Alveolar Macrophages from Septic Mice Promote Polymorphonuclear Leukocyte Transendothelial Migration via an Endothelial Cell Src Kinase/NADPH Oxidase Pathway

Abstract

Alveolar macrophages (AMφ) have been implicated in the polymorphonuclear leukocyte (PMN) recruitment to the lungs during sepsis. Using an in vivo murine model of sepsis (feces in the peritoneum), we show that peritonitis leads to increased activation of AMφ and PMN migration into pulmonary alveoli. To assess cellular mechanisms, an in vitro construct of the pulmonary vascular-interstitial interface (murine AMφ, pulmonary endothelial cells, and PMN) and a chimera approach were used. Using immunologic (Abs) and genetic blockade (CXCR2-deficient AMφ), we show that CXC chemokines in septic plasma are responsible for the activation of AMφ. The activated AMφ can promote PMN transendothelial migration, even against a concentration gradient of septic plasma, by generating platelet-activating factor and H2O2. Platelet-activating factor/H2O2 induce an oxidant stress in the adjacent endothelial cells, an event that appears to be a prerequisite for PMN transendothelial migration, since PMN migration is abrogated across Cu/Zn-superoxide dismutase overexpressing endothelial cells. Using gp91-deficient endothelial cells, we show that NADPH oxidase plays an important role in the AMφ-induced PMN transendothelial migration. Pharmacologic/small interfering RNA blockade of Src kinase inhibits AMφ-induced endothelial NADPH oxidase activation and PMN migration. Collectively, our findings indicate that the PMN transendothelial migration induced by septic AMφ is dependent on the generation of superoxide in endothelial cells via the Src kinase/NADPH oxidase signaling pathway.

Acute Lung Injury and Pulmonary Vascular Permeability: Use of Transgenic Models

“…The inability of neutrophils to produce an oxidant burst not only reduced the bacterial killing capacity of neutrophils, but also reduced the endothelial oxidant injury that was necessary for the vascular permeability increase. Wang et al (512) observed that septic plasma was responsible for activating the alveolar macrophages, which in turn promoted neutrophil transmigration across the endothelium by generating PAF and hydrogen peroxide. The oxidant stress induced by activation of endothelium was required for neutrophil attachment and transmigration and was blocked in endothelial cells overexpressing Cu/Zn SOD, or in gp91phox-deficient endothelial cells.…”

“…Neutrophil infiltration can be quantitated by counting neutrophils in the BAL fluid, or more accurately, by using morphometrics and sterology to assess global neutrophil distribution using lung histology sections (519). In a third type of sepsis model, peritonitis is produced by cecal ligation and puncture, or injection of LPS or bacteria into the peritoneum (31,87,96,149,394,512). The lung pathophysiology in peritonitis models is similar to that obtained by intravenous bacterial infusions.…”

Calpains promote neutrophil recruitment and bacterial clearance in an acute bacterial peritonitis model

“…To assess the role of calpains in the innate immune response in vivo, we subjected WT and calpain KO mice to the fecesin-peritoneum (FIP) model of acute bacterial peritonitis [40]. Colonic enterobacteria were cultured overnight prior to injection in the peritoneal cavity of WT and calpain KO mice (10 6 CFU).…”

“…Acute bacterial peritonitis was induced using a slightly modified FIP model, which was described previously [40]. Enterobacteria were obtained from the colon of a mouse and incubated overnight at 37…”

“…1C, see arrows; actin served as a loading control). Taken together, this novel model of calpain deficiency shows differences in a calpain substrate that is known to regulate innate immune responses [19].To assess the role of calpains in the innate immune response in vivo, we subjected WT and calpain KO mice to the fecesin-peritoneum (FIP) model of acute bacterial peritonitis [40]. Colonic enterobacteria were cultured overnight prior to injection in the peritoneal cavity of WT and calpain KO mice (10 6 CFU).…”