Evidence for Increased de Novo Synthesis of NAD in Immune-Activated RAW264.7 Macrophages: A Self-Protective Mechanism? 1 Ross S. Grant,* Robert Passey,† Gabrijela Matanovic,‡ George Smythe,‡ and Vimal Kapoor* ,2 *School of Physiology and Pharmacology, †Cytokine Unit, School of Pathology, and ‡Ray Williams, Biomedical Mass Spectrometry Facility, Faculty of Medicine, University of New South Wales, Sydney 2052 Australia Received May 3, 1999 The parent pyridine nucleotide NAD is the end product of oxidative tryptophan catabolism via the kynurenine pathway. Indoleamine 2,3-dioxygenase, the rate-limiting enzyme for this pathway, is in- duced by the proinflammatory cytokine interfer- on-. The aim of this study was to investigate the effect of interferon- treatment on intracellular NAD concentration in the murine macrophage cell line, RAW 264.7. A significant increase in intracellu- lar NAD concentration was observed following 24 h exposure to interferon-. This cytokine-mediated increase in NAD concentration was markedly en- hanced by the inhibition of poly(ADP-ribose) poly- merase or nitric oxide synthase or following treat- ment with the synthetic glucocorticoid dexa- methasone. NAD production was dependent on both the presence of tryptophan in the culture me- dium and on functional indoleamine 2,3-dioxygenase activity. In agreement with previous studies a marked increase in nitric oxide production was observed in these cells following activation with interferon-. These results provide evidence for the first time that de novo synthesis of NAD from tryptophan is increased concomitantly with free radical production in RAW 264.7 macrophages stimulated with interferon-. This increase in NAD biosynthesis may provide an im- proved supply of substrate to the nuclear repair en- zyme poly(ADP-ribose) polymerase assisting in DNA repair and hence cell viability. © 1999 Academic Press Key Words: NAD; interferon-; indoleamine 2,3-di- oxygenase; tryptophan; poly(ADP-ribose) polymerase; macrophages; murine; RAW 264.7. The proinflammatory cytokine interferon- (IFN-) 3 is produced mainly by activated T-lymphocytes and natural killer cells as part of the normal cellular im- mune response (1). IFN- promotes the antimicrobial activity of macrophages by enhancing production of reactive oxygen and nitrogen species (1– 4) and also induces a number of proteins, including the hemopro- tein indoleamine 2,3-dioxygenase (IDO) (5). IDO (EC 1.13.11.17) is the first and rate-limiting enzyme of tryptophan catabolism along the “kynure- nine pathway” (5) (Fig. 1). Increased catabolism through this pathway in activated macrophages (6 – 8) has been consistently observed in bacterial (9, 10), viral (10, 11), and parasitic (12) infections in vivo. A number of kynurenine pathway intermediates have been detected in the extracellular fluid of both human (13, 6, 14) and murine (15) macrophages acti- vated in vitro with IFN-. These metabolites include kynurenine, 3-hydroxyanthranilic acid, picolinic acid, and quinolinic acid. 3-hydroxyanthranilic acid is a po- tent free radical scavenger (16), while picolinic acid may work synergistically with IFN- to enhance mac- rophage NOS induction (17). Quinolinic acid (QUIN) is an N-methyl-D-aspartate receptor agonist and a poten- 1 This study was supported by grants from the R. L. Cooper Med- ical Research Foundation and the National Heart Foundation. R.S.G. is supported by a Dora Lush postgraduate scholarship from the National Health and Medical Research council of Australia. 2 To whom correspondence should be addressed. Fax: 61 2 93851099. E-mail: V.Kapoor@unsw.edu.au. 3 Abbreviations used: IFN-, interferon-; IDO, indoleamine 2,3- dioxygenase; QUIN, quinolinic acid; PARP, poly(ADP-ribsoe) poly- merase; NO ● , nitric oxide; BCS, bovine calf serum; L-NAME, N- nitro-L-arginine methyl ester hydrochloride; 3ABA, 3-aminobenz- amide; 3EBC, 3-ethoxy--carboline hydrochloride, 6CDT, 6-chloro D-tryptophan; NOS, nitric oxide synthase; CD-38, NAD, glycohydro- lase; PFB-Br, pentafluorobenzyl bromide. 0003-9861/99 $30.00 1 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 372, No. 1, December 1, pp. 1–7, 1999 Article ID abbi.1999.1381, available online at http://www.idealibrary.com on