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The Journal of Immunology, 2003, 170: 4465-4474.
Copyright © 2003 by The American Association of Immunologists

Activation with CpG-A and CpG-B Oligonucleotides Reveals Two Distinct Regulatory Pathways of Type I IFN Synthesis in Human Plasmacytoid Dendritic Cells 1

Miren Kerkmann2,*, Simon Rothenfusser2,*, Veit Hornung*, Andreas Towarowski*, Moritz Wagner*, Anja Sarris*, Thomas Giese{dagger}, Stefan Endres* and Gunther Hartmann3,*

* Department of Internal Medicine, Division of Clinical Pharmacology, University of Munich, Munich, Germany; and {dagger} Institute of Immunology, University of Heidelberg, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-{alpha} in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-{alpha} independent of the IFNR but did not affect CpG-B-induced IFN-{beta}. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-{alpha}{beta} available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-{alpha} independent of the IFNR seem to represent characteristic features of PDC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The innate immune system of vertebrates has evolved the ability to recognize unmethylated CG dinucleotides within certain sequence contexts (CpG motifs) in microbial DNA via the Toll-like receptor (TLR) 49 (1, 2, 3, 4). A close link has been found between the expression of TLR9 on certain immune cell subsets and the modulation of the immune system by CpG DNA. In humans, expression of TLR9 is restricted to B cells and to a certain subset of dendritic cells, plasmacytoid dendritic cells (PDC) (5, 6). Consequently, CpG oligonucleotides (ODN) strongly activate B cells (7, 8) and PDC (1, 9, 10). The activity of other immune cell subsets such as monocytes, NK cells, {gamma}{delta} T cells, and memory CD8 T cells is increased by CpG ODN via PDC-derived cytokines, but due to the lack of TLR9 expression in these cell subsets, there is no direct effect of CpG ODN on these cells (6, 11). As a result, CpG ODN is a potent immune adjuvant for humoral immune responses in primates (12, 13, 14, 15) and humans (15, 16). There is first evidence that CpG ODN is also active as an adjuvant for Ag-specific T cell responses in rhesus macaques (17). CpG-activated PDC strongly support priming of allogeneic T cells in vitro (9), and PDC infected with influenza virus present viral Ag to T cells (18). A better understanding of how CpG ODN modulates PDC on a cellular level will help to further characterize the biological role of PDC within the immune system.

PDC were first described as a CD11c- subset of CD4+ dendritic cells in peripheral blood (19, 20). They are identical with natural type I IFN-producing cells (IPC) (21, 22, 23) and are able to produce extremely large amounts of type I IFN upon viral infection. IPC were described for many years as a rare CD4+MHC class II (MHC-II)+ population (1:1000 within PBMC) capable of synthesizing type I IFN upon viral infection and upon stimulation with a variety of microorganisms including preparations containing microbial DNA (24, 25). So far, CpG DNA is the only defined microbial molecule recognized by PDC. Other molecules stimulate PDC via TLR7, but the natural counterpart of these molecules has not been identified yet (26). PDC play a pivotal role in the pathogenesis of certain autoimmune disorders such as lupus (27).

It has been reported that synthetic CpG ODN promote survival, activation, and maturation of PDC (e.g., ODN 2006: prototype of CpG-B), but that the induction of type I IFN production is relatively low (28, 29). Based on the ability to stimulate type I IFN in PDC, a novel type of CpG ODN was identified (CpG-A: prototype ODN 2216), which induces very high amounts of IFN-{alpha} and IFN-{beta} in PDC (400 ng/ml IFN-{alpha} in the supernatant; 5 pg/single PDC (10)) and thus seems to mimic viral infection of PDC. Both types of CpG ODN depend on recognition via TLR9 but not TLR7 (30).

So far, the regulatory pathways of type I IFN production have been examined mostly in mouse and human cell lines, and recently in monocyte-derived dendritic cells (31). IFN-{alpha} is encoded by a family of 13 structurally related genes, whereas IFN-{beta} is encoded by a single gene. Induction of type I IFNs is regulated at the level of gene transcription (32). Among nine human IRFs identified to date, IRF3 and IRF7 play essential but not identical roles in the inducible expression of type I IFN genes (33, 34). Whereas IRF3 is primarily responsible for the activation of the IFNB gene in the early phase of IFN induction, IRF7 is involved in IFNA gene activation during the late phase of type I IFN induction (35, 36). There is recent evidence that IRF5 also plays a nonredundant role in virus-induced IFN-{alpha} production (37).

In the cell types examined in the literature, the type I IFNR plays a critical role in the delayed phase of IFN induction by binding IFN-{beta} and early IFN-{alpha} isoforms (in mouse IFN-4A4 gene) in an autocrine and paracrine feedback loop. Signaling takes place through the type I IFNR complexed with Jak1 and Tyk2. These kinases phosphorylate the transcription factors Stat1 and Stat2 (38), which form hetero- and homodimers, translocate to the nucleus, and assemble with the DNA-binding protein IFN-stimulated gene factor (ISGF)3{gamma} (p48) to form the ISGF3 complex. ISGF3 functions as a transcription factor of genes containing an IFN-stimulated response element motif in their promoter. Among the IFN-stimulated genes induced through this positive feedback loop is IRF7. Induction of IRF7 protein in response to IFN and its subsequent activation by phosphorylation in response to virus-specific signals, involving two C-terminal serine residues, are thought to be required for the delayed induction of the IFNA genes (35, 36, 39).

Due to the rarity of PDC in blood and the lack of a PDC-derived cell line, information about the regulation of type I IFN in purified PDC is limited. It has been shown that IFN-{alpha} itself is a potent survival factor of PDC (40), that the level of type I IFN production by PDC is controlled by different cytokines (41), and that IFN-{alpha} production in PDC involves the mitogen-activated protein kinase p38 (18). There is recent evidence from studies with IFN-{alpha}{beta}R knockout mice that virus-induced early IFN-{alpha} production of murine PDC is independent of feedback signaling via the IFN-{alpha}{beta}R (42).

In the present study, we used CpG-A and CpG-B as tools to examine the existence of distinct regulatory pathways of type I IFN production in human PDC. We found that PDC constitutively express high levels of IRF7 and are capable of synthesizing high levels of IFN-{alpha} independent of IFNR. We show that, in PDC, unlike in other cell types, the initiation of the IFNR-dependent feedback loop depends on the type of stimulus (CpG-A vs CpG-B) and not simply the presence of IFN-{alpha}{beta}. Whereas CpG-A triggered the IFNR-dependent pathway, type I IFN induction by CpG-B was independent of the IFNR. Thus, we identified these two types of CpG ODN as model stimuli to selectively trigger the IFNR-dependent or -independent pathway of type I IFN production in PDC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Medium and reagents

IMDM cell culture medium (PAA Laboratories, Linz, Austria) supplemented with 8% human serum (BioWhittaker, Walkersville, MD), 1.5 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich, Munich, Germany) was used throughout the studies. All compounds purchased were endotoxin tested. The following ODN were provided by Coley Pharmaceutical Group (Wellesley, MA): ODN 2216 (CpG-A), 5'-ggGGGACGATCGTCgggggG-3'; and ODN 2006 (CpG-B), 5'-tcgtcgttttgtcgttttgtcgtt-3'. Sequences are from 5' to 3'; lowercase letters represent phosphorothioate linkage; uppercase letters represent phosphodiester linkage 3' of the base; and bold letters represent CpG dinucleotides. No endotoxin could be detected in ODN preparations using the Limulus amebocyte lysate assay (lower detection limit, 0.03 endotoxin U/ml; BioWhittaker, Walkersville, MD). As demonstrated in previous studies, non-CpG control ODN (i.e., ODN 2243, GC control ODN of ODN 2216) did not induce IFN-{alpha} (10) and were not included in this study due to the limited number of PDC available. ODN were used at a final concentration of 6 µg/ml as established earlier (9, 10, 43). The blood dendritic cell Ag (BDCA)-2- and BDCA-1-specific Abs were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The mouse anti-human mAb against IFN-{alpha}R chain 2 (CD118; 21385-1; blocks the IFNR function) and the nonblocking control Ab (rabbit anti-human polyclonal Ab against IFN-{alpha}R chain 2; 31385-1) were purchased from PBL Biomedical Laboratories (New Brunswick, NJ).

Isolation of PDC

Human PBMC were prepared from buffy coats provided by the blood bank of the University of Greifswald (Greifswald, Germany). Blood donors were 18- to 65-year-old healthy donors who tested negative for HIV, hepatitis B virus, and hepatitis C virus. Further exclusion criteria were manifest infections during the last 4 wk, fever, symptomatic allergies, abnormal blood cell counts, increased liver enzymes, or medication of any kind except vitamins and oral contraceptives. PBMC were obtained from buffy coats by Ficoll-Hypaque density gradient centrifugation (Biochrom, Berlin, Germany) as described (44). PDC were isolated by MACS using the BDCA-4 dendritic cell isolation kit from Miltenyi Biotec. Briefly, PDC were labeled with anti-BDCA-4 Ab coupled to colloidal paramagnetic microbeads and passed through a magnetic separation column twice (LS and RS column; Miltenyi Biotec). The purity of isolated PDC (lineage-negative, MHC-II-positive, and CD123-positive cells) was >95%. Viability was >95% as determined by trypan blue exclusion.

Cell culture of PDC

Isolated PDC were cultured in 96-well round-bottom plates (5 x 104 cells in 200 µl medium/well) in the presence of different stimuli as indicated. After incubation, the supernatant was collected for cytokine analysis, and cells were harvested for flow cytometric analysis. In some experiments, CD40 ligand (CD40L)-transfected 3TC cells (mycoplasma-negative and irradiated with 30 Gy) at a concentration of 10,000 cells/well were used as stimulus. PDC were preincubated with BDCA-2 for 20 min, and then PDC were washed and cultured with or without stimulus as described in Medium and reagents. The anti-IFNR blocking Ab was added to the cultured cells without washing step at a concentration of 20 µg/ml based on previous studies which demonstrated that this concentration completely blocks IFN-{alpha}-mediated activation of {gamma}{delta} T cells (11).

Flow cytometry

Surface Ag staining was performed as described previously (45). Fluorescence-labeled mAbs to human CD80, CD86, and HLA-DR were purchased from BD PharMingen (Heidelberg, Germany). Flow cytometric data were acquired on a BD Biosciences (Heidelberg, Germany) FACSCalibur equipped with two lasers (excitation at 488- and 635-nm wavelength). Spectral overlap was corrected by appropriate compensation. Analysis was performed on viable cells. Data were analyzed using CellQuest software (BD Biosciences).

Detection of cytokines

Because total IFN-{alpha} is comprised of 14 different isoforms, the quantity of IFN-{alpha} measured by ELISA depends on the specificity of the detection Ab for these isoforms and thus is not identical between different ELISA. For most of the experiments, the IFN-{alpha} module set Bender MedSystems (detection range, 8–500 pg/ml) was used. This ELISA detects most of IFN-{alpha} isoforms, but not IFN-{alpha} B and IFN-{alpha} F. In some experiments (indicated in the figures), IFN-{alpha} was measured with the human IFN-{alpha} multispecies ELISA (detection range, 100-5000 pg/ml; PBL Biomedical Laboratories). With this ELISA, higher levels of total IFN-{alpha} were obtained, because it detects all IFN-{alpha} species except IFN-{alpha} F. The human IFN-{beta} ELISA was from PBL Biomedical Laboratories (detection range, 250–10,000 pg/ml), and the human IL-8 ELISA (detection range, 3–200 pg/ml) and the human TNF-{alpha} ELISA (detection range, 8–500 pg/ml) were from BD PharMingen.

Real time RT-PCR

Purified PDCs were cultured for 3 or 15 h with different stimuli: IL-3 (10 ng/ml), CpG-A ODN, CpG-B ODN, or CD40L as described in Cell culture of PDC. Cells were lysed, and RNA was extracted using the total RNA isolation kit (High Pure; RAS, Mannheim, Germany). An aliquot of 8.2 µl of RNA was reverse transcribed using avian myeloblastoma virus-reverse transcriptase and oligo(dT) as primer (First Strand cDNA Synthesis kit; Roche, Mannheim, Germany). The obtained cDNA was diluted 1/25 with water, and 10 µl were used for amplification. Parameter-specific primer sets optimized for the LightCycler (RAS) were developed by and purchased from Search-LC (Heidelberg, Germany). The PCR was performed with the LightCycler FastStart DNA Sybr Green kit (RAS) according to the protocol provided in the parameter-specific kits and as described previously (6). Input was normalized by the average expression of the two housekeeping genes: {beta}-actin and cyclophilin B. The copy number was calculated from a standard curve, which was obtained by plotting known input concentrations of four different plasmids at log dilutions vs the PCR cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. Using over 300 data points, the actual copy number per microliter of cDNA was calculated as follows: X = e(-0.6553CP + 20.62).

Measurement of cytosolic calcium concentrations

After isolation, PDC were loaded with fluo 3-AM (3 µg/ml) and fura red-AM (10 µg/ml) in the presence of 0.02% of the detergent Pluronic F-127 (all from Molecular Probes, Leiden, The Netherlands) for 40 min and then washed twice as described (46, 47). Anti-BDCA-2 Ab (2.5 µg/ml; clone AC144; mouse IgG1; Miltenyi Biotec), anti-BDCA-4 Ab (2.5 µg/ml; clone AD5-17F6; mouse IgG1; Miltenyi Biotec), and anti-IFN-{alpha}R chain 2 Ab (10 µg/ml; clone MMHAR-2; mouse IgG2a; PBL Biomedical Laboratories) were added to PDC, followed by rat anti-mouse IgG1 (clone A85-1) and rat anti-mouse IgG2a (clone R19-15; both from BD Biosciences) as cross-linker. The Ca2+ ionophore ionomycin (2 µg/ml; Sigma-Aldrich, Munich, Germany) was used as a positive control. Cells were analyzed on a flow cytometer (FACSCalibur; BD Biosciences) to detect Ca2+ fluxes. Live cells were gated based on forward/side-scatter criteria. The ratio of the fluorescence intensity of the Ca2+-sensitive dyes fluo 3 (increased intensity in the presence of Ca2+) and fura red (reduced intensity in the presence of Ca2+) was used to analyze the change in cytosolic calcium concentrations vs time (time resolution, 200 ms).

Statistical analysis

Data are shown as means ± SEM. Statistical significance of differences was determined by the paired two-tailed Student’s t test and the Wilcoxon test. Differences were considered statistically significant for p < 0.05 and p < 0.01. Statistical analyses were performed using StatView 4.51 software (Abacus Concepts, Calabasas, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CpG-A shows prolonged kinetics of type I IFN production compared with CpG-B

Two distinct classes of CpG ODN have been proposed, and these two classes differ in the amount of IFN-{alpha} induced in PDC (CpG-A ODN: high IFN-{alpha} in PDC; CpG-B ODN: low IFN-{alpha} in PDC), as well as in the secondary effects on other immune cells such as T cells, NK cells, and monocytes (6, 11, 48). In the present study, we hypothesized that these two types of CpG ODN not only differ on a quantitative level regarding IFN-{alpha} induction, but also may differ on a qualitative level regarding other functional parameters of PDC biology.

Two well-established sequences, ODN 2216 (prototype of CpG-A) and ODN 2006 (prototype of CpG-B), were used throughout the study. First, we compared the amounts of IFN-{alpha} and IFN-{beta} in purified PDC incubated with CpG-B ODN and CpG-A ODN for 48 h. Although IFN-{beta} was higher in the presence of CpG-A ODN as compared with CpG-B ODN (1510 vs 615 pg/ml, n = 8; Fig. 1A), the ratio of IFN-{beta} to IFN-{alpha} was much higher for CpG-B ODN than for CpG-A ODN (IFN-{beta}:IFN-{alpha}, 1:4 for CpG-B ODN and 1:60 for CpG-A ODN; Fig. 1A). The higher percentage of IFN-{beta} of total type I IFN production with CpG-B ODN suggested that CpG-B may preferentially trigger early type I IFN production, whereas CpG-A may be able to support late type I IFN production in PDC also via the IFN-{alpha}{beta}-mediated feedback loop.



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  		FIGURE 1. IFN-{alpha} and IFN-{beta} production in PDC stimulated with CpG-B ODN or CpG-A ODN. PDC were isolated from PBMC and cultured in 96-well plates (5 x 104 cells/well) with CpG-A ODN (ODN 2216; {blacksquare}) or CpG-B ODN (ODN 2006; ) (6 µg/ml). A, Comparison of IFN-{alpha} and IFN-{beta} synthesis. After 48 h, supernatants were collected, and IFN-{alpha} and IFN-{beta} were measured by ELISA. Results are presented as means ± SEM of eight independent experiments with different donors. Values of p were as follows: IFN-{alpha}:CpG-A vs CpG-B, p < 0.001; IFN-{beta}:CpG-A vs CpG-B, p = 0.014 (t test). B, Kinetics of IFN-{alpha} production. After 12 h, the supernatant was removed, and cells were washed and recultured for an additional 12 h without stimulus. Supernatant was removed again, and cells were incubated with fresh medium for 24 h. The same was repeated at 48 h. IFN-{alpha} was measured in the supernatants by ELISA (IFN-{alpha} multispecies ELISA (PBL Biomedical Laboratories); see Materials and Methods). Results are shown as means ± range of two independent experiments. C, Comparison of CpG-A- and CpG-B-induced IFN-{beta} mRNA levels. PDC were incubated with CpG ODN for 3 or 15 h. IFN-{beta} mRNA was determined by real-time RT-PCR. The mean copy number ± SEM of four independent experiments with different donors is shown.

 
To compare the kinetics of IFN-{alpha} production in PDC, the supernatants were collected for different periods of time (0–12, 12–24, 24–48, and 48–72 h; Fig. 1B). CpG-A induced IFN-{alpha} within the first 12 h; most of the IFN-{alpha} was produced between 12 and 24 h, and considerable amounts of IFN-{alpha} were still produced after 24 h of incubation (Fig. 1B, left panel). In contrast, IFN-{alpha} production in response to CpG-B was small and was limited to the first 12 h (Fig. 1B, right panel).

Next, we sought to study the kinetics of CpG ODN-induced IFN-{beta} production in PDC. Because the total amount of IFN-{beta} induced by CpG-B was too low (see Fig. 1A) to be split up in different periods of time and still be detectable by the IFN-{beta} ELISA available (lower detection limit, 250 pg/ml), we measured the level of IFN-{beta} mRNA by real-time PCR. We found that the levels of IFN-{beta} mRNA induced by CpG-A and CpG-B at 3 h were comparable (Fig. 1C). However, at 15 h, IFN-{beta} mRNA already disappeared in PDC incubated with CpG-B, whereas it was even further increased in PDC incubated with CpG-A.

Qualitative difference in the regulation of cytokine production and surface marker expression by CpG-A and CpG-B

In addition to higher IFN-{alpha} and IFN-{beta} production by CpG-A (Fig. 1A), other parameters may exist which indicate a qualitative difference in the biological properties of both types of CpG ODN in PDC. Unlike the major differences seen for IFN-{alpha} and IFN-{beta}, TNF-{alpha} induction by CpG-B ODN and CpG-A ODN was in the same range (Fig. 2A). Induction of IL-8 was even higher by CpG-B ODN than by CpG-A ODN (Fig. 2B). Furthermore, CpG-B ODN was more active than CpG-A ODN in up-regulating costimulatory and Ag-presenting molecules on purified PDC (Fig. 3; CD80: mean fluorescence intensity (MFI), 197 vs 100; CD86: MFI, 286 vs 148; and MHC-II: MFI, 2681 vs 1899). These results clearly demonstrated that CpG-A ODN is not simply more active on PDC than CpG-B ODN, as might be inferred from type I IFN production, but that both types of CpG ODN trigger qualitatively distinct biological activities of PDC.



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  		FIGURE 2. TNF-{alpha} and IL-8 production of PDCs stimulated with CpG-A ODN or CpG-B ODN. PDC were isolated from PBMC and cultured in 96-well plates (5 x 104 cells/well) with CpG-A ODN ({blacksquare}) or CpG-B ODN () (6 µg/ml). After 48 h, supernatants were collected and assayed for TNF-{alpha} (A) (n = 10) or IL-8 (B) (n = 9) by ELISA. Results are presented as means ± SEM of independent experiments with different donors. Values of p were as follows: IL-8:no stimulus vs CpG-A, p = 0.007; IL-8:CpG-A vs CpG-B, p = 0.003 (t test).

 


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  		FIGURE 3. Activation of PDCs stimulated with CpG-A ODN or CpG-B ODN. PDC were isolated from PBMC and cultured in 96-well plates (5 x 104 cells/well) with CpG-A ODN ({blacksquare}) or CpG-B ODN () (6 µg/ml). After 48 h, the expression of CD80, CD86, and MHC-II was analyzed by flow cytometry. Results are presented as means ± SEM of nine independent experiments with different donors. Values of p were as follows: CD80:no stimulus vs CpG-A, p = 0.003; no stimulus vs CpG-B, p = 0.003; CpG-A vs CpG-B, p = 0.043 (t test); CD86:no stimulus vs CpG-A, p = 0.003; no stimulus vs CpG-B, p < 0.001; CpG-A vs CpG-B, p < 0.001 (t test); MHC-II:no stimulus vs CpG-B, p = 0.011; CpG-A vs CpG-B, p < 0.001 (t test).

 
CpG-A, but not CpG-B, triggers a positive feedback loop via the type I IFNR

It has been proposed that the early and late phases of type I IFN production can be distinguished based on the activation of the IFNR by endogenous IFN-{alpha}{beta} (constituting an autocrine feedback loop) (35). The prolonged IFN-{alpha} response induced by CpG-A (Fig. 1) suggested that CpG-A but not CpG-B may activate the positive autocrine feedback loop mediated via the IFNR. To test this hypothesis, we stimulated purified PDC with both types of CpG ODN in the presence and absence of a blocking Ab to the IFNR.

Indeed, the blockade of IFNR during stimulation with CpG-A ODN resulted in a decreased production of IFN-{alpha} (80,447 vs 36,270 pg/ml, n = 8; Fig. 4A). A control Ab had no inhibitory effect (CpG-A, 218,804, vs CpG-A and control Ab, 207,192 pg/ml, n = 2; not shown in figure). Enhanced IFN-{alpha} production via the IFNR was already detectable after 6 h but was most prominent after 10 and 24 h (Fig. 4B). The blockade of IFNR during stimulation with CpG-A ODN also resulted in decreased production of IFN-{beta} (1,392 vs 697 pg/ml, n = 5; Fig. 4C) and of TNF-{alpha} (669 vs 378 pg/ml, n = 5; Fig. 4D). In contrast, the production of IFN-{alpha}, IFN-{beta}, and TNF-{alpha} in response to CpG-B ODN was not decreased by the blockade of IFNR (Fig. 4, A, C, and D; slight increase of IFN-{alpha} and IFN-{beta}, not significant). The CpG ODN-induced IL-8 production (Fig. 4E) and up-regulation of CD80, CD86, and MHC-II (Fig. 5) were not significantly changed, indicating that these responses, in contrast to IFN-{alpha}, IFN-{beta}, and TNF-{alpha} synthesis, are not regulated via the IFNR. Together, these results demonstrated that CpG-A but not CpG-B is capable of triggering the feedback loop via the IFNR (responsible for the prolonged IFN type I production). Whereas TNF-{alpha} production seems to be enhanced via the IFNR, other parameters such as IL-8 and costimulatory molecules are independent of the IFNR. This might explain why, for these parameters, CpG-B can be more active than CpG-A (see Figs. 2B and 3).



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  		FIGURE 4. Effect of type I IFNR blockade on CpG-mediated cytokine production in PDC. PDC were isolated from PBMC and cultured in 96-well plates (5 x 104 cells/well) with CpG-A ODN ({blacksquare}) or CpG-B ODN () (6 µg/ml) in the presence or absence of type I IFNR Ab. A, After 48 h, IFN-{alpha} was determined in the supernatants (n = 8); CpG-A vs CpG-A plus IFNR Ab, p = 0.049. B, Supernatants were taken after 6, 10, or 24 h as indicated, and IFN-{alpha} was determined by ELISA (n = 3). C, After 48 h, IFN-{beta} was determined in the supernatants (n = 5; CpG-A vs CpG-A plus IFNR Ab, p = 0.043). D, After 48 h, TNF-{alpha} was determined in the supernatants (n = 5; CpG-A vs CpG-A plus IFNR Ab, p = 0.026). E, After 48 h, IL-8 was measured (n = 6). Results are presented as means ± SEM of independent experiments with different donors.

 


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  		FIGURE 5. Effect of type I IFNR blockade on CpG-mediated activation of PDC. PDC were isolated from PBMC and cultured in 96-well plates (5 x 104 cells/well) with CpG-A ODN ({blacksquare}) or CpG-B ODN () (6 µg/ml) in the presence of type I IFNR Ab. After 48 h, cells were analyzed by flow cytometry for the expression of CD80, CD86, and MHC-II. Results are presented as means ± SEM of 10 independent experiments with different donors.

 
Expression of IRF3, IRF7, and STAT1 in PDC

Whereas IRF3 is constitutively expressed in many cell lines (39), it has been reported that the expression of IRF7, required for late-phase IFN-{alpha} production, depends on the IFNR-mediated feedback loop (35, 36). Because both CpG-B ODN and CpG-A ODN induce considerable amounts of IFN-{alpha} independent of the IFNR, we hypothesized that PDC might constitutively express IRF7. Indeed, freshly isolated PDC incubated for 3 or 15 h in the absence of IL-3 and CpG ODN spontaneously expressed high levels of IRF7 mRNA (Fig. 6; note different scales used for IRF7, IRF3, and STAT1). Whereas IRF7 mRNA markedly decreased in the presence of the growth factor IL-3, both CpG-B ODN and CpG-A ODN transiently increased IRF7 expression within the first 3 h. After 15 h, IRF7 mRNA returned to the level of the control without stimulus or lower. IRF3 was also constitutively expressed in PDC but on a much lower level (Fig. 6). IRF3 was down-regulated in the presence of IL-3 but, unlike IRF7, IRF3 was unchanged or even decreased in the presence of CpG-B ODN and CpG-A ODN. These results indicated that PDC constitutively express high levels of IRF7 mRNA. However, the different quantities of IFN-{alpha} induced by CpG-B ODN and CpG-A ODN cannot be explained by different levels of IRF7 mRNA.



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  		FIGURE 6. Quantitative analysis of IRF3, IRF7, and Stat1 mRNA expression in PDC. Isolated PDC (50,000–100,000 cells) were incubated without stimulus or with IL-3, CpG-A ODN, CpG-B ODN, or CD40L for 3 h and for 15 h. Expression of IRF3, IRF7, and STAT1 mRNA was determined by quantitative real-time RT-PCR. Means ± SEM of four independent experiments with different donors are depicted.

 
STAT1 is phosphorylated by the activated IFNR. Consequently, the expression of STAT1 mRNA may affect the IFNR-mediated positive feedback loop. Because CpG-A ODN but not CpG-B ODN trigger the IFNR-mediated feedback loop, CpG-A ODN but not CpG-B ODN may up-regulate the expression of STAT1. However, we found that both CpG-B ODN and CpG-A ODN strongly increased the expression of STAT1 to similar levels after 3 and 15 h (Fig. 6). In contrast, STAT1 expression was decreased in the presence of IL-3. Together, these data indicated that the different quantities of IFN-{alpha} induced by CpG-A ODN vs CpG-B ODN cannot be explained by the regulation of mRNA expression levels of IRF3, IRF7, and STAT1.

Ligation of BDCA-2 inhibits CpG-A and CpG-B-induced IFN-{alpha}, IFN-{beta}, and TNF-{alpha} synthesis

It has been reported that ligation of the PDC-specific C-type lectin BDCA-2 with a monoclonal anti-BDCA-2 Ab rapidly increases intracellular Ca2+ levels and inhibits IFN-{alpha} production stimulated by influenza virus (49). We studied whether the BDCA-2 Ab affects CpG ODN-induced activation of PDC. The influence of BDCA-2 ligation on CpG-A- vs CpG-B-induced PDC activation (as model stimuli for the early type I IFN and the IFNR-dependent type I IFN production, respectively) may allow localization of the BDCA-2-mediated effect within the type I IFN-regulatory pathway.

Increasing concentrations of the BDCA-2 Ab inhibited CpG-A-induced IFN-{alpha} production in a dose-dependent manner (Fig. 7A), whereas a control Ab (BDCA-1) showed no effect (Fig. 7B). Unlike the blockade of IFNR, BDCA-2 Ab inhibited both CpG-A- and CpG-B-stimulated production of IFN-{alpha}, IFN-{beta}, and TNF-{alpha} (Fig. 8). CpG-induced up-regulation of costimulatory molecules (CD80 and CD86) and Ag presenting molecules (MHC-II) was not affected by the BDCA-2 Ab (not shown in figure). The inhibitory effect of the IFNR blockade was further decreased by the BDCA-2 Ab (Fig. 9), suggesting that BDCA-2 elicits a different mechanism than IFNR blockade. BDCA-2 Ab has been described as stimulating a rapid increase in intracellular calcium. We found that BDCA-2 but not the blockade of IFNR triggered intracellular calcium influx (Fig. 10). Together, these results provide evidence that ligation of BDCA-2 diminishes early type I IFN production in PDC. Therefore, the mechanism by which BDCA-2 decreases type I IFN is not dependent on the IFNR-mediated autocrine feedback loop.



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  		FIGURE 7. BDCA-2 Ab-mediated inhibition of CpG-A ODN-induced IFN-{alpha} production in PDC. A, The BDCA-2 Ab was added to isolated PDC at increasing concentrations: 0.25, 0.5, 2.5, and 20 µg/ml. Cells were incubated with the Ab for 20 min, and then cells were washed and cultured with CpG-A ODN as described in Materials and Methods. After 48 h of incubation, IFN-{alpha} was detected in the supernatant by ELISA. B, The BDCA-2 Ab or BDCA-1 Ab were added to isolated PDC in a concentration of 0.25 µg/ml. Cells were incubated with the Ab for 20 min, and then cells were washed and cultured with CpG-A ODN as described in Materials and Methods. After 48 h of incubation, IFN-{alpha} was detected in the supernatant by ELISA. Results are presented as means of two independent experiments with different donors.

 


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  		FIGURE 8. Effect of BDCA-2 Ab on CpG-stimulated cytokine production in PDC. Isolated PDC were incubated with the BDCA-2 Ab for 20 min, and then cells were washed and cultured with CpG-A ODN or CpG-B ODN for 48 h as described in Materials and Methods. Cytokines were measured by ELISA: IFN-{alpha} (BDCA-2 Ab, 2.5 µg/ml; n = 5) (A), IFN-{beta} (n = 6) (B), and TNF-{alpha} (n = 5) (C). The inhibition of cytokine synthesis by BDCA-2 is depicted as percentage ± SEM of the control without BDCA-2. *, p < 0.05; **, p < 0.01.

 


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  		FIGURE 9. Effect of simultaneous addition of BDCA-2 Ab and IFNR Ab on CpG-A ODN induced IFN-{alpha} production in PDC. PDC were isolated from PBMC and cultured in 96-well plates (5 x 104 cells/well) with CpG-A ODN (6 µg/ml) in the presence of IFNR Ab, BDCA-2 Ab, or both. After 48 h, IFN-{alpha} was measured in the supernatant by ELISA. Results are presented as means ± SEM of five independent experiments with different donors. Values of p were as follows: CpG-A plus IFNR Ab vs CpG-A plus BDCA-2 Ab plus IFNR Ab, p = 0.032; CpG-A plus BDCA-2 Ab vs CpG-A plus BDCA-2 Ab plus IFNR Ab, p = 0.022, t test.

 


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  		FIGURE 10. BDCA-2 Ab but not IFNR Ab or BDCA-4 Ab trigger intracellular calcium in PDC. PDC were stained with fluo 3 and fura red, as described in Materials and Methods, and analyzed by flow cytometry over time (x-axis). On the y-axis, the ratio of fluo 3 (FL1; increased in the presence of calcium) to fura red (FL3; decreased in the presence of calcium) is depicted. PDC were exposed to BDCA-2 Ab, BDCA-4 Ab, IFNR Ab or ionomycin (as positive control) at the time point indicated by the first arrow on the time scale. At the time point indicated by the second arrow, a secondary cross-linking rat anti-mouse IgG1 (for anti-BDCA-2 and BDCA-4) or rat anti-mouse IgG2a (anti-IFNR) was added. One of three representative experiments is shown.

 
Increased level of IFN-{alpha} induced by CpG-B in combination with CD40L is independent of the IFN-{alpha}{beta}R

To exclude the possibility that the threshold for activating the IFNR is not reached by the quantity of type I IFN induced by CpG-B, we tested whether increased type I IFN in response to CpG-B in combination with CD40L (9) is capable of initiating the IFNR-mediated feedback loop. CD40L increased the amount of IFN-{alpha} induced by CpG-B (Fig. 11A). This effect was not inhibited by the blockade of IFNR (Fig. 11A), indicating that the contribution of CD40L to IFN-{alpha} production is not mediated via the IFNR. These results demonstrate that an increased quantity of IFN-{alpha} itself (induced by CD40L and CpG-B as compared with CpG-B alone) is not sufficient to initiate the IFNR-mediated positive feedback loop.



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  		FIGURE 11. IFN-{alpha} and IFN-{beta} production of PDC stimulated with CpG-B ODN and CD40L. A, PDC were isolated from PBMC and cultured in 96-well plates (5 x 104 cells/well) with CpG-B ODN (6 µg/ml) alone or together with CD40L in the presence or absence of anti-IFNR Ab. After 48 h, IFN-{alpha} (n = 5 independent experiments) was measured in the supernatant. B, PDC were isolated from PBMC and cultured in 96-well plates (5 x 104 cells/well) with CpG-B ODN (6 µg/ml) alone or in combination with CD40L. After 48 h, IFN-{beta} (n = 4 independent experiments) was measured in the supernatant. Results are presented as means ± SEM of independent experiments with different donors.

 
Interestingly, CD40L did not increase the amount of IFN-{beta} induced by CpG-B (Fig. 11B), demonstrating that the signal provided by the ligation of CD40L cooperates selectively with the IFN-{alpha} pathway, but not with the IFN-{beta} pathway. Because CD40L did not affect the expression of IRF3, IRF7, or STAT1 mRNA (Fig. 6), the signal provided by CD40L is independent of the mRNA levels of these molecules.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The precursor of the PDC in peripheral blood is identical with the IPC, but only limited information is available on the regulation of type I IFN production in this cell type. In the present study, we demonstrate that PDC constitutively express high levels of IRF7 mRNA and are capable of producing considerable amounts of IFN-{alpha} independent of the IFNR-mediated positive autocrine feedback loop. Furthermore, we demonstrate that, depending on the type of stimulus, PDC feature two distinct regulatory pathways of type I IFN production: one pathway (early phase) that is independent of a feedback loop via the IFNR, and a second pathway in which early phase IFN-{alpha} production is amplified via an autocrine IFNR-dependent positive feedback loop resulting in prolonged kinetics of type I IFN production (late phase).

We found that two distinct classes of CpG ODN (previously defined by inducing different amounts of type I IFN in PDC) differ in their ability to trigger these pathways: CpG-B (prototype ODN 2006) selectively stimulates the early phase but is unable to initiate the IFNR-mediated feedback loop, and CpG-A (prototype ODN 2216), which triggers the late phase by activating the IFNR-mediated autocrine feedback loop. Of note, these two pathways do not simply result in different quantities of type I IFN production, but the composition of the type I IFN produced also differs. The proportion of IFN-{beta} of total type I IFN is much higher in the early phase triggered by CpG-B ODN. Future studies of different IFN-{alpha} isoforms induced by CpG-A and CpG-B may further support the concept of qualitative differences between both classes of CpG ODN.

Besides differential effects on type I IFN synthesis, CpG-A and CpG-B differ in the regulation of other parameters highlighting qualitative rather than only quantitative differences between the two biological response patterns of PDC. Although the level of TNF-{alpha} induction in PDC was in the same range for both types of CpG ODN, CpG-B was more active than CpG-A in stimulating IL-8 production and up-regulating costimulatory and Ag-presenting molecules. The fact that two qualitatively distinct response patterns of PDC can be elicited by CpG-A and CpG-B will impact on the selection of the appropriate type of CpG ODN for targeting PDC in clinical studies.

The availability of CpG-A and CpG-B as two model stimuli to elicit the IFNR-independent early or the IFNR-dependent late phase type I IFN production allowed us to study other molecules such as BDCA-2 and CD40L which are known to impact on the type I IFN pathway. The mAb against the PDC-specific C-type lectin BDCA-2 inhibits type I IFN synthesis in PDC (49). We found that BDCA-2 Ab inhibited IFN-{alpha} synthesis of both CpG-A and CpG-B. In addition, the BDCA-2 Ab further increased the inhibitory effect of IFNR blockade, and unlike IFNR blockade, stimulated an increase of intracellular calcium. Therefore, the mechanism by which BDCA-2 interacts with the IFN-{alpha} pathway is distinct and independent of the IFNR-mediated feedback loop.

Another molecule known to modulate the type I IFN production of PDC is CD40L (23). In this study, we found that CD40L enhanced the induction of IFN-{alpha} by CpG-B. Enhanced IFN-{alpha} production was not sensitive to IFNR blockade, indicating that the contribution of CD40L is independent of the IFNR-mediated feedback loop. Interestingly, IFN-{beta} production was not increased by CD40L, demonstrating that CD40L selectively amplifies the IFN-{alpha} response.

According to our data, the type of the stimulus rather than the presence of type I IFN itself determines the initiation of the IFNR-mediated feedback loop. Even increased amounts of IFN-{alpha} provided by combined stimulation with CpG-B and CD40L were not sufficient to trigger the IFNR-mediated pathway. The difference between CpG-A and CpG-B was not dependent on IRF3, IRF7, or STAT1 on the transcriptional level. It has been reported that virus-dependent kinases govern the IFNR-mediated pathway via phosphorylation of IRF3 and IRF7 (35). A recent study provides evidence that phosphorylation of IRF3 is not involved in CpG ODN-induced up-regulation of type I IFN synthesis (18). The identification of the signal that determines whether the PDC is capable of using the IFNR-mediated pathway and that is specifically activated by CpG-A but not CpG-B may lead to the distinct molecular mechanism responsible for the difference between both classes of CpG ODN. Because TLR9 is required for the activity of both classes of CpG ODN (30), a different affinity of TLR9 to CpG-A and CpG-B, possibly modulated by the engagement of different chaperons in the endosomal compartments, may be responsible for the distinct functional activities of CpG-A and CpG-B.

Even in the presence of IFNR blockade, the remaining levels of type I IFN induced by CpG-A are much higher than those with CpG-B ODN. This could be due to incomplete inhibition of IFNR (50) or due to higher activity of CpG-A upstream of IFNR. Nevertheless, in our study, CpG-B induced considerable amounts of IFN-{alpha} alone (~4 ng/ml) or in combination with CD40L (~9 ng/ml) independent of the IFNR-mediated feedback loop. This demonstrates that, although IFNR feedback contributes to the magnitude and duration of IFN-{alpha} production in CpG-A ODN-stimulated PDC, the PDC is capable of producing considerable amounts of IFN-{alpha} and IFN-{beta} independently of the IFNR.

Our observations demonstrate that, in human PDC, type I IFN expression is regulated differently than in other cell types reported previously, such as fibroblasts or monocyte-derived dendritic cells, in which IFN-{alpha} is controlled by IFNR-mediated feedback signaling (31). Our findings in human PDC are in agreement with a recent study in mice which demonstrated that, after viral infection in vivo, the subset of CD11cintCD11b-GR-1+ dendritic cells (presumably murine PDC (51)) produced IFN-{alpha} at high levels, and that early IFN-{alpha} production by these cells was largely independent of IFNR feedback signaling (42). One possible explanation for the capacity of PDC to produce IFNR-independent early IFN-{alpha} is the constitutive expression of IRF7: consistent but low IRF7 expression has been reported by Barchet et al. (42) for murine PDC isolated from unstimulated wild-type mice, and high constitutive IRF7 expression was found in our study for human PDC. It is interesting to note that murine and human PDC seem to have similar but not identical immunological functions. Besides different surface markers, murine PDC are capable of producing IL-12 in response to CpG ODN and HSV (52) or in response to murine CMV (53), whereas IL-12 production in human PDC requires costimulation with CpG ODN and CD40L (9). Future studies will show whether murine PDC, in addition to their reported IFNR-independent early IFN-{alpha} production (42), also exhibit an IFNR-mediated pathway, and whether this pathway is also controlled by the type of stimulus rather than the quantity of IFN-{alpha}{beta} available, similar to what has been demonstrated for CpG ODN 2216 and human PDC in this study.

In conclusion, our study provides the first set of data defining two distinct regulatory pathways of type I IFN synthesis in human PDC and demonstrating that these two pathways are selectively triggered by two distinct types of CpG ODN. CpG-A and CpG-B can now be used to examine the biological properties of PDC activated to display one (IFNR-dependent) or the other (IFNR-independent) pathway. Besides the quantity and the isoforms of type I IFN, a number of additional parameters that are differentially regulated by CpG-A and CpG-B, such as TNF-{alpha}, IL-8, and activation marker expression, will likely impact on the biological properties of CpG-stimulated PDC in vivo.


   Acknowledgments
 
We thank Dr. Norbert Lubenow and Prof. Andreas Greinacher from the Institute of Immunology and Transfusion Medicine at the University of Greifswald for providing blood products.


   Footnotes
 
1 G.H. is supported by grants from Deutsche Forschungsgemeinschaft (HA 2780/4-1); the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie and Coley Pharmaceutical GmbH (Langenfeld, Germany) (03-12235-6); the Friedrich-Baur-Stiftung; and the Human Science Foundation of Japan. S.E. is supported by a grant from the Deutsche Forschungsgemeinschaft (EN 169/7-1). S.R. is supported by Grant 258 of the Medical Faculty of the University of Munich. This work is part of the dissertation of M.K. at the Ludwig-Maximilians University (Munich, Germany). Back

2 M.K. and S.R. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Gunther Hartmann, Abteilung für Klinische Pharmakologie, Medizinische Klinik Innenstadt, Ziemssenstrasse 1, 80336 Munich, Germany. E-mail address: ghartmann{at}lrz.uni-muenchen.de Back

4 Abbreviations used in this paper: TLR, Toll-like receptor; PDC, plasmacytoid dendritic cell; ODN, oligonucleotide; IPC, type I IFN-producing cell; MHC-II, MHC class II; ISGF, IFN-stimulated gene factor; BDCA, blood dendritic cell Ag; CD40L, CD40 ligand; MFI, mean fluorescence intensity. Back

Received for publication September 26, 2002. Accepted for publication February 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bauer, S., C. J. Kirschning, H. Hacker, V. Redecke, S. Hausmann, S. Akira, H. Wagner, G. B. Lipford. 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. USA 98:9237.[Abstract/Free Full Text]
  2. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740.[Medline]
  3. Wagner, H.. 2001. Toll meets bacterial CpG-DNA. Immunity 14:499.[Medline]
  4. Wagner, H.. 2002. Interactions between bacterial CpG-DNA and TLR9 bridge innate and adaptive immunity. Curr. Opin. Microbiol. 5:62.[Medline]
  5. Kadowaki, N., S. Ho, S. Antonenko, R. W. Malefyt, R. A. Kastelein, F. Bazan, Y. J. Liu. 2001. Subsets of human dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens. J. Exp. Med. 194:863.[Abstract/Free Full Text]
  6. Hornung, V., S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T. Giese, S. Endres, G. Hartmann. 2002. Quantitative expression of Toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168:4531.[Abstract/Free Full Text]
  7. Bohle, B., B. Jahn-Schmid, D. Maurer, D. Kraft, C. Ebner. 1999. Oligodeoxynucleotides containing CpG motifs induce IL-12, IL-18 and IFN-{gamma} production in cells from allergic individuals and inhibit IgE synthesis in vitro. Eur. J. Immunol. 29:2344.[Medline]
  8. Hartmann, G., A. M. Krieg. 2000. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J. Immunol. 164:944.[Abstract/Free Full Text]
  9. Krug, A., A. Towarowski, S. Britsch, S. Rothenfusser, V. Hornung, R. Bals, T. Giese, H. Engelmann, S. Endres, A. M. Krieg, G. Hartmann. 2001. Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur. J. Immunol. 31:3026.[Medline]
  10. Krug, A., S. Rothenfusser, V. Hornung, B. Jahrsdörfer, S. Blackwell, Z. K. Ballas, S. Endres, A. M. Krieg, G. Hartmann. 2001. Identification of CpG oligonucleotide sequences with high induction of IFN-{alpha}/{beta} in plasmacytoid dendritic cells. Eur. J. Immunol. 31:2154.[Medline]
  11. Rothenfusser, S., V. Hornung, A. Krug, A. Towarowski, A. M. Krieg, S. Endres, G. Hartmann. 2001. Distinct CpG oligonucleotide sequences activate human {gamma}{delta} T cells via interferon-{alpha}{beta}. Eur. J. Immunol. 31:3525.[Medline]
  12. Davis, H. L., I. Suparto, R. Weeratna, S. Jumintarto, D. Iskandriati, S. Chamzah, A. Ma’ruf, C. Nente, D. Pawitri, A. M. Krieg, A. M. Heriyanto, W. Smits, D. Sajuthi. 2000. CpG DNA overcomes hyporesponsiveness to hepatitis B vaccine in orangutans. Vaccine 18:1920.[Medline]
  13. Jones, T. R., N. Obaldia, III, R. A. Gramzinski, Y. Charoenvit, N. Kolodny, S. Kitov, H. L. Davis, A. M. Krieg, S. L. Hoffman. 1999. Synthetic oligodeoxynucleotides containing CpG motifs enhance immunogenicity of a peptide malaria vaccine in Aotus monkeys. Vaccine 17:3065.[Medline]
  14. Hartmann, G., R. D. Weeratna, Z. K. Ballas, P. Payette, S. Blackwell, I. Suparto, W. L. Rasmussen, M. Waldschmidt, D. Sajuthi, R. H. Purcell, et al 2000. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164:1617.[Abstract/Free Full Text]
  15. Weiner, G. J.. 2000. The immunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides. J. Leukocyte Biol. 68:455.[Abstract/Free Full Text]
  16. Krieg, A. M.. 2002. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20:709.[Medline]
  17. Verthelyi, D., R. T. Kenney, R. A. Seder, A. A. Gam, B. Friedag, D. M. Klinman. 2002. CpG oligodeoxynucleotides as vaccine adjuvants in primates. J. Immunol. 168:1659.[Abstract/Free Full Text]
  18. Takauji, R., S. Iho, H. Takatsuka, S. Yamamoto, T. Takahashi, H. Kitagawa, H. Iwasaki, R. Iida, T. Yokochi, T. Matsuki. 2002. CpG-DNA-induced IFN-{alpha} production involves p38 MAPK-dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors. J. Leukocyte Biol. 72:1011.[Abstract/Free Full Text]
  19. O’Doherty, U., R. M. Steinman, M. Peng, P. U. Cameron, S. Gezelter, I. Kopeloff, W. J. Swiggard, M. Pope, N. Bhardwaj. 1993. Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium. J. Exp. Med. 178:1067.[Abstract/Free Full Text]
  20. O’Doherty, U., M. Peng, S. Gezelter, W. J. Swiggard, M. Betjes, N. Bhardwaj, R. M. Steinman. 1994. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology 82:487.[Medline]
  21. Galy, A., I. Christopherson, G. Ferlazzo, G. Liu, H. Spits, K. Georgopoulos. 2000. Distinct signals control the hematopoiesis of lymphoid-related dendritic cells. Blood 95:128.[Abstract/Free Full Text]
  22. Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah, S. Ho, S. Antonenko, Y. J. Liu. 1999. The nature of the principal type 1 interferon-producing cells in human blood. Science 284:1835.[Abstract/Free Full Text]
  23. Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, M. Colonna. 1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5:919.[Medline]
  24. Fitzgerald-Bocarsly, P.. 1993. Human natural interferon-{alpha} producing cells. Pharmacol. Ther. 60:39.[Medline]
  25. Chehimi, J., S. E. Starr, H. Kawashima, D. S. Miller, G. Trinchieri, B. Perussia, S. Bandyopadhyay. 1989. Dendritic cells and IFN-{alpha}-producing cells are two functionally distinct non-B, non-monocytic HLA-DR+ cell subsets in human peripheral blood. Immunology 68:488.
  26. Ito, T., R. Amakawa, T. Kaisho, H. Hemmi, K. Tajima, K. Uehira, Y. Ozaki, H. Tomizawa, S. Akira, S. Fukuhara. 2002. Interferon-{alpha} and interleukin-12 are induced differentially by Toll-like receptor 7 ligands in human blood dendritic cell subsets. J. Exp. Med. 195:1507.[Abstract/Free Full Text]
  27. Ronnblom, L., G. V. Alm. 2001. A pivotal role for the natural interferon-{alpha}-producing cells (plasmacytoid dendritic cells) in the pathogenesis of lupus. J. Exp. Med. 194:F59.
  28. Kadowaki, N., S. Antonenko, Y. J. Liu. 2001. Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c- type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN. J. Immunol. 166:2291.[Abstract/Free Full Text]
  29. Bauer, M., V. Redecke, J. W. Ellwart, B. Scherer, J. P. Kremer, H. Wagner, G. B. Lipford. 2001. Bacterial CpG-DNA triggers activation and maturation of human CD11c-, CD123+ dendritic cells. J. Immunol. 166:5000.[Abstract/Free Full Text]
  30. Hemmi, H., T. Kaisho, O. Takeuchi, S. Sato, H. Sanjo, K. Hoshino, T. Horiuchi, H. Tomizawa, K. Takeda, S. Akira. 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3:196.[Medline]
  31. Remoli, M. E., E. Giacomini, G. Lutfalla, E. Dondi, G. Orefici, A. Battistini, G. Uze, S. Pellegrini, E. M. Coccia. 2002. Selective expression of type I IFN genes in human dendritic cells infected with Mycobacterium tuberculosis. J. Immunol. 169:366.[Abstract/Free Full Text]
  32. Bisat, F., N. B. Raj, P. M. Pitha. 1988. Differential and cell type specific expression of murine {alpha}-interferon genes is regulated on the transcriptional level. Nucleic Acids Res. 16:6067.[Abstract/Free Full Text]
  33. Lin, R., Y. Mamane, J. Hiscott. 2000. Multiple regulatory domains control IRF-7 activity in response to virus infection. J. Biol. Chem. 275:34320.[Abstract/Free Full Text]
  34. Nguyen, H., J. Hiscott, P. M. Pitha. 1997. The growing family of interferon regulatory factors. Cytokine Growth Factor Rev. 8:293.[Medline]
  35. Marie, I., J. E. Durbin, D. E. Levy. 1998. Differential viral induction of distinct interferon-{alpha} genes by positive feedback through interferon regulatory factor-7. EMBO J. 17:6660.[Medline]
  36. Sato, M., N. Hata, M. Asagiri, T. Nakaya, T. Taniguchi, N. Tanaka. 1998. Positive feedback regulation of type I IFN genes by the IFN-inducible transcription factor IRF-7. FEBS Lett. 441:106.[Medline]
  37. Barnes, B. J., P. A. Moore, P. M. Pitha. 2001. Virus-specific activation of a novel interferon regulatory factor, IRF- 5, results in the induction of distinct interferon-{alpha} genes. J. Biol. Chem. 276:23382.[Abstract/Free Full Text]
  38. Darnell, J. E., Jr. 1997. STATs and gene regulation. Science 277:1630.[Abstract/Free Full Text]
  39. Au, W. C., P. A. Moore, W. Lowther, Y. T. Juang, P. M. Pitha. 1995. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc. Natl. Acad. Sci. USA 92:11657.[Abstract/Free Full Text]
  40. Ito, T., R. Amakawa, M. Inaba, S. Ikehara, K. Inaba, S. Fukuhara. 2001. Differential regulation of human blood dendritic cell subsets by IFNs. J. Immunol. 166:2961.[Abstract/Free Full Text]
  41. Gary-Gouy, H., P. Lebon, A. H. Dalloul. 2002. Type I interferon production by plasmacytoid dendritic cells and monocytes is triggered by viruses, but the level of production is controlled by distinct cytokines. J. Interferon Cytokine Res. 22:653.[Medline]
  42. Barchet, W., M. Cella, B. Odermatt, C. Asselin-Paturel, M. Colonna, U. Kalinke. 2002. Virus-induced interferon-{alpha} production by a dendritic cell subset in the absence of feedback signaling in vivo. J. Exp. Med. 195:507.[Abstract/Free Full Text]
  43. Hartmann, G., G. Weiner, A. M. Krieg. 1999. CpG DNA: a potent signal for growth, activation and maturation of human dendritic cells. Proc. Natl. Acad. Sci. USA 96:9305.[Abstract/Free Full Text]
  44. Hartmann, G., A. Krug, K. Waller-Fontaine, S. Endres. 1996. Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin. Mol. Med. 2:429.[Medline]
  45. Hartmann, G., A. Krug, M. Bidlingmaier, U. Hacker, A. Eigler, R. Albrecht, C. J. Strasburger, S. Endres. 1998. Spontaneous and cationic lipid-mediated uptake of antisense oligonucleotides in human monocytes and lymphocytes. J. Pharmacol. Exp. Ther. 285:920.[Abstract/Free Full Text]
  46. Lipp, P., E. Niggli. 1993. Ratiometric confocal Ca2+-measurements with visible wavelength indicators in isolated cardiac myocytes. Cell Calcium 14:359.[Medline]
  47. Novak, E. J., P. S. Rabinovitch. 1994. Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/fura red fluorescence ratios. Cytometry 17:135.[Medline]
  48. Verthelyi, D., K. Ishii, M. Gursel, F. Takeshita, D. Klinman. 2001. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs. J. Immunol. 166:2372.[Abstract/Free Full Text]
  49. Dzionek, A., Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, G. Gunther, I. Johnston, A. Lanzavecchia, T. Nagasaka, et al 2001. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon {alpha}/{beta} induction. J. Exp. Med. 194:1823.[Abstract/Free Full Text]
  50. Colamonici, O. R., P. Domanski. 1993. Identification of a novel subunit of the type I interferon receptor localized to human chromosome 21. J. Biol. Chem. 268:10895.[Abstract/Free Full Text]
  51. Nakano, H., M. Yanagita, M. D. Gunn. 2001. CD11c+B220+Gr-1+ cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J. Exp. Med. 194:1171.[Abstract/Free Full Text]
  52. Gilliet, M., A. Boonstra, C. Paturel, S. Antonenko, X. L. Xu, G. Trinchieri, A. O’Garra, Y. J. Liu. 2002. The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 195:953.[Abstract/Free Full Text]
  53. Dalod, M., T. P. Salazar-Mather, L. Malmgaard, C. Lewis, C. Asselin-Paturel, F. Briere, G. Trinchieri, C. A. Biron. 2002. Interferon {alpha}/{beta} and interleukin 12 responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo. J. Exp. Med. 195:517.[Abstract/Free Full Text]



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