title: Rho-kinase regulates adhesive and mechanical mechanisms of pulmonary recruitment of neutrophils in abdominal sepsis.
abstract: We hypothesized that Rho-kinase signaling plays a role in mechanical and adhesive mechanisms of neutrophil accumulation in lung. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 prior to cecal ligation and puncture (CLP). Lung levels of myeloperoxidase (MPO) and histological tissue damage were determined 6h and 24h after CLP. Expression of Mac-1 and F-actin formation in neutrophils were quantified by using flow cytometry 6h after CLP. Mac-1 expression and F-actin formation were also determined in isolated neutrophils up to 3h after stimulation with CXCL2. Labeled and activated neutrophils co-incubated with Y-27632, an anti-Mac-1 antibody and cytochalasin B were adoptively transferred to CLP mice. Y-27632 reduced the CLP-induced pulmonary injury and MPO activity as well as Mac-1 on neutrophils. Neutrophil F-actin formation peaked at 6h and returned to baseline levels 24h after CLP induction. Rho-kinase inhibition decreased CLP-provoked F-actin formation in neutrophils. CXCL2 rapidly increased Mac-1 expression and F-actin formation in neutrophils. Co-incubation with Y-27632 abolished CXCL2-induced Mac-1 up-regulation and formation of F-actin in neutrophils. Notably, co-incubation with cytochalasin B inhibited formation of F-actin but did not reduce Mac-1 expression on activated neutrophils. Adoptive transfer experiments revealed that co-incubation of neutrophils with the anti-Mac-1 antibody or cytochalasin B significantly decreased pulmonary accumulation of neutrophils in septic mice. Our data show that targeting Rho-kinase effectively reduces neutrophil recruitment and tissue damage in abdominal sepsis. Moreover, these findings demonstrate that Rho-kinase-dependent neutrophil accumulation in septic lung injury is regulated by both adhesive and mechanical mechanisms.
authors.affiliation: Department of Clinical Sciences, Section of Surgery, Malmö, Lund University, 20502 Malmö, Sweden.
authors.forename: Karzan
authors.initials: K
authors.lastname: Palani
authors.forename: Milladur
authors.initials: M
authors.lastname: Rahman
authors.forename: Zirak
authors.initials: Z
authors.lastname: Hasan
authors.forename: Su
authors.initials: S
authors.lastname: Zhang
authors.forename: Zhongquan
authors.initials: Z
authors.lastname: Qi
authors.forename: Bengt
authors.initials: B
authors.lastname: Jeppsson
authors.forename: Henrik
authors.initials: H
authors.lastname: Thorlacius
mesh_terms: D000005:Abdomen / Q000382:microbiology*; D000199:Actins / Q000737:chemistry; D000818:Animals; D001696:Biomechanical Phenomena; D002448:Cell Adhesion / Q000187:drug effects; D054426:Chemokine CXCL2 / Q000494:pharmacology; D060085:Coinfection / Q000201:enzymology / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D007416:Intestinal Perforation / Q000201:enzymology / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D008168:Lung / Q000201:enzymology / Q000276:immunology* / Q000473:pathology; D016177:Macrophage-1 Antigen / Q000378:metabolism; D008297:Male; D055595:Mechanical Phenomena; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D018805:Sepsis / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D000199:Actins; D054426:Chemokine CXCL2; D016177:Macrophage-1 Antigen; D054460:rho-Associated Kinases