Effects of anisakis simplex on nitric oxide production in J774 macrophages

Since nitric oxide (NO) sas recognized as a potent microbicidal agent, its role in host defence against intracellular parasites has been widely demons trated. Recent evidence suggests a role for NO in combating extracellular a nd multicellular pathogens. This defence activity has been demonstrated tow ard the larvae of Schistosoma mansoni, microfilariae of Onchocerca linealis , several stages of Brugia malayi and protoscoleces of Echinococcus multilo cularis. Many parasites suppress Th1 lymphocytes and directly inhibit NO pr oduction by inducing cytokines, such as IL-l, IL-10 and TGF-beta. In this s tudy, we have investigated the effects of Anisakis simplex, an enhancer of Th2-dominant responses, on NO production. We studied the effect of crude ex tracts (CE) and excretory-secretory (ES) products on the induction of induc ible nitric oxide synthase (iNOS) in bacterial lipopolysaccharide (LPS)-tre ated J774 macrophages. Stimulation of macrophages by LPS (1 mu g/ml) increa sed nitrite concentrations in the culture medium at 24 h. Go-administration of A. simplex products with LPS, dose-dependently reduced the accumulation of nitrite, Nitrite production is due to induction of iNOS, and both L-NAM E (N-G-nitro-L-arginine methyl ester) (50 mu M) and dexamethasone (10 mu M) inhibited nitrite accumulation (54.2 and 92.1% inhibition, respectively). The inhibition of nitrite production by A. simplex aas 42.1-97.8% in the ra nge 4.75-76 mu g/well (CE products) and 37.2-61.5% in the range 5-20 mu g/w ell (ES products). Cell viability assayed by the mitochondrial-dependent re duction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid e) verified that the inhibition was not due to general cellular toxicity. H owever, the effects of A, simplex, were reduced when NOS had been induced b g prior exposure to LPS and an? possible further induction was blocked by c ycloheximide, an inhibitor of protein synthesis.