Timothy R. Billiar

Tumor necrosis factor alpha inhibits hepatocyte mitochondrial respiration

Although direct cytotoxicity is a well-established phenomenon of tumor necrosis factor alpha (TNF alpha)-induced tissue damage, the intracellular events leading to cell death are still poorly understood. To study the cytotoxic effects of TNF alpha on normal parenchymal cells, rat hepatocytes were purified and incubated with various concentrations of TNF alpha. Mitochondrial respiration, total protein synthesis, and enzyme release were measured to assess metabolic performance and cell integrity. Treatment with TNF alpha suppressed mitochondrial respiration in a concentration-dependent fashion, resulting in a reduction of the activity of complex I of the respiratory chain to 67.0 +/- 3.5% of that of untreated hepatocytes by 2000 U/mL TNF alpha. Under these conditions protein synthesis and the release of intracellular enzymes were significantly increased. Both hepatocellular enzyme release and inhibition of mitochondrial respiration appear to be associated with the generation of reactive oxygen intermediates by the hepatocyte itself, because oxygen radical scavengers prevented these adverse effects of TNF alpha. Inhibition of protein synthesis by cycloheximide as well as addition of cyclic adenosine monophosphate synergistically enhanced the suppression of mitochondrial respiration by TNF alpha, resulting in complex I activity of 6.9 +/- 1.6% and 24.9 +/- 2.9% of that of untreated cells. These data indicate that inhibition of mitochondrial respiration is one of the mechanisms by which TNF alpha induces tissue injury.

Evidence that rat Kupffer cells stimulate and inhibit hepatocyte protein synthesis in vitro by different mechanisms

Kupffer cell control of hepatocyte protein synthesis may be an important mechanism involved in the regulation of normal liver function and may be one mechanism responsible for the alterations in liver function seen during sepsis. The present series of in vitro experiments compare the response to various inflammatory stimuli of hepatocytes cocultured with Kupffer cells with that of hepatocytes cultured alone. In the absence of inflammatory stimuli, Kupffer cells stimulated hepatocyte protein synthesis. Lipopolysaccharide or gentamicin-killed Escherichia coli triggered Kupffer cell-mediated inhibition of cocultured hepatocyte protein synthesis but had no effect on protein synthesis of hepatocytes cultured alone. Phorbol myristate acetate, muramyl dipeptide, and calcium ionophore had no effect on hepatocytes cultured alone but resulted in a loss of Kupffer cell-mediated stimulation of cocultured hepatocyte protein synthesis without inhibition. Addition of dexamethasone to cocultures prevented the Kupffer cell-mediated inhibition of hepatocyte protein synthesis triggered by lipopolysaccharide, but did not block Kupffer cell-mediated stimulation in the absence of lipopolysaccharide. The data suggest that Kupffer cells can stimulate and inhibit hepatocyte protein synthesis by independent mechanisms. Kupffer cells may be important regulators of hepatocellular function in health and disease.

Glutathione regulates nitric oxide synthase in cultured hepatocytes.

OBJECTIVE The authors determine the relationship between glutathione and nitric oxide (NO) synthesis in cultured hepatocytes. SUMMARY BACKGROUND DATA Glutathione is a cofactor for a number of enzymes, and its presence is essential for maximal enzyme activity by the inducible macrophage nitric oxide synthase (iNOS), which produces the reactive nitric oxide radical. Hepatocytes contain substantial quantities of glutathione, and this important tripeptide is decreased in hepatocytes stressed by ischemia/reperfusion or endotoxemia. Endotoxemia also induces the synthesis of inflammatory cytokines that result in the production of nitric oxide from hepatocytes by iNOS, suggesting that hepatocytes may be attempting to synthesize nitric oxide at times when intracellular glutathione is reduced. METHODS Hepatocytes were cultured with buthionine sulfoximine and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione. After exposure to cytokines, NO synthesis was assessed by supernatant nitrite levels, cytosolic iNOS enzyme activity, and iNOS mRNA levels. RESULTS Inhibition of glutathione synthesis with buthionine sulfoximine or inhibition of glutathione reductase activity with BCNU inhibited nitrite synthesis. Both buthionine sulfoximine and BCNU inhibited the induction of iNOS mRNA, as detected by Northern blot analysis. Exogenous glutathione increased cytokine-stimulated iNOS induction, overcame the inhibitory effects of BCNU, and increased nitrite production by intact hepatocytes, induced hepatocyte cytosol, and partially purified hepatocyte iNOS. CONCLUSIONS In cultured hepatocytes, adequate glutathione levels are required for optimal nitric oxide synthesis. This finding is predominantly due to an effect on iNOS mRNA levels, although glutathione also participates in the regulation of iNOS enzyme activity.

Risk factors for survival following surgical treatment of traumatic aortic rupture.

Linear discriminate analysis was used to determine the effects of age and Injury Severity Score on survival in 37 consecutive patients treated surgically for traumatic rupture of the thoracic aorta. Pearson product moment correlations were calculated between associated injuries and survival. The age of the injured patients was the only variable that correlated statistically with survival: the lower the patients age, the greater the chance of survival (r = 0.3535; p = 0.016). The severity of the injury, as represented by the Injury Severity Score, showed a tendency toward decreased survival with increasing Injury Severity Score (r = -0.2523; p = 0.066). Specific types of associated injuries did not correlate with survival. Survival rates were not statistically different for patients who underwent cardiopulmonary bypass compared with those in whom a temporary plastic shunt was used (chi-square = 1.72; p = 0.19). We conclude that age is the most significant factor in predicting survival in patients who undergo surgical repair of traumatic aortic rupture.

The role of intestinal flora on the interactions between nonparenchymal cells and hepatocytes in coculture.

Kupffer cells are exposed directly to a number of factors in the portal circulation that can modify or regulate their responses to septic stimuli. The gut represents a potential source of a number of these factors including endotoxin, lymphokines, and prostaglandins. We examined Kupffer cells from germfree rats and germfree rats exposed to endotoxin or bacteria via their GI tracts to determine the importance of the intestinal flora in maintaining or modulating Kupffer cell responses. Kupffer cells from germfree animals were reduced in numbers and failed to respond to LPS in Kupffer cell: hepatocyte coculture. When germfree rats were exposed to bacterial endotoxin or bacteria via the gastrointestinal tract their Kupffer cells increased in numbers to normal and the cells responded to LPS in culture. Intestinal overgrowth with Escherichia coli for 2 days activated the Kupffer cells and significantly increased Kupffer cell sensitivity to LPS. These data suggest that the environment of the gastrointestinal tract is important for normal Kupffer cell responses and that intestinal bacterial overgrowth can modify Kupffer cell responses to septic stimuli.

Role of NO and Nitrogen Intermediates in Regulation of Cell Functions

Prior to the 1980s, nitric oxide (NO) was best known as a toxic reactive free radical found in atmospheric pollutants and carcinogens [1], a by-product of microbial nitrogen metabolism [2,3] and a potent activator of the mammalian enzyme heme-containing soluble guanylate cyclase [4]. As early as 1916, evidence for a mammalian nitrogen metabolic pathway was reported with the finding that several types of animals, including humans, excreted more urinary nitrate than could be accounted for by dietary intake [5]. Further evidence was reported by Tannenbaum and co-workers [6,7] who showed that in vivo mammalian nitrate formation was substantially enhanced by administration of the immunostimulant lipopolysaccharide (LPS) [8]. Stuehr and Marletta [9] first demonstrated that murine macrophages stimulated in vitro with LPS expressed nitrogen oxide synthetic activity and produced nitrite (NO2) and nitrate (NO3 -). Simultaneously, other investigators were trying to elucidate the identity of a short-lived, diffusable endothelium-derived relaxing factor (EDRF) produced by acetylcholine-treated endothelial cells that was responsible for mediating smooth muscle cell relaxation [10]. Based on the similarities in the pharmacological properties of EDRF and NO generated from acidified nitrite, Furchgott suggested that EDRF may be NO in 1986 [11]. At the same time, Ignarro et al. also proposed that EDRF may be NO or a closely related species [12]. The following year, two independent groups of investigators [13,14] demonstrated EDRF was indeed nitric oxide (NO).

Regulation and Function of Nitric Oxide in the Liver

The inducible nitric oxide synthase (iNOS) gene is expressed in nearly every organ and cell type during endotoxemia. Previously, we have shown that a combination of cytokines synergistically activate iNOS expression in the liver, and we have cloned the first human iNOS gene from cytokine-stimulated hepatocytes. We have also shown that steriods, TGF-β, the heat shock response, and nitric oxide (NO) itself, all down-regulate iNOS expression. In vivo, we have shown that hepatic iNOS induction is differentially regulated from the typical acute-phase reactants and is not expressed as a mandatory component of the acute phase response. Thus, numerous mechanisms have evolved to regulate iNOS expression during hepatocellular injury.