Interleukin 1β-stimulated production of nitric oxide in rat hepatocytes is mediated through endogenous synthesis of interferon gamma

Abstract

Hepatocyte expression of inducible nitric oxide synthase (iNOS) and subsequent elaboration of NO is associated with multiple cellular and biochemical functions, such as inhibition of mitochondrial electron transport, activation of soluble guanylyl cyclase, regulation of glutathione synthesis, and protection against oxidative injury. 1-3 Although tumor necrosis factor ␣ (TNF-␣), interleukin 1␤ (IL-1␤), interferon gamma (IFN-␥), and endotoxin are known to synergistically induce maximal iNOS expression, investigators have recently shown that IL-1␤ alone is sufficient to induce expression of iNOS in hepatocyte cultures. [4][5][6] However, studies using other cell systems, such as the macrophage, have indicated a central role for IFN-␥ rather than IL-1␤ in the expression of iNOS. [7][8][9] Murine macrophages express iNOS in response to synergistic combinations of IFN-␥, IFN-␣, and IFN-␤ with or without endotoxin. 8,10 The murine and rat iNOS promoter sequences both have 10 copies of the IFN-␥ response element and two copies of the IFN-␣-stimulated response element. 8,11 Last, IL-1␤ is a multifunctional cytokine, affecting all cell types, and often acts in concert with other cytokines or small mediator molecules. Specifically, IL-1␤ is known to initiate expression of a variety of genes, including IFN-␣, ␤, and ␥, TNF, and IL-1 and to increase cell surface expression of IFN and TNF receptors. 12,13 On the basis of these considerations, we hypothesized that IL-1␤-induced iNOS expression in rat hepatocytes is dependent on endogenous production of IFN-␥. In a model of rat hepatocytes in primary culture, we have characterized the expression of IFN-␥ and NO synthesis in the setting of IL-1␤ stimulation. Using antisense IFN-␥ to inhibit translation of IFN-␥ messenger RNA (mRNA), we defined the role of IFN-␥ in IL-1␤-mediated NO synthesis. Finally, rat hepatocytes were transiently transfected with a plasmid reporter gene construct containing the rat hepatocyte iNOS promoter to define the action of IFN-␥ in IL-1␤ activation of the iNOS promoter. Our results suggest that IFN-␥ is a necessary but not sufficient component of the cascade required for maximal IL-1␤-associated hepatocyte expression of iNOS. MATERIALS AND METHODSRat Hepatocyte Isolation. Male Lewis rats (range, 200-300 g; Harlan Inc., Indianapolis, IN) fed water and chow ad libitum were used for hepatocyte isolation as described by Schuetz et al. 14 After anesthetization with pentobarbital sodium, the portal vein was cannulated. The liver was perfused with calcium-free Krebs' bicarbonate buffer followed by 150 mg collagenase D in 200 mL of Krebs' bicarbonate buffer containing 1.2 mmol/L CaCl 2 and 1.8% bovine serum albumin. All solutions were maintained at 37°C and aerated with 95% O 2 /5% CO 2 . The partially digested liver was excised, passed Abbreviations: iNOS, inducible nitric oxide synthase; TNF-␣, tumor necrosis factor ␣; IL-1␤, interleukin 1␤; IFN-␥, interferon gamma; mRNA, messenger RNA; DPBS, Dulbecco' s phosphate-buffered saline; PBS, phosphate-buffered s...

Study progress on mechanism of severe acute pancreatitis complicated with hepatic injury

“…Rockey and Chung (1996) found through experiment that in AP (acute pancreatitis) the endotoxin and TNF (tumor necrosis factor) stimulates the liver tissues, mainly the NO synthetase on the hepatic cells, cell Kupffer and hepatic sinusoid endothelial cell to synthesize NO. IL-I relies on the endogenous INF-γ to mediate the NO synthesis of hepatic cells (Schroeder et al, 1998). The results of sampling and analysis of the plasma of SAP patients and healthy adults show that both ET and NO are elevated in SAP patients (P<0.05) and that the elevation of the plasma ET is markedly higher than that of NO.…”

Regulation of nitric oxide synthesis in the liver

“…Interleukin-1␤ is induced not only by LPS stimulation but also by a number of other stimuli, such as Gram-positive organisms (staphylococci and streptococci), viruses, complemented components, thrombin, bile salts and androgen metabolites, and by cytokines themselves. 72 Recently, Schroeder et al 45 demonstrated that IL-1␤stimulated production in rat hepatocytes is mediated through endogenous synthesis of IFN-␥. They found that IFN-␥ is a necessary but not sufficient component of the cascade required for maximal IL-1␤-associated hepatocyte expression of iNOS.…”

Intestinal expressions of eNOSmRNA and iNOSmRNA in rats with acute liver failure

“…IL-1β can induce iNOS expression in hepatocyte by activating iNOS primer by IFN-γ, and produce a large amount of extra cellular matrix (ECM) such as hyaluronan, which induced iNOS to produce NO and form ONOO -, and aggravate tissue injury by NF-κB signal transduction when liver is damaged, moreover, stabilize microcirculation and protect against liver injury. The effect of protection or impairment on the liver mainly depends on the NO concentration and cell derivation [21][22][23] . NO induced by iNOS in endotoxemia is closely related to liver function impairment, but NO induced early by hepatocyte cNOS can protect liver tissue [24,25] .…”