ALBUMIN NITROSYLATED BY ACTIVATED MACROPHAGES POSSESSES ANTIPARASITICEFFECTS NEUTRALIZED BY ANTI-NO-ACETYLATED-CYSTEINE ANTIBODIES

Activated macrophages exert an L-arginine-dependent cytostatic effect on the extracellular parasite, Trypanosoma musculi, This effect is not observed in the absence of albumin in the culture medium but is resto red by the addition of albumin, indicating the presence of an albumin- nitric oxide (NO) adduct acting as an effector molecule, Since L-cyste ine represents a privileged target for NO, an immunochemical approach was performed using an acetylated-cysteine-ESA conjugate, This conjuga te was nitrosylated using sodium nitrite as a NO donor, Binding of NO to the conjugated haptens was assayed using spectrophotometry, It was completely abolished by mercuric chloride, confirming the presence of an S-NO bond, Polyclonal Abs were obtained after immunizing rabbits wi th S-nitroso-acetylated-cysteine (NO-ac-Cys) conjugates, Using the enz yme-linked immunosorbent assay method, Ab avidity and specificity were determined by competition experiments between NO-ac-Cys-conjugated co mpounds and other nitrosylated or non-nitrosylated compounds, The resu lting cross-reactivity ratios showed that conjugated NO-ac-Cys-BSA was the best recognized compound, These Ab were used for an in vitro stud y of the kinetics of NO-derived compounds from activated murine macrop hages. Anti-NO-ac-Cys Ab inhibited the antimicrobial effect of activat ed macrophages on the extracellular parasite, T. musculi, Moreover, th e L-arginine-dependent antiparasitic activity of supernatants from Cal mette-Guerin bacillus-activated macrophages required the presence of a lbumin and was also inhibited by anti-NO-ac-Cys Ab, showing the effect or role of S-nitroso-albumin.