Of the OSM+ cells, 29

Of the OSM+ cells, 29.88% were excluded during analysis of the data because the wavelengths of the fluorophores used to stain for CD16-APCH7 siglec 8-Alexa 647 were near each other and the rigorous compensation algorithm that we used excluded them to ensure the accuracy of the data. macrophages, T cells or B cells (n=3C5). Flow cytometric analysis of NP (n=9) showed that 5.12% of CD45+ cells were OSM+, and of the OSM+ cells, 567% were CD16+Siglec8?, indicating neutrophil lineage. Only.6.4% of CD45+ events from matched blood samples (n=5) were OSM+, suggesting that elevated OSM in CRS was locally stimulated and produced. A majority of OSM+ neutrophils expressed Arginase 1 (72.512%), suggesting a N2 phenotype. GM-CSF was elevated in nasal polyp tissue compared to control, and was sufficient to induce OSM production (p .001) in peripheral blood neutrophils cultured airway epithelium as measured by decreased epithelial resistance, increased epithelial permeability, and loss of tight junction structure. Additionally, levels of OSM in both nasal tissue from CRS patients and bronchoalveolar lavage fluid (BAL) from trans-trans-Muconic acid allergen-challenged allergic asthma patients correlated with markers of epithelial leak, suggesting that OSM may mediate barrier dysfunction in mucosal disease8. We hypothesized that OSM-induced loss of barrier function could allow the entry of various allergens, pathogens and environmental factors into the tissue where they could elicit a chronic immune response. It is important now to identify the cell type responsible for OSM production in mucosal disease in order to understand the mechanism of barrier disruption in eosinophilic mucosal disease. Neutrophil-derived OSM has been shown to contribute to the pathogenesis of many conditions including acute lung injury, asthma, breast Rabbit Polyclonal to MGST3 cancer, and rheumatoid arthritis9, 10, 11, 12. Although osteopontin, prostaglandin E2 (PGE2), follistatin-like 1 (FSTL1), complement factor 5a, and thrombin have all been shown to induce OSM expression in various cell types13, 14, 15, 16, 17, only GM-CSF was sufficient to induce OSM in neutrophils11, 18. Elbjeirami et al. showed that OSM was induced during neutrophil transendothelial migration in response to endothelial production of GM-CSF18. Queen et al. showed that co-culture of trans-trans-Muconic acid neutrophils with the breast cancer cell lines MDA-MB-231 and T-47D induced OSM production that was lost when GM-CSF was blocked11. Additionally, Miller et al, have shown that follistatin like-1 (FSTL1) was necessary for OSM induction in a mouse model of chronic asthma16. Both GM-CSF and FSTL1 have been shown to be elevated in airways disease19, 20, 21, 22, and we hypothesized that one or both of these cytokines may induce neutrophil derived OSM in CRS. Recent studies have described subtypes of polarized neutrophils, the classical, proinflammatory N1 neutrophil and the anti-inflammatory, or tumorigenic N2 neutrophil23. These N1 and N2 neutrophils have been shown to be very similar in function to their M1 and M2 macrophage counterparts23. N2 neutrophils specifically have been shown to promote tumor growth and metastasis, as well as play an important role in repair processes and the resolution of inflammation24, 25, 26. N1 and N2 neutrophil polarization has been best studied in mice, and N2 neutrophils have been shown to express the macrophage mannose receptor (MMR), arginase 1 (ARG1), chitinase-like 3 (Ym1), IL-10 and TGF, which are also characteristic markers of M2 macrophages. Both M2 trans-trans-Muconic acid macrophages and N2 neutrophils have been associated with tissue repair mechanisms25. Compared to N2, N1 neutrophils have been shown to express more proinflammatory cytokines and chemokines such as TNF, IL-1, CCL3, CCL5, IL-6 and IL-1225. Although progress is being made, N2 neutrophils in humans are much less extensively studied, and the markers used to define N2 neutrophils in mice may not all be relevant as markers for human trans-trans-Muconic acid N2 neutrophils27. Because N2 neutrophils and OSM have been shown to be important for repair processes, we wanted to test the hypothesis that N2 neutrophils were responsible for OSM production in CRS. In this manuscript we show that neutrophils are a major source of OSM producing cells in nasal polyps and neutrophils also express OSM in severe asthma. We also show that the OSM inducer, granulocyte-macrophage colony-stimulating factor (GM-CSF), was elevated in nasal polyps at levels sufficient to induce OSM production in cultured neutrophils. Methods Patients and tissue sample collection Control subjects and patients with CRS were recruited from the Allergy-Immunology and Otolaryngology clinics of the Northwestern Medicine and the Northwestern Sinus Center at Northwestern Medicine. Nasal polyps trans-trans-Muconic acid and peripheral blood were obtained during routine functional endoscopic sinus surgery from patients with CRS, as defined by.