title: Binding of CD40L to Mac-1's I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis--but does not affect immunity and thrombosis in mice.
abstract: BACKGROUND
  CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor.
  
  
  OBJECTIVE
  Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo.
  
  
  RESULTS
  CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo.
  
  
  CONCLUSIONS
  We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.
authors: Atherogenesis Research Group, Department of Cardiology, University of Freiburg, Germany.
authors: Dennis
authors: D
authors: Wolf
authors: Jan-David
authors: JD
authors: Hohmann
authors: Ansgar
authors: A
authors: Wiedemann
authors: Kamila
authors: K
authors: Bledzka
authors: Hermann
authors: H
authors: Blankenbach
authors: Timoteo
authors: T
authors: Marchini
authors: Katharina
authors: K
authors: Gutte
authors: Katharina
authors: K
authors: Zeschky
authors: Nicole
authors: N
authors: Bassler
authors: Natalie
authors: N
authors: Hoppe
authors: Alexandra Ortiz
authors: AO
authors: Rodriguez
authors: Nadine
authors: N
authors: Herr
authors: Ingo
authors: I
authors: Hilgendorf
authors: Peter
authors: P
authors: Stachon
authors: Florian
authors: F
authors: Willecke
authors: Daniel
authors: D
authors: Duerschmied
authors: Constantin
authors: C
authors: von zur Muhlen
authors: Dmitry A
authors: DA
authors: Soloviev
authors: Li
authors: L
authors: Zhang
authors: Christoph
authors: C
authors: Bode
authors: Edward F
authors: EF
authors: Plow
authors: Peter
authors: P
authors: Libby
authors: Karlheinz
authors: K
authors: Peter
authors: Andreas
authors: A
authors: Zirlik
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D050197:Atherosclerosis / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control; D001760:Bleeding Time; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology; D023201:CD40 Ligand / Q000378:metabolism*; D016466:CHO Cells; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D006224:Cricetinae; D006801:Humans; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008958:Models, Molecular; D010446:Peptide Fragments / Q000494:pharmacology / Q000627:therapeutic use; D010456:Peptides, Cyclic / Q000494:pharmacology; D010538:Peritonitis / Q000097:blood / Q000517:prevention & control; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000172:deficiency; D011993:Recombinant Fusion Proteins / Q000502:physiology; D020349:Surface Plasmon Resonance; D013927:Thrombosis / Q000209:etiology*
chemical_list: D016177:Macrophage-1 Antigen; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand