M-CSF and GM-CSF promote alveolar macrophage differentiation into multinucleated giant cells with distinct phenotypes

Multinucleated giant cells (MGC) are a hallmark of granulomatous reactions but the mechanisms that regulate their formation are unknown. To address this issue, we cultured resident alveolar macrophages (AM) from rat lung and examined the effects of defined cytokines on AM differentiation and MGC formation. The presence of MGC was found after 3 days in culture with maximal numbers obtained at 7 days and thereafter (up to 21 days). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (25-75 U/mL) stimulated the formation of MGC (up to 4-fold), whereas interleukin (IL) -3, IL-10, and interferon-gamma (IFN-gamma) had no stimulatory effect. Interestingly, MGC with distinct phenotypes were observed in AM cultures: (1) spherical MGC with 3-16 nuclei, dense cytoplasm, and lower expression of beta3 integrin (Type 1) and (2) irregular MGC with 3-30 nuclei, thin and vacuolated cytoplasm, and higher expression of beta3 integrin (Type 2). Furthermore, the actions of M-CSF and GM-CSF on AM were found to be different. GM-CSF promoted, in AM cultures, the appearance of an elongated fibroblastoid phenotype and stimulated mostly the formation of Type 2 MGC. In contrast, M-CSF did not cause significant change in the general morphology of regular AM but stimulated the appearance of both Type 1 and Type 2 MGC. Reverse transcriptase-polymerase chain reaction analysis demonstrated that, under these conditions, M-CSF induced GM-CSF gene expression in AM. In addition, neutralizing antibodies against M-CSF selectively decreased the formation of Type 1 MGC, whereas neutralizing anti-GM-CSF inhibited Type 2 formation. These data suggest that M-CSF promotes AM differentiation into Type 1 MGC, whereas GM-CSF stimulates the formation of Type 2 and that M-CSF and GM-CSF may selectively regulate in an autocrine fashion AM differentiation into distinct MGC.