The Effect of Diphtheria Toxin on Nitric Oxide Induction From RAW264.7 Murine Macrophages

Abstract: In this study the effect of diphtheria toxin (DT) on nitric oxide (NO) production from RAW 264.7 murine macrophages was investigated. Griess reagent was used to determine NO production by measuring nitrite levels in the culture supernatants. Corynebacterium diphtheriae G12/6 strain-produced DT (Limes flocculation activity and immunodiffusion assays were positive) demonstrated a dose-dependent effect on RAW 264.7 macrophages to induce NO production. L-NAME, an L-arginine analogue, inhibited NO production from DT-stimulated RAW 264.7 macrophages. At the given concentrations of DT, lipopolysaccharide (LPS or endotoxin)-induced NO production from RAW 264.7 macrophages was also inhibited. RAW 264.7 macrophage cells, incubated with various concentrations of DT for 3 days, were killed by DT in a dose dependent manner. Endotoxin contamination of DT was demonstrated with limulus amoebocyte lysate assay. Polymyxin B, frequently added to neutralize the effects of LPS in vitro, inhibited DT-induced NO production from RAW 264.7 cells. The misleading data concerning NO inducing capacity of DT was seemed to be related to endotoxin contamination. Thus, DT does not seem to be capable of inducing NO production from macrophages