Abstract P130: Inflammation-Mediated Modification Of Extracellular Matrix Generates A New Adhesive Substrate For Integrin α _D β ₂ -Dependent Macrophage Retention During Chronic Inflammation

Abstract

The accumulation of pro-inflammatory macrophages in the inflamed vascular wall is a critical step in atherogenesis. The mechanism of macrophage retention within the site of inflammation is not understood yet. High adhesion that prevents macrophage migration is one of the potential mechanisms. We previously showed that integrin α D β 2 is upregulated on pro-inflammatory macrophages, promotes macrophage retention, and contributes to atherogenesis. However, we have not identified a key ligand for α D β 2 within the tissue, since α D β 2 does not interact with major ECM proteins, collagens, and laminins. We recently found that during acute inflammation, the oxidation of docosahexaenoic acid (DHA) leads to the generation of end product carboxyethylpyrrole (CEP), which forms an adduct with fibrinogen and albumin via ε-amino group of lysines. Moreover, we revealed that macrophages adhered to CEP-modified albumin in α D β 2 -dependent manner. Now we are testing a hypothesis that DHA oxidation is a universal mechanism during chronic inflammatory diseases that promotes the generation of CEP adducts with different ECM proteins and forms α D β 2 -mediated strong anchorage of macrophages, which is critical for macrophage retention during chronic inflammation. We detected CEP-modified proteins in digested atherosclerotic lesions by western blot. In vitro DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin but not with collagen I. Using α D β 2 -transfected HEK293 cells, WT and α D -/- mouse macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading to compare with natural macrophage ligands. Using site-directed mutagenesis, we generated mutant α D I-domains and α D β 2 -transfected cells with single amino acid substitutions. Applying protein-protein binding and adhesion assays we detected one amino acid within integrin α D , K 246 , which is critical for α D β 2 binding to CEP-modified proteins. In summary, we propose a new mechanism of macrophage retention, which is based on inflammatory modifications of ECM with DHA end-product, CEP. The identification of a binding site for CEP-modified proteins within α D β 2 will help to develop a blocking reagent for the treatment of the inflammatory component of atherosclerosis.