小鼠与人肺泡巨噬细胞的分离和体外培养
Summary
该通讯描述了从人类和小鼠模型中分离和培养肺泡巨噬细胞的方法, 以供实验之用。
Abstract
肺泡巨噬细胞是末期分化的, 肺部驻留的巨噬细胞。肺泡巨噬细胞在其漫长的生命中是独一无二的, 它们在肺发育和功能方面的重要作用, 以及它们对感染和炎症的肺部局部反应。迄今为止, 没有统一的方法来识别, 隔离和处理的肺泡巨噬细胞从人和小鼠存在。这种方法是必要的研究这些重要的先天免疫细胞在各种实验设置。这里描述的方法可以很容易地被任何实验室采用, 是一种从支气管肺泡灌洗液或肺组织中获取肺泡巨噬细胞和维持它们的体外的简化方法。由于肺泡巨噬细胞主要发生在肺泡中的黏附体细胞, 所以这种方法的重点是在收获和鉴定之前逐出。肺是一种高度血管化的器官, 各种细胞类型的髓和淋巴源栖息, 相互作用, 并受肺部微环境的影响。通过使用这里描述的一组表面标记, 研究人员可以很容易和明确地区分肺泡巨噬细胞与其他白细胞, 并净化它们的下游应用。本发明的培养方法支持人和小鼠肺泡巨噬细胞用于体外生长, 并与细胞和分子研究相适应。
Introduction
肺微环境是一个独特的复杂的生态系统, 具有精心设计的空气导管和血管。吸入的空气通过气管和通过许多分支支气管和性细支气管在到达肺泡之前, 那里血液空气气体交换发生。由于与大气层的直接相互作用, 呼吸表面需要保护, 免受空气微粒和污染物的潜在有害影响。一些物理、化学和免疫屏障保护肺部。值得注意的是, 在呼吸表面部署吞噬是一项重要的一线防御系统。肺泡巨噬细胞 (AMs) 是肺吞噬的一种类型, 它们构成了肺巨噬细胞的绝大部分。顾名思义, AMs 主要定位于肺泡腔, 并作为无梗细胞持续取样周围环境, 并与肺泡上皮1进行通信。在稳态肺中, 95% 以上的吞噬在肺泡空间是 AMs2, 其组成可能会改变, 由于炎症, 感染, 或长期接触污染物。
AMs 参与范围广泛的功能, 可能是局部的肺部和/或系统的重要性。例如, AMs 对肺部的发育和最佳功能至关重要;免疫监测;和清除细胞碎片、入侵病原体和吸入微粒3,4,5,6,7。有针对性地耗尽 AMs, 损害呼吸病毒和细菌的清除,4,8。除了作为吞噬和一线防御者的肺稳态, AMs 是已知的功能作为抗原呈现细胞诱导 T 细胞免疫9, potentiating 鼻腔疫苗的功效10和肺移植后肺限制自身免疫的影响11,12。AM 功能不足已与肺肺泡支持下肺泡 (PAP) 相关联, 这是由基因突变、恶性肿瘤或感染损害肺表面活性剂清除率13, 14 的条件造成的.目前正在探索 AMs 移植治疗 PAP 1516的治疗方法。
AMs 是已知的起源于胚胎发生过程中, 并坚持在整个生命中的肺部, 而不被循环白细胞取代2,17。虽然, 在稳态肺中, am 的营业额是无法检测到的, 但在某些临床情况下, 有不同程度的 am 更替, 包括感染流感病毒4、清髓性照射18、接触内毒素19和老年20。AMs 被认为通过低级别的增生17,21进行自我更新, 但最近的一些研究声称单核细胞可以引起血管内肺巨噬细胞的数量22,23下实验条件, 但这些新转换的肺巨噬细胞的功能尚未定义在肺部疾病。此外, 在 AM 活化的背景下, 了解刺激的阈值是一个潜在的有趣的领域, 因为肺试图保持炎症信号和免疫调节机制之间的平衡。
导致免疫调节丧失的生理或病理改变对于评估各种临床环境 (例如,呼吸道感染、炎症性肺病和纤维化肺病) 是很重要的。然而, AMs 越来越被认为是肺健康的指标, 甚至是决定因素,11,24。目前, 尚无统一的协议可用于收割、鉴定和/或维持来自人类和前小鼠模型的 AMs。对 am 前体和表型缺乏共识, 缺少详细的方法是破译 AM 在肺部健康和疾病中作用的主要障碍。下面的协议提供了一个明确的标识、隔离和体外文化策略, 它将极大地促进对 am 行为的理解, 并促进有针对性的诊断和治疗研究。
Protocol
这里所描述的所有方法都已被 IACUC 医院和医疗中心的机构动物护理和使用委员会 (IRB) 和机构审查委员会批准。 1. 从小鼠支气管肺泡灌洗 (球) 液中分离 AMs 麻醉一个八周大的 C57BL/6 老鼠与氯胺酮 (87.5 毫克/千克体重) 和甲苯噻嗪 (12.5 毫克/千克体重) 鸡尾酒通过腹腔注射。当老鼠获得手术麻醉, 失去反射和肌肉松弛。 将鼠标放在解剖表面, 侧面朝上。应用眼科兽医…
Representative Results
图 1显示了用于识别鼠标 AMs 的流细胞方法。这包括分析在区分 AMs 与其他肺居民或肺浸润吞噬必要的最低限度的表面标记。需要进行差异分析, 以积极识别细胞间质巨噬细胞、树突状体细胞、中性粒细胞、单核细胞以及在肺部发生的核型肺巨噬细胞。下面的曲面标记方案可用于在AMs为 CD45+的各个单元格之间方便地区分这?…
Discussion
AMs 是长寿的肺常驻巨噬细胞, 在出生时填充肺部, 并持续超过整个生命周期26。他们的作用在肺生理学7和病理学12和他们的潜力预测肺自身免疫24已经被认可。由于 AMs 长期存在于肺部11、27中, 并且由于它们参与了免疫应答的激活和进展11,24</sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢克莱尔协助编辑手稿。DKN 由克里斯弗林基金会的研究补助金 (#2095) 支持, TM 得到国家卫生研究院 (R01HL056643 和 R01HL092514) 赠款的支持。DKN 开发了方法, 设计了研究并撰写了手稿;协助动物研究和临床样品采购;协助进行流细胞分析和细胞分选;TM 监督研究和审阅手稿。
Materials
| Non-enzymatic cell dissociating solution | Millipore-Sigma | C5789 | |
| Puralube Vet Ointment | Dechra | 620300 | |
| 22G Catheter | Terumo Medical Products | SR-OX2225CA | |
| 4-0 Non-absorbable silk braided suture | Kent Scientific | SUT-15-2 | |
| Dulbecco’s phosphate buffered saline | Corning | 21-031-CM | |
| Mouse Fc block | BD Biosciences | 553142 | |
| Lysis buffer (PureLink RNA Kit) | Thermo Fisher Scientific | 12183018A | |
| b-Mercaptoethanol | Millipore-Sigma | M6250 | |
| FACSAria II cell sorter | BD Biosciences | 644832 | |
| Ketamine (Ketathesia) | Henry Schein | 56344 | |
| Xylazine (AnaSed) | Akorn | 139-236 | |
| RPMI 1640 | Corning | 10-040-CM | |
| DMEM | Corning | 10-017-CM | |
| Liberase TL | Millipore-Sigma | 5401020001 | |
| DNase I | Millipore-Sigma | AMPD1-1KT | |
| 100μm cell strainer | Corning | 352360 | |
| Human Fc block | BD Biosciences | 564220 | |
| EDTA | Corning | 46-034-CI | |
| Countess II Automated Cell Counter | Thermo Fisher Scientific | AMQAX1000 | |
| Trypan Blue Solution | Thermo Fisher Scientific | 15250061 | |
| HEPES | Corning | 25-060-CI | |
| Fetal Bovine Serum | Atlanta Biologicals | S11150H | |
| L-929 cell line | American Type Culture Collection | ATCC, CCL-1 | |
| Penicillin/Streptomycin | Corning | 30-002-CI | |
| Sodium Pyruvate | Corning | 25-000-CI | |
| T25 Tissue culture flask | Thermo Fisher Scientific | 156367 | |
| 60 mm culture dish | Millipore-Sigma | CLS3261 | |
| 15 mL Conical tube | Corning | 352097 | |
| 50 mL Conical tube | Corning | 352098 | |
| LSRFortessa cell analyzer | BD Biosciences | 657669 | |
| FlowJo | FlowJo | v10.4 | Analysis Software |
| Anti-CD45 (Mouse) | Biolegend | 147709 | Clone I3/2.3, FITC conjugated |
| Anti-CD11b (Mouse) | Biolegend | 101228 | Clone M1/70, PerCP/Cy5.5 conjugated |
| Anti-CD11c (Mouse) | BD Biosciences | 565452 | Clone N418, BV 421 conjugated |
| Anti-I-Ab (Mouse) | Biolegend | 116420 | Clone AF6-120.1, PE/Cy7 conjugated |
| Anti-Siglec-F (Mouse) | BD Biosciences | 562757 | Clone E50-2440, PE-CF594 conjugated |
| Anti-Siglec-H (Mouse) | Biolegend | 129605 | Clone 551, PE conjugated |
| Anti-F4/80 (Mouse) | Biolegend | 123118 | Clone BM8, APC/Cy7 conjugated |
| Anti-Ly-6C (Mouse) | Biolegend | 128035 | Clone HK1.4, BV605 conjugated |
| Anti-CD64 (Mouse) | Biolegend | 139311 | Clone X54-5/7.1, BV711 conjugated |
| Anti-CD24 (Mouse) | BD Biosciences | 563115 | Clone M1/69, BV510 conjugated |
| Anti-CD103 (Mouse) | BD Biosciences | 745305 | Clone OX-62, BV650 conjugated |
| Anti-CD317 (Mouse) | Biolegend | 127015 | Clone 927, APC conjugated |
| Anti-CXCR1 (Mouse) | Biolegend | 149029 | Clone SA011F11, BV785 conjugated |
| Anti-CD45 (Human) | Biolegend | 304017 | Clone HI30, AF488 conjugated |
| Anti-CD11b (Human) | Biolegend | 101216 | Clone M1/70, PE/Cy7 conjugated |
| Anti-HLA-DR (Human) | Biolegend | 307618 | Clone L243, APC/Cy7 conjugated |
| Anti-CD169 (Human) | Biolegend | 346008 | Clone 7-239, APC conjugated |
| Anti-CD206 (Human) | Biolegend | 321106 | Clone 15-2, PE conjugated |
| Anti-CD163 (Human) | Biolegend | 333612 | Clone GHI/61, BV421 conjugated |
References
- Westphalen, K., et al. Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity. Nature. 506 (7489), 503-506 (2014).
- Guilliams, M., et al. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF. J Exp Med. 210 (10), 1977-1992 (2013).
- Cardani, A., Boulton, A., Kim, T. S., Braciale, T. J. Alveolar macrophages prevent lethal influenza pneumonia by inhibiting infection of type-1 alveolar epithelial cells. PLoS Pathog. 13 (1), e1006140 (2017).
- Ghoneim, H. E., Thomas, P. G., McCullers, J. A. Depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections. J Immunol. 191 (3), 1250-1259 (2013).
- MacLean, J. A., et al. Sequestration of inhaled particulate antigens by lung phagocytes. A mechanism for the effective inhibition of pulmonary cell-mediated immunity. Am J Pathol. 148 (2), 657-666 (1996).
- Nakamura, T., et al. Depletion of alveolar macrophages by clodronate-liposomes aggravates ischemia-reperfusion injury of the lung. J Heart Lung Transplant. 24 (1), 38-45 (2005).
- Schneider, C., et al. Alveolar macrophages are essential for protection from respiratory failure and associated morbidity following influenza virus infection. PLoS Pathog. 10 (4), e1004053 (2014).
- Pribul, P. K., et al. Alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development. J Virol. 82 (9), 4441-4448 (2008).
- Macdonald, D. C., et al. Harnessing alveolar macrophages for sustained mucosal T-cell recall confers long-term protection to mice against lethal influenza challenge without clinical disease. Mucosal Immunol. 7 (1), 89-100 (2014).
- Benoit, A., Huang, Y., Proctor, J., Rowden, G., Anderson, R. Effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (RSV). Clin Exp Immunol. 145 (1), 147-154 (2006).
- Nayak, D. K., et al. Long-term persistence of donor alveolar macrophages in human lung transplant recipients that influences donor specific immune responses. Am J Transplant. 16 (8), 2300-2311 (2016).
- Sekine, Y., et al. Role of passenger leukocytes in allograft rejection: effect of depletion of donor alveolar macrophages on the local production of TNF-alpha, T helper 1/T helper 2 cytokines, IgG subclasses, and pathology in a rat model of lung transplantation. J Immunol. 159 (8), 4084-4093 (1997).
- Borie, R., et al. Pulmonary alveolar proteinosis. Eur Respir Rev. 20 (120), 98-107 (2011).
- Greenhill, S. R., Kotton, D. N. Pulmonary alveolar proteinosis: a bench-to-bedside story of granulocyte-macrophage colony-stimulating factor dysfunction. Chest. 136 (2), 571-577 (2009).
- Happle, C., et al. Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis. Sci Transl Med. 6 (250), 250ra113 (2014).
- Suzuki, T., et al. Pulmonary macrophage transplantation therapy. Nature. 514 (7523), 450-454 (2014).
- Hashimoto, D., et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity. 38 (4), 792-804 (2013).
- Murphy, J., Summer, R., Wilson, A. A., Kotton, D. N., Fine, A. The prolonged life-span of alveolar macrophages. Am J Respir Cell Mol Biol. 38 (4), 380-385 (2008).
- Maus, U. A., et al. Resident alveolar macrophages are replaced by recruited monocytes in response to endotoxin-induced lung inflammation. Am J Respir Cell Mol Biol. 35 (2), 227-235 (2006).
- Perdiguero, G. E., et al. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature. 518 (7540), 547-551 (2015).
- Bitterman, P. B., Saltzman, L. E., Adelberg, S., Ferrans, V. J., Crystal, R. G. Alveolar macrophage replication. One mechanism for the expansion of the mononuclear phagocyte population in the chronically inflamed lung. J Clin Invest. 74 (2), 460-469 (1984).
- Misharin, A. V., et al. Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. J Exp Med. , (2017).
- Zheng, Z., et al. Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction. Sci Transl Med. 9 (394), (2017).
- Nayak, D. K., et al. Zbtb7a induction in alveolar macrophages is implicated in anti-HLA-mediated lung allograft rejection. Sci Transl Med. 9 (398), (2017).
- Misharin, A. V., Morales-Nebreda, L., Mutlu, G. M., Budinger, G. R., Perlman, H. Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung. Am J Respir Cell Mol Biol. 49 (4), 503-510 (2013).
- Kopf, M., Schneider, C., Nobs, S. P. The development and function of lung-resident macrophages and dendritic cells. Nat Immunol. 16 (1), 36-44 (2015).
- Eguiluz-Gracia, I., et al. Long-term persistence of human donor alveolar macrophages in lung transplant recipients. Thorax. 71 (11), 1006-1011 (2016).
- Yu, Y. A., et al. Flow cytometric analysis of myeloid cells in human blood, bronchoalveolar lavage, and lung tissues. Am J Respir Cell Mol Biol. , (2015).
Cite This Article
Nayak, D. K., Mendez, O., Bowen, S., Mohanakumar, T. Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages. J. Vis. Exp. (134), e57287, doi:10.3791/57287 (2018).