Abstract 19725: The CD40L/Mac-1 Dyad Recruits Leukocytes During Cardiovascular Inflammation

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Background: The integrin Mac-1 (CD11b/CD18) drives myeloid cell recruitment during inflammation in cardiovascular disease. Although integrins have generated interest as therapeutic targets in acute and chronic inflammation, they contribute to host defense, regeneration, and haemostasis. To overcome this limitation, we have designed a novel antibody that targets a distinct region within the Mac-1 major ligand-binding (I-) domain required for binding of its ligand CD40L. Here, we describe the consequences of such ligand-specific integrin therapy in mice.

Methods and Results: To generate an antibody specifically targeting the interaction of Mac-1 with CD40L, mice were immunized with a peptide containing the CD40L-binding motif EQLKKSKTL. Antibody clones were screened and tested for specificity. One clone, termed anti-M7, showed specific peptide binding and blocked the binding of Mac-1 to CD40L, but not to alternative Mac-1 ligands. Anti-M7 highly effectively limited leukocyte recruitment in vivo to the mesentery and the omentum as monitored by intravital confocal microscopy. While conventional anti-Mac-1 antibodies induced outside-in activation of the integrin, anti-M7 did not provoke MAP-kinase dependent activation and expression of inflammatory cytokines in macrophages. Following cecal-ligation and puncture (CLP) sepsis in mice, a conventional anti-Mac-1 antibody inhibited leukocyte accumulation, but potentiated bacteremia and limited overall survival. In contrast, anti-M7 limited peritoneal leukocyte accumulation, reduced bacterial titers, and improved survival during CLP. In contrast to conventional anti-Mac-1 antibodies, anti-M7 did not provoke mesenteric thrombosis.

Conclusion: We present a proof-of-concept demonstrating selective inhibition of the interaction between the leukocyte integrin Mac-1 and CD40L by a novel monoclonal antibody. This antibody shows several advantages over conventional anti-integrin therapy, and prevented inflammation and sepsis-related mortality in mice. This novel approach gives new insight into ligand-specific integrin pathways and merits further evaluation as a selective anti-inflammatory therapy in cardiovascular disease.

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