Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction.

Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.

Role of type I interferons during macrophage activation by lipopolysaccharide.

Activation of macrophages by bacterial lipopolysaccharide (LPS) is accompanied by the secretion of type I interferons (IFNs) which can act in an autocrine manner. We examined the role of type I IFNs in macrophage responses to LPS using bone marrow-derived macrophages (BMM) from IFNAR1-/- mice, which lack a component of the type I IFN receptor and do not respond to type I IFNs. We found that, unlike wild-type (WT) BMM, LPS-treated IFNAR1-/- cells failed to produce nitric oxide (NO), or express inducible NO synthase (iNOS), indicating that type I IFNs are essential for all LPS-stimulated NO production in BMM. Exogenously added type II IFN (IFNgamma) rescued these responses in LPS-treated IFNAR1-/- BMM. In contrast to effects on NO, type I IFNs negatively regulated respiratory burst activity in LPS-primed BMM. We also found that while type I IFNs mediated the anti-proliferative effects of lower concentrations of LPS, at higher concentrations LPS acted in a type I IFNs-independent manner. Finally, we report that type I IFNs are a survival factor for BMM. Despite this, the ability of LPS to also prevent apoptosis in BMM was independent of type I IFNs. These findings highlight the diverse roles of type I IFNs in mediating LPS-stimulated macrophage responses.

Type I interferons mediate the lipopolysaccharide induction of macrophage cyclin D2.

Lipopolysaccharide (LPS) is a powerful macrophage-activating agent and antimitogen. We recently showed that LPS unexpectedly induces cyclin D2 in macrophages. Since LPS stimulates macrophages to produce autocrine-acting cytokines, we examined whether LPS induction of cyclin D2 was mediated by one such type of cytokine, type I interferons (IFN). We report that bone marrow-derived macrophages (BMM) lacking a component of the type I interferon receptor (IFNAR-1) do not express cyclin D2 mRNA or protein in response to LPS stimulation (0.01-1 microg/ml for 7-30 h). Consistent with this result, addition of anti-IFN-alpha/beta neutralizing antibodies reduced levels of LPS-stimulated cyclin D2 in normal BMM. Furthermore, IFN-alpha alone induced cyclin D2 mRNA and protein in normal BMM. Thus, we have identified a new role for type I IFN in macrophages, namely, as essential mediators of LPS-stimulated cyclin D2 expression.

Macrophages--proliferation, activation, and cell cycle proteins.

Our understanding of mammalian cell proliferation has increased enormously over the past decade. A major advance has been identification and characterization of cyclins and their catalytic partners, cyclin-dependent kinases (cdks). The following brief review highlights the role of macrophages as a cell model for many of the major advances in this field. Macrophages were central to the identification of D-type cyclins and cdk4. In addition, it appears the first work showing that cell cycle proteins are the targets for anti-proliferative agents was performed in macrophages. In these latter studies, and a number of subsequent studies in other cell types, it was shown that many antimitogenic agents repressed cyclin and/or cdk expression. However, recent work in this laboratory suggests macrophage D-type cyclins may also be involved in processes other than proliferation. We have unexpectedly found that macrophages treated with lipopolysaccharide (LPS) express high levels of cyclin D2, even though LPS simultaneously represses cyclin D1 levels and potently blocks proliferation. These data, and those showing the yeast extract Zymosan A also raises cyclin D2 levels, suggest cyclin D2 plays a role in macrophage activation.

Proliferation-independent induction of macrophage cyclin D2, and repression of cyclin D1, by lipopolysaccharide.

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.

Effects of wortmannin and rapamycin on CSF-1-mediated responses in macrophages.

There are differing views regarding the roles of phosphatidylinositol 3-kinases (PI3-kinases) and p70 S6 kinase (p70s6k) in growth factor-induced cellular responses. One approach that is widely employed to investigate these roles is to use the inhibitors, wortmannin and rapamycin, respectively. This approach is used here to study the responses in macrophages to colony stimulating factor-1 (CSF-1). Wortmannin (> or = 30 nM) and rapamycin (> or = 3 nM) both weakly inhibited CSF-1-stimulated DNA synthesis in murine bone marrow-derived macrophages (BMM), suggesting that there are PI3-kinase- and p70s6k-independent pathways required for the onset of S phase; interestingly the combination of the drugs gave dramatic suppression. Inhibition of DNA synthesis by rapamycin on the BMM was much less than that observed with the CSF-1-dependent cell line, BAC1.2F5. In BMM, wortmannin suppressed CSF-1-stimulated increase in p70s6k activity indicating that PI3-kinase activity may lie upstream. In contrast to some other growth factor/cell systems, no evidence was obtained using the inhibitors for the involvement of PI3-kinase or p70s6k in CSF-1-mediated induction of c-fos mRNA expression or Erk-1 activity; in addition, no evidence was found for an involvement in the CSF-1-mediated increase in cyclin D1 expression or STAT activation. The findings reinforce the need to study the signal transduction cascades relevant to each individual growth factor and preferably not in cell lines.

G1 phase arrest of human smooth muscle cells by heparin, IL-4 and cAMP is linked to repression of cyclin D1 and cdk2.

Smooth muscle cell proliferation is a key event in the development of atherosclerosis. Inhibition of this proliferation may lead to better prevention and treatment of the disease. While a number of agents have been found to inhibit SMC proliferation, their mechanisms of action are not fully understood. We wanted to determine the effects of three physiologically relevant anti-mitogenic agents on two classes of proteins which have major roles in cellular proliferation, namely cyclins and cyclin-dependent kinases (cdks). Following stimulation with fetal calf serum (FCS), quiescent human umbilical artery smooth muscle cells (HUASMC) synthesised cyclin D1 mRNA and protein and cdk2 mRNA in the G1 phase, whereas cdc2 protein was expressed after the onset of the S phase. Heparin, a strong inhibitor of HUASMC proliferation, strongly down-modulated the levels of cyclin D1 mRNA and protein, cdk2 mRNA and cdc2 protein. Interleukin-4 (IL-4) or 8-bromo-adenosine 3',5'-cyclic monophosphate (cAMP) also lowered the levels of these cell cycle regulatory proteins, although their effects were relatively weak, reflecting their only partial inhibition of HUASMC DNA synthesis. There was specificity in the cell cycle targets of the agents since none appeared to affect the levels of cdk4 protein.

Differential regulation of cell cycle machinery by various antiproliferative agents is linked to macrophage arrest at distinct G1 checkpoints.

There is currently much interest in the mechanisms of action of antiproliferative agents and their effects on cell cycle machinery. In the present study we examined the mechanisms of action of four unrelated agents known to inhibit proliferation of CSF-1-stimulated bone marrow-derived macrophages (BMM). We report that 8-bromo-cAMP (8Br-cAMP) and lipopolysaccharide (LPS) potently reduced CSF-1-stimulated cyclin D1 protein, and cyclin-dependent kinase (cdk) 4 mRNA and protein levels, while the inhibitory effects of the Na+/ H+ antiport inhibitor 5-(N',N'-dimethyl) amiloride (DMA) and interferon gamma (IFN gamma ) were only weak. All agents repressed CSF-1-stimulated retinoblastoma protein phosphorylation. Furthermore, 8Br-cAMP and to a lesser extent IFN gamma, also reduced CSF-1-stimulated levels of E2F DNA binding activity in a macrophage cell line, BAC1.2F5. An explanation for the different effects of the agents is that 8Br-cAMP and LPS were found to arrest BMM in early/mid-G1, while IFN gamma and DMA arrested cells in late G1 or early S phase. These data indicate that (1) different antiproliferative agents can arrest the same cell type at distinct checkpoints in G1 and (2) effects of antiproliferative agents on cell cycle machinery is linked to the position at which they arrest cells in G1.

Deregulated c-myc expression overrides IFN gamma-induced macrophage growth arrest.

Induction of c-myc gene expression is an essential response to growth promoting agents, including colony-stimulating factor 1 (CSF-1). Down regulation of c-myc expression occurs in response to a variety of negative growth regulators in many cell types. However, for many of these systems the causal link between c-myc down regulation and growth arrest remains to be established. Here we show for CSF-1-dependent BAC1.2F5 mouse macrophages that interferon-gamma (IFN gamma) results in a midlate G1 phase decrease of CSF-1-dependent c-myc mRNA and subsequent cell cycle arrest. Introduction of a deregulated c-myc gene into these cells, which prevents the IFN gamma-mediated decrease in c-myc expression, overrides the cell cycle arrest and restores CSF-1-dependent growth in the presence of the cytokine. This result contrasts with the macrophage growth arrest induced by cAMP elevation, which also suppresses c-myc expression, but is not overcome by a deregulated c-myc gene. These results show that inhibition of c-myc expression is an essential component in IFN gamma-mediated cell cycle arrest and demonstrates that distinct mechanisms contribute to IFN gamma- and cAMP-mediated growth arrest in macrophages.

Interleukin-4 inhibits human smooth muscle cell proliferation.

Although proliferation of smooth muscle cells is a key event in the pathogenesis of atherosclerosis, the signals which regulate this proliferation are not fully understood. It is likely that proliferation is regulated by cytokines released by cells found in the plaque, such as T cells. In this study we report that the T cell-derived cytokine, interleukin-4 (IL-4), can inhibit proliferation of cultured human umbilical artery smooth muscle cells. Maximum inhibitory effect was achieved at IL-4 concentrations of 20 U/ml or greater. In addition, the data showed that IL-4 acted early in the G1 phase of the cell cycle, thereby preventing cells from entering S phase. The mechanism of IL-4 inhibition did not appear to involve stimulation of prostanoid synthesis since similar data were obtained when experiments were performed in the presence of a cyclooxygenase inhibitor. We propose that IL-4 may act as a protective factor released by T-cells in an atherosclerotic lesion in order to minimise the size of the plaque.

Interaction of apolipoprotein AII with the putative high-density lipoprotein receptor.

There is strong evidence to indicate that binding of HDL by cells is due to recognition of apoproteins residing on the surface of the lipoprotein by the putative HDL receptor(s). Although both of the major HDL apoproteins, AI and AII, are recognized by the putative receptor, the nature of the binding interaction and the domains of the apoproteins involved are largely unknown. Previous data from this laboratory led to the proposal of a model to explain how HDL particles containing AII interacted with the HDL receptor in a different manner as compared to HDL particles which contain apoAI but not apoAII [Vadiveloo, P. K., & Fidge, N. H. (1992) Biochem. J. 284, 145-151]. The model predicted that each chain of the apoAII homodimer contained a binding domain capable of interacting with the HDL receptor. This model was tested in the current study by preparing apoAII monomers, complexing them with phospholipid, and determining the ability of these complexes to bind to putative HDL receptors in rat liver plasma membranes (RLPM) and bovine aortic endothelial cell membranes (BAECM) by ligand blotting. The data showed that these complexes were bound by HB1 and HB2 from RLPM, and to the 110-kDa HDL binding protein from BAECM, providing critical evidence to support the model. Further investigation into the binding interaction revealed that apoAII complexed with phospholipid (apoAII-PC) bound more than delipidated apoAII, which bound more than delipidated apoAII monomers. Thus, optimum binding required the presence of lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of apoproteins AI and AII in binding of high-density lipoprotein3 to membranes derived from bovine aortic endothelial cells.

Although binding of high-density lipoproteins (HDL) to a variety of cells in culture has been widely reported, the mechanism of this binding has yet to be fully elucidated. The aim of the current studies was to explore the roles of apoproteins (apo) AI and AII in HDL3 binding to membranes derived from bovine aortic endothelial cells. Binding studies showed that HDL3 (which contains both apo AI and apo AII) and AII-HDL3 (which contain only apo AII) bound to membranes with similar affinity (44 +/- 6 and 41 +/- 9 micrograms/ml respectively) and capacity (673 +/- 97 and 969 +/- 101 ng bound/mg of membrane protein respectively). In contrast with these results, HDL3 [AI w/o AII] (which contain apo AI, but not apo AII) bound to the membranes with a significantly higher capacity (2228 +/- 206 ng bound/mg of membrane protein) and lower affinity (65 +/- 3 micrograms/ml) as compared with HDL3 or AII-HDL3. Therefore, although both apo AI and apo AII appear capable of facilitating HDL3 binding, the mechanisms involved probably differ. A model which fits the data postulates that a common receptor exists which binds both apo AI and apo AII, and that a particle containing AII can occupy up to four receptors (partly owing to each AII molecule containing two binding domains), whereas an HDL3 [AI w/o AII] particle can occupy only one.

Studies on the interaction between apolipoprotein A-II-enriched HDL3 and cultured bovine aortic endothelial (BAE) cells.

The specific binding of high-density lipoproteins (HDL) to a number of cell and membrane types has been reported. The aim of this study was to investigate the ligand specificity of HDL binding sites on bovine aortic endothelial (BAE) cells and in particular to investigate the role of apo A-II in the interaction. In order to do this we prepared AII-HDL3 particles by incubating HDL3 with apo HDL. These particles were enriched in apo A-II, contained virtually no apo A-I, and were similar to HDL3 in terms of size and lipid composition. As these particles resemble the native HDL3 structure we believe they are probably a more suitable model for investigation of ligand specificity than artificial recombinants. AII-HDL3 particles were shown to bind to cells with similar affinity and capacity as HDL3. Further experiments indicated that HDL3 and AII-HDL3 competed with each other for binding and displayed similar affinities for a common binding site(s). The results suggest that apo A-II, as well as apo A-I, play an important role in the process of HDL recognition by putative HDL receptors on endothelial cells.