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Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter.

Wang J, Sun C, Gerdes N, Liu C, Liao M, Liu J, Shi MA, He A, Zhou Y, Sukhova GK, Chen H, Cheng XW, Kuzuya M, Murohara T, Zhang J, Cheng X, Jiang M, Shull GE, Rogers S, Yang CL, Ke Q, Jelen S, Bindels R, Ellison DH, Jarolim P, Libby P, Shi GP - Nat. Med. (2015)

Bottom Line: As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney.IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression.This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. [2] State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Department of Pathophysiology, Peking Union Medical College, Tsinghua University, Beijing, China.

ABSTRACT
Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms. Interruption of IL18 action reduces atherosclerosis in mice. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe(-/-) mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe(-/-) mice or from Apoe(-/-) mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe(-/-) mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

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Na-Cl co-transporter expression and characterization. a. Immunostaining of NCC in normal human (bar: 1000 µm) and mouse aortas (bar: 50 µm). b. Immunostaining of NCC, IL18r, and CD68+ macrophages in human atherosclerotic lesions. Negative controls rabbit and mouse (not shown) IgG. Bar: 500 µm. c. Immunostaining of NCC in lesions from Apoe−/− mice. Macrophage, SMC, and EC areas were framed. Negative controls used lesions from Apoe−/−Ncc−/− mice. Bar: 200 µm, insert bar: 50 µm. d. Immunofluorescent staining of NCC and IL18r in macrophages, SMCs, and ECs from lesions from Apoe−/− mice. Bar: 50 µm. e. RT-PCR and immunostaining to detect NCC in macrophages (bar: 50 µm) and T cells (bar: 20 µm) from Apoe−/− mice. Immunostaining negative controls used macrophages from Apoe−/−Ncc−/− mice. f. Immunoblots to detect NCC in cytokine-induced mouse ECs and SMCs. NCC doublets are indicated. Positive control used NCC-transfected COS-7 cell lysate. g. IL18r immunoprecipitation followed by NCC immunoblot to detect NCC-IL18r complexes in macrophages from Apoe−/− mice. Negative control used macrophages from Apoe−/−Ncc−/− mice. h. FACS to detect biotin-IL18 and heated biotin-IL18 binding to differently transfected COS-7 cells; Scatchard plot of FITC-IL18 binding affinity on NCC-transfected COS-7 cells, and p-tyrosine immunoblot in NCC- or vector-transfected COS-7 cells treated with different treatments. Actin, protein loading. Data in panels e and h are mean ± SEM from three to six independent experiments.
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Figure 2: Na-Cl co-transporter expression and characterization. a. Immunostaining of NCC in normal human (bar: 1000 µm) and mouse aortas (bar: 50 µm). b. Immunostaining of NCC, IL18r, and CD68+ macrophages in human atherosclerotic lesions. Negative controls rabbit and mouse (not shown) IgG. Bar: 500 µm. c. Immunostaining of NCC in lesions from Apoe−/− mice. Macrophage, SMC, and EC areas were framed. Negative controls used lesions from Apoe−/−Ncc−/− mice. Bar: 200 µm, insert bar: 50 µm. d. Immunofluorescent staining of NCC and IL18r in macrophages, SMCs, and ECs from lesions from Apoe−/− mice. Bar: 50 µm. e. RT-PCR and immunostaining to detect NCC in macrophages (bar: 50 µm) and T cells (bar: 20 µm) from Apoe−/− mice. Immunostaining negative controls used macrophages from Apoe−/−Ncc−/− mice. f. Immunoblots to detect NCC in cytokine-induced mouse ECs and SMCs. NCC doublets are indicated. Positive control used NCC-transfected COS-7 cell lysate. g. IL18r immunoprecipitation followed by NCC immunoblot to detect NCC-IL18r complexes in macrophages from Apoe−/− mice. Negative control used macrophages from Apoe−/−Ncc−/− mice. h. FACS to detect biotin-IL18 and heated biotin-IL18 binding to differently transfected COS-7 cells; Scatchard plot of FITC-IL18 binding affinity on NCC-transfected COS-7 cells, and p-tyrosine immunoblot in NCC- or vector-transfected COS-7 cells treated with different treatments. Actin, protein loading. Data in panels e and h are mean ± SEM from three to six independent experiments.

Mentions: NCC is a plasma membrane ion co-transporter expressed as a homodimer in kidney distal convoluted tubules, where it participates in electrolyte homeostasis18, although other tissues express low levels of NCC5,19. We detected abundant NCC mRNA in mouse kidney, but also low levels in the heart, lung, and liver by RT-PCR (Supplementary Fig. 1). Immunostaining using an anti-human/mouse NCC polyclonal antibody revealed no NCC expression in normal human or mouse aortas (Fig. 2a), but abundant NCC expression in human fatty streaks (Supplementary Fig. 2) and advanced atherosclerotic lesions (Fig. 2b), localized to areas positive for IL18r and CD68+ macrophages. Tests of antibody specificity confirmed NCC expression in kidney distal tubules from Apoe−/− mice, but not in those from Apoe−/−Ncc−/− mice (Supplementary Fig. 3). Macrophages, SMCs, and ECs in atherosclerotic lesions from Apoe−/− mice expressed NCC (Fig. 2c). Immunofluorescent staining confirmed colocalization of NCC and IL18r in macrophages, SMCs, and ECs in lesions from Apoe−/− mice (Fig. 2d). RT-PCR demonstrated that IL18 induced NCC expression in mouse peritoneal macrophages by 65-fold, but had negligible effect on T cells. Immunostaining revealed induction of NCC by IL18 in macrophages and T cells from Apoe−/− mice (Fig. 2e). Immunoblot showed that IL18, IL1β, or TNF-α increased NCC expression in mouse ECs and SMCs, with the same molecular weight as those expressed in NCC/pcDNA3.1-transfected COS-7 cells (Fig. 2f). Immunoprecipitation with mouse anti-mouse IL18r antibody followed by immunoblot with the rabbit anti-mouse NCC antibody in macrophage lysates demonstrated an enhanced complex formation between IL18r and NCC in IL18-treated macrophages from Apoe−/− mice but not those from Apoe−/−Ncc−/− mice (Fig. 2g). To study NCC and its interaction with IL18, we subcloned NCC full-length cDNA into a pcDNA3.1 vector. Immunoblot revealed mouse NCC in NCC-transfected COS-7 cells as a doublet band around 125-kDa, due to differential protein glycosylations (Supplementary Fig. 4)18. FACS analysis demonstrated surface binding of biotin-conjugated IL18, but not heat-inactivated biotin-IL18 on NCC-transfected COS-7 cells 30 minutes after induction (Fig. 2h). FITC-conjugated IL18 binding to NCC-transfected COS-7 cells, which do not express IL18r in the presence or absence of IL18 (data not shown), showed a higher binding affinity (dissociation constant Kd = 17.3 nM) than previously reported IL18 binding on IL18r-transfected COS-1 cells (Kd = 46 nM)16 (Fig. 2h). Treatment with IL18, but not heat-inactivated IL18 for 30 minutes also increased protein tyrosine phosphorylation in NCC-transfected COS-7 cells, compared with those transfected with empty vector (Fig. 2h), indicating that IL18 interacts with NCC and yields biological consequences.


Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter.

Wang J, Sun C, Gerdes N, Liu C, Liao M, Liu J, Shi MA, He A, Zhou Y, Sukhova GK, Chen H, Cheng XW, Kuzuya M, Murohara T, Zhang J, Cheng X, Jiang M, Shull GE, Rogers S, Yang CL, Ke Q, Jelen S, Bindels R, Ellison DH, Jarolim P, Libby P, Shi GP - Nat. Med. (2015)

Na-Cl co-transporter expression and characterization. a. Immunostaining of NCC in normal human (bar: 1000 µm) and mouse aortas (bar: 50 µm). b. Immunostaining of NCC, IL18r, and CD68+ macrophages in human atherosclerotic lesions. Negative controls rabbit and mouse (not shown) IgG. Bar: 500 µm. c. Immunostaining of NCC in lesions from Apoe−/− mice. Macrophage, SMC, and EC areas were framed. Negative controls used lesions from Apoe−/−Ncc−/− mice. Bar: 200 µm, insert bar: 50 µm. d. Immunofluorescent staining of NCC and IL18r in macrophages, SMCs, and ECs from lesions from Apoe−/− mice. Bar: 50 µm. e. RT-PCR and immunostaining to detect NCC in macrophages (bar: 50 µm) and T cells (bar: 20 µm) from Apoe−/− mice. Immunostaining negative controls used macrophages from Apoe−/−Ncc−/− mice. f. Immunoblots to detect NCC in cytokine-induced mouse ECs and SMCs. NCC doublets are indicated. Positive control used NCC-transfected COS-7 cell lysate. g. IL18r immunoprecipitation followed by NCC immunoblot to detect NCC-IL18r complexes in macrophages from Apoe−/− mice. Negative control used macrophages from Apoe−/−Ncc−/− mice. h. FACS to detect biotin-IL18 and heated biotin-IL18 binding to differently transfected COS-7 cells; Scatchard plot of FITC-IL18 binding affinity on NCC-transfected COS-7 cells, and p-tyrosine immunoblot in NCC- or vector-transfected COS-7 cells treated with different treatments. Actin, protein loading. Data in panels e and h are mean ± SEM from three to six independent experiments.
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Figure 2: Na-Cl co-transporter expression and characterization. a. Immunostaining of NCC in normal human (bar: 1000 µm) and mouse aortas (bar: 50 µm). b. Immunostaining of NCC, IL18r, and CD68+ macrophages in human atherosclerotic lesions. Negative controls rabbit and mouse (not shown) IgG. Bar: 500 µm. c. Immunostaining of NCC in lesions from Apoe−/− mice. Macrophage, SMC, and EC areas were framed. Negative controls used lesions from Apoe−/−Ncc−/− mice. Bar: 200 µm, insert bar: 50 µm. d. Immunofluorescent staining of NCC and IL18r in macrophages, SMCs, and ECs from lesions from Apoe−/− mice. Bar: 50 µm. e. RT-PCR and immunostaining to detect NCC in macrophages (bar: 50 µm) and T cells (bar: 20 µm) from Apoe−/− mice. Immunostaining negative controls used macrophages from Apoe−/−Ncc−/− mice. f. Immunoblots to detect NCC in cytokine-induced mouse ECs and SMCs. NCC doublets are indicated. Positive control used NCC-transfected COS-7 cell lysate. g. IL18r immunoprecipitation followed by NCC immunoblot to detect NCC-IL18r complexes in macrophages from Apoe−/− mice. Negative control used macrophages from Apoe−/−Ncc−/− mice. h. FACS to detect biotin-IL18 and heated biotin-IL18 binding to differently transfected COS-7 cells; Scatchard plot of FITC-IL18 binding affinity on NCC-transfected COS-7 cells, and p-tyrosine immunoblot in NCC- or vector-transfected COS-7 cells treated with different treatments. Actin, protein loading. Data in panels e and h are mean ± SEM from three to six independent experiments.
Mentions: NCC is a plasma membrane ion co-transporter expressed as a homodimer in kidney distal convoluted tubules, where it participates in electrolyte homeostasis18, although other tissues express low levels of NCC5,19. We detected abundant NCC mRNA in mouse kidney, but also low levels in the heart, lung, and liver by RT-PCR (Supplementary Fig. 1). Immunostaining using an anti-human/mouse NCC polyclonal antibody revealed no NCC expression in normal human or mouse aortas (Fig. 2a), but abundant NCC expression in human fatty streaks (Supplementary Fig. 2) and advanced atherosclerotic lesions (Fig. 2b), localized to areas positive for IL18r and CD68+ macrophages. Tests of antibody specificity confirmed NCC expression in kidney distal tubules from Apoe−/− mice, but not in those from Apoe−/−Ncc−/− mice (Supplementary Fig. 3). Macrophages, SMCs, and ECs in atherosclerotic lesions from Apoe−/− mice expressed NCC (Fig. 2c). Immunofluorescent staining confirmed colocalization of NCC and IL18r in macrophages, SMCs, and ECs in lesions from Apoe−/− mice (Fig. 2d). RT-PCR demonstrated that IL18 induced NCC expression in mouse peritoneal macrophages by 65-fold, but had negligible effect on T cells. Immunostaining revealed induction of NCC by IL18 in macrophages and T cells from Apoe−/− mice (Fig. 2e). Immunoblot showed that IL18, IL1β, or TNF-α increased NCC expression in mouse ECs and SMCs, with the same molecular weight as those expressed in NCC/pcDNA3.1-transfected COS-7 cells (Fig. 2f). Immunoprecipitation with mouse anti-mouse IL18r antibody followed by immunoblot with the rabbit anti-mouse NCC antibody in macrophage lysates demonstrated an enhanced complex formation between IL18r and NCC in IL18-treated macrophages from Apoe−/− mice but not those from Apoe−/−Ncc−/− mice (Fig. 2g). To study NCC and its interaction with IL18, we subcloned NCC full-length cDNA into a pcDNA3.1 vector. Immunoblot revealed mouse NCC in NCC-transfected COS-7 cells as a doublet band around 125-kDa, due to differential protein glycosylations (Supplementary Fig. 4)18. FACS analysis demonstrated surface binding of biotin-conjugated IL18, but not heat-inactivated biotin-IL18 on NCC-transfected COS-7 cells 30 minutes after induction (Fig. 2h). FITC-conjugated IL18 binding to NCC-transfected COS-7 cells, which do not express IL18r in the presence or absence of IL18 (data not shown), showed a higher binding affinity (dissociation constant Kd = 17.3 nM) than previously reported IL18 binding on IL18r-transfected COS-1 cells (Kd = 46 nM)16 (Fig. 2h). Treatment with IL18, but not heat-inactivated IL18 for 30 minutes also increased protein tyrosine phosphorylation in NCC-transfected COS-7 cells, compared with those transfected with empty vector (Fig. 2h), indicating that IL18 interacts with NCC and yields biological consequences.

Bottom Line: As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney.IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression.This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. [2] State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Department of Pathophysiology, Peking Union Medical College, Tsinghua University, Beijing, China.

ABSTRACT
Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms. Interruption of IL18 action reduces atherosclerosis in mice. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe(-/-) mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe(-/-) mice or from Apoe(-/-) mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe(-/-) mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

Show MeSH
Related in: MedlinePlus