* p < 0

* p < 0.05. 3.4 Atropine decreases chemokinesis and chemotaxis of PBMC To ascertain if attenuation of inflammation by atropine resulted from the inability of leukocytes to respond to chemoattractants, we investigated the effects of atropine around the migratory behavior K+ Channel inhibitor of PBMC in response to chemoattractants. The role of muscarinic receptors in modulating the immune system is not clear. Vagotomy and atropinization moderate lethal anaphylaxis and histamine shock (Levy et al., 1976), and carbacholine increases the number of leukocytes in the splenic venous blood that is blocked by atropine treatment (Sandberg, 1994). Direct addition of acetylcholine to spleen cell cultures enhances the Con A-induced T-cell proliferation (Qiu et al., 1996). Therefore, activation of mAChRs may activate the immune system. However, because acetylcholine can react with both nicotinic and muscarinic receptors, it is difficult to ascertain whether the effects reflect its conversation with muscarinic and/or nicotinic receptors. In this study, we used selective muscarinic agonists/antagonists to show that while mAChR agonists stimulate, mAChR antagonists inhibit immune and inflammatory responses. These data suggest that nAChRs and mAChRs might affect the immune/inflammatory responses in an opposite manner, and may represent the yin and yang of the immune systems homeostasis. 2. Materials and Methods 2.1 Animals Male pathogen-free Lewis rats were purchased from Charles River (Wilmington, MA). Animals were housed individually in filter-top plastic cages and maintained in a 12-h light/dark cycle at 30 1C. Food and water were provided 0. 05 was considered statistically significant. 3. Results 3.1 Unlike nicotine, the K+ Channel inhibitor mAChR agonist oxotremorine enhances T-cell responses To test the hypothesis that activation of mAChRs K+ Channel inhibitor activates the immune system, rats were treated for 3 weeks with the nonselective mAChR agonist oxotremorine that does not interact with the nAChRs. Saline- and oxotremorine-treated rats were immunized with the T-cell-dependent antigen SRBC, and splenocytes from these animals K+ Channel inhibitor were tested for the antigen-specific antibody cell (AFC) response. As seen in Fig. 1A, oxotremorine treatment significantly enhanced the AFC response of spleen cells to SRBC. To determine whether oxotremorine also enhanced the T cell antigen-receptor (TCR)-mediated T-cell proliferation, spleen cells from control and oxotremorine-treated animals were cultured in the presence of anti-TCR + anti-CD28 antibodies and assayed for T-cell proliferation. CLTA Fig. 1B shows that oxotremorine treatment significantly elevated the TCR-mediated spleen cell proliferation. These results suggest that oxotremorine treatment enhances both the antigen-induced antibody response and the antigen-mediated T-cell proliferation. Open in a separate window Fig. 1 Oxotremorine treatment enhances anti-SRBC AFC and proliferation of spleen cells after ligation of the TCRLewis rats (6 animals/group) were exposed to saline (CON) or oxotremorine (OXO) for 3 weeks and immunized with SRBC intravenously as described in Material and Methods. (A) The AFC response is usually expressed as AFC/106 spleen cells. (B) Spleen cells from CON and OXO animals were cultured with previously decided optimal levels of anti-TCR + anti-CD28 (TCR). The cultures were labeled with 3H-Tdr as described in Materials and Methods, and the results are presented as mean cpm SEM. * p < 0.05. Although we and others have shown that nicotine treatment inhibits both immune and inflammatory responses (Sopori, 2002; Razani-Boroujerdi et al., 2004), to ascertain whether under the conditions of oxotremorine treatment nicotine suppresses T-cell responses, rats were treated in parallel with a nicotine concentration that produces blood nicotine/cotinine of less than a two-pack per day smoker (Geng et al., 1995), mecamylamine, or mecamylamine + nicotine. Results presented in Fig. 2 show that nicotine significantly suppressed the anti-SRBC AFC response (Fig. 2A) as well as the anti-TCR + CD28-mediated T-cell proliferation (Fig. 2B). Both responses were blocked by the nAChR antagonist mecamylamine (Fig. 2). Thus, unlike mAChR activation, activation of nAChRs suppresses the immune system and nicotinic receptor antagonists block the nicotine-induced immunosuppression. Open in a separate window Fig. 2 Nicotine inhibits T-cell-dependent immune responsesRats (6 animals/group) were treated with saline (CON), nicotine (NT), mecamylamine (MEC), or MEC +NT for 3 weeks and then immunized with SRBC intravenously as described.