The pathogenesis of inflammatory liver diseases and development of liver fibrosis involves hepatocytes as well as non-parenchymal liver cells like resident liver macrophages (Kupffer cells (KC)), Stellate cells and endothelial cells. Kupffer cells play a critical role in liver (patho)physiology and in the defense of the liver during inflammation. They constitute about 50% of non-parenchymal cells and are the largest population of tissues macrophages in the body. Infections, toxins (lipopolysacharide (LPS)), parenchymal damage and stresses stimulate the inflammatory response of Kupffer cells with the following secretion of bioactive factors, cytotoxicity, antigen processing, etc. Resident liver macrophages are the main producers of inflammatory mediators in the liver. Among them there are prostanoids (prostaglandin (PG) E2 and thromboxane (Tx) A2), cytokines (e.g. interleukin (IL)-1,-6, -10, tumor necrosis factor (TNF) α) and inorganic mediators like nitric oxide (NO). Macrophages-derived products play opposing roles in the development of liver fibrogenesis: IL-1β, TNFα, IL-6, transforming growth factor (TGF)-β and TxA2 (pro-fibrogenic mediators) promote whereas PGE2, IL-10 and nitric oxide (anti-fibrogenic mediators) suppress liver fibrogenesis. The present study shows the production of PGE2 and TxA2 by resident liver macrophages upon prolonged activation by LPS and the characterization of biosynthesis pathways. The production of PGE2 and TxA2 is followed during 24 h after stimulation of macrophages with LPS. The involvement of enzymes is measured on the RNA level (RT-PCR), protein level (Western blot analysis) and activity (activity assays), respectively. The amounts of released prostanoids are measured at time points 2, 4, 8 and 24 h after LPS stimulation. The production of PGE2 is very low without stimulation, shows a delay within the first few hours after stimulation with LPS, and thereafter linearly increases up to 24 h. TxA2 production is very low without stimulation, and increases without a time-delay after the addition of LPS. Prostanoid biosynthesis is inhibited by dexamethasone. The present study shows the involvement and regulation of the AA cascade by the following enzymes: cPLA2: is expressed in resting Kupffer cells; cPLA2 expression and phosphorylation is increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. COX-1: is expressed in resting Kupffer cells; COX-1 expression is not influenced by LPS and dexamethasone. The COX-1 inhibitor SC560 suppresses the LPS-induced production of PGE2 and TxA2 (8h and 24h), localization predominantly in membrane fraction. COX-2: is almost not expressed in resting Kupffer cells; COX-2 expression is highly increased by LPS, dexamethasone suppresses the LPS effect. The COX-2 inhibitor SC236 inhibits the production of PGE2 and TxA2 at 8h by about 77% and 20%, and at 24h by about 42% and 34%, respectively, localization predominantly in membrane fraction. mPGES-1: is almost not expressed in resting cells; mPGES-1 expression is highly increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. mPGES-2: is expressed in resting Kupffer cells; mPGES-2 expression is slightly increased by LPS, localization predominantly in membrane fraction. cPGES: is expressed in resting Kupffer cells; LPS has no effect, localization predominantly in soluble fraction. TxA2 synthase: is expressed in resting Kupffer cells; LPS and dexamethasone have no effect, localization predominantly in membrane fraction. Treatment of Kupffer cells with IL-1ß and TNF-α leads to an enhanced release of PGE2 and TxA2 and upregulate the expression of cPLA2, COX-2 and mPGES-1. IL-6 has no effect on prostanoid production. In contrast, IL-10 suppresses the LPS-induced production of PGE2 and TxA2 and expression of cPLA2, COX-2 and mPGES-1. Resting Kupffer cells release very low amounts of NO and do not express iNOS, nNOS and eNOS. LPS, TNF-α and IL-1ß upregulate NO release and the expression of iNOS whereas dexamethasone and IL-10 downregulate NO release and the expression of iNOS. PGE2 suppresses the LPS-induced release of NO but enhances the cytokine-induced release of NO. NO induces a release of PGE2. Thus, the study demonstrates a crosstalk between prostanoids, nitric oxide and cytokines in Kupffer cells under inflammatory conditions and demonstrates a possible anti-fibrogenic effect of PGE2 in the process of liver fibrogenesis.

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The pathogenesis of inflammatory liver diseases and development of liver fibrosis involves hepatocytes as well as non-parenchymal liver cells like resident liver macrophages (Kupffer cells (KC)), Stellate cells and endothelial cells. Kupffer cells play a critical role in liver (patho)physiology and in the defense of the liver during inflammation. They constitute about 50% of non-parenchymal cells and are the largest population of tissues macrophages in the body. Infections, toxins (lipopolysacharide (LPS)), parenchymal damage and stresses stimulate the inflammatory response of Kupffer cells with the following secretion of bioactive factors, cytotoxicity, antigen processing, etc. Resident liver macrophages are the main producers of inflammatory mediators in the liver. Among them there are prostanoids (prostaglandin (PG) E2 and thromboxane (Tx) A2), cytokines (e.g. interleukin (IL)-1,-6, -10, tumor necrosis factor (TNF) α) and inorganic mediators like nitric oxide (NO). Macrophages-derived products play opposing roles in the development of liver fibrogenesis: IL-1β, TNFα, IL-6, transforming growth factor (TGF)-β and TxA2 (pro-fibrogenic mediators) promote whereas PGE2, IL-10 and nitric oxide (anti-fibrogenic mediators) suppress liver fibrogenesis. The present study shows the production of PGE2 and TxA2 by resident liver macrophages upon prolonged activation by LPS and the characterization of biosynthesis pathways. The production of PGE2 and TxA2 is followed during 24 h after stimulation of macrophages with LPS. The involvement of enzymes is measured on the RNA level (RT-PCR), protein level (Western blot analysis) and activity (activity assays), respectively. The amounts of released prostanoids are measured at time points 2, 4, 8 and 24 h after LPS stimulation. The production of PGE2 is very low without stimulation, shows a delay within the first few hours after stimulation with LPS, and thereafter linearly increases up to 24 h. TxA2 production is very low without stimulation, and increases without a time-delay after the addition of LPS. Prostanoid biosynthesis is inhibited by dexamethasone. The present study shows the involvement and regulation of the AA cascade by the following enzymes: cPLA2: is expressed in resting Kupffer cells; cPLA2 expression and phosphorylation is increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. COX-1: is expressed in resting Kupffer cells; COX-1 expression is not influenced by LPS and dexamethasone. The COX-1 inhibitor SC560 suppresses the LPS-induced production of PGE2 and TxA2 (8h and 24h), localization predominantly in membrane fraction. COX-2: is almost not expressed in resting Kupffer cells; COX-2 expression is highly increased by LPS, dexamethasone suppresses the LPS effect. The COX-2 inhibitor SC236 inhibits the production of PGE2 and TxA2 at 8h by about 77% and 20%, and at 24h by about 42% and 34%, respectively, localization predominantly in membrane fraction. mPGES-1: is almost not expressed in resting cells; mPGES-1 expression is highly increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. mPGES-2: is expressed in resting Kupffer cells; mPGES-2 expression is slightly increased by LPS, localization predominantly in membrane fraction. cPGES: is expressed in resting Kupffer cells; LPS has no effect, localization predominantly in soluble fraction. TxA2 synthase: is expressed in resting Kupffer cells; LPS and dexamethasone have no effect, localization predominantly in membrane fraction. Treatment of Kupffer cells with IL-1ß and TNF-α leads to an enhanced release of PGE2 and TxA2 and upregulate the expression of cPLA2, COX-2 and mPGES-1. IL-6 has no effect on prostanoid production. In contrast, IL-10 suppresses the LPS-induced production of PGE2 and TxA2 and expression of cPLA2, COX-2 and mPGES-1. Resting Kupffer cells release very low amounts of NO and do not express iNOS, nNOS and eNOS. LPS, TNF-α and IL-1ß upregulate NO release and the expression of iNOS whereas dexamethasone and IL-10 downregulate NO release and the expression of iNOS. PGE2 suppresses the LPS-induced release of NO but enhances the cytokine-induced release of NO. NO induces a release of PGE2. Thus, the study demonstrates a crosstalk between prostanoids, nitric oxide and cytokines in Kupffer cells under inflammatory conditions and demonstrates a possible anti-fibrogenic effect of PGE2 in the process of liver fibrogenesis.