Protein array technology to investigate cytokine production by monocytes from patients with advanced alcoholic cirrhosis: An ex vivo pilot study.

AIM: In patients with advanced cirrhosis, little is known about the ability of peripheral blood monocytes to spontaneously produce signaling proteins such as cytokines. The aim of this ex vivo study was to evaluate cytokine production under baseline conditions and after stimulation by lipopolysaccharide (LPS), a toll-like receptor (TLR) agonist. METHODS: Peripheral blood monocytes were isolated from patients with advanced alcoholic cirrhosis (without ongoing bacterial infections) and normal subjects. Cells were left unstimulated or were stimulated with LPS. The abundance of 24 cytokines was measured using a filter-based, arrayed sandwich enzyme-linked immunosorbent assay (ELISA) in the supernatant of cultured monocytes. RESULTS: Cirrhotic monocytes spontaneously produced six proteins (TNF-alpha, IL-6, IL-8, MCP-1, RANTES and Gro), whereas normal monocytes produced only small amounts of IL-8 and RANTES. Analyses with the online gene set analysis toolkit WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt) found enrichment for the six proteins in the human gene ontology subcategory (http://www.geneontology.org), Kyoto Encyclopedia of Genes and Genome pathways (http://www.genome.ad.jp/kegg/) and BioCarta pathways (http://www.biocarta.com/genes/index.asp) consistent with a proinflammatory phenotype of cirrhotic monocytes resulting from activated TLR signaling. Interestingly, LPS-elicited TLR engagement further increased the production of the six proteins and did not induce the secretion of any others, in particular the anti-inflammatory cytokine IL-10. LPS-stimulated normal monocytes produced TNF-alpha, IL-6, IL-8, MCP-1, RANTES, Gro and IL-10. CONCLUSION: In patients with advanced cirrhosis, peripheral blood monocytes spontaneously produce proinflammatory cytokines, presumably in response to unrestricted TLR signaling.

Ex vivo effects of high-density lipoprotein exposure on the lipopolysaccharide-induced inflammatory response in patients with severe cirrhosis.

UNLABELLED: High-density lipoproteins (HDL) are known to neutralize lipopolysaccharide (LPS). Because patients with cirrhosis have lower HDL levels, this may contribute to LPS-induced ex vivo monocyte overproduction of proinflammatory cytokines. However, the effects of HDL on cytokine production by monocytes from patients with cirrhosis have never been studied. The aim of this study was to determine the effects of HDL on LPS-induced proinflammatory cytokine production in whole blood and isolated monocytes from patients with severe cirrhosis and controls. Plasma levels of HDL and cytokines were determined. The effects of reconstituted HDL (rHDL) on LPS-induced cytokine production in whole blood were assessed by cytokine array and on tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) production in isolated monocytes. Plasma HDL levels were significantly lower in patients with cirrhosis than in controls. Plasma levels of TNF-alpha and IL-6 were significantly higher in patients with cirrhosis than in controls. Incubation of rHDL with whole blood prevented LPS-induced TNF-alpha and IL-6 overproduction in patients with cirrhosis. LPS-induced TNF-alpha production and CD14 expression were significantly more marked in cirrhotic monocytes than in control monocytes, and both decreased significantly after rHDL incubation. LPS-induced down-regulation of scavenger receptor, class B, type I (SR-BI) expression was abolished in cirrhotic monocytes. CONCLUSIONS: This study shows that rHDL abolishes the LPS-induced overproduction of proinflammatory cytokines in whole blood from patients with severe cirrhosis. These results were confirmed in isolated monocytes from these patients. This suggests that administration of rHDL might be a useful strategy for the treatment of cirrhosis to limit LPS-induced cytokine overproduction.

Possible mechanisms involved in the discrepancy of hepatic and aortic endothelial nitric oxide synthases during the development of cirrhosis in rats.

BACKGROUND/AIM: In cirrhosis, systemic nitric oxide (NO) overproduction and hepatic NO hypoproduction lead to arterial vasodilatation and portal hypertension. The mechanisms involved in these alterations in endothelial NO synthase (eNOS)-derived NO production in hepatic and systemic vasculature remain unknown. The aim of this study was to evaluate the regulation of eNOS and its major modulators in the liver and aorta during the development of cirrhosis in rats. METHODS: Activated eNOS and Akt and expressions, and caveolin-1 (Cav-1) and scavenger receptor class B type I (SR-BI) expressions were measured before and 1, 2, 3 and 4 weeks after bile duct ligation. Plasma high-density lipoprotein (HDL) levels were measured. RESULTS: Activated aortic eNOS increased at week 1, whereas it began to decrease at week 3 in the liver. Aortic expression of Cav-1 decreased at week 3 while hepatic expression increased by four-fold. Activated aortic Akt increased progressively while in the liver it gradually decreased during the development of cirrhosis. HDL levels decreased during the first week and decreased thereafter. The hepatic expression of SR-BI decreased. CONCLUSION: This study shows that the modulation of Akt and Cav-1 is inverted in the liver and the aorta during the development of cirrhosis. In addition, decreased HDL levels may play a role in reduced hepatic eNOS activity.

Discrepant effects of inducible nitric oxide synthase modulation on systemic and splanchnic endothelial nitric oxide synthase activity and expression in cirrhotic rats.

BACKGROUND: Arterial vasodilatation, which is a major factor in the pathogenesis of the hyperkinetic circulatory state and portal hypertension in cirrhosis, is due to arterial nitric oxide (NO) overproduction secondary to endothelial NO synthase (eNOS) and inducible NOS (iNOS) upregulation. However, in cirrhosis, the respective roles of eNOS and iNOS isoforms in NO overproduction are still unknown and the effect of iNOS modulation on eNOS activity and expression has not been evaluated in the systemic or splanchnic vessels. The aim of this study was to evaluate the effects of modulating aortic and superior mesenteric arteries (SMA) iNOS on arterial eNOS activity and expression in rats with cirrhosis. METHODS: eNOS and iNOS protein expression and eNOS activity (assessed by its phosphorylation at serine 1177) were measured in the aortas and SMA in untreated and treated cirrhotic rats with lipopolysaccharide (LPS), N-iminoethyl-L-lysine (L-NIL), a selective iNOS inhibitor, and LPS plus L-NIL. RESULTS: LPS administration significantly increased eNOS and iNOS protein expression and eNOS activity in the aortas of both sham-operated and cirrhotic rats. However, in SMA, LPS administration induced a decrease in eNOS protein expression and activity and an increase in iNOS protein expression. CONCLUSION: The results of this study may explain the worsening of the hyperdynamic state in cirrhosis during septic shock by direct LPS-induced eNOS activation in large systemic vessels, and its inhibition in concomitant small splanchnic vasculature by iNOS synthesized NO.

High-density lipoprotein administration attenuates liver proinflammatory response, restores liver endothelial nitric oxide synthase activity, and lowers portal pressure in cirrhotic rats.

UNLABELLED: In patients with cirrhosis, endotoxic shock is a major complication of portal hypertension, which is related partly to intrahepatic endothelial nitric oxide synthase (eNOS) down-regulation. High-density lipoproteins (HDLs), whose plasma levels are reduced in cirrhosis, have an anti-inflammatory effect by neutralizing circulating lipopolysaccharide (LPS), and they increase eNOS activity in endothelial cells. Therefore, the aim of this study was to assess the effects of reconstituted high-density lipoprotein (rHDL) administration on the LPS-induced proinflammatory response, intrahepatic eNOS regulation, and portal hypertension in cirrhotic rats. Cirrhotic and control rats were pretreated with rHDL or saline and challenged with LPS or saline. The neutralization of LPS in HDL was assessed by the measurement of HDL-bound fluorescent LPS levels. Plasma tumor necrosis factor alpha (TNFalpha) and lipopolysaccharide binding protein (LBP) levels were measured. The expression of hepatic TNFalpha, LBP, inducible nitric oxide synthase (iNOS), and caveolin-1 (a major eNOS inhibitor) and the activity of protein kinase B (Akt; a major eNOS activator) and eNOS were determined. The portal pressure was measured. The plasma HDL levels were significantly lower in cirrhotic rats than in control rats. In cirrhotic rats, the plasma levels of HDL-bound fluorescent LPS were 50% lower than those in controls, and they were restored after rHDL administration. The plasma TNFalpha levels were significantly higher in LPS-challenged cirrhotic rats than in controls and significantly decreased after rHDL administration. rHDL administration decreased hepatic TNFalpha, LBP, iNOS, and caveolin-1 expression, restored hepatic eNOS and Akt activity, and significantly lowered the portal pressure and intrahepatic vascular resistance. CONCLUSION: In cirrhotic rats, rHDL administration decreases the hepatic proinflammatory signals induced by LPS, restores the hepatic eNOS activity, and lowers the portal pressure. This suggests that the decrease in circulating HDL in cirrhosis plays a role in the excessive proinflammatory response and intrahepatic eNOS down-regulation.

In vivo altered unfolded protein response and apoptosis in livers from lipopolysaccharide-challenged cirrhotic rats.

BACKGROUND/AIMS: Endoplasmic reticulum (ER)-related unfolded protein response (UPR) is mediated by PKR-like ER kinase (PERK), ATF6 and IRE1. PERK phosphorylates eukaryotic translation initiation factor-2alpha (eIF2alpha) to attenuate protein synthesis, including in NF-kappaB-dependent antiapoptotic proteins. We hypothesized that an altered UPR in the liver may sensitize cirrhotic livers to LPS-induced, TNFalpha-mediated apoptosis. Thus, we examined in vivo UPR and NF-kappaB activity in livers from cirrhotic and normal LPS-challenged rats. METHODS: Livers were harvested in rats that did or did not receive LPS. RESULTS: Under baseline conditions, no UPR was found in normal livers while PERK/eIF2alpha and ATF6 pathways were activated in cirrhotic livers. After LPS, in normal livers, the PERK/eIF2alpha pathway was transiently activated. ATF6 and IRE1 were activated. In cirrhotic livers, the PERK/eIF2alpha pathway remained elevated. ATF6 and IRE1 pathways were altered. LPS-induced, NF-kappaB-dependent antiapoptotic proteins increased in normal livers whereas their expression was blunted at the posttranscriptional level in cirrhotic livers. CONCLUSIONS: Cirrhotic livers exhibit partial UPR activation in the basal state and full UPR, although altered, after LPS challenge. Sustained eIF2alpha phosphorylation, a hallmark of cirrhotic liver UPR, is associated with a lack of LPS-induced accumulation of NF-kappaB-dependent antiapoptotic proteins which may sensitize cirrhotic livers to LPS/TNFalpha-mediated apoptosis.

Upregulation of TNF-alpha production signaling pathways in monocytes from patients with advanced cirrhosis: possible role of Akt and IRAK-M.

BACKGROUND/AIMS: In cirrhosis, tumor necrosis factor (TNF)-alpha overproduction is involved in both the systemic complications and progression of liver injury. Since monocytes from patients with advanced cirrhosis have an increase in lipopolysaccharide (LPS)-induced TNF-alpha production, we hypothesized that an upregulation of TNF-alpha production pathways and/or alteration of constitutive and inducible suppressor of TNF-alpha hyperproduction (protein kinase B (Akt) and interleukin-1 receptor-associated kinase (IRAK)-M, respectively) should be found in monocytes of these patients. Thus, we investigated ex vivo the signaling pathways of TNF-alpha production before and after LPS incubation in monocytes from noninfected Child-Pugh C patients with advanced cirrhosis and healthy subjects. METHODS: TNF-alpha production, expressions of intracellular TNF-alpha, toll-like receptor-4 (TLR4), IkappaB-alpha, IRAK-1, IRAK-M, mitogen-activated protein (MAP) kinases and Akt activity were measured in monocytes. RESULTS: Cirrhotic monocytes without LPS have less TLR4 expression, less IkappaB-alpha protein levels, more TNF-alpha expression, higher MAP kinase activities and decreased Akt activity than control monocytes. In cirrhotic monocytes, LPS-induced TNF-alpha hyperproduction and signaling upregulation were associated with a lack of IRAK-M induction. CONCLUSIONS: Upregulated signaling pathways of the TNF-alpha production, decreased Akt activity and a lack of IRAK-M induction may be involved in the process of cirrhotic monocyte sensitization to produce TNF-alpha.

Norfloxacin reduces aortic NO synthases and proinflammatory cytokine up-regulation in cirrhotic rats: role of Akt signaling.

BACKGROUND & AIMS: Arterial vasodilation plays a role in the pathogenesis of the complications of cirrhosis. This vasodilation is caused by the overproduction of arterial nitric oxide (NO). Bacterial translocation may be involved in NO synthase (NOS) up-regulation by activating both endothelial NOS (eNOS) and inducible NOS (iNOS). The prevention of intestinal gram-negative translocation by norfloxacin administration corrects systemic circulatory changes by decreasing NO production in cirrhosis. However, the signaling mechanisms for NO overproduction from bacterial translocation are unknown. In this study, we investigated the signal transduction pathway of bacterial translocation-induced aortic NOS up-regulation in cirrhotic rats. METHODS: Proinflammatory cytokine levels, Akt and NOS activities, eNOS phosphorylation, and NOS expressions were assessed in aorta from norfloxacin-treated and untreated cirrhotic rats. Norfloxacin was administered to reduce intestinal bacterial translocation. RESULTS: Aortic eNOS and iNOS protein expressions, Akt activity, and eNOS phosphorylation by Akt at serine 1177 were up-regulated in cirrhotic rats. Norfloxacin administration significantly decreased the incidence of gram-negative translocation and proinflammatory cytokine (tumor necrosis factor-alpha, interferon-gamma, and interleukin-6) levels; norfloxacin also decreased aortic Akt activity, eNOS phosphorylation, and NOS expressions and activities. The decrease in aortic Akt activity and NOS expressions also was obtained after colistin or anti-tumor necrosis factor-alpha antibody administration to cirrhotic rats. CONCLUSIONS: This study identifies a signaling pathway in which bacterial translocation induces aortic NOS up-regulation and thus NO overproduction in cirrhotic rats. These results strongly suggest that bacterial translocation and proinflammatory cytokines play a role in systemic NO overproduction in cirrhosis by the Akt pathway.

Exhaled nitric oxide and acute lung injury in a rat model of extracorporeal circulation.

Exhaled nitric oxide (NO) concentration, a marker of pulmonary inflammation, has been shown to be elevated in various models of acute lung injury (ALI). This study was undertaken to evaluate the pulmonary NO production in a rat model of postextracorporeal circulation (ECC) ALI. Wistar rats underwent either a partial femorofemoral ECC in normothermia for 3 h (n = 10) or a sham procedure (n = 10). The extracorporeal circuit consisted of a roller pump and a membrane oxygenator. Exhaled NO concentration was monitored with a chemiluminescence analyzer. After sacrifice, lungs were harvested for microscopic studies and to analyze the inducible nitric oxide synthase (iNOS) activity and expression (Western blot). ECC was responsible for an ALI characterized by a decreased arterial blood oxygen saturation (88.9% [51.7-94.2] vs. 93.7% [91.4-98.6] P = 0.005) and pulmonary histological changes (marked alveolar neutrophil infiltration; interstitial edema; intraalveolar hemorrhage). The lung injury score was significantly higher in the ECC group (n = 5; 3.0 [2-4]) in comparison to the sham group (n = 5; 1.0 [0-2]). Exhaled NO concentration remained stable throughout the experiment in all sham rats whereas it significantly increased in the ECC group from baseline (2 ppb [1-5]) until the end of experiment (33.5 ppb [1-47]). Lung iNOS activity and expression were also significantly increased in the ECC group. An increase in exhaled NO, however, did not correlate with the decrease in arterial oxygen pressure. ECC was responsible for an ALI in rats and for an elevated pulmonary NO production. Determination of the relationship between exhaled NO and the severity of the inflammatory process in ALI will require further studies.

Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosis.

In cirrhosis, lipopolysaccharide (LPS, a product of Gram-negative bacteria) in the blood may cause septic shock. LPS-elicited induction of arterial inducible nitric oxide synthase (iNOS) results in nitric oxide (NO)-induced vasodilation, which causes arterial hypotension and hyporeactivity to alpha(1)-adrenergic constrictors. In vitro studies have suggested that vasopressin inhibits iNOS expression in cultured vascular smooth muscle cells exposed to LPS. Thus, the aim of this study was to investigate the effects of terlipressin administration (a vasopressin analog) on in vivo LPS-induced aortic iNOS in rats with cirrhosis. LPS (1 mg/kg, intravenously) was administered followed by the intravenous administration of terlipressin (0.05 mg/kg, intravenously) or placebo 1 hour later. Arterial pressure was measured, and contractions to phenylephrine (an alpha(1)-adrenoceptor agonist), iNOS activity, and iNOS expressions (mRNA and protein) were investigated in isolated aortas. LPS-induced arterial hypotension and aortic hyporeactivity to phenylephrine were abolished in rats that received terlipressin. LPS-induced aortic iNOS activity and expression were suppressed in terlipressin-treated rats. In conclusion, in LPS-challenged rats with cirrhosis, terlipressin administration inhibits in vivo LPS-induced aortic iNOS expression. Terlipressin administration may be a novel approach for the treatment of arterial hypotension and hyporeactivity to alpha(1)-adrenergic constrictors in patients with cirrhosis and septic shock.

Prevention of gram-negative translocation reduces the severity of hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) is characterized by intrapulmonary vascular dilatations and an increased alveoloarterial oxygen difference (AaPO(2)). These abnormalities are related to augmented pulmonary nitric oxide (NO) production, dependent primarily on increases in the expression and activity of inducible NO-synthase (iNOS) within pulmonary intravascular macrophages and, to a lesser extent, of endothelial NOS (eNOS). Production of iNOS by pulmonary intravascular macrophages might be related to translocated gut bacteria present in the pulmonary circulation. To test this hypothesis, we determined whether macrophage sequestration, lung iNOS expression and activity, and HPS severity were decreased after norfloxacin was given for 5 weeks to prevent Gram-negative bacterial translocation in rats with common bile duct ligation-induced cirrhosis. Norfloxacin decreased the incidence of Gram-negative translocation from 70 to 0% and the percentage of pulmonary microvessels containing more than 10 macrophages from 52 +/- 7 to 21 +/- 8% (p < 0.01). AaPO(2) and cerebral uptake of intravenous (99m)Tc-labeled albumin macroaggregates (reflecting intrapulmonary vascular dilatations) were intermediate to those of untreated cirrhotic and sham-operated rats. The activity and expression of lung iNOS, but not eNOS, were reduced to normal. Norfloxacin may reduce HPS severity by inhibiting Gram-negative bacterial translocation, thereby decreasing NO production by pulmonary intravascular macrophages. Bacterial translocation may be the key to the pathogenesis of HPS.

Role of shear stress in aortic eNOS up-regulation in rats with biliary cirrhosis.

BACKGROUND & AIMS: In rats with portal vein stenosis, the initial cause of aortic nitric oxide (NO) overproduction might be overactivation of endothelial NO synthase (eNOS) related to increased shear stress. Cardiac output is higher in cirrhosis than in extrahepatic portal hypertension. The aims of this study were to evaluate the role of shear stress, vascular endothelial growth factor (VEGF), and cytokines in aortic eNOS up-regulation in rats with biliary cirrhosis and to compare these results with those in rats with portal vein stenosis. METHODS: NOS activities, NOS protein, heat shock protein (Hsp) 90, and VEGF expressions were studied in rat aortas. Propranolol was administered to rats with cirrhosis to reduce cardiac output and thus shear stress. RESULTS: In cirrhotic rats, the aortic eNOS protein was 3.0 and 1.7 times higher than in control and portal vein-stenosed rats, respectively. In cirrhotic rats, the Hsp90 content was 3.2 and 2.2 times higher than in control and portal vein-stenosed rats, respectively. Propranolol decreased NOS activity by 47% and eNOS and Hsp90 expression by 75% and 72%, respectively. Aortic VEGF expression was decreased in cirrhotic rats. VEGF-induced stimulation of NOS activity was greater in aortas from control rats than in aortas from portal vein-stenosed or cirrhotic rat aortas. eNOS expression was up-regulated after VEGF incubation. After lipopolysaccharide administration, eNOS expression did not change in portal vein-stenosed or cirrhotic rats. CONCLUSIONS: This study shows that in aortas from rats with biliary cirrhosis, systemic vasodilation depends mainly on eNOS up-regulation related to shear stress.

Relationship between protein kinase C alterations and nitric oxide overproduction in cirrhotic rat aortas.

BACKGROUND: Although nitric oxide (NO) overproduction and protein kinase C (PKC) alterations may play a role in systemic haemodynamic changes in cirrhotic rat aortas, the relationship between NO synthase (NOS) hyperactivation and PKC hypoactivation is unknown. Therefore, the relationships between NOS and PKC activities were studied in cirrhotic rat aortas. METHODS: The effects of NOS inhibition by Nw-nitro-L-arginine (LNNA) on the contractile response to phorbol myristate acetate (PMA), a PKC activator, were studied. The effects of NOS inhibition and those of S-nitroso-N acetyl-DL-penicillamine (SNAP), an NO donor, on PKC activity were also evaluated. The effects of PKC activation and inhibition on total NOS and inducible NOS (iNOS) activities were measured. Nitric oxide synthase inhibition caused an increase in PMA-induced contraction and an increase in PKC activity in cirrhotic rat aortas. S-nitroso-N acetyl-DL-penicillamine induced downregulation of PKC activity. Total basal aortic NOS activity was significantly higher in cirrhotic rats than in control rats and activation of PKC by PMA induced a decrease in total aortic NOS activity. Protein kinase C downregulation caused an increase in both total aortic NOS and iNOS activities only in control rats, whereas only iNOS activity increased in cirrhotic rats. CONCLUSION: In cirrhotic rat aortas, NO overproduction plays a role in the decreased PKC activation that leads to reduced aortic contraction. Overactivation of aortic NOS in cirrhotic rats may be because of, in part, the reduced PKC activity.