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HO-1 induction restores c-AMP-dependent lung epithelial fluid transport following severe hemorrhage in rats

H. Lee^*,1, M. Pespeni^*,1, J. Roux^*, P. A. Dennery, M. A. Matthay^* and J. F. Pittet^*,2

^* Laboratory of Surgical Research, Departments of Anesthesia, Surgery, and Medicine and Cardiovascular Research Institute, University of California, San Francisco, California, USA; and
Division of Neonatal and Developmental Medicine, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California, USA

²Correspondence: Department of Anesthesia, Room 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, CA 94110, USA. E-mail: pittetj{at}anesthesia.ucsf.edu

SPECIFIC AIMS

We earlier reported that stress preconditioning protects the alveolar epithelium against oxidative stress by increasing cellular levels of some constitutive and inducible stress proteins. Among the many classes of inducible stress proteins, heme oxygenase-1 (HO-1) is one of the heat shock proteins best characterized with respect to its possible protective role in lung biology. The first objective of these studies was to determine whether induction of HO-1 would attenuate release of air space NO responsible for shock-mediated failure of the lung epithelium to up-regulate vectorial fluid transport in response to catecholamines in rats. Because alveolar macrophages and lung epithelial cells have been shown to be a major source of NO production within air spaces of the lung, the second objective was to test the hypothesis that HO-1 induction would attenuate production of NO either by alveolar macrophages and/or by alveolar epithelial cells. The last objective was to determine the mechanisms involved in HO-1-mediated inhibition of endotoxin- or interferon--dependent release of NO by alveolar macrophages.

PRINCIPAL FINDINGS

1. Hemorrhagic shock decreases alveolar epithelial fluid transport
Hemorrhagic shock was induced by withdrawing 35 ± 3% of the blood volume, causing systemic arterial hypotension and metabolic acidosis. Hemorrhagic shock was associated with a failure of the alveolar epithelium to increase vectorial fluid transport in response to catecholamines. Alveolar fluid clearance did not increase in hemorrhaged rats despite adding epinephrine to the instilled protein solution in order to maximize the response of ß-adrenergic receptors (Fig. 1 ). Finally, hemorrhagic shock was associated with a moderate increase in lung endothelial permeability to protein.

View larger version (15K): [in this window] [in a new window]   Figure 1. A) Alveolar fluid clearance is shown for control rats that were left untreated or treated 16 h before onset of the experiment with i.v. hemoglobin (300 mg/kg); some animals were pretreated with s.c. SnPP (50 µmol/kg) 5 h before i.v. injection of hemoglobin; results are shown as means ± SE; *P <0.05 from control rats not receiving epinephrine into their air spaces. B)Alveolar fluid clearance is shown for hemorrhaged rats left untreated or treated 16 h before onset of hemorrhage with i.v. hemoglobin (300 mg/kg); some animals were pretreated with s.c. SnPP (50 µmol/kg) 5 h before the i.v. injection of hemoglobin; results are shown as means ± SE; *P <0.05 from hemorrhaged rats that did not receive any epinephrine into their air spaces.

2. HO-1 induction restores normal alveolar epithelial fluid transport after hemorrhagic shock
Administration of i.v. hemoglobin caused a significant increase in HO-1 activity in the lung at the time corresponding to the onset of hemorrhage in rats that underwent hemorrhagic shock. This increase in HO-1 activity was prevented by pretreating the animals 5 h prior to i.v. administration of hemoglobin with s.c. SnPP. HO-1 induction preserved the ability of the alveolar epithelium to up-regulate vectorial fluid transport in response to catecholamines in hemorrhaged rats (Fig. 1) . HO-1 induction significantly attenuated the shock-induced increase in lung endothelial permeability. HO-1 induction attenuated shock-mediated release of NO from macrophages recovered from distal air spaces of the lung of control and hemorrhaged rats, then cultured ex vivo for 24 h in the presence of increasing concentrations of LPS.

3. HO-1 induction attenuates release of NO by alveolar macrophages, but not by alveolar epithelial cells
Alveolar macrophages and alveolar epithelial cells have been shown to be a source of NO released within air spaces of the lung. MH-S cells, an alveolar macrophage cell line, were treated with COCl₂ and/or SnPP in the presence of LPS or its vehicle for 24 h. Exposure to COCl₂ caused a significant increase in HO-1 activity (Fig. 2 A). There was a 15-fold increase in production of nitrite in the medium of MH-S cell monolayers 24 h after stimulation with LPS (Fig. 2B ). LPS-mediated NO release by MH-S cells was significantly reduced by pretreatment with COCl₂ (Fig. 2B ). Similarly, there was a significant increase in iNOS protein expression in MH-S cells exposed to LPS, an effect that was attenuated by exposure to COCl₂ (Fig. 2C ). Comparable results were obtained when HO-1 activity was increased by exposing MH-S cells to hemin instead of COCl₂ (Fig. 2D ).

View larger version (27K): [in this window] [in a new window]   Figure 2. HO-1 induction reduces endotoxin-mediated NO release by MH-S cells, an alveolar macrophage cell line.A) Treatment with COCl2 increases HO-1 activity in MH-S cells. MH-S cells were incubated overnight with COCl2 (100 µM) or its vehicle; some cells were cotreated with SnPP (15 µM) or its vehicle, an inhibitor of HO-1 enzymatic activity; results are shown as the mean ± SE of 4 different experiments repeated in triplicate; *P <0.05 from controls; **P <0.05 from cell monolayers that were exposed to COCl2. B) Pretreatment with COCl2 attenuates the endotoxin-mediated release of NO by MH-S cells. MH-S cells were incubated overnight with COCl2 (100 µM) or its vehicle; some cells were cotreated with SnPP (15 µM) or its vehicle; cells were then exposed to LPS (10 ng/mL) or its vehicle for 24 h. Results are shown as the mean ± SE of 4 different experiments repeated in triplicate; *P <0.05 from controls; **P <0.05 from cells that were exposed to LPS alone. C) Pretreatment with COCl2 decreases expression of iNOS protein induced by exposure of MH-S cells to endotoxin. MH-S cells were incubated overnight with COCl2 (100 µM) or its vehicle; some cells were cotreated with SnPP (15 µM) or its vehicle; cells were then exposed to LPS (10 ng/mL) or its vehicle for 24 h; one representative experiment is shown for each experimental condition. Four additional experiments gave comparable results, as shown by densitometry analysis. Western blot analysis for ß-actin protein demonstrates equal protein loading. D) Pretreatment with hemin attenuates endotoxin-mediated release of NO by MH-S cells. MH-S cells were incubated overnight with hemin (30 µM) or its vehicle; cells were then exposed to LPS (10 ng/mL) or its vehicle for 24 h; results are shown as the mean ± SE of 4 different experiments repeated in triplicate; *P <0.05 from controls; **P <0.05 from cells that were exposed to LPS alone.

To determine whether HO-1 induction attenuates NO production in rat alveolar epithelial cells, ATII cell monolayers were treated with COCl₂ or its vehicle and with cytomix for 24 h. Exposure to COCl₂ caused significant increase in HO-1 activity. There was a 2-fold increase in production of nitrite in the medium of ATII cell monolayers 24 h after stimulation with cytomix. Coexposure with COCl₂ did not reduce the release of nitrite by polarized lung epithelial cells upon stimulation with cytomix. Increasing the dose of COCl₂ to 150 µM did not affect release of NO by lung epithelial cells exposed to cytomix. Similarly, exposure to hemin did not reduce NO release by rat lung epithelial cells. Thus, HO-1 induction significantly reduced release of NO by alveolar macrophages upon stimulation with endotoxin, but did not affect production of NO by rat ATII cells stimulated with proinflammatory cytokines.

4. HO-1 induction attenuates endotoxin-mediated NF-B activation in alveolar macrophages
The next series of experiments were designed to determine whether HO-1 induction would affect LPS-mediated activation of the NF-B pathway in MH-S cells. In control cells, addition of endotoxin resulted in translocation of NF-B subunits (p50/p65) into the nucleus and their binding to a NF-B consensus nucleotide. In contrast, prior HO-1 induction effectively blocked p50/p65 activation. Prior HO-1 induction decreased the ability of endotoxin to induce phosphorylation of IB and the p65 subunit of NF-B in MH-S cells by a p38-independent mechanism. HO-1 induction with COCl₂ in MH-S cells did not cause phosphorylation of p38 MAP kinase. These results indicate that HO-1 induction attenuates LPS-induced iNOS expression in part by inhibiting activation of the NF-B signaling pathway by endotoxin.

5. HO-1 induction reduces interferon--mediated Jak-Stat 1 activation and NO release by alveolar macrophages
The next series of experiments were designed to determine whether prior HO-1 activation would affect NO production by MH-S cells after stimulation of these cells by interferon-gamma (IFN-). IFN- is an important proinflammatory cytokine released within distal air spaces of the lung after hemorrhage or endotoxin administration. Its transcriptional effect on the iNOS promoter is mediated by the signal transducer and activator of transcription (STAT) 1. HO-1 induction by COCl₂ caused a dose-dependent decrease in IFN--induced-release of NO from MH-S cells. Phosphorylation of Stat 1 by IFN- was attenuated in MH-S cells that were incubated overnight with COCl₂ in a p38 MAP kinase-independent manner. These results indicate that HO-1 induction attenuates IFN--induced NO production in alveolar macrophages in part by inhibiting activation of the Jak-Stat 1 signaling pathway by IFN-.

CONCLUSIONS AND SIGNIFICANCE

Under pathological conditions that predispose development of acute lung injury, up-regulation of alveolar epithelial fluid transport by endogenous catecholamines is a major mechanism that prevents alveolar flooding. After severe hemorrhage, however, this protective mechanism is abolished by development of an inflammatory response that is associated with the release of radical nitrogen species within air spaces of the lung. We have shown that stress preconditioning attenuated NO-mediated oxidative stress to the alveolar epithelium after hemorrhagic shock. Stress preconditioning is associated with expression of several inducible heat shock proteins, such as Hsp 90, Hsp72, Hsp 27, or HO-1. Here, we hypothesized that prior HO-1 induction in the lung could decrease release of NO within air spaces of the lung after hemorrhagic shock. This in turn would lead to a decrease in oxidative stress experienced by the alveolar epithelium and potentially restore a physiological response of this lung barrier to catecholamines after hemorrhage. Increased HO-1 activity in the lung did result in: 1)restoration of c-AMP-dependent up-regulation of vectorial fluid transport across the alveolar epithelium; and 2)complete inhibition of lung vascular permeability associated with hemorrhagic shock.

Consistent with our earlier observations, prior HO-1 induction caused a significant attenuation of shock-mediated release of NO in the lung. NO is released by a variety of lung cells after iNOS activation by inflammatory mediators. However, alveolar macrophages and alveolar epithelial cells have been shown to be the major source of iNOS-mediated NO production within air spaces of the lung. HO-1 activation resulted in a significant decrease in the production of NO by alveolar macrophages upon stimulation with LPS. In contrast, HO-1 induction in primary cultures of rat ATII cells did not result in an attenuation of NO production upon their subsequent exposure to proinflammatory cytokines despite a 2-fold increase in HO-1 enzymatic activity in these cells. Inhibition of iNOS expression in alveolar macrophages after HO-1 activation was mediated in part by the inhibitory effect of HO-1 on the NF-B and Jak-Stat 1 signaling pathways, although these results do not exclude the possibility that induction of HO-1 may directly affect enzymatic activity of iNOS protein. These results are of potential clinical importance because a recent study of critically ill patients revealed increased production of reactive-oxygen-nitrogen intermediates in the distal air space of patients with acute lung injury. These changes were associated with impaired alveolar fluid clearance, an early predictor of prolonged duration of mechanical ventilation and higher hospital mortality in patients with acute lung injury.

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2254fje;

¹ These authors equally contributed to this work.

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