Andreas K. Nussler

Ischemic preconditioning protects from hepatic ischemia/reperfusion-injury by preservation of microcirculation and mitochondrial redox-state

BACKGROUND/AIMS Ischemic preconditioning (IP) is known to protect hepatic tissue from ischemia-reperfusion injury. However, the mechanisms involved are not fully understood yet. METHODS Using intravital multifluorescence microscopy in the rat liver, we studied whether IP exerts its beneficial effect by modulating postischemic Kupffer cell activation, leukocyte-endothelial cell interaction, microvascular no-reflow, mitochondrial redox state, and, thus, tissue oxygenation. RESULTS Portal triad cross-clamping (45 min) followed by reperfusion induced Kupffer cell activation, microvascular leukocyte adherence, sinusoidal perfusion failure (no-reflow) and alteration of mitochondrial redox state (tissue hypoxia) (P<0.05). This resulted in liver dysfunction and parenchymal injury, as indicated by decreased bile flow and increased serum glutamate dehydrogenase (GLDH) levels (P<0.05). IP (5 min ischemia and 30 min intermittent reperfusion) was capable to significantly reduce Kupffer cell activation (P<0.05), which was associated with a slight attenuation of leukocyte adherence. Further, IP markedly ameliorated sinusoidal perfusion failure (P<0.05), and, thereby, preserved adequate mitochondrial redox state (P<0.05). As a consequence, IP prevented the decrease of bile flow (P<0.05) and the increase in serum GLDH levels (P<0.05). CONCLUSIONS IP may exert its beneficial effects on hepatic ischemia-reperfusion injury by preserving mitochondrial redox state, which is guaranteed by the prevention of reperfusion-associated Kupffer cell activation and sinusoidal perfusion failure.

Selective induction of human hepatic cytochromes P450 2B6 and 3A4 by metamizole.

The pyrazolone drug metamizole is a widely used analgesic. Analysis of liver microsomes from patients treated with metamizole revealed selectively higher expression of cytochromes P450, CYP2B6 and CYP3A4 (3.8‐ and 2.8‐fold, respectively), and 2.9‐fold higher bupropion hydroxylase activity compared with untreated subjects. Further investigation of metamizole and various derivatives on different potential target genes in human primary hepatocytes demonstrated time‐ and concentration‐dependent induction by metamizole of CYP2B6 (7.8‐ and 3.1‐fold for mRNA and protein, respectively, at 100 μM) and CYP3A4 (2.4‐ and 2.9‐fold, respectively), whereas other genes (CYP2C9, CYP2C19, CYP2D6, NADPH:cytochrome P450 reductase, ABCB1, constitutive androstane receptor (CAR), pregnane X receptor (PXR)) were not substantially altered. Using reporter gene assays, we show that metamizole is not acting as a direct ligand to either PXR or CAR, suggesting a phenobarbital‐like mechanism of induction. These data warrant further studies to elucidate the drug‐interaction potential of metamizole, especially in patients with long‐term treatment.

Effects of hepatocellular iron imbalance on nitric oxide and reactive oxygen intermediates production in a model of sepsis

BACKGROUND/AIMS In mammals iron homeostasis is most important, as imbalance of iron such as iron overload may lead to severe diseases. Recently, it has been shown that the iron regulatory protein-1 is partially controlled by nitric oxide and reactive oxygen intermediates, molecules frequently seen in inflammatory events. The aim of the present study was to investigate the effects of impaired iron homeostasis on the interaction of nitric oxide, and reactive oxygen intermediate formation in hepatocytes in a model of acute inflammation. METHODS Hepatocytes isolated from Corynebacterium parvum (C parvum)-injected rats were used to examine the formation of nitrogen and oxygen intermediates by iron deprivation and iron overload in the presence of lipopolysaccharide. In addition, we investigated the RNA binding and aconitase activity of iron regulatory protein-1. RESULTS In the present study we show that iron overload in lipopolysaccharide-treated C. parvum-primed hepatocytes downregulated the RNA binding of iron regulatory protein-1 and aconitase activity. Subsequently, we observed a reduced formation of nitrite/nitrate and S-nitrosothiols but an increased production of reactive oxygen species, and hepatocellular damage. Moreover, the addition of iron to cell cultures caused a further increase in cellular damage, a drop in the cellular glutathione pool, and an increase in peroxynitrite and hydroxyl-like radicals. In contrast, addition of deferoxamine (an iron chelator) to lipopolysaccharide-treated C. parvum-primed hepatocytes protected cells by stabilizing the GSH content, maintaining the nitric oxide formation, and by reducing Fenton oxidants. CONCLUSIONS Our results show that the antioxidative effects of iron chelators prevent the formation of toxic Fenton oxidants in severe inflammatory events, which should be considered in the treatment of disorders characterized by an iron imbalance.

Influence of PMN Leukocyte-Mediated Pancreatic Damage on the Systemic Immune Response in Severe Acute Pancreatitis in Rats

The outcome of severe acute pancreatitis is determined by the development of the systemic inflammatory response and subsequent multiorgan dysfunction. Using the taurocholate-induced model of acute pancreatitis in rats, we investigated the relationship between early polymorphonuclear (PMN)-mediated pancreatic tissue damage and the systemic inflammatory response. The respiratory burst of PMN leukocytes was increased in animals with acute pancreatitis and was reduced by anti-ICAM-1 antibody and oxygen radical scavenger treatment after 24 hr. In acute pancreatitis a reduced number of peripheral helper T cells was evident, most likely due to l-selectin-mediated increased lymphocyte homing. After 24 hr the CD45RChigh/CD45RClow ratio of helper T cells, a critical factor in T cell-mediated disease was increased due to a reduction of regulatory CD45RClow cells. Only the treatment with anti-ICAM-1 mAb affected these changes, indicating that immunological changes in necrotizing pancreatitis are only in part affected by early PMN leukocyte-mediated pancreatic damage.

Inhibition of hepatic transcriptional induction of lipopolysaccharide‐binding protein by transforming‐growth‐factor β1

LPS‐binding protein (LBP) is an acute‐phase protein with the ability to bind and transfer LPS of Gram‐negative bacteria, as well as cell wall compounds of other pathogenic bacteria. This soluble pattern‐recognition molecule is present in high concentrations in serum and represents an important defense mechanism of the host. Regulation of the hepatic acute‐phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host organism. We show here that TGF‐β1, in a dose‐dependent fashion, is able to inhibit LBP transcript accumulation and LBP protein synthesis induced by IL‐6, IL‐1β and dexamethasone in hepatoma cell lines. These data were confirmed employing primary human hepatocytes, where TGF‐β1 also inhibited LBP protein synthesis. We identified and analyzed several Smad‐binding sites (Smads are major regulatory elements of TGF‐β1) within the LBP promoter, and found that one of them was active. We furthermore identified an AP‐1‐binding site clearly conferring inhibitory effects of TGF‐β1 towards LBP promoter activity, shown by gel shift and promoter mutagenesis experiments. Further elucidating the mechanism of transcriptional regulation of proteins involved in innate immune responses may potentially help to develop novel intervention strategies for the acute‐phase response, sepsis, and septic shock.

Peripheral Blood Monocytes Can Be Induced to Acquire Stem Cell-Like Properties

Adult stem or programmable cells hold great promise in diseases in which damaged or non-functional cells need to be replaced. We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into cells whose phenotypes resemble specialized cell types like hepatocytes and pancreatic beta cells. During phenotypic conversion the monocytes downregulate monocyte/macrophage differentiation markers being indicative of partial dedifferentiation and are partially reprogrammed to acquire a state of plasticity along with expression of various markers of pluripotency. These cells were termed “programmable cells of monocytic origin” (PCMOs). Current efforts focus on establishing culture conditions that increase both the plasticity and proliferation potential of PCMOs in order to be able to generate large amounts of blood-derived cells suitable for both autologous and allogeneic therapies.

NF-kappa B vermittelte NOS-2 Expression wirkt protektiv bei isolierter Dünndarmischämie

Ischamie/Reperfusion (IR) des Darmes schadigt nicht nur die betroffenen Darmabschnitte, sondern kann auch in anderen, primar nicht ischamischen Organen wie z. B. der Leber zu schweren Veranderungen fuhren. Dieser IR-Schaden kann durch Stimulation der inflammatorischen Antwort mittels IL-2 vermindert werden. In dieser Studie wurde untersucht, ob der positive Effekt der IL-2 Gabe nach intestinaler Ischamie auf einer Aktivierung des nuklearen Faktor kappa B (NFkappaB) und einer nachfolgenden vermehrten Expression von NOS-2 beruht.

Interleukin-2 vermindert die hepatische Schädigung nach isolierter Ischämie des Darmes

Introduction: Intestinal ischemia/reperfusion (IR) results in tissue destruction in the gut, but may also induce oxidative stress and cellular damage in distant organs, such as the liver. It remains unclear whether inhibition or stimulation of the inflammatory response may help to reduce tissue destruction after IR. Therefore, the effects of pro- and anti-inflammatory cytokines on oxidative stress and tissue destruction in the intestine and liver after intestinal IR were analyzed. Methods: Male Lewis rats underwent 60 min of intestinal ischemia by clamping of the superior mesenteric artery or were sham operated. Animals received an IV bolus of 40 µg/kg IL-2, IL-10 or vehicle before reperfusion. At 20 min 1 h, 4 h, and 24 h after reperfusion animals were sacrificed and blood and tissue samples were obtained for analysis of serum levels of NO- 2/NO- 3, hyaluronic acid (HA), transaminases (AST), and tissue GSH levels, as well as for RT-PCR of the inducible nitric oxide synthase (NOS-2) and hemeoxygenase-1 (HO-1) mRNA. Results: IR resulted in tissue destruction and oxidative stress in both organs indicated by elevated HA and AST serum levels and significantly reduced GSH tissue levels. Concomitantly increased expression of NOS- 2 and HO-1 mRNA was detectable in liver and intestine after IR. IL-2 administration resulted in diminished hepatic tissue destruction despite sustained reduction of GSH. IL-2 further increased NOS-2 and HO-1 mRNA expression in both organs after IR. In contrast, IL-10 resulted in increased tissue damage, but failed to increase NOS-2 or HO-1 mRNA expression in the small intestine or liver after IR. Discussion: Intestinal IR does not only result in oxidative stress in the intestine but elicits also oxidative stress in the liver. The beneficial effect of IL-2 was not due to reduction of oxidative stress, but rather due to increased expression of NOS-2 and HO-1 mRNA. In contrast, IL-10 which failed to increase NOS-2 and HO-1 mRNA expression in both organs led to increased tissue destruction after IR. Conclusions: Tissue destruction and oxidative stress in intestinal IR are not limited to the intestine. The beneficial effect of IL-2 on IR may be due to decreased neutrophil infiltration and enhanced neutralization of oxygen intermediates by nitric oxide in the intestine and liver.