Supplementary Material for: The Influence of Aspirin on Release of Eoxin C4, Leukotriene C4 and 15-HETE, in Eosinophilic Granulocytes Isolated from Patients with Asthma

<b><i>Background:</i></b> The effect of aspirin on the release of key arachidonic acid metabolites in activated eosinophils from subjects with aspirin-intolerant asthma (AIA) has not been investigated previously, despite the characteristic eosinophilia in AIA. <b><i>Methods:</i></b> Peripheral blood eosinophils were isolated from four groups of subjects: healthy volunteers (HV; n = 8), mild asthma (MA; n = 8), severe asthma (SA; n = 9) and AIA (n = 7). In the absence or presence of lysine-aspirin, eosinophils were stimulated with arachidonic acid or calcium ionophore to trigger the 15-lipoxygenase-1 (15-LO) and 5-lipoxygenase (5-LO) pathways, respectively. 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and eoxin C<sub>4</sub> (EXC<sub>4</sub>) were measured as 15-LO products and leukotriene (LT)C<sub>4</sub> as a product of the 5-LO pathway. <b><i>Results:</i></b> Activated eosinophils from patients with SA and AIA produced approximately five times more 15-HETE than eosinophils from HV or MA patients. In the presence of lysine-aspirin, eosinophils from AIA, MA and SA patients generated higher levels of 15-HETE than in the absence of lysine-aspirin. Furthermore, in the presence of lysine-aspirin, formation of EXC<sub>4</sub> was also significantly increased in eosinophils from AIA patients, and LTC<sub>4</sub> synthesis was increased both in AIA and SA patients. <b><i>Conclusions:</i></b> Taken together, this study shows an increased release of the recently discovered lipid mediator EXC<sub>4</sub>, as well as the main indicator of 15-LO activity, 15-HETE, in activated eosinophils from severe and aspirin-intolerant asthmatics, and also elevated EXC<sub>4</sub> and LTC<sub>4</sub> formation in eosinophils from AIA patients after cellular activation in the presence of lysine-aspirin. The findings support a pathophysiological role of the 15-LO pathway in SA and AIA.