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Rheumatology Advance Access originally published online on December 26, 2007
Rheumatology 2008 47(2):145-149; doi:10.1093/rheumatology/kem327
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Role of pathogenic auto-antibody production by Toll-like receptor 9 of B cells in active systemic lupus erythematosus

S. Nakano, S. Morimoto, J. Suzuki, K. Nozawa, H. Amano, Y. Tokano and Y. Takasaki

Department of Rheumatology and Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan.

Correspondence to: S. Nakano, Department of Rheumatology and Internal Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan. E-mail: soubey{at}med.juntendo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. Toll-like receptor 9 (TLR9) is a pattern-associated receptor functioning in innate immunity that may be involved in the recognition of self-antigens and the production of pathogenic auto-antibodies. Therefore, we examined the expression of TLR9 in systemic lupus erythematosus (SLE) to determine whether TLR9 is involved in the production of pathogenic auto-antibodies.

Methods. B cells were collected from patients with active SLE, and subjected to analysis of the TLR9 molecule using flow cytometry fluorescence activated cell sorting (FACS) and TLR9 mRNA by reverse-transcriptase polymerase chain reaction. SLE B cells were stimulated with CpG-ODN, and subsequent cytokine and anti-dsDNA antibody production was measured by enzyme-linked immunosorbent assay.

Results. The expression and mRNA level of TLR9 on B cells was up-regulated in SLE patients, and SLE disease activity index (SLEDAI) and CH50 were correlated with TLR9 expression on CD20+ B cells. Moreover, TLR9–CpG interaction enhanced the production of anti-dsDNA antibody and IL-10.

Conclusions. The present study demonstrated that higher expression of TLR9 on peripheral blood B cells from patients with active SLE was significantly correlated with CH50 and SLEDAI to TLR9, and induced the production of anti-dsDNA antibody and IL-10 by TLR9–CpG ligation. These results suggest that an abnormality of innate immunity plays a crucial role in the pathology of SLE, and that blockade of CpG–TLR9 interaction may be a new therapeutic approach for SLE.

KEY WORDS: SLE, B cells, Toll-like receptor 9, CpG-DNA, Anti-dsDNA antibody, IL-10, SLEDAI, CH50, Innate immunity


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Autoimmune diseases are associated with various immunological abnormalities, such as an increased number of activated B cells and auto-antibody production, and in patients with systemic lupus erythematosus (SLE) B-cell hyperactivity is a central feature [1, 2]. Although it is considered that autoimmune disease is mainly related to acquired immunity, a recent study has shown that some abnormality of the innate system may also be involved [3].

The Toll-like receptor (TLR) contributes to innate immunity, and its gene has been cloned in humans. The human TLR has 10 subunits, each recognizing a different causative factor [4, 5]. When these causative factors react with TLR, MyD88 present in the cytoplasmic compartment is activated, and nuclear localization of nuclear factor-kappa B is promoted. This in turn leads to production of inflammatory cytokines such as tumour necrosis factor-{alpha} or interleukin-6 (IL-6) [4]. TLR9 is present only in plasmacytoid dendritic cells (pDCs) and B cells, and is a receptor for microbial CpG-DNA [5]. It recognizes a single-stranded ‘CpG motif’, consisting of unmethylated CpG dinucleotides flanked by particular bases and it is not generally present in mammalian cells, including those of humans [6, 7]. However, our previous study has shown that methylation of DNA is decreased in patients with SLE, suggesting that CpG-DNA is related to the SLE pathogenesis [8]. Furthermore, a recent study has shown that anti-DNA auto-antibody production is impaired in TLR9 gene-knockout lupus-prone mice [9]. However, it has not been clarified whether TLR9 contributes directly to the pathogenesis or clinical features of human SLE.

B cells play an important role in auto-regulation of humoral immune responses, and B cells from patients with active SLE and from lupus-prone mice have an intrinsic tendency to over-react to immunological stimulation during anti-genemic challenge. This has led to a novel hypothesis regarding therapeutic approaches that might interfere with the development and progression of SLE [10]. Since TLR9 recognizes unmethylated CpG motifs characteristic of bacterial DNA and is involved in the immediate response to a wide range of microbial organisms, B cells may, in addition to their role as antibody-producing cells during the adaptive immune response, respond to pathogens in a manner associated with the innate branch of immune defence. Inducible expression of TLR9 in B cells may thus provide a link between the innate and adaptive branches of the immune system [11].

Furthermore, it has been reported that CpG-ODN can enhance TLR9 mRNA expression in activated human B cells [12]. In addition, it has been reported that TLR9 controls anti-DNA auto-antibody production in murine lupus [9].

In the present study, we examined the level of TLR9 expression on peripheral blood B cells from patients with active SLE and its correlation with clinical parameters. We also investigated whether CpG ligation of TLR9 is involved in pathogenic auto-antibody production in active SLE.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 Acknowledgements
 References
 
Patients
We obtained samples of peripheral blood from 19 SLE patients (Table 1), age- and sex-matched healthy donors and patients with rheumatoid arthritis (RA) as a disease control. Informed consent was obtained from all patients and healthy donors. Eleven blood samples were drawn from SLE patients before treatment. Eight patients had been receiving steroid therapy with prednisolone at 5–15 mg/day. All SLE patients fulfilled the 1997 revised criteria of the American College of Rheumatology (ACR) [13]. Disease activity in the SLE patients was assessed using the ACR SLE disease activity index (SLEDAI) [14].


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  		TABLE 1. Profile of patients with SLE

 
Cell preparation
For isolation of peripheral blood B cells, 5 ml of peripheral blood was labelled with 40 µl of anti-human CD20 antibody coupled with colloidal paramagnetic microbeads (Miltenyi Biotech, Bergisch-Gladbach, Germany) and isolated using AutoMACS (Miltenyi Biotech). B cells were isolated at a purity of >93%, as assessed by flow cytometric analysis.

TLR9 mRNA analysis
Total RNA was isolated from 1 x 106 B cells using an RNeasy Mini kit (QIAGEN, Valencia, CA, USA). Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed in a single 50 µl reaction volume containing 25 µl of One-step RT-PCR SYBR Green Master Mix (Applied BioSystems, Foster City, CA, USA) with 1.0 µl of AmpliTaq Gold DNA polymerase (Applied BioSystems), 0.25 µl of 40x MultiScribe reverse transcriptase (Applied BioSystems), 10 nM forward and reverse primers for TLR9 (5'-ATGGGTTTCTGCCGC and 3'-GAAGAGATGCCGCAGG) or β-actin (5'-GGACTTCGAGCAAGAGATG and 3'-AGCACTGTGTTGGCGTACA) and 5 µg of RNA. TLR9 was measured with an ABI PRISM 7500 Sequence Detection System (Applied BioSystems). TLR9 levels were normalized to β-actin for each.

Flow cytometric analysis
We carried out cell surface staining by adding 10 µl of anti-human CD20, CD80 and CD86 antibody (PharMingen, San Diego, CA, USA) conjugated with PE to the B cells (5 x 105/ml), and incubating them for 30 min on ice. For intracellular staining, we used IntraprepTM (Beckman Coulter, Miami, FL, USA) for fixation and membrization. Then, intracellular staining was performed with 5 µl of anti-human TLR9 antibody (26C593) (Imgenex, San Diego, CA, USA) conjugated with fluorescein isothiocyanate (FITC), and incubated for 30 min in accordance with the manufacturer's instructions. Two-colour analysis was then performed using FacsAria (Becton Dickinson, Mountain View, CA, USA).

Detection of anti-dsDNA antibody and cytokine
Isolated SLE B cells differentiated for 3 days were re-plated in 96-well round-bottom plates at 1 x 105 cells/well, then stimulated with medium, ODN2006 (5 µM) and ODN2216 (5 µM). Complete medium (CM) consisted of RPMI-1640 containing L-alanyl-L-glutamine dipeptide supplemented with 5–10% fetal calf serum (FCS; ICN Biomedicals, Inc.), 5 x 10–5 M 2-ME (Sigma) and antibiotics (penicillin 100 U/ml, streptomycin 100 µg/ml, GIBCO BRL). The stimulatory CpG-ODNs were ODN2006 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3') and ODN2216 (5'-GGGGGACGATCGTCGGGGG-3') [15].

Anti-dsDNA antibody (Bio-Rad) and cytokine (IL-10, R&D Systems, Minneapolis, MN, USA) concentrations were quantified by enzyme-liked immunosorbent assay (ELISA) in accordance with the manufacturer's instructions.

Statistical analysis
Statistical analysis was performed using the Mann–Whitney U-test and Pearson's correlation coefficient. Statistical significance was defined as a P-value of <0.05.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 Acknowledgements
 References
 
Expression of TLR9 on B cells from active SLE
We first examined the expression of TLR9 mRNA on B cells from patients with active SLE, and found that this was higher than on B cells from healthy donors and disease controls (P < 0.01) (Fig. 1A and B). We then examined the surface expression of TLR9 on B cells, but none was detected on B cells from any of the three groups. We then examined intracellular expression of TLR9 in B cells, and found that this was strikingly and significantly (P < 0.01) higher in CD20+ B cells from SLE patients than in those from healthy controls and disease controls by mean fluorescence intensity (MFI) (Fig. 1C and D).


Figure 1
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  		FIG. 1. Expression of TLR9 on B cells from active SLE patients and healthy donors. (A) Expression of TLR9 mRNA in active SLE patients was significantly higher than in healthy individuals (**P < 0.01). Bars show the mean ± S.D. (B) TLR9 or β-actin samples (10 µl) were used as template for RT-PCR. The products were electrophoresed on 2% Tris base, boric acid and EDTA (TBE) agarose gels containing 0.5 µg/ml ethidium bromide and visualized under ultraviolet (UV). Negative control was 50 µl of total RNA isolated from 1 x 106 CD4+ T cells. (C) Representative staining pattern of TLR9 on peripheral B cells from a healthy donor, a patient with active SLE and a patient with RA. Peripheral blood mononuclear cells were stained with monoclonal antibodies against TLR9 and CD20. (D) TLR9 molecule in B cells from patients with active SLE and healthy controls was analysed by flow cytometry. The MFI of TLR9 in active SLE patients was significantly higher than that in healthy donors and disease controls (**P < 0.01).

 
Next, we examined the relationship between TLR9 expression on CD20+ B cells from patients with active SLE and disease activity. The intracellular expression of TLR9 was significantly (P < 0.05) lower in patients whose disease activity had decreased after treatment (Fig. 2). However, the expression of TLR9 mRNA on B cells from patients with active SLE was not significantly decreased.


Figure 2
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  		FIG. 2. Decreased expression of TLR9 on B cells from active SLE patients after treatment. Post-treatment expression of TLR9 on B cells from active SLE patients. The MFI of TLR9 in post-treatment SLE patients was significantly lower than that before treatment (**P < 0.01). Pre-Tx: pre-treatment; Post-Tx: post-treatment.

 
Moreover, we investigated the relationship between the expression of TLR9 on B cells from patients with active SLE and laboratory parameters (SLEDAI, anti-dsDNA antibody, CH50), and found significant correlations with SLEDAI (Fig. 3A) (P < 0.01, R = 0.929) and CH50 by MFI (Fig. 3B) (P < 0.05, R = 0.631). However, there was no significant correlation between the expression of TLR9 mRNA and anti-dsDNA antibody.


Figure 3
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  		FIG. 3. Correlation between TLR9 molecule and active B cells from active SLE. (A) Correlation between MFI of TLR9 and SLEDAI on B cells from patients with active SLE (P < 0.01, R = 0.929). (B) Correlation between MFI of TLR9 and CH50 on B cells from patients with active SLE (P < 0.05, R = 0.631).

 
CpG induces anti-dsDNA antibody and IL-10 production
We next investigated whether TLR9–CpG interaction was related to SLE activity and its activation. ODN2006 enhanced the expression of TLR9 on B cells from patients with active SLE, and was correlated with production of anti-dsDNA antibody (Fig. 4A) (P < 0.05). Moreover, IL-10 production was significantly increased (Fig. 4B; P < 0.05) by ODN2006 ligation.


Figure 4
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  		FIG. 4. TLR9–CpG ligation enhances production of anti-dsDNA antibody and IL-10 from B cells of SLE patients. (A) ODN2006 enhanced the expression of TLR9 on SLE B cells and was correlated with higher production of anti-dsDNA antibody (P < 0.05). (B) IL-10 production was significantly increased (P < 0.05) by ODN2006 ligation. Isolated SLE B cells were stimulated with medium, ODN2006 (5 µM) or ODN2216 (5 µM). IL-10 and anti-dsDNA antibody production was measured by ELISA. NC: healthy donor.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
Acknowledgements
 References
 
In the present study, we demonstrated that higher expression of TLR9 on peripheral blood B cells from patients with active SLE was significantly correlated with CH50 and SLEDAI to TLR9, and induced anti-dsDNA antibody and IL-10 production by TLR9–CpG ligation. Although a recent study has shown that apoptosis via TLR9 on B cells may play a role in the pathogenesis of SLE [16], our present study demonstrated that production of anti-dsDNA antibody is related to SLE pathogenesis through ligation of CpG to TLR9 on B cells.

Since the TLR9 molecule is produced by mRNA and usually exists in the cytoplasm, it is difficult to examine by flow cytometric analysis. Using cytoplasmic staining and RT-PCR, the present study confirmed that the expression of TLR9 was increased on B cells from patients with active SLE, and that this was correlated with disease activity. These results suggest that innate immunity might play a role in the pathogenesis of SLE through TLR9 on B cells. TLR induced the expression of co-stimulatory molecules, as represented by CD80 and CD86, and the expression of TLR9 was correlated with that of CD86 on B cells (data not shown). Since the expression of CD86 on B cells is commonly increased in active SLE, it is possible that the increased expression of TLR9 induces the initial activation of B cells.

It has been suggested that TLR9 expression on B cells plays a role in SLE pathogenesis through the induction of anti-dsDNA antibody. As described previously, the ligand of TLR9 is CpG that is a product of abnormal methylation of DNA, and commonly recognized in SLE. A previous study has shown that methylation of DNA is decreased in SLE patients, suggesting that CpG-DNA is related to the pathogenesis of SLE [8]. Our present study showed that production of anti-dsDNA antibody occurred through ligation of CpG to TLR9 on B cells from patients with active SLE. Although the mechanism responsible for production of anti-dsDNA antibody is heterogeneous, we think that the interaction between CpG and TLR9 plays a pivotal role.

The production of anti-dsDNA antibody may not be induced directly by CpG, but may require various processes including the differentiation of B cells. In order to investigate this issue, we examined various cytokines in the culture supernatant. Surprisingly, we found that IL-10 production from patients with active SLE B cells was increased by stimulation with CpG. Although it has recently been established that IL-10 is a regulatory cytokine, it was originally reported to be a factor inducing antibody production in certain diseases, especially SLE. Indeed, injection of an IL-10 blocking antibody has been reported to suppress disease activity in lupus mice [17]. Futhermore, it has been reported that B cells from SLE patients produce IL-10 [18], and that this cytokine induces the production of anti-dsDNA antibody [17].

Abnormality of DNA methylation easily induces the transcription of mRNA from DNA, and it has been shown previously that this abnormality commonly occurs in SLE [8]. This finding suggests that the abnormal methylation induces increased transcription of the TLR9 gene, and that the CpG produced as a result reacts with the highly expressed TLR9.

The role of innate immunity in autoimmune disease is still unclear. We have demonstrated that expression of TLR9 on B cells is increased in human SLE, and that this increased expression is closely related to disease activity. We suggest that abnormality of innate immunity also plays a crucial role in the pathology of SLE, and that blockade of CpG–TLR9 interaction may be a promising new therapeutic approach for SLE.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
References
 
We are grateful to D. Douglas for assistance in preparing the manuscript.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 

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  3. Anders HJ. A Toll for lupus. Lupus (2005) 14:417–22.[Abstract/Free Full Text]
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  8. Sekigawa I, Okada M, Ogasawara H, Kaneko H, Hishikawa T, Hashimoto H. DNA methylation in systemic lupus erythematosus. Lupus (2003) 12:79–85.[Abstract/Free Full Text]
  9. Christensen SR, Kashgarian M, Alexopoulou L, Flavell RA, Akira S, Shlomchik MJ. Toll-like receptor 9 controls anti-DNA autoantibody production in murine lupus. J Exp Med (2005) 202:321–31.[Abstract/Free Full Text]
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  15. Krug A, Rothenfusser S, Hornung V, et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol (2001) 31:2154–63.[CrossRef][Web of Science][Medline]
  16. Papadimitraki ED, Choulaki C, Koutala E, et al. Expansion of toll-like receptor 9-expressing B cells in active systemic lupus erythematosus: implications for the induction and maintenance of the autoimmune process. Arthritis Rheum (2006) 54:3601–11.[CrossRef][Web of Science][Medline]
  17. Ishida H, Muchamuel T, Sakaguchi S, Andrade S, Menon S, Howard M. Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J Exp Med (1994) 179:305–10.[Abstract/Free Full Text]
  18. Miret C, Font J, Molina R, et al. Relationship of oncogenes (sFas, Bcl-2) and cytokines (IL-10, alpha-TNF) with the activity of systemic lupus erythematosus. Anticancer Res 2001. 21:3053–9.
Submitted 21 May 2007; revised version accepted 2 November 2007.
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