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Macrophage colony-stimulating factor (M-CSF or CSF-1) and granulocyte-macrophage CSF (GM-CSF) have been shown to increase human monocyte urokinase-type plasminogen-activator (u-PA) activity with possible consequences for cell migration and tissue remodeling; because monocyte u-PA activity is likely to be controlled in part also by the PA inhibitors (PAIs) made by the cell, the effect of M-CSF and GM-CSF on human monocyte PAI-2 and PAI-1 synthesis was investigated. To this end, elutriation-purified human monocytes were treated in vitro with purified recombinant human M-CSF and GM-CSF, and PAI-2 and PAI-1 antigen and mRNA levels measured by specific enzyme-linked immunosorbent assays and Northern blot, respectively. Each CSF could enhance the protein and mRNA levels of PAI-2 and PAI-1 at similar concentrations for each product. This similar regulation of monocyte PAI expression in response to the CSFs contrasted with that found for the effects of lipopolysaccharide, transforming growth factor-beta and a glucocorticoid. Therefore, PAIs may be modulating the effects of the CSFs on monocyte u-PA activity at sites of inflammation and tissue remodeling.

The regulation of PAI-1 synthesis by elutriation-purified human monocytes was studied in vitro and compared to that for PAI-2. PAI-1 formation, as measured by ELISA, was upregulated by TGF-beta (> or = 1 ng/ml) and surprisingly down-regulated by LPS (100 ng/ml), particularly in the presence of TGF-beta; LPS elevated PAI-2 levels (ELISA) while TGF-beta reduced its basal levels and those in LPS-treated cultures. Concomitant changes in mRNA expression occurred. The glucocorticoid dexamethasone (10(-7) M) elevated PAI-1 and acted in concert with TGF-beta in this regard at both the antigen and mRNA levels; interleukin-4 (IL-4) (250 pM) failed to mimic the steroid in its regulation of PAI-1 formation. Since monocyte/macrophage PA activity is likely to be important in tissue remodeling and cell migration at sites of inflammation and in fibrinolysis, it is proposed from these studies that PAI-1, as well as the usually considered PAI-2, may be involved in the negative control of PA activity in this cell type. The synthesis of each PAI appears to be independently regulated.

It is often assumed that macrophage-colony stimulating factor (M-CSF) or CSF-1, as well as granulocyte macrophage-CSF (GM-CSF), can induce inflammatory mediator production by monocytes/macrophages. We demonstrate with elutriation-purified human monocytes that, in contrast to lipopolysaccharide, recombinant human CSF-1 does not induce secretion of prostaglandin E2, interleukin-6 (IL-6), IL-1 beta, or tumor necrosis factor alpha, as measured by immunoassay; however, increased urokinase-type plasminogen activator (u-PA) activity in cell lysates and mRNA was observed. Similar results were obtained when the monocytes were treated with recombinant human GM-CSF. Such increased u-PA expression may contribute to the function of CSF-1 at sites of inflammation.

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-gamma (IFN-gamma), were unable to induce the synthesis of G-CSF. Both IL-4 (> or = 10 pM) and the glucocorticoid, dexamethasone (10(-7) M), inhibited G-CSF production in the LPS-treated monocytes; in contrast, IFN-gamma 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-gamma 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.

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.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) raised the plasminogen activator (PA) activity of cultured human monocytes. This activity was characterized to be urokinase-PA (u-PA) by incubation with specific IgG and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Increased u-PA activity reflected GM-CSF-induction of u-PA mRNA levels. The stimulatory properties of GM-CSF for monocyte PA activity differed from those of interleukin-4, which induced monocyte tissue-type PA (t-PA) activity, and of interferon-gamma (IFN-gamma), which alone was not stimulatory but augmented lipopolysaccharide-induced t-PA activity. GM-CSF alone did not stimulate detectable monocyte t-PA activity but combined with IFN-gamma to promote this activity. Plasmin formation arising from GM-CSF-induced u-PA in monocytes may contribute to the matrix turnover involved in, eg, cell migration and inflammation, and may explain some of the pathology seen in GM-CSF transgenic mice.

Urokinase-type plasminogen activator (u-PA), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) activities were measured for highly purified human monocytes cultured for 18 hr with recombinant human interleukin-3 (IL-3). IL-3 alone stimulated monocyte u-PA activity, but not TNF-alpha or IL-1 activity. However, IL-3, together with interferon-gamma (IFN-gamma), stimulated the TNF-alpha, but not IL-1, activities of monocytes from several donors. In parallel cultures, granulocyte-macrophage colony-stimulating factor (GM-CSF) behaved similarly. IL-3, like GM-CSF, synergized weakly and sometimes irregularly with lipopolysaccharide (LPS) for increased TNF-alpha and IL-1 activities. Thus, IL-3 can selectively stimulate monocyte mediator production depending on the costimulus present; however, the stimulatory properties of IL-3 vary from those of IFN-gamma and IL-4. The similarities in activity between IL-3 and GM-CSF may be explained by a common or associated IL-3/GM-CSF receptor(s), as suggested by biochemical studies.

Stimulated human monocytes/macrophages are a source of interleukin-6 (IL-6), which is a likely mediator involved in immune and inflammatory reactions. The means to control production of IL-6 by these cells could therefore have therapeutic applications. We report here, for lipopolysaccharide (LPS)-stimulated human monocytes in vitro, that the lymphokine, interferon-gamma (IFN-gamma) (100 U/ml), enhanced the level of IL-6 activity, whereas another lymphokine, interleukin-4 (IL-4) (greater than or equal to 0.1 U/ml; greater than or equal to 1.2 x 10(-11) M), suppressed it. The effects of the two lymphokines were manifested at the level of mRNA. The action of the IL-4 was similar to that of the glucocorticoid, dexamethasone, but observed at a lower molar concentration. Such regulation of monocyte IL-6 activity is similar to that found previously for interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) synthesis.

For their anti-inflammatory effects, glucocorticoids act, at least in part, by suppression of the production of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and prostaglandin E2 (PGE2) by activated monocytes/macrophages. Interleukin-4 (IL-4) also suppresses similar parameters of monocyte activation in vitro. However, contrasting effects of IL-4 and dexamethasone (Dex) on monocyte tissue-type plasminogen activator (t-PA) production suggest that these agents may operate by different pathways. We have now demonstrated that levels of IL-4 as low as 0.05-0.1 U/ml (0.6-1.2 x 10(-11)M) can augment the actions of Dex (5 x 10(-9)M) as an inhibitor of the production of monocyte pro-inflammatory mediators. These in vitro results suggest the possible supplementation of steroid therapy with low amounts of IL-4 (or an agonist) permitting the use of less steroid with concomitant reduction in steroid-associated side-effects. IL-4 can also suppress the increased release of IL-1 beta and TNF alpha by monocytes incubated with indomethacin, a non-steroidal anti-inflammatory drug.

TNF-alpha and IL-1 activities and PGE2 levels were investigated in the supernatants of highly purified human monocytes cultured for 18 h with recombinant human granulocyte-macrophage CSF (GM-CSF). GM-CSF alone did not stimulate IL-1 or TNF-alpha activities or the production of PGE2. GM-CSF with IFN-gamma, but not with LPS, consistently activated the monocytes for TNF-alpha activity. In contrast, for increased IL-1 activity, GM-CSF synergized weakly and irregularly with LPS, but not at all with IFN-gamma. For the third monocyte product investigated, GM-CSF was a weak and inconsistent inducer of PGE2 and only in the co-presence of IFN-gamma. Thus, GM-CSF can elicit different responses in human monocytes depending both on the co-stimulus as well as the monocyte product being investigated.

Stimulated human monocytes/macrophages are a source of mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and prostaglandin E2 (PGE2), which can modulate inflammatory and immune reactions. Therefore, the ability to control the production of such mediators by monocytes/macrophages may have therapeutic benefits, and it has been proposed that glucocorticoids may act in this way. Purified human monocytes, when stimulated in vitro with lipopolysaccharide (LPS) or with LPS and gamma interferon (IFN-gamma), produce TNF-alpha, IL-1, and PGE2. Cotreatment of stimulated cells with the purified human lymphokine, interleukin 4 (IL-4 greater than or equal to 0.1-0.5 unit/ml; 12-60 pM) dramatically blocked the increased levels of these three mediators; for TNF-alpha and IL-1, the inhibition was manifest at the level of mRNA. Thus, IL-4 can suppress some parameters of monocyte activation and, as for B cells, have opposite effects to IFN-gamma. The effects of IL-4 on human monocytes are similar to those obtained with the glucocorticoid dexamethasone (0.1 microM).

There have been suggestions that the production of pro-inflammatory mediators by human monocytes in response to interferon-gamma (IFN-gamma) may be controlled by changes in prostaglandins. Therefore we investigated tumour necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) activities and prostaglandin E2 (PGE2) levels in the supernatants of highly purified human monocytes cultured for 18 hr with recombinant human IFN-gamma. IFN-gamma (100 U/ml) did not stimulate monocytes isolated by counter-current centrifugal elutriation for detectable TNF alpha or IL-1 activities, or PGE2 production. However, IFN-gamma synergistically enhanced lipopolysaccharide (LPS)-induced TNF alpha and IL-1 activities. In contrast, there was no consistent change in PGE2 levels upon addition of IFN-gamma to LPS-treated monocyte cultures. The TNF alpha and IL-1 activities induced by LPS and by LPS with IFN-gamma were reduced by PGE2, and stimulated by indomethacin. As reported previously for IL-1 activities, the regulation by cyclo-oxygenase products of TNF alpha activities reflected predominantly a control of the production of immunoreactive TNF alpha, rather than the measurement of TNF alpha bio-activity. However, the addition of indomethacin or PGE2 to monocyte cultures did not change the extent of IFN-gamma synergy with LPS for increased TNF alpha and IL-1 activities. The results of this study suggest that, despite control by cyclo-oxygenase products of TNF alpha and IL-1 production in human monocytes, IFN-gamma may enhance TNF alpha and IL-1 activities independently of this regulatory mechanism. These findings are contrary to those suggested for the regulation by prostanoids of IL-1 production by murine macrophages.

There have been suggestions that the production of pro-inflammatory mediators by human monocytes in response to interferon-gamma (IFN-γ) may be controlled by changes in prostaglandins. Therefore we investigated tumour necrosis factor a (TNFα) and interleukin-1 (IL-1) activities and prostaglandin E2 (PGE2) levels in the supernatants of highly purified human monocytes cultured for 18 hr with recombinant human IFN-γ. IFN-γ (100 U/ml) did not stimulate monocytes isolated by counter-current centrifugal elutriation for detectable TNFα or IL-1 activities, or PGE2 production. However, IFN-γ synergistically enhanced lipopolysaccharide (LPS)-induced TNFα and IL-1 activities. In contrast, there was no consistent change in PGE2 levels upon addition of IFN-γ to LPS-treated monocyte cultures. The TNFα and IL-1 activities induced by LPS and by LPS with IFN-γ were reduced by PGE2, and stimulated by indomethacin. As reported previously for IL-1 activities, the regulation by cyclo-oxygenase products of TNFα activities reflected predominantly a control of the production of immunoreactive TNFα, rather than the measurement of TNFα bio-activity. However, the addition of indomethacin or PGE2 to monocyte cultures did not change the extent of IFN-γ synergy with LPS for increased TNFα and IL-1 activites. The results of this study suggest that, despite control by cyclo-oxygenase products of TNFα and IL-1 production in human monocytes, IFN-γ may enhance TNFα and IL-1 activities independently of this regulatory mechanism. These findings are contrary to those suggested for the regulation by prostanoids of IL-1 production by murine macrophages. [ABSTRACT FROM AUTHOR]