Subclinical Doses of the Nerve Gas Sarin Impair T Cell Responses through the Autonomic Nervous System Roma Kalra, Shashi P. Singh, Seddigheh Razani-Boroujerdi, Raymond J. Langley, Walter B. Blackwell, Rogene F. Henderson, and Mohan L. Sopori Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87108 Received February 8, 2002; accepted July 23, 2002 Subclinical Doses of the Nerve Gas Sarin Impair T Cell Re- sponses through the Autonomic Nervous System. Kalra, R., Singh, S. P., Razani-Boroujerdi, S., Langley, R. J., Blackwell, W. B., Henderson, R. F., and Sopori, M.L. (2002). Toxicol. Appl. Phar- macol. 184, 82– 87. The nerve gas sarin is a potent cholinergic agent, and exposure to high doses may cause neurotoxicity and death. Subclinical exposures to sarin have been postulated to contribute to the Gulf War syndrome; however, the biological effects of subclinical ex- posure are largely unknown. In this communication, evidence shows that subclinical doses (0.2 and 0.4 mg/m 3 ) of sarin admin- istered by inhalation to F344 rats for 1 h/day for 5 or 10 days inhibited the anti-sheep red blood cell antibody-forming cell re- sponse of spleen cells without affecting the distribution of lym- phocyte subpopulations in the spleen. Moreover, sarin suppressed T cell responses, including the concanavalin A (Con A) and the anti--T cell receptor (TCR) antibody-induced T cell prolifera- tion and the rise in the intracellular calcium following TCR liga- tion. These concentrations of sarin altered regional but not total brain acetylcholinesterase activity. Interestingly, serum corticoste- rone levels of the sarin-treated animals were dramatically lower than the control animals, indicating that sarin-induced immuno- suppression did not result from the activation of the hypothala- mus—pituitary–adrenal (HPA) axis. Pretreatment of animals with the ganglionic blocker chlorisondamine abrogated the inhibitory effects of sarin on spleen cell proliferation in response to Con A and anti-TCR antibodies. These results suggest that the effects of sarin on T cell responsiveness are mediated via the autonomic nervous system and are independent of the HPA axis. © 2002 Elsevier Science (USA) Key Words: organophosphates; cholinergic agents; neuroim- mune modulation; glucocorticoids; T cell mitogenesis. Organophosphorus compounds including pesticides are po- tent cholinergic compounds that annually affect approximately 3 million people worldwide and result in 220,000 deaths (WHO, 1996). Sarin (isopropyl methyl phosphonofluoridate) is a powerful organophosphorus nerve agent that irreversibly inactivates cholinesterase (ChE) activity, elevating the synaptic levels of acetylcholine (Grob and Harvey, 1958). In high doses, sarin causes seizures and even death (Marrs, 1993). Its lethality and low cost of production have made sarin the chemical of choice for terrorism. In the 1994 sarin attack of a Tokyo subway station, several people died and more than 6000 suf- fered sarin-related injuries (Nozaki et al., 1995). Humans are exposed to sarin primarily through inhalation, and subclinical sarin exposures have been proposed as a factor in the devel- opment of the Gulf War illness (Abou-Donia et al., 1996). While people in the immediate vicinity of a sarin attack may be exposed to overt neurotoxic doses of sarin, others away from the epicenter of the attack are likely to receive subclinical amounts. High levels of organophosphates such as malathion, parathion, and dichlorvos impair cellular and humoral immune responses (Street and Sharma, 1975; Desi et al., 1978). The immunological effects of subclinical doses of sarin have not been defined. Kassa et al. (2000) observed a decreased bacte- riocidal activity of peritoneal macrophages in sarin-treated rats, and some survivors of the Tokyo subway attack died from opportunistic Legionella infection (Kamimura et al., 1998), indicating that sarin may affect the immune system. However, the effects of sarin on the adaptive immune response are largely unknown. Sarin is a neuroactive agent and at subclin- ical doses affects the electrical activity of the brain (Burchfiel et al., 1976). Because the brain communicates with the im- mune system (Blalock, 1994), it is possible that sarin would influence the immune system through the CNS. In this com- munication, we show that inhalation of subclinical doses of sarin suppresses T cell responses in the rat, and that the autonomic nervous system (ANS) may play a critical role in this immunosuppression. MATERIALS AND METHODS Animals. Pathogen-free, 8-week-old male Fischer 344 rats were purchased from Harlan Sprague–Dawley Farms (Indianapolis, IN). Food and water were provided ad libitum to the animals. Approximately 10-week-old animals were used for sarin inhalation. Chlorisondamine (CHL) treatment. The ganglionic blocker CHL (Tocris, Ballwin, MO) was injected into rats (10 mg/kg body wt sc) 7 days prior to sarin exposure. This concentration of CHL blocks the behavioral responses to neuroactive substances for several months (Reuben et al., 1998). Toxicology and Applied Pharmacology 184, 82– 87 (2002) doi:10.1006/taap.2002.9497 82 0041-008X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.