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CELLULAR AND MOLECULAR
Pulmonary, Critical Care, and Sleep Medicine Section, Department of Internal Medicine (D.S.A.-G., A.A.F., A.J.H., T.A.W.), School of Allied Health Professions (S.D.S.), and Department of Biochemistry and Molecular Biology (R.G.M.), University of Nebraska Medical Center, Omaha, Nebraska; and Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska (T.A.W.)
Received November 11, 2004; accepted April 11, 2005.
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Abstract |
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(PKC-) to investigate the relationship between PKC- and C5aR availability and affinity. Western blot analysis revealed a 75-kDa lysate band from cells expressing WT and DN PKC-, but DN cells exposed to 5% CSE had no functional PKC activity. Pretreatment with Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] (PKC- inhibitor) had no effect on DN but significantly decreased WT PKC activity. Competitive binding studies conducted on either WT or DN PKC--transfected cells also revealed two classes of binding sites for C5a having different affinities. There was a significant rightward shift of the binding curve when WT cells were pretreated with Gö6976. These data suggest that C5aR is detectable on bronchial epithelial cells exposed to CSE and that exposure to CSE increases the availability of C5a binding sites. The data also indicate that PKC- may play an important role in modulating C5aR binding.
; Gerard and Gerard, 1991). These receptors are abundant in certain myeloid cells and have been detected in some nonmyeloid cells, including the liver, lung, kidney, and central nervous system (Haviland et al., 1995; Zwirner et al., 1999). Although nonmyeloid cells express constitutively low levels of C5aR (Buchner et al., 1995; Gasque et al., 1995; Haviland et al., 1995; Braun and Davis, 1998), it may be that activation of these receptors is necessary for them to invoke the effects of C5a. Currently, the functional significance of such constitutive expression of the C5aR on nonmyeloid cells is unknown.
We previously showed that human bronchial epithelial cells (HBECs) constitutively express C5aRs and that cell-surface C5aR expression is increased in the population of HBECs upon exposure to 5% cigarette smoke extract (CSE) (Floreani et al., 1998). In addition, initial exposure of HBECs to 5% CSE followed by treatment of the cells with C5a resulted in a significant increase in the release of interleukin (IL)-8 compared with HBECs exposed to CSE or C5a alone. Finally, we have reported that CSE-stimulated protein kinase C (PKC) activation is required for maximal C5a-mediated IL-8 release from HBECs (Wyatt et al., 1999).
These findings suggest that exposure of HBECs to CSE makes existing C5aRs more available on the cell surface for binding of the C5a ligand. Investigations conducted on other G protein-coupled receptors, such as the type 1 angiotensin II, 
2-adrenergic, and muscarinic receptors, suggest that G protein-coupled receptors are capable of intermolecular "cross-talk" and dimerization, which can influence the affinity of agonist binding and biologic response (Maggio et al., 1993; Hebert et al., 1996; Monnot et al., 1996). Modulation of C5aR expression in airway epithelial tissues has potentially important implications in the pathogenesis of smoking-induced airway inflammation. Based on our previous studies wherein we showed an enhanced responsiveness to C5a-mediated release of IL-6 and IL-8 in CSE-treated HBECs, it seems that CSE exposure may alter the number of C5aRs present on HBECs, the binding affinity of C5a for C5aR, or both.
In this study, we hypothesized that CSE exposure of bronchial epithelial cells modulates C5aR density and binding affinity for C5a. To test this hypothesis, we conducted whole-cell receptor binding studies on the human bronchial epithelial cell line BEAS-2B. We show herein that CSE exposure enhances C5aR-ligand binding affinity and that this effect is modulated in part by PKC- activity. In addition, our study shows the existence of two distinct classes of C5a binding sites on BEAS-2B cells. We suggest that the presence of C5aRs on airway epithelial cells may prove particularly relevant to understanding early and decisive events in cigarette smoke-induced airway inflammation.
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Materials and Methods |
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Cell Preparation. BEAS-2B cells were cultured under serum-free conditions using a 1:1 medium mixture of RPMI 1640 medium (Invitrogen) and LHC-9 (prepared from LHC basal; Biofluids) (Lechner and LaVeck, 1985). Cells were plated on type I collagen-coated 100-mm tissue culture dishes (Vitrogen 100) and grown to 90% confluence. Cells were maintained in culture at 37°C in humidified 95% air/5% CO2 for 48 to 72 h before each experiment.
PKC--Transfected BEAS-2B Cells. The tetracycline-responsive promoter expression system, as previously described (Gossen and Bujard, 1992), was used on BEAS-2B cells to produce wild-type (WT) and dominant-negative (DN) versions of PKC- (BD Biosciences Clontech, Palo Alto, CA). BEAS-2B cells were grown to 90% confluence in six-well tissue culture dishes. Cells were transfected with the pTet-On expression vector encoding reverse tetracycline transactivator protein, as well as a neomycin resistance gene using a cationic lipid technique, Lipofectamine 2000 (Invitrogen). Cells were propagated and selected for neomycin resistance using G418 (geneticin) (400 µg/ml; EMD Biosciences, San Diego, CA). Antibiotic-resistant clones were then transfected by electroporation with either the entire reading frame of the native PKC- gene (WT) or with a DN variant made by in vitro mutagenesis, substituting an alanine for lysine at position 368. Expression of PKC- from this vector was under the control of the tetracycline response element (Rosson et al., 1997).
In addition, BEAS-2B cells were simultaneously cotransfected with a plasmid encoding the "humanized" green fluorescent protein (GFP) reporter to facilitate cell selection. Dr. Dan Rosson (Lankenau Medical Research Center, Wynnewood, PA) provided all the PKC- plasmid vectors.
CSE Preparation. Cigarettes were obtained from the University of Kentucky, Tobacco-Health Research Division (unfiltered, Code 2R1; Lexington, KY). CSE was prepared as a saturated stock solution by bubbling the smoke from one research-grade 85-mm nonfiltered cigarette connected to a peristaltic pump apparatus in 25 ml of sterile LHC-9/RPMI medium as previously described (Koyama et al., 1989). A total of 0.5 ml of the CSE stock solution was then diluted with 9.5 ml of LHC-9/RPMI medium and then filter-sterilized to arrive at the 5% final concentration.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis. WT and transformed DN BEAS-2B cells were plated on type I collagen-coated six-well tissue culture dishes and grown to 90% confluence. Cells were treated with and without 5% CSE for 1 h. CSE-treated and control cells were then rinsed with cold phosphate-buffered saline and flash-frozen in phosphate-buffered saline containing protease inhibitors (1 µg/ml each of leupeptin, aprotinin, phenylmethylsulfonyl fluoride, and chymostatin).
Cell lysates were sonicated, and particulates were removed by centrifugation. Protein concentration was determined by the Bradford method (Bradford, 1976) with Bio-Rad protein reagent (Bio-Rad, Hercules, CA). Proteins were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions on a 7.5% polyacrylamide gel. The resolved proteins were electroblotted to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with Tris-buffered saline/Tween/Blotto containing 1% nonfat milk. Transferred proteins were then probed with recombinant anti-human PKC- antibody (5 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 4 h. Membranes were washed several times and incubated with 1:5000 goat/anti-rabbit IgG peroxidase conjugate for 30 min at 4°C (Rockland, Gilbertsville, PA). An enhanced chemiluminescence kit (SuperSignal, West Pico kit; Pierce, Rockford, IL) was used to visualize the blotted proteins.
PKC Activity Assay. PKC activity was determined in crude whole-cell fractions of transfected and nontransfected BEAS-2B cells. The assay used was a modification of procedures previously described (Hannun et al., 1985) and includes 900 µM PKC substrate peptide (Peninsula Laboratories, Belmont, CA), 12 mM calcium acetate, 8 µM phosphatidyl-L-serine, 30 mM dithiothreitol, 150 µM ATP, 45 mM magnesium acetate, and 10 µCi/ml [
-32P]ATP in a Tris-HCl buffer, pH 7.5. Samples (20 µl) were added to 40 µl of this reaction mixture and incubated for 15 min at 30°C. Incubations were halted by spotting 50 µl of each in phosphoric acid (75 mM) and then washed once in ethanol, dried, and counted in nonaqueous scintillant as previously described (Roskoski, 1983). Kinase activity was expressed in relationship to total cellular protein assayed and calculated in picomoles per minute per milligram.
All the samples were assayed in triplicate, and no fewer than three separate experiments (n = 3) were performed per unique parameter. Data were analyzed for significance using one-way analysis of variance, followed by Tukey's multiple comparison. Significance was assigned at p 
0.05.
Labeling of rhC5a. Iodination of C5a with Na125I was performed with the iodogen method as recommended by the manufacturer (Pierce). The average specific activity of labeled material was 29,638 cpm/ng (143.3 Ci/mmol).
Receptor Binding Assay. Cells were treated with 5% CSE for 2 h. Cells were then washed, trypsinized, washed again, and resuspended in HBSS containing HEPES and BSA (HBSS/50 mM HEPES/0.1% BSA) at 5 x 106 cells/ml. Cells were trypsinized to obtain a uniform cell suspension as previously described (Gilad et al., 1997; Jockers et al., 1999). An aliquot of cells (0.2 ml) was added to each microcentrifuge tube containing different concentrations of 125I-C5a. Triplicate samples were incubated in the absence (total binding) or presence (nonspecific binding) of a 200-fold molar excess of unlabeled ligand. Cells were then incubated for 15 h at 4°C. The cells were washed via centrifugation at 25,000g for 2 min at 4°C and then solubilized in 0.1 N NaOH for gamma counting. Protein concentrations were determined as previously described (Lowry et al., 1951).
Competitive Receptor Binding Assay. Like most G protein-coupled receptors, the C5aR (CD88) undergoes rapid phosphorylation, followed by desensitization and internalization. To avoid the rapid desensitization and internalization of C5aR, the binding studies were performed at 4°C. For the binding assay, 3 nM 125I-C5a was added to each microcentrifuge tube containing a 0.2-ml aliquot of cells in the presence or absence of unlabeled competitors. Nonspecific binding was defined as the amount of radioactivity bound in the presence of 300 nM of unlabeled C5a. The binding mixture was incubated for 15 h at 4°C, and unbound labeled material was separated from cells using a modified sucrose gradient centrifugation assay as previously described (Low and Jardine, 1986). The tips of the tubes, containing cells plus bound radiolabeled ligand, were cut off, and radioactivity was measured in a gamma counter. Unlabeled rhC5a was used as a competitor with its serial dilution of unlabeled rhC5a in the 10-17 to 10-6 M range. The percentage of total binding represents the mean of 13 assays conducted; nonspecific binding represented 20.8% of total binding (range = 12–33%). Binding data were analyzed by two-site competition nonlinear regression analysis (Prism 4.0; GraphPad Software Inc., San Diego, CA) to calculate IC50 values.
Statistical Analyses. Results were compared by one-way analysis of variance, followed by Tukey's multiple comparison. Significance was set at p < 0.05.
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Results |
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Activity in DN-Transfected Epithelial Cells. Because previous studies from our laboratory suggest that CSE exposure of HBECs activates PKC- (Wyatt et al., 1999), we investigated the relationship between PKC- and C5aR availability and affinity. We created an epithelial cell line stably transformed with WT or DN versions of PKC-. Western blot analysis confirmed a strong band at approximately 75 kDa in lysates derived from WT or the DN version of the PKC- gene (Lys-368 substituted with Ala) and cotransfected with a GFP reporter gene (Fig. 3A). The darker band expressed in the DN cells suggests the presence of PKC- protein production. Control Western blots for actin show no difference in protein loading or actin expression (unpublished data). However, when these DN cells were exposed to 5% CSE, little to no functional PKC activity was observed relative to control cells (Fig. 3B). On the other hand, WT cells exposed to 5% CSE revealed almost a 2-fold increase in PKC activity compared with WT control. As a control for stably transforming BEAS-2B, cells transfected with a GFP reporter gene were exposed to 5% CSE and revealed a 1.6-fold increased PKC- activity. The data collectively suggest that transfected BEAS-2B cells expressed PKC-; however, the DN version revealed a nonfunctional isoform of PKC-.
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Gö6976, a Specific PKC- Inhibitor, Blocks PKC Activity in WT-Transfected BEAS-2B Cells. In an attempt to identify PKC activity in stably transformed versions of the PKC-, WT- or DN-transfected BEAS-2B cells were treated with 5% CSE with or without pretreatment for 1 h with Gö6976 (1 µM), a specific PKC- inhibitor. WT cells treated alone with 5% CSE significantly stimulated PKC-, whereas DN cells revealed no stimulation of PKC- (Fig. 4). The specific PKC- inhibitor Gö6976 abrogated the CSE-mediated PKC- activation in WT cells and had no apparent effect on basal PKC- activity in DN cells.
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on CSE-Induced C5a Binding. In earlier studies, BEAS-2B cells pretreated with phorbol myristate acetate (200 ng/ml), a direct activator of PKC, had a similar binding profile to that seen in CSE-treated cells. Both treatments resulted in enhanced C5aR expression that was significantly relative to controls (1059 ± 328 cpm/5 x 106 cells and 1114 ± 212 cpm/5 x 106 cells, respectively, versus control; 263 ± 170 cpm/5 x 106 cells, p < 0.05). This similarity suggests that activation of PKC affects C5aR expression. To elucidate the relationship between PKC activation and C5aR binding, WT and DN PKC- genetic variants were treated with 5% CSE, and competition of 125I-C5a binding (3 nM) with unlabeled C5a was further examined (Fig. 5). CSE-treated WT cells had a profile similar to that observed in CSE-treated BEAS-2B cells with distinct affinities for the two classes of sites (Ki1 = 3.25 x 10-16 M; Ki2 = 1.56 x 10-9 M). CSE-treated DN cells also revealed two distinct C5a affinities with a slight shift of the curve to the right (Ki1 = 1.74 x 10-16 M; Ki2 = 8.39 x 10-9 M; p > 0.05). However, this slight shift of the curve was not significant; therefore, CSE-treated DN cells have a similar binding affinity profile to that seen in CSE-treated WT cells. To evaluate PKC- involvement in C5aR binding, CSE-treated WT cells were pretreated with Gö6976 (1 µM), a specific PKC- inhibitor. Competitive binding studies revealed a significant shift of the binding curve to the right, with two distinct C5a binding affinities (Ki1 = 3.24 x 10-14 M; Ki2 = 3.27 x 10-7 M; p < 0.05) when WT cells were pretreated with Gö6976. In addition, flow cytometric analysis revealed increased C5aR expression based on the mean fluorescence intensity (MFI) in PKC- overexpressing compared with WT (81.33 MFI versus 51.66 MFI, respectively) (manuscript in preparation). These results gave further evidence for a direct role of PKC in CSE-mediated C5aR expression. Collectively, the results suggest that smoke-induced increases in binding affinity and/or cell-surface availability of C5aR are associated with PKC- activation.
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15,000 binding sites) in cells exposed to CSE. The C5aRs on CSE-treated BEAS-2B cells bind C5a with an affinity similar to that of the C5aRs expressed on human neutrophils (Kd = 2 nM) (Chenoweth and Hugli, 1978). No C5a binding was detected on BEAS-2B cells in the absence of 5% CSE, presumably as a result of few C5aRs expressed on the surface of these cells under non-stressed conditions. An alternative explanation may be that in the absence of 5% CSE treatment, C5aRs expressed on the cell surface are unable to bind to C5a. The increased binding of C5a correlates well with our earlier flow cytometric analysis that revealed an increase in immunofluorescence for cell-surface C5aR following treatment with 5% CSE (Floreani et al., 1998). Collectively, these data suggest that enhanced functional responsiveness toward C5a may be the result of a smoke-triggered increase in the number of C5aRs and/or increased binding affinity for C5aR.
Based on the evidence that CSE increases C5aR expression and allows C5a to bind more effectively, competitive binding studies were conducted to evaluate the affinity of C5a for its receptor(s). The binding of radiolabeled C5a to the C5aR competed efficiently with unlabeled rhC5a in CSE-treated cells. Surprisingly, the competitive binding assays revealed the presence of two distinct classes of receptors for which the unlabeled ligand has distinct affinities. The saturation studies were unable to detect these two distinct sites because of the limitation of using a narrow range of radiolabeled ligand concentrations. Interestingly, the curvilinear nature of the Scatchard analysis suggested there were potentially two sites, which competition studies later confirmed. In addition, binding studies were performed on whole BEAS-2B cells rather than membranes, which are most commonly used in C5aR binding studies (Kawai et al., 1991; Konteatis et al., 1994; Vlattas et al., 1994). As a result, intact CSE-treated BEAS-2B cells revealed a biphasic response to unlabeled C5a, suggesting that the region on the C5aR responsible for binding C5a is composed of two distinct subsites. These findings support earlier studies that implicated a two-site binding motif for C5aR, which may prove to be an alternative binding paradigm for G protein-coupled receptors (Siciliano et al., 1994).
Our findings suggest that intact BEAS-2B cells are required to directly correlate ligand binding to the C5aR with signal transduction. One possibility is that exposure to CSE makes existing pools of C5aR more available for cell-surface binding with the C5a ligand. It is possible that cigarette smoke promotes molecular interactions between the transmembrane components of the C5aR that result in either cross-talk or receptor dimerization, commonly associated with G protein-coupled receptors (Maggio et al., 1993; Hebert et al., 1996). In turn, the dimerized receptor may bind C5a with higher affinity. Receptor-binding studies traditionally are done with membrane preparations to avoid the intrinsic factors that may confound ligand binding to native receptors. In doing so, the apparent monophasic response that is normally reported may mask the cells' biological response as a result of the loss of cellular integrity. Conducting whole-cell binding studies is essential to stimulate smoke interactions with the intact airway epithelium. Overall, the results revealed that CSE increases C5aR surface expression in airway epithelial cells. Our observations represent for the first time a biphasic binding profile in C5aR-bearing cells of nonmyeloid origin.
Our earlier studies confirmed that PKC activation is required for CSE-enhanced C5a-mediated release of IL-8 in bronchial epithelial cells (Wyatt et al., 1999). Kashyap et al. (2002) showed that the calcium-dependent "alpha" isoform of PKC (PKC-) mediates CSE- and C5a-stimulated IL-8 release from HBECs. We recently observed that combined CSE plus C5a treatment induced an increase in airway epithelial intercellular adhesion molecule-1 expression and cell adhesion, a process that seems to require PKC intracellular signaling (Floreani et al., 2003). Collectively, these findings suggest that PKC- activation potentiates C5aR expression and affinity for C5a, leading to the C5a-mediated stimulation of cellular responses in airway epithelial cells.
In the present work, we assessed the role of PKC- in modulating C5a binding affinity using transfected BEAS-2B cells. The binding affinity of the C5aR in WT PKC--transfected cells was similar to that measured in nontransfected BEAS-2B cells. The data also revealed that the binding affinity of the C5aR in cells expressing DN PKC- was similar to that measured in untreated WT cells when exposed to CSE. These findings were confounding because the presence of catalytically inactive PKC- was confirmed via Western blot analysis and PKC activity assays. We believe this response in the DN PKC- cells may indeed involve other PKC isoforms acting in a redundant manner. The phenotype of the cells transfected with inactive PKC- does not rule out compensatory or complementary functions for PKC members (Ron and Kazanietz, 1999; Tan and Parker, 2003). This concept of altered PKC isoform expression in DN mutant cell lines is the subject of our ongoing research and suggests the importance of direct pharmacological studies coupled with the use of mutant cell lines. When the WT BEAS-2B cells were pretreated with a selective PKC- inhibitor, the C5a binding affinity of the receptor was significantly decreased, and this was manifested by a rightward shift in the C5a competitive binding curve. In addition, PKC- activity was notably decreased when CSE-exposed WT cells were pretreated with 1 µM Gö6976. The concentration we used is pharmacologically potent and shows selectiveness of Gö6976 in airway epithelial cells (Kashyap et al., 2002). These data suggest that activation of PKC- is necessary for coupling C5a to its receptor under conditions of CSE exposure possibly by either inducing conformational changes in the C5aR or by increasing C5aR availability, which ultimately enhances C5a affinity. Both the mechanism of CSE activation of PKC- and the mechanism of PKC- modulation of the C5aR are the subject of our current investigations.
Ligand binding studies are a direct method of detecting receptor numbers and receptor affinities. Our studies show the presence of C5aR only after cells were exposed to CSE. They further confirmed that cultured cells reach a state of activation in response to CSE treatment, which induces their expression of C5aR, whereas the receptor is not detectable under resting conditions. It is well documented that C5a binds to its receptor at more than one site (Gerard et al., 1989), and there have been numerous studies using site-directed mutagenesis of C5a that have attempted to identify the residues that mediate its binding to the receptor (Bubeck et al., 1994; DeMartino et al., 1994). Although these studies have suggested different interaction points between C5a and C5aR, a hypothesis involving two major domains of C5a interacting with the receptor in two distinct regions or sites has evolved (Chenoweth and Hugli, 1980; Siciliano et al., 1994). Interestingly, our findings support such a hypothesis because binding studies revealed two distinct and relatively high affinities for C5aR. Whether it is the ability of C5a to interact with two distinct regions or sites, the data do not exclude the possible existence of another C5a-like receptor. Recent evidence proposes that the orphan receptor, C5L2, is a high-affinity C5a binding protein and may be a second receptor for C5a (Cain and Monk, 2002; Okinaga et al., 2003). Further investigation to determine whether the orphan receptor C5L2 exists on bronchial epithelial cells when exposed to CSE is required.
Modulation of C5aR activity in airway epithelial tissues has potentially important implications in understanding the pathogenesis of smoking-induced airway inflammation in vivo. The ability of CSE to increase C5aR activity seems plausible for airway epithelial cells to exhibit a gated responsiveness to C5a. These cells are in constant contact with inhaled irritants and toxins, and under normal conditions, they are relatively unresponsive to C5a. However, upon exposure to a potent inflammatory stimulus such as cigarette smoke, airway epithelial cells could modulate their responsiveness to C5a by up-regulating C5aR number or cell-surface expression. This could be beneficial in the recruitment of necessary neutrophils and possibly lymphocytes in response to smoke; however, the mechanism by which CSE modulates the C5aR is not yet clear. Unlike myeloid cells (Burg et al., 1996), limited information is available as to what regulates expression and functional responsiveness of the C5aR on airway epithelial cells. Collectively, these studies suggest a specific role for C5a and the C5aR in airway inflammation associated with cigarette smoke and other inhaled inflammatory irritants.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: C5aR, C5a anaphylatoxin receptor; HBEC, human bronchial epithelial cell; CSE, cigarette smoke extract; IL, interleukin; PKC, protein kinase C; LHC, light-harvesting complex; HBSS, Hanks' balanced salt solution; rhC5a, recombinant human C5a; BSA, bovine serum albumin; WT, wild type; DN, dominant-negative; GFP, green fluorescent protein; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; MFI, mean fluorescence intensity.
Address correspondence to: Dr. Diane S. Allen-Gipson, Pulmonary, Critical Care, and Sleep Medicine Section, University of Nebraska Medical Center, 985815 Nebraska Medical Center, Omaha, NE 68198-5815. E-mail: dallengipson{at}unmc.edu
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, WT untreated; 
, WT treated with Gö6976; and 
, PKC-