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



Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms1,2. Interruption of IL18 action reduces atherosclerosis in mice3,4. 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 kidney5. 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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Figure 1: Identification of alternative IL18-binding proteins.
Figure 2: NCC expression and characterization.
Figure 3: IL18r and NCC function in atherosclerosis.
Figure 4: NCC mediates IL18 signaling and downstream cytokine and chemokine production in macrophages, COS-7 cells and FlpIn-293 cells.


  1. 1

    Dinarello, C.A. IL-18: A TH1-inducing, proinflammatory cytokine and new member of the IL-1 family. J. Allergy Clin. Immunol. 103, 11–24 (1999).

    CAS  Article  Google Scholar 

  2. 2

    Yoshimoto, T. et al. IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-gamma production. J. Immunol. 161, 3400–3407 (1998).

    CAS  PubMed  Google Scholar 

  3. 3

    Elhage, R. et al. Reduced atherosclerosis in interleukin-18 deficient apolipoprotein E-knockout mice. Cardiovasc. Res. 59, 234–240 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Mallat, Z. et al. Interleukin-18/interleukin-18 binding protein signaling modulates atherosclerotic lesion development and stability. Circ. Res. 89, E41–E45 (2001).

    CAS  PubMed  Google Scholar 

  5. 5

    Gamba, G. et al. Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney. J. Biol. Chem. 269, 17713–17722 (1994).

    CAS  PubMed  Google Scholar 

  6. 6

    Okamura, H., Kashiwamura, S., Tsutsui, H., Yoshimoto, T. & Nakanishi, K. Regulation of interferon-gamma production by IL-12 and IL-18. Curr. Opin. Immunol. 10, 259–264 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Konishi, H. et al. IL-18 contributes to the spontaneous development of atopic dermatitis-like inflammatory skin lesion independently of IgE/stat6 under specific pathogen-free conditions. Proc. Natl. Acad. Sci. USA 99, 11340–11345 (2002).

    CAS  Article  Google Scholar 

  8. 8

    French, A.R., Holroyd, E.B., Yang, L., Kim, S. & Yokoyama, W.M. IL-18 acts synergistically with IL-15 in stimulating natural killer cell proliferation. Cytokine 35, 229–234 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Netea, M.G. et al. Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance. Nat. Med. 12, 650–656 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Sugiyama, M. et al. Deletion of IL-18 receptor ameliorates renal injury in bovine serum albumin-induced glomerulonephritis. Clin. Immunol. 128, 103–108 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Gutcher, I., Urich, E., Wolter, K., Prinz, M. & Becher, B. Interleukin 18-independent engagement of interleukin 18 receptor-alpha is required for autoimmune inflammation. Nat. Immunol. 7, 946–953 (2006).

    CAS  Article  Google Scholar 

  12. 12

    Mallat, Z. et al. Expression of interleukin-18 in human atherosclerotic plaques and relation to plaque instability. Circulation 104, 1598–1603 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Gerdes, N. et al. Expression of interleukin (IL)-18 and functional IL-18 receptor on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for atherogenesis. J. Exp. Med. 195, 245–257 (2002).

    CAS  Article  Google Scholar 

  14. 14

    Whitman, S.C., Ravisankar, P. & Daugherty, A. Interleukin-18 enhances atherosclerosis in apolipoprotein E(−/−) mice through release of interferon-gamma. Circ. Res. 90, E34–E38 (2002).

    CAS  Article  Google Scholar 

  15. 15

    Tenger, C., Sundborger, A., Jawien, J. & Zhou, X. IL-18 accelerates atherosclerosis accompanied by elevation of IFN-gamma and CXCL16 expression independently of T cells. Arterioscler. Thromb. Vasc. Biol. 25, 791–796 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Torigoe, K. et al. Purification and characterization of the human interleukin-18 receptor. J. Biol. Chem. 272, 25737–25742 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Hoshino, K. et al. Cutting edge: generation of IL-18 receptor-deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor. J. Immunol. 162, 5041–5044 (1999).

    CAS  PubMed  Google Scholar 

  18. 18

    Kunchaparty, S. et al. Defective processing and expression of thiazide-sensitive Na-Cl cotransporter as a cause of Gitelman's syndrome. Am. J. Physiol. 277, F643–F649 (1999).

    CAS  PubMed  Google Scholar 

  19. 19

    Gamba, G. et al. Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter. Proc. Natl. Acad. Sci. USA 90, 2749–2753 (1993).

    CAS  Article  Google Scholar 

  20. 20

    Schultheis, P.J. et al. Phenotype resembling Gitelman's syndrome in mice lacking the apical Na+-Cl cotransporter of the distal convoluted tubule. J. Biol. Chem. 273, 29150–29155 (1998).

    CAS  Article  Google Scholar 

  21. 21

    Gjata, M., Tase, M., Gjata, A. & Gjergji, Zh. Gitelman's syndrome (familial hypokalemia-hypomagnesemia). Hippokratia. 11, 150–153 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Morris, R.G., Hoorn, E.J. & Knepper, M.A. Hypokalemia in a mouse model of Gitelman's syndrome. Am. J. Physiol. Renal Physiol. 290, F1416–F1420 (2006).

    CAS  Article  Google Scholar 

  23. 23

    Tzanakis, I. et al. Intra- and extracellular magnesium levels and atheromatosis in haemodialysis patients. Magnes. Res. 17, 102–108 (2004).

    CAS  PubMed  Google Scholar 

  24. 24

    Hulthe, J. et al. Plasma interleukin (IL)-18 concentrations is elevated in patients with previous myocardial infarction and related to severity of coronary atherosclerosis independently of C-reactive protein and IL-6. Atherosclerosis 188, 450–454 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Blankenberg, S. et al. Interleukin-18 is a strong predictor of cardiovascular death in stable and unstable angina. Circulation 106, 24–30 (2002).

    CAS  Article  Google Scholar 

  26. 26

    Lauwerys, B.R., Renauld, J.C. & Houssiau, F.A. Synergistic proliferation and activation of natural killer cells by interleukin 12 and interleukin 18. Cytokine 11, 822–830 (1999).

    CAS  Article  Google Scholar 

  27. 27

    Brunet, A. et al. Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J. 18, 664–674 (1999).

    CAS  Article  Google Scholar 

  28. 28

    Ono, K. & Han, J. The p38 signal transduction pathway: activation and function. Cell. Signal. 12, 1–13 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Hossain Khan, M.Z. et al. Phosphorylation of Na-Cl cotransporter by OSR1 and SPAK kinases regulates its ubiquitination. Biochem. Biophys. Res. Commun. 425, 456–461 (2012).

    CAS  Article  Google Scholar 

  30. 30

    Yang, S.S. et al. Generation and analysis of the thiazide-sensitive Na+-Cl cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a model of Gitelman syndrome. Hum. Mutat. 31, 1304–1315 (2010).

    CAS  Article  Google Scholar 

  31. 31

    Pacheco-Alvarez, D. et al. The Na+:Cl cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion. J. Biol. Chem. 281, 28755–28763 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Rozansky, D.J. et al. Aldosterone mediates activation of the thiazide-sensitive Na-Cl cotransporter through an SGK1 and WNK4 signaling pathway. J. Clin. Invest. 119, 2601–2612 (2009).

    CAS  Article  Google Scholar 

  33. 33

    Griffon, N., Jeanneteau, F., Prieur, F., Diaz, J. & Sokoloff, P. CLIC6, a member of the intracellular chloride channel family, interacts with dopamine D(2)-like receptors. Brain Res. Mol. Brain Res. 117, 47–57 (2003).

    CAS  Article  Google Scholar 

  34. 34

    Heim, M.H. The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus. J. Recept. Signal Transduct. Res. 19, 75–120 (1999).

    CAS  Article  Google Scholar 

  35. 35

    De Jong, J.C. et al. Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome. J. Am. Soc. Nephrol. 13, 1442–1448 (2002).

    CAS  Article  Google Scholar 

  36. 36

    Glaudemans, B. et al. Novel NCC mutants and functional analysis in a new cohort of patients with Gitelman syndrome. Eur. J. Hum. Genet. 20, 263–270 (2012).

    CAS  Article  Google Scholar 

  37. 37

    Hoorn, E.J. et al. The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension. Nat. Med. 17, 1304–1309 (2011).

    CAS  Article  Google Scholar 

  38. 38

    Reilly, R.F. & Ellison, D.H. Mammalian distal tubule: physiology, pathophysiology, and molecular anatomy. Physiol. Rev. 80, 277–313 (2000).

    CAS  Article  Google Scholar 

  39. 39

    Miyauchi, K., Takiyama, Y., Honjyo, J., Tateno, M. & Haneda, M. Upregulated IL-18 expression in type 2 diabetic subjects with nephropathy: TGF-beta1 enhanced IL-18 expression in human renal proximal tubular epithelial cells. Diabetes Res. Clin. Pract. 83, 190–199 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Kinoshita, K. et al. Blockade of IL-18 receptor signaling delays the onset of autoimmune disease in MRL-Faslpr mice. J. Immunol. 173, 5312–5318 (2004).

    CAS  Article  Google Scholar 

  41. 41

    Sukhova, G.K. et al. Deficiency of cathepsin S reduces atherosclerosis in LDL receptor-deficient mice. J. Clin. Invest. 111, 897–906 (2003).

    CAS  Article  Google Scholar 

  42. 42

    Mach, F., Schönbeck, U., Sukhova, G.K., Atkinson, E. & Libby, P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 394, 200–203 (1998).

    CAS  Article  Google Scholar 

  43. 43

    Zhang, M.Z. et al. Role of blood pressure and the renin-angiotensin system in development of diabetic nephropathy (DN) in eNOS−/−db/db mice. Am. J. Physiol. Renal Physiol. 302, F433–F438 (2012).

    CAS  Article  Google Scholar 

  44. 44

    Bostanjoglo, M. et al. 11Beta-hydroxysteroid dehydrogenase, mineralocorticoid receptor, and thiazide-sensitive Na-Cl cotransporter expression by distal tubules. J. Am. Soc. Nephrol. 9, 1347–1358 (1998).

    CAS  PubMed  Google Scholar 

  45. 45

    Zhang, J. et al. Regulation of endothelial cell adhesion molecule expression by mast cells, macrophages, and neutrophils. PLoS ONE 6, e14525 (2011).

    CAS  Article  Google Scholar 

  46. 46

    Shi, G.P. et al. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity 10, 197–206 (1999).

    CAS  Article  Google Scholar 

  47. 47

    Riveira-Munoz, E. et al. Transcriptional and functional analyses of SLC12A3 mutations: new clues for the pathogenesis of Gitelman syndrome. J. Am. Soc. Nephrol. 18, 1271–1283 (2007).

    CAS  Article  Google Scholar 

  48. 48

    Markadieu, N. et al. A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport. Am. J. Physiol. Renal Physiol. 303, F886–F892 (2012).

    CAS  Article  Google Scholar 

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This study is supported by grants from the US National Heart, Lung, and Blood Institute (HL60942, HL81090, HL88547 to G.-P.S.; HL34636, HL80472 to P.L.) and by an American Heart Association Established Investigator Award (0840118N to G.-P.S.).

Author information




J.W. and C.S. performed most of the experiments. N.G. completed the original IL18 and IL18r mutant mouse analysis. C.L., M.L., M.A.S., A.H., Y.Z., H.C., J.Z., X.C. and Q.K. performed RT-PCR, lesion analysis, cell culture and plasma ELISA. J.L. helped with the NCC cDNA cloning. G.K.S. performed immunostaining. X.W.C., M.K., T.M. and P.L. helped with experimental design, writing and data interpretation. M.J. and G.E.S. provided the NCC mutant mice. S.R., C.-L.Y. and D.H.E. provided the NCC monoclonal antibody and performed the 293 cell experiments. S.J. and R.B. made the human NCC mutant constructs. P.J. measured plasma Mg and K. G.-P.S. designed and performed the experiments and wrote the manuscript.

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Correspondence to Guo-Ping Shi.

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The authors declare no competing financial interests.

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Wang, J., Sun, C., Gerdes, N. et al. Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter. Nat Med 21, 820–826 (2015).

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