We have examined whether certain secreted factor(s) is involved in oxidized low density lipoprotein (Ox-LDL)-induced murine macrophage growth. An antibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) effectively inhibited Ox-LDL-induced macrophage growth by >80%. Ox-LDL as well as phospholipase A 2 -treated acetylated LDL enhanced mRNA levels and protein release of GM-CSF from macrophages, while neither acetylated LDL nor lysophosphatidylcholine (lyso-PC) showed such effects. The maximal induction of GM-CSF by Ox-LDL was noted at 4 h, followed by a time-dependent decrease to a basal level within 24 h. Ox-LDL-induced macrophage growth was inhibited by 75% by replacement of the culture medium at 24 h by a fresh medium containing the same concentration of Ox-LDL, when GM-CSF had already returned to the basal level. Thus, a cytokine(s) other than GM-CSF is also expected to participate in Ox-LDL-induced macrophage growth in a later phase. The Ox-LDL-induced GM-CSF release was inhibited by calphostin C, a protein kinase C inhibitor, and was significantly reduced in macrophages from the knockout mice lacking class A, type I and type II macrophage scavenger receptors (MSR-AI/AII). These results taken together indicate that effective endocytosis of lyso-PC of Ox-LDL by macrophages through MSR-AI/AII and subsequent protein kinase C activation have led to GM-CSF release into the medium which may play a priming role in conjunction with other cytokines in Ox-LDL-induced macrophage growth.

Recent studies demonstrated that oxidized LDL (Ox-LDL) induces macrophage growth in vitro. The present study was undertaken to elucidate the intracellular signaling pathways for macrophage growth. Ox-LDL initiated a rapid and transient rise in intracellular free calcium ion and induced activation of membrane protein kinase C (PKC). Pertussis toxin completely inhibited the Ox-LDL-induced rise in free calcium ion and significantly inhibited macrophage growth by 50%. Moreover, PKC inhibitors calphostin C and H-7 significantly inhibited Ox-LDL-induced macrophage growth by 80%. On the other hand, phospholipase A2-treated acetylated LDL did not induce a rise in calcium but significantly activated PKC and led to significant macrophage growth that was significantly inhibited by calphostin C by 90%. These results suggest the presence of two intracellular signaling pathways for activation of PKC, a rise in calcium that was mediated by pertussis toxin-sensitive G protein and the internalization of lysophosphatidylcholine through the scavenger receptors. These two pathways may play an important role in Ox-LDL-induced macrophage growth.

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Oxidized low density lipoprotein (Ox-LDL) can induce macrophage proliferation in vitro. To explore the mechanisms involved in this process, we reported that activation of protein kinase C (PKC) is involved in its signaling pathway (Matsumura, T., Sakai, M., Kobori, S., Biwa, T., Takemura, T., Matsuda, H., Hakamata, H., Horiuchi, S., and Shichiri, M. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 3013-3020) and that expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) and its subsequent release in the culture medium are important (Biwa, T., Hakamata, H., Sakai, M., Miyazaki, A., Suzuki, H., Kodama, T., Shichiri, M., and Horiuchi, S. (1998) J. Biol. Chem. 273, 28305-28313). However, a recent study also demonstrated the involvement of phosphatidylinositol 3-kinase (PI3K) in this process. In the present study, we investigated the role of PKC and PI3K in Ox-LDL-induced macrophage proliferation. Ox-LDLinduced macrophage proliferation was inhibited by 90% by a PKC inhibitor, calphostin C, and 50% by a PI3K inhibitor, wortmannin. Ox-LDL-induced expression of GM-CSF and its subsequent release were inhibited by calphostin C but not by wortmannin, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by wortmannin by 50% but not by calphostin C. Ox-LDL activated PI3K at two time points (10 min and 4 h), and the activation at the second but not first point was significantly inhibited by calphostin C and anti-GM-CSF antibody. Our results suggest that PKC plays a role upstream in the signaling pathway to GM-CSF induction, whereas PI3K is involved, at least in part, downstream in the signaling pathway after GM-CSF induction.

Abstract-Glucocorticoid, an anti-inflammatory agent, inhibits the development of atherosclerosis in various experimental animal models. This is partially explained by its ability to inhibit smooth muscle cell migration and proliferation in the intima and to reduce chemotaxis of circulating monocytes and leukocytes into the subendothelial spaces. We have recently demonstrated that oxidized LDL (Ox-LDL) has a mitogenic activity for macrophages in vitro in which Ox-LDL-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) production plays an important role. Proliferation of cellular components is one of the characteristic events in the development and progression of atherosclerotic lesions. In the present study, we investigated the effects of glucocorticoids on Ox-LDL-induced macrophage growth. Dexamethasone, prednisolone, and cortisol inhibited Ox-LDL-induced thymidine incorporation into macrophages by 85%, 70%, and 50%, respectively. Ox-LDL induced a significant production of GM-CSF by macrophages, which was effectively inhibited by dexamethasone, prednisolone, and cortisol by 80%, 65%, and 50%, respectively. Dexamethasone-mediated inhibition of Ox-LDL-induced GM-CSF mRNA expression and macrophage growth was significantly abrogated by RU-486, a glucocorticoid receptor antagonist. Our results suggest that the inhibitory effects of glucocorticoids on macrophage growth may be due to the inhibition of Ox-LDL-induced GM-CSF production through transactivation of the glucocorticoid receptor. (Arterioscler Thromb Vasc Biol.