Regulatory Role of Endothelial CD47 during Leukocyte Transmigration

Abstract

The vascular endothelium plays a critical role in inflammation and leukocyte recruitment by expression of adhesion molecules and leukocyte chemoattractants. Adhesion molecules bind leukocytes and transmit signals to the endothelial cells (EC) required for transendothelial migration (TEM). An important unanswered question is how ECs integrate the signals arising from leukocyte engagement of multiple adhesion molecules. CD47, also known as Integrin Associated Protein, is a broadly expressed glycoprotein and binds thrombospondin‐1 and leukocyte expressed Signal Regulatory Proteins (SIRPα and SIRPg). Besides these “in trans” interactions, CD47 also interacts “in cis” (in the same plasma membrane) with multiple integrins. We previously reported that endothelial CD47 plays a role in TEM and transmits intracellular signals upon engagement. Interestingly, CD47 signals phenocopy those occurring after engagement of ICAM‐1. We hypothesized CD47 interacts with ICAM‐1 to form a protein complex in EC plasma membrane and that this complex binds leukocytes and transmits intracellular signals during leukocyte TEM. Here we report CD47 associates with ICAM‐1 in EC by Fluorescence Lifetime Imaging Microscopy of Förster Resonance Energy Transfer (FLIM‐FRET). Furthermore, ICAM‐1 crosslinking‐induced signaling through pyk2 and src kinases was impaired in CD47‐/‐ mouse heart ECs (MHEC) as compared to WT MHEC. This was also demonstrated in HUVEC silenced for CD47. Understanding how CD47 interacts with different molecules will clarify its role in leukocyte recruitment and EC dependent mechanisms that regulate TEM.Supported by grants from National Research Fund of Luxembourg (FNR, Award # 4800397) and NIH HL‐36028.