Background Epithelial barrier dysfunction is thought to play a role in many mucosal diseases, including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis. Objective The objective of ...this study was to investigate the role of oncostatin M (OSM) in epithelial barrier dysfunction in human mucosal disease. Methods OSM expression was measured in tissue extracts, nasal secretions, and bronchoalveolar lavage fluid. The effects of OSM stimulation on barrier function of normal human bronchial epithelial cells and nasal epithelial cells cultured at the air-liquid interface were assessed by using transepithelial electrical resistance and fluorescein isothiocyanate–dextran flux. Dual-color immunofluorescence was used to evaluate the integrity of tight junction structures in cultured epithelial cells. Results Analysis of samples from patients with CRS showed that OSM mRNA and protein levels were highly increased in nasal polyps compared with those seen in control uncinate tissue ( P < .05). OSM levels were also increased in bronchoalveolar lavage fluid of allergic asthmatic patients after segmental allergen challenge and in esophageal biopsy specimens from patients with eosinophilic esophagitis. OSM stimulation of air-liquid interface cultures resulted in reduced barrier function, as measured by decreased transepithelial electrical resistance and increased fluorescein isothiocyanate–dextran flux ( P < .05). Alterations in barrier function by OSM were reversible, and the viability of epithelial cells was unaffected. OSM levels in lysates of nasal polyps and uncinate tissue positively correlated with levels of α2 -macroglobulin, a marker of epithelial leak, in localized nasal secretions ( r = 0.4855, P < .05). Conclusions These results suggest that OSM might play a role in epithelial barrier dysfunction in patients with CRS and other mucosal diseases.
Background We have previously shown that oncostatin M (OSM) levels are increased in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS), as well as in bronchoalveolar lavage fluid, after ...segmental allergen challenge in allergic asthmatic patients. We also showed in vitro that physiologic levels of OSM impair barrier function in differentiated airway epithelium. Objective We sought to determine which hematopoietic or resident cell type or types were the source of the OSM expressed in patients with mucosal airways disease. Methods Paraffin-embedded NP sections were stained with fluorescence-labeled specific antibodies against OSM, GM-CSF, and hematopoietic cell-specific markers. Live cells were isolated from NPs and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood and cultured with the known OSM inducers GM-CSF and follistatin-like 1, and OSM levels were measured in the supernatants. Bronchial biopsy sections from control subjects, patients with moderate asthma, and patients with severe asthma were stained for OSM and neutrophil elastase. Results OSM staining was observed in NPs, showed colocalization with neutrophil elastase (n = 10), and did not colocalize with markers for eosinophils, macrophages, T cells, or B cells (n = 3-5). Flow cytometric analysis of NPs (n = 9) showed that 5.1% ± 2% of CD45+ cells were OSM+ , and of the OSM+ cells, 56% ± 7% were CD16+ Siglec-8− , indicating neutrophil lineage. Only 0.6 ± 0.4% of CD45+ events from matched blood samples (n = 5) were OSM+ , suggesting that increased OSM levels in patients with CRS was locally stimulated and produced. A majority of OSM+ neutrophils expressed arginase 1 (72.5% ± 12%), suggesting an N2 phenotype. GM-CSF levels were increased in NPs compared with those in control tissue and were sufficient to induce OSM production ( P < .001) in peripheral blood neutrophils in vitro . OSM+ neutrophils were also observed at increased levels in biopsy specimens from patients with severe asthma. Additionally, OSM protein levels were increased in induced sputum from asthmatic patients compared with that from control subjects ( P < .05). Conclusions Neutrophils are a major source of OSM-producing cells in patients with CRS and severe asthma.
Background Complement plays a major role in inflammatory diseases, but its involvement and mechanisms of activation in patients with chronic rhinosinusitis (CRS) are not known. Objectives After ...earlier studies discovering autoantibodies in patients with CRS, we sought to investigate the nature, extent, and location of complement activation in nasal tissue of patients with CRS. Specifically, we were interested in whether antibody-mediated activation through the classical pathway was a major mechanism for complement activation in patients with CRS. Methods Nasal tissue was obtained from patients with CRS and control subjects. Tissue homogenates were analyzed for complement activation products (ELISA–C5b-9, C4d, activated C1, and C5a) and major complement-fixing antibodies (Luminex). Tissue sections were stained for C5b-9, C4d, and laminin. Antibodies were purified with protein A/G columns from nasal polyps (NP), matching patient serum, and control serum and assayed for basement membrane binding by means of ELISA. Results C5b-9 levels were significantly increased in NP tissue compared with uncinate tissue (UT) of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and those with chronic rhinosinusitis without nasal polyps (CRSsNP; P < .01). Similarly, C4d levels were increased in NPs compared with UT of patients with CRSwNP, patients with CRSsNP, and control subjects ( P < .05). Activated C1 levels were also increased in NP tissue compared with UT of patients with CRSsNP and control subjects ( P < .05) and correlated with levels of C5a ( P < .01), local immunoglobulins (especially IgM, P < .0001), and anti–double-stranded DNA IgG ( P < .05). Immunofluorescence showed that C5b-9 and C4d deposition occurred linearly along the epithelial basement membrane. NP tissue extracts had significantly more anti–basement membrane antibodies than sera from patients with CRSwNP and control subjects ( P < .0001). Conclusion Levels of C5b-9, C4d, and activated C1 were significantly increased locally in NP tissue. C5b-9 and C4d were almost universally deposited linearly along the basement membrane of NP tissue. Furthermore, activated C1 levels were best correlated with local immunoglobulin and C5a levels. Together, these data suggest that the classical pathway plays a major role in complement activation in patients with CRS.
Background The polypoid form of chronic rhinosinusitis (chronic rhinosinusitis with nasal polyps CRSwNP) is a highly prevalent disease that often requires surgical intervention for treatment. Nasal ...polyps contain large quantities of B lymphocytes and immunoglobulin as well as eosinophils. Objectives The objective of this study was to investigate the expression of B cell–activating factor of the TNF family (BAFF), an important regulator of class-switch recombination and immunoglobulin production, in patients with chronic rhinosinusitis (CRS). Methods We collected nasal tissue and nasal lavage fluid from patients with CRS and control subjects. We assayed mRNA for BAFF and B-lymphocyte markers, CD20 and transmembrane activator and calcium-modulator and cyclophilin ligand interactor, by using real-time PCR, and assayed BAFF protein by using ELISA and immunohistochemistry. Results BAFF mRNA was significantly increased in nasal polyps from patients with CRSwNP ( P < .001) compared with inferior turbinate tissue from patients with CRS or healthy subjects. BAFF protein was also elevated in polypoid tissue and nasal lavage from patients with CRSwNP. Immunohistochemistry showed considerable BAFF staining in mucosal epithelial cells in nasal polyps along with unidentified cells in the lamina propria. Expression of mRNA for BAFF in sinonasal tissue was significantly correlated with CD20 and transmembrane activator and CAML interactor in sinus tissue. IgA, an immunoglobulin isotype known to activate eosinophils, was also significantly elevated in the polypoid tissue. Conclusion Overproduction of BAFF in nasal polyps may contribute to the pathogenesis of CRSwNP via the local induction of IgA and activation of eosinophils.
Background Chronic rhinosinusitis (CRS) is a heterogeneous chronic disease characterized by local inflammation of the sinonasal tissues. The pathogenesis of CRS remains controversial, but it has been ...associated with the accumulation of various immune and inflammatory cells in sinus tissue. Objectives The objective of this study was to investigate the expression of the chemokine CCL23, which is known to bind to CCR1 and recruit monocytes, macrophages, and dendritic cells, in patients with CRS. Methods We collected nasal tissue from patients with CRS and control subjects. We assayed mRNA for CCL23 by using real-time PCR and measured CCL23 protein by means of ELISA, immunohistochemistry, and immunofluorescence. Results CCL23 mRNA levels were significantly increased in nasal polyps (NPs) from patients with CRS with nasal polyps (CRSwNP; P < .05) compared with inferior turbinate and uncinate tissue from patients with CRS or control subjects. CCL23 protein levels were also increased in NPs, although these levels were not statistically significant. Immunohistochemical analysis revealed CCL23 expression in mucosal epithelial cells and inflammatory cells, but accumulation of CCL23+ inflammatory cells occurred only in NPs. Immunofluorescence data showed CCL23 colocalization with eosinophil cationic protein–positive eosinophils. The concentration of CCL23 in NPs positively correlated with the concentration of eosinophil cationic protein, suggesting that eosinophils are major CCL23-producing cells in NPs. Finally, we found that CCL23 protein levels were significantly increased in NPs from patients with CRSwNP with aspirin sensitivity. Conclusion Overproduction of CCL23 in NPs might contribute to the pathogenesis of eosinophilic CRSwNP through the recruitment of CCR1+ inflammatory cells, including monocytes and macrophages, and the amplification of local inflammation.
Conclusions These results demonstrate an elevation of epithelial, endothelial and eosinophil MPs, along with a trend toward increased levels of mast cell MP, in CRS patients, and support previous ...findings of activation of these cells in CRS.
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