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Originally published In Press as doi:10.1074/jbc.M303837200 on February 2, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17217-17223, April 23, 2004
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Activation of RAW264.7 Macrophages by Bacterial DNA and Lipopolysaccharide Increases Cell Surface DNA Binding and Internalization*

Sharon L. McCoy{ddagger}§, Stephen E. Kurtz{ddagger}§, Frances A. Hausman{ddagger}, Dennis R. Trune¶, Robert M. Bennett||, and Steven H. Hefeneider{ddagger}§||**

From the {ddagger}Department of Immunology, Veterans Affairs Medical Center, Portland, Oregon 97201, §Targeted Gene Delivery, Inc., Portland, Oregon 97201, Department of Otolaryngology, Oregon Health and Science University, Portland, Oregon 97201, and ||Department of Medicine, Division of Rheumatology, Oregon Health and Science University, Portland, Oregon 97201

Received for publication, April 11, 2003 , and in revised form, December 5, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial DNA containing unmethylated CpG motifs is a pathogen-associated molecular pattern (PAMP) that interacts with host immune cells via a toll-like receptor (TLR) to induce immune responses. DNA binding and internalization into cells is independent of TLR expression, receptor-mediated, and required for cell activation. The objective of this study was to determine whether exposure of immune cells to bacterial DNA affects DNA binding and internalization. Treatment of RAW264.7 cells with CpG oligodeoxynucleotide (ODN) for both 18 and 42 h resulted in a significant increase in DNA binding, whereas non-CpG ODN had no effect on DNA binding. Enhanced DNA binding was non-sequence-specific, inhibited by unlabeled DNA, showed saturation, was consistent with increased cell surface DNA receptors, and resulted in enhanced internalization of DNA. Treatment with Escherichia coli DNA or lipopolysaccharide (LPS) also resulted in a significant increase in DNA binding, but treatment with interleukin-1{alpha}, tumor necrosis factor-{alpha}, or phorbol 12-myristate 13-acetate had no effect on DNA binding. Soluble factors produced in response to treatment with CpG ODN or LPS did not affect DNA binding. These studies demonstrate that one consequence of activating the host innate immune response by bacterial infection is enhanced binding and internalization of DNA. During this period of increased DNA internalization, RAW264.7 cells were hypo-responsive to continued stimulation by CpG ODN, as assessed by tumor necrosis factor-{alpha} activity. We speculate the biological significance of increasing DNA binding and internalization following interaction with bacterial PAMPs may provide a mechanism to limit an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular environment.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interaction of conserved pathogen-associated molecular patterns (PAMPs),1 components of bacteria and viruses, with Toll-like receptors (TLRs), present on host innate immune cells, triggers a potent immune response (1, 2). Cell activation triggered by the interaction of PAMPs and TLRs is characterized by induction of host defense genes, production of both proinflammatory and anti-inflammatory regulatory cytokines, and the up-regulation of cell surface markers (3–9). Several bacterial PAMPs have been identified, including bacterial DNA. Bacterial DNA stimulates the proliferation of B cells and is a potent activator of macrophages and dendritic cells, eliciting an inflammatory immune response (10–13).

Host innate immune cells are activated by bacterial DNA but not mammalian DNA, because of recognition of unmethylated CpG motifs by TLR9 (4, 14). TLR9 is located intracellularly, and DNA binding and internalization are required for cell activation (15). Binding and internalization of DNA are independent of TLR9 expression, and cells that lack TLR9 bind and internalize DNA but are not activated by CpG DNA (15–18). There are numerous reports of the association of different forms of nucleic acids, such as DNA, RNA, and oligonucleotides, with cell surfaces (19–33). Bennett et al. (34) demonstrated that human leukocytes bound and internalized high molecular weight DNA by a membrane-associated protein. Receptor-mediated DNA binding on hepatic cells has been demonstrated by Emlen et al. (35), and our laboratory has reported receptor binding of DNA by murine cells (28). DNA binding to cells is saturable and inhibited by excess DNA, consistent with a receptor-mediated process (17, 36). Binding and internalization of exogenous DNA is a characteristic of most cell types and is non-sequence-restricted, and various forms (circular, linear, double-stranded, single-stranded) and types (bacterial, mammalian) of DNA bind and cross-compete with each other. The molecular characterization of the cell surface receptor responsible for the binding and internalization of exogenous DNA remains undefined (37). Because internalization of DNA is required for cell activation, we questioned whether exposure of cells to CpG DNA would affect cell surface DNA binding and internalization. In this study we demonstrate that treatment of RAW264.7 murine macrophages with CpG oligodeoxynucleotide (ODN), Escherichia coli DNA, or LPS leads to increased cell surface DNA binding. Enhanced DNA binding and internalization may represent one component of the host immune activation response, like cytokine secretion and induction of co-stimulatory molecules, that is regulated by the interaction of bacterial PAMPs and TLRs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oligodeoxynucleotides and Reagents
Nuclease-resistant phosphorylated oligodeoxynucleotides were purchased from Oligos, Inc. (Wilsonville, OR). The sequences were 5'-TCCATGACGTTCCTGACGTT-3' (CpG ODN, designated ODN1826) and 5'-TCCAATGAGCTTCCTGAGTCT-3' (non-CpG ODN, designated ODN1745) (38). Mouse IL-1{alpha} and mouse TNF-{alpha} were obtained from R&D Systems. E. coli, calf thymus DNA, PMA, and LPS were purchased from Sigma.

Cell Lines and Cultures
RAW264.7 (murine monocyte/macrophage) cells (American Type Culture Collection) were cultured at 37 °C in a 5% CO2-humidified incubator and grown in DMEM (Invitrogen) supplemented with 10% (v/v) heat-inactivated FCS, 1.5 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Treatment of Cells
RAW264.7 cells (2 x 106 cells/60-mm dish) were incubated for 24 h at 37 °C in 5% CO2 and then treated with medium, LPS, CpG ODN, or non-CpG ODN at the indicated concentrations and time periods. After treatment the cells were washed twice with phosphate-buffered saline, resuspended in DMEM containing 1% FCS, 1 mM MgCl2, and 1 mM CaCl2, and assessed for either cell surface DNA binding or DNA internalization as described below.

Cell Surface DNA Binding and Internalization
DNA, pGEM-4Z plasmid (Promega), non-CpG ODN, or CpG ODN were labeled with YOYO-1 iodide (Molecular Probes, Eugene, OR), a cyanine fluorochrome that is 100–1000 times more fluorescent in the nucleic acid-bound form than in the free form. YOYO-1-DNA binding is high affinity, preventing the migration of YOYO-1 label from one DNA molecule to another. YOYO-1 (0.2 µM) and DNA (18 µg/ml) were incubated for 1 h at room temperature in assay medium (phosphate-buffered saline (Invitrogen), 1% FCS, 1 mM MgCl2, 1 mM CaCl2,). RAW264.7 cells were grown in monolayers in T75 flasks until nearly confluent. The medium was removed, and cells were lifted with 8 mM EDTA and gentle scraping and collected into a single cell suspension. Cells were washed once with phosphate-buffered saline and resuspended at 10 x 106 cells/ml in DMEM containing 1% FCS, 1 mM MgCl2, and 1 mM CaCl2. Cells were DNased and RNased by adding 250 µg/ml deoxyribonuclease I (Worthington Biochemical) and ribonuclease A (Worthington Biochemical). After a 30-min incubation at 37 °C, EDTA (10 mM) was added to stop the action of the DNase and RNase, and the cells were washed once with phosphate-buffered saline.

Cell surface DNA Binding—Cell surface DNA binding experiments were performed in 96-well V-bottom plates (Costar). 0.5 x 106 cells and YOYO-DNA (0.39–13.3 µg/ml) were added to the wells in quadruplicate in 200 µl/well assay medium. The plates were incubated in the dark at 4 °C for 30 min, as described previously (17). Nonspecific binding was determined by preincubating cells with 100x calf thymus DNA (Sigma). After a 30-min incubation, the plates were washed twice with assay medium and resuspended in 300 µl of assay medium and 1 µg of 7-aminoactinomycin D (Molecular Probes), added to identify dead cells. Cells were incubated for 20 min on ice in the dark and analyzed by FACScan.

Internalization of DNA—Cells were resuspended in DMEM, 1% FCS, 1 mM MgCl2, and 1 mM CaCl2 (DMEM assay media). Internalization experiments were performed in 96-well V-bottom plates (Costar), with 0.5 x 106 cells and YOYO-DNA (1.35 and 3.6 µg/ml) added to the wells, in quadruplicate, in 200 µl/well DMEM assay media. Nonspecific binding was determined by preincubating cells with 200x calf thymus DNA (Sigma). The plates were incubated in the dark at 37 °C for 3 h and washed twice with assay media, and the cells were resuspended in 300 µl of assay media and 1 µg 7-aminoactinomycin D (Molecular Probes). The cells were incubated for 20 min on ice in the dark, 2.5 µl of 0.4% trypan blue stain was added to quench cell surface binding, and DNA internalization was quantified by fluorescence-activated cell sorter (FACScan, BD Biosciences).

Flow Cytometry
Cells were analyzed by flow cytometry using Cellquest software. Gates were drawn to exclude dead cells based on 7-aminoactinomycin D staining. Data obtained were geometric mean fluorescent units (F) with background autofluorescence subtracted.

Quantification of Cytokines
TNF-{alpha} levels were quantified from cell-free supernatants according to the manufacturer's directions, using commercially available enzyme-linked immunosorbent assay systems (R&D Systems).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation with CpG ODN Increases Cell Surface DNA Binding on RAW264.7 Cells—Because binding and internalization of DNA is required for cell activation by bacterial DNA (16), we questioned whether exposure of cells to CpG ODN would affect cell surface DNA binding. Previous studies from our laboratory have shown that 1 µg/ml CpG ODN induced activation of RAW264.7 cells as assessed by quantification of cytokine activity (data not shown). Therefore, this concentration of CpG ODN was examined for its effect on DNA binding. RAW264.7 cells were treated for 4, 18, or 42 h with either 1 µg/ml non-CpG ODN or CpG ODN, washed, and assessed for cell surface DNA binding by FACScan. In these experiments, YOYO-CpG ODN was used for assessment of cell surface DNA binding. Treatment with CpG ODN significantly increased cell surface DNA binding on RAW264.7 cells at both 18 and 42 h compared with treatment with non-CpG ODN (Fig. 1A). Cell surface DNA binding approximately doubled after treatment with CpG ODN for either 18 or 42 h, with the overall binding greatest after 18 h of treatment. Treatment of cells with CpG ODN for 4 h had no effect on cell surface DNA binding compared with treatment with non-CpG ODN. Using an 18-h time point, multiple doses of both CpG ODN and non-CpG ODN were incubated with RAW264.7 cells, and cell surface DNA binding was examined (Fig. 1B). RAW264.7 cells were treated with 0.5, 1, or 3.0 µg/ml non-CpG ODN or CpG ODN, washed, and assessed for cell surface DNA binding by FACScan. Treatment with CpG ODN significantly increased cell surface DNA binding in a dose-dependent manner at all three CpG ODN concentrations compared with treatment with non-CpG ODN. No dose response was seen with non-CpG-treated cells.



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  		FIG. 1.
Activation with CpG ODN increases cell surface DNA binding on RAW264.7 cells. A, RAW264.7 cells were treated for 4, 18, or 42 h with 1 µg/ml non-CpG ODN (open bars) or 1 µg/ml CpG ODN (solid bars), and cell surface binding of YOYO-DNA (3.6 µg/ml) was assessed by FACScan. Data are represented as the geometric mean fluorescence of YOYO-DNA ± S.D. B, cells were treated for 18 h with medium (hatched bar), non-CpG ODN (open bars), or CpG ODN (solid bars). ODN concentrations examined were 0.5, 1.0, and 3.0 µg/ml. Cell surface binding of YOYO-DNA (3.6 µg/ml) was assessed by FACScan, and data are represented as the geometric mean fluorescence of YOYO-DNA ± S.D.

 
Increased Cell Surface DNA Binding Is Non-sequence-specific and Consistent with Increased Receptor Expression—DNA binding to cell surfaces has been previously shown to be non-sequence-specific and receptor-mediated (17, 36). We questioned whether the enhanced DNA binding seen after treatment with CpG ODN demonstrated these same properties. Cell surface binding of three different forms of DNA (YOYO-pGEM DNA, YOYO-CpG ODN, and YOYO-non-CpG ODN) were all increased on RAW264.7 cells treated for 18 h with 1 µg/ml CpG ODN (Fig. 2). For each type of DNA examined there was an approximately 2-fold increase in cell surface binding. The intensity of cell surface DNA binding as quantified by FACScan was greatest when pGEM DNA was used because this larger DNA binds more of the fluorescent YOYO-1 dye compared with the smaller non-CpG ODN and CpG ODN. Treatment of cells with non-CpG ODN for 18 h had no effect on cell surface DNA binding compared with non-treated cells (Fig. 2). Receptor-mediated binding is characterized by inhibition with excess unlabeled ligand and saturation of ligand binding over increasing concentrations. To test for inhibition of DNA binding, RAW264.7 cells were treated for 18 h with either non-CpG ODN or CpG ODN and then treated with a 100-fold excess of unlabeled DNA before cell surface DNA binding was examined. Unlabeled DNA effectively competed for binding, reducing the fluorescence of YOYO-CpG ODN to background (Fig. 3). To test for saturation of binding, RAW264.7 cells were treated for 18 h with either non-CpG ODN or CpG ODN, and cell surface DNA binding was examined over varying concentrations of YOYO-CpG ODN (Fig. 4). Both binding curves showed saturable binding (at ~4 µg/ml YOYO-CpG ODN), with CpG ODN-treated cells saturating at a higher mean fluorescence than non-CpG ODN-treated cells; these data are consistent with increased receptor expression. Affinity of DNA binding, approximated by examining the amount of non-labeled DNA required for 50% inhibition of maximal DNA binding, showed no difference in DNA binding affinity between cells treated with either non-CpG ODN or CpG ODN (data not shown). Increased DNA binding on CpG ODN-treated cells is non-sequence-specific and is consistent with increased cell surface DNA receptors.



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  		FIG. 2.
Cell surface DNA binding is not sequence-specific. RAW264.7 cells were treated for 18 h with medium (hatched bars), 1 µg/ml non-CpG ODN (open bars), or 1 µg/ml CpG ODN (solid bars), and cell surface binding of YOYO-pGEM (3.6 µg/ml), YOYO-CpG ODN (3.6 µg/ml), or YOYO-non-CpG ODN (3.6 µg/ml) was assessed by FACScan. Data are represented as the geometric mean fluorescence of YOYO-DNA ± S.D.

 



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  		FIG. 3.
Cell surface DNA binding on RAW264.7 cells is inhibited by excess DNA. RAW264.7 cells were treated with medium (hatched bars), 1 µg/ml non-CpG ODN (open bars), or 1 µg/ml CpG ODN (solid bars) for 18 h, and cell surface binding of YOYO-DNA (3.6 µg/ml) was assessed in the presence and absence of a 100-fold excess of unlabeled calf thymus (C. T.) DNA by FACScan. Data are represented as the geometric mean fluorescence of YOYO-DNA ± S.D.

 



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  		FIG. 4.
Cell surface DNA binding curves on CpG ODN-treated RAW264.7 cells. RAW264.7 cells were treated with 1 µg/ml CpG ODN () or 1 µg/ml non-CpG ODN ({blacksquare}) for 18 h, and cell surface binding of YOYO-DNA was assessed by FACScan at varying concentrations (0.1–13.5 µg/ml). Nonspecific binding was determined by incubation at each concentration of YOYO-DNA with excess unlabeled calf thymus DNA and subtracted from total binding. Data are represented as the geometric mean fluorescence ± S.D.

 
Activation with CpG ODN Increases DNA Internalization—We next questioned whether increased cell surface DNA binding, seen after treatment with CpG ODN, would lead to enhanced internalization of DNA. RAW264.7 cells were treated for 18 h with either 1 µg/ml non-CpG ODN or CpG ODN; the cells were washed, and DNA internalization was quantified after 3 h at 37 °C by FACScan, after quenching cell surface binding. Cells treated with CpG ODN showed significant increase in the amount of YOYO-CpG ODN internalized compared with cells treated for 18 h with either medium or non-CpG ODN. An approximate 3-fold increase in internalization of DNA over background was observed in cells treated with CpG ODN, with enhanced internalization of YOYO-CpG ODN seen at both 1.35 µg/ml (Fig. 5A) and 3.6 µg/ml (Fig. 5B) YOYO-DNA. To determine whether increased DNA internalization was specific for DNA or represented nonspecific uptake, RAW264.7 cells were treated with medium, non-CpG ODN or CpG ODN, for 18 h and then treated with a 200-fold excess of unlabeled DNA before assessment of DNA internalization. In contrast to blocking of cell surface binding (Fig. 3), pretreatment of CpG ODN-activated cells with a 200-fold excess of unlabeled calf thymus DNA did not completely inhibit internalization of YOYO-CpG ODN (Fig. 5). These data suggest that enhanced DNA internalization seen after treatment of cells with CpG ODN results primarily but not exclusively from augmentation of a response that is specific for internalization of DNA.



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  		FIG. 5.
CpG ODN-treated RAW264.7 cells show increased DNA internalization. RAW264.7 cells were treated with medium (hatched bars), 1 µg/ml non-CpG ODN (open bars), or 1 µg/ml CpG ODN (solid bars) for 18 h. Internalization of YOYO-CpG ODN in the presence and absence of a 200-fold excess of unlabeled calf thymus (C. T.) DNA at 1.35 µg/ml YOYO-DNA (A) and 3.6 µg/ml YOYO-DNA (B) was assessed after 3 h at 37 °C by FACScan. Data are represented as the geometric mean fluorescence ± S.D. Cell surface DNA fluorescence was quenched prior to determining internal fluorescence.

 
Increased DNA Binding Is Seen after Cell Activation by Bacterial PAMPs but Not with Non-bacterial Cell Activators—To begin to address the mechanism by which cell surface DNA receptors are increased, RAW264.7 cells were treated with either E. coli DNA or LPS, both of which are bacterial PAMPs that are potent activators of RAW264.7 cells; however, these agents use different TLR recognition structures. RAW264.7 cells were treated with non-CpG ODN (1 µg/ml), CpG ODN (1 µg/ml), calf thymus DNA (10 µg/ml), E. coli DNA (10 µg/ml), or LPS (1 ng/ml and 3 ng/ml). Cells were treated for 18 h, and cell surface DNA binding was quantified by FACScan. As expected, treatment with CpG ODN increased cell surface DNA binding ~2-fold compared with non-CpG ODN-treated cells (Fig. 6A). Treatment with E. coli DNA and LPS significantly increased cell surface DNA binding, but the effect was not as pronounced as that seen with CpG ODN (Fig. 6A). Calf thymus DNA had no effect on DNA binding, and the result was similar to that with medium alone (Fig. 6B). We next questioned what effect non-bacterial cell activators had on cell surface DNA binding. RAW264.7 cells were treated for 18 h with non-CpG ODN (1 µg/ml), CpG ODN (1 µg/ml), TNF-{alpha} (10 ng/ml and 100 ng/ml), IL-1{alpha} (10 ng/ml and 100 ng/ml), or PMA (1 ng/ml and 10 ng/ml). Treatment of RAW264.7 cells with TNF-{alpha}, IL-1{alpha}, or PMA resulted in cell activation as evidenced by secretion of cytokines (data not shown). However, the secreted cytokine levels were increased less than 10-fold compared with an approximately 100-fold increase in secreted cytokine levels seen after treatment of RAW264.7 cells with CpG ODN, E. coli DNA, or LPS. There was a modest increase in DNA binding when cells were treated with non-bacterial agents compared with medium-treated cells; however, this increase was similar in magnitude to treatment with non-CpG ODN and did not match the increase seen after treatment with CpG ODN (Tables I and II). These data demonstrate that activating cells with these agents was not sufficient to induce increased cell surface DNA binding and suggest the involvement of bacterial PAMPs and their interaction with TLRs in mediating increased cell surface DNA binding. As further evidence of the importance of the interaction of bacterial PAMPs/TLRs, we examined the effect of CpG ODN using HEK293 cells. These cells bind and internalize DNA, but because they lack TLR9 they do not become activated on internalization of bacterial DNA. Incubation of HEK293 cells with CpG ODN specifically for human cells had no effect on cell surface DNA binding, compared with non-CpG ODN (data not shown). We have noted that cell surface DNA binding on HEK293 cells increased non-treated cells; however, no differences were seen between specific treatments (data not shown). These data are consistent with the hypothesis that increased cell surface DNA binding is one component of the cell activation profile resulting from the interaction of bacterial PAMPs and TLRs.



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  		FIG. 6.
Activation with E. coli DNA and LPS increased cell surface DNA binding on RAW264.7 cells. A, RAW264.7 cells were treated with 1 µg/ml non-CpG ODN, 1 µg/ml CpG ODN, 1 and 3 ng/ml LPS, or 10 µg/ml E. coli DNA. B, RAW264.7 cells were treated with medium, 10 µg/ml calf thymus DNA, or 10 µg/ml E. coli DNA. Cell surface binding of YOYO-DNA (3.6 µg/ml) was assessed by FACScan, and data are represented as the geometric mean fluorescence of YOYO-DNA ± S.D.

 


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  		TABLE I
Cell surface DNA binding on activated RAW264.7 cells

RAW264.7 cells were treated for 18 h at 37 °C with medium, 1 µg/ml CpG ODN, 1 µg/ml non-CpG ODN, 10 and 100 ng/ml IL-1, or 10 and 100 ng/ml TNF-{alpha}. Cell surface binding of YOYO-DNA (3.6 µg/ml) was assessed by FACScan and data represented as geometric mean fluorescence ± S.D.

 


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  		TABLE II
Cell surface DNA binding

RAW264.7 cells were treated for 18 h at 37 °C with 1 µg/ml CpG ODN, 1 µg/ml non-CpG ODN, or 1 and 10 ng/ml PMA. Cell surface binding of YOYO-DNA (3.6 µg/ml) was assessed by FACScan, and data are represented as geometric mean fluorescence ± S.D.

 
Cell-free Supernatants from Activated RAW264.7 Cells Do Not Increase Cell Surface DNA Binding Compared with Treatment with CpG ODN—We next questioned whether the increase seen after incubation with bacterial PAMPs was the result of direct interaction with PAMPs/TLRs or secondary stimulation by a soluble factor released when cells were treated by CpG ODN or LPS. RAW264.7 cells were treated for 3 h with non-CpG ODN (1 µg/ml), CpG ODN (1 µg/ml), LPS (1 ng/ml), or IL-1{alpha} (10 ng/ml), washed, and fresh media added for an additional 24 h. Cell-free supernatants were collected from treated cells and incubated with fresh non-treated cells for 18 h, and cell surface DNA binding was assessed by FACScan. Incubation of RAW264.7 cells with supernatants from CpG ODN-, LPS-, or IL-1{alpha}-treated cells showed a modest increase in cell surface DNA binding compared with supernatants from non-treated or non-CpG ODN-treated cells (Fig. 7). This effect on cell surface DNA binding did not approach the 2-fold increase seen after treatment of fresh cells for 18 h with CpG ODN (Fig. 7). Increased cell surface DNA binding does not appear to be mediated by a soluble product produced by incubation of RAW264.7 cells with CpG ODN or LPS.



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  		FIG. 7.
Cell-free supernatants from activated RAW264.7 cells do not increase cell surface DNA binding compared with treatment with CpG ODN. RAW264.7 cells were treated for 24 h with medium (hatched bar), 1 µg/ml non-CpG ODN (open bars), 1 µg/ml CpG ODN (solid bars), 1 ng/ml LPS (dotted bar) or 10 ng/ml IL-1 (checkered bar). Cell-free supernatants were collected from treated cells and incubated with fresh non-treated cells for 18 h. Cell surface binding of YOYO-DNA (3.6 µg/ml) was assessed by FACScan. Data are represented as the geometric mean fluorescence ± S.D.

 
Increased Internalization of DNA Does Not Lead to Enhanced Cytokine Secretion—The increase in cell surface DNA binding and internalization seen on RAW264.7 cells treated with CpG ODN was shown to occur between 18 and 42 h. We questioned whether cells with enhanced capacity to bind and internalize DNA would be more responsive to subsequent exposure to CpG ODN. RAW264.7 cells were treated with medium, non-CpG ODN (1 µg/ml), or CpG ODN (1 µg/ml) for 24 h, and cell-free supernatants (24-h supernatants) were collected. Treated cells were then restimulated with medium, 1 µg/ml non-CpG ODN, or 1 µg/ml CpG ODN for an additional 24 h, and a second set of cell-free supernatants (48-h supernatants) were collected, and both 24- and 48-h supernatants were assayed for TNF-{alpha} activity (Table III). As expected, 24-h supernatants from CpG ODN-treated cells showed a substantial increase in TNF-{alpha} activity (8,912 pg/ml) compared with supernatants from medium (91 pg/ml) or non-CpG ODN-treated (91 pg/ml) cells. However, 48-h supernatants from CpG ODN-treated cells that were restimulated with CpG ODN showed a significant decrease in TNF-{alpha} activity (3,181 pg/ml) compared with the 24-h supernatants (8,912 pg/ml) from CpG ODN-treated cells. Our data demonstrate that whereas exposure to CpG ODN increases cell surface DNA binding and internalization, during this period the cells are hyporesponsive to continued stimulation by CpG ODN. The biological significance of increasing cell surface DNA binding and internalization following interaction with bacterial PAMPs may be to provide a mechanism to down-regulate an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular environment.


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  		TABLE III
Cytokine secretion by activated RAW264.7 cells

RAW264.7 cells were treated with medium, non-CpG ODN (1 µg/ml), or CpG ODN (1 µg/ml) for 24 h, and cell-free supernatants (sup) were collected at 24-h. Treated cells were then restimulated with medium, 1 µg/ml non-CpG ODN, or 1 µg/ml CpG ODN for an additional 24 h, and a second set of cell-free supernatants were collected (48-h supernatants). Both 24- and 48-h supernatants were assayed for TNF-{alpha} activity by enzyme-linked immunosorbent assay.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study demonstrate that activation of macrophages with CpG ODN resulted in a 2–3-fold increase in DNA binding and internalization. Increased cell surface DNA binding was seen at both 18 and 42 h after exposure to CpG ODN and was consistent with up-regulation of cell surface DNA receptors. Binding and internalizing of non-CpG ODN did not induce this response. Exposure to the bacterial PAMPs E. coli DNA and LPS increased DNA binding but not to the level seen with CpG ODN. To address the mechanism involved in increasing cell surface DNA binding, we questioned whether cell activation induced by non-bacterial products would affect DNA binding. In contrast to exposure to bacterial PAMPs, cell activation induced by IL-1{alpha}, TNF-{alpha}, or PMA did not affect DNA binding compared with non-CpG ODN. However, these results must be interpreted with caution because cell activation as assessed by cytokine secretion was significantly more effective when RAW264.7 cells were treated with CpG ODN, E. coli DNA, or LPS compared with treatment with IL-1{alpha}, TNF-{alpha}, or PMA. Exposure of HEK293 cells, which lack TLR9 and TLR4, to E. coli DNA or LPS did not influence cell surface DNA binding compared with non-CpG ODN, consistent with a postulated requirement that increased cell surface DNA binding requires cell activation initiated via interaction of bacterial PAMPs with their appropriate TLRs. Cell-free supernatants collected from cells activated with bacterial PAMPs did not increase cell surface DNA binding when incubated with fresh RAW264.7 cells, suggesting that direct PAMP/TLR interaction is required. Collectively, these data suggest that increased cell surface DNA binding results from the specific interaction of bacterial PAMPs and TLRs, is not a component of a general cell activation response, and is not mediated by production of a soluble factor in response to cell activation. During the period of increased DNA binding and internalization, RAW264.7 cells were shown to be hypo-responsive to continued stimulation by CpG ODN. We speculate that enhanced DNA binding and internalization in response to the interaction of PAMPs/TLRs may function to down-regulate an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular environment.

Increased DNA binding and internalization seen after treatment with CpG ODN, E. coli DNA, and LPS may have application in the design of strategies for enhancing delivery of DNA-based therapies and therapeutic agents linked to DNA. Naked DNA has been used successfully in vivo for gene therapy; however, a major drawback has been its low efficiency for gene delivery (31, 39, 40). The use of cell surface DNA receptors for delivery of antigen into cells has previously been explored, and recent studies by Wagner and colleagues (36) demonstrated that linking of DNA to ovalbumin provided a significant advantage for antigen delivery compared with antigen alone. Enhanced delivery of DNA/antigen into cells was speculated to be because of binding and internalization of DNA by cell surface DNA receptors. Results of our study suggest that delivery of plasmid DNA and DNA/antigen into cells may be most efficient after first activating cells with bacterial DNA or LPS to enhance cell surface DNA binding and internalization. Our study, testing one design sequence of CpG ODN, resulted in a greater increase in cell surface DNA binding than either E. coli DNA or LPS. It may be possible to design CpG ODNs that would be more effective in enhancing cell surface DNA binding and internalization than the one used in our study. Recent studies reporting distinct stimulatory profiles specific to the class of CpG ODN and the importance of location and number of CpG motifs within a synthetic ODN to influence immunostimulatory capacity support this concept (4, 41, 42).

The initial inflammatory response mounted by innate immune cells activated by bacterial PAMPs is followed by an active down-regulation, a process requiring the involvement of several factors. Evidence for this down-regulation is shown by studies that have found that continuous or repeated stimulation of innate immune cells with bacterial PAMPs affects the function of these cells differently than the initial stimulus. Significant reduction in the production of pro-inflammatory cytokines has been observed after cells were pre-stimulated with bacterial PAMPs, incubated for 24 h, and then restimulated with the same or other PAMPs (43–47). Yeo et al. (47) suggested that the observed hypo-responsiveness of macrophages to bacterial PAMPs was not because of cell exhaustion but rather a reprogramming of the cells. Our studies show that one consequence of exposure of RAW264.7 cells to CpG ODN, bacterial DNA, or LPS is increased cell surface DNA binding. The biological significance of cells binding and internalizing both bacterial and mammalian DNA remains unknown. Bennett et al. (34) first proposed that DNA binding and internalization function as a salvage pathway for the breakdown of DNA into nucleotides with their subsequent reutilization within the cell. In certain autoimmune diseases such as systemic lupus erythematosus there is evidence of an autoantibody response to cell surface DNA receptors (48). In patients with systemic lupus erythematosus, these anti-DNA receptor antibodies function in vitro to block DNA binding to cells. One postulated consequence of this autoantibody response is impaired clearance of DNA and nucleosomes and the potential development of DNA-anti-DNA immune complexes with pathogenic significance (49). We hypothesize that enhanced DNA binding and internalization on immune cells activated by bacterial PAMPs could be a mechanism for clearance of extracellular DNA, as opposed to a means to further activate cells through increased interaction of bacterial DNA with TLR9. Up-regulation of binding to further activate a pro-inflammatory response initiated by bacterial PAMPs seems unlikely. Our data demonstrated that CpG ODN-treated RAW264.7 cells, with increased cell surface DNA binding and internalization, showed a reduced production of TNF-{alpha} in response to a secondary stimulation with CpG ODN. A potential host advantage of enhanced DNA binding and internalization would be to facilitate removal of DNA from the surrounding extracellular environment. Removal of extracellular DNA once an inflammatory response has been initiated may prevent stimulation of non-activated immune cells thereby helping to limit or contain an ongoing inflammatory response. Prolonged or chronic inflammation results when down-regulation is dysfunctional, and the uncontrolled production of pro-inflammatory cytokines can result in inflammatory responses that are harmful to the host. Recent studies (50) have demonstrated that chronic inflammation is linked to several diseases with the development of effective anti-inflammatory therapies being an area of active investigation. The results of our current study suggest that enhanced cell surface DNA binding and internalization is one component of the activation response that develops when innate immune cells encounter bacterial PAMPs in the context of TLRs. If increased DNA binding is used as a clearance mechanism to facilitate down-regulation of the inflammatory response, enhanced cell surface DNA binding and internalization may provide a potential regulatory point to target for anti-inflammatory intervention.


   FOOTNOTES
 
* This work was supported by Veterans Affairs Rehabilitation Research and Development Grant C2870R (to S. H. H.) and NIDCD Grant RO1 DC03573 (to D. R. T. and S. H. H.) and Small Business Innovation Research Grant 1R43 [PDB] DC05882-01 (to S. E. K.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Dept. of Immunology, R&D-18, Veterans Affairs Medical Center, 3710 Southwest, U. S. Veterans Hospital Rd., Portland, OR 97201. Tel.: 503-220-3428; Fax: 503-273-5351; E-mail: Hefeneid{at}OHSU.edu.

1 The abbreviations used are: PAMP, pathogen-associated molecular patterns; TLR, toll-like receptors; CpG, cytosine-phosphate-guanine; ODN, oligodeoxynucleotide; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; IL-1, interleukin 1; TNF-{alpha}, tumor necrosis factor {alpha}; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FACS, fluorescence-activated cell sorter. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Mark E. Deffebach for his helpful suggestions and review of the manuscript.



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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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