U0126, its inactive analog U0124 and SB203580 are from Calbiochem/EMD Millipore. as semaphorin-7A without affecting mRNA level suggests a unique influence by IL-3 on translation. The purpose of this study is usually to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared to IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein (RP) S6 and the upstream kinase, p90S6K. Blockade of p90S6K activation inhibited phosphorylation Nifedipine of RPS6 and IL-3-enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, phosphorylation of RPS6 and p90S6K was enhanced in human airway compared Nifedipine to circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations place IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions. and studies suggest that eosinophils trigger tissue damage, influence the immune response (1) and drive tissue fibrosis by release of toxic granule proteins, leukotrienes, cytokines and chemokines (2). In asthma, disease severity, chronicity and exacerbations are frequently associated with Nifedipine airway eosinophilia (3). Depletion of eosinophils in allergen-challenged animals reduced collagen deposition, mucus production and airway hyper-responsiveness (4, 5). In aggregate, these studies establish Rabbit Polyclonal to TRXR2 a crucial role for eosinophils in asthma pathogenesis. IL-5 regulates the differentiation, survival and function of eosinophils. The nearly unique presence of IL-5 receptor- (IL-5RA) on eosinophils made IL-5 an ideal drug target to reduce eosinophilia. Several therapeutic monoclonal antibodies (anti-IL-5 or anti-IL-5RA) are currently in phase III clinical trials (6). In asthma, these antibodies dramatically decreased peripheral blood eosinophils but had much less effect on reducing airway eosinophil numbers (~50%) (7). Yet, this decline of tissue eosinophils by anti-IL-5 therapy reduced asthma exacerbations by ~50% and decreased the use of corticosteroids in severe asthmatic subjects with previously exhibited persistent airway eosinophilia (8, 9). However, in a more general asthma populace, anti-IL-5 failed to improve symptoms and pulmonary functions (10). We have previously shown that airway eosinophils from bronchoalveolar lavage (BAL) drop their IL-5RA and do not degranulate in response to IL-5 (11), and that the upregulation and activation of 2 integrins on airway eosinophils is not affected by treatment with anti-IL-5, even though 2 integrin levels and activation state on blood eosinophils are decreased (12). These observations support the idea that other factors, besides IL-5, are important for airway eosinophil presence and activation. IL-5, GM-CSF and IL-3 initiate signaling via a common -chain receptor, and have been termed c receptor-signaling cytokines. IL-3 and GM-CSF are far more pleiotropic than IL-5, but all three are thought to have largely redundant functions on eosinophils. Mounting evidence suggest this is an oversimplification. We, as well as others have shown that IL-3 alone or associated with TNF- was more potent than IL-5 or GM-CSF to induce the production and release of proteins from eosinophils (13C15). These studies demonstrate that IL-3, IL-5 and GM-CSF have unappreciated distinct functions in eosinophil biology and by inference, asthma and allergy. IL-3 is relevant in asthma, and allergy in general, since it is usually released by activated Th-2 lymphocytes and by mast cells or basophils following IgE cross-linking (16). Serum IL-3 levels are significantly elevated in poorly controlled asthmatics and plasma Nifedipine levels are elevated in patients with asthma or airway allergy (17C19), supporting a role of IL-3 in asthma. Moreover, IL-3-positive cells are more abundant in bronchoalveolar lavage cells or activated T cells from subjects with asthma compared to control subjects, and their numbers increase with asthma severity (19). Finally, airway allergen challenge in subjects with moderate asthma leads to increased levels of IL-3 in the BAL fluid (1, 20). Recently, we have reported that human airway eosinophils expressed more of the pro-fibrotic membrane protein semaphorin-7A than blood eosinophils following an airway allergen challenge (15). Blood eosinophils treated with IL-3 but not GM-CSF or IL-5 showed an important increase of semaphorin-7A protein without changes in semaphorin-7A mRNA levels. Consistent with an effect on translation, we found more semaphorin-7A mRNA associated with polyribosomes after IL-3 signaling (15). In order to test the hypothesis that IL-3 regulates translation, we now examine the phosphorylation of ribosomal protein S6 (RPS6). RPS6 is one of the two ribosomal proteins susceptible to phosphorylation Nifedipine following cellular stimulation by cytokines (21, 22). In stromal cells, RPS6 phosphorylation is usually controlled by the mammalian target of rapamycin (mTOR) and downstream by the kinases, p70S6K1 and p70S6K2 (23). In genetically altered RPS6 (knock-in) mice with alanine substitutions at all 5 phosphorylatable serine residues, global protein synthesis was decreased in liver and mouse embryonic fibroblasts (24). These results.

U0126, its inactive analog U0124 and SB203580 are from Calbiochem/EMD Millipore