Soot-exposed mononuclear cells increase inflammatory cytokine mRNA expression and protein secretion in cocultured bronchial epithelial cells

BACKGROUND: Soot particles are air pollutants capable of inducing airway and lung parenchymal injury. Mononuclear and bronchial epithelial cells are central to the maintenance of homeostasis and inflammation in the airways. OBJECTIVES: The aim of this study was to evaluate the contribution of mononuclear cells to the release of inflammatory mediators by bronchial epithelial cells. Methods: To model the in vivo situation, an in vitro system of cocultured blood monocytes and BEAS-2B cells was established in a transwell system. Blood monocytes were exposed to soot particles (FR 101) at concentrations of up to 100 microg/10(6) cells. Inflammatory cytokine mRNA and protein concentrations were quantified in BEAS-2B mono- and BEAS-2B-BM cocultures by RT-PCR and ELISA following exposure to soot for 1 and 8 h. RESULTS: No inflammatory cytokine mRNA expression was observed in unstimulated BEAS-2B cells. IL-6 and IL-8 mRNA and protein levels showed a dose-dependent elevation in FR 101-exposed blood monocytes. In addition, both IL-6 and IL-8 mRNA expression was upregulated in cocultured BEAS-2B cells while cytokine concentrations in the blood monocyte-BEAS-2B coculture medium were significantly increased. This upregulation was likely due to a synergism of two cell populations. CONCLUSIONS: Exposure to soot particles induces an autocrine stimulation of inflammatory cytokine release by blood monocytes and BEAS-2B cells. Since IL-6 and IL-8 play a major role in the pathogenesis and persistence of bronchial inflammation, these findings may serve as a partial explanation for the aggravation of asthmatic and bronchitic symptoms after exposure to soot.     

Human polymorphonuclear leukocytes have dual effects on endothelin-1: The induction of endothelin-1 mRNA expression in vascular endothelial cells and modification of the endothelin-1 molecule

Summary The effect of human polymorphonuclear leukocytes (PMNs) on the expression of the endothelin-1 (ET-1) gene and the production of ET-1 peptide was investigated. Human PMNs were separated from venous blood with Mono-Poly Resolving Medium and activated by incubation with formyl-methionyl-lencyl-phenylalanine (FMLP) (1 μM). Then PMN suspension was added to cultured porcine endothelial cell monolayers and coincubated for various periods. Following the coincubation, ET-1 mRNA in endothelial cells was examined by Northern blotting and immunoreactive ET-1 (irET-1) peptide levels in the conditioned media were measured by an enzyme-linked immunosorbent assay (ELISA). Similar experiments were also carried out with cell-free PMN supernatant. Untreated and activated PMNs led to a 1.4-fold and 6.3-fold increase in ET-1 mRNA levels in endothelial cells, respectively, at 6 h, while irET-1 peptide levels did not significantly increase as compared with control. In contrast, when PMNs were coincubated in the presence of an Intercell chamber without direct contact, to endothelial cells, PMNs did not induce ET-1 mRNA expression in endothelial cells, and significantly decreased irET-1 peptide levels in the conditioned media. Cell-free PMN supernatant did not have all these effects on ET-1. These findings suggest that direct PMN-endothelial cell contact was essential for PMN-induced expression of the ET-1 gene and that PMNs may decrease irET-1 through some modification of the ET-1 molecule.     

Sickle erythrocytes increase prostacyclin and endothelin-1 production by cultured human endothelial cells under flow conditions

We investigated the effects of sickle erythrocytes on the production of vasotone mediators in endothelial cells (ECs) using an in vitro recirculating flow system. Sickle erythrocytes increased the EC production of two important vasoactivators, prostacyclin and endothelin-1, under venous wall shear stress conditions of 1dyncm2. The presence of interleukin-1 in the perfusion system, as a model for inflammatory cytokine effects, enhanced the overall amounts of released prostacyclin but did not affect the production of endothelin-1. This study demonstrates the effects of sickle erythrocytes on the function and metabolism of ECs under vascular flow environments. The altered production of vasoactivators may contribute to the vasotone instability and vasoocclusive crises in sickle cell anemia.     

Shear-induced changes in endothelin-1 secretion of microvascular endothelial cells

Human glomerular microvascular endothelial cell (HGMEC) culture monolayers were maintained in static culture as controls or subjected to steady laminar shear stress of 0.5, 1.0, or 1.5 N/m2. Over 25 h of shear, the cumulative secretion of ET-1 was 705.4 pg/cm2 in the control, 820.7 pg/cm2 at 0.5 N/m2, 1063.2 pg/cm2 at 1.0 N/m2, and 644.7 pg/cm2 at 1.5 N/m2. The average ET-1 secretion rate for the HGMEC monolayers exposed to 0.5, 1.0, or 1.5 N/m2 of shear stress was 32.83 +/- 2.01 pg/cm2 x h, 42.53 +/- 3.74 pg/cm2 x h, and 25.79 +/- 1.29 pg/cm2 x h, respectively. The average ET-1 secretion rate of the static controls was 28.22 +/- 3.11 pg/cm2 x h. The results showed that low shear stress (0.5 N/m2) elevated and high shear stress (1.5 N/m2) suppressed secretion of ET-1, while an intermediate level of shear stress (1.0 N/m2) led to the maximum secretion of ET-1, and furthermore, ET-1 secretion varied with the duration of shear in a nonlinear fashion, and the logistic equations may be used to describe relationship between the duration of shear and the ET-1 secretion. The major secretion period of ET-1 occurred between 5.3 and 22.3 h, with the peak secretion rate occurring at approximately 10.7-15.2 h. Our findings showed also that the major secretion period and peak secretion rate of HGMECs varied with the level of shear stress. Thus, the response of cultured human microvascular endothelial cells to shear stress differed from that of large-vessel endothelial cell cultures in terms of ET-1 secretion. In addition to the level of shear stress, the duration of shear is an important determinant in ET-1 secretion. Consequently, the heterogeneity of vascular endothelial cells and the duration of shear should both be considered in future research on the secretion of vascular endothelial cell cultures.     

Release mechanism of endothelin-1 and big endothelin-1 after stimulation with thrombin in cultured porcine endothelial cells.

Cultured porcine endothelial cells (EC) released immunoreactive endothelin-1 (ir-endothelin-1) and big endothelin-1 (ir-big endothelin-1) into the medium in a time-dependent way. Reverse-phase high-pressure liquid chromatography coupled with radioimmunoassay showed that the major component of ir-endothelin-1 corresponded to standard endothelin-1 (1-21) and that the major component of ir-big endothelin-1 corresponded to standard big endothelin-1 (porcine 1-39). This release was strongly inhibited by cycloheximide and was, therefore, related to de novo protein synthesis. The release of greater amounts was stimulated by thrombin. The protein kinase C (PKC) inhibitors from two chemical classes, H7 and staurosporine, inhibited release following such stimulation in a relatively dose-dependent way. Neither H7 nor staurosporine affected the basal release of both endothelin-1 and big endothelin-1. Phorbol myristate acetate, which activates PKC and the Ca2+ ionophore A23187, stimulated the release of ir-endothelin-1 and ir-big endothelin-1 in a dose-dependent way, respectively. In addition, the combination of both compounds had a synergistic effect. An inactive enantiomer of phorbol ester, 4 alpha-phorbol-12,13-didecanoate had no effect on the release of ir-endothelin-1 and ir-big endothelin-1. These results suggest that cultured EC release endothelin-1 and big endothelin-1 simultaneously, and that thrombin stimulates this release by a mechanism that probably involves intracellular Ca2+ mobilization and the activation of PKC.     

The endothelin-1 receptor-mediated pathway is not involved in the endothelin-1-induced defenestration of liver sinusoidal endothelial cells.

BACKGROUND/AIMS: We previously reported that endothelin (ET)-1 may be involved in the contraction of hepatic sinusoidal endothelial fenestrae (SEF). Rho has emerged as an important regulator of the actin cytoskeleton and consequently cell morphology. To clarify the role of ET receptors [endothelin A receptor (ETAR) and endothelin B receptor (ETBR)] in ET-1-induced defenestration, we studied the size of hepatic SEF under various experimental conditions. METHODS: Liver sinusoidal endothelial cells (LSECs) isolated from rat livers by collagenase perfusion were cultured and divided into four groups: control, ET-1 (10(-6) -10(-10) M)-treated, ET-1+selective ETAR antagonist (BQ610)-treated and ET-1+ETBR antagonist (BQ788)-treated groups. SEF morphology was observed by scanning electron microscopy. Protein expressions of ETAR and ETBR, Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. F-actin stress fiber formation was observed by confocal microscopy. Active Rho was measured by Ren's modification. Intracellular free Ca2+ concentration ([Ca2+]i) was measured by fluorescence digital imaging using fura-2 AM by Aqua cosmos. RESULTS: ET-1 induced a reduction in the number and size of SEF. ETAR antagonist pretreatment inhibited defenestration induced by low ET-1 concentrations (10(-8) -10(-10) M), whereas ETBR antagonist pretreatment did not block defenestration at low to high ET-1 concentrations (10(-6) -10(-10) M). F-actin stress fibers, Rho A levels and phosphorylated myosin light-chain kinase levels remained the same in various treatments. Active Rho was not detected in control and various treatments. ET-1 did not increase [Ca2+]i. Western blot showed prominent ETBR but scarce ETAR protein expression in LSECs. CONCLUSIONS: The present findings demonstrated that ETBR- and ETAR-induced contractile mechanisms are not involved in ET-1-induced defenestration, and that Rho is also not activated. Therefore, ET-1 induces hepatic defenestration by mechanisms other than receptor-mediated contraction.     

Effects of endothelin-1 on endothelial cells in the porcine coronary artery.

The effects of endothelin-1 (ET-1) on endothelial cells from the porcine coronary artery were investigated using cascade bioassay and radioimmunoassay methods. For comparison, endothelial cells from the rabbit abdominal aorta were also used. Freshly isolated endothelial cells were perfused with Krebs' solution. Liberation of vasodilatory substances was detected under bioassay conditions by measuring the relaxing activity of the perfusate on endothelium-denuded strips of the porcine coronary artery contracted with ET-1. The perfusate in the absence of ET-1 slightly inhibited the contraction, suggesting that endothelial cells spontaneously release vasorelaxing substances. After application of ET-1, the perfusate-induced relaxation drastically increased. Oxyhemoglobin completely abolished the relaxation induced by perfusate from endothelial cells of the rabbit abdominal aorta but only marginally attenuated the relaxation induced by perfusate from endothelial cells of the porcine coronary artery. In contrast, indomethacin significantly attenuated the relaxation induced by perfusate from the endothelial cells of the porcine coronary artery. In endothelium-intact strips of the porcine coronary artery, ET-1 significantly increased the concentration of 6-ketoprostaglandin F1 alpha but did not modify the cellular concentration of either cAMP or cGMP. It is concluded that ET-1 augments the release of vasorelaxing factors from endothelial cells both in the porcine coronary artery and in the rabbit abdominal aorta and that the major vasorelaxing substance derived from endothelial cells may be different in these two blood vessels. Prostaglandin I2 in the former and nitric oxide in the latter are suggested as possible candidates. The vasorelaxation induced by prostaglandin I2 may not be mediated by cAMP-dependent mechanisms in the porcine coronary artery.     

17Beta-estradiol modulates endothelin-1 expression and release in human endothelial cells

OBJECTIVE: In this study the role of 17beta-estradiol (E2) in the regulation of endothelin-1 (ET-1) mRNA expression and secretion was investigated in cultured human umbilical vein endothelial cells (HUVECs). METHODS: Endothelial cells were either deprived of or treated with 17beta-estradiol (10(-9), 10(-7) M) for 48 h. After the incubation, the effect of E2 on ET-1 gene expression was evaluated by Northern blot analysis. ET-1 release into the media was measured by radioimmunoassay after 6 h of incubation under basal conditions and upon stimulation with thrombin (4 U/ml). In addition, the cyclic guanosine 5'-monophosphate (cGMP) content of cells was assayed by immunoassay. In order to exclude the role of nitric oxide (NO) in E2-induced effects on endothelin-1 gene expression and secretion, nitric oxide synthase (NOS) inhibitor, N-nitro L-arginine methyl ester (1 mM) (L-NAME) was added to the media of some cultures. RESULTS: Incubation of HUVECs with 10(-9) and 10(-7) M E2 for 48 h resulted in a 30 and 47% inhibition of ET-1 mRNA expression, respectively. Incubation with E2 also decreased the basal and thrombin-stimulated ET-1 release while increasing the cGMP content of cells significantly. NOS inhibitor L-NAME increased the release of ET-1 from E2-incubated cells but did not alter the ET-1 release from hormone-deprived cells. However, ET-1 secretion of E2-treated cells were significantly less than the deprived ones. Northern blot analyses also demonstrated that inhibition of NOS only partly attenuated the effect of E2 on ET-1 gene expression. In the presence of L-NAME, treatment with 10(-7) M E2 caused a 12% decrease in ET-1 gene expression. CONCLUSION: The results demonstrate that E2 may play both direct and indirect role in regulation of ET-1 gene expression and production in human endothelial cells. E2-induced increase in NO but decrease in ET-1 production may partly explain the mechanism of the protective effects of the hormone on the cardiovascular system.     

Plasma endothelin-1 levels and circulating endothelial cells in patients with aortoarteritis.

To investigate the correlation between plasma endothelin-1 (ET-1), circulating endothelial cells (CECs), and the disease activity in patients with aortoarteritis. In this study, radioimmunoassay was used to measure plasma levels of ET-1 in 56 patients with aortoarteritis. Circulating endothelial cell counts were also carried out as an indicator of vessel wall lesions. The plasma levels of ET-1 and CECs in the active disease patient group were significantly higher than those in inactive patient group (p<0.001). A significant positive correlation was found between plasma ET-1 levels and erythrocyte sedimentation rates (ESRs) in patients with aortoarteritis (r=0.645, p<0.001), as well as CECs (r=0.876, p<0.001). These results suggested that the ET-1 secreted during the active stages of aortoarteritis may cause constriction and proliferation of vascular smooth muscle cells, thus contributing to the pathogenesis of luminal narrowing. The increased CECs might serve as a marker of disease activity.     

Endothelin-3 regulates endothelin-1 production in cultured human endothelial cells.

Effects of endothelin-3 on the secretion of endothelin-1 and other endothelium-derived substances were investigated in cultured human umbilical vein endothelial cells. The present binding study showed two distinct subpopulations of binding sites for endothelin-3 with higher and lower affinities in cultured human endothelial cells. Endothelin-3 caused an increase in intracellular Ca2+ and inositol 1,4,5-trisphosphate levels and activated protein kinase C in a dose-dependent manner. Endothelin-3 also caused an increase in [3H]thymidine incorporation into cellular DNA and stimulated the production of cyclic guanosine 3',5'-monophosphate, 6-ketoprostaglandin F1 alpha, and immunoreactive endothelin-1 in cultured human endothelial cells. NG-Monomethyl L-arginine (3 x 10(-4) mol/l) and indomethacin (10(-5) mol/l) enhanced endothelin-3-induced endothelin-1 production. These results suggest that endothelin-3 bound to its specific receptors and then caused phosphoinositide breakdown, subsequently mobilizing intracellular Ca2+ and leading to protein kinase C activation and the initiation of DNA synthesis, resulting in the stimulation of endothelin-1 production by human endothelial cells. Furthermore, this endothelin-1 production may be suppressed by endothelium-derived relaxing factor and prostacyclin produced in response to endothelin-3 in cultured human endothelial cells.     

Effect of endothelin-1 on cytosolic calcium ions in cultured human endothelial cells

The effects of endothelin-1 on cytosolic Ca2+ and inositol 1,4,5-triphosphate (IP3) levels were investigated in cultured, untreated human endothelial cells and in endothelial cells pretreated with anti-endothelin-1 serum for 24 h to exclude the effect of endogenous endothelin-1. Endothelin-1 was found to increase the intracellular Ca2+ level, either in the presence or absence of extracellular Ca2+, in endothelial cells pretreated with antiserum by the fura-2 fluorescence technique. IP3 levels immediately started to rise following endothelin-1 stimulation. Resting intracellular Ca2+ levels were significantly lower when the cells were pretreated with antiserum than without antiserum pretreatment. Following stimulation by endothelin-1, intracellular Ca2+ and IP3 levels in endothelial cells pretreated with antiserum increased significantly compared to those in untreated endothelial cells. Endothelin-1 also increased 45Ca influx from the extracellular space. These results suggest that endothelin-1 increases intracellular Ca2+ in endothelial cells through extracellular Ca2(+)-dependent mechanisms and by the release of Ca2+ from intracellular stores, this presumably being induced by IP3 formation.     

Eicosapentaenoic acid suppresses basal and insulin-stimulated endothelin-1 production in human endothelial cells.

cis-Polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) are the major fatty acids contained in fish oil, and are known to affect the various physiological properties of cell membranes in humans. The present study investigated the effects of polyunsaturated fatty acids on endothelin-1 (ET-1) production in human umbilical vein endothelial cells (HUVECs) and on insulin activity. After addition of various concentrations of EPA, docosahexaenoic acid, arachidonic acid, or linoleic acid to a culture medium, the concentration of ET-1 was measured using ELISA, and that of ET-1 mRNA was determined by RT-PCR. The results showed that EPA had the strongest inhibitory effect (p<0.05) on both basal ET-1 production and ET-1 mRNA levels. In addition, insulin (1 micromol/l) markedly increased ET-1 production, and EPA also significantly decreased the effect induced by insulin. Pretreatment with Ca2+ chelator EGTA (1 mmol/l), NOS inhibitor L-NAME (300 micromol/l), or calmodulin antagonist W-7 (300 micromol/l) inhibited NO production by EPA (100 micromol/l), but these pretreatments had no effect on ET-1 production by EPA. These findings suggest that EPA reduces basal and insulin-enhanced ET-1 production by inhibiting ET-1 mRNA production. These effects of EPA may contribute to its vasorelaxant and anti-atherosclerotic effects.