3= 0

3= 0.05; vs OVA plus control Ab) as effectively ENPP3 as the intact anti-Siglec-F Ab. Open in a separate window FIGURE 3 Effect of F(ab)2 fragment of anti-Siglec-F Ab on levels of eosinophils following acute OVA challenge. eosinophils. Administration of a F(ab)2 fragment of an anti-Siglec-F Ab also significantly reduced levels of eosinophilic inflammation in the lung and blood. FACS analysis exhibited increased numbers of apoptotic cells (annexin V+/CCR3+ bronchoalveolar lavage and bone marrow cells) in anti-Siglec-F Ab-treated mice challenged with OVA. The anti-Siglec-F Ab significantly reduced the number of peribronchial major basic protein+/TGF-+ cells, suggesting that reduced levels of eosinophil-derived TGF- in anti-Siglec-F Ab-treated mice contributed to reduced levels of peribronchial fibrosis. Administration of the anti-Siglec-F Ab modestly reduced levels of periodic acid-Schiff-positive mucus cells and the thickness of the easy muscle layer. Overall, these studies suggest that administration of an anti-Siglec-F Ab can significantly reduce levels of allergen-induced eosinophilic airway inflammation and features of airway remodeling, in particular subepithelial fibrosis, by reducing the production of eosinophils and increasing the number of apoptotic eosinophils in lung and bone marrow. The recruitment of bone marrow-derived eosinophils from your circulation into the airway is usually a prominent feature of allergic asthma. Important signals mediating the trafficking of eosinophils from your bone marrow to the airway include cytokines such as IL-5 that induce eosinophil proliferation (1), endothelial-induced adhesion molecules such as VCAM-1, P-selectin, and ICAM-1 that localize eosinophils to inflamed tissue sites (2, 3), and CC chemokines such as eotaxin-1 that induce the directed migration of eosinophils in the extracellular matrix (4). Once in the airway the eosinophil may contribute to the proinflammatory response by releasing preformed cytoplasmic granule mediators (i.e., major basic protein (MBP)3), newly generated lipid mediators (i.e., leukotriene C4 (LTC4)), and also transcribe an array of proinflammatory cytokines (1). Although increased levels of eosinophils and eosinophil-derived mediators have been detected in humans with asthma (5, 6), the role of the eosinophil in the pathogenesis of asthma is usually controversial (7, 8) in part because of results from recent clinical studies with anti-IL-5 that have not exhibited either reductions in late phase responses to inhalation allergen challenge in moderate asthmatics (9), nor improved symptoms and pulmonary function in moderate asthmatics (10). However, in contrast to these studies in which targeting IL-5 in asthma has been ineffective in improving symptoms or lung function, anti-IL-5 has demonstrated effectiveness in reducing levels of airway remodeling in asthma (11). For example, targeting IL-5 reduces both the quantity of eosinophils in the airway as well as features of airway remodeling in mouse models of allergen-induced airway remodeling (12), and in humans with asthma (11, 13). One of the strategies ZK824859 to limit the generation of eosinophils is usually to target receptors expressed by eosinophils that might mediate the resolution of eosinophilic inflammation. One such candidate receptor expressed by eosinophils that mediates the resolution of eosinophilic inflammation is usually Siglec-F (14, ZK824859 15). Siglec-F belongs to the CD33-related Siglec family, which is a subclass of Siglecs defined by their mutual sequence similarity (share ~50C80% sequence similarity) and clustered gene localization (chromosome 7 in mice, chromosome 19q in humans) (14). Siglec-F is usually a transmembrane receptor comprising a ligand-binding V-set domain name, three C-2 domains, a transmembrane domain name, and a cytoplasmic ITIM domain ZK824859 name (16). Of the eight mouse Siglecs and 11 human Siglecs that have been recognized, eosinophils are known to express significant levels of Siglec-F in mice (15, 17, 18), as well as its functionally convergent ortholog Siglec-8 in human eosinophils (19C21). Siglec-F, like other CD33-related Siglecs, has a tyrosine-based transmission transduction motif in its cytoplasmic tail, including a canonical ITIM motif, which is known to be ZK824859 involved in inhibitory signaling pathways in the immune system (22, 23). Support for inhibitory signaling by the cytoplasmic domain name of CD33-related Siglecs has come from studies in which Abs were used to cross-link Siglec cell surface receptors. These studies exhibited that Ab cross-linking of several CD33-related Siglecs results in inhibition of cellular-activation signals, arrest of proliferation, or induction of apoptosis (24C26). Studies of mice deficient in Siglec-F have demonstrated that they have increased levels of allergen-induced airway eosinophilic inflammation, as well as delayed resolution of airway eosinophilic inflammation following acute allergen challenge (15). These studies in Siglec-F-deficient mice suggest that Siglec-F normally functions to down-regulate eosinophilic inflammation. Therefore, in this study we have examined whether administration of an anti-Siglec-F Ab could reduce levels of airway eosinophilic inflammation and, importantly, levels ZK824859 of airway remodeling in a mouse model of chronic allergen-induced airway remodeling. Materials and Methods Effect of anti-Siglec-F Ab on levels of lung, bone marrow, and blood eosinophils Eight to 10-wk-old BALB/c mice (16 mice/group) (The Jackson Laboratory) were immunized s.c. on days.