Rho-kinase regulates adhesive and mechanical mechanisms of pulmonary recruitment of neutrophils in abdominal sepsis

Eur J Pharmacol. 2012 May 5;682(1-3):181-7. doi: 10.1016/j.ejphar.2012.02.022. Epub 2012 Feb 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Abdomen / microbiology*
  • Actins / chemistry
  • Animals
  • Biomechanical Phenomena
  • Cell Adhesion / drug effects
  • Chemokine CXCL2 / pharmacology
  • Coinfection / enzymology
  • Coinfection / immunology
  • Coinfection / metabolism
  • Coinfection / pathology
  • Gene Expression Regulation / drug effects
  • Intestinal Perforation / enzymology
  • Intestinal Perforation / immunology
  • Intestinal Perforation / metabolism
  • Intestinal Perforation / pathology
  • Lung / enzymology
  • Lung / immunology*
  • Lung / pathology
  • Macrophage-1 Antigen / metabolism
  • Male
  • Mechanical Phenomena*
  • Mice
  • Mice, Inbred C57BL
  • Neutrophils / cytology
  • Neutrophils / drug effects
  • Neutrophils / immunology*
  • Protein Multimerization / drug effects
  • Protein Structure, Quaternary
  • Sepsis / enzymology*
  • Sepsis / immunology*
  • Sepsis / metabolism
  • Sepsis / pathology
  • Signal Transduction / drug effects
  • rho-Associated Kinases / metabolism*

Substances

  • Actins
  • Chemokine CXCL2
  • Macrophage-1 Antigen
  • rho-Associated Kinases