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Inducible nitric oxide synthase and heme oxygenase-1 in the lung during lipopolysaccharide tolerance and cross tolerance

Koch, Alexander MD; Boehm, Olaf MD; Zacharowski, Paula A. PhD; Loer, Stephan A. MD; Weimann, Jörg MD; Rensing, Hauke MD; Foster, Simon J. PhD; Schmidt, Rene MD; Berkels, Reinhard PhD; Reingruber, Sonja PhD; Zacharowski, Kai MD, PhD

Laboratory Investigations
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Objective: Pretreatment with low-dose lipopolysaccharide protects cells/organs against a subsequent lethal Gram-negative (lipopolysaccharide tolerance) or Gram-positive (cross tolerance) stimulus. We determined whether this occurs in the rat lung. The involvement of inducible nitric oxide synthase and heme oxygenase-1 was evaluated.

Design: Laboratory study.

Setting: University hospital laboratory.

Subjects: Anesthetized male Wistar rats.

Interventions: To test the hypothesis, rats received saline or lipopolysaccharide (1 mg/kg). At 2, 4, 8, 16, or 24 hrs later, blood samples and lung tissue were taken to determine messenger RNA, protein concentration, and activity of inducible nitric oxide synthase and heme oxygenase-1. In additional experiments, rats were challenged with lipopolysaccharide (1 mg/kg) and subjected to Gram-negative (lipopolysaccharide) or Gram-positive (lipoteichoic acid and peptidoglycan) shock 24 hrs later. These studies were carried out in the presence and absence of inducible nitric oxide synthase or heme oxygenase-1 inhibitors (1400W or tin protoporphyrin IX). Following 6 hrs of shock, lung tissue was taken to determine lung damage and heme oxygenase-1 concentration and activity.

Measurements and Main Results: In the rat lung, lipopolysaccharide (1 mg/kg) induced a significant increase in inducible nitric oxide synthase protein at 8 hrs with a corresponding increase in plasma nitrate/nitrite at 8–16 hrs. Simultaneously, heme oxygenase-1 messenger RNA transcripts were observed at 8–16 hrs, and maximal expression of the protein followed (24 hrs). Pretreatment with low-dose lipopolysaccharide reduced myeloperoxidase activity (neutrophil infiltration) and wet-dry ratio (pulmonary edema) in the lungs of animals subjected to Gram-negative or Gram-positive shock, demonstrating tolerance. Pretreatment with low-dose lipopolysaccharide and the selective inducible nitric oxide synthase inhibitor 1400W reduced heme oxygenase-1 protein expression, and lung protection was abolished. Tin protoporphyrin IX did not affect heme oxygenase-1 expression, but heme oxygenase activity and lung protection were significantly reduced.

Conclusions: We propose that nitric oxide (most likely inducible nitric oxide synthase derived) regulates the induction of heme oxygenase-1 in the lung, which in turn plays an important part in pulmonary protection during lipopolysaccharide tolerance and cross tolerance.

From the Molecular Cardioprotection and Inflammation Group, Department of Anesthesia, Bristol University, Bristol, UK (AK, PAZ, KZ); Molecular Cardioprotection and Inflammation Group, Department of Anesthesia, University Hospital, Dusseldorf, Germany (OB, RB, KZ); Department of Anesthesia, VU University Medical Center, Amsterdam, The Netherlands (SAL, JW); Department of Anesthesia and Critical Care Medicine, University of the Saarland, Homburg, Germany (HR); Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK (SJF); Department of Anesthesiology and Critical Care Medicine, University Hospital, Freiburg, Germany (RS); and Fibrex Medical Inc., Vienna, Austria (SR). Drs. Koch and Boehm contributed equally to this work.

Supported, in part, by a grant from the Forschungskommission, University of Dusseldorf and the DFG (Za-243/8–1).

The authors have not disclosed any potential conflicts of interest.

Address requests for reprints to: Kai Zacharowski, MD, PhD, Molecular Cardioprotection & Inflammation Group, Department of Anaesthesia, BHI, Bristol Royal Infirmary, Bristol, BS2 8HW, UK. E-mail: kai.zacharowski@bristol.ac.uk

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