title: Gnb isoforms control a signaling pathway comprising Rac1, Plcβ2, and Plcβ3 leading to LFA-1 activation and neutrophil arrest in vivo.
abstract: 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 α and β 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 β2 (Plcβ2) and Plcβ3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca(2+) flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcβ2-, Plcβ3-, 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, Plcβ2, and Plcβ3 are critically involved in integrin activation and leukocyte arrest.
authors.affiliation: Department of Anaesthesiology, Intensive Care and Pain Medicine, University of Muenster, Muenster, Germany;
authors.forename: Helena
authors.initials: H
authors.lastname: Block
authors.affiliation: Department of Anaesthesiology, Intensive Care and Pain Medicine, University of Muenster, Muenster, Germany;
authors.forename: Anika
authors.initials: A
authors.lastname: Stadtmann
authors.affiliation: Department of Anaesthesiology, Intensive Care and Pain Medicine, University of Muenster, Muenster, Germany;
authors.forename: Daniel
authors.initials: D
authors.lastname: Riad
authors.affiliation: Department of Anaesthesiology, Intensive Care and Pain Medicine, University of Muenster, Muenster, Germany;
authors.forename: Jan
authors.initials: J
authors.lastname: Rossaint
authors.affiliation: Department of Anaesthesiology, Intensive Care and Pain Medicine, University of Muenster, Muenster, Germany;
authors.forename: Charlotte
authors.initials: C
authors.lastname: Sohlbach
authors.affiliation: Department of Anaesthesiology, Intensive Care and Pain Medicine, University of Muenster, Muenster, Germany;
authors.forename: Giulia
authors.initials: G
authors.lastname: Germena
authors.affiliation: Department of Pharmacology and Vascular Biology, Yale University, New Haven, CT;
authors.forename: Dianqing
authors.initials: D
authors.lastname: Wu
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA; and.
authors.forename: Scott I
authors.initials: SI
authors.lastname: Simon
authors.affiliation: La Jolla Institute for Allergy and Immunology, La Jolla, CA.
authors.forename: Klaus
authors.initials: K
authors.lastname: Ley
authors.affiliation: Department of Anaesthesiology, Intensive Care and Pain Medicine, University of Muenster, Muenster, Germany;
authors.forename: Alexander
authors.initials: A
authors.lastname: Zarbock
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D018925:Chemokines / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D004195:Disease Models, Animal; D015536:Down-Regulation; D051960:GTP-Binding Protein alpha Subunit, Gi2 / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D008070:Lipopolysaccharides / Q000009:adverse effects; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D054799:Phospholipase C beta / Q000235:genetics / Q000378:metabolism*; D011014:Pneumonia / Q000139:chemically induced / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D034741:RNA, Small Interfering / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D000954:Antigens, Surface; D018925:Chemokines; D044387:GTP-Binding Protein beta Subunits; D008070:Lipopolysaccharides; D016169:Lymphocyte Function-Associated Antigen-1; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D054799:Phospholipase C beta; D051960:GTP-Binding Protein alpha Subunit, Gi2; D020830:rac1 GTP-Binding Protein; D002118:Calcium