Abstract
Stimulatory cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and steel factor (SLF), act in a synergistic manner to stimulate the growth of hematopoietic progenitor cells, an effect also demonstrated for the growth factor-dependent human hematopoietic cell line MO7e. While little is known about the mechanisms responsible for mediating synergistic interactions of cytokines, Raf-1, a component of the MAP kinase signaling pathway, is thought to play a role in the stimulatory response evoked by several cytokines, including SLF and GM-CSF. Interferon-inducible protein-10 (IP-10) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) are members of the chemokine family of suppressive cytokines. Prior exposure of hematopoietic cells to chemokines, including IP-10 and MIP-1 alpha, inhibits the synergistic action of growth factors on stimulating cell proliferation. We report that treatment of MO7e cells with the combination of GM-CSF and SLF directly stimulates statistically significant synergistic increases in the phosphorylation and activation of Raf-1 kinase, and in cellular protein synthesis levels. Pretreatment of MO7e cells with IP-10 or MIP-1 alpha blocked synergistic growth factor action, resulting in statistically significant suppression of cell proliferation, protein synthesis, and Raf-1 phosphorylation and activation. IP-10 and MIP-1 alpha treatment also evoked significant increases in intracellular cAMP levels. Pretreatment of cells with agents which serve to raise intracellular cAMP levels, or with cAMP analogs inhibited the synergistic actions of GM-CSF and SLF in a manner similar to IP-10 and MIP-1 alpha. In addition, treatment of cells with a potent inhibitor of cAMP-dependent protein kinase A blocked the suppressive action of MIP-1 alpha and IP-10 on Raf-1 kinase activity and on MO7e cell proliferation. The ability of IP-10 and MIP-1 alpha to antagonize the synergistic action of GM-CSF and SLF appears to involve inactivation of Raf-1 and the down-regulation of protein synthesis. Our findings suggest that both MIP-1 alpha and IP-10 mediate their suppressive effects in MO7e cells by stimulating increases in cellular cAMP levels and activating protein kinase A, a mechanism we believe to be unique to these chemokines and not one applied to all growth suppressive members of the chemokine superfamily (for example, interleukin 8 and platelet factor 4).
Highlights
The 2-3-fold difference between binding affinities for MIP-1α and MIP-1β may account for the ability of excess MIP-1β to block the suppressive action of MIP-1α, since we have shown that greater concentrations of MIP-1β are required in order to block MIP-1α suppressive action (18)
We have demonstrated that GM-CSF and SLF can stimulate increases in protein synthesis in MO7e cells
By making use of protein synthesis regulation as an assay system, we have shown that pretreatment of MO7e cells with the chemokines IP-10 and MIP-1α, and IL-8 and PF4 to a lesser extent, can block the stimulatory effects of GM-CSF and SLF
Summary
Materials — Cell culture medium was purchased from Biowhittaker (Walkersville, MD). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT). Factor-starved MO7e cells were resuspended at 5 X 106 cells/ml in PBS containing 0.5% bovine serum albumin. [3H]Leucine Incorporation — Factor-starved MO7e cells were resuspended at 106/ml in leucine-free RPMI supplemented with 5 μCi/ml [3H]leucine and 0.5% FBS. Cell debris was removed by centrifugation, and supernatants were transferred to fresh microcentrifuge tubes maintained on ice. A commercially available [3H]cAMP assay kit (Amersham Corp.) was used to measure cAMP content of samples, as per kit instructions. Immunoprecipitation — Factor-starved MO7e cells were washed, resuspended in phosphate-free RPMI 1640 medium containing 0.5% bovine serum albumin, and incubated for 1 h at 37 °C. Lysates were centrifuged to remove (McCoy's) or chemokines (50 ng/ml) prior to pulse treatment of MO7e insoluble particles, and aliquots were normalized for protein content cells -/+ high specific activity [3H]Tdr. Percent GM-CSF (100 units/ml) prior to immunoprecipitation. Tris-HCl, pH 7.6), once with low LiCl buffer (0.1 M LiCl, 100 mM Tris-HCl, pH 7.6), and twice with lysis buffer. 32P-Labeled immunoprecipitates were used directly in Raf-1 kinase activity assays or analyzed by SDS-PAGE and subsequent autoradiography and immu-
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