Regulation of LFA-1-dependent inflammatory cell recruitment by Cb1-b and 14-3-3 proteins

Inside-out signaling regulation of the beta 2-integrin leukocyte function-associated antigen-1 (LFA-1) by different cytoplasmic proteins, including 14-3-3 proteins, is essential for adhesion and migration of immune cells. Here, we identify a new pathway for the regulation of LFA-1 activity by Cbl-b, an adapter molecule and ubiquitin ligase that modulates several signaling pathways. Cbl-b-1- mice displayed increased macrophage recruitment in thioglycollate-induced peritonitis, which was attributed to CbI-b deficiency in macrophages, as assessed by bone marrow chimera experiments. In vitro, Cbl-b(-1-) bone marrow-derived mononuclear phagocytes (BMDMs) displayed increased adhesion to endothelial cells. Activation of LFA-1 in Cbl-b-deficient cells was responsible for their increased endothelial adhesion in vitro and peritoneal recruitment in vivo, as the phenotype of Cbl-b deficiency was reversed in Cbl-b(-/-)LFA-1 (-/-) mice. Consistently, LFA-1-mediated adhesion of BMDM to ICAM-1 but not VLA-4-mediated adhesion to VCAM-1 was enhanced by CbI-b deficiency. Cbl-b deficiency resulted in increased phosphorylation of T758 in the beta 2-chain of LFA-1 and thereby in enhanced association of 14-3-30 protein with the beta 2-chain, leading to activation of LFA-1. Consistently, disruption of the 14-3-3/beta 2-integrin interaction abrogated the enhanced ICAM-1 adhesion of Cbl-b-1- BMDMs. In conclusion, CbI-b deficiency activates LFA-1 and LFA-1-mediated inflammatory cell recruitment by stimulating the interaction between the LFA-1 beta-chain and 14-3-3 proteins.