Epitope mapping and characterization of two endotoxin-binding motifs in leukocyte integrin I-like domain - potential application for liver sepsis therapy

Abstract

Patients with post-operative sepsis and acute liver failure are at highrisk of hospital mortality.The molecular and cellular mechanisms areattributed to the systemic release of bacterial endotoxin (or LPS) incirculation and accumulation of inflamed neutrophils and monocytesin damaged organs. Recruitment of the immune cells to the sites ofinjury and inflamed tissues are mediated by complex cell-cell and cell-extracellular matrix interactions. Integrin CD11/CD18 is one of thekey players responsible for circulation and migration of leukocytesduring immune surveillance. As such, integrin has been the primarytargets for anti-adhesion therapy to attenuate hyper-inflammatory disorders. Blockade of the integrin adhesion by anti-CD18 antibodyor antagonistic peptides can ameliorate liver damage and thus prolongorgan/graft survival. The present study aims to exploit newapproaches for preventing endotoxin-mediated liver injury in experi-mental sepsis animal models. The leukocyte integrin beta2 subunit has been identified as receptor for bacterial endotoxin, and the CD18antigen was shown to bind LPS in a dose-dependent manner. Grosstruncation mapping analysis revealed the I-like (or βA) domain to bethe LPS-recognition sites.We employed a bacterial expression systemto produce recombinant protein fragments spanning the I-Likedomain of CD18, and their LPS-binding abilities have been assayedby enzyme-linked immunosorbent assay (ELISA). Region enclosedby 190thand 319thamino-acid residues were identified to be the mostcritical sites to sustain LPS binding. Bioinformatic homology scan-ning suggested the I-like domain is composed of two putative LPS-binding sequences, herein designated as Motif A and B. Furthermore,binding affinity constant values for the CD18-LPS interaction weredetermined by BIAcore plasma resonance. Because systemic releaseof TNF-α from monocyte and lymphocyte is central to liver injury,an in vitro study of inhibition of TNF-α production from lymphocyteby recombinant CD18 peptide is also evaluated. Preliminary datasuggested that recombinant CD18 Motif B peptide could down-regulate the TNF-α transcript level in LPS-activated Jurkat T cells.The present work led us to further develop novel intervention strat-egy to endotoxemia, and evaluate the usefulness of recombinantCD18 peptide in the prophylaxis of endotoxin-mediated liver injuryin cecal ligation puncture mouse model.(Supported by Research Grants Council of Hong Kong:HKU7280/02M)