Original Contribution MECHANISM OF VITAMIN E INHIBITION OF CYCLOOXYGENASE ACTIVITY IN MACROPHAGES FROM OLD MICE: ROLE OF PEROXYNITRITE ALISON A. BEHARKA,* 1 DAYONG WU,* † MAURO SERAFINI,* 2 and SIMIN NIKBIN MEYDANI* ‡ *Nutritional Immunology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA; † Department of Immunology, Norman Bethune University of Medical Sciences, Changchun, China; and ‡ Department of Pathology, Sackler Graduate School of Biochemical Sciences, Tufts University, Boston, MA, USA (Received 31 July 2001; Accepted 14 December 2001) Abstract—Vitamin E inhibits cyclooxygenase activity in macrophages from old mice by reducing peroxynitrite production. PGE 2 is a proinflammatory mediator that has been linked to a variety of age-associated diseases such as cancer, arthritis, and cardiovascular disease. Furthermore in the aged, increased cyclooxygenase (COX)-2-mediated PGE 2 production contributes to decline in T-cell-mediated function. Previously we reported that increased macrophage PGE 2 production in the aged is due to higher COX-2 activity and that supplementation with vitamin E significantly reduced the age-associated increase in macrophage PGE 2 production posttranslationally without changing COX-2 expression. Peroxynitrite, a product of nitric oxide (NO) and superoxide (O 2 - ), increases the activity of COX without affecting its expression. Thus, we investigated if vitamin E inhibits COX activity through decreasing peroxynitrite formation. Macrophages from old mice had higher PGE 2 levels, COX activity, and NO levels than those from young mice, all of which were significantly reduced by vitamin E. When added individually, inhibitors of NO and O 2 - did not significantly reduce COX activity; however, when the inhibitors were combined, COX activity was significantly reduced in macrophages from old mice fed 30 ppm vitamin E. Increasing NO levels alone using SNAP or O 2 - levels, using X/XO, had no effect; however, increasing peroxynitrite levels using Sin-1 or X/XO + SNAP significantly increased COX activity in macrophages from old mice fed 500, but not those fed 30 ppm vitamin E. These data strongly suggest that peroxynitrite plays an important role in the vitamin E-induced inhibition of COX activity. These findings have important implications for designing interventions to reverse and/or delay age-associated dysregulation of immune and inflammatory responses and diseases associated with them. © 2002 Elsevier Science Inc. Keywords—Vitamin E, Cyclooxygenase, Macrophages, Nitric oxide, Aging, Free radicals INTRODUCTION Aging is associated with a dysregulation of immune function, the consequences of which include increased incidence of infectious, inflammatory, and neoplastic diseases as well as morbidity and mortality from them. Our laboratory, as well as those of others, has demon- strated that macrophages (M) from old humans and rodents secrete significantly more prostaglandin (PGE 2 ) than those from their younger counterparts [1–7]. This age-related increase in PGE 2 production contributes to dysregulation of the immune and inflammatory re- sponses with age. In particular, PGE 2 suppresses T-cell- mediated immunity found in the aged [8]. Furthermore, PGE 2 plays a key role in inflammation and its associated diseases such as cancer and cardiovascular diseases, in- cidence of which increases with age. The rate-limiting enzyme in prostaglandin biosynthe- sis is PGH synthetase (PGHS), also referred to as cyclo- oxygenase (COX). It catalyzes both the formation of PGG 2 from arachidonic acid via its cyclooxygenase ac- tivity and the subsequent reduction of PGG 2 to PGH 2 via Address correspondence to: Simin Nikbin Meydani, Nutritional Im- munology Laboratory, Jean Mayer Human Nutrition Research Center on Aging at Tufts University, 711 Washington St., Boston, MA 02111, USA; Tel: (617) 556-3129; Fax: (617) 556-3224; E-Mail: smeydani@ hnrc.tufts.edu. 1 Current address: University of Iowa, Veteran Administration Med- ical Center, Iowa City, IA 52246, USA. 2 Current address: Human Nutrition Unit, Antioxidant Research Lab- oratory, National Institute for Food and Nutrition Research, 00178 Rome, Italy. Free Radical Biology & Medicine, Vol. 32, No. 6, pp. 503–511, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter PII S0891-5849(01)00817-6 503