We have shown previously that stimulation of human monocytes with antibodies to CD45 induces the production of M-CSF which is strongly augmented by IL-1 beta. To determine at what molecular level IL-1 augments expression we investigated the transcriptional rate and stability of the anti-CD45 induced M-CSF message in cells stimulated in te presence and absence of IL-1. Although the initial steady-state CSF mRNA levels in anti-CD45/IL-1 treated cells were higher compared to those in cells treated with anti-CD45 alone, IL-1 had no effect on the M-CSF message half-life. Nuclear run-on assays showed that IL-1 enhanced M-CSF transcript levels through transcriptional mechanisms. The IL-1 induced enhancement of M-CSF message levels depended on de novo protein synthesis and M-CSF secretion induced IL-1 could be blocked by soluble IL-1 receptors. Both, IL-4 and 10 strongly inhibited M-CSF production by human monocytes stimulated with anti-CD45/IL-1 which was accompanied by decreased M-CSF levels. Nuclear run-on assays demonstrated that IL-4 and IL-10 decreased M-CSF message levels by repressing M-CSF gene whereas M- CSF message half-life was not affected. These data have been published. To study the presence of putative transcriptional factors involved in anti-CD45 signal transduction we have subcloned the M-CSF promotor into the bluescript vector and have generated M-CSF promotor restriction fragments that will be used in gel shift assays using nuclear extracts of human monocytes stimulated with anti-CD45. In a related project we have studied the effect of HIV-1 infection on M-CSF production by human monocytes. We found that human monocytes infected with HIV-1 and maintained in the absence of exogenous cytokines produce endogenous M-CSF at levels substantially higher than uninfected cells. Enhanced production of this cytokine was dependent on the active replication of HIV-1 within the culture system. IL-1, IL-6 TNF alpha and GM-CSF were not induced. These data are currently prepared for publication. In a separate study we are investigating signal transduction through the c-fms receptor by stimulating monocytic cell lines with M-CSF followed by immunoprecipitation with anti-phosphotyrosine antibodies and immunoblotting. We have demonstrated that M-CSF stimulation results in phosphorylation of SHC and association of SHC with GRB-2. In addition we detect proteins with molecular weights of 95 kd and 116 kd that are phosphorylated on tyrosines residues. We are underway to identify these proteins and their relationship to fms.
