Nontransformed, GM-CSF-dependent macrophage lines are a unique model to study tissue macrophage functions

Abstract

Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF-induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.

Keywords: LPS recognition; innate immunity.

Fig. 1.

MPI cells are factor-dependent, self-renewing phagocytes. (A) Giemsa staining of MPI cells, AMs, BMMs, and BMDCs. (Scale bars, 20 μm.) (B) Phagocytosis of Alexa 647-stained P. acnes (Left, yellow) in MPI cells and in mock-treated cells (Right) using confocal microscopy. Blue, DAPI-stained nuclei. (Insets) Single P. acnes- and mock-treated cells at high power. (C) Growth curve of MPI cells with GM-CSF (30 ng/mL), M-CSF (30 ng/mL), or without any growth factor.

Fig. 2.

Gene expression and reactivity to TLR ligands in MPI cells, BMMs, and BMDCs. Global gene expression using total cellular RNA of the DC line SP37A3 (SPA), BMMs, and two independently made MPI cell lines (MPI-2 and MPI-3) with microarrays. (A) Cluster analysis for all, immune response, and cell-cycle genes. (B) TNF-α and IFN-αβ levels in supernatants of MPI cells and BMMs stimulated with 0.1 μg/mL S-LPS, 0.1 μg/mL FSL-1, 8 nM CpG ODN 1668, and 25 μg/mL poly I:C. (C) TNF-α levels of WT (MPI-wt), TLR4-deficient (MPI-ΔTLR4), and TLR4-deficient human TLR4-expressing (MPI-hTLR4) MPI cells in response to LPS (0.1 μg/mL) and nickel chloride (0.5 mM).

Fig. 3.

Differences between LPS-stimulated responses of MPI cells, AMs, and BMMs. (A) Heatmap of genes differentially induced by 0.1 μg/mL LPS in BMMs and MPI cells. (B) IL-10 levels in supernatants of BMMs, MPI cells, and AMs stimulated with 0.1 μg/mL LPS. (C) Pro- and mIL-1α and IL-1β levels in lysates of MPI cells and BMMs at various time points after stimulation with 0.1 μg/mL LPS. (D) IL-1α levels in supernatants of 0.1 μg/mL LPS-stimulated BMMs, MPI cells, and AMs (Left). Western blot analysis of IL-1α isoforms immunoprecipitated from MPI cell supernatants 12 h after stimulation with 0.1 μg/mL (Right). (E) Role of IL-1α in LPS-induced lung pathology. WT and IL-1α^−/− mice were treated with 10 μg LPS intranasally, and lungs were examined histologically 2 d later (H&E staining).

Fig. 4.

Unlike BMMs, MPI cells and AMs require LBP to sense R-LPS. TNF-α response of BMMs, MPI cells, and AMs to a range of S-LPS (A) and R-LPS (B) induced in the presence of 5% serum from WT or LBP^−/− mice (A and B).

Fig. 5.

Cytokine responses to heat-killed M. tuberculosis, TDM, and Ad in MPI cells, AMs, and BMMs. IL-6, IL-1α, and IL-10 levels in supernatants of BMMs, MPI cells, and AMs stimulated with M. tuberculosis at 20 bacterial particles per cell (A), with 25 μg/mL TDM (B), or with Ad5GFP at 100 pfu/cell (C).

Fig. 6.

STAT5 is important for the self-renewing capacity and innate reactivity of MPI cells. (A) Western blot analysis of STAT5, Erk1/2, and histone H1 in cytoplasmic (cy) and nuclear (nu) extracts of GM-CSF–starved and –incubated MPI cells and in GM-CSF–starved cells expressing caSTAT5. (B) Growth curves of GM-CSF–incubated and –starved MPI cells expressing CD4 (MPI-CD4; controls) or of GM-CSF–starved cells expressing caSTAT5 and CD4 (MPI-caSTAT5). (C) IL-6 levels of MPI cells and caSTAT5 MPI cells stimulated with a range of LPS in the presence of 5% serum from WT or LBP^−/− mice. (D) IL-6 responses of GM-CSF–incubated or –starved MPI cells and GM-CSF–starved caSTAT5-expressing MPI cells to 0.1 μg/mL LPS, 0.1 μg/mL FSL-1, 25 μg/mL cord factor (TDM), 100 pfu/cell Ad5 GFP (Ad), and 10 bacterial particles per cell of heat-killed M. tuberculosis.