Sp1 and Sp3 function as key regulators of leukotriene C(4) synthase gene expression in the monocyte-like cell line, THP-1.

The goal of this study was to examine the mechanisms of leukotriene C(4) (LTC(4)) synthase gene expression in mononuclear phagocytes. Transfection of the monocyte-like cell line THP-1 with LTC(4) synthase promoter-reporter constructs demonstrated that the first 1.3 kb of the promoter mediated a 21.1-fold increase in reporter activity. Deletion analysis revealed that the region between -92 and -23 bp, which contains a signal protein (Sp)1 consensus site at -42 to -37 bp, mediated an 11.5-fold increase in reporter activity. Using a probe from -56 to -17 bp, electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and THP-1 and HeLa nuclear extracts bind to this region. Binding was eliminated by mutation of the Sp1 consensus site. Supershift EMSAs using anti-Sp1 and anti-Sp3 antibodies demonstrated that these Sp family members bind to the region. Transfection of the Sp-deficient Drosophila SL-2 cell line with a construct containing the -92 to -23 bp promoter region and Sp expression vectors revealed that Sp1 and Sp3 transactivate gene transcription. We conclude that the Sp1 site is a necessary element for LTC(4) synthase gene transcription. Sp1 and Sp3 function through this site to positively regulate transcription. Thus, we provide evidence that the LTC(4) synthase gene is transcriptionally regulated in mononuclear phagocytes.

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.

Pulmonary infiltrates, eosinophilia, and cardiomyopathy following corticosteroid withdrawal in patients with asthma receiving zafirlukast.

CONTEXT: Zafirlukast is a potent leukotriene antagonist that recently was approved for the treatment of asthma. As use of this drug increases, adverse events that occur at low frequency or in populations not studied in premarketing clinical trials may become evident. OBJECTIVE: To describe a clinical syndrome associated with zafirlukast therapy. DESIGN: Case series. PATIENTS: Eight adults (7 women and 1 man) with steroid-dependent asthma who received zafirlukast. MAIN OUTCOME MEASURES: Development of a clinical syndrome characterized by pulmonary infiltrates, cardiomyopathy, and eosinophilia following the withdrawal of corticosteroid treatment. RESULTS: The clinical syndrome developed while patients were receiving zafirlukast from 3 days to 4 months and from 3 days to 3 months after corticosteroid withdrawal. All 8 patients developed leukocytosis (range, 14.5-27.6 x 10(9)/L) with eosinophilia (range, 0.19-0.71). Six patients had fever (temperature >38.5 degrees C), 7 had muscle pain, 6 had sinusitis, and 6 had biopsy evidence of eosinophilic tissue infiltration. The clinical syndrome improved with discontinuation of zafirlukast treatment and reinitiation of corticosteroid treatment or addition of cyclophosphamide treatment. COMMENT: Development of pulmonary infiltrates, cardiomyopathy, and eosinophilia may have occurred independent of zafirlukast use or may have resulted from an allergic response to this medication. We suspect that these patients may have had a primary eosinophilic infiltrative disorder that had been clinically recognized as asthma, was quelled by steroid treatment, and was unmasked following corticosteroid withdrawal facilitated by zafirlukast.

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.

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.

Molecular cloning of the human leukotriene C4 synthase gene and assignment to chromosome 5q35.

BACKGROUND: Cysteinyl leukotrienes (LT) are mediators involved in inflammatory and allergic disorders LTC4 synthase catalyzes the first committed step in the synthesis of these inflammatory mediators, and its cellular distribution appears to be unique. MATERIALS AND METHODS: A human genomic library was screened by polymerase chain reaction (PCR) with primers that were designed based on the reported cDNA sequence for the LTC4 synthase gene. The gene was identified in one clone by Southern blotting of restriction enzyme digests, subcloning of fragments containing regions of interest, and DNA sequencing of these subclones. The transcription initiation site was determined by primer extension analysis. Chromosome location was determined by fluorescent in situ hybridization and screening of somatic cell hybrids by PCR. RESULTS: The LTC4 synthase gene is approximately 2.5 kb in length, consisting of five exons (136, 100, 71, 82, and 257 bp, respectively) and four introns (1,447, 102, 84, and 230 bp, respectively). Transcription initiation occurs at a single site 78 bp upstream of the coding region. The 5'-flanking region contains neither a TATA nor a CAAT box. The first 1 kb of the 5'-flanking region, however, contains putative DNA binding motifs for SP-1, AP-1, AP-2, ets factors, and CREB/ATF. A STAT binding motif is present in the first intron. The LTC4 synthase gene is located in the distal region of the long arm of chromosome 5 in 5q35. CONCLUSIONS: The LTC4 synthase gene does not contain elements of a typical regulated gene and may therefore contain novel regulatory elements. This gene is also located in a region on chromosome 5 that appears to play a role in allergic and inflammatory disorders, such as asthma.

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.

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.

Effects of metalloproteinase inhibitors on leukotriene A4 hydrolase in human airway epithelial cells.

Human neutrophil leukotriene A4 (LTA4) hydrolase is a zinc-containing metalloproteinase with aminopeptidase activity and can be inhibited by some metalloproteinase inhibitors. Human airway epithelial cells also contain an LTA4 hydrolase enzyme that has some novel properties, suggesting that this enzyme may be functionally and structurally unique. Thus, we questioned whether the epithelial cells were studied either intact or disrupted. Of the metalloproteinase inhibitors examined, only captopril, bestatin, and fosinoprilat had appreciable inhibitory activity for LTA4 hydrolase in disrupted epithelial cells. Concentration-inhibition curves to captopril, bestatin, and fosinoprilat revealed IC50 values of 430 microM, 7 microM, and 1 mM, respectively, for disrupted-cell LTA4 hydrolase activity. In contrast to its effects on neutrophils, 1,10-O-phenanthroline had no significant effect on disrupted epithelial cell hydrolase activity and had only minimal effects when this activity was partially purified (179-fold). LTA4 hydrolase concentration-inhibition curves examined in intact cells with captopril, bestatin, and 1,10-O-phenanthroline revealed IC50 values of 63, 70, and 920 microM, respectively. Aminopeptidase activity in disrupted epithelial cells was inhibited by amastatin, bestatin, and 1,10-O-phenanthroline (IC50 values of 500 nM, 1 microM, and 17 microM, respectively), but not by captopril at the highest concentration tested, 10 mM. These findings are in contrast to prior studies in neutrophils. When neutrophils were stimulated with A23187 after treatment with captopril, transcellular synthesis of LTB4 was inhibited more effectively than direct synthesis of leukotriene B4 (LTB4) (43.8 +/- 2.5 vs 18.5 +/- 4.7%; N = 8, P < 0.02). We conclude that LTA4 hydrolase activity of human airway epithelial cells is inhibited by some metalloproteinase inhibitors, but that the profile of inhibition is distinct from that for the neutrophil enzyme. These data provide additional information that LTA4 hydrolase in the epithelial cell is a novel enzyme, distinct from that found in the neutrophil.

Nonadrenal epinephrine-forming enzymes in humans. Characteristics, distribution, regulation, and relationship to epinephrine levels.

Animal studies indicate that nonadrenal tissues may synthesize epinephrine (E). Here we demonstrate phenylethanolamine N-methyltransferase (PNMT) and/or nonspecific N-methyltransferase (NMT) enzymatic activity in human lung, kidney, heart, liver, spleen, and pancreas. There was a significant overall correlation (r = 0.34) between tissue PNMT and E. PNMT and NMT in human tissues differed in substrate and inhibitor specificity, thermal stability, and antigenicity. By these criteria, PNMT in human lung and in human bronchial epithelial cells were indistinguishable from adrenal PNMT. PNMT and/or NMT activity were present in red blood cells (RBCs), and cancer cell lines. Human kidney, lung, and pancreas showed immunohistochemical staining with an antibody to adrenal PNMT. RBC PNMT activity was lower in males than females and was increased in hyperthyroidism and decreased in hypothyroidism. PNMT activity in a human bronchial epithelial cell line was dramatically increased by incubation with dexamethasone. E and 3H-E levels in plasma and urine during an intravenous infusion of 3H-E into humans indicated that kidney may synthesize half of urinary E. We conclude that PNMT and NMT are widely distributed in human tissues, that they may synthesize E in vivo and are influenced by glucocorticoid and thyroid hormones.

Captopril inhibits neutrophil synthesis of leukotriene B4 in vitro and in vivo.

The aim of this investigation was to determine the effects of metalloproteinase inhibitors on leukotriene (LT) A4 hydrolase in human neutrophil cytosol and to examine the effects of captopril on intact neutrophils in vitro and in vivo. Cytosolic fractions were assayed for LTA4 hydrolase and 5-lipoxygenase activity in the presence or absence of inhibitors. Only bestatin, 1,10-O-phenanthroline, captopril and fosinoprilat demonstrated significant effects. The IC50 of captopril and fosinoprilat for LTA4 hydrolase activity were 500 microM and 1 mM, respectively. No inhibition of 5-lipoxygenase activity in cytosolic fractions was detected. The effect of captopril was only minimally reversed by ZnSO4. The IC50 of captopril for inhibition of LTB4 synthesis in intact neutrophils was 63 microM. Furthermore, 5-HETE production in intact cells was diminished 25.3 +/- 8.5% in the presence of 1 mM captopril. Oral captopril inhibited stimulated LTB4 release by subsequently isolated neutrophils by 48.1 +/- 5.6% and 5-HETE release by 43.2 +/- 5.5%. Thus, captopril is an inhibitor of LTB4 synthesis in neutrophils in vitro and in vivo. However, there are differences between the potency of this drug as assessed in cytosol and intact cell studies. This study significantly extends previous reports in that it demonstrates that captopril is a more potent inhibitor of LTB4 synthesis in intact neutrophils than in cytosol and in that it demonstrates an inhibitory effect of captopril on synthesis of LTB4 by neutrophils exposed to captopril in vivo.

Characterization of human airway epithelial cell leukotriene A4 hydrolase.

We have previously shown that human airway epithelial cells contain leukotriene A4 (LTA4) hydrolase activity. To characterize this activity further, airway epithelial cells, cultured to confluence, were disrupted by sonication and were fractionated at 15,000 and 100,000 x g. Enzymatic activity was assessed by incubating fractions with 15 microM LTA4 at 37 degrees C for 15 min. LTA4 hydrolase activity was present in the 15,000 x g and the 100,000 x g supernatants and was inactivated by heating at 56 degrees C or by pronase, as is the case for neutrophil LTA4 hydrolase. However, the epithelial cell enzyme had a slower time course for product generation and demonstrated a different dose-response relationship to substrate when compared with the neutrophil. Kinetic analysis revealed nonlinear plots for epithelial data, most consistent with an enzyme that has multiple active sites. Immunoblotting, performed with anti-neutrophil LTA4 hydrolase antibody, recognized two bands in epithelial cell 15,000 x g supernatant (M(r) of 69,000 and 110,000-115,000). When resolved by gel filtration chromatography, only the M(r) 69,000 protein had enzymatic activity. Anion exchange chromatography of epithelial cell samples revealed that LTA4 hydrolase and aminopeptidase activity did not co-elute, whereas one of three peaks of aminopeptidase activity did co-elute in chromatograms of neutrophil samples. Immunoblots of proteolytic digests of partially purified M(r) 69,000 protein from epithelial cells and neutrophils revealed different immunoreactive bands. The digest of the M(r) 110,000-115,000 protein revealed no immunoreactive bands. Repeat kinetic analysis on 179-fold purified epithelial LTA4 hydrolase again revealed that it lacked significant aminopeptidase activity and retained its unique kinetic properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of human peripheral blood lymphocyte function by protoporphyrin and longwave ultraviolet light.

Modulation of immunologic effector cells by exogenous photoactive substances has been advanced as an underlying mechanism for the efficacy of various photochemotherapeutic regimens. It is also possible that endogenous photosensitizers, such as protoporphyrin, could similarly modify the function of immune cell types. We examined the effects of protoporphyrin plus longwave UV light on the ability of human PBL to proliferate in response to mitogens. Noncytotoxic dosages of protoporphyrin plus UV light suppressed PHA-stimulated proliferation of both PBMC and enriched T cells. CD8+ cells were more sensitive to this inhibitory effect than CD4+ cells. The inhibitory effect was also observed when proliferation was induced by the combination of a phorbol ester and ionomycin. Inhibition of PBMC proliferation was associated with inhibition of IL-2 secretion but proliferation was not restored with exogenous IL-2. Instead, the effect of protoporphyrin plus UV light may be on IL-2R. Cells treated with protoporphyrin and UV light did not display the increase in CD25 and the beta-chain of the IL-2R induced by PHA in control cells. In contrast to the effects of protoporphyrin and UV light on IL-2 and IL-2R alpha-chain protein expression, the accumulation of mRNA for these proteins induced by PHA was unaffected. None of the effects of protoporphyrin plus UV light on lymphocytes were observed in control experiments where cells were treated with either protoporphyrin or UV light alone. We conclude that biologically relevant dosages of protoporphyrin and UV light modify the function of circulating lymphocytes.

Leukotriene A4 hydrolase in human bronchoalveolar lavage fluid.

We examined cell-free human bronchoalveolar lavage fluid (BALF) for enzymes of the 5-lipoxygenase pathway. BALF was obtained from six patients who were active smokers and six nonsmokers. Enzymatic activity in cell-free BALF was assessed by specific assays for leukotriene (LT) A4 hydrolase, 5-lipoxygenase, and LTC4 synthase using HPLC. Only LTA4 hydrolase enzymatic activity was found. This activity ranged from 101 to 667 when expressed as picomoles of LTB4 produced per milliliter BALF. Enzymatic activity in smokers vs nonsmokers was 484 +/- 120 vs 129 +/- 32 pmol LTB4/ml BALF (mean +/- SD, P < 0.0001). There were no leukotrienes found in BALF before assay. Immunoblot analysis revealed an immunoreactive band at a relative molecular mass of 69,000 D in all samples, consistent with LTA4 hydrolase, but no evidence of 5-lipoxygenase. BALF had greater LTA4 hydrolase activity per milligram of protein than neutrophil cytosol, epithelial cell cytosol, plasma, or serum. The synthesis of LTB4 was significantly increased when neutrophils were stimulated in BALF. These data indicate the selective presence of LTA4 hydrolase in BALF which is significantly increased in smokers. This enzyme in BALF may contribute to the inflammatory response in tobacco-related lung disease.

Involvement of arachidonic acid in the chloride secretory response of intestinal epithelial cells.

The inflammatory mediator, adenosine, induces chloride secretion from the human colonic epithelial cell line, T84, in a manner apparently independent of increases in adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, or cytoplasmic Ca2+. This prompted a search for other messengers that might account for the secretory response. A possible role for arachidonic acid or a metabolite in the response to adenosine has been demonstrated 1) by showing a relationship between arachidonic acid mobilization and chloride secretion induced by the adenosine agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and 2) by determining that exogenous arachidonic acid affects T84 cell function. Addition of NECA to T84 cells induces chloride secretion and release of radioactivity from cells preloaded with [3H]arachidonic acid with similar dose dependencies. The effect of NECA on chloride secretion is inhibited by the phospholipase A2 inhibitor 4-bromophenacyl bromide or the diglyceride lipase inhibitor RG80267 but is unaffected by inhibitors of lipoxygenase or cyclooxygenase. Arachidonic acid has a small but significant effect on chloride secretion when added alone to T84 cells and synergistically enhances, as does NECA, responses to calcium-dependent secretogogues. Thus receptor-stimulated release of arachidonic acid in T84 cells may provide a second-messenger system promoting chloride secretion, in addition to calcium and cyclic nucleotides.