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Granulocyte colony-stimulating factor (G-CSF) was quantitated in the supernatants of lipopolysaccharide (LPS)-treated human monocytes by ELISA. Unlike previous reports, the lymphokines, interleukin-4 (IL-4) and interferon-γ (IFN-γ), were unable to induce the synthesis of G-CSF. Both IL-4 (⩾ 10 pM) and the glucocorticoid, dexamethasone (10-7 M), inhibited G-CSF production in the LPS-treated monocytes; in contrast, IFN-γ had a weak potentiating effect on the LPS action. Changes in antigen expression were manifested at the level of messenger RNA (mRNA). Granulocyte-macrophage (GM)-CSF in the LPS-treated monocyte supernatants was also quantitated by ELISA but its levels were somewhat lower than for G-CSF; IL-4, dexamethasone and IFN-γ had similar effects on GM-CSF levels as on G-CSF levels. The suppression of CSF production in the stimulated monocytes by IL-4 and glucocorticoid extends the list of monocyte cytokines whose levels can be down-regulated by these agents and suggests another potential anti-inflammatory and immunosuppressive function for IL-4. [ABSTRACT FROM AUTHOR]

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.

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.

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.

Using a specific enzyme-linked immunosorbent assay, plasminogen activator inhibitor-2 (PAI-2) was quantitated in cultures of human monocytes. Lipopolysaccharide (LPS) increased both extracellular and cell-associated PAI-2 levels, as well as PAI-2 mRNA measured by Northern analysis. Both the lymphokine, interleukin-4 (IL-4) (greater than or equal to 10 pmol/L), and the glucocorticoid, dexamethasone (100 nmol/L), inhibited PAI-2 formation and PAI-2 mRNA induction. Another lymphokine, interferon-gamma (IFN-gamma) (100 U/mL), as for IL-4 alone, did not stimulate PAI-2 formation; however, in contrast to IL-4, IFN-gamma did not reverse the LPS effect but could potentiate it. The suppression of PAI-2 formation by IL-4 and glucocorticoid in stimulated human monocytes extends the list of monocyte products whose synthesis can be downregulated in these cells by the two agents. The findings could have relevance to the control by monocytes/macrophages of connective tissue resorption, including that of fibrin, at sites of inflammation.

Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor-1 (CSF-1)-induced, murine bone marrow-derived macrophage (BMM) DNA synthesis even when added 8-12 h after the growth factor, a period coinciding with the G1/S-phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF-1 were examined. All three agents stimulated BMM protein synthesis; TNF alpha and LPS, but not IFN gamma, stimulated BMM Na+/H+ exchange and Na+,K(+)-ATPase activities, as well as c-fos mRNA levels. IFN gamma did not inhibit the CSF-1-induced Na+,K(+)-ATPase activity. TNF alpha and LPS inhibited both CSF-1-stimulated urokinase-type plasminogen activator (u-PA) mRNA levels and u-PA activity in BMM, whereas IFN gamma lowered only the u-PA activity. In contrast, LPS and IFN gamma, but not TNF alpha, inhibited CSF-1-induced BMM c-myc mRNA levels, the lack of effect of TNF alpha dissociating the inhibition of DNA synthesis and decreased c-myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNF alpha, IFN gamma, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge.

Transforming growth factor beta (TGF-beta) is usually associated with matrix formation and tissue repair; in contrast, cellular expression of the serine proteinase, urokinase-type plasminogen activator (u-PA) is often correlated with tissue remodeling, as well as with cell migration and transformation. We report here that purified recombinant human TGF-beta (greater than or equal to 300 pg/ml) can stimulate rapidly (within 2 h) the u-PA activity of nonrheumatoid synovial fibroblast-like cells. As for interleukin 1 (IL-1), u-PA mRNA levels are raised in response to TGF-beta, but unlike IL-1, no increase in prostaglandin E2 levels occurs. In contrast to a number of other examples in the literature, in which these two cytokines have opposing actions, TGF-beta can potentiate the action of optimal concentrations of IL-1 in enhancing u-PA expression. These effects of TGF-beta are similar to those of all-trans-retinoic acid. In addition, synovial fibroblast DNA synthesis was stimulated by TGF-beta. Because TGF-beta has been detected in the synovia of patients with rheumatoid arthritis and has been shown to reduce the collagenase levels and proliferation of synovial fibroblast-like cells, it has been proposed by others to be involved beneficially in the reparative processes occurring in arthritic lesions. However, on the basis of our findings, we propose alternative functions for this cytokine--namely, roles in the destructive events as well as in the synovial hyperplasia observed in rheumatoid joints.