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

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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.