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A modified procedure for the purification of the colony-stimulating factors (CSFs) in mouse L-cell-conditioned medium is used to isolate two forms of CSF, which are separable by reversed-phase high performance liquid chromatography with 300-A pore size supports. The specific biological activity of these CSFs (2 X 10(9) colonies/mg) was considerably higher than has been achieved by other methods. Even at high concentration (200 pM) both molecules stimulated predominantly more macrophage than granulocyte colonies; however, the less hydrophobic form appeared to stimulate the formation of more pure granulocytic colonies. Almost twice as much of the less hydrophobic CSF was recovered from L-cell-conditioned medium. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both forms of L-cell CSF had apparent molecular masses of approximately 70,000 daltons. However, on reduction with 2-mercaptoethanol, while both forms generated a 39,000-dalton subunit, the less hydrophobic form also yielded a 32,000-dalton subunit. Storage of either form of L-cell CSF at pH 2.1, in the presence of acetonitrile or isopropanol, destroyed the biological activity. Electrophoretic analysis of the L-cell CSFs stored under these conditions indicated that this was associated with a spontaneous dissociation of the CSF dimer into the inactive subunits. There was some charge heterogeneity (pI 3.5-4.7) indicating different degrees of glycosylation. The unique N-terminal amino acid sequences of both forms of CSF were the same: (Lys-Glu-Val-Ser-Glu-His-X-Ser-His-Met-Ile-Gly-Asn). Thus, the polypeptide chains appear to be identical for the subunits of both forms of L-cell CSF.

D-type cyclins are induced in response to mitogens and are essential and rate-limiting for G1 phase progression in normal mammalian cells. Macrophages proliferating in response to colony-stimulating factor-1 (CSF-1) express cyclin D1 and to a lesser extent cyclin D2 but not cyclin D3. Previously we showed that the macrophage-activating agent lipopolysaccharide (LPS) blocks CSF-1-induced proliferation and cyclin D1 expression in macrophages. Here we report upon the effect of LPS on expression of cyclin D2 in normal mouse bone marrow-derived macrophages (BMM). Unexpectedly we found that this anti-mitogen raised levels of CSF-1-stimulated cyclin D2 mRNA and protein. Furthermore, LPS alone induced cyclin D2 but not cyclin D1. Inhibition of the MEK/ERK (MAPK/ERK kinase/extracellular signal-regulated kinase) mitogen-activated protein kinase pathway repressed LPS-induced cyclin D2 mRNA, whereas inhibition of the p38 mitogen-activated protein kinase enhanced expression. However, in contrast to cyclin D1, cyclin D2 in bone marrow-derived macrophages did not appear to be regulated by protein kinase A pathways. The present data (a) show elevation of a D-type cyclin in the absence of proliferation, (b) demonstrate inverse regulation of two distinct D-type cyclins under identical conditions, and (c) suggest that cyclin D2 plays a role in macrophage activation by LPS.