Reactive oxygen species (ROS), including hydrogen peroxide

Transcription

1 Macrophages Generate Reactive Oxygen Species in Response to Minimally Oxidized Low-Density Lipoprotein Toll-Like Receptor 4 and Spleen Tyrosine Kinase Dependent Activation of NADPH Oxidase 2 Yun Soo Bae, Jee Hyun Lee, Soo Ho Choi, Sunah Kim, Felicidad Almazan, Joseph L. Witztum, Yury I. Miller Abstract Oxidative modification of low-density lipoprotein (LDL) plays a causative role in the development of atherosclerosis. In this study, we demonstrate that minimally oxidized LDL (mmldl) stimulates intracellular reactive oxygen species (ROS) generation in macrophages through NADPH oxidase 2 (gp91phox/nox2), which, in turn, induces production of RANTES and migration of smooth muscle cells. Peritoneal macrophages from gp91phox/nox2 / mice or J774 macrophages in which Nox2 was knocked down by small interfering RNA failed to generate ROS in response to mmldl. Because mmldl-induced cytoskeletal changes were dependent on Toll-like receptor (TLR)4, we analyzed ROS generation in peritoneal macrophages from wild-type, TLR4 /, or MyD88 / mice and found that mmldlmediated ROS was generated in a TLR4-dependent, but MyD88-independent, manner. Furthermore, we found that ROS generation required the recruitment and activation of spleen tyrosine kinase (Syk) and that mmldl also induced phospholipase PLC 1 phosphorylation and protein kinase C membrane translocation. Importantly, the phospholipase C 1 phosphorylation was reduced in J774 cells expressing Syk-specific short hairpin RNA. Nox2 modulated mmldl activation of macrophages by regulating the expression of proinflammatory cytokines interleukin-1, interleukin-6, and RANTES. We showed that purified RANTES was able to stimulate migration of mouse aortic smooth muscle cells and addition of neutralizing antibody against RANTES abolished the migration of mouse aortic smooth muscle cells stimulated by mmldl-stimulated macrophages. These results suggest that mmldl induces generation of ROS through sequential activation of TLR4, Syk, phospholipase C 1, protein kinase C, and gp91phox/nox2 and thereby stimulates expression of proinflammatory cytokines. These data help explain mechanisms by which endogenous ligands, such as mmldl, can induce TLR4-dependent, proatherogenic activation of macrophages. (Circ Res. 2009;104: ) Key Words: minimally oxidized LDL reactive oxygen species NADPH oxidase 2 RANTES atherosclerosis Reactive oxygen species (ROS), including hydrogen peroxide and superoxide anion, are generally considered cytotoxic. 1 However, in recent years, many reports have demonstrated that intracellular ROS, produced in mammalian cells in response to the activation of various receptors, also serve as important second messengers in cell signaling. 1 The major source of ROS in phagocytes is an NADPH oxidase complex. 2 It is composed of 6 protein components: 2 transmembrane flavocytochrome b proteins (gp91phox and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac). The catalytic protein in this complex is gp91phox, recently renamed as Nox2. The NH 3 -terminal region of gp91phox/nox2 consists of transmembrane domains that bind 2 heme groups, and the COOH-terminal region contains NADPH/FAD binding sites. The cytosolic component of the complex, p47phox, plays a critical role in an assembly of the whole NADPH oxidase complex on activation of resting phagocytic cells. p47phox is extensively phosphorylated by protein kinase (PK)C and then helps recruit p67phox, p40phox, and Rac. The translocation of the cytosolic components to the transmembrane component gp91phox results in the formation of a stable molecular structure of NADPH oxidase, which extracts an electron from NADPH that passes through FAD and heme groups and finally reduces an oxygen molecule to make a superoxide anion. We and others demonstrated that Nox isozymes can be activated by Toll-like receptors (TLRs), which are key regulators of innate immunity. 3,4 TLRs recognize pathogenassociated molecular patterns on the surface of pathogens, as well as altered host proteins and lipoproteins and stimulate inflammatory signaling pathways. 5,6 We reported that the stimulation of TLR4 with lipopolysaccharide (LPS) induces Original received June 10, 2008; revision received November 14, 2008; accepted December 4, From the Department of Life Sciences (Y.S.B., J.H.L., S.K.), Ewha Womans University, Seoul, Korea; and Department of Medicine (S.H.C., F.A., J.L.W., Y.I.M.), University of California, San Diego. Correspondence to Yun Soo Bae, Department of Life Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemoon-Gu, Seoul , Korea. baeys@ewha.ac.kr 2009 American Heart Association, Inc. Circulation Research is available at DOI: /CIRCRESAHA

2 Bae et al mmldl-dependent ROS Generation 211 Figure 1. mmldl stimulates ROS generation in J774 macrophages. A, J774 cells were incubated for 10 minutes with indicated concentrations of mmldl, followed by a 10 minutes of incubation with DCF-DA. The generation of ROS (H 2 O 2 ) was then monitored by FACS analysis as an increase in DCF fluorescence. Fluorescence was analyzed in cells with excitation at 488 nm and emission at 530 nm. B, J774 cells were incubated with equal amounts (50 g/ml) of native LDL (nldl), mmldl, or OxLDL for 10 minutes, and ROS generation was measured as in A. ROS generation and nuclear factor (NF)- B activation and that this process is mediated by the interaction of TLR4 with Nox4. 4 Moreover, we have demonstrated that Nox4- dependent ROS generation plays an important role in LPSinduced proinflammatory cytokine production by endothelial cells (ECs) and activation, leading to adhesion molecule expression. 7 These results suggest that EC-generated ROS may play an important role in the process of atherogenesis because infiltration of blood vessel intima by leukocytes, aided by the expression of chemokines and adhesion molecules, is an initial and rate-limiting step in the development of atherosclerotic lesions. 8 Indeed, increased levels of superoxide have been found in atherosclerotic lesions in human coronary arteries of explanted hearts, which were accompanied by increased expression of gp91phox and p22phox in phagocytic cells and of Nox4 in nonphagocytic cells in the lesions. 9 Oxidation of low-density lipoprotein (LDL) is a major pathogenic factor in the development of atherosclerosis. 10 Extensively oxidized LDL (OxLDL) accumulates in atherosclerotic lesions and activates macrophages and other vascular cells. The resulting chronic inflammation in the vascular wall makes atherosclerotic plaques vulnerable to rupture, leading to acute cardiovascular events. Because TLR4 and TLR2 are key regulators of inflammation and MyD88 is an adaptor molecule essential for the TLR4- and TLR2-mediated signaling, several laboratories studied atherogenesis in TLR4-, TLR2-, and MyD88-deficient mice. In general, these knockout mice have no obvious phenotype, but they are unresponsive to specific microbial TLR ligands. When crossed to apolipoprotein (apo)e / or LDLR / mice and fed a high-fat diet, TLR4-, TLR2-, and MyD88-deficient mice developed less atherosclerosis than apoe / or LDLR / controls We have developed and characterized a model of minimally oxidized LDL (mmldl), which interacts with CD14 and activates cytoskeletal rearrangements in macrophages and induces secretion of certain cytokines via TLR4. 14,15 Because mmldl activates TLR4 in macrophages and TLR4 mediates Nox4 activation in ECs, we asked whether mmldl induces TLR4-mediated activation of Nox2, the predominant NADPH oxidase in macrophages. In this report, we demonstrate that mmldl stimulates ROS generation in macrophages via activation of TLR4 and a subsequent signaling cascade involving Syk, phospholipase (PL)C, PKC, and Nox2. Moreover, we report that the production and functional role of specific proinflammatory cytokines in response to mmldl requires the presence of Nox2. Materials and Methods Detailed information on materials, animals, primary macrophages, cell culture and immunoblot analysis, LDL isolation and modification, generation of a retrovirus containing small interfering (si)rna against Nox2, Syk short hairpin (sh)rna transient transfection, measurements of ROS, yeast 2-hybrid, subcellular fractionation, immunoblotting, and immunostaining are described in the expanded Material and Methods section, available in the online data supplement at Results mmldl Stimulates ROS Generation in J774 Macrophages Our earlier results indicate that LPS activation of TLR4 stimulates ROS generation in ECs 4 and that mmldl activates macrophages via TLR4. 14 Thus, we reasoned that mmldl may stimulate TLR4-dependent ROS generation in macrophages. The intracellular levels of ROS in J774 cells, a murine macrophage-like cell line, were determined by measuring the oxidation of 2,7 -dichlorofluorescein diacetate (DCF-DA) to DCF using fluorescence-activated cellsorting (FACS) analysis. The exposure of J774 cells to the indicated concentrations of mmldl resulted in increased ROS generation in a dose-dependent manner (Figure 1A). Equal concentrations of extensively oxidized LDL (OxLDL) induced ROS generation as well, although less than mmldl, whereas native LDL was inactive (Figure 1B). To identify the specific species of ROS generated, we used an adenovirus expressing mutant catalase with the COOH-terminal KANL peroxisomal targeting sequence deleted (pad5-cmv- Cat P, deleted K 524 ANL 527 ). This mutation results in the Cat P localization in the cytosol instead of its targeting to the peroxisomes compartment, and the enzyme retains its catalytic activity. 16,17 Expression of mutant catalase (Cat P) in J774 cells

3 212 Circulation Research January 30, 2009 resulted in inhibited DCFH oxidation, indicating that the generated ROS species are effectively reduced. Therefore, it appears that hydrogen peroxide is a dominant component of the ROS generated (Figure I in the online data supplement). Gp91phox/Nox2 Is Responsible for mmldl-induced ROS Generation Several lines of evidence indicate that ROS generation is mediated by an NADPH oxidase complex in phagocytic cells, in which gp91phox/nox2 is the major catalytic component. 18 First, we explored whether gp91phox/nox2 was involved in the mmldl-dependent ROS generation in J774 cells. We generated a retrovirus encoding a sirna specific to gp91phox/nox2. J774 cells infected with the retrovirusgp91phox/nox2 sirna exhibited a marked reduction in the expression of endogenous gp91phox/nox2 (Figure 2B) and failed to generate ROS in response to mmldl, whereas cells infected with the control virus generated a robust ROS response (Figure 2A). Next, we explored whether peritoneal resident macrophages from gp91phox/ Nox2 knockout (gp91phox/nox2 / ) mice would have a blunted ROS response to mmldl. Indeed, gp91phox/nox2- deficient macrophages, identified as CD11b-positive cells in the peritoneal cell lavage, exhibited significantly reduced ROS levels in response to mmldl compared to wild-type macrophages (Figure 2C). These results indicate that gp91phox/nox2 is responsible for mmldl-induced ROS generation in macrophages. mmldl-induced ROS Generation Is Mediated by TLR4 and Syk Peritoneal macrophages from TLR4 knockout mice, stimulated with mmldl, failed to generate ROS, whereas macrophages from MyD88 knockout mice showed a normal ROS response (Figure 2D), indicating that mmldl-induced ROS generation depends on the presence of TLR4 but not MyD88. We previously showed that LPS-induced ROS generation and NF- B activation in HEK293T cells was mediated by a direct interaction of TLR4 with Nox4. 7 In this study, we found that, unlike Nox4, the COOH-terminal domain of Nox2 displayed only a weak interaction with TLR4 (Figure 2E), which is unlikely to account for the Nox2 activation. These results suggest that rather than a direct interaction, TLR4 activates Nox2 via a complex signaling pathway. Because MyD88 was not involved in the mmldl-induced ROS generation, we searched for a kinase that would associate with TLR4 in response to mmldl and activate a signaling cascade leading to the Nox2-depenent ROS generation. We found that in J774 macrophages stimulated with mmldl, spleen tyrosine kinase (Syk) coimmunoprecipitated with TLR4 (Figure 3A and supplemental Figure IIA). In addition, Syk was phosphorylated in response to mmldl (Figure 3B and supplemental Figure II, B). To confirm the functional connection between Syk and TLR4, TLR4 shrna was transfected into J774 cells and the phosphorylation of Syk was examined in a FACS assay. The TLR4 knockdown ( 60%) abolished mmldl-induced Syk phosphorylation (supplemental Figure III). Next, we examined whether Syk regulates gp91phox/nox2 and ROS generation. Pretreatment of J774 cells with piceatannol, a pharmacological inhibitor of Syk, reduced ROS generation in response to mmldl in a dose-dependent manner (Figure 4A). To provide specific evidence for a role of Syk, J774 cells were transfected with a Syk-specific shrna or a control vector for 48 hours. Syk protein expression was significantly reduced, and the Syk-deficient cells failed to generate ROS in response to mmldl, whereas cells transfected with control shrna exhibited normal ROS levels (Figure 4B). These results indicate that Syk is a key upstream regulatory molecule in mmldl-induced ROS generation. Activation of Nox2 requires its interaction with GTP-bound Rac. Indeed, mmldl induced Rac activation in control J774 cells but not in Syk knockdown cells (supplemental Figure IV). These results support the hypothesis that Syk regulates mmldlinduced Nox2 activation in macrophages. Activation of PLC 1 and PKC by mmldl A protein tyrosine kinase activity is known to phosphorylate PLC 1 at Y783, which, in turn, attracts PKC to the membrane, where it is activated. The sequential activation of PLC 1 and PKC leads to Nox2 activation and ROS generation. 19 Thus, we tested whether mmldl-induced Syk activation stimulates PLC 1 phosphorylation. Treatment of J774 cells with mmldl resulted in a concentration-dependent increase in tyrosine phosphorylation of PLC 1 (Figure 5A and supplemental Figure V, A). Transfection of J774 cells with Syk-specific shrna abolished mmldl-induced tyrosine phosphorylation of PLC 1 compared to that seen with control shrna (Figure 5B and supplemental Figure V, B). A PKC-dependent phosphorylation of p47phox leads to its association with and activation of gp91phox/nox2. We analyzed mmldl-dependent activation of PKC in terms of its membrane translocation using a subcellular fractionation assay and an immunocytochemical analysis. J774 cells were stimulated with mmldl and then the cytosol and plasma membranes were separated (see Materials and Methods). mmldl stimulated translocation of PKC from cytosol to the plasma membrane in a time-dependent manner (Figure 5C). This result was confirmed by examining PKC distribution in cells stimulated with mmldl, which resulted in PKC translocation from the cytosol to the membrane and its concentration in areas of actin polymerization (Figure 5D). Thus, mmldl-induced activation of Syk and PLC stimulates PKC activation in macrophages, leading to Nox2- dependent ROS generation. Gp91phox/Nox2-Dependent Expression of Proinflammatory Cytokines in Response to mmldl Because proinflammatory cytokines play an important role in atherogenesis, we next analyzed the time-dependent cytokine expression in peritoneal macrophages from wild-type or Nox2 knockout mice (Figure 6). Quantitative real-time PCR demonstrated that mmldl induced expression of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-2, MIP-1, interleukin (IL)-1, RANTES, tumor necrosis factor (TNF)-, IL-10, and IL-6, which all peaked 2 hours

4 Bae et al mmldl-dependent ROS Generation 213 Figure 2. Effect of Nox2 depletion on mmldl-induced ROS generation. A, J774 cells were infected with either a retrovirus encoding Nox2-specific sirna or a retrovirus encoding scrambled sirna. After 48 hours, cells were stimulated with mmldl for 10 minutes, and the generation of H 2 O 2 was monitored by FACS as in Figure 1. Data are means SE of mean fluorescence intensity from 3 independent experiments. *P 0.005, comparison to the value of control group. B, Nox2 mrna expression was assessed in total RNA by RT-PCR as described in Materials and Methods. GAPDH served as the loading control. C and D, Resident peritoneal macrophages were obtained from C57BL/6 wild-type, Nox2 /, MyD88 /, and TLR4 / mice stained with a phycoerythrin-labeled CD11b antibody to select for macrophages and then incubated with mmldl (50 g/ml) for 10 minutes, followed by a 10-minute incubation with DCF-DA. DCF fluorescence was then measured by FACS in the population of CD11b-positive cells. Data are means SE of mean fluorescence intensity from 3 independent experiments. **P , comparison to the value of the control group; ***P 0.01, wild-type vs TLR4 / macrophages. E, Interactions of the COOHterminal region of TLR4 with the COOHterminal domains of Nox1, Nox2, Nox3, or Nox4 were estimated using a yeast 2-hybrid assay (see Materials and Methods). The intensity of blue color indicates the expression levels of LacZ and corresponds to the affinity of TLR4 binding with Nox isozymes. poststimulation. Remarkably, mmldl-induced expression of RANTES, IL-1, and IL-6 was significantly reduced in Nox2 / macrophages compared to wild type, whereas the expression of MCP-1, MIP-1, MIP-2, and TNF- was unchanged or nonsignificantly reduced. In contrast, the expression of antiinflammatory IL-10 in Nox2 / macrophages tended to increase, but the difference was not statistically significant. To corroborate the results with primary Nox2 / macrophages, we tested the cytokine expression in Nox2 knockdown J774 cells stimulated with mmldl and found that the expression of IL-6, IL-1, and RANTES was Nox2-dependent, although the expression levels in J774 cells were significantly lower than in primary macrophages (supplemental Figure VI). These experiments with Nox2-deficient macrophages suggest that mmldl stimulates both Nox2-dependent and -independent expression of cytokines and that the cellular redox state selectively regulates specific macrophage functions. Stimulation of Mouse Aortic Smooth Muscle Cell Migration by RANTES Peritoneal macrophages from wild-type or Nox2 / mice were plated in the bottom chamber of Transwells and stimulated with mmldl. Upper chambers with mouse aortic smooth muscle cells (MASMCs) were subsequently assembled with the bottom chambers to induce migration of MASMCs. Wild-type macrophages stimulated by mmldl led to a significantly higher level of MASMC migration

5 214 Circulation Research January 30, 2009 Figure 3. Syk association with TLR4 and phosphorylation of Syk. J774 cells were incubated with mmldl for the indicated times, and the incubations were terminated by the addition of a lysis buffer. Cell lysates were subjected to immunoprecipitation with an antibody against Syk, and then the immune complexes were directly subjected to immunoblot analysis with an antibody against TLR4 (A) or phosphotyrosine (4G10) (B). Total cell lysates were used for the control of Syk and TLR4 load. IP indicates immunoprecipitation; IB, immunoblot. compared to Nox2 / (Figure 7A), suggesting that mmldl stimulated macrophage secretion of a potent chemokine in an Nox2-dependent manner. Because our gene expression data above demonstrated that RANTES expression was Nox2- dependent (Figure 6 and supplemental Figure VI), we examined whether RANTES was responsible for the MASMC migration. First, we demonstrated that the secretion of RANTES protein was induced by mmldl in wild-type, but not Nox2 /, macrophages (Figure 7B). Addition of recombinant RANTES to the culture system induced MASMC migration (Figure 7C). Furthermore, when a neutralizing antibody against RANTES was added to the lower chamber with mmldlstimulated macrophages, the migration of MASMCs was suppressed to the control level (Figure 7D). These data collectively suggest that the mmldl-induced, Nox2- mediated RANTES expression in macrophages is a likely mechanism leading to SMC migration. Discussion Although the minimal degree of oxidation that occurs in our mmldl preparation is insufficient to trigger its binding to scavenger receptors, 14 we and others have demonstrated that mmldl has numerous biological effects. 14,15,20,21 Furthermore, we have shown that a number of these biological effects depends, at least in part, on CD14 binding and TLR4-mediated signaling. 14,15 In addition, we have recently shown that oxidized cholesteryl esters found in mmldl and in murine atherosclerotic lesions are responsible, in part, for its impact on macrophage function. 22 In this study, we report an important new biological effect of mmldl, namely the ability to stimulate ROS generation in macrophages via activation of Nox2, and that this was TLR4-dependent but MyD88-independent. Furthermore, we identify Syk as a necessary component linking the TLR4 activation by mmldl with the intracellular activation of PLC, PKC, Rac, and Nox2 (Figures 2 through 6 and supplemental Figures II through V). Previously, we reported that TLR4 directly interacts with and activates the Nox4 isozyme in human aortic endothelial cells (HAECs) and HEK293 cells, leading to the generation of ROS. 4,7 Nox4 is mainly expressed in fibroblasts and epithelial and endothelial cells, whereas expression of Nox2 is restricted mainly to hematopoietic cells. In this report, we confirm that peritoneal macrophages and J774 cells predominantly express Nox2. Whereas TLR4 directly binds to Nox4 in HAECs, a yeast 2-hybrid study detected only a weak binding of Nox2 to TLR4 in macrophages (Figure 2E), which is, based on our experience with the yeast 2-hybrid system, unlikely to have any biological importance. Thus, TLR4-mediated activation of Nox2 in macrophages likely occurs by a pathway distinct from that observed for TLR4 and Nox4 in ECs. Because mmldl-induced Nox2 activation in macrophages was MyD88-independent (Figure 2D) and because the TLR4 and Nox2 direct interaction was insignificant (Figure 2E), we sought a signaling pathway that would link TLR4 with Nox2. A canonical mechanism of Nox2 activation requires activated PKC. In turn, PKC activation is regulated Figure 4. Role of Syk in mmldl-induced ROS generation. A, J774 cells were pretreated for 30 minutes with indicated concentrations of the Syk inhibitor piceatannol and then incubated with 50 g/ml mmldl for an additional 10 minutes. ROS was measured as in Figure 1. Data are means SE of mean fluorescence intensities from 3 independent experiments. *P 0.02, **P vs control. B, J774 cells were transfected with either Sykspecific shrna or control shrna. After 48 hours, the cells were stimulated with mmldl (50 g/ml) for 10 minutes, and ROS were measured. Data are means SE of mean fluorescence intensities from 5 independent experiments. *Comparison to the value of control group, P C, The Syk knockdown was confirmed in cell lysates subjected to Western blot analysis with antibodies against Syk and -actin (loading control).

6 Bae et al mmldl-dependent ROS Generation 215 Figure 5. mmldl-induced activation of PLC 1 and membrane translocation of PKC. A, J774 cells were stimulated with mmldl (25 or 50 g/ml) for 10 minutes. Cell lysates from each sample were then prepared and subjected to Western blot analysis with antibodies to PLC 1 or phospho-specific PLC 1 (Y783). B, J774 cells were transfected with either Syk-specific shrna or control shrna. After 48 hours, the cells were stimulated with mmldl (50 g/ml) for 10 minutes. Cell lysates from each sample were then subjected to Western blot analysis with antibodies against Syk, PLC 1, or phospho-specific PLC 1 (Y783). C, J774 cells were stimulated with mmldl (50 g/ml) for the indicated times and subjected to cellular fractionation (see Materials and Methods). Density fractions were immunoblotted with antibodies against PKC, -actin (cytosolic marker), and 2 -integrin (membrane marker). D, J774 cells were incubated with medium alone or medium plus mmldl (50 g/ml) for 30 minutes. PKC localization was visualized with a primary anti-pkc antibody and a secondary fluorescein isothiocyanate labeled antibody. Cells were also stained with tetramethylrhodamine B isothiocyanate phalloidin and Hoechst to visualize F-actin and nuclei, respectively. by a PLC -catalyzed hydrolysis of PIP2 (phosphatidylinositol 4,5-bisphosphate) into IP3 (inositol 1,4,5-triphosphate) and DAG (diacylglycerol), the latter being a specific PKC activator. 19,21,23 Syk is among the tyrosine kinases that phosphorylates Tyr783 of PLC. Syk has been mainly implicated in lymphocyte development, integrin signaling pathways and in regulation of phagocytosis Recent studies suggested that Syk may be constitutively associated with TLR4 in monocytic cells and that the TLR4-Syk interaction can be stimulated by LPS in neutrophils. 25,26 Remarkably, our data demonstrated that the mmldl induced activation of TLR4 in macrophages led to a similar recruitment of Syk to TLR4 and Syk phosphorylation (Figure 3). In turn, Syk was required for mmldl-induced PLC activation and ROS generation (Figures 4 and 5). In support of this pathway, we demonstrated that mmldl induced the membrane translocation of PKC (Figure 5C and 5D), a necessary step in Nox2 activation. Thus, our results suggest that the ROS generation by mmldl involves the sequential activation of TLR4, Syk, PLC, PKC, and Nox2 (Figure 8). Several groups have reported that ROS play an important role in NF- B dependent inflammatory processes. 28,29 For example, we demonstrated that TLR4/Nox4-mediated ROS generation in HAECs was necessary for LPS-induced NF- B activation and expression of IL-8, MCP-1, and intercellular adhesion molecule Likewise, in macrophages, mmldl induced the expression of a number of cytokines, such as MCP-1, MIP-2, TNF-, and IL-6, but only the MIP-2 expression was clearly MyD88- dependent. 15 In the present study, we used primary peritoneal macrophages from wild-type and Nox2 / mice to determine the ability of mmldl to induce cytokine expression. In these studies, mmldl induced 8 different cytokines (MCP-1, MIP-2, TNF-, IL-6, IL-1, RANTES, and IL-10) (Figure 7). From this set, 3 of them (IL-1, RANTES, and IL-6) were significantly downregulated in Nox2 / macrophages, implying that their regulation was redox-dependent. The results showing that the MIP-2 expression was not significantly affected in Nox2 / cells (Figure 6) and that the ROS generation was MyD88- independent (Figure 2D) agree with our previous data showing that MIP-2 expression was MyD88-dependent. 15 These data suggest that the mmldl engagement of TLR4 stimulates at least 2 independent pathways, one via MyD88 and another mediated by Syk, the latter leading to Nox2 activation and the redox-sensitive expression of IL-1, RANTES, and IL-6 (Figure 8). Our data demonstrate that mmldl activates TLR4, which, in turn, initiates an intracellular signaling cascade, leading to Nox2-mediated ROS generation, culminating in the secretion of IL-1, IL-6, and RANTES, which are proinflammatory and likely proatherogenic. IL-6 has been implicated in athero-

7 216 Circulation Research January 30, 2009 Figure 6. mmldl-induced expression of cytokines in peritoneal resident macrophages. Peritoneal resident macrophages were isolated from wild-type and Nox2 / mice and stimulated with mmldl (50 g/ml) or media only for the indicated times. Total RNA was isolated, reverse-transcribed, and quantified by real-time PCR with respective primers specific for each cytokine and GAPDH. The data are presented as fold increases in mrna levels in mmldl-stimulated cells over the levels in nonstimulated cells. *P 0.05 wild-type vs Nox2 / macrophages. genesis. IL-6 expression is significantly reduced in carriers bearing a TLR4 ASP299GLY polymorphism, which is associated with a significantly lower frequency of myocardial infarction compared to the control population. 30 Both MyD88-dependent and MyD88-independent pathways may stimulate IL-6 expression. 31 In this study, we provide evidence that the Nox2-mediated ROS generation is MyD88- independent (Figure 2) and that this Nox2 activity regulates the IL-6 expression (Figure 6). RANTES and IL-1 have also been shown to have a functional role in atherogenesis. Kirii et al have demonstrated that atherosclerotic lesion size of IL-1 / apoe / mice is significantly smaller than that in IL-1 / apoe / mice. 32 Through its binding to CCR5, RANTES stimulates both the recruitment and transendothelial migration of leukocytes to inflammation sites, thereby facilitating the development of inflammatory vascular lesions. 33 We demonstrated that mmldl-induced Nox2 activity regulated the production of RANTES in macrophages (Figures 6 and 7B). Remarkably, RANTES antagonists prevent progression of even established atherosclerotic lesions in LDLR / mice. 34,35 This underscores the relevance and importance of our findings that mmldl-induced Nox2 ac-

8 Bae et al mmldl-dependent ROS Generation 217 Figure 7. RANTES-dependent MASMC migration. A, MASMCs ( cells in 100 L of media) were added to the upper chamber and peritoneal macrophages from wild-type or Nox2 / mice in the lower chamber. First, macrophages were activated with media alone or 50 g/ml mmldl for 2 hours, and then the upper and lower chambers were assembled. After an 18-hour incubation, the membrane in the upper chamber was recovered, fixed, and stained, and the number of migrated cells was counted. Data are from 5 independent experiments. *P 0.05, mmldl/wt vs mmldl/ Nox2 /. B, Analysis of RANTES protein secretion into cell culture media following macrophage activation with mmldl (50 g/ml) for 0, 1, 2, 4, or 6 hours. C, MASMCs ( cells in 100 L of media) and RANTES (0, 100, 250, or 500 pg/ml) were added to the upper chamber and lower chamber, respectively, and incubated for 18 hours, and the numbers of migrated cells were counted. D, Blocking experiments were performed by incubating the mmldl-stimulated macrophages (wild type) with 100 ng/ml the neutralizing antibody against RANTES (R&D Systems) or with the isotypematched control antibody in the lower chamber. The number of migrated cells was determined as above. Data are from 3 independent experiments. *P 0.05 vs mmldl/anti-rantes antibody. tivity stimulated the production of RANTES by macrophages (Figures 6 and 7B), which, in turn, induced MASMC migration (Figure 7A). The specificity of this mmldl/nox2 effect was shown in the experiments with Nox2 / macrophages and with an anti-rantes antibody, which both blocked the MASMC migration (Figure 7A and 7D). Recently, Yamada et al reported that expression of RANTES was regulated by Syk in nasal fibroblast, 36 which would agree with our results placing Syk upstream of the RANTES expression. Taken together, these results suggest that the mmldl-initiated Figure 8. A proposed signaling pathway of mmldl-induced, TLR4/Syk-dependent ROS generation, and proinflammatory cytokine expression. redox signaling may be involved in the processes of vascular inflammation and atherosclerosis. Although one would predict that Nox2 would be proinflammatory, and thus proatherogenic, conclusive experimental data to support such a role in vivo are not currently available. The role of p47phox or gp91phox have been evaluated in various murine models by examining whole body knockouts in the background of apoe deficiency In one study, the knockout of p47phox decreased lesion formation in the whole aorta, but not at the aortic root. 37 In a second study, disruption of p47phox led to no changes in lesion formation at the aortic ring. 38 Yet, in a third study, gp91phox knockout led to lowered plasma cholesterol levels, yet no measured decreases in atherosclerosis, 39 which, in the setting of lowered plasma cholesterol, might even be interpreted as enhanced lesion formation. The reasons for these differences are not clear but could be related to a differential impact of NADPH oxidase on different stages of lesion development or to its role in different cell types. 40 Tissue-specific knockouts will be required to evaluate the latter possibility. In conclusion, we present a signaling mechanism by which mmldl stimulates ROS generation in macrophages. The interaction of mmldl with TLR4 results in the activation of Syk tyrosine kinase, leading to phosphorylation of PLC 1, which, in turn, induces PKC, activation of Rac, and Nox2 activation, resulting in the expression of proinflammatory cytokines IL-1, IL-6, and RANTES (Figure 8). Because mmldl is likely a component of atherosclerotic lesions, these data define mechanisms by which endogenous ligands, such as mmldl, can activate TLR4-dependent signaling mechanisms in macrophages that lead to proatherogenic effect.

9 218 Circulation Research January 30, 2009 Sources of Funding This work was supported by the National Core Research Center program (grant R ) and World Class University program of The Ministry of Education, Science and Technology/ Korea Science and Engineering Foundation through Ewha Womans University; Korea Health 21 R&D Project grant A of the Ministry of Health & Welfare; Seoul R&D Program grant 10527; National Research Laboratory Program grant ROA ; NIH grant HL (to Y.I.M.); American Heart Association Grant N (to Y.I.M.); and a grant from the Leducq Fondation (J.L.W.). Y.S.B. is a recipient of International Exchange Program of LG YeonAm Foundation, and S.K. is a recipient of BK21 scholarship. None. Disclosures References 1. Rhee SG. Cell signaling. H2O2, a necessary evil for cell signaling. Science. 2006;312: Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007;87: Zhang X, Shan P, Jiang G, Cohn L, Lee PJ. Toll-like receptor 4 deficiency causes pulmonary emphysema. J Clin Invest. 2006;116: Park HS, Jung HY, Park EY, Kim J, Lee WJ, Bae YS. Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B. J Immunol. 2004;173: Kawai T, Akira S. TLR signaling. Semin Immunol. 2007;19: Bjorkbacka H. Multiple roles of Toll-like receptor signaling in atherosclerosis. Curr Opin Lipidol. 2006;17: Park HS, Chun JN, Jung HY, Choi C, Bae YS. Role of NADPH oxidase 4 in lipopolysaccharide-induced proinflammatory responses by human aortic endothelial cells. Cardiovasc Res. 2006;72: Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. 2006;354: Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Sorescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor WR, Griendling KK. Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation. 2002;105: Glass CK, Witztum JL. Atherosclerosis. the road ahead. Cell. 2001;104: Bjorkbacka H, Kunjathoor VV, Moore KJ, Koehn S, Ordija CM, Lee MA, Means T, Halmen K, Luster AD, Golenbock DT, Freeman MW. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Nat Med. 2004;10: Michelsen KS, Wong MH, Shah PK, Zhang W, Yano J, Doherty TM, Akira S, Rajavashisth TB, Arditi M. Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Proc Natl Acad Sci USA. 2004;101: Mullick AE, Tobias PS, Curtiss LK. Modulation of atherosclerosis in mice by Toll-like receptor 2. J Clin Invest. 2005;115: Miller YI, Viriyakosol S, Binder CJ, Feramisco JR, Kirkland TN, Witztum JL. Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells. J Biol Chem. 2003;278: Miller YI, Viriyakosol S, Worrall DS, Boullier A, Butler S, Witztum JL. Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages. Arterioscler Thromb Vasc Biol. 2005;25: Purdue PE, Lazarow PB. Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence. J Cell Biol. 1996;134: Bai J, Rodriguez AM, Melendez JA, Cederbaum AI. Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury. J Biol Chem. 1999;274: Babior BM. NADPH oxidase. Curr Opin Immunol. 2004;16: Choi H, Leto TL, Hunyady L, Catt KJ, Bae YS, Rhee SG. Mechanism of angiotensin II-induced superoxide production in cells reconstituted with angiotensin type 1 receptor and the components of NADPH oxidase. J Biol Chem. 2008;283: Patricia MK, Kim JA, Harper CM, Shih PT, Berliner JA, Natarajan R, Nadler JL, Hedrick CC. Lipoxygenase products increase monocyte adhesion to human aortic endothelial cells. Arterioscler Thromb Vasc Biol. 1999;19: Boullier A, Li Y, Quehenberger O, Palinski W, Tabas I, Witztum JL, Miller YI. Minimally oxidized LDL offsets the apoptotic effects of extensively oxidized LDL and free cholesterol in macrophages. Arterioscler Thromb Vasc Biol. 2006;26: Harkewicz R, Hartvigsen K, Almazan F, Dennis EA, Witztum JL, Miller YI. Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized LDL. J Biol Chem. 2008;283: Rhee SG. Regulation of phosphoinositide-specific phospholipase C. Ann Rev Biochem. 2001;70: Mocsai A, Zhou M, Meng F, Tybulewicz VL, Lowell CA. Syk is required for integrin signaling in neutrophils. Immunity. 2002;16: Arndt PG, Suzuki N, Avdi NJ, Malcolm KC, Worthen GS. Lipopolysaccharide-induced c-jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-kinase and Syk-mediated pathways. J Biol Chem. 2004;279: Chaudhary A, Fresquez TM, Naranjo MJ. Tyrosine kinase Syk associates with Toll-like receptor 4 and regulates signaling in human monocytic cells. Immunol Cell Biol. 2007;85: Palacios EH, Weiss A. Distinct roles for Syk and ZAP-70 during early thymocyte development. J Exp Med. 2007;204: Gloire G, Legrand-Poels S, Piette J. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol. 2006;72: Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J. 1991;10: Candore G, Aquino A, Balistreri CR, Bulati M, Di Carlo D, Grimaldi MP, Listi F, Orlando V, Vasto S, Caruso M, Colonna-Romano G, Lio D, Caruso C. Inflammation, longevity, and cardiovascular diseases: role of polymorphisms of TLR4. Ann N Y Acad Sci. 2006;1067: Bagchi A, Herrup EA, Warren HS, Trigilio J, Shin HS, Valentine C, Hellman J. MyD88-dependent and MyD88-independent pathways in synergy, priming, and tolerance between TLR agonists. J Immunol. 2007; 178: Kirii H, Niwa T, Yamada Y, Wada H, Saito K, Iwakura Y, Asano M, Moriwaki H, Seishima M. Lack of interleukin-1beta decreases the severity of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 2003;23: Duma L, Haussinger D, Rogowski M, Lusso P, Grzesiek S. Recognition of RANTES by extracellular parts of the CCR5 receptor. J Mol Biol. 2007;365: Veillard NR, Kwak B, Pelli G, Mulhaupt F, James RW, Proudfoot AE, Mach F. Antagonism of RANTES receptors reduces atherosclerotic plaque formation in mice. Circ Res. 2004;94: Braunersreuther V, Steffens S, Arnaud C, Pelli G, Burger F, Proudfoot A, Mach F. A novel RANTES antagonist prevents progression of established atherosclerotic lesions in mice. Arterioscler Thromb Vasc Biol. 2008;28: Yamada T, Fujieda S, Yanagi S, Yamamura H, Inatome R, Sunaga H, Saito H. Protein-tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on RANTES production. J Immunol. 2001;166: Barry-Lane PA, Patterson C, van der Merwe M, Hu Z, Holland SM, Yeh ET, Runge MS. p47phox is required for atherosclerotic lesion progression in ApoE( / ) mice. J Clin Investi. 2001;108: Hsich E, Segal BH, Pagano PJ, Rey FE, Paigen B, Deleonardis J, Hoyt RF, Holland SM, Finkel T. Vascular effects following homozygous disruption of p47(phox): an essential component of NADPH oxidase. Circulation. 2000;101: Kirk EA, Dinauer MC, Rosen H, Chait A, Heinecke JW, LeBoeuf RC. Impaired superoxide production due to a deficiency in phagocyte NADPH oxidase fails to inhibit atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2000;20: Cathcart MK. Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages: contributions to atherosclerosis. Arterioscler Thromb Vasc Biol. 2004;24:23 28.