographed in the UV illuminator Gel Doc 2000 (BioRad). 2.8. Radial enzyme diffusion – total nuclease activity A 1% agarose gel in 1× Dnase reaction buffer was prepared and 150 l heat-denatured salmon sperm DNA (Invitrogen Corp., Carlsbad, CA, 10 g/ml) and 50 l EtBr (1 g/ml) was added. A standard curve was prepared by Dnase1 starting at 0.1 U/l and five steps of five fold serial dilutions. Serum protein concentrations were measured by bicinchoninic acid (BCA) protein assay (Pierce Biochemicals, Rockford, IL, USA) and normalized for total protein content. Three l standard dilutions or normalized serum samples were added to the wells. The gels were incubated in darkness at 37 ◦ C for 18 h. The gels were then photographed in the UV illuminator Gel Doc 2000 (BioRad) and the area of cleared circles of
Unpaired two tailed t-test was performed to compare the means of two sets of measurements. One-way Anova test was performed to compare three or more groups. Spearman correlation analyses were done to see if there could be any connection between chromatin levels and anti-dsDNA antibodies production. 3. Results 3.1. Anti-dsDNA antibody titers and chromatin levels are inversely correlated in circulation of human SLE patients, RA patients and normal healthy controls (NHC) First, we analyzed if serum levels of circulating DNA and antidsDNA antibody correlated with each other in sera from 99 SLE patients, 57 RA patents and 44 NHC (Fig. 2A–C, respectively). The results revealed a significant variance in the anti-dsDNA antibody levels and in the chromatin concentrations (Fig. 2D, for chromatin concentrations). A biplot of the two parameters revealed a negative correlation (R = −0.219, p = 0.0018) demonstrating that high anti-dsDNA antibody titer correlated with low levels of DNA in the circulation (Fig. 2E). 3.2. Chromatin levels in circulation of (NZBxNZW)F1 mice are negatively correlated with anti-dsDNA antibody titers as determined in a longitudinal study Chromatin concentrations estimated by qPCR in sera of BALB/c mice and B/W mice at different ages revealed substantial individual variances (Fig. 3A and B, for BALB/c and B/W mice, respectively). Chromatin levels in sera from BALB/c mice were significantly higher compared with sera from B/W mice producing anti-dsDNA antibodies (Fig. 3C, p = 0.0428). Mice with antibody positive sera were divided into groups based on immune complex deposits in MM only or deposition also in GBM. This group distinction caused an increase in the significantly inverse correlation between circulating DNA levels and anti-dsDNA antibody positive sera taken from mice with MM deposits compared to BALB/c (Fig. 3D, p = 0.0308). Circulating DNA levels were not correlated with anti-dsDNA antibody titers when analyzing all sera from BALB/c and B/W mice irrespective whether producing anti-dsDNA antibodies or not (Fig. 3E). However, in anti-dsDNA antibody positive sera, levels of circulating chromatin were inversely correlated with amount of anti-dsDNA antibodies produced (Fig. 3F). 3.3. Chromatin levels correlate negatively with anti-dsDNA antibodies production, but not with total serum nuclease activity during progression of murine lupus nephritis Considerable individual differences of serum DNA levels were observed between the BALB/c and B/W mice in the different age groups (Fig. 3B). Therefore, sera from five (NZBxNZW)F1 mice taken at different time points from age of 12 w.o. to the development of end-stage renal disease were analyzed by (i) anti-dsDNA antibodies as detected by ELISA, (ii) real-time PCR to measure chromatin levels, and (iii) total serum nuclease activity as determined by radial diffusion nuclease assay. Anti-dsDNA antibody production profile for each mouse is presented in Fig. 4A. Serum chromatin concentrations for each individual mouse demonstrated a cyclical pattern
182
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
Fig. 2. Quantification of anti-dsDNA antibodies and chromatin levels in sera from SLE, RA and NHC. Anti-dsDNA antibodies and chromatin concentrations were measured in native sera and in proteinase K treated sera from SLE patients (A), RA patients (B) and normal healthy donors (NHC) (C) respectively. A comparison of chromatin levels in the three different groups demonstrated a significant lower amount in sera from SLE patients than in RA patients and NHC (unpaired t-test p = 0.0018 and p < 0.0001, respectively, D). A Spearman correlation analysis on all subjects demonstrated a negative correlation of R = −0.219 (p = 0.018) were high amounts of anti-dsDNA antibodies correlated to lower concentrations of chromatin (E).
with decreasing levels at the end point (Fig. 4B). The activity of circulating total nucleases showed the same cyclical pattern as chromatin levels (Fig. 4C). Chromatin levels plotted against antidsDNA antibody levels revealed an inverse correlation between the 2 parameters (Fig. 4D). The chromatin levels showed no correlation with total serum nuclease activity (Fig. 4E). Dnase1 zymography corresponded to the observed total nuclease activity (data not shown). DNA levels measured in the circulation of BALB/c mice from 22 to 36 w.o. was significantly higher than DNA levels in the circulation of B/W mice of the same age, in which presence of anti-dsDNA antibodies correlated inversely with DNA concentration (Fig. 5A and B). There were no significant differences in the levels of total nuclease activity in sera from B/W compared to sera from BALB/c (Fig. 5C). Interestingly, the levels of total nuclease activity in sera were more stable in BALB/c than in sera of B/W mice (Fig. 5C). 4. Discussion In this study, we determined the impact of three parameters that may in an interdependent manner have impact on devel-
opment of SLE and lupus nephritis. These parameters are levels of circulating DNA, anti-dsDNA antibodies and serum nucleases. One perception from this study is reduced DNA concentration in sera from SLE patients compared to RA patients and NHC, and an inverse correlation between DNA concentration and anti-dsDNA antibody levels. This is in contrast to results published by other groups [22,24,25,27,39–42]. For example, Amoura et al. measured significantly higher levels of nucleosomes in plasma of 13 of 58 SLE patients compared to the healthy individuals [24]. Similar results were obtained using the Picogreen assay to quantify DNA in sera and plasma of SLE patients [25]. However, none of the 13 patients with elevated DNA concentration had detectable antinucleosome or anti-dsDNA antibodies. Interestingly, Amoura et al. demonstrated that there was an inverse correlation between DNA concentration, anti-nucleosome antibodies and anti-dsDNA antibodies when the entire population of 58 SLE patients was compared [24]. Bengtsson et al. measured DNA levels in circulating immune complexes by a quantitative immunochemical assay and found a decrease in DNA concentration at severe SLE flares [43]. Notably, they demonstrated an inverse correlation between immunecomplexed DNA levels and anti-dsDNA antibody concentrations
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
183
Fig. 3. Circulating chromatin levels in groups of BALB/c control mice and B/W mice are presented. Sera taken consecutively every second week in groups of 3 were analyzed for levels of circulating chromatin (A and B for BALB/c and B/W mice respectively). There was a significant difference in serum chromatin concentrations in sera from BALB/c mice compared to anti-dsDNA positive B/W sera (C, unpaired t-test p = 0.0428). Dividing the anti-dsDNA positive sera into sera from mice with MM deposits or GBM deposits the significant difference between BALB/c and B/W with MM deposits increased (D, p = 0.0308). The chromatin levels in all BALB/c and B/W sera were not correlated with anti-dsDNA antibody levels (E).
measured by the following methods: Critidiae Luciliae assay, ELISA, plasmid DNA-based assay and Farr assay. The inverse relationship between DNA and anti-dsDNA antibodies in plasma from lupus patients was also observed by Coubrey-Hoyer et al. [22]. However many patients had high levels of plasma DNA and anti-dsDNA antibodies without clinical nephritis. This suggests that other factors than the presence of chromatin and anti-dsDNA antibodies is important in the initiation of glomerular damage in SLE. Analyzing serum levels of circulating chromatin over time in B/W mice similarly revealed a weak inverse correlation with the production of anti-dsDNA antibodies. Due to the design of the clinical part of our study, we have not been able to address if similar time dependent dynamics are present in patients with SLE. However, since there seem to be a cyclical increase and decrease in the amount of circulating chromatin measured in mice, this may be important to investigate as this may explain the flares of the disease often seen in SLE.
Our findings in SLE patients demonstrating an inverse correlation between anti-dsDNA antibody levels and circulating concentrations of DNA were confirmed in lupus prone B/W mice. High levels of anti-dsDNA antibodies corresponded to reduced levels of DNA in the circulation. Since this was limited to mice producing anti-dsDNA antibodies, reduced levels of DNA in circulation also correlated with progression of lupus and lupus nephritis. However, there was no significant differences in levels of circulating DNA between anti-dsDNA antibody negative B/W and anti-dsDNA positive B/W, in contrast to the inverse correlation between anti-dsDNA antibodies and circulating DNA between BALB/c and B/W mice. This discrepancy may be explained by masking the production of anti-dsDNA antibodies by the formation of immune complexes and deposition in the kidney [13]. A masked increase of circulating DNA may be explained by the deposition of the immune complexes containing chromatin. It is reasonable
184
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
Fig. 4. Anti-dsDNA antibody levels, chromatin concentrations and total nuclease activity in sera of five B/W mice taken every week from 12 w.o. until development of proteinuria. Anti-dsDNA antibodies were measured by ELISA and individual levels for each mouse (T1–5) are shown in (A). The individual mean ± SEM of circulating chromatin in sera of the five mice taken at different time points (age) demonstrated a cyclical pattern with decreasing amount at end point (B). The total nuclease activity demonstrated the same cyclical pattern over time (C). A Spearman correlation analysis demonstrated a negative correlation R = −0.306, p = 0.003, were high amounts of anti-dsDNA antibodies correlated to lower concentrations of chromatin (D). There was no correlation between chromatin concentration and total nuclease activity, R = 0.125, p = 0.267 (E).
to assume that immune complex deposition in the glomeruli stimulates an inflammatory cascade characterized by complement activation and neutrophil granulocytes and macrophage infiltration [44]. Progressive inflammation can cause release of chromatin from the tissue and cells [45], and may explain the higher chromatin concentration in sera from mice with GBM deposits in contrast to levels in sera from B/W mice with immune complex deposits only located in MM. In line with this, Williams et al. demonstrated that the levels of circulating nucleosomes were raised in SLE patients with very active central nervous system and renal involvement [46]. However, this is in contrast to our overall results and to previously results from Bengtsson et al. showing decreased levels of circulating nucleosomes during flares of the disease [43]. The inverse correlation between DNA concentration and antidsDNA antibodies in the circulation demonstrated in this study, as
Fig. 5. Chromatin levels and total nuclease activity measured consecutively in the circulation of BALB/c and B/W mice. Serum levels of chromatin in sera from BALB/c mice taken from the age of 22–36 weeks were compared with levels in sera from age-matched B/W mice (A). There was a significantly higher amount of circulating chromatin fragments in sera from BALB/c mice compared to sera from B/W mice (B) (unpaired t-test p < 0.0001). There was no significant difference between the total nuclease activity in sera taken over time from BALB/c and B/W mice (C).
well as by others [22,43,47–49] suggest that anti-dsDNA antibodies influence the levels of circulating nucleosomes. Anti-dsDNA antibodies may form immune complexes with chromatin fragments in the circulation which are possible cleared by a combination of phagocytosis and by deposition in e.g. the kidneys. Such a process indicates that initial events in progressive lupus nephritis involve production of anti-dsDNA/nucleosome antibodies that bind available nucleosomes in circulation, and capture of the formed immune complexes by mesangial cells through binding to Fc␥R [50] with subsequent release to the mesangial matrix. In this microenvironment the immune complexes may trigger inflammatory pathways having detrimental effects on glomeruli with renal failure as the
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
end result. Subsequent shut down of renal nucleases may further facilitate this process [13,51]. Thus, progressive lupus nephritis may have the origin in the fatal meeting of 2, by themselves nonpathogenic factors, anti-dsDNA antibodies and circulating DNA. As single factors, they are epiphenomenons with no pathogenic impact; together they start the processes that threaten the function of the kidneys. This hypothesis has its origin in a recent longitudinal study of progressive lupus nephritis in B/W mice [13]. We have recently published data on the effect of heparin treatment leading to enhanced nucleosome degradation and reduced chromatin deposition within the MM and the GBM of glomeruli of (NZBXNZW)F1 mice [52]. Heparin binds to chromatin and makes it more susceptible for degradation by both proteases and nucleases. This treatment led to delayed production of anti-dsDNA antibodies and reduced immune complex deposition within the mesangial matrix and glomerular basement membrane in heparin treated B/W mice compared to untreated animals. The important observation in this study was that increased variation of circulating chromatin levels in heparin treated mice were observed compared to non-treated control mice. A prevention of the development of lupus nephritis could thus hypothetically be achieved by hindering the process of immune complex formation by anti-dsDNA antibodies and chromatin in the circulation or the deposition of them to the mesangial matrix and GBM can be hindered. This makes the immune complexes or the deposition of them a possible target for treatment of lupus nephritis.
Acknowledgments We are grateful for the anti-dsDNA mAb from Tony N Marion. Berit Tømmerås, Natalya Seredkina and Annica Hedberg are appreciated for their technical help. This work was funded by the Foundation for Health and Rehabilitation through the Norwegian Rheumatology Organization (project 2008/2/0229) (KF), Northern Norway Regional Health Authoroty medical Research Progema (OPR, Grant # SFP-100-04, SFP 100-04) and the Novo Nordisk Foundation (SJ). This study was approved by the Ethical committee in Copenhagen.
References [1] Lahita R. The clinical presentation of systemic lupus erythematosus. In: Lahitha R, editor. Systemic lupus erythematosus. San Diego: Academic Press; 1999. p. 325–36. [2] Fismen S, Hedberg A, Fenton KA, Jacobsen S, Krarup E, Kamper AL, et al. Circulating chromatin–anti-chromatin antibody complexes bind with high affinity to dermo-epidermal structures in murine and human lupus nephritis. Lupus 2009;18:597–607. [3] Balow JE, Boumpas DT, Austin III HA. Systemic lupus erythematosus and the kidney. In: Lahita RG, editor. Systemic lupus erythematosus. New York: Elsevier Academic Press; 2004. p. 877–912. [4] Manson JJ, Ma A, Rogers P, Mason LJ, Berden JH, d. van V, et al. Relationship between anti-dsDNA, anti-nucleosome and anti-alpha-actinin antibodies and markers of renal disease in patients with lupus nephritis: a prospective longitudinal study. Arthritis Res Ther 2009;11:R154. [5] Fritzler MJ, Rattner JB, Luft LM, Edworthy SM, Casiano CA, Peebles C, et al. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F. Autoimmun Rev 2011;10:194–200. [6] Minota S, Jarjour WN, Suzuki N, Nojima Y, Roubey RA, Mimura T, et al. Autoantibodies to nucleolin in systemic lupus erythematosus and other diseases. J Immunol 1991;146:2249–52. [7] Gomez-Puerta JA, Burlingame RW, Cervera R. Anti-chromatin (antinucleosome) antibodies: diagnostic and clinical value. Autoimmun Rev 2008;7:606–11. [8] Kalaaji M, Mortensen E, Jorgensen L, Olsen R, Rekvig OP. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells. Am J Pathol 2006;168:1779–92. [9] Kalaaji M, Fenton KA, Mortensen ES, Olsen R, Sturfelt G, Alm P, et al. Glomerular apoptotic nucleosomes are central target structures for nephritogenic antibodies in human SLE nephritis. Kidney Int 2007;71:664–72.
185
[10] Mjelle JE, Rekvig OP, Fenton KA. Nucleosomes possess a high affinity for glomerular laminin and collagen IV and bind nephritogenic antibodies in murine lupus-like nephritis. Ann Rheum Dis 2007;66:1661–8. [11] Kramers C, van Bruggen MCJ, Walgreen B, Elema JD, Kallenberg CGM, van den Born J, et al. Nucleosomes and histones are present in glomerulardeposits in human lupus nephritis. J Am Soc Nephrol 1995;6:425. [12] Malide D, Londono I, Russo P, Bendayan M. Ultrastructural localization of DNA in immune deposits of human lupus nephritis. Am J Pathol 1993;143:304– 11. [13] Fenton K, Fismen S, Hedberg A, Seredkina N, Fenton C, Mortensen ES, et al. Anti-dsDNA antibodies promote initiation, and acquired loss of renal Dnase1 promotes progression of lupus nephritis in autoimmune (NZBxNZW)F1 mice. PLoS One 2009;4:e8474. [14] Fenton KA, Tommeras B, Marion TN, Rekvig OP. Pure anti-dsDNA mAbs need chromatin structures to promote glomerular mesangial deposits in BALB/c mice. Autoimmunity 2010;43:179–88. [15] van Bruggen MC, Kramers C, Walgreen B, Elema JD, Kallenberg CG, van den Born J, et al. Nucleosomes and histones are present in glomerular deposits in human lupus nephritis. Nephrol Dial Transplant 1997;12:57–66. [16] Herrmann M, Voll RE, Zoller OM, Hagenhofer M, Ponner BB, Kalden JR. Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. Arthritis Rheum 1998;41:1241–50. [17] Baumann I, Kolowos W, Voll RE, Manger B, Gaipl U, Neuhuber WL, et al. Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus. Arthritis Rheum 2002;46:191–201. [18] Gaipl US, Munoz LE, Grossmayer G, Lauber K, Franz S, Sarter K, et al. Clearance deficiency and systemic lupus erythematosus (SLE). J Autoimmun 2007;28:114–21. [19] Liou LB, Chao WJ. Changing cytokine patterns in systemic lupus: A prospective longitudinal study. J Microbiol Immunol Infect 2010;43:18–25. [20] Emlen W, Mannik M. Clearance of circulating DNA–anti-DNA immune complexes in mice. J Exp Med 1982;155:1210–5. [21] Mannik M. Mechanisms of tissue deposition of immune complexes. J Rheumatol Suppl 1987;14(Suppl. 13):35–42. [22] Coubrey-Hoyer A, Okarma TB, Holman HR. Partial purification and characterization of plasma DNA and its relation to disease activity in systemic lupus erythematosus. Am J Med 1984;77:23–34. [23] Steinman CR. Circulating DNA in systemic lupus erythematosus. Isolation and characterization. J Clin Invest 1984;73:832–41. [24] Amoura Z, Piette JC, Chabre H, Cacoub P, Papo T, Wechsler B, et al. 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 1997;40:2217–25. [25] Chen JA, Meister S, Urbonaviciute V, Rodel F, Wilhelm S, Kalden JR, et al. Sensitive detection of plasma/serum DNA in patients with systemic lupus erythematosus. Autoimmunity 2007;40:307–10. [26] Sano H, Morimoto C. DNA isolated from DNA/anti-DNA antibody immune complexes in systemic lupus erythematosus is rich in guanine-cytosine content. J Immunol 1982;128:1341–5. [27] Raptis L, Menard HA. Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus. J Clin Invest 1980;66:1391–9. [28] Holdenrieder S, Stieber P, Chan LY, Geiger S, Kremer A, Nagel D, et al. Cellfree DNA in serum and plasma: comparison of ELISA and quantitative PCR. Clin Chem 2005;51:1544–6. [29] Umetani N, Kim J, Hiramatsu S, Reber HA, Hines OJ, Bilchik AJ, et al. Increased integrity of free circulating DNA in sera of patients with colorectal or periampullary cancer: direct quantitative PCR for ALU repeats. Clin Chem 2006;52:1062–9. [30] Umylny B, Presting G, Efird JT, Klimovitsky BI, Ward WS. Most human Alu and murine B1 repeats are unique. J Cell Biochem 2007;102:110–21. [31] Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, et al. Initial sequencing and comparative analysis of the mouse genome. Nature 2002;420:520–62. [32] Tokuyasu KT, Singer SJ. Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol 1976;71:894–906. [33] Haugbro K, Nossent JC, Winkler T, Figenschau Y, Rekvig OP. Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity. Ann Rheum Dis 2004;63:386–94. [34] Rekvig OP, Moens U, Sundsfjord A, Bredholt G, Osei A, Haaheim H, et al. Experimental expression in mice and spontaneous expression in human SLE of polyomavirus T-antigen. A molecular basis for induction of antibodies to DNA and eukaryotic transcription factors. J Clin Invest 1997;99:2045–54. [35] Krishnan MR, Jou NT, Marion TN. Correlation between the amino acid position of arginine in VH-CDR3 and specificity for native DNA among autoimmune antibodies. J Immunol 1996;157:2430–9. [36] Detter JC, Nguyen QA, Kingsmore SF. Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR. Nucleic Acids Res 1998;26:4091–2. [37] Umetani N, Hiramatsu S, Hoon DS. Higher amount of free circulating DNA in serum than in plasma is not mainly caused by contaminated extraneous DNA during separation. Ann NY Acad Sci 2006;1075:299–307. [38] Rosenthal AL, Lacks SA. Nuclease detection in SDS–polyacrylamide gel electrophoresis. Anal Biochem 1977;80:76–90.
186
M.H. Jørgensen et al. / Immunology Letters 138 (2011) 179–186
[39] Morimoto C, Sano H, Abe T, Homma M, Steinberg AD. Correlation between clinical activity of systemic lupus erythematosus and the amounts of DNA in DNA/anti-DNA antibody immune complexes. J Immunol 1982;129:1960–5. [40] Licht R, van Bruggen MCJ, Oppers-Walgreen B, Rijke TPM, Berden JHM. Plasma levels of nucleosomes and nucleosome-autoantibody complexes in murine lupus – effects of disease progression and lipopolysaccharide administration. Arthritis Rheum 2001;44:1320–30. [41] Tsai CY, Yu CL, Huang DF, Hsieh SC, Tsai YY, Tsai ST. The elevation of plasma DNA in patients with systemic lupus erythematosus is attributable to increased DNA release and defective DNA binding of mononuclear cells. Zhonghua Yi Xue Za Zhi (Taipei) 1994;54:291–9. [42] Atamaniuk J, Hsiao YY, Mustak M, Bernhard D, Erlacher L, Fodinger M, et al. Analysing cell-free plasma DNA and SLE disease activity. Eur J Clin Invest 2010. [43] Bengtsson A, Nezlin R, Shoenfeld Y, Sturfelt G. DNA levels in circulating immune complexes decrease at severe SLE flares-correlation with complement component C1q. J Autoimmun 1999;13:111–9. [44] Albrightson CR. Role of eicosanoids in renal disease. In: Goldstein RS, editor. Mechanisms of injury in renal disease and toxicity. CRC Press; 1994. p. 63–102. [45] Jiang N, Pisetsky DS. The effect of inflammation on the generation of plasma DNA from dead and dying cells in the peritoneum. J Leukoc Biol 2005;77:296–302.
[46] Williams Jr RC, Malone CC, Meyers C, Decker P, Muller S. Detection of nucleosome particles in serum and plasma from patients with systemic lupus erythematosus using monoclonal antibody 4H7. J Rheumatol 2001;28:81–94. [47] Steinman CR. Circulating DNA in systemic lupus erythematosus. Association with central nervous system involvement and systemic vasculitis. Am J Med 1979;67:429–35. [48] Nezlin R, Dayan M, Zinger H, Mozes E. DNA levels in immune complexes circulating in mice with induced systemic lupus erythematosus. Immunol Lett 1999;67:85–90. [49] Nezlin R. A quantitative approach to the determination of antigen in immune complexes. J Immunol Methods 2000;237:1–17. [50] Bergtold A, Gavhane A, D’Agati V, Madaio M, Clynes R. FcR-bearing myeloid cells are responsible for triggering murine lupus nephritis. J Immunol 2006;177:7287–95. [51] Seredkina N, Zykova SN, Rekvig OP. Progression of murine lupus nephritis is linked to acquired renal Dnase1 deficiency and not to up-regulated apoptosis. Am J Pathol 2009;175:97–106. [52] Hedberg A, Fismen S, Fenton KA, Fenton C, Osterud B, Mortensen ES, et al. Heparin exerts a dual effect on lupus nephritis by enhancing enzymatic chromatin degradation and preventing chromatin-binding in glomerular membranes. Arthritis Rheum 2010.