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Originally published online as doi:10.1189/jlb.0405208 on August 4, 2005

Published online before print August 4, 2005
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(Journal of Leukocyte Biology. 2005;78:930-936.)
© 2005 by Society for Leukocyte Biology

The effects of CpG DNA on HMGB1 release by murine macrophage cell lines

Weiwen Jiang*, Jianhua Li{dagger}, Margot Gallowitsch-Puerta{dagger}, Kevin J. Tracey{dagger} and David S. Pisetsky*,{ddagger},1

* Division of Rheumatology and Immunology, Department of Medicine, Duke University, Durham, North Carolina;
{dagger} Laboratory of Biomedical Science, North Shore University Hospital-New York University School of Medicine, Manhasset; and
{ddagger} Medical Research Services, Durham Veterans Affairs Medical Center, North Carolina

1Correspondence: 151G Durham VA Medical Center, 508 Fulton Street, Durham, NC 27705. E-mail: piset001{at}mc.duke.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA containing cytosine-guanine dinucleotide (CpG) motifs (CpG DNA) has potent immunostimulatory activities that resemble those of lipopolysaccharide (LPS) in its effects on the innate immune system. Among its activities, LPS can induce the release of high mobility group protein (HMGB1) by macrophages, a dual function molecule that can mediate the late effects of LPS. To determine whether CpG DNA can also induce HMGB1 release, the effects of a synthetic CpG oligonucleotide (ODN) on HMGB1 release from RAW 264.7 and J774A.1 cells were assessed by Western blotting of culture supernatants. Under conditions in which the CpG ODN activated the cell lines, as assessed by stimulation of tumor necrosis factor {alpha} and interleukin-12, it failed to cause HMGB1 release into the media. Although unable to induce HMGB1 release by itself, the CpG ODN nevertheless potentiated the action of LPS. With RAW 264.7 cells, lipoteichoic acid and polyinosinic-polycytidylic acid, like LPS, stimulated HMGB1 release as well as cytokine production. These results indicate that the effects of CpG DNA on macrophages differ from other ligands of Toll-like receptors and may lead to a distinct pattern of immune cell activation in the context of infection or its use as an immunomodulatory agent.

Key Words: oligonucleotide • lipopolysaccharide • Toll-like receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA is a complex macromolecule whose immunological properties vary with species origin. As shown in vitro as well as in vivo, DNA from bacteria has potent immunostimulatory activities, which resemble those of lipopolysaccharide (LPS) [1 , 2 ]. These activities depend on sequence motifs that center on unmethylated cytosine-guanine dinucleotide (CpG) dinucleotides, and DNA bear these motifs known as CpG DNA [3 ]. As these motifs occur more commonly in bacterial than mammalian DNA, they represent an immune recognition system based on DNA sequence. Along with other bacterial molecules that serve as pathogen-associated molecular patterns (PAMPs), CpG DNA can stimulate innate immunity by interaction with pattern recognition receptors known as Toll-like receptors (TLRs) [4 ].

Although CpG DNA and LPS induce responses in common, they nevertheless differ in their signaling pathways and range of activities. Thus, CpG DNA acts through TLR9, and LPS acts through TLR4 [5 , 6 ]. It is important that TLR9 activation occurs at an intracellular site, with access to DNA requiring internalization and passage through an endosomal compartment [7 ]. In contrast, TLR4 is a surface receptor. Another difference between activation by CpG DNA and LPS concerns the relative induction, directly or indirectly, of various cytokines and other mediators, including glucocorticoids [8 ]. The basis for these differences is not known, as LPS and CpG DNA, despite use of different TLRs, nevertheless involve similar downstream signaling molecules such as myeloid differentiaton factor 88 [9 ].

Among molecules whose expression is modulated by LPS, high-mobility group (HMG)B1 serves as a late mediator of its action. HMGB1 is a nonhistone nuclear molecule with dual function. Inside the cell, HMGB1 plays a role in chromatin structure and transcriptional activity [10 11 12 ]. Outside the cell, HMGB1 serves as a cytokine and induces an array of inflammatory responses such as those of LPS [13 ]. The release of HMGB1 from the cell during activation involves hyperacetylation, translocation from the nucleus, and secretion from vesicles [14 ]. A central role of HMGB1 in inflammation comes from studies in animal models showing that inhibition of HMGB1 by antibody treatment can attenuate sepsis and collagen-induced arthritis [15 , 16 ].

To explore further the activity of CpG DNA in immunity, we have investigated whether CpG DNA can function like LPS and can induce HMGB1 release from macrophages. For this purpose, we have compared the effects of a synthetic CpG ODN and LPS using the murine macrophage cell lines RAW 264.7 and J774A.1 as models. HMGB1 release from cells was assessed by Western blotting of supernatants as well as confocal microscopy. In studies presented herein, we show that activation of macrophages by CpG DNA, unlike that of LPS, does not induce the translocation and release of HMGB1. Nevertheless, CpG DNA can act synergistically with LPS to induce the cellular release of this mediator. Together, these results indicate important differences in the consequences of TLR activation and distinguish CpG DNA from LPS in its potential for stimulating innate immunity and inducing inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and cell culture
The murine macrophage-like cell lines, RAW 264.7 and J774A.1, were purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 media (Invitrogen, San Diego, CA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. A synthetic phosphorothioate CpG oligonuecleotide (ODN), denoted ODN1826, was used as a source of CpG [17 ]. This ODN was purchased from the Midland Certified Reagent Co. (Midland, TX) and has the following sequence: 5'-TCCATGACGTTCCTGACGTT-3'. The control ODN, SAK1, has the sequence 5'-TCCATGAGCTTCCTGAGTCT-3'. Other stimulants tested include LPS from Escherichia coli 0111:B4 (Sigma Chemical Co., St. Louis, MO), lipoteichoic acid (LTA) from Bacillus subtilis (InvivoGen, San Diego, CA), and polyinosinic-polycytidylic [poly (I:C); Invivogen]. For RAW 264.7 and J774A.1, cells were plated in six-well culture plates for 2–3 h, washed twice with Opti-MEM (Invitrogen), and stimulated with different TLR ligands in Opti-MEM for 24 h. Supernatants were collected and assayed for cytokines or HMGB1.

Western blotting
Supernatants described above were collected after 24 h of stimulation and concentrated by Centricon YM-10 (Millipore, Billerica, MA). The volume of the concentrated supernatants was adjusted to ~70 µl, and samples were resolved on 4–12% NuPAGE® Tris-Bis sodium dodecyl sulfate-polyacrylamide gel (Invitrogen). Protein was transferred to polyvinylidene difluoride membranes (Invitrogen), blocked with 5% dry milk in Tris-buffered saline-Tween, and blotted with rabbit anti-HMGB1 polyclonal antibody. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) followed by Super Signal® West Femto substrate (Pierce, Rockford, IL). Images were captured by exposing the membrane to a charged-coupled deivce camera (FluorChem8900, Alpha Innotech, San Leandro, CA).

Enzyme-linked immunosorbent assay (ELISA)
To determine cytokine levels in cultured RAW 264.7 cells, supernatants were collected at 24 h after stimulation. Cytokine [tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL)-12] concentrations in culture media were determined by ELISA. Capture antibodies, anti-TNF-{alpha} (R&D Systems, Minneapolis, MN) or anti-IL-12 p40/p70 (BD PharMingen, San Diego, CA), were coated overnight at 4°C on Immunlon® 96-well plates. After three washes, samples were added in duplicate and incubated at room temperature for 2 h. Biotinylated anti-TNF-{alpha} (R&D Systems) or anti-IL-12 p40/p70 detection antibodies (BD PharMingen) were added and incubated for an additional 2 h at room temperature. Avidin-conjugated HRP was added with 30 min incubation at room temperature followed by 0.015% 3,3',5,5'-tetramethylbenzidine, 0.01% H2O2in 0.1 M citrate buffer, pH 4.0, substrate until color development. Three phosphate-buffered saline (PBS) washes were performed between steps, and plates were read at optical density of 650 using an automated microtiter plate reader.

Confocal imaging
After 24 h stimulation, cells were washed twice with ice-cold PBS, scraped off the plates, and replated in eight-well chamber slides in RPMI 1640 supplemented with 10% FBS. Cells were allowed to adhere for 2–3 h, washed twice with ice-cold PBS. Cells are then fixed and permeablized using Cytofix/Permeable kit (BD PharMingen), followed by incubation with rabbit-anti-HMGB1 antibody and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Images were captured by a confocal laser-scanning microscope (Zeiss LSM 510, Carl Zeiss, Oberkochen, Germany).

Endotoxin assays
A commercial chromogenic limulus amebocyte lysate (LAL) test (BioWhittaker, Walkersville, MD) was used to determine endotoxin levels in the TLR ligands. To further determine the effects of endotoxin on HMGB1 release, polymixin B was added in the cultures stimulated with different TLR ligands. Supernatants were collected at 20–24 h after stimulation and were subjected to Western blotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of CpG DNA on HMGB1 release
In these studies, we have investigated whether CpG DNA, like LPS, can induce the release of HMGB1 from macrophages. For this purpose, we stimulated RAW 264.7 and J774A.1 cells with a CpG ODN and LPS and measured HMGB1 release by Western blotting. As shown in Figure 1A , LPS induced the release of HMGB1 in RAW 264.7 cells, confirming previously reported studies [13 , 18 ]. In contrast, ODN1826, a CpG phosphorothioate ODN, failed to induce HMGB1 release over a wide range of concentrations (Fig. 1B) . To confirm the activity of ODN1826, TNF-{alpha} and IL-12 levels were also measured. As shown in Figure 1C , the levels of cytokines induced by ODN1826 were similar to those of LPS. This stimulatory effect was sequence-specific, as the control ODN lacking a CpG motif was inactive. Similar results were obtained with the J774A.1 cell line, although background levels of extracellular HMGB1 were higher than those observed with the RAW 264.7 cells (Fig. 1A) .



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  		Figure 1. The effect of CpG DNA and LPS on HMGB1 release. RAW 264.7 and J774.A1 cells were stimulated with medium, LPS (0.5 µg/ml), SAK1 (1.5 µM), and 1826 (1.5 µM) for 24 h. Culture medium was collected at 24 h and subjected to Western blotting (A) and cytokine ELISA assay (C; RAW 264.7 cells only). The effects of increasing doses of LPS and 1826 on RAW 264.7 cell release of HMGB1 release into culture medium were also assayed by Western blotting (B).

 
The effects of CpG DNA on HMGB1 release induced by LPS
These results suggest that CpG DNA, unlike LPS, does not cause HMGB1 release despite its ability to activate macrophages. A failure to induce HMGB1 release appears to be common to TLR9 ligands, as similar results were obtained with E. coli DNA, and a "second generation" CpG ODN modified chemically to enhance activity (data not shown). To investigate whether CpG DNA has any effect on HMGB1 release, RAW 264.7 cells were stimulated with LPS and CpG DNA to determine any interaction between the two compounds. As data shown in Figure 2A , although LPS at a concentration of 0.025 µg/ml failed to induce HMGB1 release, combination with CpG DNA caused this release. This effect was specific, as the inactive ODN did not cause HMGB1 release in the presence of the low concentration of LPS. The HMGB1 release induced by the combination of the two compounds appears to correlate with a synergistic cytokine production (Fig. 2B) . ODN1826 also dose-dependently augmented HMGB1 release in the presence of suboptimal concentrations of LPS (Fig. 2C) .



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  		Figure 2. Synergistic effects of CpG DNA and LPS on HMGB1 release. RAW 264.7 cells were stimulated with a suboptimal dose of LPS (0.025 µg/ml), SAK1, or 1826 (1.5 µM) or a combination of DNA and LPS for 24 h. Supernatants were collected and assayed for HMGB1 (A) and cytokine (B). To determine a dose range for CpG DNA-induced synergy in HMGB1 release, 1826, at a concentration ranging from 0.015 to 1.5 µM, was added to RAW 264.7 cell culture medium containing a low-dose LPS (0.025 µg/ml). Twenty-four hours after the stimulation, culture medium was subject to Western blotting (C).

 
To evaluate further the effects of LPS and CpG DNA on HMGB1 release, confocal microscopy was used. As shown by other investigators [18 ], LPS causes the translocation of HMGB1 from nucleus to the cytoplasm for eventual secretion, a process in which nuclear staining of HMGB1 is reduced. As shown in Figure 3 , in control RAW 264.7 cells, HMGB1 staining is distributed throughout the cell rather than showing exclusive nuclear localization. The staining of nucleus and cytoplasm appears to be a feature of this cell line, as shown in other studies [18 ]. In contrast, in cells stimulated with LPS, nuclear staining is absent in a portion of the cells. In contrast, the translocation of HMGB1 is not observed with cells stimulated with control ODN, ODN1826, or low-dose LPS; nuclear translocation was observed, however, with the combination of LPS and ODN1826. These results confirm findings with Western blotting and provide further evidence for the differences in stimulation by LPS and CpG DNA.



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  		Figure 3. HMGB1 translocation in RAW 264.7 cells by confocal microscopy. Stimulated RAW 264.7 cells were plated on chamber slides and allowed to attach. Cells were fixed and permeabilized, followed by rabbit anti-HMGB1 and anti-rabbit IgG conjugated with Alexa Fluor 488.

 
The effects of LTA and double-stranded (ds)RNA on HMGB1 release
As CpG and LPS differed in the induction of HMGB1 release, other TLR ligands were studied to investigate more fully the range of triggers for this response. For this purpose, LTA and poly (I:C) were studied. LTA, a cell-wall product of a Gram-positive organism, interacts with TLR2, and the synthetic dsRNA, poly (I:C), interacts with TLR3 [19 , 20 ]. As shown in Figure 4A , LTA stimulates HMGB1 release in RAW 264.7 cells in a dose-dependent way; similarly, poly (I:C) induces strong HMGB1 release even at the lowest dose (0.25 µg/ml) tested (Fig. 4A) . Like LPS, both ligands induce TNF-{alpha} production in association with HMGB1 release and differ from CpG DNA in this property (Fig. 4B) . Confirming the Western blot, confocal microcopy demonstrated HMGB1 translocation (Fig. 4C) , although translocation was only observed in some cells in the cultures.



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  		Figure 4. The effects of TLR2 and TLR3 stimulation on HMGB1 release. RAW 264.7 cells were stimulated with LTA and poly (I:C) at varying concentrations for 24 h. Culture medium was collected and analyzed by Western blotting (A) and cytokine ELISA assays (B). Stimulated RAW 264.7 cells were plated on chamber slides, fixed, permeablized, and stained for HMGB1, and translocation was visualized by confocal microscopy (C).

 
To show that the stimulation of HMGB1 by LTA and poly (I:C) was specific for these receptors and did not result from the contamination of LPS, additional experiments were performed. As shown by the LAL assay, the LTA used in this study had a low amount of endotoxin (0.1 EU/ml). This amount is more than 1000 times less than the amount of LPS needed in this study to cause HMGB1. Furthermore, the addition of polymixin B to the culture inhibited LPS-induced HMGB1 release but not LTA-induced HMGB1 release (Fig. 5 ). Similar results were obtained with poly (I:C). These results indicate that HMGB1 secretion, by LTA and poly (I:C), reflects the direct action of these compounds rather than LPS contamination. Together, these results indicate that stimulation of TLR9 by CpG DNA differs from stimulation of TLR2, -3, and -4 in the translocation and release of HMGB1.



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  		Figure 5. The effects of polymixin B on HMGB1 release stimulated by LPS, LTA, or poly (I:C). RAW 264.7 cells were stimulated with different TLR ligands as above in the presence of polymixin B. Supernatants were collected and analyzed by Western blotting.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results presented herein suggest striking differences in the stimulation of innate immunity by CpG DNA compared with LPS as well as LTA and poly (I:C). Whereas stimulation by CpG DNA failed to induce HMGB1 release from macrophages, the other PAMPs tested induced the release of this molecule into the supernatant as well as caused its translocation from the nucleus. The failure of CpG DNA to cause HMGB1 release did not reflect a defect in immune stimulation of the cell lines tested, as under the conditions of these experiments, CpG DNA induced the expression of TNF-{alpha} and IL- 12. As such, these findings suggest selective stimulation of inflammatory mediator release by different PAMPs, a finding that is relevant to the activation of innate immunity in bacterial infection as well as settings in which PAMPs may be used as adjuvants or immune modulators.

As shown by studies in vitro and in vivo, HMGB1 serves as a late mediator of the action of LPS and displays a wide array of immunostimulatory actions, which includes the induction of TNF-{alpha}, IL-1, and nitric oxide (NO) [21 22 23 ]. These actions become manifest when HMGB1 exits the cell into the external milieu, where it displays its alternative function as a cytokine. This action reflects the activity of a structural domain of HMGB1—the B box [24 ]. It is interesting that another structural domain—the A box—can inhibit responses of HMGB1 [16 ]. Although the signaling pathways elicited by HMGB1 are not fully defined, there is evidence that the triggering occurs via the receptor for advanced glycation end-products [25 ] as well as TLR2 and TLR4 [26 ]. The importance of this pathway for immune activation is evident in studies demonstrating that HMGB1 levels are elevated in patients with sepsis [13 ] and that anti- HMGB1 antibodies can block sepsis and inflammation in animal models [16 ].

In these studies, we have used a synthetic oligonucleotide as a model for CpG DNA. As shown in many studies, this ODN is a potent immune stimulator, which can activate cells via TLR9 and elicit a wide array of inflammatory responses that include B cell activation and stimulation of proinflammatory mediators including cytokines and NO [3 , 27 , 28 ]. Nevertheless, this ODN failed to induce release of HMGB1 and, moreover, failed to cause the translocation of this molecule from the nucleus. Other TLR9 ligands, including a natural DNA as well as a second-generation immunomer, had similar behavior as the ODN, although these sources of CpG DNA induced cytokine production (data not shown). These findings suggest that TLR9 ligands, in general, are incapable of inducing HMGB1 release from macrophages.

Although CpG DNA alone did not cause HMGB1 release, it nevertheless potentiated the action of LPS. Thus, in the presence of CpG DNA, the doses of LPS required for HMGB1 release were reduced dramatically. Synergism between CpG DNA and LPS is also manifest in other responses such as the production of TNF-{alpha} and IL-12. Together, these findings indicate that CpG DNA and LPS, although leading to similar signaling events such as the activation of mitogen-activated protein kinases and nuclear factor-{kappa}B, nevertheless differ in the ultimate effects of stimulation. Furthermore, CpG DNA differs from ligands of TLR2 and TLR3, which, like LPS, can induce cytokine expression and HMGB1 release. In the context of infection, although CpG alone may not trigger HMGB1 release, it nevertheless may act in concert with other PAMPs to augment and prolong this response.

The differences between stimulation by CpG DNA and LPS in the HMGB1 release are notable, given their categorization as PAMPs and postulated roles in innate immunity. Previous studies have also demonstrated that CpG DNA and LPS are not equivalent in their action. Thus, LPS induces significant release of glucocorticoids under conditions in which CpG DNA is much less effective in this response, despite similar induction of cytokines [8 , 29 ]. Furthermore, in this setting in vivo, LPS, but not CpG DNA, can induce extensive lymphocyte apoptosis, which can culminate into the release of nucleosomes into the blood [8 ]. Such cell loss may contribute to the immune suppression that occurs during sepsis.

Previous studies about the induction of shock by CpG DNA involved mice treated with galactosamine [30 ]. This sugar inhibits transcription in the liver and renders mice extraordinarily sensitive to the effects of TNF-{alpha}. In mice treated with galactosamine, LPS can stimulate shock at doses 1000 less than those required for an untreated animal. Although CpG DNA can induce shock in galactosamine-treated mice, the effect may result from an enhanced effect of the TNF induced. In the intact mouse, however, although CpG DNA can stimulate TNF production, the other conditions necessary for shock may not be present. As such, stimulation by CpG DNA may be well-tolerated. In this conceptualization, CpG DNA may be "safer" than other "danger" molecules.

Another potential difference between CpG ODN and other TLR ligands on the induction of HMGB1 release concerns the effects of these agents on apoptosis. Thus, in these culture systems, RAW 264.7 cell activation by LPS, LTA, and dsRNA is accompanied by an increase in apoptosis as assessed by activation of caspase 3, although CpG DNA does not cause this response (W. Jiang and D. S. Pisetsky, preliminary observations). Although HMGB1 release has been considered a property of necrotic but not apoptotic cells, this issue has been investigated extensively only in HeLa cells [31 ]. It is possible therefore that certain cell types (e.g., macrophages), undergoing apoptosis or secondary necrosis, release HMGB1, accounting for the differences among TLR ligands in this response. This issue is currently under investigation.

CpG ODN has been investigated as an adjuvant as well as immunomodulator to enhance host defense in the setting of infection [32 , 33 ]. As suggested by the current studies as well as those previously published, CpG DNA may lead to a unique pattern of immune activation characterized by induction of cytokines in the absence of HMGB1 release as well as other immune effects, including lymphocyte apoptosis [8 ] and glucocorticoid induction [8 , 29 ]. Although this pattern may be beneficial for an adjuvant, its role in the stimulation of innate immunity is speculative.

While this manuscript was being prepared, Dumitriu et al. [34 ] reported that CpG ODN induced human plasmacytoid DCs to release HMGB1, a result in contrast to our findings with macrophages. Among differences between our studies and theirs are the cell type, species, maturation stage, and use of cell lines as opposed to cells grown from peripheral blood. Furthermore, upon CpG ODN stimulation, plasmacytoid dendritic cells produce large amounts of interferon-{alpha}, and macrophages produce IL-12 and NO [35 ]. These mediators may determine whether HMGB1 is released. In this regard, although activation via different TLRs may induce similar signaling pathways, it is also possible that depending on cell type, CpG DNA may trigger a unique secondary signal, as yet to be identified, which prevents the release of HMGB1. Future studies will define the pathways by which triggering different TLRs leads to HMGB1 release and the interaction of these pathways occurring during infection.


   ACKNOWLEDGEMENTS
 
This work is supported by Veterans Affairs Medical Research Service, National Institutes of Health (NIH) Grant AI44808, and a grant from the Lupus Research Institute. W. J. is supported by NIH Training Grant T32 EB01630. The authors thank Dr. Farshid Guilak and Robert A. Nielsen for their technical support with confocal imaging.

Received April 20, 2005; revised June 29, 2005; accepted July 5, 2005.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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