Marginating肺白细胞的选择性收获
Summary
Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.
Abstract
Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.
Introduction
白细胞粘附到肺部的毛细血管（ 即 marginating肺（MP），白细胞）1被示出与来自其他免疫区室（ 例如 ，循环，脾，骨髓）的白细胞显示出不同的白细胞组成和独特活性2- 4。例如，MP-白细胞表现出较高的天然杀伤（NK）细胞的细胞毒性对各种肿瘤细胞相比，循环和脾NK细胞，以及分化信使RNA（mRNA）水平和升高亲和抗炎性细胞因子的分泌。细胞的组合物也由循环白细胞分化为MP-白细胞具有相比循环白细胞（50％对30％，）先天/适应性免疫的比例较高。本文提出的方法的目标是使MP-白细胞选择性收割，为了研究这个重要和独特的免疫隔室（细胞群），以及elucIDATE对这些特定单元的各种操作（ 例如 ，免疫激活）的影响。
为了理解这个独特群体的意义，要注意的是，免疫系统能够控制通过细胞介导的免疫（CMI）的体内功能循环肿瘤细胞，微转移和残余疾病是很重要的。尽管免疫系统的先例未能控制原发肿瘤这种能力是显而易见的，并且在癌症患者和动物模型5 的体内证据充分的支持。重要的是，这些研究结果通常与在体外和离体研究，其中报告，大多数自体肿瘤细胞是抗细胞毒性通过从人类和动物的循环血液样品中的白细胞不一致（由细胞毒性测定法测定）6,7。这种差异可能是由于不同的白细胞群，如前述的体内存在上述的MP白细胞种群，特别是其活化NK细胞3的亚群。实际上，同基因的肿瘤细胞（MADB106），这被认为是对循环和脾白细胞抗性，却显示出了由MP-NK细胞-3,8-被裂解。因此，转移至的fischer344（F344）大鼠肺中据称“NK抗性'MADB106细胞通过MP-NK细胞，但不被循环或脾NK细胞，其通常研究了给予它们易于访问的控制。
纯化和活性的MP-白细胞通过从肺，其基于肺组织研磨或生物降解9白细胞的标准收获方式无法访问。相比于组织处理方法我们的方法有两个主要的优点。首先，将灌注方法选择性地收割的MP-白细胞，来自其他小区从肺实质，间质性起源，和支气管肺泡COMPAR将它们分离tments。其次，灌注技术更好保持完整性和MP-白细胞的生理环境，不像研磨和生物处理方法损害细胞，改变它们的形态，并诱导生产和调节免疫活性和特异性地抑制NK细胞的各种因素释放细胞毒性10。
肺是癌症转移和用于各种感染性疾病的主要靶器官。所有的循环恶性细胞和感染细胞穿过肺毛细血管，在那里他们需要变形和毛细血管内皮细胞和白细胞居民互动。在这些条件下，循环细胞可以容易地由驻留MP-白细胞定位。因此它似乎有活化的白细胞在这种免疫隔室生物学有利的，并且在不同的生物，实验和临床研究这种独特的MP-人口是重要的。这是值得要注意，SY通过各种生物响应改性剂stemic免疫激活（ 例如 ，聚肌胞苷酸（聚I：C）或C型的CpG寡脱氧核苷酸（CpG基-C ODN））已经显示激活MP-白细胞比循环白细胞3以上， 8,11。
Protocol
Representative Results
Discussion
本文提供的方法使得能够选择性收获和MP-白细胞的独特群体的研究。相比循环或脾的白细胞中，MP人口的特征在于白细胞亚群的一个独特的组合物，更高的活化水平，各种细胞因子的更高的释放，和亲和抗炎细胞因子2,3的更高的mRNA水平。具体地，我们已经表明，MP-NK细胞比循环NK细胞对各种靶细胞3,4-多种细胞毒性，并表达更高水平的干扰素γ的。总的来说，我们的结果表明，MP-NK白…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者这项工作是由NIH / NCI补助＃R01CA172138（与SBE）的支持。
Materials
| Autoclud Peristaltic pump | |||
| Butterfly needle | OMG | 26G | |
| Butterfly needle | OMG | 21G*3/4" | |
| Syringe | Pic solution | 1 ml | |
| Syringe | Pic solution | 2.5 ml | |
| Syringe | Pic solution | 5 ml | |
| Syringe | Pic solution | 10 ml | |
| Syringe | Pic solution | 25 ml | |
| Blunted-edged forceps | |||
| Scissors | |||
| hemostat | |||
| Tissue forceps | |||
| Heparin sodium porcine mucosa (perservative free) | Sigma Aldrich |
Referências
- Kuebler, W. M., Goetz, A. E. The marginated pool. Eur Surg Res. 34, 92-100 (2002).
- Benish, M., et al. The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics. Immunol Res. 58, 28-39 (2014).
- Melamed, R., et al. The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells. J Immunother. 33, 16-29 (2010).
- Shakhar, G., et al. Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis. Ann Surg Oncol. 14, 841-852 (2007).
- Smyth, M. J., Godfrey, D. I., Trapani, J. A. A fresh look at tumor immunosurveillance and immunotherapy. Nat Immunol. 2, 293-299 (2001).
- Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. Role of NK cells in tumour growth and metastasis in beige mice. Nature. 284, 622-624 (1980).
- Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med. 155, 1823-1841 (1982).
- Melamed, R., et al. Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor. Brain Behav Immun. 19, 114-126 (2005).
- O’Dea, K. P., et al. Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia. Am J Respir Crit Care Med. 172, 1119-1127 (2005).
- Sorski, L., Melamed, R., Ben-Eliyahu, A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy. , (2014).
- Levi, B., et al. Continuous stress disrupts immunostimulatory effects of IL-12). Brain Behav Immun. 25, 727-735 (2011).
