Kesimer Mehmet, Scull Margaret, Brighton Brian, DeMaria Genevieve, Burns Kimberlie, O'Neal Wanda, Pickles Raymond J, Sheehan John K
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
FASEB J. 2009 Jun;23(6):1858-68. doi: 10.1096/fj.08-119131. Epub 2009 Feb 3.
Airway mucus forms the structural basis of the local innate immune defense mechanism. It is an integrated, active, viscoelastic gel matrix evolved to protect the exposed lung from physical, chemical, and pathological erosion. Exosomes are biologically active vesicles secreted by different cell types including epithelial, hematopoietic, and some tumor cells. They are also present in some biological fluids such as serum, urine, breast milk, and bronchoalveolar lavage fluid. In this study, we demonstrate for the first time that exosome-like vesicles with antiviral properties are present in human tracheobronchial epithelial (HTBE) cell culture secretions. These vesicles have been isolated by differential centrifugation and are characterized further by mass spectrometry, flow cytometry, immunoblotting, electron microscopy, and light-scattering methods. HTBE vesicles exhibited characteristic exosomal size (30-100 nm) and morphology (cup-shaped) with a buoyant density in sucrose of 1.12-1.18 g/ml. Biochemical characterization further revealed typical surface, cytoskeletal, and cytoplasmic proteins characteristic of exosomes, including the multivesicular and late endosomal membrane markers Tsg101 and CD63. The presence of RNA was also observed. The epithelial mucins MUC1, MUC4, and MUC16 also contributed to the vesicles' structure. Notably, alpha-2,6-linked sialic acid was associated with these mucin molecules and subsequent functional analysis showed that these vesicles have a neutralizing effect on human influenza virus, which is known to bind sialic acid. Taken together, these findings suggest that airway epithelial cells release exosome-like vesicles and that these structures may be involved in diverse physiological processes in airway biology, including innate mucosal defense.
气道黏液构成了局部固有免疫防御机制的结构基础。它是一种整合的、具有活性的、粘弹性凝胶基质，进化而来以保护暴露的肺部免受物理、化学和病理侵蚀。外泌体是由包括上皮细胞、造血细胞和一些肿瘤细胞在内的不同细胞类型分泌的生物活性囊泡。它们也存在于一些生物流体中，如血清、尿液、母乳和支气管肺泡灌洗液。在本研究中，我们首次证明人气管支气管上皮（HTBE）细胞培养分泌物中存在具有抗病毒特性的类外泌体囊泡。这些囊泡已通过差速离心法分离，并通过质谱、流式细胞术、免疫印迹、电子显微镜和光散射方法进行了进一步表征。HTBE囊泡呈现出特征性的外泌体大小（30 - 100nm）和形态（杯状），在蔗糖中的浮力密度为1.12 - 1.18g/ml。生化表征进一步揭示了外泌体典型的表面、细胞骨架和细胞质蛋白，包括多囊泡和晚期内体膜标记物Tsg101和CD63。还观察到了RNA的存在。上皮黏蛋白MUC1、MUC4和MUC16也对囊泡结构有贡献。值得注意的是，α - 2,6 - 连接的唾液酸与这些黏蛋白分子相关，随后的功能分析表明这些囊泡对已知结合唾液酸的人类流感病毒具有中和作用。综上所述，这些发现表明气道上皮细胞释放类外泌体囊泡，并且这些结构可能参与气道生物学中的多种生理过程，包括固有黏膜防御。
