Establishment of non-transformed, continuously growing, alternatively activated mouse macrophage cell lines

巨噬细胞是先天免疫系统的重要组成部分，在防御入侵病原体方面发挥着关键作用。巨噬细胞可以通过细胞和组织对感染的反应产生的化学信使(细胞因子)来切换到执行不同的功能。一些细胞因子将巨噬细胞转换成一种类型(M2)，这种类型对寄生虫病和伤口愈合有益，但在某些细菌感染和哮喘等过敏性疾病中起负面作用。最近的研究表明，与大多数其他类型的巨噬细胞不同，肺M2巨噬细胞可以在体内增殖。由于M2巨噬细胞的关键作用，为了开发更有效的感染治疗方法，对M2巨噬细胞有更多的了解是重要的。对M2巨噬细胞的很大一部分研究是在小鼠系统中进行的，在小鼠系统中，各种突变株的可获得性使了解特定的途径成为可能。在这里，研究人员完全依赖于使用活的动物。M2巨噬细胞直接从器官中分离和研究，或在体外从骨髓中培养和分化。在所有这些程序中获得的细胞寿命有限，因此需要反复使用动物。这项建议的目的是建立永久生长的、交替激活的(M2)巨噬细胞系，以减少目前用于提供此类细胞的动物数量。最近，我们建立了一种新的小鼠肺巨噬细胞(MPI细胞)体外模型。与现有的系统相比，这些原始的、未转化的细胞可以从各种小鼠品系中无限生长，并且在含有生长因子的情况下几乎可以无限数量地获得。因此，它们的使用可以使以前有问题的巨噬细胞实验变得可行。例如，通过支气管肺泡灌洗技术，可以从一只小鼠身上获得数量非常有限的肺巨噬细胞(大约30万个细胞)。相比之下，我们通常从一个75cm2的组织培养瓶中获得2000万-3000万个MPI细胞。我们的最新数据表明，IL-4或IL-13处理可以诱导MPI细胞出现类似M2的表型。这些发现与哮喘患者有关，在哮喘患者中，M2巨噬细胞占主导地位，肺部病毒感染的频率更高，后果更严重。因此，在这个试点项目中，我们希望建立MPI细胞培养作为M2肺巨噬细胞的忠实模型，用于重要的医学研究。用细胞因子IL-4处理的MPI细胞将被表征，并与现有的交替激活的巨噬细胞、小鼠骨髓来源的巨噬细胞和用这些细胞因子处理的肺泡巨噬细胞进行比较。我们将研究目前在交替激活的骨髓来源的巨噬细胞中研究的相关细菌病原体对交替激活的MPI细胞的感染。我们还将分析该系统中空气传播病毒感染的特征，因为实验数据表明，M2巨噬细胞与这些微生物在哮喘等过敏性呼吸道疾病中发挥着重要作用。完成这一项目可以大幅减少用作交替激活巨噬细胞来源的动物数量。此外，拟议系统的建立将揭示生物医学科学中的新机制。通过为高通量筛查和药物测试提供新的工具，它也可能为治疗重要疾病开辟新的前景。

Macrophages are crucial components of the innate immune system and play key roles in defence against invading pathogens. Macrophages can be switched to perform different functions by chemical messengers (cytokines) produced by cells and tissues in response to infection. Some cytokines switch macrophages to a type (M2) that are beneficial in parasitic diseases and wound healing but play negative roles in certain bacterial infections, and in allergic diseases such as asthma. Recent studies indicate that, in contrast to most other macrophage types, lung M2 macrophages can proliferate in vivo. Due to their key roles and to develop more effective therapies for infections, it is important to understand more about M2 macrophages. A significant part of research on M2 macrophages are done in the mouse system where the availability of various mutant strains makes the understanding of specific pathways possible. Here, researchers rely entirely on the use of living animals. M2 macrophages are isolated and studied directly from organs or grown and differentiated from bone-marrow in vitro. The cells obtained in all of these procedures have a limited life-span, thus requiring the repeated use of animals. The aim of this proposal is to establish permanently growing, alternatively activated (M2) macrophage cell lines in order to reduce the number of animals currently used to provide such cells. Recently we established a novel in vitro model of mouse lung macrophages (MPI cells). In contrast to existing systems, these primary, non-transformed cells can be grown indefinitely from various mouse strains and are available in practically unlimited numbers when cultured with growth factors. Thus, their use can make macrophage experiments that have been previously problematic feasible. For example, a very limited number of lung macrophages, (approximately 300, thousand cells) can be obtained from one mouse by the technique of broncho-alveolar lavage. In comparison, we regularly obtain 20-30 million MPI cells from a single 75 cm2 tissue culture flask.Our most recent data show that IL-4 or IL-13 treatment induces an M2 like phenotype in the MPI cells. These findings have relevance to patients with asthma, where M2 macrophages are predominant, and a higher frequency and more serious outcome of pulmonary virus infections are experienced. Therefore, in this pilot project we want to establish MPI cell cultures as a faithful model of M2 lung macrophages for medically important studies. MPI cells treated with the cytokine IL-4 will be characterized and compared to existing models of alternatively activated macrophages, mouse bone marrow derived macrophages and lung alveolar macrophages treated with these cytokines. We will study the infection of alternatively activated MPI cells with relevant bacterial pathogens currently studied in alternatively activated bone marrow derived macrophages. We will also analyze the characteristics of airborne virus infection in this system because experimental data suggest an important role for M2 macrophages with these microbes in allergic airway diseases, such as asthma.Accomplishment of this project can drastically reduce the number of animals used as a source of alternatively activated macrophages. Furthermore, the establishment of the proposed system would reveal novel mechanisms in biomedical science. By providing a new tool for high throughput screening and drug testing it may open new perspectives for the treatment of important diseases as well.