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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 29, 2005 as doi:10.1096/fj.04-2368fje. |
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* INSERM U 700 and U 683, Faculté de Médecine Xavier Bichat, Université Paris VII, Paris, France;
Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, UK;
Service de Biochimie, Hôpital Lariboisière, Université Paris VII, Paris, France;
Service de Biochimie, Hôpital du Kremlin Bicêtre, Université Paris XI, Paris, France; and
‘|| ||Département d’Anesthésie- Réanimation, Hôpital Charles Nicole, Rouen, France
2 Correspondence: INSERM U700, Faculté X. Bichat, BP416, Paris 75870, France. E-mail: jbb2{at}bichat.inserm.fr
SPECIFIC AIMS
This study is devoted to the investigation of a potential protective role for bilirubin, one of the products of heme degradation by the enzyme heme oxygenase, in endotoxic shock. The rationale is that the antioxidant and cytoprotective properties of bilirubin could protect against endotoxic shock, a high mortality pathologic condition.
Several years ago, Stocker and co-workers demonstrated that bile pigments are potent scavengers of free radicals in vitro. Since then, it has been shown that bilirubin interacts with other biological processes, such as the ones involved in protein phosphorylation and nitric oxide (NO) signaling. The protective effect of bilirubin against various types of cellular damage (oxidative and nonoxidative) has been demonstrated in vitro and in vivo using different experimental models and conditions, such as ischemia-reperfusion injury and organ graft rejection. These effects are not restricted to animal experimental models since different epidemiological studies revealed a beneficial action of bilirubin against the development of cardiovascular disease and cancer. Given these broad protective properties of bilirubin, we hypothesized that this bile pigment could contribute in counteracting septic shock, a condition characterized by uncontrolled inflammatory responses to an infectious agent which ultimately leads to hypotension, multiple organ failure and, in a high percentage of cases, to death.
In the first part of the study, we examined the effects elicited by Escherichia coli endotoxin (lipopolysaccharide, LPS) administration to Gunn rats, an animal model that can be used to study the importance of bilirubin in protection against pathophysiological conditions. Gunn rats are a mutant Wistar strain with an autosomal recessive deficiency of glucuronyl transferase, the enzyme responsible for the conjugation of bilirubin to a more soluble and excretable form. As a result of this deficiency, homozygous rats become hyperbilirubinemic, whereas heterozygous animals have a normal concentration of bilirubin in plasma. It must be noted, however, that the responses of Gunn rats to LPS could be modulated by the chronic effects imposed by the hyperbilirubinemic state. Therefore, in the second part of the study, we investigated the effect elicited by endotoxin administration to Sprague-Dawley rats rendered acutely hyperbilirubinemic by a continuous intravenous infusion of exogenous bilirubin. Finally, in the third part of the study, we verified the in vitro potential protective effects of bilirubin in a murine macrophages cell line (RAW 264.7) activated with LPS, and further characterized the mechanism(s) involved in bilirubin protection.
PRINCIPAL FINDINGS
1. Homozygous jaundiced Gunn rats, which display high plasma bilirubin levels, were significantly more resistant to endotoxin-induced hypotension and death than nonjaundiced rats
2. Sprague-Dawley rats subjected to exogenous bilirubin administration better tolerated the LPS challenge, with a reduced hypotension and decreased mortality
3. No significant differences in systemic and local oxidative stress parameters or in circulating inflammatory cytokines (IL-6, TNF-
, and IL-10) were detected between hyperbilirubinemic and normal rats
However, in both Gunn rats and bilirubin-injected Sprague Dawley rats, the LPS-stimulated production of nitric oxide (NO) was significantly decreased, as was the renal, myocardial, and aortic protein expression of inducible NO synthase (NOS2). Furthermore, an inverse and statistically significant correlation (r2=0.7, P<0.01) was found between plasmatic bilirubin and NOx levels.
4. NOS2 protein expression and activity as well as nitrite levels were also reduced in LPS-stimulated murine macrophages incubated with bilirubin at concentrations similar to the one found in the serum of hyperbilirubinemic animals, showing a direct effect of the bile pigment on NOS2 expression
5. Attenuation of NOS2 expression by bilirubin was probably secondary to inhibition of NAD(P)H oxidase since 1) Pharmacological inhibition of NAD(P)H oxidase attenuated NOS2 induction by LPS; 2) bilirubin decreased NAD(P)H oxidase activity in vivo and in vitro; and 3) in vitro NOS2 down-regulation by bilirubin was reversed by addition of NAD(P)H
Protein expression of NAD(P)H oxidase components (gp91phox, p22phox and p47phox) was not modified by pretreatment with bilirubin in vivo and in vitro, suggesting that bilirubin interfered with NAD(P)H oxidase activation rather than decreasing protein expression. This phenomenon was independent of p38 and p42/44 mitogen-activated protein kinases (MAPK) pathway.
CONCLUSIONS AND SIGNIFICANCE
To the best of our knowledge, the present results show that bilirubin has beneficial effects in endotoxic shock. The protective mechanisms are probably related to an attenuation of NOS2 induction in cardiovascular and other tissues, an effect that is secondary to inhibition of NAD(P)H oxidase activity. This is a new mechanism that extends our knowledge on the cytoprotective and antioxidant properties of bilirubin. The most likely mechanism explaining the protective effect of bilirubin against mortality in animals challenged with LPS could be related to the attenuation of endotoxin-induced hypotension, since shock is a main determinant of mortality in endotoxemia. Our results show that the hypotension typically observed after endotoxin challenge was prevented in both hyperbilirubinemic in vivo models. This effect was not related to attenuation of either oxidative stress or inflammation, as described in other experimental conditions, since the levels of antioxidant enzymes, malondialdehyde, and proinflammatory cytokines such as IL-6 and TNF- or the anti-inflammatory cytokine IL-10 were not affected by bilirubin. However, the protective effect of bilirubin could be related to a decreased expression and activity of NOS2 observed in the cardiovascular tissues (myocardium and aorta), a main mediator of LPS-induced hypotension. A relationship between the increase in bilirubin levels and attenuation of the NO pathway is further supported by the strong negative correlation between plasmatic bilirubin and nitrite/nitrate levels observed in the Sprague-Dawley rats. A direct effect of bilirubin on NOS2 expression and activity is reinforced by in vitro results obtained in murine macrophages which show that concentrations of bilirubin in the range of those found in the serum of hyperbilirubinemic animals significantly attenuated NOS2 protein expression and activity as well as nitrite formation in culture media. NOS2 was the only isoform of NOS affected by bilirubin, since the expression and activity of the constitutive NOS1 and NOS3 isoforms in vivo and in vitro were unchanged by the bile pigment. Different mechanism(s) could explain the attenuation of NOS2 induction by bilirubin. We investigated the role of inhibition of NAD(P)H oxidase because 1) bilirubin inhibits NAD(P)H oxidase activity in vitro in a neutrophil cell-free system, and 2) activation of NAD(P)H oxidase has been shown to be involved in NOS2 induction. Small and highly controlled superoxide anion production by NAD(P)H oxidases involved in cell results of the present study showed that bilirubin significantly decreased NAD(P)H oxidase activity in vivo in the aorta of Sprague-Dawley rats and in vitro in RAW 264.7 macrophages. This effect very likely contributes to the attenuation of NOS2 expression by bilirubin since inhibition of NAD(P)H oxidase by two different pharmacological compounds (diphenylene iodinium and apocynin) significantly reduced NOS2 expression in RAW 264.7 macrophages. In vitro NOS2 down-regulation by bilirubin was reversed by addition of NAD(P)H. The decrease in NAD(P)H oxidase activity by bilirubin was independent of a decrease in protein expression of components of the NAD(P)H oxidase system (gp91phox, p47phox and p22phox). It is plausible to suggest that bilirubin interferes with the activation process of the enzyme, a phenomenon that would explain the inhibition of NAD(P)H oxidase activity observed as soon as 1 h after incubation with the bile pigment. In this context, and given the properties of bilirubin to interfere with protein phosphorylation, we evaluated its effect on phosphorylation of MAPK pathways participating in the oxidase activation, such as p38 and p42/44. In vitro experiments showed no change in p38 and p42/44 phosphorylation by bilirubin, excluding this mechanism of action. Bilirubin did not modify the expression of HO-1, which is known to modulate NAD(P)H oxidase and NOS2 expression/activity. Modulation of other pathways of NAD(P)H oxidase activation, such as phosphorylation of p47phox, could be another plausible mechanism explaining the reduction in NAD(P)H oxidase activity by bilirubin. Further experiments are needed to explore the mechanism(s) of this phenomenon. However, irrespective of the exact mechanism(s) involved in the inhibition of NAD(P)H oxidase activity and in addition to the attenuation of NOS2 induction elicited by the bile pigment, these findings are important as they highlight the inherent properties of bilirubin to protect against endotoxic shock and because redox signaling by NAD(P)H oxidase in vessels is known to significantly contribute to the refractory vasodilation after LPS inoculation.
This study demonstrates that high bilirubin concentrations significantly protect against LPS-mediated toxicity by reducing mortality and preventing the hypotension typical of endotoxic shock. The protective effect we found did not involve inhibition of the classical inflammatory mediators (i.e. TNF- and IL-6). Our results show, however, that the beneficial effects of bilirubin on endotoxic shock are probably related to an attenuation of NOS2 induction in cardiovascular and other tissues, secondary to inhibition of NAD(P)H oxidase activity. This is a new mechanism extending our knowledge on the protective effects of bilirubin. Heme depletion and carbon monoxide generation, both concomitant with the production of bilirubin after HO induction, also impair NAD(P)H oxidase expression and/or activity, pointing out that this enzyme is a main target for the signal transduction mechanisms elicited by the HO system.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2368fje;
1 These authors contributed equally to this work. 
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