Activation of RAW264.7 Macrophages by Bacterial DNA and Lipopolysaccharide Increases Cell Surface DNA Binding and Internalization

Abstract

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-1α, tumor necrosis factor-α, 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-α 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. 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-1α, tumor necrosis factor-α, 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-α 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. The interaction of conserved pathogen-associated molecular patterns (PAMPs), 1The 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-α, tumor necrosis factor α; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FACS, fluorescence-activated cell sorter. components of bacteria and viruses, with Toll-like receptors (TLRs), present on host innate immune cells, triggers a potent immune response (1Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (3958) Google Scholar, 2Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (6247) Google Scholar). 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(35Emlen W. Rifai A. Magilavy D. Mannik M. Am. J. Pathol. 1988; 133: 54-60PubMed Google Scholar), and our laboratory has reported receptor binding of DNA by cells S.H. Cornell K.A. Brown L.E. Bakke A.C. McCoy S.L. Bennett R.M. Cornell K.A. Clin. Immunol. Immunopathol. 1992; 63: 245-251Crossref PubMed Scopus (114) Google Scholar). DNA binding to cells is and inhibited by DNA, consistent with a S.L. Hausman F.A. Deffebach M.E. Bakke A. Merkens L.S. Bennett R.M. Hefeneider S.H. J. Immunol. Methods. 2000; 241: 141-146Crossref PubMed Scopus (9) Google Scholar, T. A. Hochrein H. F. M. Bauer S. Lipford G.B. Vabulas R.M. Wagner H. Eur. J. Immunol. 2002; 32: PubMed Scopus Google Scholar). Binding and internalization of DNA is a of cell and is and forms and of DNA bind and with The molecular of the cell surface receptor for the binding and internalization of DNA Merkens L.S. T. A.C. McCoy S.L. Deffebach M.E. Bennett R.M. Hefeneider S.H. J. 2000; PubMed Scopus Google Scholar). internalization of DNA is required for cell whether exposure of cells to CpG DNA affect cell surface DNA binding and internalization. this study demonstrate that treatment of RAW264.7 macrophages with CpG oligodeoxynucleotide Escherichia coli DNA, or LPS to increased cell surface DNA binding. Enhanced DNA binding and internalization may one of the host immune activation and induction of that is by the interaction of bacterial PAMPs and were from The were ODN, and ODN, A.K. Krieg A.M. J. Immunol. 1998; Google Scholar). and were from E. calf DNA, PMA, and LPS were from RAW264.7 cells were in a and in with FCS, and RAW264.7 cells were for h in and with LPS, CpG ODN, or non-CpG ODN the and treatment the cells were with in containing FCS, and and assessed for cell surface DNA binding or DNA internalization as DNA, non-CpG ODN, or CpG ODN were with a that is in the nucleic in the binding is high the of from one DNA to and DNA were for h in FCS, RAW264.7 cells were in in The was and cells were with and and into a cell were with and in containing FCS, and were and by and a was to the of the and and the cells were with Cell surface DNA surface DNA binding were in cells and were to the in in The were in the for as S.L. Hausman F.A. Deffebach M.E. Bakke A. Merkens L.S. Bennett R.M. Hefeneider S.H. J. Immunol. Methods. 2000; 241: 141-146Crossref PubMed Scopus (9) Google Scholar). binding was by cells with calf DNA a the were with and in of and of to were for on in the and by of were in DMEM, FCS, and were in with cells and and to the in in binding was by cells with calf DNA The were in the for h and with and the cells were in of and The cells were for on in the of was to cell surface binding, and DNA internalization was by fluorescence-activated cell were by were to cells on were with were from to the with CpG ODN Cell DNA Binding on RAW264.7 binding and internalization of DNA is required for cell activation by bacterial DNA H. Mischak H. Miethke T. Liptay S. Schmid R. Sparwasser T. Heeg K. Lipford G.B. Wagner H. EMBO J. 1998; 17: 6230-6240Crossref PubMed Scopus (586) Google Scholar), whether exposure of cells to CpG ODN affect cell surface DNA binding. studies from our laboratory have that CpG ODN activation of RAW264.7 cells as assessed by of not this of CpG ODN was for effect on DNA binding. RAW264.7 cells were for or 42 h with non-CpG ODN or CpG ODN, and assessed for cell surface DNA binding by ODN was used for of cell surface DNA binding. Treatment with CpG ODN increased cell surface DNA binding on RAW264.7 cells both 18 and 42 h with treatment with non-CpG ODN Cell surface DNA binding treatment with CpG ODN for 18 or 42 with the binding 18 h of Treatment of cells with CpG ODN for h had no effect on cell surface DNA binding with treatment with non-CpG an of both CpG ODN and non-CpG ODN were with RAW264.7 cells, and cell surface DNA binding was RAW264.7 cells were with or non-CpG ODN or CpG ODN, and assessed for cell surface DNA binding by Treatment with CpG ODN increased cell surface DNA binding in a CpG ODN with treatment with non-CpG response was with Cell DNA Binding and with binding to cell surfaces has been to and S.L. Hausman F.A. Deffebach M.E. Bakke A. Merkens L.S. Bennett R.M. Hefeneider S.H. J. Immunol. Methods. 2000; 241: 141-146Crossref PubMed Scopus (9) Google Scholar, T. A. Hochrein H. F. M. Bauer S. Lipford G.B. Vabulas R.M. Wagner H. Eur. J. Immunol. 2002; 32: PubMed Scopus Google Scholar). We whether the enhanced DNA binding treatment with CpG ODN demonstrated Cell surface binding of different forms of DNA DNA, ODN, and were increased on RAW264.7 cells for 18 h with CpG ODN of DNA was an increase in cell surface binding. The of cell surface DNA binding as by was DNA was used because this DNA of the with the non-CpG ODN and CpG Treatment of cells with non-CpG ODN for 18 h had no effect on cell surface DNA binding with cells Receptor-mediated binding is characterized by with unlabeled and of binding increasing for of DNA binding, RAW264.7 cells were for 18 h with non-CpG ODN or CpG ODN and with a of unlabeled DNA cell surface DNA binding was DNA for binding, the of ODN to for of binding, RAW264.7 cells were for 18 h with non-CpG ODN or CpG ODN, and cell surface DNA binding was of ODN binding showed binding with CpG cells a non-CpG are consistent with increased receptor of DNA binding, by the of DNA required for of DNA binding, showed no in DNA binding cells with non-CpG ODN or CpG ODN not DNA binding on CpG cells is and is consistent with increased cell surface DNA surface DNA binding on RAW264.7 cells is inhibited by DNA. RAW264.7 cells were with non-CpG ODN or CpG ODN for 18 and cell surface binding of was assessed in the and of a of unlabeled calf DNA by are as the of surface DNA binding on CpG RAW264.7 RAW264.7 cells were with CpG ODN or non-CpG ODN for 18 and cell surface binding of was assessed by binding was by of with unlabeled calf DNA and from binding. are as the with CpG ODN DNA whether increased cell surface DNA binding, treatment with CpG ODN, to enhanced internalization of DNA. RAW264.7 cells were for 18 h with non-CpG ODN or CpG the cells were and DNA internalization was h by cell surface binding. with CpG ODN showed significant increase in the of ODN internalized with cells for 18 h with or non-CpG increase in internalization of DNA was in cells with CpG ODN, with enhanced internalization of ODN both and determine whether increased DNA internalization was for DNA or RAW264.7 cells were with non-CpG ODN or CpG ODN, for 18 h and with a of unlabeled DNA of DNA internalization. to of cell surface binding of CpG cells with a of unlabeled calf DNA did not internalization of ODN These that enhanced DNA internalization treatment of cells with CpG ODN but not from of a response that is for internalization of DNA. DNA Binding Cell by Bacterial PAMPs but with Cell to the mechanism by cell surface DNA receptors are RAW264.7 cells were with E. coli DNA or LPS, both of are bacterial PAMPs that are potent of RAW264.7 different TLR recognition RAW264.7 cells were with non-CpG ODN CpG ODN calf DNA E. coli DNA or LPS and were for 18 and cell surface DNA binding was by treatment with CpG ODN increased cell surface DNA binding with non-CpG cells Treatment with E. coli DNA and LPS increased cell surface DNA binding, but the effect was not as as that with CpG ODN DNA had no effect on DNA binding, and the was to that with We effect cell had on cell surface DNA binding. RAW264.7 cells were for 18 h with non-CpG ODN CpG ODN and and or and Treatment of RAW264.7 cells with TNF-α, or resulted in cell activation as by of not the were increased with an increase in treatment of RAW264.7 cells with CpG ODN, E. coli DNA, or There was a increase in DNA binding cells were with with this increase was in to treatment with non-CpG ODN and did not the increase treatment with CpG ODN and These demonstrate that activating cells with was not to induce increased cell surface DNA binding and the of bacterial PAMPs and interaction with TLRs in increased cell surface DNA binding. of the of the interaction of bacterial the effect of CpG ODN These cells bind and internalize DNA, but because lack TLR9 not activated on internalization of bacterial DNA. of cells with CpG ODN for human cells had no effect on cell surface DNA binding, with non-CpG ODN not We have that cell surface DNA binding on cells increased no were not These are consistent with the that increased cell surface DNA binding is one of the cell activation from the interaction of bacterial PAMPs and surface DNA binding on activated RAW264.7 ODN ODN in a surface DNA ODN ODN in a from RAW264.7 Cell DNA Binding with Treatment with CpG whether the increase with bacterial PAMPs was the of interaction with or stimulation by a factor cells were by CpG ODN or RAW264.7 cells were for h with non-CpG ODN CpG ODN LPS or and for an were from cells and with cells for 18 and cell surface DNA binding was assessed by of RAW264.7 cells with from CpG or cells showed a increase in cell surface DNA binding with from or non-CpG cells effect on cell surface DNA binding did not the increase treatment of cells for 18 h with CpG ODN cell surface DNA binding not to by a produced by of RAW264.7 cells with CpG ODN or of DNA to Enhanced increase in cell surface DNA binding and internalization on RAW264.7 cells with CpG ODN was to 18 and 42 We whether cells with enhanced to bind and internalize DNA to exposure to CpG RAW264.7 cells were with non-CpG ODN or CpG ODN for and were cells were with non-CpG ODN, or CpG ODN for an and a of were and both and were for from CpG cells showed a increase in with from or non-CpG from CpG cells that were with CpG ODN showed a significant in with the from CpG demonstrate that whereas exposure to CpG ODN cell surface DNA binding and internalization, this period the cells are to continued stimulation by CpG The biological significance of increasing cell surface DNA binding and internalization following interaction with bacterial PAMPs may to provide a mechanism to an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular by activated RAW264.7 in a The of the present study demonstrate that activation of macrophages with CpG ODN resulted in a increase in DNA binding and internalization. cell surface DNA binding was both 18 and 42 h exposure to CpG ODN and was consistent with up-regulation of cell surface DNA Binding and of non-CpG ODN did not induce this to the bacterial PAMPs E. coli DNA and LPS increased DNA binding but not to the with CpG the mechanism in increasing cell surface DNA binding, whether cell activation by affect DNA binding. to exposure to bacterial cell activation by TNF-α, or did not affect DNA binding with non-CpG with because cell activation as assessed by was RAW264.7 cells were with CpG ODN, E. coli DNA, or LPS with treatment with TNF-α, or of cells, lack TLR9 and to E. coli DNA or LPS did not cell surface DNA binding with non-CpG ODN, consistent with a that increased cell surface DNA binding cell activation via interaction of bacterial PAMPs with from cells activated with bacterial PAMPs did not increase cell surface DNA binding with RAW264.7 cells, that interaction is that increased cell surface DNA binding from the interaction of bacterial PAMPs and is not a of a cell activation and is not by production of a factor in response to cell activation. During the period of increased DNA binding and internalization, RAW264.7 cells were to hypo-responsive to continued stimulation by CpG We speculate that enhanced DNA binding and internalization in response to the interaction of may to an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular environment. DNA binding and internalization treatment with CpG ODN, E. coli DNA, and LPS may have in the of for enhancing of and to DNA. DNA has been used in for a has been for R.M. Antisense Res. Dev. 1993; 3: 235-241Crossref PubMed Scopus (57) Google Scholar, G. 2003; PubMed Scopus Google Scholar, H. 2003; PubMed Scopus Google Scholar). The of cell surface DNA receptors for of into cells has been and studies by Wagner and T. A. Hochrein H. F. M. Bauer S. Lipford G.B. Vabulas R.M. Wagner H. Eur. J. Immunol. 2002; 32: PubMed Scopus Google Scholar) demonstrated that of DNA to a significant for with Enhanced of into cells was to because of binding and internalization of DNA by cell surface DNA of our study that of DNA and into cells may activating cells with bacterial DNA or LPS to cell surface DNA binding and internalization. one of CpG ODN, resulted in a increase in cell surface DNA binding E. coli DNA or may to CpG that in enhancing cell surface DNA binding and internalization the one used in our studies to the of CpG ODN and the of and of CpG motifs a ODN to this (4Krieg A.M. Annu. Rev. Immunol. 2002; 20: 709-760Crossref PubMed Scopus (2239) Google Scholar, D.M. D. Clin. Exp. Immunol. 2003; 133: PubMed Scopus Google Scholar, K. C. S. P. J. G. J. 2003; PubMed Scopus Google Scholar). 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We that enhanced DNA binding and internalization on immune cells activated by bacterial PAMPs a mechanism for clearance of extracellular DNA, as to a to cells increased interaction of bacterial DNA with of binding to a response by bacterial PAMPs demonstrated that CpG RAW264.7 cells, with increased cell surface DNA binding and internalization, showed a production of in response to a stimulation with CpG host of enhanced DNA binding and internalization to of DNA from the extracellular environment. of extracellular DNA an inflammatory response has been may stimulation of immune cells to limit or an ongoing inflammatory or is and the production of in inflammatory that are to the studies L.M. Z. Nature. 2002; PubMed Scopus Google Scholar) have demonstrated that is to with the of anti-inflammatory an of The of our study that enhanced cell surface DNA binding and internalization is one of the activation response that innate immune cells bacterial PAMPs in the of increased DNA binding is used as a clearance mechanism to of the inflammatory enhanced cell surface DNA binding and internalization may provide a regulatory to for anti-inflammatory We E. Deffebach for and of the