The Journal of Immunology Nicotine Inhibits Fc«RI-Induced Cysteinyl Leukotrienes and Cytokine Production without Affecting Mast Cell Degranulation Through a7/a9/a10-Nicotinic Receptors Neerad C. Mishra,* Jules Rir-sima-ah,* R. Thomas Boyd, † Shashi P. Singh,* Sravanthi Gundavarapu,* Raymond J. Langley,* Seddigheh Razani-Boroujerdi,* and Mohan L. Sopori* Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC 4 . Cysteinyl leuko- trienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high- affinity receptor for IgE (Fc«RI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using a-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) a7, a9, and a10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for ‡8h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A 2 activity and the PI3K/ERK/NF-kB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kB. The suppressive effect of nicotine on the late-phase response was blocked by the a7/a9-nAChR antagonists methyllycaconitine and a-bungarotoxin, as well as by small interfering RNA knockdown of a7-, a9-, or a10-nAChRs, suggesting a functional interaction between a7-, a9-, and a10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This “hybrid” receptor might serve as a target for novel antiallergic/antiasthmatic therapies. The Journal of Immunology, 2010, 185: 588–596. T he prevalence and severity of atopic diseases, including allergic asthma, rhinitis, and eczema, have increased dra- matically in recent years (1–5). Allergic diseases involve the allergen-induced Th2 response characterized by the production of Th2 cytokines, including IL-4, -5, and -13, which are critical in the development of the allergic response. Mast cells are critical tissue-based effector cells that mediate IgE-dependent allergic responses (6–8). Mast cells express IgERs (FcεRI), and binding of an allergen to IgE-FcεR1 induces the release of three classes of proinflammatory mediators: 1) preformed granule-associated chemical mediators; 2) newly synthesized arachidonic acid metab- olites, such as leukotrienes (LTs); and 3) proinflammatory cyto- kines, including TNF-a and Th2 cytokines (6–8). Among these mediators, the cysteinyl LTs (cysLTs) exert a number of patho- physiological effects of allergic asthma, including proliferation and contraction of bronchial smooth muscle cells, mucus secretion, inflammatory cell migration, and increased vascular permeability (9–11). Indeed, cysLTs are important indicators of allergic asthma severity (12–14). Several reports suggested an inverse correlation between ciga- rette smoking and the development of allergic diseases (15, 16). Smoking increases the risk for various diseases, including lung infections that may stem from the immunosuppressive effects of nicotine (NT) in cigarette smoke (17). Linneberg et al. (16) reported that smoking was negatively associated with the in- cidence of allergic sensitization, which is consistent with another population-based study that concluded that cigarette smokers were less likely to develop allergic sensitization during an 8-y follow-up period (15). Several cross-sectional studies also reported a lower incidence of aeroallergen sensitization among current smokers than among never-smokers; even past smokers were less likely to be sensitized than never-smokers (18–22). NT, the major constituent of cigarette smoke, suppresses adap- tive and inflammatory immune responses (23–25); recently, we demonstrated that NT pretreatment attenuated some parameters of ragweed- and house dust mite-induced allergic asthma in Brown Norway rats by primarily suppressing leukocytic infiltra- tion and the production of LTs and Th2 cytokines/chemokines, without affecting the allergen-induced hexosaminidase/histamine release in the lung (26). Thus, in addition to its effects on T cells (27) and macrophages (24), NT affects the mast cell function in the lung (26), and the presence of nicotinic acetylcholine receptors (nAChRs) on murine bone marrow-derived mast cells (28) and *Immunology and Asthma Division, Lovelace Respiratory Research Institute, Albu- querque, NM 87108; and † Department of Neuroscience, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210 Received for publication July 10, 2009. Accepted for publication April 23, 2010. This work was supported in part by National Institutes of Health Grants RO1 DA017003, RO1 DA04208-17, and RO1 DA04208S. Address correspondence and reprint requests to Dr. Mohan L. Sopori, Lovelace Re- spiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM, 87108. E-mail address: msopori@lrri.org Abbreviations used in this paper: a-BTX, a-bungarotoxin; [Ca 2+ ] i , intracellular free Ca 2+ concentration; CON, control; cPLA 2 , cytosolic phospholipase A 2 ; CS, cromoglycate sodium; cysLT, cysteinyl leukotriene; LT, leukotriene; LTC 4 , leukotriene C 4 ; MLA, methyllycaconitine; nAChR, nicotinic acetylcholine receptor; NT, nicotine; qPCR, quantitative PCR; RBL cells, RBL-2H3 cells; siRNA, small interfering RNA. Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902227