Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
J Immunol Methods. 2012 Jan 31;375(1-2):100-10. doi: 10.1016/j.jim.2011.09.013. Epub 2011 Oct 2.
We developed a flow cytometry-based assay to simultaneously quantify multiple leukocyte populations in the marginated vascular, interstitial, and alveolar compartments of the mouse lung. An intravenous injection of a fluorescently labeled anti-CD45 antibody was used to label circulating and marginated vascular leukocytes. Following vascular flushing to remove non-adherent cells and collection of broncho-alveolar lavage (BAL) fluid, lungs were digested and a second fluorescent anti-CD45 antibody was added ex vivo to identify cells not located in the vascular space. In the naïve mouse lung, we found about 11 million CD45+ leukocytes, of which 87% (9.5 million) were in the vascular marginated compartment, consisting of 17% NK cells, 17% neutrophils, 57% mononuclear myeloid cells (monocytes, macrophage precursors and dendritic cells), and 10% T cells (CD4+, CD8+, and invariant NKT cells). Non-vascular compartments including the interstitial compartment contained 7.7×10(5)cells, consisting of 49% NK cells, 25% dendritic cells, and 16% other mononuclear myeloid cells. The alveolar compartment was overwhelmingly populated by macrophages (5.63×10(5)cells, or 93%). We next studied leukocyte margination and extravasation into the lung following acid injury, a model of gastric aspiration. At 1 h after injury, neutrophils were markedly elevated in the blood while all other circulating leukocytes declined by an average of 79%. At 4 h after injury, there was a peak in the numbers of marginated neutrophils, NK cells, CD4+ and CD8+ T cells and a peak in the number of alveolar NK cells. Most interstitial cells consisted of DCs, neutrophils, and CD4+ T cells, and most alveolar compartment cells consisted of macrophages, neutrophils, and NK cells. At 24 h after injury, there was a decline in the number of all marginated and interstitial leukocytes and a peak in alveolar neutrophils. In sum, we have developed a novel assay to study leukocyte margination and trafficking following pulmonary inflammation and show that marginated cells comprise a large fraction of lung leukocytes that increases shortly after lung injury. This assay may be of interest in future studies to determine if leukocytes become activated upon adherence to the endothelium, and have properties that distinguish them from interstitial and circulating cells.
我们开发了一种基于流式细胞术的检测方法，可同时定量分析小鼠肺部边缘血管、间质和肺泡腔中的多种白细胞群体。静脉注射荧光标记的抗 CD45 抗体，用于标记循环和边缘血管中的白细胞。血管冲洗去除非黏附细胞并收集支气管肺泡灌洗液（BAL）后，将肺消化并在体外添加第二种荧光抗 CD45 抗体，以识别不在血管腔内的细胞。在初始状态的小鼠肺部，我们发现约有 1100 万个 CD45+白细胞，其中 87%（950 万个）位于边缘血管腔中，包括 17%的 NK 细胞、17%的中性粒细胞、57%的单核髓样细胞（单核细胞、巨噬细胞前体和树突状细胞）和 10%的 T 细胞（CD4+、CD8+和不变 NKT 细胞）。包括间质腔在内的非血管腔含有 770 万个细胞，其中包括 49%的 NK 细胞、25%的树突状细胞和 16%的其他单核髓样细胞。肺泡腔中主要是巨噬细胞（563 万个细胞，占 93%）。接下来，我们研究了酸损伤（胃抽吸模型）后白细胞向肺部边缘的黏附和渗出。损伤后 1 小时，血液中的中性粒细胞显著升高，而所有其他循环白细胞平均下降 79%。损伤后 4 小时，边缘中性粒细胞、NK 细胞、CD4+和 CD8+T 细胞数量达到峰值，肺泡 NK 细胞数量也达到峰值。大多数间质细胞由 DC、中性粒细胞和 CD4+T 细胞组成，大多数肺泡腔细胞由巨噬细胞、中性粒细胞和 NK 细胞组成。损伤后 24 小时，所有边缘和间质白细胞数量减少，肺泡中性粒细胞数量达到峰值。总之，我们开发了一种新的检测方法来研究肺部炎症后白细胞的边缘黏附和迁移，并表明边缘细胞构成了肺部白细胞的很大一部分，在肺损伤后不久就会增加。这种检测方法在未来的研究中可能会很有趣，以确定白细胞在黏附于内皮时是否被激活，并且具有与间质和循环细胞区分开来的特性。
