Experimental and Molecular Pathology 97 (2014) 590–599
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IL-10 negatively regulates oxLDL-P38 pathway inhibited macrophage emigration☆ Hong Yang a,b,⁎, Hai peng Liu b,c, Dong Weng b, Bao xue Ge a,b,c,⁎ a b c
Department of Microbiology and Immunology, Tongji University School of Medicine, Shanghai 200092, China Clinical Translational Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200092, China Shanghai TB Key Laboratory, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China
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Article history: Received 22 October 2014 Accepted 28 October 2014 Available online 29 October 2014 Keywords: Macrophage Emigration IL-10 β-Arrestin2 CCR7 P38 Atherosclerosis
a b s t r a c t The effect of IL-10 on macrophage migration was investigated, including the analysis of protein expression, cytokine secretion and activation of the MAPKs and NF-kB pathway. The expression of endogenic IL-10 was downregulated in macrophage stimulated with oxLDL for indicated time. Exogenous IL-10 reversed oxLDL-inhibited chemotactic macrophage numbers by 48.18 ± 4.93% (3 h), 64.8 ± 5.61% (6 h) and 63.66 ± 3.05% (12 h), and pretreated with SB203580 (P38 signaling inhibitor) in macrophage, oxLDL could not inhibit macrophage emigration. IL-10 significantly decreased oxLDL-mediated increase of SR-A expression, and pretreatment with βarrestin2 RNAi in macrophage could not change oxLDL-induced SR-A expression. IL-10 also significantly decreased oxLDL-mediated increase of β-arrestin2 expression, and pre-treated with SR-A RNAi in macrophage, oxLDL could not induce the increase of β-arrestin2 expression. However, IL-10 significantly reversed oxLDLmediated inhibition of CCR7 expression about 95.78 ± 0.99% (mRNA) and 80.06 ± 19.46% (protein), and pretreated with P38 inhibitor SB203580 in macrophage, oxLDL could not decrease CCR7 expression. IL-10 inhibited oxLDL-mediated inhibition of MMP9 secretion about 74.02 ± 22.35%, and pretreated with P38 inhibitor SB203580 in macrophage, oxLDL could not decrease MMP9 secretion. Treatment with oxLDL increased P38phosphorylation by 31.88 ± 2.79%, 40.24 ± 5.69% and 30.93 ± 4.66% at 15, 30 and 60 min, respectively, whereas the effect of IL-10 on the expression of phosphorylated P38 was reversed by 49.49 ± 12.12%, 70.93 ± 16.14% and 47.62 ± 6.00% in up-indicated time-points, respectively. From these data, we speculated oxLDL-SR-A-βarrestin2-P38-MMP9/CCR7 could play a critical role in the macrophages migration, which was blocked by IL10 through inhibiting oxLDL uptake. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Coronary artery disease, resulting from atherosclerosis, is the leading cause of death in the Western world (Feig and Fisher, 2013). Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophage in the artery wall (van Gils et al., 2012). Foam cells can
Abbreviations: oxLDL, oxidative low density lipoprotein; SR-A, scavenge receptor; IL-10, recombinant human interleukin-10; MIP-1, macrophage inflammatory protein-1; CCR7, cysteine–cysteine chemokine receptor 7; CCR5, cysteine–cysteine chemokine receptor 5; TNF-α, tumor necrosis factor-α; MMP9, matrix metalloproteinase; IL-17, interleukin-17; IFN-γ, interferon-γ. ☆ Authors' contributions: HY and BXG conceived the study. HY performed experiments, data analysis and wrote the manuscript. HPL provided helpful discussions. DW provided technical assistance with the experiments. All authors read and approved the final manuscript. ⁎ Corresponding authors at: Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, China. E-mail addresses: [email protected] (H. Yang), [email protected] (H. Liu), [email protected] (D. Weng), [email protected] (B. Ge).
http://dx.doi.org/10.1016/j.yexmp.2014.10.008 0014-4800/© 2014 Elsevier Inc. All rights reserved.
be induced by oxidized low density lipoprotein (oxLDL) in human monocyte-derived macrophage, which represents a major source of foam cells in atherogenesis (Ramkhelawon et al., 2013). Reversal of hyperlipidemia with a genetic switch favorably affects the content and inflammatory state of macrophage in atherosclerotic plaques (Feig et al., 2011a; Pagler et al., 2011). During atherosclerosis regression foam cell emigration is a very important clinical goal. Rapid regression ensued in a process involving emigration of plaque macrophage-derived foam cells to regional and systemic lymph nodes (Feig and Fisher, 2013; Yang et al., 2013). The macrophage migration exposed in oxLDL was altered (Choi et al., 2010). Diminished oxLDL uptake was mechanistically linked to resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression (MéndezBarbero et al., 2013). β-Arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways (Watari et al., 2013a). β-Arrestins are
H. Yang et al. / Experimental and Molecular Pathology 97 (2014) 590–599
scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts (Porter et al., 2010). The scavenger receptor might regulate cell migration and chemotaxis by β-arrestin2 via the MAPKs/NF-kB pathways (Watari et al., 2013a; Ehrlich et al., 2013; Lipfert et al., 2013). CCR7, a known factor in dendritic cell migration, was observed a significant increase in CCR7 mRNA in CD68+ cells from both the atorvastatin and rosuvastatin treated mice associated with emigration of CD68 + cells from plaques (Feig et al., 2011b). MMP9 play important roles in the migration of splenic T cells, B cells, macrophages/dendritic cells and smooth muscle cells (SMC) (Lötzer et al., 2010; Goldklang et al., 2012). It was reported that the reduction in atherosclerosis was accompanied by decreased plasma levels of interleukin-1alpha and tumor necrosis factor alpha, and preceded by increased anti-inflammatory cytokine interleukin-10 (Shen et al., 2010a). Previous work in the literature led us to propose a novel signaling pathway, oxLDL-SR-A-β-arrestin2-MAPKs/NF-kB-MMP9/CCR7, which may play a critical role in emigration of macrophages, and to investigate whether the anti-inflammatory cytokine IL-10, could block atherosclerotic progression and provide a protective role. The aim of this study was to characterize the impact of IL-10 on macrophage migration with regards to the expression of scavenger receptor, β-arrestin and CCR, cytokine secretion and activation of MAPKs/NF-kB. 2. Materials and methods 2.1. Mice, peritoneal macrophage isolation and treatments C57/BL6 mice were obtained from the Jackson laboratory, all female mice were maintained in the Shanghai Laboratory Animal Center (SLAC, Shanghai, China) and used at 8 weeks of age. Peritoneal macrophages were isolated as described previously (Liu et al., 2013). Briefly, mice received i.p. 2 mL of 4% thioglycollate medium (Sigma-Aldrich). After 3 days, cells harvested by peritoneal lavage with cold PBS were allowed to adhere for 2 h. Subsequently, RPMI 1640 medium was changed to remove non-adherent cells. The remaining adherent monolayer cells were used as primary peritoneal macrophages. All cells were cultured at 37 °C in a humidified incubator with 5% CO2. All animal studies were approved by the Institutional Animal Care and Use Committee of Tongji Unversity. Then cells were separated into four different stimulated groups with carrier control, only with 40 mg/L oxLDL (Sigma, St. Louis, USA), with 40 mg/L oxLDL and 20 μg/L IL-10 (Sigma), and with 40 mg/L oxLDL and 20 μg/L IL-10 (Sigma) and SB203580 (10 μM) /βarrestint2 RNAi (30nM)/SR-A RNAi (30 nM) for 24 h. The optimal doses and incubation times for the responses to oxLDL or IL-10 were determined in previous experiments (Yang et al., 2011a; Yang and Chen, 2011a; Yang et al., 2011b). The expression of IL-10 protein was assayed in macrophage only treated with 40 mg/L oxLDL and co-treated with oxLDL and 10 μM SP600125/PD98059/PDTC for indicated time. There was a pre-incubation for 30 min with each inhibitor. All The sequence-specific RNA interference designed as follows: 5′-GGA CCA GGG-3′ and 5′-CUG UUC UCC UUG CGA UGU ACU UGU C-3′ for βarrestint2 RNAi and 5′-GCA ACU GAC CAA AGA CUU A-3′ and 5′-UAA GUC UUU GGU CAG UUG C-3′ for SR-A RNAi. SiPORT Amine Transfection Agent 5 μL was used for transient transfection in 2 × 106 macrophage for 48 h before all other stimuli. 2.2. Chemotaxis assay Chemotaxis experiments were performed using a modified Boyden chamber assay (Corning Life Sciences, NY, USA). Cells were washed and resuspended in RPMI-1640 medium supplemented with 10 mmol/L HEPES, 2 mmol/L L-glutamine, and 1% BSA (Sigma-Aldrich). The chemoattractant MIP-1 (PeproTech EC) (Woszczek et al., 2008) was resuspended in the same medium and added to the lower wells for four groups. Cells (2 × 105) were added to the upper wells and separated by a polycarbonate filter with 5-μm pores (Corning). Usually, the in vitro
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transwell migration assay stops at 3–4 h later. The number of cells that migrated to the lower surface of the filter was counted after 3 h, 6 h and 12 h of incubation.
2.3. RNA isolation and quantitative real-time RT-PCR (qRT-PCR) After oxLDL treatment for 0 h, 2 h, 6 h, 9 h, 12 h and 24 h, or for 6 h of the four cell groups described above, total RNA was extracted from the cells by using the Trizol (Invitrogen, Carlsbad, USA) reagent. cDNA was generated from the RNA using Reverse Transcriptase XL (AMV), ribonuclease inhibitor, dNTP mixture and oligo d (T) 18 purchased from TaKaRa (Dalian, China). The cDNA was subsequently analyzed in the qPCR run on the Rotor Gene 3000 instrument (Corbett Research Co, Australia) using SYBR Premix Ex TaqTM (Dalian, China) and the sequence-specific PCR primers designed as follows: 5′-AAT GGA TTT GGA CGC ATT GGT-3′ and 5′-TTT GCA CTG GTA CGT GTT GAT-3′ for GAPDH (NM_008085); 5′-TTG TCG CGT TTG CTC CCA TT-3′ and 5′GAA GGG CTT GGC AGT TCT G-3′ for IL-10 (NM_008348); 5′-TGG AGG AGA GAA TCG AAA GCA-3′ and 5′-CTG GAC TGA CGA AAT CAA GGA A3′ for SR-A (NM_001113326); 5′-ATG GGC TGT GAT CGG AAC TG-3′ and 5′-TTT GCC ACG TCA TCT GGG TTT-3′ for CD36 (NM_001159556); 5′-TCT GAC TTT CGG AAA GAC CTG T-3′ and 5′-TCT TGA TGA GTC GCT CCT GTA G-3′ for β-Arrestin1 (NM_177231); 5′-AGT CGA GCC CTA ACT GCA AG-3′ and 5′-ACG AAC ACT TTC CGG TCC TTC-3′ for βArrestin2 (NM_145429); 5′-ATG GAT TTT CAA GGG TCA GTT CC-3′ and 5′-CTG AGC CGC AAT TTG TTT CAC-3′ for CCR5 (NM_009917); and 5′-CAG GTG TGC TTC TGC CAA GAT-3′ and 5′-GGT AGG TAT CCG TCA TGG TCT-3′ for CCR7 (NM_007719). The cycle number represented the relative quantity of the specific template when the fluorescence of the amplified gene product first reached a preset threshold (Ct). Transcripts of the housekeeping gene GAPDH in the same runs were used for normalization. The amplification of the control (GAPDH) and the test amplicons (SR-A, CD36, β-arrestin1, β-arrestin2, CCR5 and CCR7) showed very similar efficiencies over the concentration range tested. Treatment effects on gene expression are reported as a fold change of specific target mRNA in the cells incubated with different stimuli versus control cells incubated in parallel with carrier. The gene expression levels were calculated by the delta delta CT method: fold change = 2−Δ(ΔCt), where ΔCt = Ct (specific transcript) − Ct (housekeeping transcript) and Δ(ΔCt) = ΔCt (treatment) − ΔCt (control).
2.4. Protein extraction and Western blotting After the 6 h incubation with different stimuli, proteins from the four cell treatment groups were extracted using the KeyGen MembraneCytosol Protein Extraction Kit (Nanjing KeyGen Biotechnology, Nanjing, China) according to the manufacturer's protocol. The protein concentrations were determined using the Bradford method. Aliquots of partially purified membrane-bound or cytosolic protein (20 μg/lane) were subjected to 10% SDS-polyacrylamide gel electrophoresis in the presence of a reducing reagent, transferred onto nitrocellulose filters and incubated with goat anti-SR-A and anti-CD36 polyclonal antibody (I-20 and L17, 1:150 dilution, Santa Cruz Biotechnology, Santa Cruz, USA), with rabbit anti-β-arrestin1 and anti-β-arrestin2 monoclonal antibody (D8O3J and C16D9; 1:1000) (Cell Signaling Technology Inc., USA), with rabbit anti-CCR5 and rabbit anti-CCR7 monoclonal antibody (E164 and Y59; 1:5,000) (Abcam). Primary antibody binding was visualized by using IRDye® 800CW labeled donkey anti-goat and goat anti-rabbit IgG (1:15,000) with the Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, USA). The internal control, GAPDH, was detected by incubating with rabbit anti-GAPDH antibody (Immunogen Life Science & Technology Co. Ltd. Shanghai, China), followed by incubation with the IRDye® 800CW labeled goat anti-rabbit IgG antibody.
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2.5. Macrophage cytokine secretion
anti-p-ERK, rabbit anti-p-P65 (1:1000, Cell Signaling Technology Inc.). Primary antibody binding was visualized using IRDye® 800CW labeled goat anti-rabbit IgG (1:15,000) on an Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, USA).
The supernatants of macrophage cultures were harvested and stored at −80 °C. The levels of IL10, TNF-α, MMP9, IL-17 and IFN-γ were determined using cytokine-specific ELISA kits (R&D, Minneapolis, USA), according to the manufacturer's instructions.
2.7. Statistical analysis 2.6. Analysis of MAPKs and NF-kB activation All experiments were repeated three times. Data were expressed as the mean ± SEM, and were compared using one-way ANOVA by Fisher's least significant difference (LSD) analysis or paired Student's t tests, as appropriate. Differences were considered statistically significant when p b 0.05.
The method was similar to that used for Western blotting analysis, except that the protein was extracted following incubation for 0, 5, 15, 30, 60 and 90 min. The protein was transferred onto nitrocellulose filters and incubated with rabbit anti-p-P38, rabbit anti-p-JNK, rabbit
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Fig. 1. IL-10 expression and the chemotaxis to MIP-1 were measured in macrophage with different stimuli for indicated hours. (A). IL-10 mRNA expression was measured by qRT-PCR in macrophage under oxLDL stimuli for indicated hours, and GADPH levels were used as loading controls.(B–E) IL-10 protein expression was measured by ELISA under oxLDL stimuli with DMSO (gray line) or different signaling inhibitor (black line): such as P38 signaling inhibitor SB203580 (B), JNK signaling inhibitor SP600125 (C), ERK signaling inhibitor PD98059 (D), and P65 signaling inhibitor PDTC (E) for indicated hours. (F) The chemotaxis to MIP-1 was measured in macrophage treated with carrier control (white bar), with only oxLDL (gray bar), with oxLDL + IL-10 (black bar), and with oxLDL + SB203580 (dot bar) for indicated hours. Data are shown as the mean ± SEM from three separate experiments. Asterisk (*) and hash (#) symbols indicate indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group, # with oxLDL group vs. with oxLDL + SB203580 group).
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3. Results
that the variation trend of IL-10 protein expression were the same in macrophage only treated with oxLDL compared with co-treated with oxLDL and JNK inhibitor SP600125 (Fig. 1 C) or ERK inhibitor PD98059 (Fig. 1D) or P65 inhibitor PDTC (Fig. 1E). Fig. 1 F showed exogenous IL-10 reversed oxLDL-inhibited macrophage emigration by 48.18 ± 4.93% (3 h), 64.8 ± 5.61% (6 h) and 63.66 ± 3.05% (12 h), (black bar). And pretreated with SB203580 (P38 signaling inhibitor) in macrophage, oxLDL could not inhibit macrophage emigration (dot bar). Therefore, IL-10, as an anti-inflammatory cytokine, abrogated oxLDL-mediated inhibition of chemotaxis, and this effect was timedependent in macrophage. These data suggested that IL-10 might play a regulatory role through oxLDL-P38 signaling pathway in macrophage migration.
3.1. The expression of endogenic IL-10 was down-regulated in macrophage treated with oxLDL, and exogenous IL-10 reversed oxLDL-P38-pathwayinhibited macrophage emigration in time-dependent manner Fig. 1 A showed that the expression of endogenic IL-10 mRNA was down-regulated in macrophage stimulated with oxLDL for indicated time. At 6 h there is a decrease about 40%, at 12 h it is 66%, which is no change at 24 h. Fig. 1 B showed the expression of endogenic IL-10 protein had the same variation trend compared with that of IL-10 mRNA, but IL-10 protein expression had no change in macrophage treated with oxLDL and SB203580 (P38 inhibitor). Fig. 1 C–E showed
B Relative expression of CD36 mRNA
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Fig. 2. SR-A and CD36 expressions were measured in macrophage with different stimuli. (A and B) The mRNA levels of SR-A and CD36 were detected by qRT-PCR in macrophage treated with carrier control, only with oxLDL, with oxLDL + IL-10, and with oxLDL + β-arrestint2 RNAi, and GADPH levels were used as loading controls. (C and D) The protein levels of SR-A and CD36 were detected by Western blotting in macrophage according to up-indicated groups, and GADPH levels were used as loading controls. (E and F) Relative expression levels of SR-A and CD36 are presented as the mean ± SEM optical densities from three separate experiments. Asterisk (*) and indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group).
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3.2. IL-10 inhibited the oxLDL-induced increase of SR-A expression in macrophage
directly inhibited the oxLDL-induced increase of SR-A expression in macrophage, which was not through β-arrestin2.
We next determined the effect of IL-10 on the expression of the scavenge receptor A (SR-A) and CD36, which are the two main receptors for oxLDL. As shown in Fig. 2A, C and E, IL-10 inhibited oxLDLinduced increase of SR-A expressions about 43.93 ± 4.01% (mRNA) and 33.91 ± 7.57% (protein) in macrophage. And pretreated with βarrestin2 RNAi in macrophage, oxLDL had the same effect on SR-A expression compared with that only treatment with oxLDL. However, as shown in Fig. 2B, D and F, IL-10 did not alter oxLDL-induced increase of CD36 levels. And pre-treatment with β-arrestin2 RNAi, oxLDL also could not change of the CD36 expression in macrophage compared with that only treated with oxLDL. These results showed that IL-10
3.3. IL-10 blocked the oxLDL-mediated increase of β-arrestin2 expression in macrophage Expression of the important MAPKs regulatory molecule, βarrestin1 and β-arrestin2, was also analyzed. As shown in Fig. 3A, C and E, the mRNA and protein levels of β-arrestin1 were increased in oxLDL treated group, which was not blocked by IL-10 or SR-A inhibitor. However, as shown in Fig. 3B, D and F, IL-10 blocked oxLDL-induced increase of β-arrestin2 expressions about 43.49 ± 8.63% (mRNA) and 45.44 ± 9.45% (protein) in macrophage. And in macrophage pretreated with SR-A RNAi, oxLDL could not increase β-arrestin2 expression. These
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Fig. 3. β-Arrestin1 and β-arrestin2 expressions were measured in macrophage with different stimuli. (A and B) The mRNA levels of β-arrestin1 and β-arrestin2 were detected by qRT-PCR in macrophage treated with carrier control, only with oxLDL, with oxLDL + IL-10, and with oxLDL + SR-A RNAi, and GAPDH levels were used as loading controls. (C and D) The protein levels of β-arrestin1 and β-arrestin2 were detected by Western blotting in macrophage according to up-indicated groups, and GAPDH levels were used as loading controls. (E and F) Relative expression levels ofβ-arrestin1 and β-arrestin2 are presented as the mean ± SEM optical densities from three separate experiments. Asterisk (*) and hash (#) symbols indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group, # with oxLDL group vs. with oxLDL + SR-A RNAi group).
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results suggested that IL-10 blocked the effects of oxLDL on β-arrestin2 expression in macrophage through inhibiting oxLDL uptake, which was mediated by SR-A.
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with P38 inhibitor SB203580, oxLDL could not decrease CCR7 expression. These results demonstrated that IL-10 counteracted the oxLDLP38-signaling-pathway-mediated inhibition of CCR7 expression in macrophage.
3.4. IL-10 reversed the oxLDL-mediated inhibition of CCR7 expression in macrophage 3.5. IL-10 significantly abrogated the oxLDL-mediated decreases in the levels of MMP9 in macrophage
To determine whether the chemokine receptors CCR5 and CCR7 are involved in emigration in this study, the expression levels of CCR5 and CCR7 were examined under a range of conditions by Western blotting. As shown in Fig. 4A, C and E, the mRNA and protein levels of CCR5 were increased in oxLDL treated group, which was not blocked by IL10 and SB203580. However, as shown in Fig. 4B, D and F, IL-10 inhibited oxLDL-mediated inhibition of CCR7 expressions about 95.78 ± 0.99% (mRNA) and 80.06 ± 19.46% (protein). And in macrophage pretreated
Macrophages secrete a wide spectrum of cytokines and chemokines (Sprague and Khalil, 2009). Therefore, we examined the effect of IL-10 on the cytokine profile of macrophage treated with oxLDL. The levels of MMP9, TNF-α, IFN-γ and IL-17 were examined by ELISA. Most of these factors are involved in the stimulation of mononuclear cells in atherogenesis (Sprague and Khalil, 2009).
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Fig. 4. CCR5 and CCR7 expressions were measured in macrophage with different stimuli. (A and B) The mRNA levels of CCR5 and CCR7 were detected by qRT-PCR in macrophage treated with carrier control, only with oxLDL, with oxLDL + IL-10, and with oxLDL + SB203580, and GAPDH levels were used as loading controls. (C and D) The protein levels of βarrestin1 and β-arrestin2 were detected by Western blotting in macrophage according to up-indicated groups, and GAPDH levels were used as loading controls. (E and F) Relative expression levels of CCT5 and CCR7 are presented as the mean ± SEM optical densities from three separate experiments. Asterisk (*) and hash (#) symbols indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group, # with oxLDL group vs. with oxLDL + SB203580 group).
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Fig. 5. Cytokines secretion were measured in macrophage with different stimuli. The concentrations of TNF-α (A), MMP9 (B), IL-17 (C) and INF-γ (D) were measured by specific sandwich ELISA in macrophage treated with carrier control, only with oxLDL, with oxLDL + IL-10, and with oxLDL + SB203580. Data represent the mean ± SEM of three separate experiments from 1 × 106 cells. Asterisk (*) and hash (#) symbols indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group, # with oxLDL group vs. with oxLDL + SB203580 group).
As shown in Fig. 5A, and C, the levels of TNF-α and IL-17 were increased in oxLDL treated groups, which was not blocked by IL-10 and SB203580. However, as shown in Fig. 5B, IL-10 inhibited oxLDLmediated inhibition of MMP9 secretion about 74.02 ± 22.35%. And in macrophage pretreated with P38 inhibitor SB203580, oxLDL could not decrease MMP9 secretion. However, as shown in Fig. 5D, the levels of IFN-γ were not significantly altered in four differently treated groups.
3.6. IL-10 suppressed the oxLDL-mediated decrease in P38 phosphorylation in macrophage As shown in Fig. 6A, the levels of phosphorylated P38, JNK, ERK and P65 in macrophage were detected by Western blotting at 0, 5, 15, 30, 60 and 90 min. Fig. 6B showed IL-10 suppressed the oxLDL-mediated decrease of P38 phosphorylation in macrophage for indicated time (black bar). At 15 min there is an increase about 39%, at 30 min it is 40%, at 60 min it is 41%. Fig. 6C–E showed that IL-10 could not change the oxLDL-induced phosphorylation of JNK (Fig. 6C, black bar) or ERK (Fig. 6D, black bar) or P65 (Fig. 6E, black bar).
4. Discussion Chronic inflammatory diseases such as atherosclerosis were characterized by an accumulation of macrophages. It had been previously shown that mouse atherosclerosis regression involves monocytederived (CD68 +) cell emigration from plaques (Feig et al., 2010). Cholesterol-lowering therapy leaded to plaque stabilization or regression in human atherosclerosis, characterized by reduced macrophage content (Potteaux et al., 2011). Macrophage cholesterol efflux might have a role in facilitating emigration of macrophages from lesions during regression (Murphy et al., 2012). HDL promoted rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells (Yang and Chen, 2011b; Feig et al., 2011c). Here, we demonstrate that the expressions of endogenic IL-10 (anti-inflammatory cytokine) and MIP-1 induced chemotaxis were significantly inhibited in macrophage treated with oxLDL through P38 signaling pathway and in a time-dependent manner. There was IL-10 basic secretion at time 0 h. However, exogenous IL-10 reversed the effects on macrophage migration, which might attenuate atherogenesis. These results were coincident with the report that the reduction in atherosclerosis
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Fig. 6. Phosphorylation of MAPKs and NF-kB was measured in macrophage with different stimuli for indicated times. The phosphorylation levels of P38 (A), JNK (B), ERK (C) and P65 were detected by Western blotting in macrophage only treated with oxLDL and treated with oxLDL + IL-10 for 0, 5, 15, 30, 60 and 90 min, and GAPDH levels were used as loading controls. Asterisk (*) symbols indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group).
was accompanied by decreased plasma levels of interleukin-1alpha and tumor necrosis factor alpha, and preceded by increased antiinflammatory cytokine interleukin-10 (Shen et al., 2010b). Scavenger receptors promoted atherosclerosis; therefore, we hypothesized that the multifunctional adaptor protein, β-arrestin2, might regulate this pathological process. The results of our study showed that treatment with oxLDL in macrophage significantly increased SR-A, CD36, β-arrestin1 and β-arrestin2 expression levels, but decreased CCR5 and CCR7 expression. However, IL-10 reversed the effects of oxLDL on the expression of SR-A, and pretreatment with
β-arrestin2 RNAi in macrophage oxLDL also increase SR-A expression. These results showed that in macrophage oxLDL was not through βarrestin2 to increase SR-A expression. IL-10 inhibited oxLDL-induced β-arrestin2 expressions in macrophage, and pretreated with SR-A RNAi in macrophage, oxLDL could not increase β-arrestin2 expression. These results suggested that oxLDL was through SR-A to increase βarrestin2 expression in macrophage. IL-10 reversed oxLDL-mediated decrease of CCR7 expression, and in macrophage pretreated with P38 inhibitor SB203580, oxLDL could not decrease CCR7 expression. These results demonstrated that the oxLDL-mediated inhibition of CCR7
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expression in macrophage was through P38 signaling pathway. Above three results suggested that the regulation axis was oxLDL-SR-A-βarrestin2-P38-CCR7. There was similar report that the alternate CXCR7 (scavenger chemokine receptor) ligand, I-TAC/CXCL11, activated ERK and AKT through β-arrestin in astrocytes (Odemis et al., 2012). Feig et al. also showed that atherosclerosis regression in mice involves macrophage emigration from plaques and is dependent on the chemokine receptor CCR7 (a mediator of leukocyte emigration and a factor functionally required for regression) (Feig and Fisher, 2013; Yang et al., 2013; Feig et al., 2010; Potteaux et al., 2011; Feig et al., 2011c; Trogan et al., 2006). Therefore, IL-10 may exhibit some of its clinical benefits by accelerating the emigration of macrophage-derived foam cells, thereby promoting the regression of atherosclerosis through directly inhibiting oxLDL uptake. In terms of the analysis of cytokine secretion and the associated signal transduction pathway, we focused our studies on MMP9, TNF-α, IL17, IFN-γ, MAPKs and NF-kB, because of their important roles in aspects of macrophage migration (Yuan et al., 2010; Choi et al., 2012; Ben et al., 2013; Watari et al., 2013b). Our results showed that treatment with oxLDL in macrophage significantly increased TNF-α, IL-17 and P38 phosphorylation levels, but decreased MMP9. IL-10 and SB203580 suppressed the effects of oxLDL on the secretion of MMP9 and P38 phosphorylation. In aortas of atherosclerotic rabbits, down-regulation of 6 pro-inflammatory genes, TNF-α, MCP-1, IL-1β, IL-18, MMP-9, MMP-12 and up-regulation of the anti-inflammatory IL-10 gene were observed (Bulgarelli et al., 2013). Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group (de Oliveira et al., 2009). The P38 inhibitor, SB203580, suppressed the effects of oxLDL on expression levels of CCR7, MMP9, IL-10 and macrophage chemotaxis.
Consistently, IL-10 also reversed all of the above effects of oxLDL on macrophages. In summary, this was the first report to establish a novel signaling pathway, oxLDL-SR-A-β-arrestin2-P38-MMP9/CCR7, which played a critical role in macrophage emigration (Fig. 7). The data suggested that activation of P38 was a key component of the oxLDL-SR-A-β-arrestin2-P38-MMP9/CCR7 signal transduction cascade, which was blocked by IL-10 through inhibiting oxLDL uptake. However, it had been reported that β-arrestin2, MMP9, TNF-α and PI3K phosphorylation aggravated atherosclerosis through mechanisms involving dendritic cell (DC) proliferation and migration, which are critical in the later phase and progression of atherosclerosis and restenosis (Yang et al., 2013). From these findings we suggested that SR-A, βarrestin2, CCR7, MMP9 and p38 might play an important role in the initiation of atherosclerotic progression. The notion that P38 activity facilitated macrophage-derived foam cell emigration suggested that cell migration might be a potential target for preventing or treating vascular diseases, such as atherosclerosis and restenosis.
Conflict of interest The authors have no conflicts of interest to disclose.
Acknowledgments This work was supported by grants from the Health Bureau of Shanghai (Grant No. 20124253) awarded to HY, the National Natural Science Foundation of China (Grant No. 81371776) awarded to HY, and the National Basic Research Program of China (973 Program 2012CB578100) awarded to BXG.
o oxL LD DL IL--10 0 R-A A SR stiin2 β-arres 2 ma acroph hag ge
p p-P P38 8 cyttop pla asm CR R7,, M 0 CC 9, IIL--10 MM MP9 ucle eu us n nu n etion sec s cre CR R7,, M CC 0 9, ILI -10 MM MP9
macrrop ma pha ag ge e em mig gra atio on n
egress sion pla aque e re
Fig. 7. The proposed signaling pathways and mechanism by which IL-10 regulates the macrophage emigration to promote plaque regression. In macrophage, engagement of SR-A by oxLDL enhanced β-arrestin2 expression, leading to an increase in P38 phosphorylation, which in turn, downregulates the expression of CCR7 and secretion of MMP9. IL-10 inhibits this process, thereby promoting macrophage migration.
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IL-10 negatively regulates oxLDL-P38 pathway inhibited macrophage emigration☆ Hong Yang a,b,⁎, Hai peng Liu b,c, Dong Weng b, Bao xue Ge a,b,c,⁎ a b c
Department of Microbiology and Immunology, Tongji University School of Medicine, Shanghai 200092, China Clinical Translational Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200092, China Shanghai TB Key Laboratory, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Received 22 October 2014 Accepted 28 October 2014 Available online 29 October 2014 Keywords: Macrophage Emigration IL-10 β-Arrestin2 CCR7 P38 Atherosclerosis
a b s t r a c t The effect of IL-10 on macrophage migration was investigated, including the analysis of protein expression, cytokine secretion and activation of the MAPKs and NF-kB pathway. The expression of endogenic IL-10 was downregulated in macrophage stimulated with oxLDL for indicated time. Exogenous IL-10 reversed oxLDL-inhibited chemotactic macrophage numbers by 48.18 ± 4.93% (3 h), 64.8 ± 5.61% (6 h) and 63.66 ± 3.05% (12 h), and pretreated with SB203580 (P38 signaling inhibitor) in macrophage, oxLDL could not inhibit macrophage emigration. IL-10 significantly decreased oxLDL-mediated increase of SR-A expression, and pretreatment with βarrestin2 RNAi in macrophage could not change oxLDL-induced SR-A expression. IL-10 also significantly decreased oxLDL-mediated increase of β-arrestin2 expression, and pre-treated with SR-A RNAi in macrophage, oxLDL could not induce the increase of β-arrestin2 expression. However, IL-10 significantly reversed oxLDLmediated inhibition of CCR7 expression about 95.78 ± 0.99% (mRNA) and 80.06 ± 19.46% (protein), and pretreated with P38 inhibitor SB203580 in macrophage, oxLDL could not decrease CCR7 expression. IL-10 inhibited oxLDL-mediated inhibition of MMP9 secretion about 74.02 ± 22.35%, and pretreated with P38 inhibitor SB203580 in macrophage, oxLDL could not decrease MMP9 secretion. Treatment with oxLDL increased P38phosphorylation by 31.88 ± 2.79%, 40.24 ± 5.69% and 30.93 ± 4.66% at 15, 30 and 60 min, respectively, whereas the effect of IL-10 on the expression of phosphorylated P38 was reversed by 49.49 ± 12.12%, 70.93 ± 16.14% and 47.62 ± 6.00% in up-indicated time-points, respectively. From these data, we speculated oxLDL-SR-A-βarrestin2-P38-MMP9/CCR7 could play a critical role in the macrophages migration, which was blocked by IL10 through inhibiting oxLDL uptake. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Coronary artery disease, resulting from atherosclerosis, is the leading cause of death in the Western world (Feig and Fisher, 2013). Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophage in the artery wall (van Gils et al., 2012). Foam cells can
Abbreviations: oxLDL, oxidative low density lipoprotein; SR-A, scavenge receptor; IL-10, recombinant human interleukin-10; MIP-1, macrophage inflammatory protein-1; CCR7, cysteine–cysteine chemokine receptor 7; CCR5, cysteine–cysteine chemokine receptor 5; TNF-α, tumor necrosis factor-α; MMP9, matrix metalloproteinase; IL-17, interleukin-17; IFN-γ, interferon-γ. ☆ Authors' contributions: HY and BXG conceived the study. HY performed experiments, data analysis and wrote the manuscript. HPL provided helpful discussions. DW provided technical assistance with the experiments. All authors read and approved the final manuscript. ⁎ Corresponding authors at: Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, China. E-mail addresses: [email protected] (H. Yang), [email protected] (H. Liu), [email protected] (D. Weng), [email protected] (B. Ge).
http://dx.doi.org/10.1016/j.yexmp.2014.10.008 0014-4800/© 2014 Elsevier Inc. All rights reserved.
be induced by oxidized low density lipoprotein (oxLDL) in human monocyte-derived macrophage, which represents a major source of foam cells in atherogenesis (Ramkhelawon et al., 2013). Reversal of hyperlipidemia with a genetic switch favorably affects the content and inflammatory state of macrophage in atherosclerotic plaques (Feig et al., 2011a; Pagler et al., 2011). During atherosclerosis regression foam cell emigration is a very important clinical goal. Rapid regression ensued in a process involving emigration of plaque macrophage-derived foam cells to regional and systemic lymph nodes (Feig and Fisher, 2013; Yang et al., 2013). The macrophage migration exposed in oxLDL was altered (Choi et al., 2010). Diminished oxLDL uptake was mechanistically linked to resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression (MéndezBarbero et al., 2013). β-Arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways (Watari et al., 2013a). β-Arrestins are
H. Yang et al. / Experimental and Molecular Pathology 97 (2014) 590–599
scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts (Porter et al., 2010). The scavenger receptor might regulate cell migration and chemotaxis by β-arrestin2 via the MAPKs/NF-kB pathways (Watari et al., 2013a; Ehrlich et al., 2013; Lipfert et al., 2013). CCR7, a known factor in dendritic cell migration, was observed a significant increase in CCR7 mRNA in CD68+ cells from both the atorvastatin and rosuvastatin treated mice associated with emigration of CD68 + cells from plaques (Feig et al., 2011b). MMP9 play important roles in the migration of splenic T cells, B cells, macrophages/dendritic cells and smooth muscle cells (SMC) (Lötzer et al., 2010; Goldklang et al., 2012). It was reported that the reduction in atherosclerosis was accompanied by decreased plasma levels of interleukin-1alpha and tumor necrosis factor alpha, and preceded by increased anti-inflammatory cytokine interleukin-10 (Shen et al., 2010a). Previous work in the literature led us to propose a novel signaling pathway, oxLDL-SR-A-β-arrestin2-MAPKs/NF-kB-MMP9/CCR7, which may play a critical role in emigration of macrophages, and to investigate whether the anti-inflammatory cytokine IL-10, could block atherosclerotic progression and provide a protective role. The aim of this study was to characterize the impact of IL-10 on macrophage migration with regards to the expression of scavenger receptor, β-arrestin and CCR, cytokine secretion and activation of MAPKs/NF-kB. 2. Materials and methods 2.1. Mice, peritoneal macrophage isolation and treatments C57/BL6 mice were obtained from the Jackson laboratory, all female mice were maintained in the Shanghai Laboratory Animal Center (SLAC, Shanghai, China) and used at 8 weeks of age. Peritoneal macrophages were isolated as described previously (Liu et al., 2013). Briefly, mice received i.p. 2 mL of 4% thioglycollate medium (Sigma-Aldrich). After 3 days, cells harvested by peritoneal lavage with cold PBS were allowed to adhere for 2 h. Subsequently, RPMI 1640 medium was changed to remove non-adherent cells. The remaining adherent monolayer cells were used as primary peritoneal macrophages. All cells were cultured at 37 °C in a humidified incubator with 5% CO2. All animal studies were approved by the Institutional Animal Care and Use Committee of Tongji Unversity. Then cells were separated into four different stimulated groups with carrier control, only with 40 mg/L oxLDL (Sigma, St. Louis, USA), with 40 mg/L oxLDL and 20 μg/L IL-10 (Sigma), and with 40 mg/L oxLDL and 20 μg/L IL-10 (Sigma) and SB203580 (10 μM) /βarrestint2 RNAi (30nM)/SR-A RNAi (30 nM) for 24 h. The optimal doses and incubation times for the responses to oxLDL or IL-10 were determined in previous experiments (Yang et al., 2011a; Yang and Chen, 2011a; Yang et al., 2011b). The expression of IL-10 protein was assayed in macrophage only treated with 40 mg/L oxLDL and co-treated with oxLDL and 10 μM SP600125/PD98059/PDTC for indicated time. There was a pre-incubation for 30 min with each inhibitor. All The sequence-specific RNA interference designed as follows: 5′-GGA CCA GGG-3′ and 5′-CUG UUC UCC UUG CGA UGU ACU UGU C-3′ for βarrestint2 RNAi and 5′-GCA ACU GAC CAA AGA CUU A-3′ and 5′-UAA GUC UUU GGU CAG UUG C-3′ for SR-A RNAi. SiPORT Amine Transfection Agent 5 μL was used for transient transfection in 2 × 106 macrophage for 48 h before all other stimuli. 2.2. Chemotaxis assay Chemotaxis experiments were performed using a modified Boyden chamber assay (Corning Life Sciences, NY, USA). Cells were washed and resuspended in RPMI-1640 medium supplemented with 10 mmol/L HEPES, 2 mmol/L L-glutamine, and 1% BSA (Sigma-Aldrich). The chemoattractant MIP-1 (PeproTech EC) (Woszczek et al., 2008) was resuspended in the same medium and added to the lower wells for four groups. Cells (2 × 105) were added to the upper wells and separated by a polycarbonate filter with 5-μm pores (Corning). Usually, the in vitro
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transwell migration assay stops at 3–4 h later. The number of cells that migrated to the lower surface of the filter was counted after 3 h, 6 h and 12 h of incubation.
2.3. RNA isolation and quantitative real-time RT-PCR (qRT-PCR) After oxLDL treatment for 0 h, 2 h, 6 h, 9 h, 12 h and 24 h, or for 6 h of the four cell groups described above, total RNA was extracted from the cells by using the Trizol (Invitrogen, Carlsbad, USA) reagent. cDNA was generated from the RNA using Reverse Transcriptase XL (AMV), ribonuclease inhibitor, dNTP mixture and oligo d (T) 18 purchased from TaKaRa (Dalian, China). The cDNA was subsequently analyzed in the qPCR run on the Rotor Gene 3000 instrument (Corbett Research Co, Australia) using SYBR Premix Ex TaqTM (Dalian, China) and the sequence-specific PCR primers designed as follows: 5′-AAT GGA TTT GGA CGC ATT GGT-3′ and 5′-TTT GCA CTG GTA CGT GTT GAT-3′ for GAPDH (NM_008085); 5′-TTG TCG CGT TTG CTC CCA TT-3′ and 5′GAA GGG CTT GGC AGT TCT G-3′ for IL-10 (NM_008348); 5′-TGG AGG AGA GAA TCG AAA GCA-3′ and 5′-CTG GAC TGA CGA AAT CAA GGA A3′ for SR-A (NM_001113326); 5′-ATG GGC TGT GAT CGG AAC TG-3′ and 5′-TTT GCC ACG TCA TCT GGG TTT-3′ for CD36 (NM_001159556); 5′-TCT GAC TTT CGG AAA GAC CTG T-3′ and 5′-TCT TGA TGA GTC GCT CCT GTA G-3′ for β-Arrestin1 (NM_177231); 5′-AGT CGA GCC CTA ACT GCA AG-3′ and 5′-ACG AAC ACT TTC CGG TCC TTC-3′ for βArrestin2 (NM_145429); 5′-ATG GAT TTT CAA GGG TCA GTT CC-3′ and 5′-CTG AGC CGC AAT TTG TTT CAC-3′ for CCR5 (NM_009917); and 5′-CAG GTG TGC TTC TGC CAA GAT-3′ and 5′-GGT AGG TAT CCG TCA TGG TCT-3′ for CCR7 (NM_007719). The cycle number represented the relative quantity of the specific template when the fluorescence of the amplified gene product first reached a preset threshold (Ct). Transcripts of the housekeeping gene GAPDH in the same runs were used for normalization. The amplification of the control (GAPDH) and the test amplicons (SR-A, CD36, β-arrestin1, β-arrestin2, CCR5 and CCR7) showed very similar efficiencies over the concentration range tested. Treatment effects on gene expression are reported as a fold change of specific target mRNA in the cells incubated with different stimuli versus control cells incubated in parallel with carrier. The gene expression levels were calculated by the delta delta CT method: fold change = 2−Δ(ΔCt), where ΔCt = Ct (specific transcript) − Ct (housekeeping transcript) and Δ(ΔCt) = ΔCt (treatment) − ΔCt (control).
2.4. Protein extraction and Western blotting After the 6 h incubation with different stimuli, proteins from the four cell treatment groups were extracted using the KeyGen MembraneCytosol Protein Extraction Kit (Nanjing KeyGen Biotechnology, Nanjing, China) according to the manufacturer's protocol. The protein concentrations were determined using the Bradford method. Aliquots of partially purified membrane-bound or cytosolic protein (20 μg/lane) were subjected to 10% SDS-polyacrylamide gel electrophoresis in the presence of a reducing reagent, transferred onto nitrocellulose filters and incubated with goat anti-SR-A and anti-CD36 polyclonal antibody (I-20 and L17, 1:150 dilution, Santa Cruz Biotechnology, Santa Cruz, USA), with rabbit anti-β-arrestin1 and anti-β-arrestin2 monoclonal antibody (D8O3J and C16D9; 1:1000) (Cell Signaling Technology Inc., USA), with rabbit anti-CCR5 and rabbit anti-CCR7 monoclonal antibody (E164 and Y59; 1:5,000) (Abcam). Primary antibody binding was visualized by using IRDye® 800CW labeled donkey anti-goat and goat anti-rabbit IgG (1:15,000) with the Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, USA). The internal control, GAPDH, was detected by incubating with rabbit anti-GAPDH antibody (Immunogen Life Science & Technology Co. Ltd. Shanghai, China), followed by incubation with the IRDye® 800CW labeled goat anti-rabbit IgG antibody.
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2.5. Macrophage cytokine secretion
anti-p-ERK, rabbit anti-p-P65 (1:1000, Cell Signaling Technology Inc.). Primary antibody binding was visualized using IRDye® 800CW labeled goat anti-rabbit IgG (1:15,000) on an Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, USA).
The supernatants of macrophage cultures were harvested and stored at −80 °C. The levels of IL10, TNF-α, MMP9, IL-17 and IFN-γ were determined using cytokine-specific ELISA kits (R&D, Minneapolis, USA), according to the manufacturer's instructions.
2.7. Statistical analysis 2.6. Analysis of MAPKs and NF-kB activation All experiments were repeated three times. Data were expressed as the mean ± SEM, and were compared using one-way ANOVA by Fisher's least significant difference (LSD) analysis or paired Student's t tests, as appropriate. Differences were considered statistically significant when p b 0.05.
The method was similar to that used for Western blotting analysis, except that the protein was extracted following incubation for 0, 5, 15, 30, 60 and 90 min. The protein was transferred onto nitrocellulose filters and incubated with rabbit anti-p-P38, rabbit anti-p-JNK, rabbit
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Fig. 1. IL-10 expression and the chemotaxis to MIP-1 were measured in macrophage with different stimuli for indicated hours. (A). IL-10 mRNA expression was measured by qRT-PCR in macrophage under oxLDL stimuli for indicated hours, and GADPH levels were used as loading controls.(B–E) IL-10 protein expression was measured by ELISA under oxLDL stimuli with DMSO (gray line) or different signaling inhibitor (black line): such as P38 signaling inhibitor SB203580 (B), JNK signaling inhibitor SP600125 (C), ERK signaling inhibitor PD98059 (D), and P65 signaling inhibitor PDTC (E) for indicated hours. (F) The chemotaxis to MIP-1 was measured in macrophage treated with carrier control (white bar), with only oxLDL (gray bar), with oxLDL + IL-10 (black bar), and with oxLDL + SB203580 (dot bar) for indicated hours. Data are shown as the mean ± SEM from three separate experiments. Asterisk (*) and hash (#) symbols indicate indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group, # with oxLDL group vs. with oxLDL + SB203580 group).
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3. Results
that the variation trend of IL-10 protein expression were the same in macrophage only treated with oxLDL compared with co-treated with oxLDL and JNK inhibitor SP600125 (Fig. 1 C) or ERK inhibitor PD98059 (Fig. 1D) or P65 inhibitor PDTC (Fig. 1E). Fig. 1 F showed exogenous IL-10 reversed oxLDL-inhibited macrophage emigration by 48.18 ± 4.93% (3 h), 64.8 ± 5.61% (6 h) and 63.66 ± 3.05% (12 h), (black bar). And pretreated with SB203580 (P38 signaling inhibitor) in macrophage, oxLDL could not inhibit macrophage emigration (dot bar). Therefore, IL-10, as an anti-inflammatory cytokine, abrogated oxLDL-mediated inhibition of chemotaxis, and this effect was timedependent in macrophage. These data suggested that IL-10 might play a regulatory role through oxLDL-P38 signaling pathway in macrophage migration.
3.1. The expression of endogenic IL-10 was down-regulated in macrophage treated with oxLDL, and exogenous IL-10 reversed oxLDL-P38-pathwayinhibited macrophage emigration in time-dependent manner Fig. 1 A showed that the expression of endogenic IL-10 mRNA was down-regulated in macrophage stimulated with oxLDL for indicated time. At 6 h there is a decrease about 40%, at 12 h it is 66%, which is no change at 24 h. Fig. 1 B showed the expression of endogenic IL-10 protein had the same variation trend compared with that of IL-10 mRNA, but IL-10 protein expression had no change in macrophage treated with oxLDL and SB203580 (P38 inhibitor). Fig. 1 C–E showed
B Relative expression of CD36 mRNA
A *
C
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ox xLD DL(1 100mg g/l) Il-10((20 μg//l ) β-A Arrestin2 RN NAi
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SR R-A A GA APDH H
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+ + -
+ + + CD D36 6 GA APD DH
F
E *
Fig. 2. SR-A and CD36 expressions were measured in macrophage with different stimuli. (A and B) The mRNA levels of SR-A and CD36 were detected by qRT-PCR in macrophage treated with carrier control, only with oxLDL, with oxLDL + IL-10, and with oxLDL + β-arrestint2 RNAi, and GADPH levels were used as loading controls. (C and D) The protein levels of SR-A and CD36 were detected by Western blotting in macrophage according to up-indicated groups, and GADPH levels were used as loading controls. (E and F) Relative expression levels of SR-A and CD36 are presented as the mean ± SEM optical densities from three separate experiments. Asterisk (*) and indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group).
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3.2. IL-10 inhibited the oxLDL-induced increase of SR-A expression in macrophage
directly inhibited the oxLDL-induced increase of SR-A expression in macrophage, which was not through β-arrestin2.
We next determined the effect of IL-10 on the expression of the scavenge receptor A (SR-A) and CD36, which are the two main receptors for oxLDL. As shown in Fig. 2A, C and E, IL-10 inhibited oxLDLinduced increase of SR-A expressions about 43.93 ± 4.01% (mRNA) and 33.91 ± 7.57% (protein) in macrophage. And pretreated with βarrestin2 RNAi in macrophage, oxLDL had the same effect on SR-A expression compared with that only treatment with oxLDL. However, as shown in Fig. 2B, D and F, IL-10 did not alter oxLDL-induced increase of CD36 levels. And pre-treatment with β-arrestin2 RNAi, oxLDL also could not change of the CD36 expression in macrophage compared with that only treated with oxLDL. These results showed that IL-10
3.3. IL-10 blocked the oxLDL-mediated increase of β-arrestin2 expression in macrophage Expression of the important MAPKs regulatory molecule, βarrestin1 and β-arrestin2, was also analyzed. As shown in Fig. 3A, C and E, the mRNA and protein levels of β-arrestin1 were increased in oxLDL treated group, which was not blocked by IL-10 or SR-A inhibitor. However, as shown in Fig. 3B, D and F, IL-10 blocked oxLDL-induced increase of β-arrestin2 expressions about 43.49 ± 8.63% (mRNA) and 45.44 ± 9.45% (protein) in macrophage. And in macrophage pretreated with SR-A RNAi, oxLDL could not increase β-arrestin2 expression. These
B
A
#
*
C
D
oxL LDL L(10 00m mg/l) Il-1 10(2 20 μg/l μ l) SR R-A RN NAi(100 0uM M)
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00uM) SR R-A A RN NAi(10 ox xLD DL(1 100 0mg g/l)
+ + +
Il--10((20 μg g/l )
+ -
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β-ar β rre estiin2 2
β rre β-ar estiin1 1
G GAP PD DH
G DH GAP
F Relative expression of β-Arrestin1 β Arrestin1 protein
E 1 08 0.8
#
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06 0.6 04 0.4 02 0.2 0
Fig. 3. β-Arrestin1 and β-arrestin2 expressions were measured in macrophage with different stimuli. (A and B) The mRNA levels of β-arrestin1 and β-arrestin2 were detected by qRT-PCR in macrophage treated with carrier control, only with oxLDL, with oxLDL + IL-10, and with oxLDL + SR-A RNAi, and GAPDH levels were used as loading controls. (C and D) The protein levels of β-arrestin1 and β-arrestin2 were detected by Western blotting in macrophage according to up-indicated groups, and GAPDH levels were used as loading controls. (E and F) Relative expression levels ofβ-arrestin1 and β-arrestin2 are presented as the mean ± SEM optical densities from three separate experiments. Asterisk (*) and hash (#) symbols indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group, # with oxLDL group vs. with oxLDL + SR-A RNAi group).
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results suggested that IL-10 blocked the effects of oxLDL on β-arrestin2 expression in macrophage through inhibiting oxLDL uptake, which was mediated by SR-A.
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with P38 inhibitor SB203580, oxLDL could not decrease CCR7 expression. These results demonstrated that IL-10 counteracted the oxLDLP38-signaling-pathway-mediated inhibition of CCR7 expression in macrophage.
3.4. IL-10 reversed the oxLDL-mediated inhibition of CCR7 expression in macrophage 3.5. IL-10 significantly abrogated the oxLDL-mediated decreases in the levels of MMP9 in macrophage
To determine whether the chemokine receptors CCR5 and CCR7 are involved in emigration in this study, the expression levels of CCR5 and CCR7 were examined under a range of conditions by Western blotting. As shown in Fig. 4A, C and E, the mRNA and protein levels of CCR5 were increased in oxLDL treated group, which was not blocked by IL10 and SB203580. However, as shown in Fig. 4B, D and F, IL-10 inhibited oxLDL-mediated inhibition of CCR7 expressions about 95.78 ± 0.99% (mRNA) and 80.06 ± 19.46% (protein). And in macrophage pretreated
Macrophages secrete a wide spectrum of cytokines and chemokines (Sprague and Khalil, 2009). Therefore, we examined the effect of IL-10 on the cytokine profile of macrophage treated with oxLDL. The levels of MMP9, TNF-α, IFN-γ and IL-17 were examined by ELISA. Most of these factors are involved in the stimulation of mononuclear cells in atherogenesis (Sprague and Khalil, 2009).
B
A
#
0..9 0..6 0..3 0
C Il-1 10(2 20 μ μg/ll ) SB B203 358 80(1 100 0uM M)
E
Relative expression of CCR7 mRNA
Relative expression of CCR5 mRNA
1..2
oxL LDL L(10 00m mg//l)
*
1.5 5
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1.2 2 0.9 9 0.6 6 0.3 3 0
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oxLD DL(100 0mg g/l) Il--10 0(20 0 μg g/l ) SB20 035 580(10 00uM M)
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+ -
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+ + -
C R5 CCR
CC CR R7
G PDH GAP
H GAP PDH
F
#
*
Fig. 4. CCR5 and CCR7 expressions were measured in macrophage with different stimuli. (A and B) The mRNA levels of CCR5 and CCR7 were detected by qRT-PCR in macrophage treated with carrier control, only with oxLDL, with oxLDL + IL-10, and with oxLDL + SB203580, and GAPDH levels were used as loading controls. (C and D) The protein levels of βarrestin1 and β-arrestin2 were detected by Western blotting in macrophage according to up-indicated groups, and GAPDH levels were used as loading controls. (E and F) Relative expression levels of CCT5 and CCR7 are presented as the mean ± SEM optical densities from three separate experiments. Asterisk (*) and hash (#) symbols indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group, # with oxLDL group vs. with oxLDL + SB203580 group).
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A
B 6 MMP-9(ng/ml)
6
4.5
TNF-α(ng/ml)
*
3
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1.5
1.5
0
0
C
D 0.8
3.5 2.8
0.4 0.2 0
IFN-γ (ng/ml)
IL-17(ng/ml)
0.6
2.1 1.4 0.7 0
Fig. 5. Cytokines secretion were measured in macrophage with different stimuli. The concentrations of TNF-α (A), MMP9 (B), IL-17 (C) and INF-γ (D) were measured by specific sandwich ELISA in macrophage treated with carrier control, only with oxLDL, with oxLDL + IL-10, and with oxLDL + SB203580. Data represent the mean ± SEM of three separate experiments from 1 × 106 cells. Asterisk (*) and hash (#) symbols indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group, # with oxLDL group vs. with oxLDL + SB203580 group).
As shown in Fig. 5A, and C, the levels of TNF-α and IL-17 were increased in oxLDL treated groups, which was not blocked by IL-10 and SB203580. However, as shown in Fig. 5B, IL-10 inhibited oxLDLmediated inhibition of MMP9 secretion about 74.02 ± 22.35%. And in macrophage pretreated with P38 inhibitor SB203580, oxLDL could not decrease MMP9 secretion. However, as shown in Fig. 5D, the levels of IFN-γ were not significantly altered in four differently treated groups.
3.6. IL-10 suppressed the oxLDL-mediated decrease in P38 phosphorylation in macrophage As shown in Fig. 6A, the levels of phosphorylated P38, JNK, ERK and P65 in macrophage were detected by Western blotting at 0, 5, 15, 30, 60 and 90 min. Fig. 6B showed IL-10 suppressed the oxLDL-mediated decrease of P38 phosphorylation in macrophage for indicated time (black bar). At 15 min there is an increase about 39%, at 30 min it is 40%, at 60 min it is 41%. Fig. 6C–E showed that IL-10 could not change the oxLDL-induced phosphorylation of JNK (Fig. 6C, black bar) or ERK (Fig. 6D, black bar) or P65 (Fig. 6E, black bar).
4. Discussion Chronic inflammatory diseases such as atherosclerosis were characterized by an accumulation of macrophages. It had been previously shown that mouse atherosclerosis regression involves monocytederived (CD68 +) cell emigration from plaques (Feig et al., 2010). Cholesterol-lowering therapy leaded to plaque stabilization or regression in human atherosclerosis, characterized by reduced macrophage content (Potteaux et al., 2011). Macrophage cholesterol efflux might have a role in facilitating emigration of macrophages from lesions during regression (Murphy et al., 2012). HDL promoted rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells (Yang and Chen, 2011b; Feig et al., 2011c). Here, we demonstrate that the expressions of endogenic IL-10 (anti-inflammatory cytokine) and MIP-1 induced chemotaxis were significantly inhibited in macrophage treated with oxLDL through P38 signaling pathway and in a time-dependent manner. There was IL-10 basic secretion at time 0 h. However, exogenous IL-10 reversed the effects on macrophage migration, which might attenuate atherogenesis. These results were coincident with the report that the reduction in atherosclerosis
H. Yang et al. / Experimental and Molecular Pathology 97 (2014) 590–599
A
oxxLD DL+ +IL L-10
oxL o LDL 0
5
15 1
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5
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3 30
60
90 (m min n) p-P3 38 p-JN NK
p ERK K p-E 5 p-P P65
G PDH H GAP
B
C
*
*
*
D
E
Fig. 6. Phosphorylation of MAPKs and NF-kB was measured in macrophage with different stimuli for indicated times. The phosphorylation levels of P38 (A), JNK (B), ERK (C) and P65 were detected by Western blotting in macrophage only treated with oxLDL and treated with oxLDL + IL-10 for 0, 5, 15, 30, 60 and 90 min, and GAPDH levels were used as loading controls. Asterisk (*) symbols indicated p b 0.05 (* with oxLDL group vs. with oxLDL + IL-10 group).
was accompanied by decreased plasma levels of interleukin-1alpha and tumor necrosis factor alpha, and preceded by increased antiinflammatory cytokine interleukin-10 (Shen et al., 2010b). Scavenger receptors promoted atherosclerosis; therefore, we hypothesized that the multifunctional adaptor protein, β-arrestin2, might regulate this pathological process. The results of our study showed that treatment with oxLDL in macrophage significantly increased SR-A, CD36, β-arrestin1 and β-arrestin2 expression levels, but decreased CCR5 and CCR7 expression. However, IL-10 reversed the effects of oxLDL on the expression of SR-A, and pretreatment with
β-arrestin2 RNAi in macrophage oxLDL also increase SR-A expression. These results showed that in macrophage oxLDL was not through βarrestin2 to increase SR-A expression. IL-10 inhibited oxLDL-induced β-arrestin2 expressions in macrophage, and pretreated with SR-A RNAi in macrophage, oxLDL could not increase β-arrestin2 expression. These results suggested that oxLDL was through SR-A to increase βarrestin2 expression in macrophage. IL-10 reversed oxLDL-mediated decrease of CCR7 expression, and in macrophage pretreated with P38 inhibitor SB203580, oxLDL could not decrease CCR7 expression. These results demonstrated that the oxLDL-mediated inhibition of CCR7
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expression in macrophage was through P38 signaling pathway. Above three results suggested that the regulation axis was oxLDL-SR-A-βarrestin2-P38-CCR7. There was similar report that the alternate CXCR7 (scavenger chemokine receptor) ligand, I-TAC/CXCL11, activated ERK and AKT through β-arrestin in astrocytes (Odemis et al., 2012). Feig et al. also showed that atherosclerosis regression in mice involves macrophage emigration from plaques and is dependent on the chemokine receptor CCR7 (a mediator of leukocyte emigration and a factor functionally required for regression) (Feig and Fisher, 2013; Yang et al., 2013; Feig et al., 2010; Potteaux et al., 2011; Feig et al., 2011c; Trogan et al., 2006). Therefore, IL-10 may exhibit some of its clinical benefits by accelerating the emigration of macrophage-derived foam cells, thereby promoting the regression of atherosclerosis through directly inhibiting oxLDL uptake. In terms of the analysis of cytokine secretion and the associated signal transduction pathway, we focused our studies on MMP9, TNF-α, IL17, IFN-γ, MAPKs and NF-kB, because of their important roles in aspects of macrophage migration (Yuan et al., 2010; Choi et al., 2012; Ben et al., 2013; Watari et al., 2013b). Our results showed that treatment with oxLDL in macrophage significantly increased TNF-α, IL-17 and P38 phosphorylation levels, but decreased MMP9. IL-10 and SB203580 suppressed the effects of oxLDL on the secretion of MMP9 and P38 phosphorylation. In aortas of atherosclerotic rabbits, down-regulation of 6 pro-inflammatory genes, TNF-α, MCP-1, IL-1β, IL-18, MMP-9, MMP-12 and up-regulation of the anti-inflammatory IL-10 gene were observed (Bulgarelli et al., 2013). Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group (de Oliveira et al., 2009). The P38 inhibitor, SB203580, suppressed the effects of oxLDL on expression levels of CCR7, MMP9, IL-10 and macrophage chemotaxis.
Consistently, IL-10 also reversed all of the above effects of oxLDL on macrophages. In summary, this was the first report to establish a novel signaling pathway, oxLDL-SR-A-β-arrestin2-P38-MMP9/CCR7, which played a critical role in macrophage emigration (Fig. 7). The data suggested that activation of P38 was a key component of the oxLDL-SR-A-β-arrestin2-P38-MMP9/CCR7 signal transduction cascade, which was blocked by IL-10 through inhibiting oxLDL uptake. However, it had been reported that β-arrestin2, MMP9, TNF-α and PI3K phosphorylation aggravated atherosclerosis through mechanisms involving dendritic cell (DC) proliferation and migration, which are critical in the later phase and progression of atherosclerosis and restenosis (Yang et al., 2013). From these findings we suggested that SR-A, βarrestin2, CCR7, MMP9 and p38 might play an important role in the initiation of atherosclerotic progression. The notion that P38 activity facilitated macrophage-derived foam cell emigration suggested that cell migration might be a potential target for preventing or treating vascular diseases, such as atherosclerosis and restenosis.
Conflict of interest The authors have no conflicts of interest to disclose.
Acknowledgments This work was supported by grants from the Health Bureau of Shanghai (Grant No. 20124253) awarded to HY, the National Natural Science Foundation of China (Grant No. 81371776) awarded to HY, and the National Basic Research Program of China (973 Program 2012CB578100) awarded to BXG.
o oxL LD DL IL--10 0 R-A A SR stiin2 β-arres 2 ma acroph hag ge
p p-P P38 8 cyttop pla asm CR R7,, M 0 CC 9, IIL--10 MM MP9 ucle eu us n nu n etion sec s cre CR R7,, M CC 0 9, ILI -10 MM MP9
macrrop ma pha ag ge e em mig gra atio on n
egress sion pla aque e re
Fig. 7. The proposed signaling pathways and mechanism by which IL-10 regulates the macrophage emigration to promote plaque regression. In macrophage, engagement of SR-A by oxLDL enhanced β-arrestin2 expression, leading to an increase in P38 phosphorylation, which in turn, downregulates the expression of CCR7 and secretion of MMP9. IL-10 inhibits this process, thereby promoting macrophage migration.
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