Indian Journal of Experimental Biology Vol. 41, April 2003, pp. 290-295 Effect of ascorbic acid on stimulatory status of activated mouse peritoneal phagocytes Rachna Agarwal, A K Tripathi & Asit K Chakrabarty* Department of Biochemistry, University College of Medical Sciences & GTB Hospital, Dilshad Garden, Delhi 110095, India Received 24 September 2002; revised 21 January 2003 Mouse peritoneal macrophages (MPM) when elicited by the antioxidant ascorbic acid have been found to be signifi- cantly stimulatory, exhibiting marked alteration at the cellular and enzyme levels. Alterations recorded were as follows - cel- lular yield per mouse, their protein content. lysosomal acid hydrolase levels and capability to phagocyte, all were signifi- cantly enhanced. The new stimulant was observed to produce no synergistic action on MPM when thioglycollate, BCG or endotoxin along with the same stimulated the latter. Levels of antioxidants like ascorbic acid and glutathione were found to be enhanced in elicited macrophages whereas superoxide dismutase levels varied when the three above stimulators were administered. However, the ascorbic acid elicited cells showed an increase in glutathione levels and a decrease in SOD le v- els but no change in total intracellular ascorbic acid level s. Further, though ascorbic acid interaction enhanced the phago- cy tic capability of MPM as compared to resident cells, no significant boosting of phagocytic process cou ld be observed when each of three stimulators co upled with ascorbic acid was used for macrophage elicitation. Macrophages exposed in vivo to exogenous inducers of inflammation such as thioglycollate (Tg), BCG, endotoxin etc. are reported to show enhanced phago- cytic, metabolic and secretory activities. These elic- ited cells show changes like heightened capacity to synthesize and release a variety of intracellular hydro- lytic enzymes and decreased levels of 5' nucleotidase as compared to resident cells l - 4 • In activated macro- phages increased bactericidal and tumoricidal activity has been attributed to the generation of superoxide anions, hydrogen peroxide and hydroxyl radicals 5 . 6 . Macrophages also possess a built-in protective mechanism to offset the newly produced toxic oxygen metabolites produced during activation via respiratory burst 7 • This protective mechanism results in the en- hanced synthesis of antioxidant enzymes like super- oxide dismutase (SOD), catalase and glutathione per- oxidase. Besides these ascorbic acid (A A) and glu- tathione (GSH) being important antioxidants serve protective function against oxidative damage in macrophages. Studies done in subjects with AA defi- ciency show decrease in GSH: GSSG ratio during oxidative stress 8 . Thus GSH and AA appear to be an integrated antioxidant system. AA not only enhances bacterial phagocytosis by macrophages but also pro- tects phagocytes from auto-oxidation by virtue of its *Correspondent author: Fax: 0091-11-2290495 E-mail: dbmi @ucms.ernet.in antioxidant propert/· IO . Therefore, our interest in the present study was to reveal the effect of stimulated macrophages on the antioxidant system. As AA, one of the important antioxidants is known to influence the immune system, we attempted to determine the functional enhancement, if any, of stimulated macro- phages in relation to their cellular physiology when AA was additionally administered to the experimental animals with or without stimulators. Materials and Methods Elicitation of macrophages Peritoneal cells were harvested by lavage from normal BALB/c mice and also from the mice injected intraperitoneally 3 days earlier with one of these stimulators - 1 ml 5% thioglycollate broth (Oifco), freeze dried BCG suspension (BCG Vaccine Lab ., Chennai) containing 1 x 10 6 cells or 30 /J.g endotoxin (E. coli stereotype 055:B5, Sigma). Along with these stimulators, each experimental mouse was given I mg of ascorbic acid (1 ml of 0.1 % ascorbic acid dissolved in saline) daily for 3 days. Macrophages purified by glass adherence were counted by using a haemocy- to meter. Viability of the cells was also done . Enzyme assays Acid phosphatase activity in macrophages was as- sayed using 4-nitrophenyl phosphate as substrate 3 . Cathepsin 0 was determined by following hydrolysis