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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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Identification of alternative IL18-binding proteins. a. Aortic root lesion intima, Mac-3+ macrophage, CD4+ T cell, and α-actin–positive SMC areas in Apoe−/−Il18r−/− and Apoe−/− mice. n=7–10 per group. b. FACS of ECs from Apoe−/−Il18r−/− and Apoe−/− mice after binding with biotin-IL18 with and without excessive non-labeled IL18, followed by incubation with PE-streptavidin. c. P-tyrosine immunoblot in ECs treated with and without IL18 or heat-inactivated IL18 for 30 minutes. Actin, loading control. d. IL18-binding protein identification in ECs from Apoe−/−Il18r−/− mice. SDS-PAGE silver staining detected IL18-bound proteins in three sequential elutes from anti-IL18 antibody-bound protein-A agarose beads pre-incubated with cell lysates from ECs treated with or without IL18.
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Figure 1: Identification of alternative IL18-binding proteins. a. Aortic root lesion intima, Mac-3+ macrophage, CD4+ T cell, and α-actin–positive SMC areas in Apoe−/−Il18r−/− and Apoe−/− mice. n=7–10 per group. b. FACS of ECs from Apoe−/−Il18r−/− and Apoe−/− mice after binding with biotin-IL18 with and without excessive non-labeled IL18, followed by incubation with PE-streptavidin. c. P-tyrosine immunoblot in ECs treated with and without IL18 or heat-inactivated IL18 for 30 minutes. Actin, loading control. d. IL18-binding protein identification in ECs from Apoe−/−Il18r−/− mice. SDS-PAGE silver staining detected IL18-bound proteins in three sequential elutes from anti-IL18 antibody-bound protein-A agarose beads pre-incubated with cell lysates from ECs treated with or without IL18.

Mentions: IL18 polarizes Th1 cells1,2 and induces production of inflammatory cytokines, chemokines, and vascular adhesion molecules2,6–8. Prior studies with mice deficient in IL18 (Il18−/−), its receptor IL18r (Il18r−/−), or soluble receptors demonstrate the contribution of IL18 to several inflammatory diseases9–11. Atherosclerotic lesions but not normal human aortas express high levels of IL18 and IL18r in macrophages, T cells, endothelial cells (ECs), and smooth-muscle cells (SMCs)12,13. In atherosclerosis-prone apolipoprotein E–deficient (Apoe−/−) mice, the absence of IL18 or blockade of IL18 signaling reduces atherosclerosis and lesion inflammation3,4. Intraperitoneal administration of IL18 in Apoe−/− mice enhances lesion burden and inflammation14,15. IL18r is a heterodimer with a low IL18-binding affinity16. Absence of IL18r leads to reduced NK cell IFN-γ production and impaired Th1 cell signaling and differentiation17. Yet, Apoe−/− and Apoe−/−Il18r−/− littermates showed no differences in atherogenesis. In Apoe−/− mice, the presence or absence of IL18r did not affect aortic root intimal area, or lesion macrophage, CD4+ T-cell, or SMC contents (Fig. 1a) — suggesting IL18 action on target cells that is independent of, or in addition to, the known IL18r. Mouse ECs from Apoe−/− and Apoe−/−Il18r−/− mice incubated with biotinylated IL18, showed no differences in cell surface IL18 binding, competable by a 5~10-fold excess of unlabeled IL18 (Fig. 1b). ECs from Apoe−/− and Apoe−/−Il18r−/− mice showed a similar pattern of protein tyrosine phosphorylation after 30 minutes of incubation with IL18, as detected by immunoblot with anti-phospho (p)-tyrosine antibody. Heat-inactivated IL18 did not elicit these activities (Fig. 1c). These data pointed to the presence of alternative molecules that mediate cell-surface IL18 binding and downstream signaling. Incubation of ECs from Apoe−/−Il18r−/− mice with IL18 for 15 minutes followed by immunoprecipitation with an anti-mouse IL18 polyclonal antibody identified possible cell-surface IL18-binding molecules. Silver-stained SDS-PAGE showed two major bands between 85 to 150 kDa in series eluates from IL18-treated ECs (Fig. 1d). Mass spectrometry identified a 125-kDa Na-Cl co-transporter (NCC) in addition to multiple matrix protein fragments (data not shown).


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)

Identification of alternative IL18-binding proteins. a. Aortic root lesion intima, Mac-3+ macrophage, CD4+ T cell, and α-actin–positive SMC areas in Apoe−/−Il18r−/− and Apoe−/− mice. n=7–10 per group. b. FACS of ECs from Apoe−/−Il18r−/− and Apoe−/− mice after binding with biotin-IL18 with and without excessive non-labeled IL18, followed by incubation with PE-streptavidin. c. P-tyrosine immunoblot in ECs treated with and without IL18 or heat-inactivated IL18 for 30 minutes. Actin, loading control. d. IL18-binding protein identification in ECs from Apoe−/−Il18r−/− mice. SDS-PAGE silver staining detected IL18-bound proteins in three sequential elutes from anti-IL18 antibody-bound protein-A agarose beads pre-incubated with cell lysates from ECs treated with or without IL18.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Identification of alternative IL18-binding proteins. a. Aortic root lesion intima, Mac-3+ macrophage, CD4+ T cell, and α-actin–positive SMC areas in Apoe−/−Il18r−/− and Apoe−/− mice. n=7–10 per group. b. FACS of ECs from Apoe−/−Il18r−/− and Apoe−/− mice after binding with biotin-IL18 with and without excessive non-labeled IL18, followed by incubation with PE-streptavidin. c. P-tyrosine immunoblot in ECs treated with and without IL18 or heat-inactivated IL18 for 30 minutes. Actin, loading control. d. IL18-binding protein identification in ECs from Apoe−/−Il18r−/− mice. SDS-PAGE silver staining detected IL18-bound proteins in three sequential elutes from anti-IL18 antibody-bound protein-A agarose beads pre-incubated with cell lysates from ECs treated with or without IL18.
Mentions: IL18 polarizes Th1 cells1,2 and induces production of inflammatory cytokines, chemokines, and vascular adhesion molecules2,6–8. Prior studies with mice deficient in IL18 (Il18−/−), its receptor IL18r (Il18r−/−), or soluble receptors demonstrate the contribution of IL18 to several inflammatory diseases9–11. Atherosclerotic lesions but not normal human aortas express high levels of IL18 and IL18r in macrophages, T cells, endothelial cells (ECs), and smooth-muscle cells (SMCs)12,13. In atherosclerosis-prone apolipoprotein E–deficient (Apoe−/−) mice, the absence of IL18 or blockade of IL18 signaling reduces atherosclerosis and lesion inflammation3,4. Intraperitoneal administration of IL18 in Apoe−/− mice enhances lesion burden and inflammation14,15. IL18r is a heterodimer with a low IL18-binding affinity16. Absence of IL18r leads to reduced NK cell IFN-γ production and impaired Th1 cell signaling and differentiation17. Yet, Apoe−/− and Apoe−/−Il18r−/− littermates showed no differences in atherogenesis. In Apoe−/− mice, the presence or absence of IL18r did not affect aortic root intimal area, or lesion macrophage, CD4+ T-cell, or SMC contents (Fig. 1a) — suggesting IL18 action on target cells that is independent of, or in addition to, the known IL18r. Mouse ECs from Apoe−/− and Apoe−/−Il18r−/− mice incubated with biotinylated IL18, showed no differences in cell surface IL18 binding, competable by a 5~10-fold excess of unlabeled IL18 (Fig. 1b). ECs from Apoe−/− and Apoe−/−Il18r−/− mice showed a similar pattern of protein tyrosine phosphorylation after 30 minutes of incubation with IL18, as detected by immunoblot with anti-phospho (p)-tyrosine antibody. Heat-inactivated IL18 did not elicit these activities (Fig. 1c). These data pointed to the presence of alternative molecules that mediate cell-surface IL18 binding and downstream signaling. Incubation of ECs from Apoe−/−Il18r−/− mice with IL18 for 15 minutes followed by immunoprecipitation with an anti-mouse IL18 polyclonal antibody identified possible cell-surface IL18-binding molecules. Silver-stained SDS-PAGE showed two major bands between 85 to 150 kDa in series eluates from IL18-treated ECs (Fig. 1d). Mass spectrometry identified a 125-kDa Na-Cl co-transporter (NCC) in addition to multiple matrix protein fragments (data not shown).

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