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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

We aim to investigate the additive value of B cell-activating factor (BAFF) when added to oligodeoxynucleotides (ODN)-activated B cells with respect to TLR-9, CD69, MHC-II expression, IL-6 and IL-10 secretion and B cell cycling. Therefore, B cells from healthy individuals were incubated under the following conditions: (1) B cells with medium, (2) B cells with ODN 0.5 μm, (3) B cells with BAFF 20 μm and (4) B cells with both ODN 0.5 μm and BAFF 20 μm. We found that addition of BAFF did not enhance the expression of TLR-9, CD69 and MHC-II in ODN-activated B cells. Incubation of B cells with BAFF and ODN together leads to a marked elevation of IL-6 and IL-10 levels compared to ODN alone. Synthesis and mitosis were higher in B cells stimulated by BAFF than in B cells stimulated by ODN. These findings suggest that both BAFF and TLR-9 contribute independently to B cell function.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Increased activity of the innate immune system, especially overexpression of Toll-like receptor 9 (TLR-9), plays a major role in the development of autoimmunity. It is well established that TLR-9 is expressed at very low levels in human naive B cells, but TLR-9 mRNA is rapidly upregulated by B cell receptor triggering. This process mainly occurs in memory B cells [1]. Stimulation of CD27+ memory B cells by TLR-9 using its ligand, unmethylated CpG DNA, is considered a homoeostatic mechanism that maintains adaptive long-term B cell memory in the absence of antigen [2].

Patients with systemic lupus erythematosus (SLE) frequently have B cell lymphopoenia (with naïve B cells being more affected), higher levels of early plasma, pregerminal centre cells and expansion of autoreactive B cell subpopulations in peripheral blood. Circulating peripheral CD19+ B cells from patients with SLE express high levels of CD80, CD86 and CD71+ compared with healthy controls [3–5].

Recent studies have revealed that TLR-9 is overexpressed in peripheral blood B cells isolated from patients with active SLE. In these patients, TLR-9 expansion was found to be correlated with SLE disease activity index and anti-double-stranded DNA (anti-dsDNA) antibody production [6, 7]. Stimulation of TLR-9 in B cells, using a synthetic ligand that mimics bacterial, viral and parasitic DNA, the unmethylated CpG oligodeoxynucleotides (ODN-CpG), induces proliferation, cytokine secretion, such as IL-10, IL-6, IL-12, and up-regulation of costimulatory molecules, such as CD40 and CD86 [8–10]. IL-10 was shown to be of a special importance in SLE with a potent stimulatory effect on B lymphocytes, leading to their proliferation, differentiation and autoantibodies production [11–14].

Therefore, it seems that activation of B cells isolated from healthy individuals, by ODN-CpG leads to some of the peripheral B cells phenotype and functional changes which were reported in patients with SLE. B cell-activating factor (BAFF) is an important regulator of peripheral B cell survival, maturation, immunoglobulin production and immunoglobulin class-switch recombination [15]. It is a member of the tumour necrosis factor (TNF) family, expressed by T cells, monocytes/macrophages, dendritic cells and epithelial cells [16]. B cell-activating factor trimmers interact with three TNF-R family members, TACI (CD267, TNFRSF13B), BAFF-R (also known as BR3, CD268, or TNFRSF17), and B cell maturation antigen (BCMA; CD269, TNFRSF13C) [17, 18]. Increased release of BAFF may lead to the development of B cell autoreactivity, especially in patients with genetic susceptibility. The purpose of the present study is to evaluate the additive value of BAFF when added to ODN-CpG-activated B cells, obtained from healthy voluntaries. The main aim of this study was to see if this addition could increase expression of TLR-9, activation markers, and cytokines production.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Study group.  Twenty five healthy voluntaries from the medical staff of Bnai Zion Medical Center served as the study group. Informed consent was obtained, and the study was approved by the local Helsinki committee at Bnai Zion Medical Center, Haifa, Israel.

Cells.  Peripheral blood mononuclear cells (PBMCs) were isolated on Lymphoprep (Axis-Shield, Oslo, Norway). Purified B lymphocyte fraction was collected by positive selection using CD22 MicroBeads and magnetic cell separation kit (MACS system; Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions, achieving >95% purity. CD22 cells were cultured in 96-well plates (Corning Glass Works, Corning, NY, USA), 1 × 106 cells/ml, in RPMI-1640 medium supplemented with 10% FCS, 1% glutamine, 1% penicillin and 1% streptomycin for 24 h. To assess BAFF effect, and its possible co-effect with ODN, B cells were cultured with 0.5 μm ODN (ODN 2006 human type B; Invivogen, San Diago, CA, USA), or with 20 μm BAFF (R&D Systems, Minneapolis, MN, USA) or with BAFF and ODN, all conditions in the presence of 50 U/ml rHIL-4 (Biological Industries, Biet – haemek, Israel). Cells cultured in medium alone served as a control. The working concentration of ODN, 0.5 μm, was determined after dose–effect study in which B cells were stimulated by ODN at concentrations of 0, 0.25, 0.5 and 1 μm, and the end point of TLR-9 expression was determined by flow cytometry analysis (Fig. 1).

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Figure 1.  Dose–response of the ODN effect on TIR-9 expression in memory B cells. Purified B cells (1 × 106/ml) were incubated with ODN at concentrations of 0 (control), 0.25, 0.5 and 1 μm, and after 24 h, TLR-9 expression was determined by flow cytometry analysis in CD19+CD27+ cells. These results are representative of three independent experiments. The expression of TLR-9 was significantly increased after 24 h of stimulation with ODN at concentration of 0.5 and 1 μm compared to the control (5.9 ± 0.45 versus 7.2 ± 0.7 MFI, P < 0.05, 5.9 ± 0.45 versus 7.0 ± 0.87 MFI, P < 0.05, respectively). The MFI expression of TLR-9 was not changed significantly following stimulation with ODN at concentration 0.25 μm. Additionally, MFI expression of TLR-9 was higher significantly following incubation of B cells with ODN 0.5 versus 0.25 μm (7.2 ± 0.7 versus 6.1 ± 0.6 MFI, P < 0.05). Data are given in mean ± SEM.

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Flow cytometry analysis.  Following 24 h of culturing, memory B cells (CD19+, CD27+) and naïve B cells (CD19+, CD27) were analysed for the expression of TLR-9, CD 69 and MHCII. B lymphocytes were stained for extracellular markers with anti-human CD19 (clone: cJ3–119), CD27 (clone: 1A4CD27) and CD69 (clone: TP1.55.3) or MHCII Abs (clone: Immu-357) (Immunotech, Beckman coulter co. Marseille, France). Isotype-matched IgG was used as a negative control. For the detection of intracellular TLR-9, cells were fixed and permeablized with cell permeabilization kit (Caltag Lab, An Der Grub, Austria) and then stained with anti-PE-conjugated anti-TLR-9 monoclonal antibodies (clone: 26C593.2) (R&D systems). These markers were assessed on CD27high B cells subset, which are considered to express high levels of TLR-9. Data were acquired using Flow Cytometry (FC 500; Beckman coulter co., Marseille, France). All markers were expressed in mean flow cytometry intensity (MFI), with results given in mean ± SEM.

Cell cycle analysis.  For analysis of cell cycle using propidium iodide, purified B cells were cultured with 0.5 μm ODN (ODN 2006 human type B; Invivogen), or with 20 μm BAFF (R&D Systems) or with BAFF and ODN, all conditions in the presence of 50 U/ml rHIL-4 (Biological Industries). Following 24 h of the indicated stimulations, B cells were incubated for 10 min at room temperature with propidium iodide solution of 200 μl (a buffer containing 0.3% (w/v) saponin, 50 μg/ml RNAse, 5 mm EDTA pH = 8 and 5 μg/ml propidium iodide (Sigma-Aldrich, Rehovot, Israel) and were analysed for cell cycle by Flow Cytometry.

Cytokines.  Levels of IL-6 and IL-10 in cell medium, produced by B cells cultured under the indicated conditions, were quantified using a sandwich ELISA kits (Quantikine ELISA Kit for IL-6 and DuoSet for IL-10; both R & D Systems).

Statistical analysis.  Comparison of TLR-9, CD 69 and MHCII expression in memory B cells, IL-10 and IL-6 supernatant levels between the four matched groups with differing culture conditions, was performed using Analysis of Variance (anova) for repetitive measures test. When the anovaP values were significant, a paired student’s t-test with a Bonferroni correction was performed to evaluate the statistical difference between the individual groups. Two tailed P values of 0.05 or less were considered to be statistically significant. If not mentioned otherwise, data are given in mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The effect of 20 μm BAFF alone or added to 0.5 μm ODN on the expression of TLR-9, MHCII and CD69 on B cells

Stimulation of memory B cells (CD19+, CD27+) by ODN for 24 h induced, as expected, expression of CD69 (Fig. 2A), MHCII (Fig. 2B) and TLR-9 (Fig. 2C) (14.1 ± 1.4 versus 9.2 ± 0.6 MFI, P < 0.0001, 5.5 ± 0.5 versus 4.7 ± 0.3 MFI, P < 0.0001 and 6.2 ± 0.6 versus 5 ± 0.5 MFI, P < 0.001, respectively). Expression of CD69, MHCII and TLR-9 was not affected following stimulation with BAFF alone. Addition of BAFF to ODN-activated B cells did not result in further increased expression of these markers in memory B cells (Fig. 2). A representative experiment of TLR-9 and CD69 expressions in memory B cells in the different conditions is shown in Fig. 3. Regarding naïve B cells (CD19+, CD27), stimulation by ODN for 24 h induced elevated expression of TLR-9 (1.27 ± 0.6 versus 2.2 ± 1.4 MFI, P < 0.0001) and CD69 (6.5 ± 1.1 versus 12.6 ± 4.1 MFI, P < 0.0001). Interestingly, BAFF alone also increased the expression of TLR-9 (1.27 ± 0.6 versus 2.2 ± 1.4 MFI, P < 0.0001) and CD69 (6.5 ± 1.1 versus 13.2 ± 4.1 MFI, P < 0.0001). Addition of BAFF to ODN-activated B cells did not result in further increased expression of TLR-9 or CD69. The MFI expression of MHCII was not changed following stimulation with ODN, BAFF or both.

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Figure 2.  Expression of CD69, MHCII and TLR-9 was not affected following stimulation with B cell-activating factor (BAFF) 20 μm alone or BAFF 20 μm together with ODN 0.5 μm. Purified B cells (1 × 106/ml) were incubated alone with medium or stimulated with ODN 0.5 μm or BAFF 20 μm or combination of ODN and BAFF for 24 h. Afterwards, cells were collected, washed and stained with labelled monoclonal antibodies (mAbs), for the detection of extracellular markers, with anti-human CD19, CD27 and CD69 or MHCII mAbs. For the detection of intracellular TLR-9, cells were fixed and permeablized and then stained with anti-PE-conjugated anti-TLR-9 monoclonal antibodies and anti-human CD19, CD27 mAbs. These markers were assessed on CD27high B cells subset, which are considered to express high levels of TLR-9. Results are expressed in mean fluorescence intensity (mean ± SEM). Expression of CD69 (A), MHCII (B) and TLR-9 (C) was significantly increased after 24 h of stimulation with ODN, whereas CD69, MHCII and TLR-9 expression was not affected following stimulation with BAFF alone or upon ODN addition.

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image

Figure 3.  A representative experiment demonstrating the effect of ODN 0.5 μm, B cell-activating factor (BAFF) 20 μm and combination of ODN 0.5 μm and BAFF 20 μm on the expression of TLR-9 in memory B cells obtained from healthy individual. Isolated B cells (Panel A) were cultured (1 × 106/ml) for 24 h in different conditions; afterwards, B cells were triple stained for the expression of TLR-9 in memory B cells (CD19+, CD27+) (Panel B). Stimulation of B cells by ODN 0.5 μm induced expression of TLR-9 on memory B cells [7.76 (Panel D) versus 4.21 MFI (Panel C)]. Expression of TLR-9 was not affected following stimulation with BAFF 20 μm (Panel E). Combination of BAFF 20 μm and ODN 0.5 μm did not result in further significant increased expression of TLR-9 compare to ODN 0.5 μm in memory B cells (Panel F).

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IL-6 secretion was further increased following B lymphocytes stimulation by BAFF 20 μm added to 0.5 μm ODN

Incubation of isolated B cells with ODN for 24 h resulted in a statistically significant increase in the secretion of IL-6 (35.6 ± 2.7 versus 0.3 ± 0.2 pg/ml, P < 0.0001). However, when these B cells were incubated with BAFF together with ODN, a marked further increase in IL-6 secretion was documented, which was significantly higher than that achieved upon stimulation by each ODN or BAFF alone (50.4 ± 3.8 versus 35.6 ± 2.7 pg/ml, P < 0.0001 and 50.4 ± 3.8 versus 6.9 ± 2.5 pg/ml, P < 0.0001, respectively) (Fig. 4A).

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Figure 4.  IL-6 and IL-10 secretion was increased following B lymphocytes stimulation by B cell-activating factor (BAFF) 20 μm added to ODN 0.5 μm. Purified B cells (1 × 106/ml) were incubated alone with medium or stimulated with ODN 0.5 μm or BAFF 20 μm or combination of ODN and BAFF for 24 h. Afterwards, supernatants were removed from cells and analysed for IL-6 and IL-10 levels by ELISA. Results are expressed in pg/ml (mean ± SEM). (A) IL-6 secretion by B cells was significantly higher when these cells were stimulated by BAFF together with ODN than when these cells were stimulated by ODN or BAFF alone (50.4 ± 3.8 versus 35.6 ± 2.7 pg/ml, P < 0.0001 and 50.4 ± 3.8 versus 6.9 ± 2.5 pg/ml, P < 0.0001, respectively). (B) IL-10 secretion by B cells was significantly higher when these cells were stimulated by BAFF together with ODN than when these cells were stimulated by ODN or BAFF alone (24.6 ± 6.1 versus 10.8 ± 4.6 pg/ml, P < 0.01 and 24.6 ± 6.1 versus 0.3 ± 0.2 pg/ml, P < 0.0001, respectively).

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IL-10 secretion was additionally increased following B lymphocytes stimulation by BAFF 20 μm added to ODN 0.5 μm

Incubation of isolated B cells with ODN 0.5 μm for 24 h resulted in increased secretion of IL-10 (10.8 ± 4.6 pg/ml versus undetectable level), while incubation with BAFF alone did not result in any increase in IL-10 secretion. A marked increase in IL-10 secretion was documented when B cells were incubated with BAFF together with ODN, being significantly higher when compared with that achieved by incubation of B cells with ODN or BAFF alone (24.6 ± 6.1 versus 10.8 ± 4.6 pg/ml, P < 0.01 and 24.6 ± 6.1 versus 0.3 ± 0.2 pg/ml, P < 0.0001, respectively) (Fig. 4B).

Stimulation of B cells by BAFF 20 μm and ODN 0.5 μm together induced DNA synthesis and mitosis

Following 24 h of stimulation by ODN, BAFF and ODN together with BAFF, B cells were analysed for cell cycle. Synthesis and mitosis of B cells were significantly higher when B cells were stimulated by BAFF than by ODN (28.9 ± 2.7% versus 23.8 ± 2.2%, P = 0.01). Both, synthesis and mitosis were not further increased upon stimulation with both BAFF and ODN together (28.9 ± 2.7% versus 31.7 ± 3.6%, P = ns) (Fig. 5).

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Figure 5.  B cell-activating factor (BAFF) 20 μm stimulated DNA synthesis and mitosis in B cells which were not further increased upon addition of ODN 0.5 μm. Purified B cells (1 × 106/ml) were incubated with ODN 0.5 μm or BAFF 20 μm or combination of ODN and BAFF for 24 h. Afterwards, for cell cycle analysis, cells were collected, washed and incubated for 10 min at room temperature with propidium iodide solution 200 μl. Synthesis and mitosis of B cells were increased following 24 h of stimulation by BAFF when compared to stimulation by ODN (28.9 ± 2.7% versus 23.8 ± 2.2%, P = 0.01). Both synthesis and mitosis were not further increased upon incubation with both BAFF and ODN together (28.9 ± 2.75% versus 31.7 ± 3.6%, P = ns).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Both TLR-9 and BAFF were found to be important participants in the pathogenesis of autoimmune diseases especially in SLE. B cell-activating factor has been found to affect B cell lineage at different stage of development and to function as a key survival cytokine for B cells. Overproduction of BAFF promotes survival of B cells, including autoreactive B cells, whereas inhibition of BAFF results in autoreactive B cell apoptosis [19, 20]. TLR-9 is expressed at very low levels in human naive B cells, but it is rapidly upregulated by BCR triggering and is expressed at high constitutive levels in memory B cells [2]. The majority of TLR-9 is present within the endosomal compartment. However, several groups have, in addition, demonstrated the presence of TLR-9 on the surface of cells such as activated B cells, LPS-stimulated human peripheral blood mononuclear cells and B cells isolated from human tonsils [21]. Cognasse F et al., further identified two distinct subpopulations of B cells, CD19+ CD27 CD23+ and CD19+CD27hi CD80+ cells, that expressed higher levels of membrane TLR-9 than other subpopulations. These subpopulations expressed high levels of membrane TLR-9 and exhibited a strong in vitro response to binding a relevant CpG motif by secreting high levels of IL-6 [22]. There is ample evidence that ODN activate memory human B cells which, in contrast to naïve B cells, express high levels of TLR-9. Recently, it was shown that ODN can also promote expansion, proliferation and survival of human naïve B cells by radioprotective 105 (RP105), a TLR-like molecule. RP105 forms a complex with MD-1 and can promote these changes only in human naïve B cells and not memory contributing potentially to innate host defence against microbes [23].

Recently, Miles K et al., found that TLR-9 can also mediate immunoregulatory signals. They demonstrated that DNA-containing complexes on the surface of apoptotic cells are sensed by B regulatory cells and delivered to TLR-9-containing endosomes, where they induce IL-10 secretion, which modulates the severity of ongoing immune responses [24]. The effect of ODN-CpG on memory B cells was further elucidated by, Henn AD et al., who found upregulation of antibody production pathways in the CD27hi memory B cells and upregulation of NF-kB activation pathways in the CD27low subset preceding differentiation into a plasmablast phenotype. Additionally, undivided activated ODN-CpG memory B cells showed active transcription of antigen presentation, cytokine secretion and co-stimulation genes [25].

Previous studies showed that TLR-9 stimulation increases BAFF-receptor, TACI and BCMA expression on B cells, which allows these cells to be more responsive to both BAFF and APRIL [26–28]. In this study, we wondered whether BAFF could possibly enhance the expression of TLR-9 in B cells. First, as previously has been shown by us and others, ODN increased expression of TLR-9 on memory B cells [29, 30]. However, the addition of BAFF to naïve B cells or to ODN-activated B cells did not influence expression of TLR-9 on memory B cells. B cell-activating factor, in contrast to ODN, did not enhance the expression of CD69 or MHC-II on memory B cells or on ODN-activated B cells. B cell-activating factor significantly increased the production of IL-6 and IL-10 when added to ODN-activated B cells, but not when added to isolated peripheral B cells. As it previously was demonstrated, levels of both IL-10 and IL-6 were found to be increased in serum of patients with SLE. IL-10 specifically has a potent stimulatory effect on B lymphocytes, leading to their proliferation, differentiation and autoantibodies production [11–14]. It was shown that peripheral blood B cells and monocytes obtained from patients with SLE produce high levels of IL-10, and this overproduction was found to correlate positively with disease activity [31–35]. Both IL-6 and IL-6 receptors were also found to be highly produced/expressed by B cells obtained from patients with SLE and were in association with the secretion of large amounts of immunoglobulins. Thus, neutralization of IL-6 leads to a significant decrease in this spontaneous immunoglobulins production, which was restored with exogenous IL-6 [13, 36, 37].

Our finding of the additive effect of BAFF on ODN-activated B cells, namely its contributory role in increasing IL-6 and IL-10 secretion, is important for understanding how increased serum BAFF in SLE increases autoimmunity and disease activity. The precise mechanism by which BAFF induces the overproduction of these cytokines is beyond the scope of this study. Other studies supported the importance of these cytokines in this regard by showing that when IL-6 or IL-10 was neutralized by different treatments, SLE disease activity was attenuated [38, 39]. In addition, we found that BAFF further increases the synthesis and mitosis of ODN-activated B cells, which possibly could increase the inflammatory process. As a consequence of the important role that BAFF plays in autoimmune diseases, especially in SLE, anti-BAFF treatment was developed to inhibit the continuous stimulation of autoreactive B cells [40–44]. On 9 March 2011, the US Food and Drug Administration approved belimumab, a fully human anti-BAFF monoclonal antibody, as a new B cell-specific treatment for SLE. In a recent phase III, multicentre, randomized placebo-controlled trial, Belimumab plus standard therapy significantly improved SLE responder index (SRI) rate, reduced SLE disease activity and severe flares and was generally well tolerated by patients with SLE [44].

In conclusion, we offer an additional explanation to the high levels of IL-6 and IL-10 in patients with SLE. We, hereby, demonstrated that production of IL-6 and IL-10 by ODN-activated B cells is significantly higher than by BAFF-activated B cells. However, a further significant increase in production of these two cytokines is achieved when B cells are signalled by both ODN and BAFF together. These findings further support the rational of treating patients with SLE with anti-BAFF treatment. Further studies are needed to investigate the precise mechanism through which BAFF influence the expression of IL-6 and IL-10 by ODN-activated B cells.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References