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

• Gnb isoforms are centrally involved in Rac1-dependent chemokine-induced LFA-1 activation. • Plcb2 and Plcb3 function nonredundantly to produce inositol triphosphate with subsequent calcium flux leading to LFA-1 activation. 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 a and b subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstreamsignaling, relatively little is knownabout 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, andGNB5) 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 toRas-relatedC3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipaseCb2 (Plcb2) and Plcb3. They act nonredundantly to produce inositol triphosphate–mediated intracellular Ca flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcb2-, Plcb3-, 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, Plcb2, and Plcb3 are critically involved in integrin activation and leukocyte arrest. (Blood. 2016;127(3):314-324)