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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 4, 2004 as doi:10.1096/fj.03-0643fje. |
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* Division of Pulmonary, Allergy and Critical Care Medicine, and
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA;
Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria; and
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
2Correspondence: University of Pittsburgh School of Medicine, Montefiore University Hospital, Rm. 628 NW, Pittsburgh, PA 15213, USA. E-mail: otterbeinl{at}upmc.edu
SPECIFIC AIMS
Carbon monoxide (CO) at low concentrations exerts potent anti-inflammatory effects and prevents lipopolysaccharide (LPS)-induced lung and liver injury in rats. Our aim in this study was to evaluate the expression of inducible nitric oxide synthase (iNOS)/NO in the lung and liver following administration of LPS and whether CO and/or NO play a role in preservation of the cells in each tissue.
PRINCIPAL FINDINGS
1. CO provides protection against lethal endotoxemia
We show here that exposure to a low concentration of CO, one of the by-products of heme catabolism by heme oxygenase-1 (HO-1), can substitute for HO-1 in conferring anti-inflammatory effects. Administration of LPS [50 mg/kg intravenously (i.v.)] to rats exposed to room air resulted in 80% mortality within 20 h of LPS injection. A 1 h pretreatment with CO (250 ppm) before injection of LPS resulted in a more than 75% improvement in survival. We demonstrate that as little as 10 ppm administered continuously as a 1-h pretreatment or if initiated 1 h post-LPS administration was also significantly beneficial (Fig. 1
).
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2. CO modulates LPS-induced cytokine production in vivo
LPS was administered to rats at 3 mg/kg i.v. to evaluate the acute inflammatory response that mimics more closely a sepsis-like syndrome in humans. This dose induces an acute-phase response and concomitant lung injury. CO (1 h, 250 ppm) was administered as a pretreatment only. LPS-induced generation of serum pro- and anti-inflammatory cytokines was significantly modulated in those animals pretreated with CO: TNF-
and IL-6 were reduced by more than 50%, and there was a more than twofold augmentation in IL-10 production, the prototypical anti-inflammatory cytokine. In the therapeutic protocol where CO was initiated 1 h post-LPS, CO similarly decreased IL-6, augmented IL-10, and also reduced LPS-induced neutrophil alveolitis, yet had no effect on TNF-, as might be expected as this proinflammatory cytokine peaks in the serum 1 h after LPS.
3. Effect of CO on LPS-induced lung and liver injury
Although exposure to CO can impart potent anti-inflammatory properties, the functional outcomes in an endotoxic shock model have not been evaluated. Administration of LPS to rats results in a time-dependent increase in pulmonary edema, leukocyte infiltration (primarily neutrophils) with cell damage, and tissue injury. To evaluate the effects of CO on these parameters, animals were administered the sublethal dose of endotoxin, and a bronchoalveolar lavage was performed 24 h later. Those animals exposed to CO showed a marked and significant reduction in LPS-induced neutrophil alveolitis (>75%, P<0.05), protein, and lactate dehydrogenase (LDH) accumulation and nitrite levels (as a measure of NO generation). These indices are measures not only of inflammation (polymorphonuclear neutrophil, nitrite) but also of alterations in lung endothelial/epithelial barrier function and cellular injury (protein, LDH). As endotoxin affects multiple organs, we evaluated the effects of CO on LPS-induced liver injury. Rats were pretreated with CO and then administered the lethal dose of LPS. Serum alanine aminotransferase (ALT) levels were measured as a marker of liver [hepatocyte (HC)] damage. Those animals that were exposed to CO showed a significant reduction in serum ALT to baseline levels (60±5 IU/ml in LPS-treated vs. 18±7 in CO/LPS-treated animals; P<0.02). These data combined with those relating to the lung reveal for the first time the effects of CO on functional parameters of organ preservation.
4. CO modulates LPS-induced cytokine production in rat lung macrophages and protects against cell death in HC
To assess whether CO modulates the inflammatory cytokines in the same manner in vitro, we exposed freshly isolated rat macrophages obtained by BAL to 1 µg/ml LPS. This induced a time-dependent secretion of TNF-, IL-6, and IL-10. CO significantly decreased LPS-induced proinflammatory cytokine production (>80% IL-6 and TNF-; P<0.01) and simultaneous fourfold augmentated IL-10 production over LPS-treated cells exposed to air (P<0.05). Similar effects were observed in the rat macrophage cell line NR8383 (data not shown). HC treated with TNF-/actinomycin D undergo cell death. We tested whether CO would prevent cell death and mimic what we observed in vivo. CO-treated HC showed a >85% survival rate vs. <50% survival in air-treated controls (P<0.05). This cytoprotective effect of CO is potentially the mechanism accounting for the lower serum ALT levels in vivo and very likely parallel to the lesser liver damage as observed in the in vivo model.
5. CO exerts reciprocal effects on LPS-induced iNOS/NO expression in vitro and in vivo
In our present study, we hypothesized that protection initiated by CO might involve NO because of the well-documented role for iNOS/NO in the inflammatory response and tissue injury following LPS. To examine whether CO had an effect on modulating LPS-induced iNOS, nitrite/nitrate accumulation was determined in vitro in the supernatant of rat lung macrophages and HC, as well as in vivo in serum of LPS-treated rats. Nitrite and nitrate levels were evaluated over time in the serum, peaking 24 h following LPS administration. In vitro in macrophages and HC, peak nitrite levels occurred 16 h after LPS. In vitro in macrophages and in vivo in the serum, CO significantly decreased LPS-induced nitrite/nitrate production vs. that observed in rats treated with LPS and exposed to air. In contrast, exposure of HC to CO in the presence of LPS resulted in a significant augmentation of nitrite production. iNOS enzyme expression was assessed to correlate with nitrite/nitrate levels (Fig. 2
). In the presence of CO, LPS-induced iNOS expression was ablated in the lung and AM
(Fig. 2A-C
). In contrast, CO, which correlated directly with nitrite/nitrate production, augmented LPS-induced iNOS expression in the liver as well as in the isolated HC.
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6. CO exerts reciprocal effects on LPS-induced nuclear factor (NF)-
B/inhibitor of B activation in lung and liver
Further investigations into the cellular mechanisms by which CO differentially regulates iNOS expression in the lung and liver examined the effects on NF-B activation, as it is a principal transcription factor involved in the regulation of iNOS expression. Similar to iNOS, CO inhibited LPS-induced activation in the lung and simultaneously augmented LPS-induced activation in the liver. These findings emphasize the complex biological effects of this gas molecule as well as lend insight into the specificity of its actions among organ systems.
CONCLUSIONS AND SIGNIFICANCE
In the present study, we tested the effects of CO in a LPS model in rats. CO suppressed the systemic, proinflammatory response and protected the lung and liver, as well as isolated cells from each of these two organs. Most importantly, CO pretreatment modulated production of NO differentially in these two organs. The up-regulation in the liver and the suppression in the lung likely in each case protected the organ, given the adverse effects of NO in the lung and the protective effects in the liver. Exposure to CO for just 1 h before LPS was sufficient to prevent lung and liver injury that develops over the ensuing 8–24 h. We believe that exposure to CO directs the cell toward an anti-inflammatory, cytoprotective phenotype, such that upon addition of LPS, there is greater resistance to cellular damage and tissue injury.
Our study further delineates the apparently important physiologic inter-relationship between CO and NO and the two enzymes that lead to the generation of these gases. The fact that CO differentially regulates the generation of NO in different tissues but in each to the benefit of the cells in that tissue suggests that CO is a component of the natural, protective system in those organs. Future studies are focused on evaluating a direct relationship between CO and iNOS in terms of cellular and tissue cytoprotection. We have recently shown that the therapeutic effects of CO in a model of hepatitis induced by TNF- and D-galactosamine involve the requisite induction of iNOS and production of NO. Our present findings in the liver may be in concert with the earlier ones, although the down-regulation of iNOS in the lung speaks to the complexity of these protective mechanisms.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0643fje; 
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). CO (250 ppm) administered as a 1-h pretreatment only before LPS (•). CO (10 ppm) administered as a 1-h pretreatment and continuously thereafter (
). CO (10 ppm) administered 1 h post-LPS and then continuously (
). n = 8–10 rats/group. B–D) Serum cytokines measured at 1 h [tumor necrosis factor 
