Beta-amyloid peptide induces tumor necrosis factor-alpha and nitric oxide production in murine macrophage cultures

We investigated the effect of beta-amyloid peptide (betaA) on the activation of the murine-derived monocyte/macrophage J774 cell-line. BetaA induced tumor necrotic factor-alpha (TNF alpha) in these cells in a dose-dependent manner. Incubation of cells with betaA slightly increased nitric oxide (NO) production, an effect that was significantly enhanced by the addition of interferon-gamma (IFN gamma). Substitution of betaA4 with TFN alpha and incubation of the cultures with IFN gamma resulted in significant NO production, although this was lower than that obtained in the presence of the peptide. Incubation of cultures with a monoclonal antibody (mAb) against TNF alpha abrogated NO production. Our results suggest that betaA4-induced TNF alpha production is a crucial event in the activation of peripheral macrophages.     

Mechanisms of action of endothelin-1 in rat adrenal

Displacement curves of 125I-Endothelim-1 (ET-1) binding to rat adrenal cells with unlabeled ET-1, and the ET-1 receptor-related peptides sarafotoxin and BQ-123, show that rat adrenal cortex possess, as its bovine counterpart, two different receptors to ET-1 named ET-A and ET-B. Binding of ET-1 to its rat adrenal receptors stimulates i) aldosterone production, in vivo and in vitro ii) calcium influx, which is mediated through voltage dependent- and receptor operated- calcium channels, iii) cholesterol uptake, iv) stimulation of Na+/K+-ATPase and iv) diacylglycerol production. While the last effect is mediated through ET-A receptors the others involve binding of ET-1 to ET-B receptors. Finally, ouabain potentiates the ET-1-mediated stimulation of aldosterone production, suggesting that the effect of the peptidic hormone on Na+/K+-ATPase could act as a negative feedback mechanism.     

Chemokine production and adhesion molecule expression by neural cells exposed to IL-1, TNF alpha and interferon gamma

We investigated the effect of TNF alpha, IL-1alpha and IFN gamma on two neuroblastoma (NB) cell lines (SK-N-SH and SK-N-MC). These lines responded differentially to IL-1alpha, TNF alpha and IFN gamma for MCP-1 and IL-8 production and expression of the ICAM-1 and VCAM-1 adhesion molecules. None of the cytokines induced MCP-1 or IL-8 on SK-N-MC cells. Both chemokines were produced in response to IL-1alpha by SK-N-SH cells, while TNF alpha induced mainly MCP-1 production. Addition of IFN gamma decreased IL-8, but not MCP-1 production. These responses correlated with monocyte and neutrophil chemotactic activity in NB culture supernatants. This activity was neutralized by antibodies to IL-8 and MCP-1. The expression of ICAM-1 on SK-N-MC was up-regulated by TNF alpha or IFN gamma, while IL-1alpha also upregulated ICAM-1 on SK-N-SH cells. VCAM-1 expression on SK-N-SH was induced by IL-1alpha and TNF alpha and IFN gamma synergized with TNF alpha in this respect on both NB cell lines. These results suggest that mechanisms for chemokine production and VCAM-1 and ICAM-1 upregulation by inflammatory cytokines differ and IFN gamma, in conjunction with TNF alpha, stimulate neural cell responses (high MCP-1 and VCAM-1 and decreased IL-8) favouring mononuclear cell recruitment.     

Some experiences with hospital-based psychiatric aftercare over ten years

Smooth muscle cells represent a significant percentage of the total cells in the airway but their contribution to the inflammatory response seen in airway disease has not been studied. Hence, we have looked at the release of the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in response to bacterial lipopolysaccharide (LPS) and the pro-inflammatory cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). Human airway smooth muscle (HASM) cells released GM-CSF under basal conditions (45.4 +/- 13.1 pg ml-1) that was significantly enhanced by IL-1 beta and TNF alpha with a maximal effect seen at 10 ng ml-1 (1.31 +/- 0.07 ng ml-1 and 0.72 +/- 0.16 ng ml-1, respectively). In contrast, neither LPS nor IFN gamma produced a significant increase in GM-CSF release. However, HASM cells exposed to IL-1 beta, TNF alpha and IFN gamma generated more GM-CSF than that evoked by any cytokine alone (2.2 +/- 0.15 ng ml-1). The release of GM-CSF elicited by the cytokine mixture was inhibited by cycloheximide and dexamethasone. These data suggest that HASM cells might play an active part in initiating and/or perpetuating airway inflammation in addition to controlling airway calibre.     

Modulation by endothelin-1 of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells: interaction of endothelin-1 with cytokines

The interaction of endothelin-1 (ET-1) with either interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on the release of tissue plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was investigated in a culture system of vascular endothelial cells derived from human umbilical vein. The t-PA:Ag release was significantly decreased by either IL-1 beta or TNF alpha; ET-1 intensified the suppressive effect of the cytokines. In contrast, PAI-1:Ag release was significantly increased by either IL-1 beta or TNF alpha; ET-1 significantly reduced the stimulatory effect of the cytokines. The data suggest that endothelial cell-mediated fibrinolysis may be modulated by ET-1.     

Interleukin-13 is a more potent inhibitor of the expression of inducible nitric oxide synthase in smooth muscle cells than in macrophages: a comparison with interleukin-4 and interleukin-10

This study compares the effects of interleukin (IL)-13, a cytokine with anti-inflammatory properties, with those of IL-4 or IL-10 on the expression of inducible nitric oxide synthase (iNOS) protein and activity in 1) a murine macrophage cell line (J774.2) activated with lipopolysaccharide (LPS) and 2) rat aortic smooth muscle cells (RASM) activated with LPS plus interferon-gamma. Pretreatment of macrophages with IL-4 or IL-13 caused a similar, concentration-dependent inhibition of the formation of nitrite and the expression of iNOS protein elicited by LPS. In contrast, IL-13 was a much more potent inhibitor of the formation of nitrite and the expression of iNOS protein in activated RASM than IL-4. IL-10 caused only a small, but significant, inhibition of the nitrite formation induced by LPS in macrophages and RASM. Pretreatment of J774.2 macrophages, but not of RASM, with the phosphatidylinositol-3-kinase inhibitor, wortmannin (10-100 nM), attenuated the inhibition by either IL-13 or IL-4 of the LPS-induced increase in nitrite in a dose-related fashion. Thus, IL-13 is more potent than IL-4 in preventing the expression of iNOS protein and activity in activated RASM, whereas IL-13 and IL-4 are equipotent in inhibiting the expression of iNOS protein and activity in J774.2 macrophages.     

Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin 1

The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single-cell fluorescence. Regardless of the duration of HSC culture, ET-1 caused a BQ-123-sensitive but IRL-1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETA but not ETB receptors. HSCs in early culture ("quiescent HSCs") were mildly responsive to ET-1: the ET-1 concentration required to obtain a [Ca2+]i transient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET-1 concentrations above 40 nmol/L. With culture time, -smooth muscle actin (-SMA) expression increased, as did the ET-1 sensitivity of cells, resulting in a shift of the RC50 value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET-1 sensitivity was higher in -SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]i response to extracellular uridin 5'-triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+ stores, which could be mobilized by ET-1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET-1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to -adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET-1-mobilizable Ca2+ store is contained in and is smaller than the Ca2+ pool, which is mobilized by phenylephrine or UTP.     

Tumour-derived, endocrine, exogenous and therapeutic factors differentially modulate cytokine secretion in whole blood cell culture

Following our previous results which showed that TGF-beta 1 suppressed the secretion of certain cytokines, we investigated the effects of different endogenous and exogenous factors on cytokine secretion in whole blood cell culture by using an enzyme-linked immunosorbent assay (ELISA) for measurement of cytokine concentrations. Several molecules including dexamethasone, noradrenaline (NA) and ethanol differentially inhibited mitogen-induced cytokine secretion. Dexamethasone and noradrenaline suppressed secretion of IL-2, IFN alpha, IFN gamma, TNF alpha, IL-1 alpha and IL-1 beta. beta-Endorphin and Leu-Enkephalin had no significant influence on cytokine secretion. Suppression of cytokine secretion by TGF-beta 1 was further intensified significantly and dose dependently by addition of noradrenaline. GM-CSF stimulated the secretion of IL-1 alpha, IL-1 beta and TNF gamma, but had no influence on the secretion of IL-2, IFN alpha and IFN gamma. G-CSF, IL-3 and SCF did not significantly influence secretion of all cytokines tested. Thus, endogenous and exogenous factors differentially influence cytokine secretion by immunocompetent cells.     

The cytotoxic effect of endotoxin on bone marrow cells in zinc deficient rats

The release of monokines such as Tumor Necrosis Factor a (TNF alpha), Interleukin-1 beta (IL-1 beta) and Interleukin-6 (IL-6) by activated monocytes/macrophages is an important step in the immune as well as in the inflammatory response. In this study the production of TNF alpha, IL-1 beta and IL-6 by human monocytes (HM) and peripheral blood mononuclear cells (PBMC) was evaluated after HHV-6 infection. Our results demonstrate that HHV-6 can selectively regulate monokine synthesis, in a time-dependent manner. Moreover, we observed a different response closely related to the cellular population (HM or PBMC) examined. The hypothesis we evaluated was that IFN gamma is an important factor triggering the activation of HHV-6 infected human monocytes, to release monokines.     

Interferon-gamma potentiates interleukin (IL)-6 and tumor necrosis factor-alpha but not IL-1beta induced by endotoxin in the brain

Because interferon-gamma (IFN gamma) is present in the central nervous system during neurologic diseases associated with inflammation, its effect on endotoxin-induced cytokines was studied. Cerebrospinal fluid (CSF) and serum levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF alpha), their messenger RNA expression in brain areas (hypothalamus, hippocampus, and striatum) and in spleen were evaluated 2 and 8 h after endotoxin [lipopolysaccharide (LPS), 25 microg/rat i.c.v.], IFN gamma (2.5 microg/rat i.c.v.) or after their coadministration in rats. CSF and serum IL-1beta levels were increased by LPS alone and IFN gamma coadministration did not furtherly increase them. IFN gamma potentiated LPS effect on IL-6 and TNF alpha levels in both CSF and serum. LPS and IFN-gamma coadministration did not alter IL-1beta messenger RNA expression induced by LPS in brain areas and in spleen, but it potentiated that of IL-6 and TNF alpha. The present in vivo data show that i.c.v. coadministration of LPS and IFN gamma results in a potentiation of cytokine production (IL-6 and TNF alpha) which may trigger a cascade of events relevant to neurodegenerative processes. This action is independent of IL-1beta because the production of this cytokine is not altered by IFN gamma treatment.     

Teratocarcinosarcoma of the paranasal sinuses: a clinicopathologic and immunohistochemical study

We observed endothelin (ET)-induced contractile responses on prostatic and epididymal segments, as well as the facilitation of an electrically stimulated tone on prostatic segments of isolated rat vas deferens. In both segments, the selective ET(B)-receptor agonists, IRL 1620 and sarafotoxin S6c, produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of contraction potencies (pD2 value) was ET-1 = ET-2 > ET-3 > sarafotoxin S6c = IRL 1620. The maximum responses of ET-induced contractions in the prostatic segments were larger than those in the epididymal segments. The contractile response to ET-3 was antagonized by pretreatment for 30 min with BQ-123 (10 nM), a selective ET(A) receptor antagonist, and BQ-788 (1 microM), a selective ET(B) receptor antagonist. The contractile responses to ET-1 were antagonized by pretreatment with BQ-123 (10 microM), but not with BQ-788 (1 microM). The ET-3-induced facilitation on the twitch response to electrical stimulation in the prostatic segment of the vas deferens was antagonized by BQ-123 (0.1 microM) and BQ-788 (1 microM). The ET-1-induced facilitation was antagonized by pretreatment with BQ-123 (3 microM), but not with BQ-788 (10 microM). These results suggest that in rat vas deferens the ET(A) receptors are divided into BQ-123-sensitive ET(A1) and BQ-123-insensitive ET(A2) subtypes, and the production of a contractile response of smooth muscle as well as the facilitation of neurotransmission are accomplished through mediation by ET(A1)- and ET(A2)-subtypes.     

Cloning of Drosophila MCM homologs and analysis of their requirement during embryogenesis

Modification of blood flow by endothelin-1 (ET-1) was examined in the s.c. HSN fibrosarcoma and compared to normal tissues of anaesthetised CBH/CBi rats. The ET receptor subtypes involved in the response were investigated using the ET(A) and ET(B) receptor antagonists BQ-610 and BQ-788, respectively. Blood flow and vascular resistance were determined using the uptake of radiolabelled iodo-antipyrine (125I-IAP). BQ-610 or BQ-788 was infused for 30 min prior to blood flow determination. ET-1 was administered 15 min into the infusion time. BQ-610 and BQ-788 infused alone did not modify any vascular parameters. Tumour blood flow increased slightly following ET-1, contrasting with most normal tissues, in which blood flow was reduced. Vascular resistance increased in all tissues, including the tumour. Neither antagonist significantly modified the ET-1-induced changes in tumour blood flow or vascular resistance, whereas in the majority of normal tissues BQ-610 attenuated and BQ-788 potentiated the vascular resonse to ET-1. Our results show that the HSN tumour vasculature is only weakly responsive to ET- 1 and antagonism of its effects by BQ-610 and BQ-788. This contrasts with the majority of normal tissues, in which ET- 1 induces an intense vasoconstriction.     

Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma

Endothelins (ETs) are potent bronchoconstrictor agents postulated to contribute to the pathophysiology of asthma and other respiratory disorders. An increase in both the expression and release of immunoreactive (ir) ETs was reported in bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic patients. We investigated whether dexamethasone (DEX), a potent anti-inflammatory and anti-asthmatic drug, regulates the basal and stimulated release of ETs from guinea-pig cultured tracheal epithelial cells. These airway epithelial cells spontaneously release ET-1 over 24 h. When incubated in the presence of 10(-7) and 10(-6) M DEX for 24 h, basal production of ET-1 decreased by 32 and 29%. Lipopolysaccharide (LPS; 1, 5, 10 micrograms/mL), tumor necrosis factor-alpha (TNF alpha; 5, 10 ng/mL), and interleukin-1 beta (IL-1 beta; 1, 5 ng/mL) significantly increased the basal release of ET-1 after 24 h. When these cells were pretreated with DEX (10(-7) M) for a 24-h period, then incubated in the presence of LPS (10 micrograms/mL), TNF alpha (10 ng/mL), or IL-1 beta (1 ng/mL) for another 24 h, the stimulated release of ET-1 was inhibited by 48, 31, and 38%, respectively. At 10(-6) M, DEX decreased the stimulated release by 45, 37, and 46%, respectively. The present results show that DEX can regulate the basal release and inhibit the pro-inflammatory cytokine-stimulated production of ET-1 from guinea-pig cultured tracheal epithelial cells. They suggest that the beneficial effect of glucocorticoids in asthma may be related to the inhibition of ET synthesis.     

The contribution of "time-of-flight" MRI-angiography in the study of neurovascular interactions (hemifacial spasm and trigeminal neuralgia)

Rat tracheal epithelial cells were cultured and the effects of LPS and TNF alpha on cell morphology, rate of proliferation and NO synthase activity were studied. NO synthase activity was determined by measuring the accumulation of 3H-L-citrulline during incubation of confluent monolayer with 3H-L-arginine. In untreated cells no significant 3H-L-citrulline formation was detected, and bradykinin and the calcium ionophore A 23187 failed to stimulate 3H-L-citrulline formation excluding a constitutively expressed, calcium-dependent NO synthase activity. After culturing the cells for 18 h in the presence of LPS (10 micrograms/ml) and TNF alpha (500 U/ml) a marked formation of 3H-L-citrulline could be detected, which was largely inhibited by N(G)-monomethyl-L-arginine (L-NMMA) indicating the induction of NO synthase activity which could be prevented by dexamethasone. Exposure of confluent monolayer to LPS and TNF alpha for up to 4 days resulted in a reduction in cell density by 20% within 1 to 2 days and in additional marked changes in cell morphology. The normal honeycomb-like structure of the culture was lost and a considerable number of cells developed "dendritic' outgrowths. These morphological changes as well as the reduction in cell density was attenuated by dexamethasone, but not by the NO synthase inhibitor L-NMMA. The rate of cell proliferation was determined in non-confluent cultures 24 h after passage by determination of the incorporation of tritium into DNA during 24 h of incubation with 3H-thymidine. 3H-thymidine incorporation was reduced by about 40-45% when LPS or TNF alpha was present during exposure to 3H-thymidine, and by about 65%, when LPS and TNF alpha were present in combination. Neither L-NMMA nor dexamethasone significantly affected the 3H-thymidine incorporation nor the inhibitory effects of LPS and TNF alpha. In conclusion, airway epithelial cells are markedly affected by LPS and TNF alpha and the various responses (changes in the cell morphology, inhibition of cell proliferation and induction of NO synthase activity) appear to be caused by different (dexamethasone-sensitive and -insensitive), cellular mechanisms. An enhanced formation of endogenous NO may not be responsible for the observed morphological changes or the inhibition of cell proliferation.     

Dendritic cell development: multiple pathways to nature''s adjuvants

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca2+]i in Bergmann glial cells in cerebellar slices acutely isolated from 20-25 day-old mice. The intracellular calcium concentration ([Ca2+]i) was monitored using Fura-2-based [Ca2+]i microfluorimetry. The ET-triggered [Ca2+]i transients were mimicked by ETB receptor agonist BQ-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca(2+)-free extracellular solution and the ET-triggered [Ca2+]i elevation was blocked by 500 nM thapsigargin indicating that the [Ca2+]i was released from InsP3-sensitive intracellular pools. The ET-triggered [Ca2+]i increase in Ca(2+)-free solution was shorter in duration. Restoration of normal extracellular [Ca2+] briefly after the ET application induced a second [Ca2+]i increase indicating the presence of a secondary Ca2+ influx which prolongs the Ca2+ signal. Pre-application of 100 microM ATP or 10 microM noradrenaline blocked the ET response suggesting the involvement of a common Ca2+ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca2+ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ETB receptors which induce the generation of intracellular [Ca2+]i signals by activation of Ca2+ release from InsP3-sensitive intracellular stores followed by a secondary Ca2+ influx.     

Research on the mechanism of endothelin inflammatory effects on human mesangial cells

OBJECTIVE: To investigate the mechanism of endothelin (ET) inflammatory effects on human mesangial cells (HMC). METHODS: The following experiments were performed on cultured HMC after ET-1 stimulation: (1) the expression of tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) itself messenger ribonucleic acid (mRNA) was determined by Northern Blot analysis; (2) the TNF alpha concentration was tested with radioimmunoassay; the IL-1 activity was assayed by the enhancement of thymocyte proliferation in response to mitogen; the surface expression of ICAM-1 and VCAM-1 was measured with cell enzyme linked immunoadsorbent assay (ELISA) analysis. RESULTS: ET-1 (10(-7) mol/L) induced the following changes on HMC: (1) up-regulation of the expression of TNF alpha mRNA and protein; (2) up-regulation of the expression of ICAM-1 and VCAM-1 mRNA and protein; (3) up-regulation of the expression of ET-1 itself mRNA. However, the expression of IL-1 mRNA and protein was not changed. CONCLUSIONS: ET-1 can stimulate HMC to produce TNF alpha, ICAM-1 and VCAM-1, and thereby induce inflammatory effects. ET-1 can also stimulate HMC to up-regulate the expression of ET-1 itself, so as to amplify inflammatory effects. So, ET-1 is actually an inflammatory mediator and may play an important role in the pathogenesis of glomerulonephritis.