Gnb isoforms control a signaling pathway comprising Rac1, Plc beta 2, and Plc beta 3 leading to LFA-1 activation and neutrophil arrest in vivo

Block H, Stadtmann A, Riad D, Rossaint J, Sohlbach C, Germena G, Wu D, Simon SI, Ley K, Zarbock A

Zusammenfassung

Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the alpha and beta subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C beta 2 (Plc beta 2) and Plc beta 3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca2+ flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plc beta 2-, Plc beta 3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plc beta 2, and Plc beta 3 are critically involved in integrin activation and leukocyte arrest.