Role of cytokines in alveolar macrophage differentiation.

Abstract

The effects of macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on rat alveolar macrophage (AM) differentiation in vitro were investigated in this study. Both M-CSF and GM-CSF triggered AM differentiation and multinucleated giant cell (MGC) formation. Morphological analysis further demonstrated that there were two distinct variants of MGC. Type 1 MGC normally contained 3 to 8 nuclei and appeared as a large round cell and type 2 MGC contained a higher number of nuclei (up to 30) and displayed irregular, elongated shapes. We also observed that a greater proportion of type 2 MGC expressed $\beta\sb3$ integrin, thus bringing additional evidence for differences between type 1 and type 2 MGC. Assessment of the relative proportion of type 1 and type 2 MGC indicated that M-CSF induced the formation of both types of MGC to a similar extent and GM-CSF induced predominantly the formation of type 2 MGC. Experiments with anti-M-CSF or anti-GM-CSF antibody to neutralize and cross-block the effects of M-CSF and GM-CSF further confirmed that M-CSF is associated with type 1 MGC formation whereas GM-CSF is responsible for type 2 MGC formation. Type 2 MGC seen in M-CSF treated groups may result from endogenous production of GM-CSF induced by M-CSF. This is supported by RT-PCR experiments in which M-CSF was shown to stimulate GM-CSF mRNA expression. Molecular phenotyping of a set of cytokines known to participate in inflammation and AM regulation was performed using RT-PCR at various times (up to 5 days) of AM differentiation. AM freshly obtained by BAL (0 time) did not show mRNA expression of TNF-$\alpha,$ IL-1$\alpha$ and IL-6, indicating these proinflammatory cytokines are not constitutively expressed by rat AM. Compared to the controls, both M-CSF and GM-CSF increased mRNA expression of TNF-$\alpha$ and IL-1$\alpha,$ suggesting these 2 cytokines are involved in M-CSF or GM-CSF induced AM differentiation. A significant increase of IL-6 mRNA expression was observed only in GM-CSF treated groups and the expression appeared early and persistently at all time points studied. Experiments with exogenous IL-6 and antibody to IL-6 receptor further indicated that IL-6 is involved in type 2 MGC formation. TGF-$\beta$ mRNA was constitutively expressed by rat AM and further enhanced by M-CSF and GM-CSF. Results from exogenous TGF-$\beta$ suggested that this cytokine favors formation of type 1 MGC over type 2 MGC. Cytoplasmic expression of TNF-$\alpha,$ PDGF and TGF-$\beta$ was investigated using immunocytochemical procedures. MGC were found to be able to express these cytokines, suggesting that MGC are a functional population rather than merely dead-end cells. Interestingly, type 1 MGC always showed higher levels of these cytokines than type 2 MGC, suggesting that type 1 MGC may be functionally more active than type 2 MGC.