No abstract

Rationale CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and haemostasis 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. Methods and 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. cM7, a cyclisized version optimized for in vivo use, 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.

Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.