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Originally published online as doi:10.1189/jlb.0204089 on October 20, 2004

Published online before print October 20, 2004
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(Journal of Leukocyte Biology. 2005;77:71-79.)
© 2005 by Society for Leukocyte Biology

Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9-dependent and -independent pathways

Kei Yasuda, Yoshiyuki Ogawa, Ikuko Yamane, Makiya Nishikawa and Yoshinobu Takakura1

Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Japan

1 Correspondence: Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29, Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan. E-mail: takakura{at}pharm.kyoto-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, we showed that bacterial DNA and vertebrate DNA/cationic liposome complexes stimulate potent inflammatory responses in cultured mouse macrophages. In the present study, we examined whether endocytosis and subsequent acidification are associated with these responses. The endocytosis inhibitor, cytochalasin B, reduced tumor necrosis factor {alpha} (TNF-{alpha}) production by a plasmid DNA (pDNA)/cationic liposome complex. The endosomal acidification inhibitor, monensin, inhibited cytokine production by pDNA or a calf thymus DNA/liposome complex. These results suggest, similarly to CpG motif-dependent responses, that endocytosis and subsequent endosomal acidification are also required for these inflammatory responses. It is intriguing that another inhibitor of endosomal acidification, bafilomycin A, stimulated the production of TNF-{alpha} mRNA and its protein after removal of the pDNA/liposome complex and inhibitors, although it inhibited the release of interleukin-6. Similar phenomena were observed in the activation of macrophages by CpG oligodeoxynucleotide, calf thymus DNA, and Escherichia coli DNA complexed with liposomes. Moreover, bafilomycin A also induced a high degree of TNF-{alpha} release after stimulation with naked pDNA. These results suggest that bafilomycin A increases TNF-{alpha} production induced by DNA at the transcriptional level via an as-yet unknown mechanism. Furthermore, we investigated the contribution of Toll-like receptor 9 (TLR9), the receptor of CpG motifs, to the cell activation by the DNA/cationic liposome complex using the macrophages from TLR9–/– mice. We observed a reduced inflammatory cytokine release from macrophages of TLR9–/– mice compared with wild-type mice. However, the cytokine production was not completely abolished, suggesting that the DNA/cationic liposome complex can induce macrophage activation via TLR9-dependent and -independent pathways.

Key Words: macrophages • CpG motifs • tumor necrosis factor (TNF)-{alpha} • gene therapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Unmethylated CpG sequences (CpG motifs) in bacterial DNA, but not in vertebrate DNA, are recognized by the immune system as a danger signal [1 , 2 ]. When macrophages or dendritic cells (DC) take up CpG DNA, it is recognized by Toll-like receptor 9 (TLR9), which is one of the pattern recognition receptors [3 ]. TLR9 is present in the intracellular compartment [4 ], and inflammatory cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}), interleukin-6 (IL-6), and IL-12 are secreted. These cytokines significantly influence DNA-based therapies in different ways. In gene therapy, cytokine production generally seems inappropriate, as these inflammatory cytokines significantly reduce transgene expression in target cells through their direct cytotoxicity and promoter attenuation [5 , 6 ]. Conversely, they are essential for the efficacy of DNA vaccination, as these cytokines can enhance the immune responses, and the balance of these cytokines profoundly affects the nature of the immune responses [7 , 8 ].

Cationic liposomes are often used for easy and efficient transfection of plasmid DNA (pDNA) in vitro and in vivo. Several recent studies have shown that intravenous (i.v.) administration of a pDNA/cationic liposome complex leads to systemic gene expression especially in the lung. However, pDNA/cationic liposome complexes are well known to induce high amounts of inflammatory cytokines in vivo [9 10 11 12 ]. When delivered intranasally, pDNA/liposome complexes have a marked toxic effect on the lung [12 ]. Empty pDNA complexed with liposomes can produce a potent antitumor effect [13 ]. Even when inflammation is not critical, gene expression using a pDNA/liposome complex is only transient [14 ]. Qin et al. [5 ] have shown that interferon-{gamma} (IFN-{gamma}) and TNF-{alpha} inhibit gene expression by promoter attenuation. In vitro gene expression of lung endothelial cells was reduced by TNF-{alpha} at low concentrations even when no obvious toxicity was observed [15 ]. We have demonstrated that tissue macrophages play an important role in cytokine induction following i.v. injection of pDNA cationic liposome formulations [16 ]. The important role of immunostimulatory effects mediated by the CpG motif in gene therapy and DNA vaccination has been clearly defined. However, most of the in vitro studies focusing on the mechanisms of activation mediated by CpG DNA have been carried out using naked phosphorothioate CpG oligodeoxynucleotide (CpG S-ODN) or naked bacterial DNA in combination with macrophage cell lines.

We have studied the in vivo disposition characteristics of naked pDNA and found that the liver nonparenchymal cells, probably Kupffer cells (liver-resident macrophages), play an important role [17 , 18 ]. Further in vitro studies using cultured mouse peritoneal macrophages have demonstrated that a specific receptor, such as the class A scavenger receptor, may be involved in the endocytotic uptake of naked pDNA by macrophages [19 , 20 ]. Conversely, pDNA/cationic liposome complexes should be taken up by macrophages via a nonspecific mechanism based on electrostatic interaction. pDNA/cationic liposome complexes and naked CpG-ODN have been assumed to induce immune responses via similar mechanisms. However, we have demonstrated that not only bacterial DNA but also vertebrate calf thymus DNA complexed with cationic liposomes induce inflammatory cytokines from murine macrophages [21 ].

In the light of these findings, the present study was undertaken to characterize the inflammatory responses by a DNA/cationic liposome complex in mouse peritoneal macrophages in vitro. We examined whether endocytosis and endosomal acidification are also required for the inflammatory responses to pDNA or calf thymus DNA complexed with liposomes in macrophages. In addition, we examined whether this macrophage activation induced by the DNA/cationic liposome complex is dependent on TLR9.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
RPMI-1640 medium and Hanks’ balanced salt solution were obtained from Nissui Pharmaceutical (Tokyo, Japan). Cytochalasin B, chloroquine, monensin, Escherichia coli DNA, and calf thymus DNA were purchased from Sigma Chemical Co. (St. Louis, MO). Bafilomycin A was purchased from Wako (Tokyo, Japan). LipofectAMINE plusTM (LAplus or LA) reagent and Opti-modified Eagle’s medium (MEM) were purchased from Invitrogen (Carlsbad, CA). Dextran sulfate (molecular weight, 500,000) and Triton X-114 were purchased from Nacalai Tesque (Kyoto, Japan).

Cell cultures
Male Institute for Cancer Research (ICR; 5 weeks) mice or C3H/HeJ mice [lipopolysaccharide (LPS) nonresponder; 5 weeks] were purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals (Japan). C57BL/6 mice were obtained from Harlan Winkelmann (Borchen, Germany). TLR9–/– mice and littermate wild-type C57BL/6 mice were used at 8–12 weeks of age. Resident macrophages were collected from the peritoneal cavity of unstimulated mice with RPMI-1640 medium. Cells were washed, suspended in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (1.2 µg/ml), and then plated on 24-well culture plates (Falcon, Becton Dickinson, Lincoln Park, NJ) at a density of 5 x 105 cells/well for the activation experiments. For confocal microscopic observations, cells were plated on a cover glass in 12-well culture plates at a density of 5 x 105 cells/well. After a 2-h incubation at 37°C in 5% CO2–95% air, adherent macrophages were washed three times with RPMI-1640 medium to remove nonadherent cells and then cultured under the same conditions for 24 h. RAW264.7 cells were cultured with RPMI-1640 medium supplemented with 10% FBS, penicillin G (100 U/ml), and streptomycin (100 µg/ml). They were then plated on 24-well culture plates at a density of 5 x 105 cells/ml and cultured for 24 h. Peritoneal macrophages from TLR9–/– mice or littermate wild-type C57BL/6 mice were plated on 96-well culture plates at a density of 1 x 106 cells/well.

pDNA
pcDNA3 vector was purchased from Invitrogen. The cytomegalovirus promoter-luciferase (pCMV-Luc)-encoding firefly Luc gene was constructed as described previously [22 ]. pcDNA3 has 26 5'-Pur-Pur-CpG-Pyr-Pyr-3' sequences including two GACGTT sequences reported to be the most potent CpG motifs for mice [23 ]. pDNA was purified using an Endo-freeTM plasmid Giga kit (Qiagen, Valencia, CA). Methylated-CpG pDNA was synthesized by methylation of pDNA (pCMV-Luc) with 1 unit CpG methylase (New England Biolabs, Beverly, MA) per µg pDNA for 24 h at 37°C. The methylated-CpG pDNA was tested for digestion with HpaII (Takara, Kyoto, Japan) to confirm methylation. pDNA mobility was analyzed by 1% agarose gel electrophoresis.

Purification of DNA
To minimize the activation by contaminated LPS, DNA samples were purified extensively with Triton X-114, a nonionic detergent. Extraction of endotoxin from pDNA, methylated-CpG pDNA, E. coli DNA, and calf thymus DNA samples was performed according to previously published methods [24 , 25 ] with slight modifications. DNA samples were purified by extraction with phenol:chloroform isoamyl alcohol (25:24:1) and ethanol precipitation. DNA (10 mg) was diluted with 20 ml pyrogen-free water, and then 200 µl Triton X-114 was added followed by mixing. The solution was placed on ice for 15 min and incubated for 15 min at 55°C. Subsequently, the solution was centrifuged for 20 min at 25°C, 600 g. The upper phase was transferred to a new tube, 200 µl Triton X-114 was added, and the previous steps were repeated three or more times. The activity of LPS was measured by Limulus amebocyte lysate (LAL) assay using the Limulus F single test kit (Wako). After purification using the Endo-freeTM plasmid Giga kit, 1 µg/ml pDNA was found to contain 0.01–0.05 EU/ml endotoxin. After Triton X-114 extraction, the endotoxin levels of DNA samples could no longer be determined by LAL assay; i.e., 1 µg/ml DNA contained less than 0.001 EU/ml. Without extraction of endotoxin by Triton X-114, 100 µg/ml naked pDNA, which contains 1–5 EU/ml endotoxin, could release 521 ± 73 pg/ml TNF-{alpha} over 24 h.

ODNs
Phosphorothioate ODNs were purchased from GENSET (Paris, France). The sequences of CpG S-ODN 1668 are 5'-TCC ATG ACG TTC CTG ATG CT-3', a proven activator of murine-immune cells as described previously [26 , 27 ]. Phosphorothioate non-CpG-ODN 1720 (5'-TCC ATG AGC TTC CTG ATG CT-3') was used as a control.

Cationic liposome formulation
LAplus complexes were prepared according to the manufacturer’s instructions. In brief, DNA or dextran sulfate was diluted with 75 µl Opti-MEM, and Plus reagent was added at a concentration of 1.2 µl per 1 µl DNA. LA was diluted in 75 µl Opti-MEM. After a 15-min incubation, the LA solution was added to the mixture containing DNA and Plus reagent. After a 15-min incubation, complex was added to the cells. In the case of the liposome formulation for TLR9–/– mice, DNA was diluted with 100 µl serum-free RPMI. LA was diluted in 100 µl serum-free RPMI medium, and then the DNA solution and LA solution were mixed. After a 15-min incubation, 200 µl RPMI medium containing 10% fetal calf serum was added to the DNA/LA complex solution.

Cytokine secretion
Mouse macrophages, resident peritoneal macrophages from ICR and CH3/HeJ mice, and RAW264.7 were washed three times with 0.5 ml RPMI 1640 before use. Cells were incubated for 2 h with 0.3 ml of the solutions containing the DNA/LAplus complex. Then, the cells were washed with RPMI 1640 and incubated with RPMI 1640 containing 10% FBS for specified periods up to 48 h. In the case of the inhibition experiments, cells were incubated with the medium containing an inhibitor alone at various concentrations for 30 min and were then incubated with the medium containing DNA/liposome formulations together with the inhibitor. After 2 h, the cells were washed and incubated with growth medium. At the indicated times, the supernatants were collected for enzyme-linked immunosorbent assay (ELISA) and kept at –80°C. In the case of TLR9–/– mice, cells were incubated in 100 µl complete medium, and 100 µl DNA/liposome complex was added to the cells. After 8 h, the supernatants were collected for ELISA. The levels of TNF-{alpha} and IL-6 in the supernatants were determined by the AN’ALYSATM immunoassay system (Genzyme, Minneapolis, MN)

Confocal microscopy
pCMV-Luc was labeled using a Fasttag fluorescein-labeled (FL) labeling kit according to the manufacturer’s instructions (Vector Laboratories, Burlinghame, CA). Cells were washed three times and incubated with the medium containing the FL-pDNA/LAplus complex. After a 1- or 3-h incubation at 4°C or 37°C, the cells were washed four times and fixed with 1% paraformaldehyde for 1 h. The cells were examined by confocal microscopy (MRC-1024, Bio-Rad, Hercules, CA). When the effect of inhibitors was examined, the cells were treated in the same manner as described above.

RNase protection assay (RPA)
Total RNA was extracted from cells using TRIzol (Invitrogen) and subjected to RPA. Detection of mouse cytokines was performed with the RiboQuant multiprobe RPA system (PharMingen, San Diego, CA). The multiprobe template set involved mCK-3b: TNF-ß, lymphotoxin-ß, TNF-{alpha}, IL-6, IFN-{gamma}, transforming growth factor (TGF)-ß1, TGF-ß2, TGF-ß3, migration inhibitory factor (MIF), L32, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used for in vitro transcription using the T7 RNA polymerase to direct the synthesis of highly specific, [32P]-labeled antisense RNA mixtures. Each template set was transcribed using the Riboprobe system (Promega, Madison, WI) in the presence of [32P]-uridine triphosphate (3000 Ci/mmol, NEN, Boston, MA). Total RNA (20 µg) was hybridized with [32P]-labeled antisense RNAs at 56°C overnight and then subjected to RNase treatment. Protected fragments were precipitated and separated on a 5% acrylamide gel. The gel was dried and sensitized to an X-ray film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytokine production induced by cationic liposomes with DNA or dextran sulfate
Previously, we reported that pDNA and calf thymus DNA complexed with cationic liposomes induced cytokines [21 ]. However, it is not clear whether DNA itself is required for this activation of macrophages. Therefore, we used another polyanion, dextran sulfate, and made a complex with LAplus. Highly purified pDNA or calf thymus DNA without liposome could not activate peritoneal macrophages of ICR mice as shown previously (Fig. 1 ; ref. [28 ]). With liposomes, pDNA and calf thymus DNA could induce almost the same amount of TNF-{alpha} and IL-6. Conversely, the dextran sulfate/LAplus complex could not stimulate the induction of cytokines, although naked dextran sulfate could. Naked pDNA as well as LAplus complexes with pDNA and calf thymus DNA stimulated macrophage cell line RAW264.7 cells to produce TNF-{alpha} and IL-6 as shown before [28 ] (data not shown). Peritoneal macrophages seem less sensitive to CpG DNA than the RAW264.7 cell line. These cytokines were also induced from RAW264.7 cells upon stimulation with naked dextran sulfate. However, the dextran sulfate/LAplus complex did not activate them (data not shown). LAplus alone could not induce significant amounts of cytokines (Fig. 1) . These results suggested that the cationic liposome complex required DNA to activate macrophages.



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  		Figure 1. Cytokine secretion induced by polyanions from peritoneal macrophages of ICR mice. The cells were incubated with naked DNA (100 µg/ml), naked dextran sulfate (100 µg/ml), or the DNA/LAplus complex (2.5:5 µg/well) or dextran sulfate/LAplus complex (2.5:5 µg/ml) for 8 h. After incubation, culture supernatant was collected, and the levels of TNF-{alpha} (A) or IL-6 (B) were determined by ELISA. Each result represents the mean ± SD (n=3).

 
Effect of endocytosis and endosomal acidification inhibitors on the uptake or cellular localization of pDNA
The mechanism of naked CpG DNA immunostimulation appears to depend on the internalization, as CpG-ODN linked to a solid support has no stimulatory effect, and non-CpG-ODN inhibits the responses of CpG-ODN [27 ]. Endosomal acidification of DNA is also required for the CpG DNA-mediated activation. Inhibitors of endosomal acidification, such as bafilomycin A, chloroquine, and monensin, abolish the immune responses by CpG motifs [29 , 30 ]. To examine whether these responses are also required for the cytokine production induced by pDNA complexed with cationic liposomes, we studied the effect of endosomal acidification inhibitors, such as bafilomycin A, chloroquine, and monensin, and an endocytosis inhibitor, cytochalasin B. These inhibitors may affect the cellular uptake and subsequent intracellular localization of pDNA. Therefore, confocal microscopic studies were carried out using the FL-pDNA/LAplus complex with inhibitors in macrophages from ICR mice. In the control cells, intense signals derived from FL-pDNA were observed mostly in vesicular structures, and some fluorescence diffused into the cytosol (Fig. 2 ). No apparent changes were observed for bafilomycin A and chloroquine (Fig. 2B and 2C) . In contrast, cytochalasin B significantly inhibited the uptake of the FL-pDNA/LAplus complex (Fig. 2D) . Monensin inhibited the diffusion of the FL-pDNA/LAplus complex (Fig. 2E) .



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  		Figure 2. Effect of endocytosis and endosomal acidification inhibitors on uptake and cellular localization of the FL-pDNA/LAplus complex in macrophages of ICR mice. The cells were preincubated without inhibitor (A) with 250 nM bafilomycin A (B), 2.5 µg/ml chloroquine (C), 10 µg/ml cytochalasin B (D), or 10 µM monensin (E) for 30 min and were then incubated with the FL-pDNA/LAplus complex (2.5:5 µg/ml) in the absence or presence of the same inhibitor. After a 3-h incubation, the cells were washed and scanned by confocal microscopy.

 
Effect of endosomal acidification and endocytosis inhibitors on cytokine release
Bafilomycin A, cytochalasin B, chloroquine, and monensin significantly reduced the amount of TNF-{alpha} release induced by naked CpG-ODN 1668 from macrophages [21 ]. The effect of these inhibitors on TNF-{alpha} release induced by the pDNA/LAplus complex was examined in resident peritoneal macrophages from ICR and C3H/HeJ mice (LPS nonresponder) and a macrophage cell line RAW264.7. In these experiments, inhibitors were washed out after a 2-h incubation because of the cytotoxicity of the complexes, and cytokine determination was carried out after an additional 6 h incubation. Cytochalasin B slightly reduced the TNF-{alpha} release induced by the pDNA/LAplus complex from the macrophages of ICR mice (Fig. 3A ). Monensin significantly reduced the TNF-{alpha} secretion by the pDNA/LAplus complex from the macrophages of ICR and C3H/HeJ mice and to a lesser extent, from RAW264.7 (Fig. 3A 3B 3C) . TNF-{alpha} release was slightly reduced by chloroquine in RAW264.7 (Fig. 3B) . These endocytosis or endosomal acidification inhibitors alone showed no induction of TNF-{alpha} over the concentration range tested in these experiments (data not shown).



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  		Figure 3. Effect of inhibitors on TNF-{alpha} release by the pDNA/LAplus complex from peritoneal macrophages of ICR mice (A), mouse macrophage cell line RAW264.7 cells (B), or peritoneal macrophages of C3H/HeJ mice (LPS nonresponder; C). The cells were incubated with or without various inhibitors, cytochalasin B (hatched bar), chloroquine (shaded bar), or monensin (solid bars), for 30 min and were then incubated with the pDNA/LAplus complex (2.5:5 µg/well) in the presence or absence of inhibitors. After a 2-h incubation, liposomes were removed, and growth medium was added to the macrophages. The supernatants were collected 8 h after incubation with liposomes. TNF-{alpha} levels were determined by ELISA. Each result represents the mean ± SD (n=3). Differences in the cytokine levels in the samples treated with DNA only and DNA + inhibitors (cytochalasin B, chloroquine, and monensin) were statistically analyzed by the Welch t- test. *, P < 0.05; **, P < 0.01.

 
Further experiments were carried out to see whether only the pDNA/LAplus complex or other DNA/LAplus complexes were also affected by endosomal acidification. After an 8-h incubation, the methylated-CpG pDNA/LAplus complex stimulated peritoneal macrophages of C3H/HeJ mice to produce TNF-{alpha}, as shown previously [20 ]. Monensin reduced this TNF-{alpha} induction (Fig. 4A ). Similar results were observed in the TNF-{alpha} release stimulated by E. coli DNA or the calf thymus DNA/LAplus complex (Fig. 4B) . These results suggested that TNF-{alpha} production by the DNA/LAplus complex was inhibited by endosomal acidification inhibitors.



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  		Figure 4. Effect of an endosomal acidification inhibitor on TNF-{alpha} release by pDNA or methylated pDNA (A) or E. coli DNA or calf thymus DNA (B), complexed with LAplus from macrophages of C3H/HeJ mice. The cells were incubated for 30 min without inhibitor or with monensin (10 µM, solid bars). Then the cells were incubated with the DNA/LAplus complex (2.5:5 µg/well) in the presence or absence of the same inhibitor. After a 2-h incubation, liposomes were removed, and growth medium was added to the macrophages. The supernatants were collected 8 h after incubation with liposomes. TNF-{alpha} levels were determined by ELISA. Each result represents the mean ± SD (n=3). Differences in the cytokine levels in the samples treated with the DNA/LAplus complex alone and the DNA/LAplus complex + monensin were statistically analyzed by the Welch t- test. *, P < 0.05; **, P< 0.01.

 
Effect of bafilomycin A on cytokine release induced by the DNA/LAplus complex
Next, we examined the effect of bafilomycin A, another inhibitor of endosomal acidification, on cytokine release by the DNA/LAplus complex. It was unexpected that bafilomycin A induced TNF-{alpha} production by the pDNA/LAplus complex from peritoneal macrophages and RAW264 cells, and this cytokine increased in a time-dependent manner (Fig. 5A and 5C ). Conversely, cytokine IL-6 production induced by the pDNA/LAplus complex was inhibited by bafilomycin A (Fig. 5B and 5D) . Bafilomycin A alone did not induce TNF-{alpha} production from peritoneal macrophages or RAW cells (data not shown). These results indicate that bafilomycin A stimulates TNF-{alpha} secretion induced by DNA/liposome formulation. To examine whether these results are also observed with other types of DNA, TNF-{alpha} release induced by pDNA, E. coli DNA, or calf thymus DNA complexed with LAplus after 8 h was determined. Bafilomycin A potently stimulated the TNF-{alpha} release induced by the DNA/LAplus complex from peritoneal macrophages of ICR mice (Fig. 6A ). Similar results were observed in methylated-CpG pDNA or CpG-ODN complexed with LAplus from peritoneal macrophages of C3H/HeJ mice (data not shown). However, IL-6 release was reduced by bafilomycin A (Fig. 6B) . Similar results were observed in RAW264.7 cells after an 8-h incubation (data not shown).



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  		Figure 5. Time-course of cytokine secretion by the pDNA/LAplus complex from resident macrophages of ICR mice (A, B) or RAW264.7 cells (C, D). The cells were incubated with or without various inhibitors, bafilomycin A, for 30 min and were then incubated with the pDNA/LAplus complex (2.5:5 µg/well) in the presence or absence of inhibitors. After a 2-h incubation, liposomes were removed, and growth medium was added to the macrophages. The supernatants were collected at the indicated time after incubation with liposomes, and TNF-{alpha} levels were determined by ELISA. Each result represents the mean ± SD (n=3). Differences in the cytokine levels in the samples treated with the DNA/LAplus complex alone and the DNA/LAplus complex + bafilomycin were statistically analyzed by the Welch t- test. *, P < 0.05; **, P < 0.01.

 


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  		Figure 6. Effect of bafilomycin A on TNF-{alpha} or IL-6 release by the DNA/LAplus complex from resident macrophages of ICR mice. The cells were incubated for 30 min in the presence or absence of bafilomycin A (250 nM, solid bars). Then, the cells were incubated with pDNA, E. coli DNA, or calf thymus DNA (2.5 µg/well) complexed with LAplus (5 µg/well) with or without inhibitors. After a 2-h incubation, liposomes were removed, and growth medium was added to the macrophages. The supernatants were collected 8 h after incubation with liposomes. The cytokine concentrations were measured by ELISA. Each result represents the mean ± SD (n=3). Differences in the cytokine levels in the samples treated with the DNA/LAplus complex alone and the DNA/LAplus complex + bafilomycin were statistically analyzed by the Welch t- test. **, P < 0.01.

 
Bafilomycin A-increased TNF-{alpha} mRNA production
Among these inhibitors, only bafilomycin A increased TNF-{alpha} release by the DNA/LAplus complex. To examine how bafilomycin A affects the transcription or translation of TNF-{alpha}, we investigated the mRNA production of TNF-{alpha}. Figure 7 shows the expression of mRNA in peritoneal macrophages after stimulation with the pDNA/LAplus complex. The amount of TNF-{alpha} mRNA induced by the pDNA/LAplus complex from macrophages of ICR mice was increased by bafilomycin A, and this increase was time-dependent (Fig. 7B) . No mRNA expression of IL-6 was detected, although IL-6 protein was released from the cells (Fig. 6) . IFN-ß mRNA was detected at 2 h, and this was reduced by bafilomycin A treatment, and it was undetectable at 8 h.



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  		Figure 7. Cytokine gene expression measured by RPA. (A) Peritoneal macrophages of ICR mice were treated with or without bafilomycin A for 30 min. Then, the pDNA/LAplus complex was added to the cells in the presence or absence of bafilomcyin A. After 2 h, liposomes were removed, and growth medium was added to the cells. At the indicated time, total RNA (20 µg/lane) was extracted from the cells and subjected to RPA (A). The intensity of each protected band was normalized according to the intensity of the band of GAPDH (B).

 
Effect of bafilomycin A on TNF-{alpha} production induced by naked pDNA
Bafilomycin A and other inhibitors of endosomal acidification inhibit TNF-{alpha} release induced by naked CpG-ODN 1668 [29 ]. However, in our study, after removal of the DNA/LAplus complex and bafilomycin A, TNF-{alpha} was increased, as shown in Figure 5 . Therefore, the effect of these inhibitors on cytokine release induced by naked pDNA from RAW264.7 cells was examined. pDNA and inhibitors were incubated and then washed after a 2-h incubation. TNF-{alpha} production was monitored up to 24 h. TNF-{alpha} was induced by naked pDNA and was reduced after washout. Monensin inhibited the TNF-{alpha} production. Bafilomycin A also inhibited TNF-{alpha} release up to 2 h. However, after 2 h, it enhanced TNF-{alpha} production (Fig. 8 ).



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  		Figure 8. Time-course of cytokine secretion induced by naked pDNA from RAW264.7 cells. Cells were incubated with or without inhibitors for 30 min. Then, naked pDNA (10 µg/ml) was added to the cells in the presence or absence of inhibitors. After 2 h, DNA was washed, and growth medium was added to the macrophages. Supernatants were collected at the time indicated. Cytokine concentrations were determined by ELISA. Each result represents the mean ± SD (n=3). Differences in the cytokine levels in the samples treated with the DNA/LAplus complex alone and the DNA/LAplus complex + bafilomycin or monensin were statistically analyzed by the Welch t- test. **, P < 0.01.

 
TLR9-dependent and -independent pathways are involved in macrophage activation by DNA/cationic liposomes
In general, the difference between bacterial DNA and vertebrate DNA is that the former has many unmethylated CpG motifs, and the latter does not. These CpG motifs are reported to be recognized by an intracellular receptor TLR9 [31 ]. To clarify whether the immunoactivation induced by the vertebrate calf thymus DNA/cationic liposome complex is TLR9- dependent, we used the peritoneal macrophages from TLR9–/– mice and control mice, and measured cytokine production.

Naked pDNA or calf thymus DNA could not induce TNF-{alpha} production (Fig. 9 ), as shown previously [28 ]. When pDNA was complexed with LA, the peritoneal macrophages released TNF-{alpha}. This cytokine release was significantly reduced in the macrophages from TLR9–/– mice, indicating that the cytokine induction is dependent on TLR9. However, it is interesting that the cytokine production was not completely abolished. Moreover, calf thymus DNA, which should not be a ligand of TLR9, also stimulated the macrophages from TLR9–/– mice, although the amount of TNF-{alpha} was less compared with that from control wild-type mice. Phosphorothioate CpG 1668, a typical TLR9 ligand, did not induce cytokine production from the cells of TLR9–/– mice. Both of the macrophages did not respond to LA alone. These results suggest that TLR9-dependent and -independent pathways are involved in the macrophage activation induced by the DNA/cationic liposome complex.



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  		Figure 9. Cytokine production induced by the DNA/cationic liposome complex from peritoneal macrophages from normal and TLR9–/– mice. Naked pDNA (10 µg/ml) or the DNA/LA complex (10:20 µg/ml) was added to the cells. After 8 h, supernatants were collected at the time indicated. Cytokine concentrations were determined by ELISA. Each result represents the mean ± SD (n=2). The results are the average of duplicate determinations and are typical of two experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many in vivo studies in mice have shown that pDNA/cationic liposome complexes stimulate potent cytokine production [11 , 15 , 32 33 34 35 36 ]. However, the immune responses evoked by these complexes at a cellular level are poorly understood and seem to be similar to that of naked DNA with CpG motifs. Few in vitro studies using cultured cells have been reported [37 38 39 ].

Previously, we investigated the immune response induced by the DNA/cationic liposome complex and found that bacterial pDNA and vertebrate calf thymus DNA stimulate murine macrophages [21 ]. In this study, we showed that DNA is essential for this activation, as the dextran sulfate/cationic liposome complex could not induce any cytokine release from macrophages. Therefore, we investigated the activation mechanism of the DNA/cationic liposome complex and compared it with the activation mechanism of naked CpG-ODN or naked bacterial DNA. CpG-ODN is reported to require endocytosis to induce immunoactivation [28 , 30 , 40 ]. Intracellular TLR9 recognizes the CpG motifs [4 ]. Naked DNA, including pDNA and ODN, is taken up by macrophages via receptor-mediated mechanisms, which are still unknown. Conversely, the DNA/cationic liposome complex seems to be internalized into the cells via nonspecific mechanisms based on electrical interactions. Therefore, we investigated whether endocytosis was also required for the cytokine release induced by the DNA/cationic liposome complex. Cytochalasin B, an endocytosis inhibitor, causes depolymerization of actin filaments and blocks endocytosis and phagocytosis [41 ]. The effect of cytochalasin B was straightforward. The TNF-{alpha} release induced by the pDNA/LAplus complex, which is supposed to be taken up by adsorptive endocytosis, was inhibited (Fig. 3A) . Reduced uptake in the presence of this inhibitor was confirmed by confocal microscopy (Fig. 2E) . These results indicate that endocytosis is also essential for the immunoactivation induced by the DNA/cationic liposome complex.

Next, we investigated whether acidification of the endosomal compartment was also essential for the immune response by the DNA/LAplus complex, as it has been reported to be required for CpG-ODN [29 , 30 ]. Three types of endosomal acidification inhibitors were used: Bafilomycin A is a specific inhibitor of vacuolar-type H+-ATPase [42 ], monensin is a Na+/H+ ionophore, and chloroquine is a weak base [43 ]. Monensin exhibited an inhibitory effect on TNF-{alpha} and IL-6 release induced by the pDNA/LAplus complex. Restricted intracellular diffusion by monensin after internalization may be an indication of this (Fig. 1) . Chloroquine slightly suppressed TNF-{alpha} production, and bafilomycin A inhibited IL-6 release. These results show that the pDNA/LAplus complex required endosomal acidification. Moreover, these immune responses are independent of the type of DNA, as monensin reduced the TNF-{alpha} and IL-6 production induced by E. coli DNA, methylated DNA, or calf thymus DNA complexed with the LAplus complex, and bafilomycin A reduced IL-6 production (Fig. 5) .

CG sequences are suppressed in vertebrate DNA and are highly methylated compared with bacterial DNA. TLR9 recognizes these differences, namely unmethylated CpG motifs. Therefore, in principle, calf thymus DNA would not be recognized by TLR9. However, our results show that bacterial pDNA and vertebrate calf thymus DNA can induce TLR9-dependent and -independent activation of macrophages when these DNA are complexed with liposomes. The TLR9-dependent or -independent mechanism is not fully understood. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is reported to be another receptor for immunostimulatory CpG DNA [44 ]; however, the study using the DNA-PKcs-deficient mice shows that this protein does not recognize immunostimulatory CpG DNA [45 ]. One possible explanation of macrophage activation by calf thymus DNA/cationic liposome complexes is that calf thymus DNA has very few unmethylated CpG sequences [46 ]. The human genome has ~45,000 unmethylated CpG sequences (CpG islands), and the mouse genome has 37,000 CpG islands [47 ]. The limited uptake and subsequent degradation of naked calf thymus DNA may account for the inability to induce a significant macrophage activation by the naked form [46 ]. In fact, 40–50% of 0.1 µg/ml naked pDNA was associated with resident peritoneal macrophages or RAW264.7 cells, and ~30% of pDNA was degraded after a 3-h incubation. [25 ]. Complexation with liposome would increase DNA uptake and prevent DNA degradation, consequently enhancing the availability of CpG motifs in vertebrate DNA. Another possibility is that a non-CpG motif can induce activation when DNA is complexed with liposome. Tousignant et al. [48 ] have shown that i.v. injection of non-CpG-ODN/cationic liposome complexes can induce systemic IL-12 production. This ODN contains GATC sequences, and the inversion of AT to TA reduces the activity.

Other studies also support our observation. Double-stranded mouse genomic DNA can induce activation of the bone marrow-derived DC when it was transfected with FuGENE, another cationic lipid [49 ]. Zhao et al. [50 ] challenged the i.v. injection of pDNA/cationic liposome into TLR9–/– mice and showed that there was TLR9-independent toxicity at high amounts of the pDNA/cationic liposome complex, although a dramatic reduction in toxicity was observed. This finding also agrees with our results in the present study. Further investigation is required to identify the unknown mechanism of TLR9-dependent or -independent activation of macrophages.

Bafilomycin A showed unexpected effects on TNF-{alpha} release. If the DNA/LAplus complex requires endosomal acidification, bafilomycin A should inhibit cytokine production. However, bafilomycin A significantly enhanced the production of TNF-{alpha}, although it inhibited IL-6 release. This inhibitor did not affect the distribution of the pDNA/LAplus complex, as no apparent change was observed in the intracellular localization of FL-pDNA (Fig. 2) . It also increased when macrophages were stimulated by the LAplus complex with E. coli DNA or calf thymus DNA (Fig. 6) . Moreover, it increased TNF-{alpha} production by naked pDNA after removal of DNA (Fig. 8) . These results indicate that bafilomycin A increases the degree of TNF-{alpha} release. The amount of TNF-{alpha} mRNA increased following bafilomycin A treatment, although the level of IFN-ß expression was reduced (Fig. 7) . Therefore, bafilomycin A affects the signal transduction before TNF-{alpha} mRNA production induced by the DNA/LAplus complex or the stability of mRNA. Bidani and Heming [51 ] reported that bafilomycin A increased TNF release from LPS-activated alvaeolar macrophages. Bafilomycin A is known to block vacuolar-type H+-ATPase on endosomal and plasma membranes, which not only leads to inhibition of endosomal acidification but also to significant cytosolic acidification [42 , 52 , 53 ]. Moreover, the production of TNF-{alpha} is under post-transcriptional control. An adenine and uridine-rich element (ARE) in the 3'-untranslated region of TNF-{alpha} transcripts is an important determinant of post-transcriptional control [54 ]. Furthermore, unlike other cytokines, TNF-{alpha} is a membrane-binding protein and becomes soluble following proteolytic cleavage by TNF-{alpha}-converting enzyme (TACE) [55 ]. Therefore, there is the possibility that bafilomycin A changes the cytosolic pH and affects ARE or TACE. The detailed mechanisms underlying these phenomena await further investigation.

In conclusion, the present study has demonstrated that DNA complexed with cationic liposomes can induce CpG motif-independent activation to produce TNF-{alpha} and IL-6 in cultured resident peritoneal macrophages from mice or RAW264.7 cells. Endocytosis and endosomal acidification is also required for cytokine production by DNA complexed with cationic liposomes in cultured resident peritoneal macrophages from mice or RAW264.7 cells. Moreover, the DNA/cationic liposome complex stimulates mouse peritoneal macrophages in a TLR9-dependent and -independent manner.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Sciences and Technology, Japan. The authors thank Professor Hermann Wagner (Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany), who kindly provided TLR9–/– mice, which were originally donated by Professor Sizuo Akira (Osaka University, Japan). The authors also thank Assistant Professor Satoshi Tanaka (Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan) for his helpful advice about the RPA.

Received February 16, 2004; revised September 17, 2004; accepted September 23, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Krieg, A. M. (2002) CpG motifs in bacterial DNA and their immune effects Annu. Rev. Immunol. 20,709-760[CrossRef][Medline]
  2. Wagner, H. (1999) Immunobiology of bacterial CpG-DNA Current Topics in Microbiology and Immunology 247 Springer Berlin, Germany.
  3. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., Akira, S. (2000) A Toll-like receptor recognizes bacterial DNA Nature 408,740-745[CrossRef][Medline]
  4. Ahmad-Nejad, P., Hacker, H., Rutz, M., Bauer, S., Vabulas, R. M., Wagner, H. (2002) Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments Eur. J. Immunol. 32,1958-1968[CrossRef][Medline]
  5. Qin, L., Ding, Y., Pahud, D. R., Chang, E., Imperiale, M. J., Bromberg, J. S. (1997) Promoter attenuation in gene therapy: interferon-{gamma} and tumor necrosis factor-{alpha} inhibit transgene expression Hum. Gene Ther. 8,2019-2029[Medline]
  6. Ghazizadeh, S., Carroll, J. M., Taichman, L. B. (1997) Repression of retrovirus-mediated transgene expression by interferons: implications for gene therapy J. Virol. 71,9163-9169[Abstract]
  7. Leitner, W. W., Ying, H., Restifo, N. P. (1999) DNA and RNA-based vaccines: principles, progress and prospects Vaccine 18,765-777[CrossRef][Medline]
  8. Krieg, A. M., Kline, J. N. (2000) Immune effects and therapeutic applications of CpG motifs in bacterial DNA Immunopharmacology 48,303-305[CrossRef][Medline]
  9. Paillard, F. (1999) CpG: the double-edged sword Hum. Gene Ther. 10,2089-2090[CrossRef][Medline]
  10. Alton, E. W., Geddes, D. M., Gill, D. R., Higgins, C. F., Hyde, S. C., Innes, J. A., Porteous, D. J. (1998) Towards gene therapy for cystic fibrosis: a clinical progress report Gene Ther. 5,291-292[CrossRef][Medline]
  11. Freimark, B. D., Blezinger, H. P., Florack, V. J., Nordstrom, J. L., Long, S. D., Deshpande, D. S., Nochumson, S., Petrak, K. L. (1998) Cationic lipids enhance cytokine and cell influx levels in the lung following administration of plasmid:cationic lipid complexes J. Immunol. 160,4580-4586[Abstract/Free Full Text]
  12. Yew, N. S., Zhao, H., Wu, I. H., Song, A., Tousignant, J. D., Przybylska, M., Cheng, S. H. (2000) Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs Mol. Ther. 1,255-262[CrossRef][Medline]
  13. Whitmore, M., Li, S., Huang, L. (1999) LPD lipopolyplex initiates a potent cytokine response and inhibits tumor growth Gene Ther. 6,1867-1875[CrossRef][Medline]
  14. Scheule, R. K., St, G. J., Bagley, R. G., Marshall, J., Kaplan, J. M., Akita, G. Y., Wang, K. X., Lee, E. R., Harris, D. J., Jiang, C., Yew, N. S., Smith, A. E., Cheng, S. H. (1997) Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung Hum. Gene Ther. 8,689-707[Medline]
  15. Tan, Y., Li, S., Pitt, B. R., Huang, L. (1999) The inhibitory role of CpG immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo Hum. Gene Ther. 10,2153-2161[CrossRef][Medline]
  16. Sakurai, F., Terada, T., Yasuda, K., Yamashita, F., Takakura, Y., Hashida, M. (2002) The role of tissue macrophages in the induction of proinflammatory cytokine production following intravenous injection of lipoplexes Gene Ther. 9,1120-1126[CrossRef][Medline]
  17. Kawabata, K., Takakura, Y., Hashida, M. (1995) The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake Pharm. Res. 12,825-830[CrossRef][Medline]
  18. Yoshida, M., Mahato, R. I., Kawabata, K., Takakura, Y., Hashida, M. (1996) Disposition characteristics of plasmid DNA in the single-pass rat liver perfusion system Pharm. Res. 13,599-603[CrossRef][Medline]
  19. Takagi, T., Hashiguchi, M., Mahato, R. I., Tokuda, H., Takakura, Y., Hashida, M. (1998) Involvement of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages Biochem. Biophys. Res. Commun. 245,729-733[CrossRef][Medline]
  20. Takakura, Y., Takagi, T., Hashiguchi, M., Nishikawa, M., Yamashita, F., Doi, T., Imanishi, T., Suzuki, H., Kodama, T., Hashida, M. (1999) Characterization of plasmid DNA binding and uptake by peritoneal macrophages from class A scavenger receptor knockout mice Pharm. Res. 16,503-508[CrossRef][Medline]
  21. Yasuda, K., Ogawa, Y., Kishimoto, M., Takagi, T., Hashida, M., Takakura, Y. (2002) Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes Biochem. Biophys. Res. Commun. 293,344-348[CrossRef][Medline]
  22. Nomura, T., Yasuda, K., Yamada, T., Okamoto, S., Mahato, R. I., Watanabe, Y., Takakura, Y., Hashida, M. (1999) Gene expression and antitumor effects following direct interferon (IFN)- {gamma} gene transfer with naked plasmid DNA and DC-chol liposome complexes in mice Gene Ther. 6,121-129[CrossRef][Medline]
  23. Klinman, D. M., Yi, A. K., Beaucage, S. L., Conover, J., Krieg, A. M. (1996) CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon {gamma} Proc. Natl. Acad. Sci. USA 93,2879-2883[Abstract/Free Full Text]
  24. Cotten, M., Baker, A., Saltik, M., Wagner, E., Buschle, M. (1994) Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus Gene Ther. 1,239-246[Medline]
  25. Hartmann, G., Krieg, A. M. (1999) CpG DNA and LPS induce distinct patterns of activation in human monocytes Gene Ther. 6,893-903[CrossRef][Medline]
  26. Sparwasser, T., Miethke, T., Lipford, G., Erdmann, A., Hacker, H., Heeg, K., Wagner, H. (1997) Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor-{alpha}-mediated shock Eur. J. Immunol. 27,1671-1679[Medline]
  27. Krieg, A. M., Yi, A. K., Matson, S., Waldschmidt, T. J., Bishop, G. A., Teasdale, R., Koretzky, G. A., Klinman, D. M. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation Nature 374,546-549[CrossRef][Medline]
  28. Yasuda, K., Kawano, H., Yamane, I., Ogawa, Y., Yoshinaga, T., Nishikawa, M., Takakura, Y. (2004) Restricted cytokine production from mouse peritoneal macrophages in culture in spite of extensive uptake of plasmid DNA Immunology 111,282-290[CrossRef][Medline]
  29. Yi, A. K., Tuetken, R., Redford, T., Waldschmidt, M., Kirsch, J., Krieg, A. M. (1998) CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species J. Immunol. 160,4755-4761[Abstract/Free Full Text]
  30. Hacker, H., Mischak, H., Miethke, T., Liptay, S., Schmid, R., Sparwasser, T., Heeg, K., Lipford, G. B., Wagner, H. (1998) CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation EMBO J. 17,6230-6240[CrossRef][Medline]
  31. Wagner, H. (2004) The immunobiology of the TLR9 subfamily Trends Immunol. 25,381-386[CrossRef][Medline]
  32. Li, S., Wu, S. P., Whitmore, M., Loeffert, E. J., Wang, L., Watkins, S. C., Pitt, B. R., Huang, L. (1999) Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors Am. J. Physiol. 276,L796-L804
  33. Macfarlane, D. E., Manzel, L. (1999) Immunostimulatory CpG-oligodeoxynucleotides induce a factor that inhibits macrophage adhesion J. Lab. Clin. Med. 134,501-509[CrossRef][Medline]
  34. Gursel, M., Tunca, S., Ozkan, M., Ozcengiz, G., Alaeddinoglu, G. (1999) Immunoadjuvant action of plasmid DNA in liposomes Vaccine 17,1376-1383[CrossRef][Medline]
  35. Yew, N. S., Wang, K. X., Przybylska, M., Bagley, R. G., Stedman, M., Marshall, J., Scheule, R. K., Cheng, S. H. (1999) Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes Hum. Gene Ther. 10,223-234[CrossRef][Medline]
  36. Rudginsky, S., Siders, W., Ingram, L., Marshall, J., Scheule, R., Kaplan, J. (2001) Antitumor activity of cationic lipid complexed with immunostimulatory DNA Mol. Ther. 4,347-355[CrossRef][Medline]
  37. Sonehara, K., Saito, H., Kuramoto, E., Yamamoto, S., Yamamoto, T., Tokunaga, T. (1996) Hexamer palindromic oligonucleotides with 5'-CG-3' motif(s) induce production of interferon J. Interferon Cytokine Res. 16,799-803[Medline]
  38. Roman, M., Martin, O. E., Goodman, J. S., Nguyen, M. D., Sato, Y., Ronaghy, A., Kornbluth, R. S., Richman, D. D., Carson, D. A., Raz, E. (1997) Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants Nat. Med. 3,849-854[CrossRef][Medline]
  39. Pisetsky, D. S., Reich, C. F. (1999) The influence of lipofectin on the in vitro stimulation of murine spleen cells by bacterial DNA and plasmid DNA vectors J. Interferon Cytokine Res. 19,1219-1226[CrossRef][Medline]
  40. Manzel, L., Macfarlane, D. E. (1999) Lack of immune stimulation by immobilized CpG-oligodeoxynucleotide Antisense Nucleic Acid Drug Dev. 9,459-464[Medline]
  41. Cooper, J. A. (1987) Effects of cytochalasin and phalloidin on actin J. Cell Biol. 105,1473-1478[Free Full Text]
  42. Bowman, E. J., Siebers, A., Altendorf, K. (1988) Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells Proc. Natl. Acad. Sci. USA 85,7972-7976[Abstract/Free Full Text]
  43. Mellman, I., Fuchs, R., Helenius, A. (1986) Acidification of the endocytic and exocytic pathways Annu. Rev. Biochem. 55,663-700[CrossRef][Medline]
  44. Chu, W., Gong, X., Li, Z., Takabayashi, K., Ouyang, H., Chen, Y., Lois, A., Chen, D. J., Li, G. C., Karin, M., Raz, E. (2000) DNA-PKcs is required for activation of innate immunity by immunostimulatory DNA Cell 103,909-918[CrossRef][Medline]
  45. Ishii, K. J., Takeshita, F., Gursel, I., Gursel, M., Conover, J., Nussenzweig, A., Klinman, D. M. (2002) Potential role of phosphatidylinositol 3 kinase, rather than DNA-dependent protein kinase, in CpG DNA-induced immune activation J. Exp. Med. 196,269-274[Abstract/Free Full Text]
  46. Stacey, K. J., Young, G. R., Clark, F., Sester, D. P., Roberts, T. L., Naik, S., Sweet, M. J., Hume, D. A. (2003) The molecular basis for the lack of immunostimulatory activity of vertebrate DNA J. Immunol. 170,3614-3620[Abstract/Free Full Text]
  47. Cross, S. H., Lee, M., Clark, V. H., Craig, J. M., Bird, A. P., Bickmore, W. A. (1997) The chromosomal distribution of CpG islands in the mouse: evidence for genome scrambling in the rodent lineage Genomics 40,454-461[CrossRef][Medline]
  48. Tousignant, J. D., Zhao, H., Yew, N. S., Cheng, S. H., Eastman, S. J., Scheule, R. K. (2003) DNA sequences in cationic lipid:pDNA-mediated systemic toxicities Hum. Gene Ther. 14,203-214[CrossRef][Medline]
  49. Ishii, K. J., Suzuki, K., Coban, C., Takeshita, F., Itoh, Y., Matoba, H., Kohn, L. D., Klinman, D. M. (2001) Genomic DNA released by dying cells induces the maturation of APCs J. Immunol. 167,2602-2607[Abstract/Free Full Text]
  50. Zhao, H., Hemmi, H., Akira, S., Cheng, S. H., Scheule, R. K., Yew, N. S. (2004) Contribution of Toll-like receptor 9 signaling to the acute inflammatory response to nonviral vectors Mol. Ther. 9,241-248[Medline]
  51. Bidani, A., Heming, T. A. (1995) Effects of bafilomycin A1 on functional capabilities of LPS-activated alveolar macrophages J. Leukoc. Biol. 57,275-281[Abstract]
  52. McKinney, L. C., Moran, A. (1995) Regulation of intracellular pH in J774 murine macrophage cells: H+ extrusion processes Am. J. Physiol. 268,C210-C217
  53. Swallow, C. J., Grinstein, S., Sudsbury, R. A., Rotstein, O. D. (1993) Relative roles of Na+/H+ exchange and vacuolar-type H+ ATPases in regulating cytoplasmic pH and function in murine peritoneal macrophages J. Cell. Physiol. 157,453-460[CrossRef][Medline]
  54. Anderson, P. (2000) Post-transcriptional regulation of tumour necrosis factor {alpha} production Ann. Rheum. Dis. 59,i3-i5[Free Full Text]
  55. Black, R. A. (2002) Tumor necrosis factor-{alpha} converting enzyme Int. J. Biochem. Cell Biol. 34,1-5[CrossRef][Medline]



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