1 Center for Biologics Evaluation and Research/Food and Drug Administration, Bethesda, Maryland, USA,
2 NIAMS, National Institutes of Health, Bethesda, Maryland, USA and
3 II. Medical Clinic, University of Kiel, Germany
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Abstract |
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Methods. Secretion of cytokines and IgM, cell proliferation and up-regulation of co-stimulatory molecules were evaluated in PBMC from SLE patients (n=24) and normal controls (n=24) after stimulation with synthetic oligodeoxynucleotides (ODN) containing CpG motifs.
Results. Up-regulation of co-stimulatory molecules and the secretion of interferon- and interleukin-6 (IL-6) in response to CpG ODN was significantly reduced in monocytes and dendritic cells from SLE patients. Secretion of interferon-
by natural killer (NK) cells was also reduced. In contrast, the IgM and IL-10 response of B cells to CpG ODN was normal.
Conclusion. Monocytes, dendritic cells and NK cells from SLE patients respond abnormally to CpG ODN stimulation, which may contribute to the cytokine imbalance observed in SLE.
KEY WORDS: SLE, Innate immune system, CpG oligodeoxynucleotide, Toll-like receptor, Dendritic cells.
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Introduction |
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It is well established that bacterial DNA containing unmethylated CpG motifs' are inherently immunostimulatory, triggering B cells to proliferate and secrete IgM and IL-10, monocytes/dendritic cells to secrete IL-6 and interferon-, and NK cells to produce interferon-
[5]. Several findings suggest that bacterial DNA may play a role in the development and/or progression of SLE. First, lupus patients have increased levels of free DNA and DNAanti-DNA immune complexes in their serum. These immune complexes contain hypomethylated CpG-rich DNA [68], leading Sato et al. [8] to speculate that such DNA might be of bacterial origin. Second, CpG-rich DNA may persist for prolonged periods in lupus patients due to a decrease in DNase activity [9, 10]. Third, animal studies show that CpG DNA stimulates autoantibody production [11, 12]. Finally, Pisetsky et al. reported that the anti-DNA autoantibodies in SLE patients cross-react with bacterial and mammalian DNA [13]. We therefore hypothesized that peripheral blood mononuclear cells (PBMC) from SLE patients might respond abnormally to in vitro stimulation with CpG oligodeoxynucleotides (ODN).
Two different types of synthetic ODN containing unmethylated CpG ODN have been shown to reproduce the immunostimulatory properties of bacterial DNA on PBMC from normal healthy individuals [5]. K-type ODN have phosphorothioate backbones containing multiple TCGTT or TCGTA motifs. These ODN trigger B-cell proliferation and the secretion of IL-6, IL-10 and IgM. By comparison, D-type ODN have a mixed phosphodiesterphosphorothioate backbone, a single purine (Pu) pyrimidine (Py) CG PuPy motif flanked by self-complementary bases, and a 3' poly-G tail. D ODN primarily stimulate human dendritic and NK cells to produce interferon- and -
, while increasing surface expression of MHC class II and various co-stimulatory molecules on monocytes and dendritic cells [5].
This study evaluated the in vitro response of PBMC from SLE patients to these two types of CpG ODN. In comparing the response of PBMC from SLE patients with those from healthy controls, we found that monocytes, dendritic cells and NK cells from SLE patients hypo-responded to CpG ODN, whereas B cells responded normally. The implications of these findings are discussed.
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Patients and methods |
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Patients were diagnosed with SLE using established criteria [14, 15]. PBMC from a first set of 16 patients were used to assess IgM and cytokine secretion and proliferation induced by CpG ODN. The average Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) of these patients was 9.7 (range 023). Ten of these 16 patients were receiving hydroxychloroquine (200 mg twice daily) and 14 of 16 were receiving prednisone with an average dose of 16.5±8.5 mg per day (Table 1). Eight of 16 patients had also received other immunosuppressive drugs within the past 2 months, including methotrexate (n=2), azathioprine (n=1), cyclosporin (n=2), mycophenolate (n=1) or cyclophosphamide (n=2). PBMC from a second set of patients with similar characteristics (n=8, average SLEDAI 8.8, range 216) and receiving hydroxychloroquine (n=5) and/or prednisone (n=5) were used for flow cytometry analysis. The baseline characteristics of both patient groups were comparable as shown in Table 1
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Mononuclear cell preparation
PBMC were separated by density gradient centrifugation over Ficoll-Hypaque as described [4]. Cells were washed three times and cultured in 96-well plates in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 1.5 mM L-glutamine, 1 mM HEPES, 0.1 nM sodium pyruvate and 50 µM 2-mercaptoethanol in a 5% CO2 in air incubator at 37°C. PBMC were stimulated with 13 µM CpG ODN or with phytohemagglutinin (1 µg/ml, GIBCO, Grand Island, NY). The cells were incubated for 72 h at 4x105 cells/well to generate supernatants or 8x104 cells/well for proliferation assays. Cell viability after 72 h was >95% as assessed by trypan blue. The concentrations of ODN used (1 µM for K ODN, 3 µM for D ODN), as well as the incubation period were chosen from doseresponse curves with PBMC from healthy donors [5].
Oligodeoxynucleotides
ODN were synthesized at the Center for Biologics Evaluation and Research Core Facility. All had less than <0.1 EU/ml endotoxin per mg of ODN as assessed by LAL-Assay (BioWhittaker, Walkersville, Maryland). PBMC were stimulated with the panel of D ODN (D19: GGTGCATCGATGCAGGGGGG, D29: GGTGCACCGGTGCAGGGGGG and D28: GGTGCGTCGATGCAGGGGGG) or K ODN (K3: ATCGACTCTCGAGCGTTCTC, K23: TCGAGCGTTCTC and K123:TCGTTCGTTCTC). CGs are underlined, bases in bold type are phosphorodiester and bases in normal type are phosphorothioate. Control ODN had the same size, backbone and flanking sequence of stimulatory ODN, but lacked the active CpG dinucleotide (CG was switched to GC or TG).
Cytokine and Ig levels
Cytokine and Ig levels in culture supernatants were evaluated by enzyme-linked immmunosorbent assay (ELISA) as previously described [5]. Briefly, Immunolon 2 microtitre plates (Dynex Inc. Chantilly, VA) were coated with monoclonal antibodies to IL-6 (R&D, Minneapolis, Minnesota), interferon- (Endogen, Woburn, Maine), IL-10 (Endogen, Woburn, Maine), interferon-
(PBL-Biomedical Laboratory, New Brunswick, New Jersey) or human Ig (Serotec, Oxford, England) and blocked with phosphate buffered saline/bovine serum albumin (PBS-BSA). Cytokine and Ig levels in culture supernatants were detected colorimetrically using biotin-labelled antibodies to IL-6 (Biosource, Camarillo, California), interferon-
, IL-10, interferon-
or IgM (Boehringer, Mannheim, Germany) followed by phosphatase-conjugated avidin and a phosphatase-specific colorimetric substrate (Pierce, Rockford, Illinois). Standard curves were generated using recombinant cytokine or purified IgM. All assays were performed in duplicate or triplicate and the mean±S.D. of the log-normalized data from all three D ODN and K ODN determined. The detection limits of the assays were: 5 pg/ml for interferon-
and IL-10, 20 pg/ml for IL-6 and 50 U/ml for interferon-
. When cytokine or Ig levels were below assay sensitivity, the lower limit of detection was used to calculate the stimulation indices. Stimulation indices were calculated by the formula: (stimulated cells background)/(unstimulated cells background).
Cell proliferation
A total of 8x104 cells were stimulated for 68 h with 13 µM CpG or control ODN, then pulsed with 1 µCi of [3H]-thymidine and harvested 4 h later.
Flow cytometry
PBMC were stimulated for 24 h with 3 µM D, K or control ODN. Cells were then harvested, fixed (Fix&Perm, Caltag, Burlingame, California), washed and stained with phycoerithrin-labelled anti-CD19 or anti-CD11c, cytochrome-labelled anti-CD40, and fluoroscein isothiocyanate (FITC)-labelled anti-HLA-DR (Pharmingen, San Diego, California). A total of 20 000 events were analysed per sample using a FACScan flow cytometer (Becton Dickinson, San Jose, California) and CellQuest software.
Toll-like receptor 9 (TLR-9) reverse transcription polymerase chain reaction (RT-PCR)
Total RNA from human PBMC was extracted with TRIzol (Gibco BRL, Gaithersburg, MD) according to the manufacturer's protocol: 5 µg of total RNA was reverse-transcribed using Superscript-II (Gibco BRL, Gaithersburg, MD) in first strand buffer (50 mM Tris-HCl, pH 7.5; 75 mM KCl and 2.5 mM MgCl2), containing 25 µg/ml oligo-(dT)1218, 2 mM dNTP and 10 mM DTT.
The reaction was conducted at 42°C for 1 h. One microlitre of each cDNA sample was subjected to PCR (94°C for 30 s, 62°C for 30 s, 72°C for 60 s) for 30 cycles followed by a 10 min extension at 72°C using Taq polymerase (Promega, Madison, WI) and 0.5 µM of the following primers: human TLR-9: CTTCGGGGGCAGCTGGAGGAGT and ACAGGCAGGCAGAGGTGAGGTGAGT (483 bp); and human GAPDH: ACCACCATGGAGAAGGCTGG and CTCAGTGTAGCCCAGGATGC (527 bp).
Statistical analysis
The data were not normally distributed and therefore non-parametric two-tailed tests were used. Differences between lupus and control PBMC were evaluated using the MannWhitney U-test. Differences in their response to medium, D, K and control ODN were evaluated using non-parametric ANOVA (KruskalWallis with Dunnett's post-test comparisons method). Correlations between ODN responsiveness, disease activity (SLEDAI) and patient treatment were analysed using the Spearman correlation. For all tests P<0.05 was considered significant in a two-tailed comparison.
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Results |
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Effect of CpG ODN on B cells from SLE patients
K ODN trigger B cells from healthy subjects to proliferate, secrete IgM and IL-10, and up-regulate cell surface expression of HLA-DR and co-stimulatory molecules (such as CD40) [18]. We next assessed whether the magnitude of the B-cell response to CpG ODN in PBMC from SLE patients was altered. Under optimal stimulatory conditions, no significant difference in the reactivity of lupus compared with normal B cells was observed. K ODN induced a 43-fold increase in IgM production by normal PBMC, and an equivalent 36-fold increase by lupus PBMC (Fig. 4A). The increase in IL-10 production was 3.6-fold in controls and 3.7-fold in SLE PBMC (Fig. 4B
). Although the proliferative response of normal PBMC triggered by K ODN exceeded that of SLE cells (17-fold vs 11-fold), this difference was not statistically significant (Fig. 4
C).
Further evidence that the response of lupus B cells to CpG stimulation was comparable with that of healthy subjects derived from phenotypic studies of activation marker expression. Consistent with previous reports, K ODN induced a 1.8-fold increase in HLA-DR expression on CD19+ cells. That increase was comparable to the 1.6-fold increase in HLA-DR expression on B cells from SLE patients (data not shown). Similarly, K ODN triggered a 4-fold increase in CD40 expression on normal B cells and a 3.7-fold increase on SLE B cells. These findings suggest that B cells from lupus patients conserve their ability to respond to CpG DNA.
TLR-9 mRNA expression
CpG ODN-mediated cell activation is mediated by TLR-9 expressed by B cells and plasmacytoid dendritic cells (pDC) [19]. To assess whether the reduction in cellular responsiveness to CpG ODN stimulation was due to a reduction in TLR-9 expression, RT-PCR was performed on PBMC from three SLE patients. As shown in Fig. 5, their expression levels were similar to those found on PBMC from healthy subjects. This demonstrates that the reduction in the magnitude of the response to CpG ODN is not due to a systemic defect in TLR-9 expression.
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Discussion |
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Bacterial DNA expresses CpG motifs that are recognized as a pathogen-associated molecular pattern (PAMP) by the innate immune system. This recognition is mediated by TLR-9, and initiates a rapid inflammatory response [19]. Studies indicate that the innate immune response of SLE patients can be defective [20]. Scheinecker et al. [1] found a lower frequency of dendritic cells in PBMC from SLE patients compared with healthy controls. Moreover, dendritic cells in peripheral blood have reduced expression of CD40 and HLA-DR and reduced T-cell stimulatory capacity in mixed lymphocyte reactions. In vitro, monocytes from SLE patients show evidence of diminished phagocytic capacity [21] and impaired maturation into dendritic cells with low expression of CD40 and HLA-DR on their cell surface when cultured with granulocytemacrophage colony-stimulating factor (GM-CSF) and IL-4 [2].
Despite the presence of CpG-rich DNAanti-DNA immune complexes that elicit the secretion of interferon- by dendritic cells from normal donors, recent studies document that the number of interferon-
-producing plasmacytoid dendritic cells in the peripheral blood is reduced in SLE patients [22, 23]. The current study extends these findings by showing a reduced response of monocytes and dendritic cells from SLE patients to activation by CpG ODN.
The maturation and activation of dendritic cells and NK cells is intimately integrated. Both the up-regulation of CD40 on the cell surface of dendritic cells and their secretion of interferon- promote NK cell activation and the secretion of interferon-
. Conversely, activated NK cells contribute to the maturation of dendritic cells [24]. Previous studies have shown that in SLE patients, the number and lytic ability of NK cells in peripheral blood are reduced compared with NK cells from healthy controls [3, 20, 25, 26]. In addition, the number of PBMC secreting interferon-
in the absence of stimulation is reduced in these patients [4, 27]. The current results extend those findings by showing that the interferon-
production by SLE PBMC in response to CpG ODN stimulation is diminished. This suggests that the response of NK cells may be impaired, since they serve as a major source of interferon-
[5]. The reduced ability of NK cells to secrete interferon-
in response to CpG ODN is most likely secondary to a reduction in CD40 expression or interferon-
secretion by plasmacytoid dendritic cells [19].
The response of SLE monocytes, dendritic cells and NK cells to CpG ODN did not correlate with medication or disease activity as measured by the SLEDAI. This lack of correlation might be due to small sample size, since this study was not designed to detect such a correlation. However, reduced responses to CpG stimulation were also seen in patients whose disease was stable and in remission, suggesting that the abnormal response to CpG-induced immune activation is a sign of immunopathology in SLE that is independent from disease activity.
It is unclear whether the abnormal response of monocytes, dendritic cells and NK cells from lupus patients to CpG ODN reflects intrinsic cellular abnormalities, acclimatization to chronic exposure to stimulatory DNA in vivo or homeostatic efforts to reduce responsiveness to external stimuli. A systemic abnormality in CpG ODN recognition does not appear to be involved since B cells from SLE patients retain their responsiveness to CpG ODN (as measured by proliferation, up-regulation of CD40 and secretion of IgM and IL-10). Furthermore, as shown in Fig. 5, the expression of TLR-9 on PBMC from SLE patients is comparable with that of healthy controls. However, since the TLR-9 expression was studied on PBMC (where B cells outnumber pDC), we cannot exclude the possibility of an expression defect in a subpopulation of cells.
Regardless of the mechanism, the inability of dendritic cells to respond normally to CpG DNA may contribute to the unbalanced cytokine milieu (reduced interferon- and an increased ratio of IL-10:interferon-
-secreting cells) described in these patients [4, 2729]. Thus, efforts to identify the abnormalities responsible for defective CpG responsiveness of SLE PBMC may shed light on the aetiopathogenesis of this disease.
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Acknowledgments |
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This work was supported by an educational grant of the II. Medical Clinic, University of Kiel, Germany in part by an appointment to the Research Participation Program at CBER administered by the Oak Ridge Institute for Science and Education.
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Notes |
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References |
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