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Oxidized phospatidylcholine but not native phosphatidylcholine inhibits inducible nitric oxide synthase in RAW 264.7 macrophages
Life Sciences 78 (2006) 1586 – 1591 www.elsevier.com/locate/lifescie
Oxidized phospatidylcholine but not native phosphatidylcholine inhibits inducible nitric oxide synthase in RAW 264.7 macrophages Roswitha Friedl *, Ingeborg Pichler, Paul Spieckermann, Thomas Moeslinger Medical University of Vienna, Institute of Physiology, Schwarzspanierstrasse 17, A-1090 Vienna, Austria Received 15 April 2005; accepted 19 July 2005
Abstract This study was designed to compare the effects of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and native PAPC on the inducible nitric oxide synthase (iNOS) in the macrophage cell line RAW 264.7. Macrophages stimulated by bacterial lipopolysaccharide (1 Ag/ml) were incubated with increasing amounts of native or oxidized PAPC (oxPAPC, 10 – 20 Ag/ml). Cells incubated with oxPAPC showed a dose-dependent inhibition of inducible nitric oxide synthesis, as well as reduced iNOS protein expression and mRNA levels. Additionally, chromatin immunoprecipitation assay revealed that oxPAPC reduced the interaction of the active NF-nB subunit p65 with the iNOS promoter region when compared to native PAPC. D 2005 Elsevier Inc. All rights reserved. Keywords: Oxidized phospholipids; Atherosclerosis; Inducible nitric oxide synthase; Murine macrophages
Introduction Phosphatidylcholine (Lecithin, PhC) is the most abundant phospholipid in humans and is the prominent structural element of the cell membrane bilayers. PhC is also the principal phospholipid present in low density lipoprotein (LDL). Oxidative stress occurring in processes like acute and chronic inflammation leads to formation of reactive oxygen species (ROS) by phagocytic cells and subsequently to oxidation of LDL (Jerlich et al., 2003; Memon et al., 2000). OxLDL accumulation has been demonstrated at sites of chronic inflammation such as atherosclerosis (Chisolm and Steinberg, 2000; Miller et al., 2003). One of oxLDL’s biological activities in this context is the ability to serve as ligand for scavenger receptors of macrophages, which had been demonstrated for scavenger receptor class B type 1 (GillotteTaylor et al., 2001) and CD36 (Podrez et al., 2002). This process leads to the formation of foam cells, a hallmark in progression of atherosclerosis. The two most abundant molecular species of phosphatidylcholines present in LDL containing readily oxidizable fatty
* Corresponding author. Tel.: +43 1 4277 621 30; fax: +43 1 4277 9621. E-mail address: [email protected] (R. Friedl). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.07.035
acid constituents at their sn-2-position are: 1-palmitoyl-2arachidonyl-sn-glycero-3-phosphorylcholine (PAPC, (Bochkov et al., 2002) and 1-palmitoyl-2-linoleoyl-sn-3-phosphorylcholine (PLPC, (Podrez et al., 2002). Three prominent products of minimally oxidized PAPC have been identified (Watson et al., 1997): 1-palmitoyl-2-(5oxovaleroyl)-sn-glycero-3-phosphocholine (POV-PC), 1-palmitoyl-2-(glutaroyl)-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(epoxyisoprostane)-sn-glycero-3-phosphocholine (PEIPC). Nitric oxide (NO) is synthesized from l-arginine by the larginine-nitric oxide pathway (Palmer et al., 1988) and is converted to nitrite and nitrate in oxygenated solutions (Marletta et al., 1988). A family of enzymes, termed the nitric oxide synthases (NOS), catalyze the formation of NO and citrulline from l-arginine, O2, and NADPH (Marletta et al., 1993). The constitutive NOS isoforms (NOS-1 and NOS-3) produce low levels of NO as a consequence of increased intracellular Ca2+ (Nathan et al., 1994). By contrast, the inducible isoform of NOS (NOS-2 or iNOS; EC 1.14.13.39) generates large amounts of NO upon stimulation over a prolonged period of time through a Ca2+ independent pathway (Xie et al., 1992). Inducible NOS expression has been observed in many cells, including murine macrophages (Hibbs et al., 1988), endothelial cells (Gross et al., 1991), smooth muscle
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cells (Beasley et al., 1991), and cardiac myocytes (Schulz et al., 1992). Human iNOS is most readily observed in monocytes or macrophages from patients with infectious or inflammatory disease (MacMicking et al., 1997). Several studies demonstrated the inhibition of iNOS by oxLDL (Dulak et al., 1999; Liu et al., 1998; Yang et al., 1994, 1996). Further, it could be demonstrated that the protein moiety of oxLDL had no effect on NO production whereas the lipid moiety inhibited NO production to about the same extent as intact oxLDL (Liu et al., 1998). This study was aimed at the question whether oxPAPC is capable of inhibiting iNOS and compared it to the effect of native PAPC on LPS-stimulated macrophages. Materials and methods Cell culture All reagents were obtained from Sigma, unless otherwise noted. The mouse monocyte/macrophage cell line RAW 264.7 (ATCC TIB 67) was cultured in DMEM (Dulbeccos modified eagle’s medium) supplemented with 10% FCS, 25 mM HEPES, 100 U penicillin/ml and 100 Ag streptomycin/ml at 37 -C, 5% CO2, and 95% humidity. Cells between passage 7 and 30 were stimulated with lipopolysaccharide from E. coli (LPS; 1 Ag/ml) and incubated either with native or oxidized PAPC (10 – 20 Ag/ml) for 24 h. LPS and PAPC were added at the same time. Five milligrams of PAPC (lyophilized powder) was redissolved in 1 ml of PBS, pH 4, and the solution was exposed to atmospheric oxygen in a sterile surrounding at RT for 6 – 8 h. This stock solution was used as oxPAPC stock in our experiments. Nitrite analysis Nitrite was determined spectrophotometrically in supernatants by using the Griess reagent. Absorbance was measured at 550 nm with baseline correction at 650 nm and nitrite concentration was determined using sodium nitrite as a standard (Green et al., 1982). Protein determination Protein was determined according to the method of Bradford (Bradford et al., 1976) using bovine serum albumin as standard. Western blotting RAW 264.7 cells were treated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 – 20 Ag/ml) for 24 h. Cells were lysed in ice-cold lysis buffer (25 mM monosodium phosphate (pH 7.4), 75 mM NaCl, 5 mM EDTA, 1% Triton X-100, and a protease inhibitor mix) and centrifuged at 50,000g for 20 min at 4 -C. The cytosolic proteins (20 Ag per lane) were separated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins were trans-
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ferred to nitrocellulose filters, and then immunoblotted either with a rabbit anti-iNOS polyclonal antibody or with a rabbit anti-p65-polyclonal antibody. Control blots were done with anti-actin antibody. Anti-rabbit AP-conjugated antibody was used as a secondary antibody at a dilution of 1:7500. Finally, the blots were incubated with 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) reagent (Promega) for 10 – 15 min. Semiquantitative RT-PCR RAW 264.7 cells were treated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 –20 Ag/ml) for 24 h. Total RNA was extracted from the cells using a commercially available kit (Promega) and then subjected to semiquantitative RT-PCR using iNOS and beta-actin primers as described previously (Friedl et al., 2000). Chromatin immunoprecipitation ChIP experiments were performed using the ChIP Assay Kit from Upstate Biotechnology Inc. and following the instruction manual. 1 106 RAW 264.7 cells were seeded per 10 cm dish and treated with or without LPS (1 Ag/ml) and native or oxPAPC (20 Ag/ml) for 24 h. To cross-link DNA with proteins, formaldehyde (1%) was added to the culture medium and cells were incubated at room temperature for 10 min. Glycine (final concentration 0.125 M) was then added for 5 min to stop the cross-linking reaction. Cells were washed twice in PBS, scraped, and lysed in lysis buffer (1% SDS, 10 mM Tris – HCl, pH 8.0, with 1 mM PMSF, pepstatin A, and aprotinin) for 30 min at 4 -C. After sonication (5 times, 10 s each) to yield an average DNA fragment size of 500 bp, the DNA fragments cross-linked to the proteins were enriched by immunoprecipitation with an antibody specific for p65 (Santa Cruz). After reversal of the cross-links and DNA purification, the extent of enrichment was monitored by PCR amplification using the following primers specific to the NF-nB binding sites located in the socalled region I of the murine iNOS promoter that regulates LPS-induced expression of the iNOS gene (Lowenstein et al., 1993): region I for (5V-CATGAGGATACACCACAGAG), region I back (5V-AAGACCCAAGCGTGAGGAGC). As a positive control (input), sonicated nuclear lysates from LPSstimulated cells underwent reverse cross-link and phenol/ chloroform extraction and were subjected to PCR. For negative control, a no antibody reaction was used in immunoprecipitation against dilute sonicated nuclear lysate from LPS-stimulated cells. PCR products were fractionated and visualized on 2% agarose TBE gel containing ethidium bromide. Each experiment was repeated for at least 3 times. Preparation of nuclear extracts RAW 264.7 cells were treated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 –20 Ag/ml) for 24 h. Cells were harvested and pelleted at 450g for 5 min
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at 4 -C. Nuclear proteins were extracted using a kit obtained from Sigma (St. Louis, MO, USA) and following the manufacturer’s manual as described previously (Friedl et al., 2000).
analyses were performed by use of ANOVA followed by Student’s t tests for unpaired data. Statistical significance was defined as P < 0.05. Results
Data analysis Effect of native vs. oxidized PAPC on NO synthesis Each experimental result as shown in the figures is the mean T SD for at least three measurements. When SD is not displayed, it is smaller than the size of the symbol. Statistical
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Activated RAW 264.7 cells released large amounts of nitrite into the culture medium (156 T 11.2 nmol nitrite/mg protein). Incubation of activated RAW 264.7 cells with increasing amounts of oxidized PAPC was associated with a dosedependent reduction in NO production (a reduction to 38.9% of uninhibited nitrite level at 10 Ag/ml and 9.6% at 20 Ag/ml). Probably due to oxidation processes of the originally natively added PAPC during the 24 h of incubation in the cell culture wells, a slight reduction in nitrite production was also observed in wells incubated with native PAPC (81.31% resp. 71.65%) (Fig. 1A). To exclude that oxPAPC interferes with detection of nitrite by the Griess reaction or influences the stability of NO, we incubated the NO donor linsidomine (0.5 mM) in the presence or absence of oxPAPC (10 – 20 Ag/ml) for 24 h at room temperature. PAPC had no statistically significant effect on the measured nitrite levels (data not shown). We conclude therefore that PAPC does not scavenge NO or interfere with the detection of nitrite by the Griess reaction.
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iNOS protein was reduced in a dose-dependent manner by increasing amounts of oxPAPC (10 – 20 Ag/ml). In contrast, beta-actin (43 kDa) levels remained unchanged during incubations with oxPAPC. This shows that oxPAPC induced iNOS
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Fig. 1. RAW 264.7 cells were stimulated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 – 20 Ag/ml) for 24 h. LPS and PAPC were added at the same time. 1: unstimulated RAW 264.7 cells; 2 – 6: cells stimulated with 1 Ag/ml LPS; 3: cells incubated with 10 Ag/ml of native PAPC; 4: cells incubated with 20 Ag/ml of native PAPC; 5: cells incubated with 10 Ag/ ml of oxidized PAPC; 6: cells incubated with 20 Ag/ml of oxidized PAPC. (A) Measurement of nitrite release. Culture media were collected and assayed for nitrite after 24 h as described in the Materials and methods section. Nitrite levels were related to protein levels determined by the Bradford assay. (B) Immunoblotting against iNOS. Upper panel: the 130 kDa band of iNOS, lower panel: the 43 kDa band of beta-actin. (C) Quantification of immunoblot data by densitometry using the NIH Image software. (D) Semiquantitative PCR. Unstimulated cells (lane 1) served as negative control. Semiquantitative estimation was done by comparing mRNA expression of iNOS to beta-actin represented by the amount of PCR product formed. (E) Quantification of PCR data by densitometry using the NIH Image software.
R. Friedl et al. / Life Sciences 78 (2006) 1586 – 1591 1
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Fig. 2. Effect of native vs. oxidized PAPC on nuclear p65 protein level from LPS-stimulated cells. Unstimulated RAW 264.7 cells served as negative control (lane 1). Lanes 2 – 6 show nuclear extracts from cells that were stimulated with LPS, lanes 3 – 6 were treated with either native or oxidized PAPC as given in Fig. 1. Nuclear proteins were collected 24 h after treatment followed by Western immunoblotting as described in the Materials and methods section. The blot shown is representative of 3 similar experiments.
inhibition is not associated with a generalized decrease in protein expression. iNOS protein was reduced to 21.5% for 10 Ag/ml oxPAPC, and to 6.4% for 20 Ag/ml oxPAPC when compared to the amount of beta-actin protein using densitometry (Fig. 1B and C). Effects of native vs. oxidized PAPC on iNOS mRNA expression A dose-dependent reduction of iNOS mRNA was seen when cells were incubated with oxPAPC (Fig. 1D, lanes 5 and 6). Actin mRNA levels remained unchanged (Fig. 1D and E). Effect of native vs. oxidized PAPC on nuclear p65 protein level from LPS-stimulated cells To elucidate the mechanism of inhibition, we next determined whether oxPAPC could inhibit the translocation of p65, an active subunit of the transcription factor NF-nB. Western blotting of nuclear extracts against p65 was done. Unstimulated RAW264.7 cells served as negative control. No difference in the extent of p65 translocation to the nuclear protein fraction can be seen between untreated and PAPC-treated stimulated cells. The blot shown is representative of 3 similar experiments (Fig. 2). Effect of oxPAPC on binding of p65 to the iNOS promoter region We next investigated if the interaction of p65 with its target region on the iNOS promoter region is affected by oxPAPC or not. ChIP assays were done with an antibody specific for p65. For the detection of DNA associated with proteins, PCR primers specific for an iNOS promoter region that is known to contain LPS-related responsive elements, including a binding site for NF-nB, were used (Lowenstein et al., 1993). Stimulated cells were incubated with either native or oxPAPC. Cells incubated with oxPAPC resulted in reduced PCR signals compared to cells incubated with native PAPC. At least 3 independent experiments led to this result (Fig. 3). Discussion The focus on oxidized lipoproteins, and especially oxLDL, started with the consistent conviction that these molecules can be designated the main culprits in the development of atherosclerotic lesions and are responsible for the induction
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of vascular inflammation (Chisolm and Steinberg, 2000; Nilsson et al., 1992; Witztum et al., 1994). oxPAPC itself has recently been shown to trigger atherogenic inflammation by induction of proinflammatory chemokines (Furnkranz et al., 2005). It has already been shown some time ago that oxLDL inhibits the inducible isoform of the NO synthases (Dulak et al., 1999; Huang et al., 1999; Thai et al., 1995; Yang et al., 1994). NO released by iNOS in turn seems to play a role in fighting the adverse effects of lipid uptake of macrophages leading to atherosclerotic lesion. One example is a study (Hemmrich et al., 2003) showing that blocking of iNOS expression in primary endothelial cell cultures by antisenseoligos leads to a strong decrease in the expression of the protective stress response genes bcl-2, vascular endothelial growth factor, and heme oxygenase-1 (HO-1). The authors suggest that this might contribute to endothelial dysfunction and death during oxidative stress. Another study using iNOS-deficient mice demonstrated that lack of iNOS resulted in elevated blood pressure, elevated serum cholesterol levels and higher incidence of atherosclerotic plaques (Ihrig et al., 2001). With our study we wanted to address the question, whether oxPAPC is capable of inhibiting iNOS. As PAPC is one of the main components of LDL and is likely to be affected by oxidative stress, we wished to shed light on the underlying principles of oxLDL mediated iNOS inhibition. Indeed, it could be clearly demonstrated that it is the oxidized form of PAPC that leads to a reduction in nitrite, iNOS protein and iNOS mRNA. The promoter region of iNOS is known to host several transcription factor binding sites and it had been demonstrated that upon LPS stimulation NF-nB is activated (Lowenstein et al., 1993). In our study, Western blotting of nuclear extracts showed that p65 translocation was unaffected by native and oxPAPC. In contrast, ChIP experiments revealed that incubation with oxPAPC resulted in reduced interaction of p65 with the murine iNOS promoter region compared to cells incubated with native PAPC. This points to the assumption that the
anti-p65 LPS native PAPC oxPAPC
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Fig. 3. Effect of oxPAPC on binding of p65 to the iNOS promoter region. RAW 264.7 cells were stimulated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 – 20 Ag/ml) for 24 h. ChIP assays were done with antibody specific for p65 to immunoprecipitate NF-nB subunit p65 cross-linked with DNA fragments. 1: untreated cells. 2: cells stimulated with LPS. 3: stimulated cells incubated with 20 Ag/ml of native PAPC. 4: stimulated cells incubated with 20 Ag/ml of oxPAPC. 5: ‘‘no antibody’’ control on stimulated cells. 6: input control on stimulated cells.
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observed inhibition of NO production by oxPAPC is at least partially NF-nB-mediated. Conclusion Our experiments clearly demonstrated the ability of oxPAPC to inhibit inducible nitric oxide synthesis of LPSstimulated macrophages, whereas native PAPC produced no significant downregulation of nitrite levels. Concomitantly, mRNA and protein levels of stimulated macrophages treated with oxPAPC were reduced and native PAPC had only an insignificant effect. This clearly showed that inhibition of iNOS can be ascribed to oxidized PAPC as occurring in states of oxidative stress, but not to native PAPC. In addition, it could be demonstrated that oxidized PAPC reduced binding of p65 to the iNOS promoter region, suggesting that oxPAPC interferes with the NF-nB transcription regulation pathway. Acknowledgments This work was funded by the regular endowment for academic institutions from the Austrian government. References Beasley, D., Schwartz, J.H., Brenner, B.M., 1991. Interleukin 1 induces prolonged l-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. Journal of Clinical Investigation 87 (2), 602 – 608. Bochkov, V.N., Kadl, A., Huber, J., Gruber, F., Binder, B.R., Leitinger, N., 2002. Protective role of phospholipid oxidation products in endotoxininduced tissue damage. Nature 419 (6902), 77 – 81. Bradford, M.M., MacMicking, J., Xie, Q.W., Nathan, C., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 (1), 248 – 254. Chisolm, G.M., Steinberg, D., 2000. The oxidative modification hypothesis of atherogenesis: an overview. Free Radical Biology & Medicine 28 (12), 1815 – 1826. Dulak, J., Polus, M., Guevara, I., Hartwich, J., Wybranska, I., Krzesz, R., Dembinska-Kiec, A., 1999. Oxidized low density lipoprotein inhibits inducible nitric oxide synthase, GTP cyclohydrolase I and transforming growth factor beta gene expression in rat macrophages. Journal of Physiology and Pharmacology 50 (3), 429 – 441. Friedl, R., Brunner, M., Moeslinger, T., Spieckermann, P.G., 2000. Testosterone inhibits expression of inducible nitric oxide synthase in murine macrophages. Life Sciences 68 (4), 417 – 429. Furnkranz, A., Schober, A., Bochkov, V.N., Bashtrykov, P., Kronke, G., Kadl, A., Binder, B.R., Weber, C., Leitinger, N., 2005. Oxidized phospholipids trigger atherogenic inflammation in murine. Arteriosclerosis, Thrombosis, and Vascular Biology, 251 – 256. Gillotte-Taylor, K., Boullier, A., Witztum, J.L., Steinberg, D., Quehenberger, O., 2001. Scavenger receptor class B type I as a receptor for oxidized low density lipoprotein. Journal of Lipid Research 42 (9), 1474 – 1482. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., MacMicking, J., Xie, Q.W., Nathan, C., 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Analytical Biochemistry 126 (1), 131 – 138. Gross, S.S., Jaffe, E.A., Levi, R., Kilbourn, R.G., 1991. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank – order of potency characteristic of activated
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Oxidized phospatidylcholine but not native phosphatidylcholine inhibits inducible nitric oxide synthase in RAW 264.7 macrophages Roswitha Friedl *, Ingeborg Pichler, Paul Spieckermann, Thomas Moeslinger Medical University of Vienna, Institute of Physiology, Schwarzspanierstrasse 17, A-1090 Vienna, Austria Received 15 April 2005; accepted 19 July 2005
Abstract This study was designed to compare the effects of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and native PAPC on the inducible nitric oxide synthase (iNOS) in the macrophage cell line RAW 264.7. Macrophages stimulated by bacterial lipopolysaccharide (1 Ag/ml) were incubated with increasing amounts of native or oxidized PAPC (oxPAPC, 10 – 20 Ag/ml). Cells incubated with oxPAPC showed a dose-dependent inhibition of inducible nitric oxide synthesis, as well as reduced iNOS protein expression and mRNA levels. Additionally, chromatin immunoprecipitation assay revealed that oxPAPC reduced the interaction of the active NF-nB subunit p65 with the iNOS promoter region when compared to native PAPC. D 2005 Elsevier Inc. All rights reserved. Keywords: Oxidized phospholipids; Atherosclerosis; Inducible nitric oxide synthase; Murine macrophages
Introduction Phosphatidylcholine (Lecithin, PhC) is the most abundant phospholipid in humans and is the prominent structural element of the cell membrane bilayers. PhC is also the principal phospholipid present in low density lipoprotein (LDL). Oxidative stress occurring in processes like acute and chronic inflammation leads to formation of reactive oxygen species (ROS) by phagocytic cells and subsequently to oxidation of LDL (Jerlich et al., 2003; Memon et al., 2000). OxLDL accumulation has been demonstrated at sites of chronic inflammation such as atherosclerosis (Chisolm and Steinberg, 2000; Miller et al., 2003). One of oxLDL’s biological activities in this context is the ability to serve as ligand for scavenger receptors of macrophages, which had been demonstrated for scavenger receptor class B type 1 (GillotteTaylor et al., 2001) and CD36 (Podrez et al., 2002). This process leads to the formation of foam cells, a hallmark in progression of atherosclerosis. The two most abundant molecular species of phosphatidylcholines present in LDL containing readily oxidizable fatty
* Corresponding author. Tel.: +43 1 4277 621 30; fax: +43 1 4277 9621. E-mail address: [email protected] (R. Friedl). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.07.035
acid constituents at their sn-2-position are: 1-palmitoyl-2arachidonyl-sn-glycero-3-phosphorylcholine (PAPC, (Bochkov et al., 2002) and 1-palmitoyl-2-linoleoyl-sn-3-phosphorylcholine (PLPC, (Podrez et al., 2002). Three prominent products of minimally oxidized PAPC have been identified (Watson et al., 1997): 1-palmitoyl-2-(5oxovaleroyl)-sn-glycero-3-phosphocholine (POV-PC), 1-palmitoyl-2-(glutaroyl)-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(epoxyisoprostane)-sn-glycero-3-phosphocholine (PEIPC). Nitric oxide (NO) is synthesized from l-arginine by the larginine-nitric oxide pathway (Palmer et al., 1988) and is converted to nitrite and nitrate in oxygenated solutions (Marletta et al., 1988). A family of enzymes, termed the nitric oxide synthases (NOS), catalyze the formation of NO and citrulline from l-arginine, O2, and NADPH (Marletta et al., 1993). The constitutive NOS isoforms (NOS-1 and NOS-3) produce low levels of NO as a consequence of increased intracellular Ca2+ (Nathan et al., 1994). By contrast, the inducible isoform of NOS (NOS-2 or iNOS; EC 1.14.13.39) generates large amounts of NO upon stimulation over a prolonged period of time through a Ca2+ independent pathway (Xie et al., 1992). Inducible NOS expression has been observed in many cells, including murine macrophages (Hibbs et al., 1988), endothelial cells (Gross et al., 1991), smooth muscle
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cells (Beasley et al., 1991), and cardiac myocytes (Schulz et al., 1992). Human iNOS is most readily observed in monocytes or macrophages from patients with infectious or inflammatory disease (MacMicking et al., 1997). Several studies demonstrated the inhibition of iNOS by oxLDL (Dulak et al., 1999; Liu et al., 1998; Yang et al., 1994, 1996). Further, it could be demonstrated that the protein moiety of oxLDL had no effect on NO production whereas the lipid moiety inhibited NO production to about the same extent as intact oxLDL (Liu et al., 1998). This study was aimed at the question whether oxPAPC is capable of inhibiting iNOS and compared it to the effect of native PAPC on LPS-stimulated macrophages. Materials and methods Cell culture All reagents were obtained from Sigma, unless otherwise noted. The mouse monocyte/macrophage cell line RAW 264.7 (ATCC TIB 67) was cultured in DMEM (Dulbeccos modified eagle’s medium) supplemented with 10% FCS, 25 mM HEPES, 100 U penicillin/ml and 100 Ag streptomycin/ml at 37 -C, 5% CO2, and 95% humidity. Cells between passage 7 and 30 were stimulated with lipopolysaccharide from E. coli (LPS; 1 Ag/ml) and incubated either with native or oxidized PAPC (10 – 20 Ag/ml) for 24 h. LPS and PAPC were added at the same time. Five milligrams of PAPC (lyophilized powder) was redissolved in 1 ml of PBS, pH 4, and the solution was exposed to atmospheric oxygen in a sterile surrounding at RT for 6 – 8 h. This stock solution was used as oxPAPC stock in our experiments. Nitrite analysis Nitrite was determined spectrophotometrically in supernatants by using the Griess reagent. Absorbance was measured at 550 nm with baseline correction at 650 nm and nitrite concentration was determined using sodium nitrite as a standard (Green et al., 1982). Protein determination Protein was determined according to the method of Bradford (Bradford et al., 1976) using bovine serum albumin as standard. Western blotting RAW 264.7 cells were treated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 – 20 Ag/ml) for 24 h. Cells were lysed in ice-cold lysis buffer (25 mM monosodium phosphate (pH 7.4), 75 mM NaCl, 5 mM EDTA, 1% Triton X-100, and a protease inhibitor mix) and centrifuged at 50,000g for 20 min at 4 -C. The cytosolic proteins (20 Ag per lane) were separated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins were trans-
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ferred to nitrocellulose filters, and then immunoblotted either with a rabbit anti-iNOS polyclonal antibody or with a rabbit anti-p65-polyclonal antibody. Control blots were done with anti-actin antibody. Anti-rabbit AP-conjugated antibody was used as a secondary antibody at a dilution of 1:7500. Finally, the blots were incubated with 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) reagent (Promega) for 10 – 15 min. Semiquantitative RT-PCR RAW 264.7 cells were treated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 –20 Ag/ml) for 24 h. Total RNA was extracted from the cells using a commercially available kit (Promega) and then subjected to semiquantitative RT-PCR using iNOS and beta-actin primers as described previously (Friedl et al., 2000). Chromatin immunoprecipitation ChIP experiments were performed using the ChIP Assay Kit from Upstate Biotechnology Inc. and following the instruction manual. 1 106 RAW 264.7 cells were seeded per 10 cm dish and treated with or without LPS (1 Ag/ml) and native or oxPAPC (20 Ag/ml) for 24 h. To cross-link DNA with proteins, formaldehyde (1%) was added to the culture medium and cells were incubated at room temperature for 10 min. Glycine (final concentration 0.125 M) was then added for 5 min to stop the cross-linking reaction. Cells were washed twice in PBS, scraped, and lysed in lysis buffer (1% SDS, 10 mM Tris – HCl, pH 8.0, with 1 mM PMSF, pepstatin A, and aprotinin) for 30 min at 4 -C. After sonication (5 times, 10 s each) to yield an average DNA fragment size of 500 bp, the DNA fragments cross-linked to the proteins were enriched by immunoprecipitation with an antibody specific for p65 (Santa Cruz). After reversal of the cross-links and DNA purification, the extent of enrichment was monitored by PCR amplification using the following primers specific to the NF-nB binding sites located in the socalled region I of the murine iNOS promoter that regulates LPS-induced expression of the iNOS gene (Lowenstein et al., 1993): region I for (5V-CATGAGGATACACCACAGAG), region I back (5V-AAGACCCAAGCGTGAGGAGC). As a positive control (input), sonicated nuclear lysates from LPSstimulated cells underwent reverse cross-link and phenol/ chloroform extraction and were subjected to PCR. For negative control, a no antibody reaction was used in immunoprecipitation against dilute sonicated nuclear lysate from LPS-stimulated cells. PCR products were fractionated and visualized on 2% agarose TBE gel containing ethidium bromide. Each experiment was repeated for at least 3 times. Preparation of nuclear extracts RAW 264.7 cells were treated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 –20 Ag/ml) for 24 h. Cells were harvested and pelleted at 450g for 5 min
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at 4 -C. Nuclear proteins were extracted using a kit obtained from Sigma (St. Louis, MO, USA) and following the manufacturer’s manual as described previously (Friedl et al., 2000).
analyses were performed by use of ANOVA followed by Student’s t tests for unpaired data. Statistical significance was defined as P < 0.05. Results
Data analysis Effect of native vs. oxidized PAPC on NO synthesis Each experimental result as shown in the figures is the mean T SD for at least three measurements. When SD is not displayed, it is smaller than the size of the symbol. Statistical
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Activated RAW 264.7 cells released large amounts of nitrite into the culture medium (156 T 11.2 nmol nitrite/mg protein). Incubation of activated RAW 264.7 cells with increasing amounts of oxidized PAPC was associated with a dosedependent reduction in NO production (a reduction to 38.9% of uninhibited nitrite level at 10 Ag/ml and 9.6% at 20 Ag/ml). Probably due to oxidation processes of the originally natively added PAPC during the 24 h of incubation in the cell culture wells, a slight reduction in nitrite production was also observed in wells incubated with native PAPC (81.31% resp. 71.65%) (Fig. 1A). To exclude that oxPAPC interferes with detection of nitrite by the Griess reaction or influences the stability of NO, we incubated the NO donor linsidomine (0.5 mM) in the presence or absence of oxPAPC (10 – 20 Ag/ml) for 24 h at room temperature. PAPC had no statistically significant effect on the measured nitrite levels (data not shown). We conclude therefore that PAPC does not scavenge NO or interfere with the detection of nitrite by the Griess reaction.
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iNOS protein was reduced in a dose-dependent manner by increasing amounts of oxPAPC (10 – 20 Ag/ml). In contrast, beta-actin (43 kDa) levels remained unchanged during incubations with oxPAPC. This shows that oxPAPC induced iNOS
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Fig. 1. RAW 264.7 cells were stimulated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 – 20 Ag/ml) for 24 h. LPS and PAPC were added at the same time. 1: unstimulated RAW 264.7 cells; 2 – 6: cells stimulated with 1 Ag/ml LPS; 3: cells incubated with 10 Ag/ml of native PAPC; 4: cells incubated with 20 Ag/ml of native PAPC; 5: cells incubated with 10 Ag/ ml of oxidized PAPC; 6: cells incubated with 20 Ag/ml of oxidized PAPC. (A) Measurement of nitrite release. Culture media were collected and assayed for nitrite after 24 h as described in the Materials and methods section. Nitrite levels were related to protein levels determined by the Bradford assay. (B) Immunoblotting against iNOS. Upper panel: the 130 kDa band of iNOS, lower panel: the 43 kDa band of beta-actin. (C) Quantification of immunoblot data by densitometry using the NIH Image software. (D) Semiquantitative PCR. Unstimulated cells (lane 1) served as negative control. Semiquantitative estimation was done by comparing mRNA expression of iNOS to beta-actin represented by the amount of PCR product formed. (E) Quantification of PCR data by densitometry using the NIH Image software.
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Fig. 2. Effect of native vs. oxidized PAPC on nuclear p65 protein level from LPS-stimulated cells. Unstimulated RAW 264.7 cells served as negative control (lane 1). Lanes 2 – 6 show nuclear extracts from cells that were stimulated with LPS, lanes 3 – 6 were treated with either native or oxidized PAPC as given in Fig. 1. Nuclear proteins were collected 24 h after treatment followed by Western immunoblotting as described in the Materials and methods section. The blot shown is representative of 3 similar experiments.
inhibition is not associated with a generalized decrease in protein expression. iNOS protein was reduced to 21.5% for 10 Ag/ml oxPAPC, and to 6.4% for 20 Ag/ml oxPAPC when compared to the amount of beta-actin protein using densitometry (Fig. 1B and C). Effects of native vs. oxidized PAPC on iNOS mRNA expression A dose-dependent reduction of iNOS mRNA was seen when cells were incubated with oxPAPC (Fig. 1D, lanes 5 and 6). Actin mRNA levels remained unchanged (Fig. 1D and E). Effect of native vs. oxidized PAPC on nuclear p65 protein level from LPS-stimulated cells To elucidate the mechanism of inhibition, we next determined whether oxPAPC could inhibit the translocation of p65, an active subunit of the transcription factor NF-nB. Western blotting of nuclear extracts against p65 was done. Unstimulated RAW264.7 cells served as negative control. No difference in the extent of p65 translocation to the nuclear protein fraction can be seen between untreated and PAPC-treated stimulated cells. The blot shown is representative of 3 similar experiments (Fig. 2). Effect of oxPAPC on binding of p65 to the iNOS promoter region We next investigated if the interaction of p65 with its target region on the iNOS promoter region is affected by oxPAPC or not. ChIP assays were done with an antibody specific for p65. For the detection of DNA associated with proteins, PCR primers specific for an iNOS promoter region that is known to contain LPS-related responsive elements, including a binding site for NF-nB, were used (Lowenstein et al., 1993). Stimulated cells were incubated with either native or oxPAPC. Cells incubated with oxPAPC resulted in reduced PCR signals compared to cells incubated with native PAPC. At least 3 independent experiments led to this result (Fig. 3). Discussion The focus on oxidized lipoproteins, and especially oxLDL, started with the consistent conviction that these molecules can be designated the main culprits in the development of atherosclerotic lesions and are responsible for the induction
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of vascular inflammation (Chisolm and Steinberg, 2000; Nilsson et al., 1992; Witztum et al., 1994). oxPAPC itself has recently been shown to trigger atherogenic inflammation by induction of proinflammatory chemokines (Furnkranz et al., 2005). It has already been shown some time ago that oxLDL inhibits the inducible isoform of the NO synthases (Dulak et al., 1999; Huang et al., 1999; Thai et al., 1995; Yang et al., 1994). NO released by iNOS in turn seems to play a role in fighting the adverse effects of lipid uptake of macrophages leading to atherosclerotic lesion. One example is a study (Hemmrich et al., 2003) showing that blocking of iNOS expression in primary endothelial cell cultures by antisenseoligos leads to a strong decrease in the expression of the protective stress response genes bcl-2, vascular endothelial growth factor, and heme oxygenase-1 (HO-1). The authors suggest that this might contribute to endothelial dysfunction and death during oxidative stress. Another study using iNOS-deficient mice demonstrated that lack of iNOS resulted in elevated blood pressure, elevated serum cholesterol levels and higher incidence of atherosclerotic plaques (Ihrig et al., 2001). With our study we wanted to address the question, whether oxPAPC is capable of inhibiting iNOS. As PAPC is one of the main components of LDL and is likely to be affected by oxidative stress, we wished to shed light on the underlying principles of oxLDL mediated iNOS inhibition. Indeed, it could be clearly demonstrated that it is the oxidized form of PAPC that leads to a reduction in nitrite, iNOS protein and iNOS mRNA. The promoter region of iNOS is known to host several transcription factor binding sites and it had been demonstrated that upon LPS stimulation NF-nB is activated (Lowenstein et al., 1993). In our study, Western blotting of nuclear extracts showed that p65 translocation was unaffected by native and oxPAPC. In contrast, ChIP experiments revealed that incubation with oxPAPC resulted in reduced interaction of p65 with the murine iNOS promoter region compared to cells incubated with native PAPC. This points to the assumption that the
anti-p65 LPS native PAPC oxPAPC
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Fig. 3. Effect of oxPAPC on binding of p65 to the iNOS promoter region. RAW 264.7 cells were stimulated with LPS (1 Ag/ml) and incubated either with native or oxidized PAPC (10 – 20 Ag/ml) for 24 h. ChIP assays were done with antibody specific for p65 to immunoprecipitate NF-nB subunit p65 cross-linked with DNA fragments. 1: untreated cells. 2: cells stimulated with LPS. 3: stimulated cells incubated with 20 Ag/ml of native PAPC. 4: stimulated cells incubated with 20 Ag/ml of oxPAPC. 5: ‘‘no antibody’’ control on stimulated cells. 6: input control on stimulated cells.
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observed inhibition of NO production by oxPAPC is at least partially NF-nB-mediated. Conclusion Our experiments clearly demonstrated the ability of oxPAPC to inhibit inducible nitric oxide synthesis of LPSstimulated macrophages, whereas native PAPC produced no significant downregulation of nitrite levels. Concomitantly, mRNA and protein levels of stimulated macrophages treated with oxPAPC were reduced and native PAPC had only an insignificant effect. This clearly showed that inhibition of iNOS can be ascribed to oxidized PAPC as occurring in states of oxidative stress, but not to native PAPC. In addition, it could be demonstrated that oxidized PAPC reduced binding of p65 to the iNOS promoter region, suggesting that oxPAPC interferes with the NF-nB transcription regulation pathway. Acknowledgments This work was funded by the regular endowment for academic institutions from the Austrian government. References Beasley, D., Schwartz, J.H., Brenner, B.M., 1991. Interleukin 1 induces prolonged l-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. Journal of Clinical Investigation 87 (2), 602 – 608. Bochkov, V.N., Kadl, A., Huber, J., Gruber, F., Binder, B.R., Leitinger, N., 2002. Protective role of phospholipid oxidation products in endotoxininduced tissue damage. Nature 419 (6902), 77 – 81. Bradford, M.M., MacMicking, J., Xie, Q.W., Nathan, C., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 (1), 248 – 254. Chisolm, G.M., Steinberg, D., 2000. The oxidative modification hypothesis of atherogenesis: an overview. Free Radical Biology & Medicine 28 (12), 1815 – 1826. Dulak, J., Polus, M., Guevara, I., Hartwich, J., Wybranska, I., Krzesz, R., Dembinska-Kiec, A., 1999. Oxidized low density lipoprotein inhibits inducible nitric oxide synthase, GTP cyclohydrolase I and transforming growth factor beta gene expression in rat macrophages. Journal of Physiology and Pharmacology 50 (3), 429 – 441. Friedl, R., Brunner, M., Moeslinger, T., Spieckermann, P.G., 2000. Testosterone inhibits expression of inducible nitric oxide synthase in murine macrophages. Life Sciences 68 (4), 417 – 429. Furnkranz, A., Schober, A., Bochkov, V.N., Bashtrykov, P., Kronke, G., Kadl, A., Binder, B.R., Weber, C., Leitinger, N., 2005. Oxidized phospholipids trigger atherogenic inflammation in murine. Arteriosclerosis, Thrombosis, and Vascular Biology, 251 – 256. Gillotte-Taylor, K., Boullier, A., Witztum, J.L., Steinberg, D., Quehenberger, O., 2001. Scavenger receptor class B type I as a receptor for oxidized low density lipoprotein. Journal of Lipid Research 42 (9), 1474 – 1482. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., MacMicking, J., Xie, Q.W., Nathan, C., 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Analytical Biochemistry 126 (1), 131 – 138. Gross, S.S., Jaffe, E.A., Levi, R., Kilbourn, R.G., 1991. Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank – order of potency characteristic of activated
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