Abstract
Atherosclerosis is characterized by chronic inflammation of the arterial wall due to chemokine-driven mononuclear cell recruitment1,2,3,4. Activated platelets can synergize with chemokines to exacerbate atherogenesis; for example, by deposition of the chemokines platelet factor-4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium5,6,7,8,9. Homo-oligomerization is required for the recruitment functions of CCL5, and chemokine heteromerization has more recently emerged as an additional regulatory mechanism, as evidenced by a mutual modulation of CXCL8 and CXCL4 activities and by enhanced monocyte arrest resulting from CCL5-CXCL4 interactions10,11,12,13. The CCL5 antagonist Met-RANTES reduces diet-induced atherosclerosis9,14; however, CCL5 antagonism may not be therapeutically feasible, as suggested by studies using Ccl5-deficient mice which imply that direct CCL5 blockade would severely compromise systemic immune responses, delay macrophage-mediated viral clearance and impair normal T cell functions15,16. Here we determined structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt proinflammatory CCL5-CXCL4 interactions, thereby attenuating monocyte recruitment and reducing atherosclerosis without the aforementioned side effects. These results establish the in vivo relevance of chemokine heteromers and show the potential of targeting heteromer formation to achieve therapeutic effects.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Hansson, G.K. Inflammation, atherosclerosis, and coronary artery disease. N. Engl. J. Med. 352, 1685–1695 (2005).
Weber, C., Zernecke, A. & Libby, P. The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models. Nat. Rev. Immunol. 8, 802–815 (2008).
Zernecke, A., Shagdarsuren, E. & Weber, C. Chemokines in atherosclerosis: an update. Arterioscler. Thromb. Vasc. Biol. 28, 1897–1908 (2008).
Charo, I.F. & Taubman, M.B. Chemokines in the pathogenesis of vascular disease. Circ. Res. 95, 858–866 (2004).
Weber, C. Platelets and chemokines in atherosclerosis: partners in crime. Circ. Res. 96, 612–616 (2005).
Weyrich, A.S. & Zimmerman, G.A. Platelets: signaling cells in the immune continuum. Trends Immunol. 25, 489–495 (2004).
von Hundelshausen, P. et al. RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium. Circulation 103, 1772–1777 (2001).
Huo, Y. et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat. Med. 9, 61–67 (2003).
Schober, A. et al. Deposition of platelet RANTES triggering monocyte recruitment requires P-selectin and is involved in neointima formation after arterial injury. Circulation 106, 1523–1529 (2002).
Baltus, T., Weber, K.S., Johnson, Z., Proudfoot, A.E. & Weber, C. Oligomerization of RANTES is required for CCR1-mediated arrest but not CCR5-mediated transmigration of leukocytes on inflamed endothelium. Blood 102, 1985–1988 (2003).
Proudfoot, A.E. et al. Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. USA 100, 1885–1890 (2003).
Nesmelova, I.V. et al. Platelet factor 4 and interleukin-8 CXC chemokine heterodimer formation modulates function at the quaternary structural level. J. Biol. Chem. 280, 4948–4958 (2005).
von Hundelshausen, P. et al. Heterophilic interactions of platelet factor 4 and RANTES promote monocyte arrest on endothelium. Blood 105, 924–930 (2005).
Veillard, N.R. et al. Antagonism of RANTES receptors reduces atherosclerotic plaque formation in mice. Circ. Res. 94, 253–261 (2004).
Makino, Y. et al. Impaired T cell function in RANTES-deficient mice. Clin. Immunol. 102, 302–309 (2002).
Tyner, J.W. et al. CCL5–CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection. Nat. Med. 11, 1180–1187 (2005).
Linton, M.F., Atkinson, J.B. & Fazio, S. Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation. Science 267, 1034–1037 (1995).
Sachais, B.S. et al. Elimination of platelet factor 4 (PF4) from platelets reduces atherosclerosis in C57Bl/6 and apoE−/− mice. Thromb. Haemost. 98, 1108–1113 (2007).
Rajagopal, P., Waygood, E.B., Reizer, J., Saier, M.H. & Jr & Klevit, R.E. Demonstration of protein-protein interaction specificity by NMR chemical shift mapping. Protein Sci. 6, 2624–2627 (1997).
Nesmelova, I.V., Idiyatullin, D. & Mayo, K.H. Measuring protein self-diffusion in protein–protein mixtures using a pulsed gradient spin-echo technique with WATERGATE and isotope filtering. J. Magn. Reson. 166, 129–133 (2004).
Mayo, K.H. & Chen, M.J. Human platelet factor 4 monomer-dimer-tetramer equilibria investigated by 1H NMR spectroscopy. Biochemistry 28, 9469–9478 (1989).
Clore, G.M. & Gronenborn, A.M. Three-dimensional structures of alpha and beta chemokines. FASEB J. 9, 57–62 (1995).
Sticht, H. et al. Solution structure of the human CC chemokine 2: a monomeric representative of the CC chemokine subtype. Biochemistry 38, 5995–6002 (1999).
Kim, K.S., Rajarathnam, K., Clark-Lewis, I. & Sykes, B.D. Structural characterization of a monomeric chemokine: monocyte chemoattractant protein-3. FEBS Lett. 395, 277–282 (1996).
Ilyina, E., Milius, R. & Mayo, K.H. Synthetic peptides probe folding initiation sites in platelet factor-4: stable chain reversal found within the hydrophobic sequence LIATLKNGRKISL. Biochemistry 33, 13436–13444 (1994).
Sorensen, L.N. & Paludan, S.R. Blocking CC chemokine receptor (CCR) 1 and CCR5 during herpes simplex virus type 2 infection in vivo impairs host defence and perturbs the cytokine response. Scand. J. Immunol. 59, 321–333 (2004).
Anders, H.J. et al. CC chemokine ligand 5/RANTES chemokine antagonists aggravate glomerulonephritis despite reduction of glomerular leukocyte infiltration. J. Immunol. 170, 5658–5666 (2003).
Paoletti, S. et al. A rich chemokine environment strongly enhances leukocyte migration and activities. Blood 105, 3405–3412 (2005).
Crown, S.E., Yu, Y., Sweeney, M.D., Leary, J.A. & Handel, T.M. Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding. J. Biol. Chem. 281, 25438–25446 (2006).
Weber, C. & Koenen, R.R. Fine-tuning leukocyte responses: towards a chemokine 'interactome'. Trends Immunol. 27, 268–273 (2006).
Hackeng, T.M., Griffin, J.H. & Dawson, P.E. Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. Proc. Natl. Acad. Sci. USA 96, 10068–10073 (1999).
Halden, Y. et al. Interleukin-8 binds to syndecan-2 on human endothelial cells. Biochem. J. 377, 533–538 (2004).
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
Johnson, B.A. & Blevins, R.A. NMRView: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).
Chung, C.W., Cooke, R.M., Proudfoot, A.E. & Wells, T.N. The three-dimensional solution structure of RANTES. Biochemistry 34, 9307–9314 (1995).
Skelton, N.J., Aspiras, F., Ogez, J. & Schall, T.J. Proton NMR assignments and solution conformation of RANTES, a chemokine of the C–C type. Biochemistry 34, 5329–5342 (1995).
Duma, L., Häussinger, D., Rogowski, M., Lusso, P. & Grzesiek, S. Recognition of RANTES by extracellular parts of the CCR5 receptor. J. Mol. Biol. 365, 1063–1075 (2007).
Nesmelova, I.V., Sham, Y., Gao, J. & Mayo, K.H. CXC and CC chemokines form mixed heterodimers: association free energies from molecular dynamics simulations and experimental correlations. J. Biol. Chem. 283, 24155–24166 (2008).
Brooks, B.R. et al. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217 (1983).
Zernecke, A. et al. SDF-1α/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells. Circ. Res. 96, 784–791 (2005).
Braunersreuther, V. et al. Ccr5 but not Ccr1 deficiency reduces development of diet-induced atherosclerosis in mice. Arterioscler. Thromb. Vasc. Biol. 27, 373–379 (2007).
Bernhagen, J. et al. MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat. Med. 13, 587–596 (2007).
Acknowledgements
This study was supported by grants from the Deutsche Forschungsgemeinschaft (WE1913/5-2, WE1913/7-1 to C.W., KO2948/1-1 to R.R.K., HU1618/1-1 to P.v.H., ZE827/1-1 to A.Z. and FOR809 to R.R.K., P.v.H., A.Z. and C.W.), the Interdisciplinary Center for Clinical Research “Biomat” within the Medical Faculty of RWTH Aachen University (TV-B112 and TV-B113 to R.R.K. and C.W.), Nederlandse Organisatie voor Wetenschappelijk Onderzoek (VIDI 917.36.372 to T.M.H.) and the US National Institutes of Health (National Research Service Award training grant HL 07062 to I.V.N.). We thank S. Meiler, S. Winkler, J. Tupiec, S. Knarren, M. Garbe, S. Wilbertz, D. Suylen and W. Adriaens for technical assistance. Computer resources were provided by the Minnesota Supercomputing Institute (University of Minnesota). NMR instrumentation was provided with funds from the US National Science Foundation (BIR-961477), the University of Minnesota Medical School and the Minnesota Medical Foundation. Met-RANTES was provided by P. Nelson (University of Munich). NMR chemical-shift assignments for the CCL5 monomer state were provided by S. Grzesiek (University of Basel).
Author information
Authors and Affiliations
Contributions
R.R.K. designed the peptides; designed, supervised and conducted experiments; and wrote the paper. P.v.H. designed and conducted experiments. I.V.N. designed, conducted and analyzed NMR studies. A.Z. designed, conducted and supervised animal experiments. E.A.L. conducted animal experiments. A.S. and B.K.K. expressed and purified recombinant proteins. A.M.P. conducted biophysical experiments. M.A.K. supplied transgenic mice. S.R.P. conducted viral clearance experiments in mice. A.J.K. designed, supervised and analyzed biophysical experiments. T.M.H. synthesized peptide inhibitors. K.H.M. designed, supervised and analyzed NMR studies and wrote the paper. C.W. designed and supervised the study and wrote the paper.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figs. 1–6 and Supplementary Methods (PDF 434 kb)
Rights and permissions
About this article
Cite this article
Koenen, R., von Hundelshausen, P., Nesmelova, I. et al. Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. Nat Med 15, 97–103 (2009). https://doi.org/10.1038/nm.1898
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nm.1898
This article is cited by
-
Platelet-derived exerkine CXCL4/platelet factor 4 rejuvenates hippocampal neurogenesis and restores cognitive function in aged mice
Nature Communications (2023)
-
A new obligate CXCL4–CXCL12 heterodimer for studying chemokine heterodimer activities and mechanisms
Scientific Reports (2022)
-
Immune cell-mediated features of non-alcoholic steatohepatitis
Nature Reviews Immunology (2022)
-
Stable CAD patients show higher levels of platelet-borne TGF-β1 associated with a superior pro-inflammatory state than the pro-aggregatory status; Evidence highlighting the importance of platelet-derived TGF-β1 in atherosclerosis
Journal of Thrombosis and Thrombolysis (2022)
-
Platelets fine-tune effector responses of naïve CD4+ T cells via platelet factor 4-regulated transforming growth factor β signaling
Cellular and Molecular Life Sciences (2022)