HEPATOLOGY Elsewhere JACQUELYN MAHER, EDITOR
ADVISORY COMMITTEE LAURIE DELEVE, Los Angeles, CA DAVID CRABB, Indianapolis, IN ADRIAN DIBISCEGLIE, St. Louis, MO EMMET KEEFFE, Palo Alto, CA JOEL LAVINE, San Diego, CA MICHAEL NATHANSON, New Haven, CT DON ROCKEY, Durham, NC DWAIN THIELE, Dallas, TX
San Francisco General Hospital Building 40, Room 4102 1001 Potrero Avenue San Francisco, CA 94110
GIVE ME iNOS OR GIVE ME DEATH
Sass G, Koerber K, Bang R, Guehring H, Tiegs G. Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice. J Clin Invest 2001;107:439-447. Reprinted with permission. ABSTRACT
Concanavalin A (Con A) causes severe TNF-␣–mediated and IFN-␥–mediated liver injury in mice. In addition to their other functions, TNF-␣ and IFN-␥ both induce the inducible nitric oxide (NO) synthase (iNOS). Using different models of liver injury, NO was found to either mediate or prevent liver damage. To further elucidate the relevance of NO for liver damage we investigated the role of iNOS-derived NO in the Con A model. We report that iNOS mRNA was induced in livers of Con A–treated mice within 2 hours, with iNOS protein becoming detectable in hepatocytes as well as in Kupffer cells within 4 hours. iNOSⴚ/ⴚ mice were protected from liver damage after Con A treatment, as well as in another TNF-␣–mediated model that is inducible by LPS in D-galactosamine–sensitized (GalN-sensitized) mice. iNOSdeficient mice were not protected after direct administration of recombinant TNF-␣ to GalN-treated mice. Accordingly, pretreatment of wild-type mice with a potent and specific inhibitor of iNOS significantly reduced transaminase release after Con A or GalN/LPS, but not after GalN/TNF-␣ treatment. Furthermore, the amount of plasma TNF-␣ and of intrahepatic TNF-␣ mRNA and protein was significantly reduced in iNOSⴚ/ⴚ mice. Our results demonstrate that iNOS-derived NO regulates proinflammatory genes in vivo, thereby contributing to inflammatory liver injury in mice by stimulation of TNF-␣ production. COMMENTS
In the liver, where hepatocytes and immune cells coexist in harmonious balance to regulate many of the important metabolic functions of the body, cellular battles of life and death are waged at times with powerful weapons of defense and attack. One potent weapon is nitric oxide (NO), a radical molecule produced by nitric oxide synthase (NOS). As a soluble gas, NO can stealthily pass through membranes from cell to cell and interact with multiple proteins and reactive oxygen species. These interactions send ripples of signaling information through the cellular and organ milieu that can tip the scales of homeostasis towards cell death or proliferation. Nitric oxide, the 1992 Science journal molecule of the year, seems to be everywhere. In 1998, Furchgott, Murad, and Ignarro won the Nobel Prize in Physiology or Medicine for uncovering the role of NO in the cardiovascular system. Problems and controversies have grown from observations that
NO can have widely disparate effects, depending on its local concentration and the nature of its target proteins and cells. In the quickly expanding world of apoptosis, multiple roles for NO have been identified, that either block or induce cell death.1 As they are in many other tissues and organs, the cytotoxic and cytoprotective roles of NO in liver are both complex and controversial. Many in vivo studies suggest that NO and inducible nitric oxide synthase (iNOS) protect against hepatocyte apoptosis.2,3 However, in the article highlighted above, Sass et al. show convincingly that “iNOS-derived NO regulates proinflammatory genes in vivo, thereby contributing to inflammatory liver injury in mice by stimulation of tumor necrosis factor-␣ (TNF-␣) production.”4 The investigators examined NO in two models of liver injury, one involving intravenous injection of the T-cell mitogenic plant lectin concanvalin A (Con A) and the other intraperitoneal injection of D-galactosamine with lipopolysaccharide (GalN/LPS). Both treatments induced fulminant hepatitis in wild-type mice within 8 hours, coincident with hepatic upregulation of iNOS. Strikingly, knockout mice deficient in the iNOS gene displayed profoundly less Con A– or GalN/LPS-induced liver damage, as measured by transaminase release into plasma and TUNEL staining of liver tissue for DNA fragmentation. These findings were also reproduced by pretreatment of wild-type mice with the iNOS inhibitor NG-iminoethyl-L-lysine (LNIL). Sass et al. went on to show that iNOS is necessary for Con A–induced increases in plasma TNF-␣ and interferon gamma (IFN-␥), as well as the time-dependent rise in hepatic expression of TNF-␣ mRNA and protein. When TNF-␣ was administered intravenously with intraperitoneal GalN instead of LPS, both wild-type mice pretreated with L-NIL and iNOSdeficient mice displayed liver damage comparable to control mice. The investigators concluded that soluble TNF-␣ bypassed the need for iNOS-mediated production of TNF-␣, and damaged the liver directly. These results suggest that iNOS is critical for up-regulation of the cytokines TNF-␣ and IFN-␥ in inflammatory-mediated liver injury. Other models of liver injury have also highlighted the detrimental effects of iNOS. In a model of hemorrhagic shock, iNOS inhibition or deficiency prevented upregulation of the inflammatory cytokines interleukin 6 (IL-6) and granulocytecolony stimulating factor (G-CSF), as well as the transcription factors nuclear factor-B (NF-B) and signal transducer and activator of transcription-3 (STAT3). These changes were associated with a marked reduction of lung and liver injury.5 In a warm ischemia-reperfusion model of liver injury, iNOS knockout mice showed decreased levels of plasma transaminases, suggesting that local iNOS production contributes to hepatic injury.6 Taken together with this recent report, these
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data suggest that iNOS is essential for initiation of the inflammatory response and liver damage in select models involving hepatic inflammation. The damaging consequences of iNOS contrast with observations in other in vivo models, that NO is a potent inhibitor of hepatocyte cell death. For example, inhibition of iNOS or targeted disruption of the iNOS gene increases hepatic apoptosis in endotoxemia3 and following partial hepatectomy.7 A liver-selective NO donor was shown to almost completely suppress hepatocyte apoptosis in the wellestablished TNF/GalN model in rats.2 Dissecting out the factors that permit NO to exert diverse actions in the same organ is difficult. However, two important points were mentioned in the Discussion that may provide some clues. First, although Con A treatment induced iNOS in both hepatocytes and Kupffer cells, GalN/LPS treatment increased iNOS expression only in Kupffer cells. It could be that Kupffer cell– derived NO contributes more to the proinflammatory effects than hepatocyte-derived NO. iNOS-dependent production of TNF-␣ by Kupffer cells may then lead to the damage of the adjacent hepatocyte. NO is a potent inhibitor of TNF-mediated toxicity in hepatocytes, through the upregulation of protective proteins8 and by the inhibition of caspasedependent events via S-nitrosylation of caspases and poorly understood effects of cGMP.9,10 We can only speculate why NO did not protect the hepatocytes in the article by Sass et al.4 One explanation is that NO levels within the hepatocyte were inadequate to block death; it is also possible that the hepatocytes were under additional redox stress such that the chemical fate of NO did not favor protection. For example, it is known that one factor that predicts NO protection is the content of nonheme iron within the cell.11 The second point revealed by the investigators was that by immunofluorescent staining, iNOS was absent in TUNELpositive liver cells after Con A administration. This suggests a cell-specific regional protection in the liver where hepatocytes that could produce iNOS were protected. It would be interesting to know whether proximity of the hepatocytes to the portal triad correlated with concentrations of reactive oxygen species and cell death by TUNEL staining. The in vivo experiments by Sass et al. extend our insight beyond in vitro cultures of single cell types into the more complex relationships at the organ level. Understanding the relationship between hepatocytes and immune cells in the liver has profound implications for liver transplantation, regeneration, hepatitis, and liver failure. We commend them for showing that iNOS, likely from Kupffer cells, produces NO, which upregulates TNF-␣ production that damages hepato-
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cytes. After intraperitoneal injection of Con A or GalN/LPS, hepatocytes may not produce enough NO to protect against this cytokine assault, hence the resultant liver damage and release of cellular enzymatic contents into the plasma. This hypothesis is not contradictory to our previous results that show hepatoprotection by NO and iNOS expression in hepatocytes. Under specific pathologic conditions, perhaps the cells will proclaim, “Give me iNOS and give me death.” PETER K. M. KIM, M.D. TIMOTHY R. BILLIAR, M.D. Department of Surgery Laboratories University of Pittsburgh Medical Center Pittsburgh, PA REFERENCES 1. Kim PKM, Zamora R, Petrosko P, Billiar TR. The regulatory role of nitric oxide in apoptosis. Int Immunopharmacol 2001;1:1421-1441. 2. Saavedra JE, Billiar TR, Williams DL, Kim YM, Watkins SC, Keefer LK. Targeting nitric oxide (NO) delivery in vivo. Design of a liver- selective NO donor prodrug that blocks tumor necrosis factor-alpha- induced apoptosis and toxicity in the liver. J Med Chem 1997;40:1947-1954. 3. Ou J, Carlos TM, Watkins SC, Saavedra JE, Keefer LK, Kim YM et al. Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, ICAM- 1 expression, and neutrophil accumulation during endotoxemia. Nitric Oxide 1997;1:404-416. 4. Sass G, Koerber K, Bang R, Guehring H, Tiegs G. Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice. J Clin Invest 2001;107:439-447. 5. Hierholzer C, Harbrecht B, Menezes JM, Kane J, MacMicking J, Nathan CF, Peitzman AB, et al. Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock. J Exp Med 1998;187:917-928. 6. Lee VG, Johnson M, Baust J, Laubach V, Watkins S, Billiar TR. The roles of iNOS in liver ischemia-reperfusion injury. Shock 2001 (in press). 7. Rai RM, Lee FY, Rosen A, Yang SQ, Lin HZ, Koteish A, Liew FY, et al. Impaired liver regeneration in inducible nitric oxide synthase deficient mice. Proc Natl Acad Sci U S A 1998;95:13829-13834. 8. Kim YM, de Vera ME, Watkins SC, Billiar TR. Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-alpha-induced apoptosis by inducing heat shock protein 70 expression. J Biol Chem 1997; 272:1402-1411. 9. Kim YM, Talanian RV, Billiar TR. Nitric oxide inhibits apoptosis by preventing increases in caspase-3- like activity via two distinct mechanisms. J Biol Chem 1997;272:31138-31148. 10. Li J, Yang S, Billiar TR. Cyclic nucleotides suppress tumor necrosis factor alpha-mediated apoptosis by inhibiting caspase activation and cytochrome c release in primary hepatocytes via a mechanism independent of Akt activation. J Biol Chem 2000;275:13026-13034. 11. Kim YM, Chung HT, Simmons RL, Billiar TR. Cellular non-heme iron content is a determinant of nitric oxide- mediated apoptosis, necrosis, and caspase inhibition. J Biol Chem 2000;275:10954-10961.