Phenotypic Stability of Self-Replicating Murine Alveolar Macrophage Model Prolonged by the Addition of the Cytokine TGFbeta

RATIONALE.Occupational exposure to crystalline silica (cSiO 2 ) is associated with multiple airway diseases and etiologically linked to development of autoimmune disease.Elucidating cSiO 2 's effects on alveolar macrophages (AM), a primary line of defense against inhaled particles, is crucial for understanding cSiO 2 -induced toxicity.An obstacle to in vitro mechanistic studies of AMs is their paucity in a single mouse (1E5 cells).Previously, our lab has employed Max Planck Institute (MPI) cells as a self-replicating AM surrogate.MPI cells are obtained by culturing fetal liver monocytes with the cytokine GM-CSF.While these cells are initially similar to AMs in morphology and gene expression, they lose this phenotype after 1 month in culture.We hypothesized that supplementing cell culture media with TGF-beta, recently identified as critical to the development and maintenance of AMs in the lungs, will prolong an AM-like phenotype in MPI cells.METHODS.We compared MPI cells cultured with 30 ng/mL GM-CSF alone (control) to cells with 30 ng/mL GM-CSF and 20 ng/mL TGF-beta (TGF-beta-treated).Expression of AM marker genes and proteins was assessed by qPCR and flow cytometry, respectively.Morphology was assessed by light and scanning electron microscopy.To evaluate functional changes, we cultured control and TGF-beta MPI cells with cSiO 2 (50 and 100 μg/cm 2 ).cSiO 2 phagocytosis was assessed by observation of cSiO 2free areas surrounding cSiO 2 -engorged cells.RESULTS.After approximately 1 month, TGF-beta-treated MPI cells retained an ovoid morphology with numerous outer plasma membrane ruffles, while control MPI cells adopted a more fusiform morphology with loss of membrane ruffling.Similarly, control cells lost expression of AM-identifying genes Siglecf, Marco, and Pparg and began expressing Cd14, used to identify monocyte-derived macrophages recruited to the lung.Expression of SiglecF and CD14 was confirmed by flow cytometry.Remarkably, we observed little change in expression of these markers in TGF-beta-treated cells, even up to 4 months in culture.When assessing the functional implications of these changes on the ability to phagocytose cSiO 2, we found no difference between control and TGF-beta-treated, recently differentiated MPI cells (cultured <1 month).However, when we assessed MPI cells cultured >1 month, we observed that TGF-beta-treated cells effectively phagocytosed cSiO 2 while control cells exhibited poor phagocytosis.CONCLUSION.Our results suggest that culturing MPI cells with TGF-beta allows these cells to represent ex vivo AMs for an extended period of time.Their high isolation yield and self-replicating capabilities make them an attractive and accessible AM model for large scale mechanistic studies.