Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in monocytes in vitro via granulocyte macrophage colony-stimulating factor and interleukin 3.

The aim of this study was to examine the role of lymphocytes in regulating expression of the 5-lipoxygenase pathway in monocytes. When monocytes were cultured over a period of days with lymphocytes, calcium ionophore-stimulated 5-lipoxygenase activity was enhanced. If lymphocytes alone were activated with lectins and their supernatants added to monocytes, stimulated 5-lipoxygenase activity was increased, whereas supernatants from lymphocytes cultured without lectins had no effect. Increased immunoreactive protein and mRNA for 5-lipoxygenase and 5-lipoxygenase activating protein were present in cells conditioned with lectin-activated lymphocyte supernatants. The effect of activated-lymphocyte supernatants could be mimicked by either GM-CSF or IL-3, but there was no additive effect with both cytokines. Both GM-CSF and IL-3 were present in the supernatant from lectin-activated lymphocytes at concentrations above their ED50, but were undetectable in the supernatant from nonactivated lymphocytes. The effect of lectin-activated lymphocyte supernatant could be inhibited by neutralizing antibodies to both cytokines, but not to either cytokine alone. We conclude that lymphocytes can regulate the expression of 5-lipoxygenase in monocytes, over a period of days, via the release of soluble factors, primarily GM-CSF and IL-3.

Dexamethasone increases expression of 5-lipoxygenase and its activating protein in human monocytes and THP-1 cells.

The aim of this study was to assess the effect of dexamethasone on 5-lipoxygenase pathway expression in human peripheral blood monocytes and the acute monocytic leukemia cell line, THP-1. Cells were conditioned over a period of days with dexamethasone, at concentrations relevant in vivo, to study the effect of the glucocorticoid on calcium-ionophore-stimulated 5-lipoxygenase product and arachidonic acid release. The effect of dexamethasone on levels of immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and its activating protein (5-LAP) was also assessed. Dexamethasone increased the stimulated release of 5-lipoxygenase products from both monocytes and THP-1 cells in a dose-dependent fashion. The increase in product generation was not due to changes in the availability of arachidonic acid. However, immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP were increased by conditioning with dexamethasone. There was no apparent effect of the glucocorticoid on LTA4-hydrolase-immunoreactive protein levels or specific activity. We conclude that dexamethasone increases 5-lipoxygenase pathway expression in both monocytes and in THP-1 cells. This effect is due, at least in part, to increases in immunoreactive protein and steady-state messenger RNA encoding for 5-lipoxygenase and 5-LAP. These results suggest a role for glucocorticoids in the regulation of 5-lipoxygenase pathway expression in mononuclear phagocytes.

Human monocytes lose 5-lipoxygenase and FLAP as they mature into monocyte-derived macrophages in vitro.

Previous studies in mononuclear phagocytes have shown that macrophages have substantially greater 5-lipoxygenase activity than monocytes and that this is associated with greater amounts of 5-lipoxygenase and its activating protein (FLAP). The aim of this study was to examine the effect of mononuclear phagocyte maturation in vitro on 5-lipoxygenase expression. At baseline, monocytes had significant 5-lipoxygenase activity, but then lost all detectable 5-lipoxygenase activity over 7 days. Immunoblot and Northern blot analysis revealed that immunoreactive protein and mRNA for both 5-lipoxygenase and FLAP were significantly decreased over time. These studies demonstrate that in vitro differentiation of monocytes into a macrophage phenotype is not accompanied by the enhanced expression of 5-lipoxygenase and FLAP seen in macrophages derived from in vivo sources. In fact, baseline expression of 5-lipoxygenase and FLAP by monocytes is lost in vitro. These studies have clear implications for the use of cultured monocytes as a model of macrophages, and they also further our understanding of the regulation of the 5-lipoxygenase pathway.

TGF-beta increases leukotriene C4 synthase expression in the monocyte-like cell line, THP-1.

The goal of this study was to determine whether cytokines modulate leukotriene C4 (LTC4) synthase expression in mononuclear phagocytes. A panel of cytokines was surveyed for changes in LTC4 synthase mRNA in THP-1 cells. TGF-beta1, -2, and -3 had significant stimulatory effects. The addition of TGF-beta resulted in a time-dependent increase in LTC4 synthase mRNA at 6 h, which persisted through 48 h. Furthermore, this conditioning resulted in an increase in immunoreactive protein for LTC4 synthase through 7 days. TGF-beta conditioning of cells resulted in a time- and dose-dependent increase in stimulated LTC4 synthase activity. Following transient transfection of THP-1 cells with a promoter-reporter construct containing 1.2 kb of the LTC4 synthase promoter, TGF-beta treatment resulted in a 2-fold increase in reporter activity. Conditioning with TGF-beta did not prolong the half-life of LTC4 synthase mRNA, as assessed by RNase protection assays in actinomycin D-treated cells. Cycloheximide exposure experiments revealed that new protein synthesis was not required for the observed stimulatory effect of TGF-beta on LTC4 synthase mRNA. We conclude that LTC4 synthase expression is increased at a transcriptional level by TGF-beta in mononuclear phagocytes.

Leukotriene B4 costimulates 5-lipoxygenase activity in neutrophils via increased 5-lipoxygenase translocation.

The goal of this investigation was to assess the effect of leukotriene B4 (LTB4) on 5-lipoxygenase activity and to examine the possible mechanisms of this effect. Exogenous LTB4 significantly increased the release of endogenous LTB4 from A-23187-stimulated neutrophils. The 5-lipoxygenase product release from A-23187-stimulated neutrophils decreased in the presence of an LTB4 receptor antagonist, suggesting that LTB4 has a receptor-mediated, autocrine effect on 5-lipoxygenase activity. Neutrophil 5-lipoxygenase activity increased significantly as cell density increased. In the presence of exogenous LTB4, no significant change in [14C]arachidonic acid release from neutrophils was observed. Exogenous LTB4 increased the amount of immunoreactive 5-lipoxygenase protein detected in the nuclear fraction of disrupted cells. LTB4 receptor antagonism decreased the amount of immunoreactive 5-lipoxygenase detected in the nuclear fraction. Thus LTB4 exerts an autocrine, receptor-mediated, costimulatory effect on 5-lipoxygenase activity. This feedback appears to have biological significance and involves enhanced 5-lipoxygenase translocation to the nuclear membrane.

Activated lymphocytes increase expression of 5-lipoxygenase and its activating protein in THP-1 cells.

The aim of this study was to investigate the regulation of the 5-lipoxygenase pathway of arachidonic acid metabolism by lymphocytes using the monocyte-like cell line, THP-1. When THP-1 cells were incubated over 4-7 days in 10% supernatant from lectin-activated human lymphocytes, their capacity to synthesize 5-lipoxygenase products was significantly increased. In contrast, the supernatant from nonactivated lymphocytes had no effect. The increase in capacity to synthesize 5-lipoxygenase products was mimicked by the addition of either granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-3. These increases in synthetic capacity reflected increased enzymatic activity. Increased immunoreactive protein and mRNA for the enzymes 5-lipoxygenase and 5-lipoxygenase-activating protein were also found in cells conditioned with activated lymphocyte supernatants. Furthermore, the increase in mRNA for both enzymes was not blocked by cycloheximide, suggesting that the effect on steady-state mRNA levels does not require the synthesis of new protein. The increase in mRNA could be reproduced by GM-CSF. We conclude that lymphocytes can regulate the expression of 5-lipoxygenase in THP-1 cells over a period of days via the release of soluble factors.