The present report describes studies on: a) control mechanisms of GM-CSF production by EL-4 thymomoa T cells as compared to that of other lymphokines, and b) the regulation of differentiation of myeloid cells by G-CSF. EL-4 cells when stimulated by mitogens produce several lymphokines like IL-2, IL-3 and GM-CSF. Cyclosporin A (CsA) inhibits the production of IL-2 and IL-3 but not that of GM-CSF. Supernatants from mitogen stimulated EL-4 cells were fractionated by anion exchange chromatography and in the absence of CsA two peaks of activity, representing GM-CSF and IL-3 were identified. In contrast, only a single peak of activity identified as GM-CSF was detected in the presence of CsA. In additional experiments, Northern blots of poly(A+)RNA isolated from mitogen stimulated EL-4 cells in the presence and absence of CsA were hybridized with GM-CSF and IL-2 cDNA probes. Expression of the GM-CSF gene was detected independent of CsA while the expression of the IL-2 gene was inhibited by CsA. These data suggest a different control mechanism for GM-CSF production than that for IL-2 and IL-3.G-CSF induces differentiation of M1 murine myeloid leukemia cells into mature granulocytes and macrophages and also causes an accumulation of the cells in the G1 phase of the cell cycle. We examined therefore, whether synchronization of M1 cells in G1 could have an effect on G-CSF-induced differentiation as quantitated by expression of Fc receptors (FcR) and lysozyme activity. Cells were arrested in early G1 by density inhibition in the absence of serum and in late G1 by aphidicolin. Cells synchronized in early G1, when stimulated with G-CSF, showed an enhanced expression of FcR and lysozyme activity. Eighty percent of the cells expressed FcR 18 hours after addition of G-CSF, while in exponentially growing cells this percentage was reached 72 hours after addition of G-CSF. Cells synchronized in late G1 did not show enhanced expression of differentiation markers. These results imply that with respect to G-CSF induced differentiation, G1 phase can be dissected into an early permissive and a later non-permissive stage.
