Pothoven Kathryn L, Norton James E, Suh Lydia A, Carter Roderick G, Harris Kathleen E, Biyasheva Assel, Welch Kevin, Shintani-Smith Stephanie, Conley David B, Liu Mark C, Kato Atsushi, Avila Pedro C, Hamid Qutayba, Grammer Leslie C, Peters Anju T, Kern Robert C, Tan Bruce K, Schleimer Robert P
Division of Allergy-Immunology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Ill.
Department of Otolaryngology, Northwestern University Feinberg School of Medicine, Chicago, Ill.
J Allergy Clin Immunol. 2017 Jun;139(6):1966-1978.e9. doi: 10.1016/j.jaci.2016.10.039. Epub 2016 Dec 18.
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.
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.
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.
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 CD16Siglec-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).
Neutrophils are a major source of OSM-producing cells in patients with CRS and severe asthma.
我们之前已经表明，慢性鼻-鼻窦炎（CRS）患者鼻息肉（NP）中的抑瘤素M（OSM）水平升高，以及变应性哮喘患者在节段性变应原激发后支气管肺泡灌洗液中的OSM水平也升高。我们还在体外表明，生理水平的OSM会损害分化的气道上皮的屏障功能。
我们试图确定在黏膜气道疾病患者中表达的OSM的造血细胞或驻留细胞类型。
用针对OSM、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和造血细胞特异性标志物的荧光标记特异性抗体对石蜡包埋的NP切片进行染色。从NP和匹配的血液样本中分离活细胞进行流式细胞术分析。从全血中分离中性粒细胞，并用已知的OSM诱导剂GM-CSF和类卵泡抑素1进行培养，然后测量上清液中的OSM水平。对来自对照受试者、中度哮喘患者和重度哮喘患者的支气管活检切片进行OSM和中性粒细胞弹性蛋白酶染色。
在NP中观察到OSM染色，显示与中性粒细胞弹性蛋白酶共定位（n = 10），而与嗜酸性粒细胞、巨噬细胞、T细胞或B细胞的标志物不共定位（n = 3 - 5）。对NP（n = 9）的流式细胞术分析显示，5.1% ± 2%的CD45细胞是OSM阳性，在OSM阳性细胞中，56% ± 7%是CD16Siglec-8，表明为中性粒细胞谱系。来自匹配血液样本（n = 5）的CD45事件中只有0.6 ± 0.4%是OSM阳性，这表明CRS患者中升高的OSM水平是局部刺激产生的。大多数OSM阳性中性粒细胞表达精表达精氨酸酶1（72.5% ± 12%），表明为N2表型。与对照组织相比，NP中的GM-CSF水平升高，并且足以在体外诱导外周血中性粒细胞产生OSM（P <.001）。在重度哮喘患者的活检标本中也观察到OSM阳性中性粒细胞水平升高。此外，与对照受试者相比，哮喘患者诱导痰中的OSM蛋白水平升高（P <.05）。
中性粒细胞是CRS和重度哮喘患者中产生OSM的细胞的主要来源。
