Analysis of the signal transduction in the induction of nitric oxide synthase by lipoteichoic acid in macrophages

Abstract

1 This study investigates the signal transduction mechanisms leading to the enhanced formation of nitric oxide (NO) due to the induction of NO synthase (iNOS) in murine J774.2 macrophages in culture activated with lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus. 2 LTA (10 Mg ml-') caused within 24 h an enhanced accumulation of nitrite (an indicator of NO biosynthesis) in the supernatant of J774.2 macrophages which was prevented by the non-selective NOS inhibitor N0-monomethyl-L-arginine (L-NMMA; IC50: 35yM) or by the iNOS-selective NOS inhibitor, aminoethyl-isothiourea (AE-ITU; ICso: 6 gM). The inhibition of nitrite formation afforded by these agents was prevented by excess L-arginine (3-30 mM), but not by D-arginine (3-30 mM). Furthermore, the degree of iNOS inhibition was similar when these NOS inhibitors were added to the macrophages 10 h after LTA. 3 Pretreatment of J774.2 macrophages with cyclohexamide or dexamethasone prevented the enhanced formation of nitrite caused by LTA. This inhibition did not occur when dexamethasone or cyclohexamide were added to the cells 10 h after LTA. The increase in nitrite formation stimulated by LTA (10 Mg ml-') was not affected by polymyxin B (0.05-0.5 pg ml-'), an agent which binds and inactivates endotoxin. 4 A specific inhibitor of phosphatidylcholine-phospholipase C (PC-PLC), D609, prevented the increase in nitrite formation (IC50 = 20 pg ml-') caused by LTA. The inhibition afforded by D609 was significantly smaller when this agent was added to the cells 10 h after LTA. 5 The structurally distinct tyrosine kinase inhibitors, erbstatin, genistein, and tyrphostin AG126 prevented the formation of nitrite caused by LTA. The inhibition afforded by these compounds was significantly attenuated when they were added to the cells 10 h after LTA. In contrast, daidzein or tyrphostin A-1, which are inactive analogues of genistein and tyrphostin (up to a concentration of 10 pM) did not affect the nitrite formation caused by LTA. 6 Inhibitors of the activation of the nuclear transcription factor NF-KB such as pyrrolidine dithiocarbamate (PDTC; an antioxidant and a metal chelator), butylated hydroxyanisole (BHA; an antioxidant), L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), calpain inhibitor I (both IKBprotease inhibitors), or rotenone (an antioxidant which inhibits electron transport) prevented the nitrite formation stimulated by LTA. The inhibition afforded by these agents was significantly smaller when they were added to the macrophages 10 h after LTA. TPCK. 8 Thus, the signal transduction leading to the expression of iNOS protein and activity caused by LTA in murine J774.2 macrophages involves (i) the activation of PC-PLC, (ii) phosphorylation of tyrosine kinase, and (iii) the activation of the transcription factor NF-KB.

Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells

“…PDTC, an antioxidant and NF-B inhibitor, strongly inhibited LPS-mediated NO/iNOS induction and NF-B activation when cells were cultured under 25 mM glucose conditions, as we and others previously reported (13,46). However, LPS-mediated NO/iNOS induction was not significantly altered by PDTC under 5 mM glucose conditions.…”

Role of nitric oxide in the circulatory failure and organ injury in a rodent model of Gram‐positive shock

“…before administration of this iNOS inhibitor, 64+4 JuM, n =6) was not significantly different from the ones measured at 120 min in rats which had received LTA + PepG only. The baseline plasma levels of nitrite + nitrate were not significantly influenced by dexamethasone or aminoguanidine in rats treated with saline rather than LTA + PepG ( spiratory dysfunction, as indicated by a fall in Pao2, ai rises in the serum levels of GOT (10 fold) and GPT (1( which are biochemical markers of hepatocellular inji various species including the rat (see Baue, 1993 iNOS protein and activity caused by LTA in cultured macrophages (Kengatharan et al, 1996a) and in the rat (De Kimpe et al, 1995a). Here we demonstrate that dexamethasone prevented the expression of iNOS activity in homogenates of aorta, liver, lung and kidney; and of iNOS protein in the lung of rats challenged with LTA + PepG.…”

“…Therefore, this study was designed to evaluate whether an enhanced formation of NO by iNOS contributes importantly to the circulatory failure and MODS caused by the co-administration of LTA plus PepG in the anaesthetized rat. We have, therefore, investigated the effects of (i) dexamethasone, which prevents the induction of iNOS by endotoxin (Radomski et al, 1990) or LTA (Kengatharan et al, 1996a), and (ii) aminoguanidine, a selective inhibitor of iNOS activity (Corbett et al, 1992;Griffiths et al, 1993;Misko et al, 1993) on these adverse effects caused by LTA plus PepG in the anaesthetized rat. Preliminary accounts of this study have been presented to the British Pharmacological Society (Kengatharan et al, 1996b,c).…”

“…In contrast to endotoxic shock, we know relatively little about the mechanisms by which Gram-positive bacteria, which lack endotoxin, cause shock and MODS. However, lipoteichoic acid (LTA) from Staphylococcus aureus causes the induction of a calciumindependent isoform of nitric oxide (NO) synthase (iNOS) in vascular smooth muscle cells and macrophages in vitro and the signal transduction pathway leading to the expression of iNOS by LTA in cultured macrophages (J774.2) involves the activation of tyrosine kinase and the nuclear transcription factor NFKB (Auguet et al, 1992;Cunha et al, 1993;De Kimpe et al, 1995a;Kengatharan et al, 1996a). Staphylococcus aureus LTA causes circulatory failure as well the induction of iNOS protein and activity in the rat lung (De Kimpe et al, 1995a).…”

Calpain Inhibitor I Reduces the Development of Acute and Chronic Inflammation

“…5,6 This calpain-dependent step of the activation of NF-B is abolished by calpain inhibitor I. [7][8][9][10] Thus, calpain inhibitor I prevents the expression of many NF-B-dependent genes, including those for iNOS [11][12][13] and COX-2. 14,15 In addition to proteins regulated by NF-B, the generation of superoxide anions, hydroxyl radicals, and peroxynitrite also plays a pivotal role in the tissue injury associated with inflammation.…”