We have demonstrated that in a guinea pig model, organophosphorus pesticides (OPs) cause airway hyperreactivity that is dose-related and associated with the functional loss of autoinhibitory muscarinic M2 receptors that normally limit acetylcholine release from parasympathetic nerves that innervate airway smooth muscle. We recently reported that sensitization to antigen alters the mechanisms underlying OP-induced airway hyperreactivity to involve IL-5-dependent mechanisms in the sensitized but not the non-sensitized guinea pig. How OPs cause neuronal M2 dysfunction in the non-sensitized animal is not known but our preliminary data indicate that this effect is not mediated by cholinesterase inhibition or direct antagonistic interactions with neuronal M2 receptors. Rather, OPs appear to influence neuronal M2 receptor function indirectly via effects on macrophages since depletion of macrophages using liposome-encapsulated clodronate protects against OP-induced airway hyperreactivity. It is our hypothesis that OPs activate macrophages to upregulate expression and release of inflammatory cytokines previously shown to cause M2 receptor dysfunction in various models of airway hyperreactivity. We propose four Aims to test this hypothesis. In Aim 1, we will use in vivo physiological measurements to confirm that macrophages mediate airway hyperreactivity caused by OPs and determine whether their role changes over time, as has been observed for eosinophils in ozone-induced airway hyperreactivity. Aim 2 will utilize macrophages isolated from bronchoalveolar lavage collected from OP-treated versus untreated guinea pigs guinea pigs to examine the effect of OPs on macrophage expression and release of inflammatory cytokines implicated in airway hyperreactivity. In Aim 3, we will use primary nerve cell cultures to determine whether OP- induced macrophage mediators interact with nerves directly to alter M2 receptor expression or function or the structural plasticity of nerves. Aim 4 will confirm the in vivo physiological relevance of OP-induced macrophage mediators identified in aims 2 and 3. Mechanistic studies are critical to developing preventive and therapeutic approaches for OP-induced airway hyperreactivity, which are likely to differ between sensitized (allergic) and non-sensitized individuals, and for determining the risks to human health posed by OP exposures. The public health implications of these studies are significant in light of the increasing prevalence of asthma, the wide spread exposure of humans and especially children to OPs and the credible threat of terrorist use of OP pesticides and nerve agents. PUBLIC HEALTH RELEVANCE: Recent epidemiological studies suggest a link between exposure to organophosphorus pesticides (OPs) and asthma. Our previous work confirms this link by demonstrating that OPs cause airway hyperreactivity, a major symptom of asthma, in a guinea pig model. The goal of this project is to elucidate the mechanism(s) by which OPs cause airway hyperreactivity, which will be critical to developing effective preventive and therapeutic approaches to OP-induced airway hyperreactivity. Given the documented widespread exposure to OPs not only in the U.S. but worldwide, and the credible threat of terrorist use of OPs, the proposed work is of significant public health relevance.