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D Steinhilber

Publications and source records attributed to D Steinhilber.

At least 37 records · Page 2Linked to original sources
PubMed1996

Purification of transforming growth factor beta 1 from human platelets.

Transforming growth factor-beta (TGF beta) is a growth and differentiation factor which can be released from many cell types. In previous studies, platelets were identified as a rich source of TGF beta. Here we present a rapid and convenient method for TGF beta purification from human platelets which includes acid-ethanol extraction and gelfiltration, cation exchange and reversed phase chromatography. All purification steps are performed under acidic conditions to prevent adsorption of TGF beta to the vial walls. In addition, volatile solvents and buffers were used which allowed easy removal of solvent and salt by lyophilization. Using this method pure TGF beta can be easily obtained in high yield (370 micrograms) from 20 units of platelet concentrate.

Arachidonate 5-Lipoxygenase↗
PubMed1995

The nuclear receptor for melatonin represses 5-lipoxygenase gene expression in human B lymphocytes.

The two subtypes of retinoid Z receptor (RZR alpha and beta) and the three splicing variants of retinoid orphan receptor (ROR alpha 1, alpha 2, and alpha 3) form a subfamily within the superfamily of nuclear hormone receptors. Very recently we found that the pineal gland hormone melatonin is a natural ligand of RZR alpha and RZR beta. Ligand-induced transcriptional control is therefore proposed to mediate physiological functions of melatonin in the brain where RZR beta is expressed, but also in peripheral tissues, where RZR alpha was found. However, no natural RZR responding genes have been identified yet. Here, we report that a response element in the promoter of 5-lipoxygenase binds specifically RZR alpha and ROR alpha 1, but not ROR alpha 2 and alpha 3. 5-Lipoxygenase is a key enzyme in the biosynthesis of leukotrienes, which are known to be allergic and inflammatory mediators. We could show that the activity of the whole 5-lipoxygenase promoter as well as of the RZR response element fused to the heterologous thymidine kinase promoter could be repressed by melatonin. The hormone down-regulated the expression of 5-lipoxygenase about 5-fold in B lymphocytes, which express RZR alpha. In contrast, 5-lipoxygenase mRNA levels were not affected in differentiated monocytic and granulocytic cell lines, which do not express RZR alpha. This indicates that 5-lipoxygenase is the first natural RZR alpha responding gene. Furthermore, our results open up a new perspective in understanding the involvement of melatonin in inflammatory and immunological reactions.

Arachidonate 5-Lipoxygenase↗
PubMed1995

Sequential induction of 5-lipoxygenase gene expression and activity in Mono Mac 6 cells by transforming growth factor beta and 1,25-dihydroxyvitamin D3.

5-Lipoxygenase (5-LO; EC 1.13.11.34) activity in the human monocytic cell line Mono Mac 6 was upregulated by combined treatment with transforming growth factor beta 1 (TGF-beta) and 1,25-dihydroxyvitamin D3 (VD3). In undifferentiated cells, 5-LO enzyme activity was undetectable. After the addition of TGF-beta plus VD3, the activity of intact cells was 800 ng per 10(6) cells--500 times more than the assay detection limit. Also 5-LO protein and mRNA expression were induced > 128-fold and 64-fold, respectively, as compared to undifferentiated cells. Both TGF-beta and VD3 were required for these prominent responses. Either agent alone gave small amounts of 5-LO protein and mRNA but very low 5-LO activities. After the addition of TGF-beta and VD3, the induction of 5-LO protein was obvious after 1 day, but the increase in activity was delayed and did not appear until the second day. Pretreatment of cells with TGF-beta or VD3 alone for 2 days led to 5-LO protein expression but very low enzyme activity. Addition of the lacking second inducer was required for full induction of 5-LO protein expression and for upregulation of enzyme activity. Partial purification of 5-LO from Mono Mac 6 cells and recombination with soluble cellular proteins from different sources indicated the presence of cytosolic factors that affect the activity of 5-LO.

Arachidonate 5-Lipoxygenase↗
PubMed1994

On the induction of 5-lipoxygenase expression and activity in HL-60 cells: effects of vitamin D3, retinoic acid, DMSO and TGF beta.

Induction of 5-LO activity in DMSO differentiated HL-60 cells, by the serum protein TGF beta, was partially dependent on the presence of serum lipids (Steinhilber, D., Hoshiko, S., Grunewald, J., Rådmark, O., and Samuelsson, B. (1993) Biochim. Biophys. Acta 1178, 1-8). Here we demonstrate that the serum lipid fraction can be substituted by picomolar concentrations of 1,25-dihydroxyvitamin D3 (VD3). A high concentration of VD3 (24 nM) gave a 4-fold induction of 5-LO mRNA, a 14-fold increase in 5-LO protein, and a 38-fold upregulation of the 5-LO activity of intact HL-60 cells after differentiation in the presence of DMSO and serum proteins for 4 days. Also VD3 alone gave a substantial upregulation of 5-LO protein expression and activity. On the other hand, TGF beta alone was a poor inducer of the 5-LO pathway, the presence of a differentiation inducer (retinoic acid, DMSO or VD3) was required. The most prominent induction of 5-LO protein expression and activity in cell homogenates and intact cells was observed when VD3 and TGF beta were combined.

Arachidonate 5-Lipoxygenase↗
PubMed1994

5-Lipoxygenase: enzyme expression and regulation of activity.

5-Lipoxygenase catalyzes the transformation of arachidonic acid to leukotriene A4. This unstable intermediate can be converted to leukotriene B4 by LTA4-hydrolase or to leukotriene C4 by LTC4-synthase. Leukotrienes are involved in host defense reactions and play an important role in inflammatory diseases like asthma, inflammatory bowel disease and arthritis. The capability to release leukotrienes is restricted to a few cell types. Under pathophysiological conditions, leukotrienes are released from granulocytes, mast cells or macrophages. During hematopoiesis the competence of these cells for leukotriene biosynthesis is supposed to be upregulated. In mature cells, 5-lipoxygenase activity is tightly regulated and seems to be under the control of additional cellular components. One cellular component, a membrane-bound peptide termed FLAP, which is necessary for 5-LO activity in intact cells has been recently identified. Inhibitors of FLAP function prevent translocation of 5-lipoxygenase from cytosol to the membrane and inhibit 5-LO activation. Thus, the understanding of the regulatory mechanisms of cellular leukotriene biosynthesis provides new concepts for the development of antiinflammatory drugs. This review focuses on the regulation of gene expression and activity of 5-lipoxygenase.

Animals↗
PubMed1993

Serum factors regulate 5-lipoxygenase activity in maturating HL60 cells.

5-Lipoxygenase activity in DMSO-differentiated HL60 cells is regulated by human serum. The serum effect depended on the differentiation state of the cells. For a stimulatory effect to occur, it was required that the cells had been treated with DMSO before addition of serum. After this regimen, the HL60 cells acquired the same high 5-LO activity as found for human neutrophils isolated from peripheral blood (about 9-times higher than for HL60 cells treated only with DMSO). On the other hand, when serum was added together with DMSO and present during the entire differentiation period (seven days), or withdrawn after the first four days, the 5-LO activity did not increase. 5-LO activity of HL60 cells covaried with the expression of the CD14 molecule, a marker for myeloid cell maturation which was recently identified as a receptor for the complex of LPS and LPS-binding protein. These serum effects on 5-LO activity were only observed for intact cells. The prominent increase in 5-LO activity induced by serum was not concomitant with similar changes in the expression of 5-LO or 5-LO-activating protein (FLAP), as judged from analyses of immunoreactive protein and mRNA. Also, the high 5-LO activity induced by serum was rather insensitive to the drug MK886 under our standard assay conditions, which included addition of exogenous arachidonic acid (40 microM). The results indicate that additional cellular components of importance for 5-LO activity in HL60 cells become operative after serum treatment, and that mere expression of 5-LO and FLAP is insufficient for high 5-LO activity in intact cells.

Arachidonate 5-Lipoxygenase↗
PubMed1993

Transforming growth factor beta upregulates 5-lipoxygenase activity during myeloid cell maturation.

Transforming growth factor beta (TGF beta) increased the arachidonate 5-lipoxygenase (5-LO; EC 1.13.11.34) activity in HL-60 cells induced to granulocytic differentiation by dimethyl sulfoxide. The presence of a factor in human serum that caused a similar increase was recently demonstrated. Several observations indicate that the serum factor consists of isoforms of TGF beta. Heat-treated serum and TGF beta both resulted in approximately 10-fold increased 5-LO activity of HL-60 cells, antiserum to TGF beta neutralized the 5-LO-increasing activity in serum, and physical properties of the serum factor (lipophilic nature, alkaline pI, stability to heat and acid) coincided with those of TGF beta. The pattern of activity of native and heat-treated sera is compatible with activation of a latent form of TGF beta in serum. This activity was specific for TGF beta, since none of several other cytokines could increase 5-LO activity in differentiating HL-60 cells. However, granulocyte/macrophage-colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha enhanced the effect of TGF beta. The most prominent effects of TGF beta, whether alone or together with GM-CSF, were observed for 5-LO activity in intact cells (10-fold or 30-fold induction, respectively). 5-LO protein levels were less affected (up to 2- or 5-fold, respectively, as judged from Western blots). There was no appreciable effect of TGF beta, or a combination of TGF beta and GM-CSF, on 5-LO mRNA expression.

5-Lipoxygenase-Activating Proteins↗
PubMed1993

A heat stable serum factor upregulates 5-lipoxygenase activity in HL-60 cells, modulation by TNF alpha or GM-CSF.

5-Lipoxygenase (5-LO) activity in differentiating HL-60 cells was upregulated by a heat stable protein in human serum. Hematopoietic cytokines were evaluated for this effect on 5-lipoxygenase activity, none of the compounds tested could replace serum. However, TNF alpha or GM-CSF were able to augment the effect of heat treated serum, giving 5-LO activities that (per cell) were higher than for granulocytes isolated from peripheral blood. The serum factor primarily upregulated 5-LO activity. The amounts of 5-LO protein, or mRNA for 5-LO or 5-lipoxygenase activating protein (FLAP), were less affected.

5-Lipoxygenase-Activating Proteins↗
PubMed1992

On the expression and regulation of 5-lipoxygenase in human lymphocytes.

The expression of arachidonate 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) and the 5-lipoxygenase-activating protein (FLAP) genes in human tonsillar B cells and lymphoblastoid B-cell lines was demonstrated at the transcriptional level by reverse transcription-PCR analysis. Also, five lymphoblastoid T-cell lines were investigated and found to express the FLAP gene but not the 5-lipoxygenase gene, suggesting that the transcriptional regulation of these two genes is different. Western blot analysis of the cytosolic proteins from a lymphoblastoid B-cell line with an antiserum raised against purified human leukocyte 5-lipoxygenase revealed an immunoreactive band that comigrated with recombinant human 5-lipoxygenase. Intact B cells produced very low amounts of leukotriene B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid, in comparison to the amounts formed by sonicates of these cells. However, preincubation of intact lymphoblastoid B cells with the glutathione-depleting agents azodicarboxylic acid bis(dimethylamide) or 1-chloro-2,4-dinitrobenzene prior to the addition of the calcium ionophore A23187 and arachidonic acid led to similar amounts of leukotriene B4 as were formed by sonicated cells. In contrast, the glutathione synthesis inhibitor buthionine sulfoximine diminished the cellular level of glutathione by greater than 90% but did not influence the production of leukotriene B4 or 5-hydroxyeicosatetraenoic acid in intact cells. These results demonstrate that certain drugs affecting the redox status can stimulate the cryptic 5-lipoxygenase activity in intact lymphoblastoid B cells but that the mechanism of this activation is unclear and appears not to be directly related to intracellular glutathione levels.

5-Lipoxygenase-Activating Proteins↗
PubMed1991

Determination of picomole amounts of lipoxins C4, D4 and E4 by high-performance liquid chromatography with electrochemical detection.

A method for the sensitive determination of the sulphopeptide lipoxins (LXs) C4, D4 and E4 by high-performance liquid chromatography with subsequent electrochemical detection is described. The best results were obtained when the analysis was carried out with the solvent system methanol-water-trifluoroacetic acid (66:34:0.008, v/v/v). The acquired half-wave potentials were different for all investigated compounds: +1.18 V for LXC4 +1.3 V for LXD4 and +1.25 V for LXE4. The detection limits of LXC4, LXD4 and LXE4, based on a signal-to-noise ratio of 3:1, were found to be 200-700 fmol. Although sulphopeptide lipoxins possess a high molar absorptivity, electrochemical detection still is three times more sensitive than ultraviolet detection.

Arachidonic Acids↗
PubMed1991

Human B lymphocytes possess 5-lipoxygenase activity and convert arachidonic acid to leukotriene B4.

Incubation of cell sonicates from monoclonal B cells with arachidonic acid led to the formation of leukotriene (LT) B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In contrast, stimulation of intact B cells with the calcium ionophore A23187 +/- arachidonic acid did not, under similar conditions, lead to formation of LTB4. The identification of these products was based on reverse phase- and straight phase-HPLC analysis, UV-spectroscopy and gas chromatography-mass spectrometry. Cell sonicates of highly enriched human tonsillar B lymphocytes also converted arachidonic acid to LTB4 and 5-HETE. Activation of these cells with B cell mitogen and cytokines for three days led to an upregulation of 5-lipoxygenase activity. This study provides evidence for the biosynthesis of LTB4 from arachidonic acid in B cell lines and in normal human tonsillar B lymphocytes.

Arachidonate 5-Lipoxygenase↗
PubMed1990

Effects of novel antifungal azole derivatives on the 5-lipoxygenase and cyclooxygenase pathway.

5-Lipoxygenase and cyclooxygenase catalyze the first steps of the conversion of arachidonic acid into leukotrienes and prostanoids, respectively. Leukotrienes are important mediators of inflammation and thromboxanes are mainly involved in platelet functions. The effects of the new antimycotic drugs, itraconazole and fluconazole, on the 5-lipoxygenase pathway in human polymorphonuclear leukocytes (PMNL), on the eicosanoid metabolism in platelets, platelet aggregation and on cyclooxygenase activity were investigated and compared with miconazole and ketoconazole. Itraconazole inhibited the formation of 5-lipoxygenase metabolites in human PMNL (IC50 = 2 x 10(-6) mol/l), while it had no effect on cyclooxygenase and platelet aggregation at concentrations up to 10(-4) mol/l and 10(-5) mol/l, respectively. Fluconazole was ineffective in all assays. Miconazole inhibited thromboxane synthase (IC50 = 1 x 10(-5) mol/l) and platelet aggregation (IC50 = 3 x 10(-5) mol/l). Ketoconazole was less active in this respect (IC50 for platelet aggregation = 2 x 10(-4) mol/l). All compounds did not affect cyclooxygenase activity in concentrations up to 10(-4) mol/l in the pure enzyme assay. These results indicate that among the tested azoles two compounds show remarkable effects. Miconazole inhibits thromboxane synthesis and itraconazole is a potent inhibitor of leukotriene formation which has only minor effects on the cyclooxygenase pathway. This finding is of considerable interest regarding the application of itraconazole during immunosuppressive therapy and suggests further studies of its potential antiphlogistic properties.

Antifungal Agents↗
PubMed1989

New series of lipoxins isolated from human eosinophils.

Granulocytes from human eosinophilic donors were incubated with arachidonic acid or 15-hydroxyeicosatetraenoic acid (15-HETE) and stimulated with the ionophore A23187. The eicosanoids were extracted with reversed-phase cartridges and subjected to RP-HPLC analysis. When extracts from eosinophil-enriched populations were analysed and compared with extracts from human neutrophils, three additional peaks were detected which coeluted with 15-hydroxy-delta 13-trans-15H derivatives of leukotriene C4, D4 and E4 in different HPLC systems. The recorded absorbance spectra of the eluted compounds and the standards were identical and showed a maximum at 307 nm which is characteristic for a conjugated tetraene system with a bathochromic shift by the sulfur moiety in alpha-position to the tetraene system. The compound which coeluted with the 15-hydroxy-LTC4 standard was treated with gamma-glutamyltransferase and converted to the corresponding leukotriene D4 derivative. The results indicate that interaction between the 5- and 15-lipoxygenase pathways leads to the formation of a new series of arachidonic acid metabolites in human eosinophils. Since the biosynthetic route is similar to that of lipoxin A4 and lipoxin B4, we suggest the trivial names lipoxin C4, D4 and E4.

Arachidonic Acid↗