ANALYSIS OF THE SIGNAL-TRANSDUCTION IN THE INDUCTION OF NITRIC-OXIDE SYNTHASE BY LIPOTEICHOIC ACID IN MACROPHAGES

1 This study investigates the signal transduction mechanisms leading t o the enhanced formation of nitric oxide (NO) due to the induction of NO synthase (iNOS) in murine J774.2 macrophages in culture activated w ith lipoteichoic acid (LTA), a cell wall component of the gram-positiv e bacterium Staphylococcus aureus. 2 LTA (10 mu g ml(-1)) caused withi n 24 h an enhanced accumulation of nitrite (an indicator of NO biosynt hesis) in the supernatant of J774.2 macrophages which was prevented by the non-selective NOS inhibitor N-G-monomethyl-L-arginine (L-NMMA; IC 50: 35 mu M) or by the iNOS-selective NOS inhibitor, aminoethyl-isothi ourea (AE-ITU; IC50: 6 mu M). The inhibition of nitrite formation affo rded 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 inhibiti on was similar when these NOS inhibitors were added to the macrophages 10 h after LTA. 3 Pretreatment of J774.2 macrophages with cyclohexami de or dexamethasone prevented the enhanced formation of nitrite caused by LTA. This inhibition did not occur when dexamethasone or cyclohexa mide were added to the cells 10 h after LTA. The increase in nitrite f ormation stimulated by LTA (10 mu g ml(-1)) was not affected by polymy xin B (0.05-0.5 mu g ml(-1)), an agent which binds and inactivates end otoxin. 4 A specific inhibitor of phosphatidylcholine-phospholipase C (PC-PLC), D609, prevented the increase in nitrite formation (IC50 = 20 mu g ml(-1)) caused by LTA. The inhibition afforded by D609 was signi ficantly smaller when this agent was added to the cells 10 h after LTA . 5 The structurally distinct tyrosine kinase inhibitors, erbstatin, g enistein, and tyrphostin AG126 prevented the formation of nitrite caus ed by LTA. The inhibition afforded by these compounds was significantl y attenuated when they were added to the cells 10 h after LTA. In cont rast, daidzein or tyrphostin A-1, which are inactive analogues of geni stein and tyrphostin (up to a concentration of 10 mu M) did not affect the nitrite formation caused by LTA. 6 Inhibitors of the activation o f the nuclear transcription factor NF-kappa B such as pyrrolidine dith iocarbamate (PDTC; an antioxidant and a metal chelator), butylated hyd roxyanisole (BHA; an antioxidant), L-1-tosylamido-2-phenylethyl chloro methyl ketone (TPCK), calpain inhibitor I (both I kappa B-protease inh ibitors), or rotenone (an antioxidant which inhibits electron transpor t) prevented the nitrite formation stimulated by LTA. The inhibition a fforded by these agents was significantly smaller when they were added to the macrophages 10 h after LTA. 7 Incubation of J774.2 cells with LTA over 24 h resulted in the expression of iNOS protein (130 kDa) as identified by Western blot analysis. The expression of iNOS protein by LTA was significantly attenuated by cyclohexamide, D609, tyrphostin A G126, PDTC or by 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) phosphorylati on of tyrosine kinase, and (iii) the activation of the transcription f actor NF-kappa B.