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Critical Care |

Mutual Reciprocal Regulation of Neutrophils and Pulmonary Microvascular Endothelial Cells Through Inducible Nitric Oxide Synthase-Dependent Mechanisms In Murine Sepsis

Sanjay Mehta*, MD; Lefeng Wang, PhD; Ravi Taneja, MBBS; Habib Moshref Razavi, MD; Cedrin Law, MS; Chris Gillis, MD; Marta Rohan, MD
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Lawson Health Research Institute, UWO, London, ON, Canada


Chest. 2012;142(4_MeetingAbstracts):299A. doi:10.1378/chest.1390601
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Abstract

SESSION TYPE: New Insights into ARDS/Lung Injury

PRESENTED ON: Monday, October 22, 2012 at 04:00 PM - 05:30 PM

PURPOSE: The interaction of neutrophils and pulmonary microvascular endothelial cells (PMVEC) is central to the pathophysiology of sepsis-induced acute lung injury (ALI). Nitric oxide produced by inducible NO synthase (iNOS) contributes importantly to sepsis/ALI. Here, we assess the specific contributions of neutrophil vs PMVEC iNOS in a murine model of sepsis-ALI.

METHODS: Sepsis was induced by cecal-ligation/perforation, and 4-hrs later, ALI characterized by increases in pulmonary neutrophil infiltration (tissue myeloperoxidase activity, bronchoalveolar lavage neutrophils), microvascular leak of Evans blue (EB) dye-labeled albumin, oxidant stress (8-isoprostane levels), and PMVEC death by intravital videomicroscopy.

RESULTS: Septic ALI was neutrophil dependent, as pretreatment with anti-CD18 before cecal-ligation/perforation significantly (P<0.05) attenuated septic increases in pulmonary myeloperoxidase (9±1 vs. 35±5 mU/mg protein), lavage neutrophils (0.5%±0.2% vs. 2.1%±0.6%), microvascular EB-albumin leak (1.3±0.3 vs. 2.6±0.7 ug EB/g/minute), 8-isoprostane content (115±16 vs. 74±15 pg/mg protein), and septic PMVEC death. The specific role of neutrophil iNOS was assessed by creating neutrophil-iNOS chimeric mice: iNOS+/+ versus iNOS-/- mice were bone-marrow depleted by irradiation and selectively reconstituted with iNOS+/+ versus iNOS-/- neutrophils. Cecal-ligation/perforation resulted in significant septic ALI in +to- neutrophil-iNOS chimeric mice (selectively iNOS+/+ neutrophils), but not in -to+ neutrophil-iNOS chimeric mice (selectively iNOS-/- neutrophils). There were no significant differences between iNOS+/+ and iNOS-/- neutrophils in vitro in phagocytosis, respiratory burst, CD11a/b/CD18 surface expression, or apoptosis rates (by microscopy and FACS annexin-V binding) under naïve or septic conditions. However, neutrophil apoptosis was inhibited by transmigration only across iNOS+/+ PMVEC (1.6±0.3%) but not across iNOS-/- PMVEC (4.3±1%, p<0.05).

CONCLUSIONS: Neutrophils and specifically neutrophil iNOS contribute importantly to murine septic ALI and PMVEC injury in vivo. Moreover, PMVEC iNOS inhibits the apoptosis of neutrophils transmigrating across PMVEC monolayers. We conclude that there is mutual, reciprocal iNOS-dependent regulation of neutrophils and PMVEC during neutrophil-PMVEC interactions in sepsis-ALI.

CLINICAL IMPLICATIONS: Pharmacological manipulation of these interactions may offer a novel therapeutic approach in patients with sepsis-ALI.

DISCLOSURE: The following authors have nothing to disclose: Sanjay Mehta, Lefeng Wang, Ravi Taneja, Habib Moshref Razavi, Cedrin Law, Chris Gillis, Marta Rohan

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Lawson Health Research Institute, UWO, London, ON, Canada

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