EXOGENOUS AND ENDOGENOUS NITRIC-OXIDE ATTENUATES TUMOR-NECROSIS-FACTOR SYNTHESIS IN THE MURINE MACROPHAGE CELL-LINE RAW-264.7

Effects of TNF on nitric oxide (NO) production have been well document ed in a variety of experimental and clinical settings, as for example, in septic shock. Investigations focusing on an inverse relation of NO on TNF synthesis are rare. Previously we could demonstrate that exoge nous NO-releasing agents suppress LPS-induced TNF production in human PBMC. We now investigate whether a regulatory role on TNF synthesis co uld also be ascribed to endogenous NO. This was studied in the murine macrophage cell line RAW 264.7, which is able to express both TNF and the inducible NO synthase. NO production was determined by measuring n itrite with Griess reagents. TNF formation was quantified by L929 cyto toxicity. We found a suppression of LPS-induced TNF synthesis by the e xogenous addition of NO-releasing agents in the murine cell line, as p reviously observed in human cells. The application of NO synthase inhi bitors led to a decrease in NO production, associated with an increase in TNF synthesis. TNF production increased from a base line (stimulat ion with 1 mu g/ml LPS alone) of 20.8 ng/ml to 36.3 ng/ml (means of si x experiments) in the presence of the NO synthase inhibitor N-G-monome thyl L-arginine (100 mu M). Similiar results were obtained with anothe r NO synthase inhibitor, N-G-nitro L-arginine-methylester. Lack of L-a rginine in the medium resulted in a threefold increase in LPS-stimulat ed TNF synthesis compared with medium containing the usual concentrati on of 1 mM L-arginine. Restitution of L-arginine but not of D-arginine reversed this increase in TNF synthesis in a dose-dependent manner. T o our knowledge these results indicate for the first time a negative f eedback by endogenous NO on TNF synthesis in vitro. This finding may b e relevant in pathophysiologic processes in which both TNF and NO are formed and in experimental therapies aiming at changes of NO concentra tions.