Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils
EXPERIMENTAL CELL RESEARCH
Authors: Carmo, Livia A. S.; Dias, Felipe F.; Malta, Kassia K.; Amaral, Katia B.; Shamri, Revital; Weller, Peter F.; Melo, Rossana C. N.
Abstract
Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results: STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean +/- SEM of 3.9 +/- 0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-alpha) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-alpha-stimulated eosinophils. Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. (C) 2015 Elsevier Inc. All rights reserved.
Prognostic factors for recurrence after endoscopic sinus surgery for chronic rhinosinusitis with nasal polyps
AURIS NASUS LARYNX
Authors: Nakayama, Tsuguhisa; Asaka, Daiya; Kanaya, Hiroaki; Kuboki, Akihito; Haruna, Shin-ichi
Abstract
Objective: In this study, we aimed to clarify the prognostic factors affecting the ethmoid condition duHng a long-term follow-up after endoscopic sinus surgery in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Methods: Thirty-six patients with CRSwNP underwent surgery from December 2008 to February 2012. All surgeries were performed by one surgeon, and all patients were followed up for at least 2 years postoperatively. We investigated the association of postoperative endoscopic findings with clinical parameters, mucosal eosinophil count and mRNA expression of CCL11, IL-5, and IFN-gamma in nasal polyps. Results: Seventeen patients (47.2%) had severe mucosal edema, and the patency of each sinus was not confirmed during the >2-year follow-up. The mucosal eosinophil count and two eosinophil-associated factors, namely the CCL11 and IL-5 mRNA levels, were higher in the severe mucosal edema group than in the control group. The severe mucosal edema group was divided into two subgroups: the steroid-responsive and-resistant groups. Five patients (13.9%) had frank polyp formation because the oral steroids were less effective. The mucosal eosinophil count was significantly different among the four groups, including the control group (p = 0.001); however, the CCL11, 11-5, and LPN-gamma mRNA levels were not significantly different. Although the IL-5 mRNA level was not significantly different among the four groups, it tended to increase when the sinus condition worsened. In the severe mucosal edema group, a higher IL-5 mRNA level was associated with earlier severe mucosal edema in the ethmoid cavity. Conclusion: The IL-5 mRNA level is associated with the time of severe edema formation in the ethmoid cavity. This finding permits early intervention on the postoperative course and would prevent polyp recurrence. (C) 2016 Elsevier Ireland Ltd. All rights reserved.