Blunted stress responses in delayed type hypersensitivity in mice lacking the neuronal isoform of nitric oxide synthase Staci D. Bilbo a , Andrew K. Hotchkiss b , Silvana Chiavegatto a,1 , Randy J. Nelson b, * ,2 a Department of Psychological and Brain Sciences, The Johns Hopkins University, Baltimore, MD 21218, USA b Departments of Psychology and Neuroscience, The Ohio State University, Columbus, OH 43210, USA Received 29 January 2003; received in revised form 17 April 2003; accepted 23 April 2003 Abstract Nitric oxide (NO) is implicated in inflammation and hypothalamic – pituitary responses to immune stimuli; however, the specific role of NO from neurons during stress-induced immune responses remains unspecified. We measured antigen-specific delayed-type-hypersensitivity (DTH) responses in the skin of wild-type (WT) and neuronal nitric oxide synthase knockout (nNOS À / À ) mice at baseline and after 2 h of restraint. Baseline corticosterone concentrations were higher in nNOS À / À than WT mice. However, stress-induced increases in corticosterone were dampened in nNOS À / À mice, and restraint suppressed DTH only in WT animals. Furthermore, WT mice lost more body mass after stress, and exhibited more anxiety-like behavior in the open field, than nNOS À / À mice. Neuronal NO appears to be involved in the neuroendocrine-immune response to stress, perhaps via glucocorticoid regulation. D 2003 Elsevier B.V. All rights reserved. Keywords: HPA axis; Inflammation; Glucocorticoids; Leukocyte trafficking; Anxiety; Open field 1. Introduction Nitric oxide (NO) was initially identified as an endoge- nous regulator of blood vessel tone (Furchgott and Zawadzki, 1980). More recently, NO has also been pro- posed to serve as a neurotransmitter (Baranano et al., 2001; Baranano and Snyder, 2001). NO is an atypical neurotrans- mitter because it exists as an endogenous gas at body temperature and possesses several biochemical properties of a free radical. With a half-life of < 5 s, NO is extremely labile and difficult to measure and manipulate. Consequent- ly, many studies have investigated NO indirectly by manip- ulating its synthetic enzyme, nitric oxide synthase (NOS). NOS transforms arginine into NO and citrulline. Three distinct isoforms of NOS have been characterized: (1) in neural tissue (nNOS; NOS-1), (2) in macrophages (iNOS; NOS-2), and (3) in the endothelial tissue of blood vessels (eNOS; NOS-3) (Baranano and Snyder, 2001), although they are not restricted to these locations. Suppres- sion of NO formation by either elimination of arginine or by use of N-methyl-L-arginine, a potent NOS inhibitor, affects all three isoforms of NOS. Specific inhibitors of the various NOS isoforms have been developed (Southan et al., 1997; Southan and Szabo, 1996). Another approach to examine the role of specific NOS isoforms is to develop specific gene knockout mice (Huang, 2000). Mice missing the gene for nNOS (nNOS À / À ) display enlarged stomachs because of abnormalities in pyloric relaxation (Huang et al., 1993), impaired ovulation (Klein et al., 1998), hypertrophic dilated bladders, and increased urinary frequency (Burnett et al., 1997), and are highly aggressive (Nelson et al., 1995). Several studies have implicated NO in the modulation of corticotrophic releasing hormone (CRH) secretion from the hypothalamus. nNOS is localized throughout discrete brain areas, including the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus (Huang et al., 1993). These nuclei represent the major source of neurons contain- ing CRH (Kawano et al., 1988; Liposits et al., 1986; Owens and Nemeroff, 1991). Intracerebroventricular or peripheral injection of the general NOS inhibitor, N-nitro-L-arginine- methyl ester (L-NAME), attenuates stress-induced adreno- 0165-5728/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-5728(03)00175-9 * Corresponding author. Department of Psychology, The Ohio State University, Townshend Hall, 1885 Neil Avenue, Columbus, OH 43210, USA. Tel.: +1-614-538-9540; fax: +1-614-451-3116. E-mail addresses: bilbo.1@osu.edu (S.D. Bilbo), schiaveg@usp.br (S. Chiavegatto), rnelson@osu.edu (R.J. Nelson). 1 Current address: Institute of Psychiatry and Heart Institute (InCor), School of Medicine, University of Sao Paulo, Eneas de Carvalho Aguiar, 44, 10th floor, 05403-904, Sao Paulo, SP, Brazil. 2 Tel.:614-538-9526; fax: 614-451-3116. www.elsevier.com/locate/jneuroim Journal of Neuroimmunology 140 (2003) 41 – 48