Cystic fibrosis therapy--where we are and how we got there.
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Adult↗
Biomedical subjects
D C Gruenert
Publications and source records attributed to D C Gruenert.
Gene targeting of CFTR DNA in CF epithelial cells.
A goal of cystic fibrosis (CF) gene therapy is correction of the mutant CF transmembrane conductance regulator (CFTR) gene with wild-type (wt) DNA sequences to restore normal CFTR protein and function. Experiments with wtCFTR cDNA expression vectors have shown that the Cl ion transport phenotype associated with CF can be corrected to resemble that in normal cells. An alternative to cDNA-based gene therapy strategies is one that corrects endogenous mutant sequences by targeted replacement with the wt homologue. To test whether such a strategy was feasible, a small fragment homologous replacement (SFHR) strategy was used to replace specific genomic sequences in human epithelial cells. Small fragments of genomic wtCFTR DNA were transfected into transformed CF epithelial cells. Replacement by exogenous CFTR DNA at the appropriate genomic locus and its expression as mRNA was indicated by: (1) allele-specific polymerase chain reaction (PCR) amplification of genomic DNA and mRNA-derived cDNA; and (2) hybridization of PCR products with allele-specific probes. In addition, the functional activity of CFTR protein was determined by whole cell patch clamp. Southern hybridization and patch clamp analyses suggested that approximately 1 in 100 CF cells underwent a homologous replacement event that resulted in intact Cl transport.
Cell Line↗
Episomal expression of wild-type CFTR corrects cAMP-dependent chloride transport in respiratory epithelial cells.
The isolation of the gene responsible for the Cl- ion transport defect in cystic fibrosis (CF) has provided important information about the relationship between the disease pathology and the underlying genetic and biochemical mechanisms. In addition, new areas of investigation and therapy are now possible. Most notably, the isolation of the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR) has been led to the development of different gene therapy strategies. To circumvent possible complications due to insertional mutagenesis and virally induced immune responses, we have employed Epstein-Barr virus (EBV)-based expression vectors for correction of the cAMP-dependent Cl- transport defect associated with CF. A CFTR-containing expression construct under the regulation of the Rous sarcoma virus (RSV) long terminal repeat (LTR) (pREP5/CFTR) was introduced into transformed human airway epithelial cells defective in cAMP-dependent Cl-transport. Transfected cells were assayed for Cl- ion transport by (36)Cl- efflux, SPQ, and patch clamp and showed restoration of intact cAMP-dependent transport. CFTR transcription from pREP5/CFTR was detected by Northern hybridization. The level of response to agonists appeared to be dependent on the level of CFTR expression. When cells were tested for functional expression of CFTR after removal of selection pressure, they showed a continuous decrease in responsiveness to forskolin as a function of time after removal of selection. This decrease correlated with a loss of CFTR mRNA in the loss of the PREP5/CFTR. After 12 to 15 weeks growth without selection both cAMP-dependent Cl- transport and plasmid-derived CFTR mRNA were not detectable. However, it was still possible to rescue cAMP-dependent Cl- transport in these transfected cells by reselection suggesting the presence of the CFTR containing plasmid in a portion of the cells. Analysis of DNA indicated that the pREP5/CFTR vector copy number was reduced from 30 copies per cell with continuous selection, to approximately 0.3 copies per cell after 20 weeks without hygromycin B.
Cells, Cultured↗
Overexpression of annexin V in cystic fibrosis epithelial cells from fetal trachea.
In this report, we investigated the expression of annexins I, II, V, and VI by Northern and Western blot analysis in four cell lines isolated from human fetal tracheae. Two cell lines were obtained from normal fetuses and the two others from fetuses with cystic fibrosis (CF). One CF fetus was heterozygous for the S549N and N1303K substitutions, whereas the other was homozygous for the delta F508 deletion. We found that the four annexins are always coexpressed. The expression of annexins I, II, and VI was the same in the four cell lines. In contrast, that of annexin V was significantly higher in CF than in normal cells. These observations demonstrate that annexins I, II, V, and VI are independently regulated in tracheal epithelial cell lines. Moreover, they suggest that the overexpressed annexin V, a Ca2+ channel, might profoundly modify Ca2+ transport across the membranes of CF cells.
Actins↗
Cholesterol enhances cationic liposome-mediated DNA transfection of human respiratory epithelial cells.
Cationic liposome transfection reagents are useful for transferring polynucleotides into cells, and have been proposed for human pulmonary gene therapy. The effect of adding cholesterol to cationic lipid preparations has been tested by first formulating the cationic lipid N-[1-(2,3-dioleoyloxy)propyl-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI) with varying amounts of dioleoylphosphatidylethanolamine (DOPE) and cholesterol. Cholesterol was found to enhance lipid-mediated transfection in both the respiratory epithelial cells and mouse fibroblasts. These findings will facilitate nucleic acid transfection of many cell types including differentiated epithelial cell monolayers, and therefore may be useful for examining gene regulation in various cell types and for developing pulmonary gene therapy.
3T3 Cells↗
Epithelial cell specific properties and genetic complementation in a delta F508 cystic fibrosis nasal polyp cell line.
Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a "tight epithelium" in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, delta F508 homozygote, cell line that has tight junctions pre-crisis. The cells (sigma CFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl- transport as measured by efflux of radioactive Cl-, short circuit current (Isc), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K(+)- and Ca(+2)-dependent Cl- transport. Measurement of 36Cl- efflux following stimulation with A23187 and ionomycin indicate a Ca(+2)-dependent Cl- transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26 degrees C for 48 h there is no indication of cAMP-dependent Cl- as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells.
Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.
Adenosine Triphosphate↗
Culture and transformation of human airway epithelial cells.
The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. Recent advances in culturing primary epithelial cells and the development of transformed airway epithelial cell lines have been particularly important in enhancing our understanding of the pathology associated with cystic fibrosis and lung cancer. The establishment of conditions that enhance the proliferative capacity of airway epithelial cells in primary culture was the first technical hurdle overcome in the development of in vitro culture systems. Research is now being geared toward the development of cell culture conditions that facilitate the expression in culture of the differentiated characteristics found in the native epithelium. Aside from the advances that have been made in defining the growth media and extracellular matrixes that enhance the expression of differentiated features, the use of an air-liquid interface has been a significant advance in the culture of airway epithelial cells. The implementation of the in vitro cell culture systems that have now been established and the research into optimizing the conditions for the growth of airway epithelial cells have been and will continue to be essential in the development of therapies for airway disease.
Animals↗
CFTR gene transfer corrects defective glycoconjugate secretion in human CF epithelial tracheal cells.
We demonstrate that in immortalized normal human tracheal epithelial cells (NT-1 and 56FHTE8o-) 14C-labeled glycoconjugate secretion may be regulated independently by agonists of the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. In contrast, in immortalized cystic fibrosis (CF) human tracheal epithelial cells (CFT-1 and CFT-2), regulation is defective for agonists specific for the PKA but not for the PKC pathway. To characterize the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in regulated glycoconjugate secretion, we examined the effect of adenovirus-mediated gene transfer of CFTR to CF and control cells. Forty-eight hours after infection, at a multiplicity of infection of 50 plaque-forming units per cell, high levels of CFTR mRNA were detected by reverse transcription-polymerase chain reaction, and de novo synthesis of CFTR protein was demonstrated by immunoblotting. Gene transfer to CF cells restored defective adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretion not only of chloride but also of glycoconjugates. Taken together, these results argue for a role for CFTR in cAMP-mediated glycoconjugate secretion.
Adenoviridae↗
Volume-sensitive chloride currents in four epithelial cell lines are not directly correlated to the expression of the MDR-1 gene.
It has been shown recently that heterologous expression of human MDR-1 gene, which is responsible for multidrug resistance during cancer therapy, causes appearance of volume-sensitive Cl- currents, thus suggesting that the product of the MDR-1 gene (the P-glycoprotein) has a Cl- channel activity (Valverde, M. A., Diaz, M., Sepulveda, M. A., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992) Nature 355, 830-833). In the present work, we have tested four epithelial cell lines both for the expression of MDR-1 gene and for the presence of volume-sensitive Cl- currents. LoVo/H and LoVo/Dx cells derive from a human colon adenocarcinoma, the latter cell line being resistant to high concentrations of the antitumoral drug doxorubicin. 9HTEo- cells were obtained by transformation of human tracheal epithelium. The 9HTEo-/Dx cell line was established from these cells by selection in doxorubicin. As expected, higher levels of P-glycoprotein expression were detected in LoVo/Dx and 9HTEo-/Dx by means of reverse transcriptase polymerase chain reaction technique, indirect immunofluorescence, and Western immunoblot assays. In contrast with these data, the size of swelling-induced Cl- current was the same in the sensitive cell line and in its drug-resistant counterpart. Actually, the Cl- conductance of 9HTEo- and 9HTEo-/Dx was 4-fold higher than that of either LoVo/H or LoVo/Dx cells. This indicates that the amplitude of this conductance is not directly related to the expression of the MDR-1 gene.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
cAMP-dependent activation of ion conductances in bronchial epithelial cells.
The cAMP-dependent activation of Cl- channels was studied in a bronchial epithelial cell line (16HBE14o-) in fast and slow whole-cell, and cell-attached patch-clamp experiments. The cells are known to express high levels of cystic fibrosis transmembrane conductance regulator mRNA and protein. Isoproterenol, forskolin and histamine (all 10 mumol/l) reversibly and significantly depolarized the membrane voltage (Vm) and increased the whole-cell Cl- conductance significantly by 34.0 +/- 0.9 (n = 3), 18.1 +/- 2.7 (n = 50), and 25 +/- 4.5 (n = 37) nS respectively. The effect of histamine was blocked by cimetidine (10 mumol, n = 5) but not by diphenhydramine (10 mumol/l, n = 4), which suggests binding of histamine to H2 receptors. The forskolin-induced current was not inhibited significantly by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (0.5 mmol/l, n = 9) nor glibenclamide (10 mumol/l, n = 3) and had an anion-permeability sequence of Cl = Br- > I- (n = 9). In cell-attached recordings forskolin (10 mumol/l) increased the conductance of the patched membrane from 65.5 +/- 13.6 pS to 150.8 +/- 33.2 pS (n = 30). Although the conductance was increased significantly, clear ion channel events occurring in parallel with the current activation were not detected in the cell-attached membrane. In 4 out of 30 cell-attached recordings single-channel currents were observed. These channels, with a single-channel conductance of about 6 pS, were already active before forskolin was added. No effect of forskolin on the channel amplitude, open probability or kinetics of these channels was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Bronchi↗
Ca(2+)- and swelling-induced activation of ion conductances in bronchial epithelial cells.
The present study was performed to examine Ca(2+)-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (Vte) and resistance (Rte) were measured in confluent monolayers. Resting cells had a membrane voltage (Vm) of -36 +/- 1.1 mV (n = 137) which was mainly caused by K+ and Cl- conductances and to a lesser extent by a Na+ conductance. Vte was apical-side-negative after stimulation. Equivalent short-circuit (Isc = Vte/Rte) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1 - 10 mumol/l). and ATP (0.1 - mumol/l). The histamine-induced Is was blocked by either basolateral diphenhydramine (0.1 mmol/l, n = 4) or apical cimetidine (0.1 mmol/l, n = 4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K+ conductance and hyperpolarized Vm. This effect was mimicked by the Ca2+ ionophore ionomycin (1 mumol/l, n = 11). Inhibition of the bradykinin-induced Isc by the blocker HOE140 (1 mumol/l, n = 3) suggested the presence of a BK2 receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP approximately ATP > ITP > GTP approximately CTP approximately [beta, gamma-methylene] ATP approximately 2-methylthio-ATP = 0 and was obtained in Isc measurements and patch-clamp recordings. This suggests the presence of a P2u receptor. Hypotonic cell swelling activated both Cl- and K+ conductances.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗
Stimulation of chloride secretion by P1 purinoceptor agonists in cystic fibrosis phenotype airway epithelial cell line CFPEo-.
1. P1 purinoceptor agonists like adenosine have been shown to stimulate Cl- transport in secretory epithelia. In the present study, we investigated whether P1 agonist-induced Cl- secretion is preserved in cystic fibrosis airway epithelium and which signalling mechanism is involved. The effects of purinoceptor agonists on Cl- secretion were examined in a transformed cystic fibrosis airway phenotype epithelial cell line, CFPEo-. 2. Addition of adenosine (ADO; 0.1-1 mM) markedly increased 125I efflux rate. The rank order of potency of purinoceptor agonists in stimulating 125I efflux was ADO > AMP > ADP approximately equal to ATP. A similar order of potency was seen in transformed cystic fibrosis nasal polyp cells, CFNPEo- (ADO > ATP > AMP > ADP). These results are consistent with the activation of Cl- secretion via a P1 purinoceptor. 3. The P1 agonists tested (at 0.01 and 0.1 mM) revealed a rank order of potency of 5'-N-ethylcarboxamine adenosine (NECA) > 2-chloro-adenosine (2-Cl-ADO) > R-phenylisopropyl adenosine (R-PIA). 4. The known potent A2 adenosine receptor (A2AR) agonist, 5'-(N-cyclopropyl) carboxamidoadenosine (CPCA, 2 microM) but not the A1 adenosine receptor agonist, N6-phenyl adenosine (N6-phenyl ADO, 10 microM) markedly increased 125I efflux rate (baseline, 5.9 +/- 2.0% min-1, + CPCA, 10.9 +/- 0.6% min-1; P < 0.01). The stimulant effect of CPCA (10 microM) was abolished by addition of the A2AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (100 microM; reported K(i) = 11 +/- 3 microM). These results favour the involvement of A2AR. 5. ADO (0.1-mM) and CPCA (2 microM) both induced a marked increase in intracellular [Ca2+] ([Ca2+]i); the effect of the latter was again abolished by pretreatment of the cells with DMPX. By contrast, N6-phenyl ADO did not affect [Ca2+]i. 6. In patch-clamp experiments, ADO (1 mM) induced an outwardly-rectified whole-cell Cl- current (baseline, 2.5 +/- 0.8 pA pF-1, + ADO, 78.4 +/- 23.8 pA pF-1; P < 0.02), which was largely inhibited in cells internally perfused with a selective inhibitory peptide of the multifunctional Ca2+/calmodulin-dependent protein kinase, CaMK [273-302] (20 microM), as compared to a control peptide, CaMK [284-302]. Addition of BAPTA (10 mM), a Ca2+ chelator, to the perfusion pipette also abolished the ADO-elicited Cl- current. 7. In conclusion, our results suggest that A2AR participates in regulation of airway C1 secretion via aCa2+-dependent signalling pathway, which involves CaMK and appears to be at least partially conserved in cystic fibrosis airway epithelial cells.
Adenine Nucleotides↗
Heterotrimeric G proteins, vesicle trafficking, and CFTR Cl- channels.
Previously (E.M. Schwiebert, N. Kizer, D. C. Gruenert, and B. A. Stanton, Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992), we showed that heterotrimeric G proteins regulate adenosine 3',5'-cyclic monophosphate (cAMP)-activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in human airway epithelial cells. The goal of the present study was to test the hypothesis that heterotrimeric G proteins regulate vesicle trafficking and exocytosis and that these events are critical for cAMP activation of CFTR-mediated Cl- secretion. We report that cAMP stimulates exocytosis and CFTR Cl- conductance (GCl) in normal but not in CF cells. Stimulation of the heterotrimeric G protein G alpha i-2 inhibited cAMP-activated CFTR GCl and exocytosis in normal cells. In contrast, inhibition of G alpha i-2 stimulated exocytosis and allowed cAMP to stimulate CFTR GCl in cells isolated from patients with cystic fibrosis (CF). Brefeldin A and nocodazol prevented cAMP-induced exocytosis and also blocked cAMP stimulation of CFTR GCl in normal airway epithelial cells. Our studies suggest that the heterotrimeric G protein G alpha i-2 regulates CFTR GCl in human airway epithelial cells by modulating vesicle trafficking and the delivery of CFTR Cl- channels from an intracellular vesicular pool to the plasma membrane. Inhibition of G alpha i-2 may be a useful therapeutic approach to target mutant delta F508 CFTR Cl- channels from an intracellular vesicular pool to the plasma membrane and thereby correct defective Cl- secretion in CF airway epithelial cells.
Calcium↗
CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.
A major limitation in the study of vectorial ion transport, secretion, and differentiated function in the human airway epithelium has been the lack of suitable cell culture systems. Progress in this direction has been made through the transformation of primary cultured epithelial cells. However, these transformants tend to lose differentiated properties with increasing serial passage, particularly following crisis. The successful establishment of a postcrisis SV40 large T-antigen transformed epithelial cell line derived from human bronchial epithelium is described. This cell line, 16HBE14o-, retains differentiated epithelial morphology and functions. Cell cultures show the presence of tight junctions and cilia, and monolayers generate transepithelial resistance, as measured in Ussing chambers, and retain beta-adrenergic stimulation of cAMP-dependent chloride ion transport, measured either by 36Cl- efflux or as short-circuit current in Ussing chambers. The cells also increase chloride transport in response to bradykinin or calcium ionophore. In addition, 16HBE14o- cells express levels of both the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein readily detectable by Northern and Western hybridization analysis, respectively. These cells provide a valuable resource for studying the modulation of CFTR and its role in regulation of chloride ion transport in human airway epithelium as well as other aspects of human airway cell biology.
Bronchi↗
ATP and A1 adenosine receptor agonists mobilize intracellular calcium and activate K+ and Cl- currents in normal and cystic fibrosis airway epithelial cells.
The effect of purinergic compounds on [Ca2+]i and membrane currents of cell lines derived from the airway epithelium of normal and cystic fibrosis individuals has been investigated. 2-Chloroadenosine (2-CADO), as well as other agonists of the A1 adenosine receptors, causes a transient elevation of cytosolic [Ca2+] that is antagonized by the A1 adenosine receptor antagonist 8-cyclopentyl-1,3 dipropylxanthine (DPCPX). ATP is also effective, but at a lower extent. The [Ca2+]i increase induced by 2-CADO and ATP is abolished by preincubation with phorbol 12-myristate 13-acetate and the Ca(2+)-ATPase inhibitor thapsigargin. This latter result suggests that purinergic agonists mobilize Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores. Pertussis toxin completely inhibits the effect of 2-CADO, whereas only it partially affects that of ATP, suggesting the involvement of different types of G proteins. Perforated patch clamp experiments carried out in both current clamp and voltage clamp modes show that 2-CADO and ATP activate K(+)- and Cl(-)-selective membrane currents, with a mechanism inhibited by preincubation with DPCPX and thapsigargin. These data indicate that activation of adenosine A1 receptor, in a similar way to ATP receptor, causes [Ca2+]i increase and ion channels activation through a transduction mechanism that is not impaired in cystic fibrosis airway epithelial cells.
2-Chloroadenosine↗
Mechanism of inhibition of cAMP-dependent epithelial chloride secretion by phorbol esters.
In T84 cells, we investigated how stimulation of protein kinase C leads to an inhibition of cAMP-dependent chloride secretion. Specifically, we tested the hypothesis that the inhibition was caused by loss of the cystic fibrosis transmembrane regulator (CFTR), an apical membrane chloride channel. As described by others (Trapnell, B. C., Zeitlin, P. L., Chu, C.-S., Yoshimura, K., Nakamura, H., Guggino, W. B., Bargon, J., Banks, T. C., Dalemans, W., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1991) J. Biol. Chem. 266, 10319-10323), we found that treatment with the phorbol ester, phorbol myristate acetate (PMA), reduced CFTR mRNA levels by approximately 80% with a t 1/2 of approximately 2 h. Chloride secretion, measured as forskolin-induced short circuit current, was also abolished by PMA with a t 1/2 of approximately 2 h. Levels of mature glycosylated CFTR measured by Western blotting also declined to 50 +/- 8% (n = 7) of control after a 12-h PMA treatment. However, a 12-h exposure to PMA did not affect the forskolin-stimulated efflux of 125I into high potassium medium, a measure of apical membrane CFTR activity. We conclude that increased turnover of apical membrane CFTR in PMA-treated cells compensates for the decline in anion channel numbers. By contrast to its lack of effect on 125I effluxes, PMA reduced the cAMP-induced increase in 86Rb efflux, suggesting that it inhibits chloride secretion mainly by an action on basolateral potassium channels.
Blotting, Western↗
Asbestos exposure increases human bronchial epithelial cell fibrinolytic activity.
Chronic exposure to asbestos fibers results in fibrotic lung disease. The distal pulmonary epithelium is an early target of asbestos-mediated injury. Local plasmin activity may be important in modulating endoluminal inflammatory responses in the lung. We studied the effects of asbestos exposure on cell-mediated plasma clot lysis as a marker of pericellular plasminogen activation. Exposing human bronchial epithelial (HBE) cells to 100 micrograms/ml of asbestos fibers for 24 h resulted in increased plasma clot lysis. Fibrinolytic activity was augmented in a dose-dependent fashion, was not due to secreted protease, and occurred only when there was direct contact between the plasma clot and the epithelial monolayer. Further analysis showed that asbestos exposure increased HBE cell-associated urokinase-type plasminogen activator (uPA) activity in a time-dependent manner. The increased cell-associated PA activity could be removed by acid washing. The increase in PA activity following asbestos exposure required new protein synthesis because it was abrogated by treatment with either cycloheximide or actinomycin D. Therefore, asbestos exposure increases epithelial-mediated fibrinolysis by augmenting expression of uPA activity at the cell surface by mechanisms that require new RNA and protein synthesis. These observations suggest a novel mechanism whereby exposure of the distal epithelium to inhaled particulates may result in a chronic inflammatory response that culminates in the development of fibrotic lung disease.
Asbestos↗