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1.
Am J Physiol Renal Physiol ; 285(4): F748-57, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12799307

ABSTRACT

The progression of renal disease correlates strongly with hypertension and the degree of proteinuria, suggesting a link between excessive Na+ reabsorption and exposure of the proximal tubule to protein. The present study investigated the effects of albumin on cell growth and Na+ uptake in primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml) increased cell proliferation to 134.1 +/- 11.8% (P < 0.001) of control levels with no change in levels of apoptosis. Exposure to 0.1 and 1.0 mg/ml albumin increased total 22Na+ uptake to 119.1 +/- 6.3% (P = 0.005) and 115.6 +/- 5.3% (P < 0.006) of control levels, respectively, because of an increase in Na+/H+ exchanger isoform 3 (NHE3) activity. This was associated with an increase in NHE3 mRNA to 161.1 +/- 15.1% (P < 0.005) of control levels in response to 0.1 mg/ml albumin. Using confocal microscopy with a novel antibody raised against the predicted extracellular NH2 terminus of human NHE3, we observed in nonpermeabilized cells that exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cell surface to 115.4 +/- 2.7% (P < 0.0005) and 122.4 +/- 3.7% (P < 0.0001) of control levels, respectively. This effect was paralleled by significant increases in NHE3 in the subplasmalemmal region as measured in permeabilized cells. These albumin-induced increases in expression and activity of NHE3 in PTC suggest a possible mechanism for Na+ retention in response to proteinuria.


Subject(s)
Albumins/pharmacology , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/metabolism , Sodium-Hydrogen Exchangers/metabolism , Apoptosis/drug effects , Cell Division/drug effects , Cells, Cultured , Fluorescent Antibody Technique , Humans , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/physiology , Microscopy, Confocal , Osmolar Concentration , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sodium/pharmacokinetics , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/genetics
2.
Eur J Med Res ; 6(10): 422-32, 2001 Oct 29.
Article in English | MEDLINE | ID: mdl-11698229

ABSTRACT

Filtered proteins play a role in the pathogenesis of renal interstitial inflammation and fibrosis. At least part of these effects are mediated by the nuclear factor kappaB (NF-kappaB). Receptor-mediated endocytosis of proteins like albumin in renal proximal tubular cells is in part dependent on Na superset+/H superset+ exchanger (NHE) isoform 3. We tested the hypothesis that pharmacological inhibition of NHE-3 reduces albumin-induced NF-kappaB activation - and therefore albumin-induced renal interstitial inflammation and fibrosis - using established proximal tubular cell lines (OK and LLC-PK1). 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) or HOE694 were used to inhibit NHE. Albumin endocytosis was determined by fluorometric analysis of FITC-BSA uptake. Electromobility gel shift assays were performed in order to determine the NF-kappaB-specific DNA-binding activity. EIPA reduced albumin uptake in OK and LLC-PK1 cells and HOE694 decreased albumin uptake in LLC-PK1 cells, with IC subset50 values corresponding to NHE-3 inhibition. Furthermore, albumin-induced increases in NF-kappaB DNA-binding activity were partially inhibited by EIPA in OK and LLC-PK1 cells. HOE694 at a concentration of 100 micromol/l similarly decreased albumin-induced NF-kappaB DNA-binding activity. Cytosolic acidification by propionic acid did not prevent BSA-induced activation of NF-kappaB. Inhibition of BSA endocytosis by chlorpromazine decreased NF-kappaB activation. NHE-dependent albumin endocytosis induces an increase in NF-kappaB-specific protein activity in renal proximal tubular cells in culture, which is decreased by EIPA and HOE694. Thus, inhibition of albumin uptake might be a therapeutical strategy to prevent albumin-induced NF-kappaB activation and albumin-associated inflammatory or fibrotic renal pathomechanisms in vivo.


Subject(s)
Amiloride/analogs & derivatives , Kidney Tubules, Proximal/metabolism , NF-kappa B/metabolism , Serum Albumin, Bovine/metabolism , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Amiloride/pharmacology , Animals , Cell Line , Chlorpromazine/pharmacology , DNA/metabolism , Endocytosis/drug effects , Guanidines/pharmacology , LLC-PK1 Cells , Opossums , Serum Albumin, Bovine/pharmacology , Sulfones/pharmacology , Swine , Tetradecanoylphorbol Acetate/pharmacology
3.
Eur J Med Res ; 6(6): 247-58, 2001 Jun 28.
Article in English | MEDLINE | ID: mdl-11432793

ABSTRACT

Albuminuria represents one of the most unfavourable diagnostic factors for the prognosis of nephropathies. We investigated albumin-induced NF-kappaB activation and the potential contribution of tyrosine kinase and protein kinase C. Therefore we exposed proximal tubule-derived human (IHKE-1), opossum (OK) and porcine (LLC-PK1) cell lines to serum albumin at concentrations of 10 - 500 mg/l. DNA-binding activity of NF-kappaB increased concentration-dependently in the presence of albumin. In OK and LLC-PK1 cells, NF-kappaB activity increased during the first 45 min and reached a plateau thereafter. In IHKE-1, cells NF-kappaB activity reached a plateau after 90 min with a maximum at 180 min exposure to albumin. The albumin-induced increase in NF-kappaB DNA-binding activity was inhibited by herbimycin A (tyrosine kinase inhibitor) and BIM (protein kinase C inhibitor). Reporter gene assays demonstrated that albumin stimulates NF-kappaB mediated reporter gene activation in LLC-PK1 cells, which was partially inhibited by herbimycin A and BIM. Our data indicate, that albumin exposure induces a rapid increase in NF-kappaB protein activity in renal proximal tubule cells of different species via a tyrosine kinase- and protein kinase C-dependent pathway, at concentrations occurring during mild glomerular injury.


Subject(s)
Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , NF-kappa B/metabolism , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Serum Albumin, Bovine/pharmacology , Albuminuria/metabolism , Animals , Base Sequence , Benzoquinones , Cell Line , DNA/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Genes, Reporter/drug effects , Humans , Indoles/pharmacology , Kinetics , LLC-PK1 Cells , Lactams, Macrocyclic , Maleimides/pharmacology , NF-kappa B/genetics , Opossums , Protein Kinase C/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinones/pharmacology , Rifabutin/analogs & derivatives , Serum Albumin, Bovine/administration & dosage , Species Specificity , Swine , Transcriptional Activation
4.
Kidney Int ; 58(4): 1523-33, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11012887

ABSTRACT

BACKGROUND: Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule. METHODS: In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting. RESULTS: OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA. CONCLUSION: The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.


Subject(s)
Albumins/pharmacokinetics , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Animals , Binding, Competitive , Biological Transport/drug effects , Biological Transport/physiology , Cell Membrane/chemistry , Cell Membrane/metabolism , Cells, Cultured , Endocytosis/drug effects , Endocytosis/physiology , Epithelial Cells/chemistry , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Fluorescein-5-isothiocyanate/analysis , Fluorescein-5-isothiocyanate/pharmacokinetics , Gene Products, vpr/pharmacology , Heymann Nephritis Antigenic Complex , Immunohistochemistry , Intrinsic Factor/pharmacology , Membrane Glycoproteins/analysis , Microscopy, Immunoelectron , Opossums , Receptors, Cell Surface/analysis , Serum Albumin, Bovine/analysis , Serum Albumin, Bovine/pharmacokinetics , Vitamin B 12/pharmacology
5.
Pflugers Arch ; 440(4): 521-9, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10958336

ABSTRACT

The ubiquitous nephritogenic and carcinogenic fungal metabolite ochratoxin A (OTA) has been shown to interact with renal cell function at low nanomolar concentrations. This is possibly brought about through changes in cellular pH (pHc) homeostasis and mitochondrial function. We assessed the effect of nanomolar concentrations of OTA on pHc homeostasis and the possible involvement of mitochondria using immortalized human kidney epithelial (IHKE1) cells. Within seconds OTA evoked a decrease of pHc with a threshold concentration of 0.1 nmol/l, followed by a sustained alkalinization. Acidification was the same in bicarbonate and non-bicarbonate Ringer solution. When Ca2+ entry across the plasma membrane was prevented, virtually no OTA-induced pH changes could be observed. Inhibition of Na+/H+-exchange (NHE, Na+-free solution) and H+-ATPase (bafilomycin A1) did not reduce the OTA-induced acidification. By contrast, determination of NHE activity as a function of pHc revealed that OTA stimulates NHE (maximal flux increases) in a Ca2+-dependent manner. OTA exposure did not increase lactic acid production, indicating that anaerobic glycolysis was not enhanced. Inhibiting complexes I, III and IV of the mitochondrial electron transport chain (ETC) with rotenone, antimycin A and CN- prevented the OTA-induced acidification almost completely. Completely inhibiting F1FO-ATPsynthase with oligomycin reduced the effect of OTA by approximately equal 50%. In addition, OTA induced a hyperpolarization of the mitochondrial membrane potential (psim) in a Ca2+-dependent manner. Furthermore, OTA exposure resulted in a mitochondria-dependent increase of the cellular ATP content. We conclude that OTA activates mitochondria and NHE by interfering with cellular Ca2+ homeostasis. Stimulation of mitochondrial metabolism leads to enhanced "proton production". Anaerobic glycolysis is not enhanced.


Subject(s)
Kidney/metabolism , Mitochondria/drug effects , Ochratoxins/pharmacology , Antimycin A/pharmacology , Calcium/metabolism , Calcium/pharmacology , Cell Line, Transformed , Cyanides/pharmacology , Cytosol/metabolism , Electron Transport/drug effects , Enzyme Inhibitors/pharmacology , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Glycolysis , Homeostasis , Humans , Hydrogen-Ion Concentration , Kidney/ultrastructure , Lactic Acid/metabolism , Mitochondria/physiology , Ochratoxins/administration & dosage , Ochratoxins/toxicity , Oligomycins/pharmacology , Proton-Translocating ATPases/antagonists & inhibitors , Rotenone/pharmacology , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sodium-Hydrogen Exchangers/metabolism
6.
Respiration ; 67(3): 291-7, 2000.
Article in English | MEDLINE | ID: mdl-10867598

ABSTRACT

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.


Subject(s)
Air Pollutants/adverse effects , Blood Proteins/metabolism , Bronchi/immunology , Carbon/adverse effects , Epithelial Cells/metabolism , Inflammation Mediators/analysis , Monocytes/immunology , Adult , Bronchi/cytology , Cells, Cultured , Coculture Techniques , Cytokines/analysis , Cytokines/genetics , Enzyme-Linked Immunosorbent Assay , Female , Humans , Interleukin-6/analysis , Interleukin-8/analysis , Male , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity
7.
Scand J Work Environ Health ; 26(1): 71-7, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10744181

ABSTRACT

OBJECTIVES: In lungs of asbestos-exposed persons alveolar and interstitial macrophages are able to release genotoxic substances such as reactive oxygen intermediates. It is unknown whether reactive oxygen intermediates released by macrophages are able to induce DNA (deoxyribonucleic acid) strand lesions in neighboring bronchial epithelial cells. METHODS: A co-culture (transwell) system was established which allows exposure of human blood monocytes cultured on a polycarbonate membrane within a distance of 1 mm of a monolayer of the bronchial epithelial cell line BEAS-2B. RESULTS: Exposure of blood monocytes to chrysotile B (100 microg/10(6)cells) caused an up to 2.8-fold increase in DNA strand lesions in co-cultured BEAS-2B cells measured by alkaline elution when compared with the levels of control cells after 1, 3, 24, and 48 hours. The main DNA damage thus occurred as early as within 1 hour of incubation, corresponding to the time course of the release of reactive oxygen intermediates by blood monocytes determined by chemiluminescence. The maximum release of reactive oxygen intermediates (3.2-fold increase over control values) was measured after 30 minutes of exposure of blood monocytes to chrysotile B. The addition of catalase (200 U/ml) or desferoxamine (100 microM) to the culture medium blocked almost completely the induction of DNA strand lesions in this system (maximum 85%). CONCLUSIONS: Exposure of blood monocytes to chrysotile B results in an increase in the release of reactive oxygen intermediates and induces DNA strand lesions in neighboring bronchial epithelial cells.


Subject(s)
Asbestos, Serpentine/toxicity , Bronchi/drug effects , DNA Damage , DNA/drug effects , Monocytes/drug effects , Adult , Antioxidants/pharmacology , Bronchi/cytology , Cell Line , Coculture Techniques , Epithelial Cells/cytology , Epithelial Cells/drug effects , Female , Humans , Luminescent Measurements , Male , Monocytes/metabolism , Reactive Oxygen Species
8.
J Biol Chem ; 274(51): 36774-80, 1999 Dec 17.
Article in English | MEDLINE | ID: mdl-10593986

ABSTRACT

14-3-3 proteins play a regulatory role in a diverse array of cellular functions such as apoptosis, regulation of the cell cycle, and regulation of gene transcription. The phytotoxin fusicoccin specifically induces association of virtually any 14-3-3 protein to plant plasma membrane H(+)-ATPase. The 14-3-3 binding site in the Arabidopsis plasma membrane H(+)-ATPase AHA2 was localized to the three C-terminal residues of the enzyme (Tyr(946)-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr(947) (K(D) = 88 nM) and was in practice irreversible in the presence of fusicoccin (K(D) = 7 nM). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr(947). We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.


Subject(s)
Arabidopsis/metabolism , Proteins/metabolism , Proton-Translocating ATPases/metabolism , Signal Transduction , Tyrosine 3-Monooxygenase , 14-3-3 Proteins , Amino Acid Sequence , Cell Membrane/metabolism , Molecular Sequence Data , Phosphorylation , Threonine , Tyrosine , Valine
9.
J Physiol ; 520 Pt 3: 709-21, 1999 Nov 01.
Article in English | MEDLINE | ID: mdl-10545138

ABSTRACT

1. Receptor-mediated endocytosis is an important mechanism for transport of macromolecules and regulation of cell-surface receptor expression. In renal proximal tubules, receptor-mediated endocytosis mediates the reabsorption of filtered albumin. Acidification of the endocytic compartments is essential because it interferes with ligand-receptor dissociation, vesicle trafficking, fusion events and coat formation. 2. Here we show that the activity of Na+-H+ exchanger isoform 3 (NHE3) is important for proper receptor-mediated endocytosis of albumin and endosomal pH homeostasis in a renal proximal tubular cell line (opossum kidney cells) which expresses NHE3 only. 3. Depending on their inhibitory potency with respect to NHE3 and their lipophilicity, the NHE inhibitors EIPA, amiloride and HOE694 differentially reduced albumin endocytosis. The hydrophilic inhibitor HOE642 had no effect. 4. Inhibition of NHE3 led to an alkalinization of early endosomes and to an acidification of the cytoplasm, indicating that Na+-H+ exchange contributes to the acidification of the early endosomal compartment due to the existence of a sufficient Na+ gradient across the endosomal membrane. 5. Exclusive acidification of the cytoplasm with propionic acid or by removal of Na+ induced a significantly smaller reduction in endocytosis than that induced by inhibition of Na+-H+ exchange. 6. Analysis of the inhibitory profiles indicates that in early endosomes and endocytic vesicles NHE3 is of major importance, whereas plasma membrane NHE3 plays a minor role. 7. Thus, NHE3-mediated acidification along the first part of the endocytic pathway plays an important role in receptor-mediated endocytosis. Furthermore, the involvement of NHE3 offers new ways to explain the regulation of receptor-mediated endocytosis.


Subject(s)
Endocytosis/physiology , Kidney Tubules, Proximal/physiology , Receptors, Cell Surface/physiology , Serum Albumin/metabolism , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Binding Sites/drug effects , Cell Membrane/metabolism , Cytosol/drug effects , Cytosol/metabolism , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Epithelial Cells/physiology , Guanidines/pharmacology , Hydrogen-Ion Concentration/drug effects , Kidney Tubules, Proximal/cytology , Lysosomes/drug effects , Lysosomes/metabolism , Opossums , Propionates/pharmacology , Sodium/metabolism , Sulfones/pharmacology
10.
Lung ; 177(6): 343-54, 1999.
Article in English | MEDLINE | ID: mdl-10541885

ABSTRACT

Alveolar macrophages (AM) play a decisive role in the immunologic defense system of the lung and in inflammatory pulmonary pathomechanisms. AM and blood monocytes (BM) were exposed to chrysotile B, soot FR 101, and Printex 90 (P 90). We evaluated the reactive oxygen intermediate (ROI) release of AM and BM after particle exposure. ROI release was measured by chemiluminescence. Thirty-minute exposure caused a significant (up to 2.5-fold) increase in ROI release of AM (100 micrograms/10(6) cells) compared with control experiments (p < 0.01). Identical exposure conditions for BM resulted in a similar reaction pattern (maximum 2.2-fold increase in ROI release; p < 0.05). After a 90-min particle exposure at concentrations of 10 and 100 micrograms/10(6) cells, we investigated the steady-state level of p50/p105 mRNA encoding for the precursor protein of the p50 subunit of nuclear factor kappa B (NF-kappa B) by semiquantitative reverse transcription-polymerase chain reaction. One hundred micrograms Chrysotile B, FR 101, or P 90 induced a significant maximum 4.0-fold up-regulation of NF-kappa B gene expression in AM and a 3.3-fold up-regulation in BM (p < 0.05). The addition of superoxide dismutase (200 U/ml) to particle- and fiber-exposed macrophages resulted in inhibition of ROI release and a decrease in NF-kappa B mRNA expression (70%). NF-kappa B is an important transcription factor involved in the regulation of numerous genes (e.g., for inflammatory cytokines, and cytokine receptors). These cytokines are supposed to be involved in inflammatory pathomechanisms in bronchial epithelial cells, which result, for example, in chronic obstructive pulmonary disease. Our results suggest that particle-induced ROI release is associated with an increase in NF-kappa B (p50/p105) mRNA steady-state level.


Subject(s)
Asbestos, Serpentine/adverse effects , Carbon/adverse effects , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/metabolism , NF-kappa B/drug effects , RNA, Messenger/drug effects , Reactive Oxygen Species/metabolism , Adult , Aged , Asbestos, Serpentine/administration & dosage , Bronchoalveolar Lavage Fluid/cytology , Carbon/administration & dosage , Cells, Cultured , Dose-Response Relationship, Drug , Female , Gene Expression Regulation/drug effects , Humans , Luminescent Measurements , Male , Middle Aged , Monocytes/drug effects , Monocytes/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
11.
Eur J Med Res ; 4(7): 257-63, 1999 Jul 28.
Article in English | MEDLINE | ID: mdl-10425262

ABSTRACT

Some pulmonary diseases like bronchitis or asthma bronchiale are mediated by inflammatory mechanisms in bronchial epithelial cells. Alveolar macrophages are located directly in the surrounding of these cells, so that we suppose an interaction between epithelial cells and macrophages regarding to the release of inflammatory mediators. For measuring the contribution of macrophages to the release of inflammatory mediators by bronchial epithelial cells, we established an in vitro model of co-cultured blood monocytes (BM) and BEAS-2B cells in a transwell system (Costar). BM were exposed to Chrysotile B and soot particle FR 101 in a concentration of 100 microg/10(6) cells. After up to 90 min exposure time ELISA, EMSA (electromobility shift assay) and RT-PCR were used to measure protein tyrosine kinase activity, protein activity of NF-kappaB and cytokine (IL-1beta, IL-6, TNF-alpha) specific mRNA levels in BEAS-2B cells. We observed an increase in protein tyrosine kinase activity (up to 1.8 +/- 0.5-fold) and NF-kappaB protein activity in BEAS-2B cells after particle or fibre exposure of co-cultured BM. Consecutive IL-1beta-, IL-6- and TNF-alpha-mRNA were elevated (up to 1.9 +/- 0.58-fold). Protein tyrosine kinase activity, NF-kappaB activity, and the synthesis of cytokine-specific mRNA were inhibited by antioxidants. These data suggest a ROI-dependent NF-kappaB mediated transcription of inflammatory cytokines in bronchial epithelial cells.


Subject(s)
Bronchi/metabolism , Cytokines/genetics , Monocytes/metabolism , NF-kappa B/metabolism , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/genetics , Reactive Oxygen Species , Adult , Asbestos, Serpentine , Base Sequence , Bronchi/cytology , Bronchi/enzymology , Carbon , Coculture Techniques , DNA Primers , Epithelial Cells/enzymology , Epithelial Cells/metabolism , Female , Humans , Inflammation Mediators/metabolism , Male , Monocytes/enzymology , RNA, Messenger/metabolism
12.
Eur J Med Res ; 4(2): 59-66, 1999 Feb 25.
Article in English | MEDLINE | ID: mdl-10066641

ABSTRACT

Soot particles, asbestos fibres and irritant gas are common air pollutants which are able to induce lung and airway pulmonary injury. The aim of this study was to investigate the effect of a simultaneous NO2 and particle or fibre exposure on the proinflammatory specific mRNA expression and protein secretion of human alveolar macrophages (AM) in comparison to only particle or fibre exposed AM. AM were simultaneously exposed to FR 101, P 90, TiO2 or Chrysotile B at a concentration of 100 microg/10(6) cells and to NO2 at a concentration of 1.0 ppm for 30 min. Particle or fibre exposure of the AM was continued in humidified air at 5% CO2 and 37 degrees C for an additional hour (harvesting of total RNA) or additional 7 hrs (harvesting of culture supernatant). The mRNA expression of the proinflammatory cytokines IL-1beta, IL-6, IL-8 and TNF-alpha of NO2-particle/fibre co-exposed AM and only particle or fibre exposed AM was detected using specific RT-PCR. IL-1beta-, IL-6-, IL-8- and TNF-alpha-specific protein secretion was measured by ELISA. Cytotoxicity was detected by lactatedehydrogenase quantification in the culture supernatant. We observed an increased IL-1beta-, IL-6-, IL-8- and TNF-alpha-specific mRNA expression of particle or fibre exposed AM, which was decreased after an additional NO2 exposure. Also the particle or fibre exposure induced significant increase in IL-1beta-, IL-6-, IL-8 and TNF-alpha-release of AM which was decreased after an additional NO2 exposure (p <0.031). The relative cytotoxicity of the NO2-particle/fibre co-exposure was higher than the particle or fibre induced cytotoxicity, but mostly <10%. Therefore it is concluded that particle or fibre exposure may result in an increase in proinflammatory cytokine release by AM, which may be decreased by toxic NO2 due to the oxidative potential (e.g. lipidperoxydation) of this irritant gas. Particle, asbestos fibre and irritant gas exposure may induce airway and pulmonary injury by the activation of AM and consecutive proinflammatory cytokine release.


Subject(s)
Air Pollutants/pharmacology , Asbestos, Serpentine/pharmacology , Cytokines/biosynthesis , Gene Expression Regulation/drug effects , Irritants/pharmacology , Macrophages, Alveolar/drug effects , Nitrogen Dioxide/pharmacology , RNA, Messenger/biosynthesis , Titanium/pharmacology , Aged , Air Pollutants/toxicity , Asthma/etiology , Bronchial Neoplasms/pathology , Bronchoalveolar Lavage Fluid , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Small Cell/pathology , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Drug Synergism , Female , Humans , Inflammation , Interleukin-1/biosynthesis , Interleukin-1/genetics , Interleukin-1/metabolism , Interleukin-6/biosynthesis , Interleukin-6/genetics , Interleukin-6/metabolism , Interleukin-8/biosynthesis , Interleukin-8/genetics , Interleukin-8/metabolism , Irritants/toxicity , Lung Neoplasms , Macrophages, Alveolar/metabolism , Male , Middle Aged , Nitrogen Dioxide/toxicity , Particle Size , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
13.
Lung ; 177(1): 9-19, 1999.
Article in English | MEDLINE | ID: mdl-9835630

ABSTRACT

Indoor air pollutants may cause inflammatory changes of the airways and adjacent pulmonary tissue. After phagocytosis of inhaled particles alveolar macrophages (AM) release chemotactic mediators capable of attracting inflammatory cells into the lung tissue. To evaluate these mechanisms further peripheral blood mononuclear cells (PBMNC) and human AM (freshly recovered from the lower respiratory tract) were exposed to the indoor particles Soot FR 101 and Printex 90, the asbestos fiber Chrysotile B, and titanium dioxide (TiO2) at concentrations of 10 or 50 microg/10(6) cells for up to 8 h. The migration of granulocytes into the conditioned supernatants of AM and PBMNC was quantified by chemotaxis assay in a Boyden chamber. Granulocyte migration increased by 42.3 +/- 25.8% (Chrysotile B), 64. 6 +/- 18.3% (FR 101), 74.2 +/- 16.5% (P 90), and 86.7 +/- 25.6% (TiO2) in AM-conditioned supernatants (p < 0.05). Qualitative, Interleukin (IL)-8 specific reverse transcriptase-polymerase chain reaction was performed after exposure of AM or PBMNC to Chrysotile B, FR 101, P 90, and TiO2 at concentrations of 10 and 50 microg/10(6) cells for 90 min. Each of the tested particles caused an increase in IL-8-specific mRNA expression of AM or PBMNC after particle exposure compared with the unexposed control. To find out if IL-8, the most powerful granulocyte chemokine, is involved, supernatants were preincubated with anti-IL-8. Granulocyte migration decreased by up to 35 +/- 15% (50 ng/ml anti-IL-8) and 41.5 +/- 16% (100 ng/ml anti-IL-8) (p < 0.0625) in AM-conditioned supernatants. Pretreatment of the granulocytes with human IL-8 decreased by up to 59 +/- 18% (10 ng/ml) (p < 0.0625) in AM-conditioned supernatants. Similar reaction patterns were observed using anti-IL-8-pretreated supernatants of particle-exposed PBMNC. In conclusion, indoor air pollutants may promote inflammatory changes in the lung via IL-8 release by alveolar macrophages.


Subject(s)
Air Pollutants/pharmacology , Interleukin-8/biosynthesis , Macrophages, Alveolar/drug effects , Adult , Aged , Air Pollution, Indoor , Bronchoalveolar Lavage Fluid/cytology , Chemotaxis, Leukocyte/drug effects , Female , Humans , Leukocytes, Mononuclear/drug effects , Macrophages, Alveolar/metabolism , Male , Polymerase Chain Reaction , RNA, Messenger/genetics
14.
Eur J Med Res ; 3(9): 432-8, 1998 Sep 17.
Article in English | MEDLINE | ID: mdl-9737890

ABSTRACT

Soot FR 101, Printex 90 and Chrysotile B are frequently found in indoor air pollutants phagocytized by alveolar macrophages (AM) involved in inflammatory pulmonary processes as, e.g. in cytokine secretions. The transcription factor NF-kappaB has a role in the trans-duction pathway of proinflammatory cytokines like IL-1beta, IL-6 and TNF-alpha. We therefore investigated whether the transcription factor NF-kappaB and subsequent inflammatory cytokine secretions by AM are induced by exposure to these particles compared to the inert TiO2. AM were incubated for 90 min at particle concentrations of up to 100 microg/10(6) cells. Sequential reverse transcription and semiquantitative cDNA amplification (RT-PCR) was used to measure NF-kappaB and cytokine mRNA expressions. Compared to control exposures these particles induced an up to 4.6-fold increase in gene expression of the transcription factor NF-kappaB (p < 0.01), resulting in up to 12.9-fold enhanced transcription rates of IL-1beta, IL-6 and TNF-alpha (p <0.05). The particles and fibre dependent increases in mRNA reached maximum levels at 90 min post exposure. After an exposure time of 8 hrs, IL-1beta, IL-6 and TNF-alpha proteins, measured by enzyme-linked immunosorbent assays (ELISA), were significant elevated in supernatants of AM, revealing an up to 30.5-fold increase in TNF-alpha secretion rates (p <0.01). Our results suggest that exposure of human AM to soot FR 101, Printex 90, TiO2 and Chrysotile B induce the transcription and production of proinflammatory cytokines via NF-kappaB and may play an important role in the pathogenesis of airway disease and lung parenchymal injury.


Subject(s)
Asbestos, Serpentine/toxicity , Carbon/toxicity , Cytokines/genetics , Macrophages, Alveolar/drug effects , Titanium/toxicity , Transcription, Genetic , Enzyme-Linked Immunosorbent Assay , Humans , In Vitro Techniques , Inflammation Mediators/metabolism , Macrophages, Alveolar/metabolism , NF-kappa B/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
15.
Eur J Med Res ; 1(9): 425-8, 1996 Jun 25.
Article in English | MEDLINE | ID: mdl-9353242

ABSTRACT

Activated monocytes secrete tumor necrosis factor-alpha (TNF-alpha), whose inflammatory and fibroblast activating characteristics may play a role in the maintenance of pulmonary inflammatory processes and subsequent fibrosis. Human alveolar macrophages (AM) and peripheral blood mononuclear cells (PBMNC) were exposed to soot particles or asbestos fibres in concentrations ranging from 5-50 micrograms/1 x 10(6) cells for 8 hrs in RPMI medium. A culture was established with the exposed monocytes and the remaining cells were used to determine TNF-alpha. TNF-alpha was quantified by commercial ELISA-kits. 8 hrs exposure to soot particles and asbestos fibres induced a significant increase in spontaneous TNF-alpha release (p < 0.05). Cytotoxicity of monocytes was checked by trypan blue exclusion and lactate dehydrogenase assay, noted values ranging from 0.5%-16.2%.


Subject(s)
Asbestos/toxicity , Carbon/toxicity , Macrophages, Alveolar/immunology , Monocytes/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Bronchoalveolar Lavage Fluid/cytology , Cell Survival/drug effects , Cells, Cultured , Female , Humans , Lung Neoplasms/surgery , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/pathology , Male , Middle Aged , Mineral Fibers , Monocytes/drug effects , Monocytes/pathology , Neoplasm Staging , Tumor Necrosis Factor-alpha/metabolism
16.
J Clin Microbiol ; 12(2): 220-5, 1980 Aug.
Article in English | MEDLINE | ID: mdl-7229008

ABSTRACT

Several oxidase reagents and commercial products were evaluated as to their efficacy in detecting oxidase activity in species of the genus Pasteurella. Recommendations are made concerning the reagent of choice for determining oxidase activity in the genus Pasteurella. Recommendations are made also concerning the use of commercial products and their efficacy in detecting oxidase activity in this genus.


Subject(s)
Indicators and Reagents , Oxidoreductases/analysis , Pasteurella/enzymology , Animals , Cattle , Humans , Naphthols , Pasteurella/classification , Phenylenediamines , Poultry , Swine , Tetramethylphenylenediamine
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