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Am J Physiol Lung Cell Mol Physiol 293: L1163-L1170, 2007. First published August 24, 2007; doi:10.1152/ajplung.00103.2007
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Feedlot dust stimulation of interleukin-6 and -8 requires protein kinase C{varepsilon} in human bronchial epithelial cells

Todd A. Wyatt,1,2 Rebecca E. Slager,1 Jane DeVasure,1 Brent W. Auvermann,3 Michael L. Mulhern,4 Susanna Von Essen,1 Tracy Mathisen,1 Anthony A. Floreani,1 and Debra J. Romberger1,2

1Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, Omaha; 2Research Service, Department of Veterans Affairs Medical Center, Omaha; 3Texas A & M Agricultural Research & Extension Center, Amarillo, Texas; and 4Department of Ophthalmology and Visual Sciences, Nebraska Medical Center, Omaha, Nebraska

Submitted 16 March 2007 ; accepted in final form 21 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Individuals exposed to dusts from concentrated animal feeding operations report increased numbers of respiratory tract symptoms, and bronchoalveolar lavage samples from such individuals demonstrate elevated lung inflammatory mediators, including interleukin (IL)-8 and IL-6. We previously found that exposure of bronchial epithelial cells to hog barn dusts resulted in a protein kinase C (PKC)-dependent increase in IL-6 and IL-8 release. We hypothesized that cattle feedlot dusts would also generate bronchial epithelial interleukin release in vitro. To test this, we used interleukin ELISAs and direct PKC isoform assays. We found that a dust extract from cattle feedlots [feedlot dust extract (FLDE)] augments PKC activity of human bronchial epithelial cells in vitro. A 5–10% dilution of FLDE stimulated a significant release of IL-6 and IL-8 at 6–24 h in a PKC-dependent manner vs. control medium-treated cells. An increase in PKC{alpha} activity was observed with 1 h of FLDE treatment, and PKC{varepsilon} activity was elevated at 6 h of FLDE exposure. The PKC{alpha} inhibitor, Gö-6976, did not inhibit FLDE-stimulated IL-8 and IL-6 release. However, the PKC{varepsilon} inhibitor, Ro 31-8220, effectively inhibited FLDE-stimulated IL-8 and IL-6 release. Inhibition of FLDE-stimulated IL-6 and IL-8 was confirmed in a dominant-negative PKC{varepsilon}-expressing BEAS-2B cell line but not observed in a PKC{alpha} dominant negative BEAS-2B cell line. These data support the hypothesis that FLDE exposure stimulates bronchial epithelial IL-8 and IL-6 release via a PKC{varepsilon}-dependent pathway.

feedlot dust extract

CONCENTRATED ANIMAL FEEDING OPERATIONS (CAFOs) generate inhalable dusts that are the focus of numerous health concerns (6, 10, 23, 26). Such airborne dusts include endotoxin and other microbial products as well as ammonia, hydrogen sulfide, and a variety of volatile organic compounds (6). Increased medical complaints are more common in populations living near some CAFOs (30). Symptoms of respiratory disease and lung function test abnormalities have been described in workers employed in CAFOs, but the impact of CAFO dusts on ambient public exposure is not defined. Furthermore, the type of animal involved in the feeding operation may likely be important in the airborne agents generated and the effect on public health. CAFOs where the animals are located inside buildings such as hogs, poultry, and dairy cattle have been investigated more thoroughly than CAFOs where animals are located outdoors.

Clinical treatment of CAFO dust exposure is of importance to the general population. Simulation studies suggest that when CAFO workers comprise 15–45% of the community, human influenza cases are increased by 42–86% (22). Overall, in the United States, an increased rate of asthma among school children has occurred, and a recent study suggests that children in schools near CAFOs have a significantly increased prevalence of physician-diagnosed asthma (24). Some studies have suggested that dairy farmers' exposure to cattle dust leads to increased airborne particulate matter exposure and the potential for long-term development of chronic lung disease (4, 5).

Occupational exposures are responsible for ~15% of chronic obstructive pulmonary disease (COPD) (2). COPD is a disease syndrome characterized by chronic bronchitis, chronic asthma, and emphysema. COPD involves airway remodeling in response to chronic lung inflammatory events. While the mechanisms are complex and poorly defined, one hallmark of COPD is the chronic recruitment of inflammatory cells through the production of cytokines such as IL-6 and IL-8 (18). A principal source of IL-6 and IL-8 production in the airways is the bronchial epithelial cell (14, 27).

Previously, we examined the effect of CAFO dust from hog confinement facilities on airway epithelial cell proinflammatory cytokine production. Hog barn dust stimulated a significant production and release of IL-6 and IL-8 in an endotoxin-independent manner (20). We also found that the release of these interleukins was regulated via the action of protein kinase C (PKC). In addition, we observed that hog barn dust extract enhanced the adhesion of lymphocytes to airway epithelial cells in a PKC-dependent manner (15). BEAS-2B cells also have been used in a model for soil-derived, dust-induced proinflammatory cytokine release (29). Thus exposure to organic dusts derived from hog CAFOs demonstrates an in vitro potential for stimulating airway inflammation. It is likely that exposure to other CAFOs could elicit proinflammatory airway responses as well, although the effect of dusts from cattle feedlots has not been extensively studied either in vivo or in vitro.

We hypothesized that dusts generated from cattle feedlots would also stimulate interleukin release in bronchial epithelial cells in a PKC-dependent manner. To test this hypothesis, we measured interleukin production in normal human bronchial epithelial cells as well as in bronchial epithelial cell lines that express a dysfunctional specific PKC isoenzyme. Our findings suggest that cattle feedlot dusts have the potential to elevate proinflammatory cytokines in human airway cells.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of feedlot dust extract. Airborne particulate matter was obtained on 8" x 10" glass-fiber filters that were exposed in high-volume, total suspended particulate (TSP) samplers (General Metal Works, Smyrna, GA) deployed downwind of large cattle feedlots across the Great Plains. Samplers were operated for 3–5 h at air flow rates of ~1.13 cubic meters per minute (m3/min) during the well-documented "evening dust peak" (28), a diurnal phenomenon characteristic of cattle feedlots. Filters were preconditioned before exposure in a drying oven for ~18 h at 90°C. Following exposure in the high-volume sampler, filters were again conditioned in the drying oven and weighed on an analytical balance (model AG245; Mettler-Toledo, Columbus, OH) to determine the time-averaged mass concentration of TSP represented by the samples. Filters were folded exposed-side-in and archived in paper envelopes. No records of sampler locations were retained.

The dust was extracted from the filters by placing sections of the filters into 50-ml conicals containing 40 ml of distilled H2O. Conicals were agitated for 72 h. The solution was frozen at –80°C until lyophilized. Feedlot dust extract (FLDE) was prepared by mixing 0.1 g of lyophilized material in 10 ml of Hanks' balanced salt solution (HBSS; Biosource International, Camarillo, CA) without calcium. The mixture was mixed on a stir plate, incubating at room temperature for 1 h and clarified twice by centrifugation (2,000 g). The second supernatant was filter-sterilized and used as 100% fresh FLDE for the subsequent experiments. Endotoxin levels in FLDE were assayed using the limulus amebocyte lysate assay commercially available (Sigma, St. Louis, MO) (19).

Cell culture. Normal human bronchial epithelial (NHBE) cells were purchased from Clonetics (Cambrex, East Rutherford, NJ). NHBE cells were passaged no more than six times before use in experiments. Additionally, BEAS-2B cells (American Type Culture Collection, Manassas, VA), a simian virus 40-transformed human bronchial epithelial cell line, were also used. Airway epithelial cells from both sources were plated on type I collagen (Vitrogen 100; Collagen, Palo Alto, CA)-coated dishes and maintained in culture in serum-free medium at 37°C in 5% CO2-95% air. A mixture (2:1) of Laboratory for Human Carcinogenesis/Roswell Park Memorial Institute (LHC-9/RPMI 1640) media was used to support the growth of the BEAS-2B cells under serum-free conditions, as previously described (20). NHBE cells were grown under serum-free conditions using a 1:1 mixture of Bronchial Epithelial Cell Basal Medium (Cambrex) and DMEM. Confluent monolayers of cells were pretreated for 1 h with or without PKC inhibitors followed by 1–24 h of exposure to 1–10% FLDE at 37°C.

Interleukin assay. After cell treatment conditions, media supernatants were collected and assayed for the concentration of interleukins released using a sandwich ELISA (20). Flat-bottomed 96-well polystyrene ELISA plates (Thermo Electron, Milford, MA) were coated with 200 µl/well of purified (goat) anti-human IL-8 antibody diluted 1:500 (R&D Systems, Minneapolis, MN) or IL-6 antibody diluted 1:1,000 in Voller's buffer (pH 9.6) overnight at 4°C. After three washings in PBS-Tween 20 (PBS-Tween), undiluted culture supernatants and human recombinant IL-8 or IL-6 standards (R&D Systems) were applied to the plates and incubated at room temperature for 2 h. Plates were again washed three times with PBS-Tween and incubated with (rabbit) anti-human IL-8 antibody diluted 1:500 (Endogen, Woburn, MA) or IL-6 antibody (Calbiochem, La Jolla, CA) diluted 1:1,000 in PBS Tween/BLOTTO (0.2% instant nonfat milk in PBS-Tween) for 1 h. After three washes, human serum-absorbed peroxidase-conjugated (goat) anti-rabbit IgG (Rockland, Gilbertsville, PA) was added at 1:2,000 (IL-6) or 1:1,000 (IL-8) in PBS-Tween/BLOTTO for 45 min. The plates were again washed three times, and 200 µl/well of peroxidase substrate [10 ng/ml orthophenylenediamine (Sigma), 0.003% H2O2 in dH2O] was added. The reaction was terminated with 27.5 µl/well of 8 M sulfuric acid, and plates were read at 490 nm in an automated ELISA reader (Bio-Rad, Hercules, CA). Culture supernatants were also assayed for IL-4, -10, -13, -17, and -18, as well as IL-6 and IL-8, utilizing the SearchLight array (Pierce Biotechnology, Rockford, IL), a multiplexed sandwich ELISA.

Remaining cells from the above treatment conditions were assayed for mRNA expression changes for IL-6 and IL-8.

IL-6 and IL-8 mRNA expression. Cells were grown to near confluence on collagen-coated six-well tissue culture dishes and maintained in serum-free LHC-9/RPMI 1640 (1:1) growth medium. Cells (1.5 x 106/condition) were challenged with 1% or 5% FLDE dissolved in growth medium for 1, 2, 6, or 24 h. At the end of the challenge period, cell layers were rinsed twice with HBSS and trypsinized at 37°C, and cell pellets were suspended in RNAlater (Applied Biosystems, Foster City, CA) and stored at –20°C until analyzed. Total RNA was extracted from ~7.5 x 105 cells/condition using the MagMAX-96 Total RNA Isolation kit (Applied Biosystems) with contaminating genomic DNA removed by DNase treatment. Quality of the isolated RNA was measured spectrophotometrically (ND-1000; NanoDrop Technologies, Wilmington, DE), and the nucleic acid-to-protein (260/280) ratios fell between 1.8 and 2.3 for all samples. Reverse transcription was performed using 40 ng of purified mRNA template, 1x RT buffer, 5.5 mM MgCl2, 500 mM deoxyNTP mixture, 2.5 mM random hexamers, 0.4 U/ml RNase inhibitor, and 1.25 U/ml reverse transcriptase (Applied Biosystems Gene Expression Assay kit). Thermal cycling conditions recommended by the manufacturer were as follows: incubation at 25°C for 10 min, transcription at 48°C for 30 min, and enzyme inactivation at 95°C for 5 min. Real-time PCR was then employed to quantify human IL-6 and IL-8 message. IL-6 (hs00174131_m1)- and IL-8 (hs00174103_m1)-specific primer/probe sets were combined with TaqMan Universal Master Mix and buffer and were subjected to 40 cycles in an Applied Biosystems 7500 Real Time PCR System. Thermal cycling parameters included two set-up steps at 50°C for 2 min and 95°C for 10 min followed by 40 denaturing and annealing cycles (95°C for 15 s and 60°C for 1 min each). Fluorescence values were recorded for each sample. To control for unequal loading of mRNA, all values were normalized to endogenous ribosomal RNA, and final results were expressed as fold change compared with unmanipulated control samples.

Cell viability assay. Media supernatant (50 µl) from cell monolayers treated with FLDE, PKC inhibitors, or media alone were sampled for cell viability assays. In addition, confluent 60-mm dishes were lysed as a positive control for lactate dehydrogenase (LDH) release. Cell viability was determined by a commercially available kit (Sigma) to measure LDH release according to the manufacturer's instructions.

PKC isoform assay. After media supernatants were removed from treated cells, the cell monolayers were flash frozen in cell lysis buffer as described (36). The cells were scraped with a cell lifter, sonicated, and centrifuged at 10,000 g for 30 min at 4°C. The supernatant was removed (cytosolic fraction), and the pellet was resuspended in cell lysis buffer containing 0.01% Triton X-100 and sonicated again (particulate fraction). PKC isoform activity was determined in crude whole cell cytosolic and particulate fractions of NHBE and BEAS-2B similar to methods described previously (32, 36). Airway epithelial cells contain the {alpha}, beta, {delta}, {varepsilon}, and {zeta} PKC isoforms (1). To measure PKC isoform activity specifically, 24 µg/ml PMA, 30 mM dithiothreitol, 150 µM ATP, 45 mM Mg-acetate, a PKC{varepsilon} isoform-specific substrate peptide, and 10 µCi/ml [{gamma}-32P]ATP were mixed in a Tris·HCl buffer (pH 7.5). Chilled (4°C) cell lysate (cytosolic or particulate) samples (20 µl) were added to 40 µl of the reaction mix and incubated for 15 min at 30°C. This mixture (60 µl) was then spotted onto P-81 phosphocellulose papers (Whatman, Clinton, NJ) to halt incubation, and papers were subsequently washed five times in 75 mM phosphoric acid for 5 min, washed once in 100% ethanol for 1 min, dried, and counted in nonaqueous scintillant (National Diagnostics, Atlanta, GA). PKC activity was expressed in relation to the total amount of cellular protein assayed as picomoles of phosphate incorporated per minutes per milligram.

Cloning and transfection of human PKC mutants in airway epithelial cells. Mutant PKC{alpha}-expressing cells were generated on BEAS-2B cells to produce dominant-negative (DN) versions of PKC{alpha} using the tetracycline-responsive promoter expression system (Clontech, Palo Alto, CA), as previously described (7). BEAS-2B cells were grown to 90% confluency in six-well tissue culture dishes. Cells were transfected with the pTet-On expression vector encoding reverse tetracycline transactivator protein (rtTA) as well as a neomycin resistance gene using a cationic lipid technique, Lipofectamine-2000 (Invitrogen, Carlsbad, CA). Cells were propagated and selected for neomycin resistance using G418 (400 µg/ml; Calbiochem, San Diego, CA). Antibiotic-resistant clones were then transfected by electroporation with a DN variant made by in vitro mutagenesis, substituting an alanine for lysine at position 368. Expression of PKC{alpha} from this vector was under the control of the tetracycline response element (21). In addition, BEAS-2B cells were simultaneously cotransfected with a plasmid encoding the "humanized" green fluorescent protein (GFP) reporter to facilitate cell selection. All PKC{alpha} plasmid vectors were generously provided by Dr. Dan Rosson (Lankenau Medical Research Center, Wynnewood, PA).

To generate DN PKC{varepsilon}, mutation within the ATP-binding site of PKC{varepsilon} was created at amino acid position 437 using site-directed mutagenesis, rendering the kinase inactive (9, 12, 17, 31). The PKC DN cDNA was similarly cloned into the Clontech pEGFP-N1 vector, a gift of Dr. Christer Larsson. This construct was transfected using Lipofectamine 2000 into live BEAS-2B cells. Stable transfectants were selected for 3 wk with media containing 200 µg/ml G418. Isolated stable clones were extracted using Corning cloning cylinders (Corning, Acton, MA), and GFP-positive clones were sorted by fluorescence-activated cell sorting to enrich the population of transfected cells.

Reagents. To assess the effect of chemical inhibition of various isoforms of PKC, confluent, submerged cell monolayers were treated with the PKC{alpha} inhibitor Gö-6976 (1 µM), the PKC{zeta} inhibitor rottlerin (20 µM), the PKC{zeta} inhibitor myristylated PKC{zeta} (1 µM), or the novel PKC inhibitor Ro 31-8220 (BioMol, Plymouth Meeting, PA). Optimal PKC{varepsilon} activity inhibition occurred at 10 µM Ro 31-8220 as previously determined (25). Specific PKC isoform substrate peptides were for PKC{varepsilon} (ERMRPRKRQGSVRRRV; Calbiochem), PKC{alpha} (VRKRTLRRL; Bachem, Torrance, CA), PKC{delta} (Calbiochem), and PKC{zeta} (SIYRRGSRRWRKL; Biosource, Camarillo, CA). All other reagents not specified were purchased from Sigma.

Statistical analysis. All quantitative experiments were performed in triplicate, and each experiment was repeated a total of three times. All data were analyzed using GraphPad Prism (version 4.00 for Windows; GraphPad Software, San Diego, CA) and represented as means ± SE. Therefore, each data point graphically presented represents n = 9 to generate the standard error of the mean. Data were analyzed for statistical significance using ANOVA. Significance was accepted at the 95% confidence interval.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The effect of FLDE exposure on proinflammatory cytokine release in NHBE cells was examined. Confluent monolayers of NHBE cells were exposed to increasing concentrations of FLDE from 1–10%, and supernatant media was collected 1–24 h. The media was subsequently assayed for IL-6 and IL-8 using ELISAs specific for these interleukins. A significant increase in IL-6 release was detected in response to 1% and 5% FLDE (P < 0.05) at 6 h, but an additional significant (P < 0.05) elevation in IL-6 was observed in response to 24-h treatment with 1% and 5% FLDE (Fig. 1A). Likewise, 1% and 5% FLDE stimulated significant increases in IL-8 release at 6- and 24-h treatment (Fig. 1B). These increases in interleukin protein release were temporally preceded by a rapid elevation of mRNA for both IL-6 and IL-8 at 1-h exposure to either 1% or 5% FLDE (Fig. 1, A and B, insets). A purified concentration of endotoxin equivalent to that in our preparation of 10% FLDE failed to stimulate any release of IL-6 or IL-8 in our assays (data not shown). No significant media release of IL-4, -10, -13, -17, or -18 was detected in response to any concentration or time point of FLDE assayed (data not shown). No cell death was observed in response to 10% FLDE (data not shown). These data demonstrate that an extract of cattle feedlot dust can elicit the specific release of IL-6 and IL-8 from the bronchial epithelium.


Figure 1
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  		Fig. 1. Feedlot dust extract (FLDE) stimulates IL-6 and IL-8 mRNA production and protein release in a concentration-dependent manner in normal human bronchial epithelial cells. FLDE (1% in gray bars; 5% in black bars) stimulated a significant (*P < 0.05 vs. media control) release of both IL-6 (A) and IL-8 (B) at 6 h of treatment. At 24-h treatment, both 1% and 5% FLDE were significantly (**P < 0.05) increased over the 6-h treatment. By 1-h treatment with 1% and 5% FLDE, increases in mRNA for both IL-6 (A, inset) and IL-8 (B, inset) were detected.

 
Because our previous studies revealed that CAFO dust obtained from hog confinement facilities stimulated the release of IL-6 and IL-8 in a PKC-dependent manner (20), we assayed the effect of FLDE on PKC activity in NHBE cells. FLDE-stimulated isoenzyme activity was assayed for PKC{alpha}, PKC{delta}, PKC{varepsilon}, and PKC{zeta}, isoforms previously identified in airway epithelium (34). A rapid activation of PKC{alpha} was observed after a 1-h exposure with 1% and 5% FLDE (Fig. 2A). A subsequent activation of PKC{varepsilon} was observed after a 6-h exposure with 1% and 5% FLDE (Fig. 2B). No significant activation of PKC{delta} or PKC{zeta} was observed under these same treatment conditions (data not shown). These data reveal that FLDE activates PKC{alpha} and PKC{varepsilon} concurrent with the release of IL-6 and IL-8.


Figure 2
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  		Fig. 2. FLDE activates protein kinase C (PKC) in a concentration-dependent manner in normal human bronchial epithelial cells. PKC{alpha} is significantly (*P < 0.05 vs. media control) activated by 1–5% FLDE at 1-h exposure (A), whereas PKC{varepsilon} is significantly activated at 6-h FLDE exposure to 1–5% FLDE (B). PKC{alpha} activity is significantly elevated at 1 h by 5% vs. 1% (*P < 0.05).

 
The impact of pharmacological inhibition of PKC on FLDE-stimulated release of interleukins was assayed next. NHBE cells were pretreated for 1 h in the presence or absence of 1 µM of the PKC{alpha}-specific inhibitor, Gö-6976. We previously found this concentration of Gö-6976 to be effective at inhibiting PKC{alpha} activity in airway epithelium (35). Gö-6976 did not inhibit IL-6 (Fig. 3A) or IL-8 (Fig. 3C) release after a 6-h exposure to 5% FLDE. IL-6 and IL-8 release was also not significantly decreased in the presence of Gö-6976 after a 24-h exposure to FLDE (data not shown). Direct assay of NHBE PKC{alpha} activity levels demonstrated that FLDE failed to activate PKC{alpha} in the presence of Gö-6976 (Fig. 3E). Next, NHBE cells were pretreated for 1 h in the presence or absence of 10 µM of the novel PKC class inhibitor Ro 31-8220. We previously used this concentration of Ro 31-8220 to effectively inhibit PKC{varepsilon} activity in bronchial epithelium (25). Ro 31-8220 significantly inhibited IL-6 (Fig. 3B) and IL-8 (Fig. 3D) release after a 6- and 24-h exposure to 5% FLDE. Likewise, the direct assay of NHBE PKC{varepsilon} activity levels demonstrated that FLDE failed to activate PKC{varepsilon} in the presence of Ro 31-8220 (Fig. 3F). Interestingly, the PKC{delta} inhibitor rottlerin and the PKC{zeta} inhibitor myristolated PKC{zeta} inhibitor peptide failed to inhibit FLDE-stimulated release of IL-6 or IL-8, even though these inhibitors both decreased their respective isoform activities (data not shown). These data show that whereas FLDE activates both PKC{alpha} and PKC{varepsilon}, only the inhibition of PKC{varepsilon} results in the inhibition of IL-6 and IL-8 release.


Figure 3
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  		Fig. 3. Inhibition of PKC{varepsilon}, but not PKC{alpha}, blocks FLDE-stimulated increases in IL-6 and IL-8 in normal human bronchial epithelial cells. Preincubation for 1 h with 1 µM Gö-6976 (Gö), a PKC{alpha}-specific inhibitor, before stimulation with 5% FLDE did not inhibit the stimulated release of IL-6 (A) or IL-8 (C) even though Gö-6976 effectively blocked FLDE-stimulated increases in PKC{alpha} (E). Preincubation for 1 h with 1 µM Ro 31-8220 (Ro), a PKC{varepsilon} inhibitor, before stimulation with 5% FLDE inhibited the stimulated release of IL-6 (B) and IL-8 (D). Ro 31-8220 also inhibited FLDE-stimulated increases in PKC{varepsilon} (F). *P < 0.05 vs. media control.

 
Because pharmacological kinase inhibitors can lack specificity or induce unexpected side effects, we created cell lines from the immortalized BEAS-2B cell line that express a DN PKC{alpha} or PKC{varepsilon} to confirm our results using inhibitor drugs. To characterize these BEAS-2B mutant cell lines, we performed direct kinase activity and translocation assays. The DN PKC{alpha} cell line made in BEAS-2B cells demonstrated a significant reduction in calcium-activated PKC{alpha} activity, whereas the DN PKC{varepsilon} cell line retained a calcium-activated PKC{alpha} response similar to that of wild-type BEAS-2B cells (Fig. 4A). Conversely, a significant decrease in total PKC{varepsilon} activity was detected in the DN PKC{varepsilon} cell line, but not in the DN PKC{alpha} cells (Fig. 4B). Functionally, no stimulated translocation of PKC{varepsilon} from cytosol to membrane in response to the PKC-activating phorbol ester, PMA, was observed in DN PKC{varepsilon} cells (Fig. 4C). These control experiments characterize these DN cell lines to be functionally lacking their specific PKC isoform activities and demonstrate their usefulness in assaying specific PKC{alpha}- and PKC{varepsilon}-stimulated responses.


Figure 4
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  		Fig. 4. Dominant negative (DN) PKC-expressing cell lines are isoform specific. Cell lines DN for PKC{alpha} and PKC{varepsilon} (DN-PKC{alpha} and DN-PKC{varepsilon}) were constructed in an immortalized human bronchial epithelial cell line (BEAS-2B). Wild-type (WT) cells demonstrated a 4.5-fold increase in total PKC{alpha} activity in response to 10 µM CaCl2 (A). Calcium-stimulated PKC{alpha} activity was present in DN-PKC{varepsilon} cells, but not in DN-PKC{alpha} cells (*P < 0.05 vs. WT control). DN-PKC{varepsilon} cells demonstrated a significant (*P < 0.05) decrease in PKC{varepsilon} activity compared with WT or DN-PKC{alpha} cells (B). As visualized by confocal microscopy, no stimulated translocation of PKC{varepsilon} from cytosol to membrane in response to 15 min of 20 ng/ml of the PKC-activating phorbol ester, PMA, was observed in DN-PKC{varepsilon} cells (C).

 
Using these isoform-specific PKC DN BEAS-2B cell lines, we examined the effect of FLDE on the stimulated release of IL-6 and IL-8. Treatment with 5% FLDE for 6 and 24 h stimulated a significant IL-6 release in wild-type BEAS-2B cells (Fig. 5A). The DN PKC{alpha}-expressing cell line demonstrated no differences from these wild-type cells in response to FLDE. Similarly, the FLDE-stimulated IL-8 release, although decreased, remained significantly stimulated in PKC{alpha} DN-expressing cells (Fig. 5C). Direct PKC activity assay revealed that PKC{alpha} was not activated by FLDE in the DN PKC{alpha} cells (Fig. 5E). However, in the DN PKC{varepsilon}-expressing cell line, a significant reduction in IL-6 (Fig. 5B) and IL-8 (Fig. 5D) in response to 6- and 24-h FLDE was observed in contrast to the levels of stimulated IL-6 and IL-8 in wild-type BEAS-2B at the same time points. Again, direct PKC isoform activity assays in these same cells showed no PKC{varepsilon} activation in response to FLDE in PKC{varepsilon} DN cells (Fig. 5F). These data confirm that the FLDE-stimulated release of IL-6 and IL-8 requires the activation of PKC{varepsilon} in bronchial epithelial cells.


Figure 5
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  		Fig. 5. FLDE fails to stimulate IL-6 and IL-8 release in a bronchial epithelial cell line (BEAS-2B) expressing a DN PKC{varepsilon}. No differences in 5% FLDE-stimulated release of IL-6 are observed between WT BEAS-2B cells and a DN-PKC{alpha}-expressing cell line (A). A significant, although reduced, amount of IL-8 is released in response to 5% FLDE-stimulated DN-PKC{alpha} cells (C). Significant inhibition of 5% FLDE-stimulated IL-6 (B) and IL-8 release (D) is observed in a DN-PKC{varepsilon}-expressing cell line. FLDE fails to activate PKC{alpha} or PKC{varepsilon} in the DN cell lines (E and F). #P < 0.01 vs. media control; *P < 0.05 vs. media control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our findings suggest, for the first time, that PKC{varepsilon} regulates the stimulation of IL-6 and IL-8 release in response to FLDE in NHBE and BEAS-2B cells. Using direct measurements of interleukin release and kinase activation, we found that both a PKC{varepsilon} inhibitor as well as expression of DN PKC{varepsilon} blocks the stimulation of IL-6 and IL-8 release by extracted feedlot dust. We used two approaches to confirm that the DN-transfected constructs expressed in the BEAS-2B cell line were indeed capable of altering the function of our target enzyme. First, we directly assayed the kinase activity of the DN cells. Because the classic PKC isoforms require calcium binding to the regulatory region of the enzyme before catalytic activation can occur, we assayed the total in vitro PKC{alpha} activity in the presence and absence of calcium. Unlike PKC from the wild-type BEAS-2B cells, PKC from the DN PKC{alpha} cell line failed to respond to exogenous calcium. Second, we assayed the translocation of activated kinase to the membrane in both wild-type and PKC{varepsilon} DN cells. It is established that phorbol esters bind to the diacylglycerol binding site of the regulatory region and induce PKC{varepsilon} to translocate to the cell membrane. Therefore, we tagged both our wild-type and DN PKC{varepsilon} constructs with a GFP so that the localization of PKC{varepsilon} could be visualized by confocal microscopy. In this manner, we demonstrated that wild-type GFP-PKC{varepsilon}-expressing cells rapidly translocated to the cell membrane in response to phorbol ester, but DN GFP-PKC{varepsilon}-expressing cells failed to perform this enzyme activation function. Furthermore, GFP had no affect on kinase activity. Therefore, we have provided evidence that the DN cells used in our study indeed have functionally inhibited PKC isoform activity before using them in a novel assay involving FLDE.

It is established that airway epithelial cells contain the PKC isoforms {alpha}, beta, {delta}, {varepsilon}, and {zeta}. Both the classic calcium-dependent isoform PKC{alpha} as well as the novel isoform PKC{delta} have been implicated in the regulation of interleukin release in airway epithelial cells. PKC{alpha} has been reported to regulate stretch-induced IL-8 secretion in A549 cells (37) and cigarette smoke (33) or aldehyde-stimulated IL-8 release (35) in bronchial epithelial cells. PKC{delta} has been reported to regulate TNF{alpha}-stimulated IL-8 release (16) in the 16HBE14o– human bronchial cell line. It was therefore surprising to us to find that neither PKC{alpha} nor PKC{delta} activation was responsible for FLDE-stimulated IL-6 and IL-8 release in bronchial epithelium. The difference in isoform activation times made it attractive to speculate that PKC{alpha} was activated early (1 h) by FLDE, and this activation led to a sequential activation of PKC{varepsilon} at 6 h. However, we found that although FLDE was associated with the activation of PKC{alpha}, similar to the effect that hog barn dust has on bronchial epithelial cells (20), PKC{alpha} activation was not associated with FLDE-stimulated IL-6 or IL-8 release.

Our findings also differ from previous reports that IL-8 is stimulated via the activation of PKC{alpha} in that we observe no change in FLDE-stimulated IL-6 and IL-8 whether we use normal cells treated with the PKC{alpha} inhibitor or mutant cells that express a DN PKC{alpha}. This observation may be unique to FLDE stimulation. However, we observed no inhibition of FLDE-stimulated IL-6 or IL-8 in response to the PKC{delta} inhibitor rottlerin even though rottlerin effectively inhibited PKC{delta} activity in NHBE and BEAS-2B. In fact, we have been unable to block the stimulated IL-8 release in response to cigarette smoke, aldehyde-adducted proteins, or TNF{alpha} by inhibiting PKC{delta} (data not shown). This finding is in contrast with those of Page et al. (16) where DN PKC{delta}-expressing cells were shown to block TNF-stimulated IL-8 release, although direct measurements of PKC{delta} activity were not performed in that study.

PKC{varepsilon} regulates diverse functions in the airway epithelium. PKC{varepsilon} has been shown to increase PMA-stimulated migration and cell shape changes in human alveolar A549 cells (3). PKC{varepsilon} has been reported to facilitate cystic fibrosis transmembrane regulator activity via RACK1 binding in the human tracheal Calu-3 cell line (13) as well as the regulation of the sodium, potassium-ATPase in rat alveolar cells (8). We have recently implicated PKC{varepsilon} in the attachment of ciliated epithelial cells to the basal epithelium (25). Clearly, this isoenzyme plays many roles in airway epithelial signaling depending on the specific cell type and stimulatory agent involved. Adding to this complexity is the observation that interleukin release can be mediated by other PKC isoforms, such as the stimulation of cigarette smoke-induced IL-8 release via PKC{alpha} (11). Due to the nature of auto-downregulation in response to activation of PKC, it is not surprising that multiple PKC isoforms would demonstrate overlapping functions. Under such a scenario, the stimulation of proinflammatory cytokines may take place in cells exposed to cigarette smoke via PKC{alpha} after such a cytokine response by FLDE leading to downregulated PKC{varepsilon} has occurred. Such a scenario of dual cigarette smoke and organic dust exposure would be an interesting hypothesis to test using in vivo models.

Studies of the signal transduction mechanisms regulating airway inflammation in response to inhaled agricultural occupational dusts have important clinical implications. We have observed that PKC isoenzymes, especially PKC{varepsilon}, are critically involved in FLDE stimulation of epithelial cell IL-8 and IL-6 release, suggesting that the isoenzymes may represent novel targets for therapies aimed at modulating organic dust-induced airway inflammation. Dusts from different types of animal confinement feeding operations cause similar respiratory symptoms, but different dusts may elicit inflammatory mediator release using multiple signaling pathways. Defining how dusts are different in terms of cell signaling and additionally what components of dusts are responsible for these differences may assist in improved monitoring of occupational settings.

It is important to relate the dust material used in this study with realistic exposure conditions. Sweeten et al. (28) reported net downwind concentrations (5 h averaging time) of TSP ranging from 97 to 1,687 µg/m3 and averaging 700 ± 484 µg/m3 (mean ± 1 SD) on three commercial cattle feed yards in the Texas panhandle (net downwind concentrations are computed as the downwind concentration minus the upwind, or background, concentration, and they reflect the net increase in aerosol concentration attributable to the source area). Their measurements were taken between 15 and 61 m downwind of the feed pens. The corresponding 5-h concentrations of PM10 (particles having an aerodynamic equivalent diameter of 10 µm or less) ranged from 11 to 866 µg/m3 with an average of 285 ± 214 µg/m3. Actual TSP concentrations measured in the monitoring events from which the present samples were taken ranged from 100 to 4,000 µg/m3 at a distance of ~15 m downwind from the source boundary. In Sweeten et al. (28), as well as the present study, samples were collected along or near the center line of the plume at ground level under atmospheric conditions of very high to extreme dust potential. For the samples analyzed in the present study, those conditions included low afternoon relative humidity (<25%), high afternoon temperatures (>32°C), and low wind speed (<6.7 m/s).

In summary, an extract of dust obtained from ambient air downwind of cattle feeding operations stimulates airway epithelial cells' release of inflammatory cytokines, IL-8 and IL-6. We have also determined that this dust augments epithelial cell PKC{alpha} and PKC{varepsilon} activation. Using both isoenzyme inhibitors and DN epithelial cells for PKC{alpha} and PKC{varepsilon}, we observed that PKC{varepsilon} is critical in the regulation of feedlot dust-induced IL-8 and IL-6. Further studies are needed to define the component(s) of cattle feedlot dust responsible for stimulation of IL-8 and IL-6 via a PKC{varepsilon}-dependent pathway.


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This work was supported by grants from the Department of Veterans Affairs (Merit Review grant to T. A. Wyatt) and the Centers for Disease Control/National Institute for Occupational Safety and Health (CDC/NIOSH; OH008539 to D. J. Romberger). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of CDC/NIOSH.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. J. Romberger, Research Service, VA Nebraska Western Iowa Health Care System (Omaha Div.), 4101 Woolworth Ave., Omaha, NE 68105 (e-mail: dromberg{at}unmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Alpert SE, Walenga RW, Mandal A, Bourbon N, Kester M. 15-HETE-substituted diglycerides selectively regulate PKC isotypes in human tracheal epithelial cells. Am J Physiol Lung Cell Mol Physiol 277: L457–L464, 1999.[Abstract/Free Full Text]
  2. Boschetto P, Quintavalle S, Miotto D, Lo Cascio N, Zeni E, Mapp CE. Chronic obstructive pulmonary disease (COPD) and occupational exposures. J Occup Med Toxicol 1: 11, 2006.[CrossRef][Medline]
  3. Busso N, Masur SK, Lazega D, Waxman S, Ossowski L. Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells. J Cell Biol 126: 259–270, 1994.[Abstract/Free Full Text]
  4. Cathomas RL, Bruesch H, Fehr R, Reinhart WH, Kuhn M. Organic dust exposure in dairy farmers in an alpine region. Swiss Med Wkly 132: 174–178, 2002.[Medline]
  5. Dalphin JC, Maheu MF, Dussaucy A, Pernet D, Polio JC, Dubiez A, Laplante JJ, Depierre A. Six year longitudinal study of respiratory function in dairy farmers in the Doubs province. Eur Respir J 11: 1287–1293, 1998.[Abstract]
  6. Donham KJ, Haglind P, Peterson Y, Rylander R. Environmental and health studies in swine confinement buildings. Am J Ind Med 10: 289–293, 1986.[ISI][Medline]
  7. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 5547–5551, 1992.[Abstract/Free Full Text]
  8. Guerrero C, Pesce L, Lecuona E, Ridge KM, Sznajder JI. Dopamine activates ERKs in alveolar epithelial cells via Ras-PKC-dependent and Grb2/Sos-independent mechanisms. Am J Physiol Lung Cell Mol Physiol 282: L1099–L1107, 2002.[Abstract/Free Full Text]
  9. Hong DH, Petrovics G, Anderson WB, Forstner J, Forstner G. Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-epsilon. Am J Physiol Gastrointest Liver Physiol 277: G1041–G1047, 1999.[Abstract/Free Full Text]
  10. Iversen M, Kirychuk S, Drost H, Jacobson L. Human health effects of dust exposure in animal confinement buildings. J Agric Saf Health 6: 283–288, 2000.[Medline]
  11. Kashyap R, Floreani AA, Heires AJ, Sanderson SD, Wyatt TA. Protein kinase C-alpha mediates cigarette smoke extract- and complement factor 5a-stimulated interleukin-8 release in human bronchial epithelial cells. J Investig Med 50: 46–53, 2002.[ISI][Medline]
  12. Li RC, Ping P, Zhang J, Wead WB, Cao X, Gao J, Zheng Y, Huang S, Han J, Bolli R. PKCepsilon modulates NF-kappaB and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes. Am J Physiol Heart Circ Physiol 279: H1679–H1689, 2000.[Abstract/Free Full Text]
  13. Liedtke CM, Yun CH, Kyle N, Wang D. Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor. J Biol Chem 277: 22925–22933, 2002.[Abstract/Free Full Text]
  14. Martin LD, Rochelle LG, Fischer BM, Krunkosky TM, Adler KB. Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators. Eur Respir J 10: 2139–2146, 1997.[Abstract]
  15. Mathisen T, Von Essen SG, Wyatt TA, Romberger DJ. Hog barn dust extract augments lymphocyte adhesion to human airway epithelial cells. J Appl Physiol 96: 1738–1744, 2004.[Abstract/Free Full Text]
  16. Page K, Li J, Zhou L, Iasvoyskaia S, Corbit KC, Soh JW, Weinstein IB, Brasier AR, Lin A, Hershenson MB. Regulation of airway epithelial cell NF-kappa B-dependent gene expression by protein kinase C delta. J Immunol 170: 5681–5689, 2003.[Abstract/Free Full Text]
  17. Pass JM, Zheng Y, Wead WB, Zhang J, Li RC, Bolli R, Ping P. PKC{varepsilon} activation induces dichotomous cardiac phenotypes and modulates PKC{varepsilon}-RACK interactions and RACK expression. Am J Physiol Heart Circ Physiol 280: H946–H955, 2001.[Abstract/Free Full Text]
  18. Patel IS, Roberts NJ, Lloyd-Owen SJ, Sapsford RJ, Wedzicha JA. Airway epithelial inflammatory responses and clinical parameters in COPD. Eur Respir J 22: 94–99, 2003.[Abstract/Free Full Text]
  19. Rojas-Corona RR, Skarnes R, Tamakuma S, Fine J. The Limulus coagulation test for endotoxin. A comparison with other assay methods. Proc Soc Exp Biol Med 132: 599–601, 1969.[Medline]
  20. Romberger DJ, Bodlak V, Von Essen SG, Mathisen T, Wyatt TA. Hog barn dust extract stimulates IL-8 and IL-6 release in human bronchial epithelial cells via PKC activation. J Appl Physiol 93: 289–296, 2002.[Abstract/Free Full Text]
  21. Rosson D, O'Brien TG, Kampherstein JA, Szallasi Z, Bogi K, Blumberg PM, Mullin JM. Protein kinase C-alpha activity modulates transepithelial permeability and cell junctions in the LLC-PK1 epithelial cell line. J Biol Chem 272: 14950–14953, 1997.[Abstract/Free Full Text]
  22. Saenz RA, Hethcote HW, Gray GC. Confined animal feeding operations as amplifiers of influenza. Vector Borne Zoonotic Dis 6: 338–346, 2006.[CrossRef][ISI][Medline]
  23. Schwartz DA, Donham KJ, Olenchock SA, Popendorf WJ, Van Fossen DS, Burmeister LF, Merchant JA. Determinants of longitudinal changes in spirometric function among swine confinement operators and farmers. Am J Respir Crit Care Med 151: 47–53, 1995.[Abstract]
  24. Sigurdarson ST, Kline JN. School proximity to concentrated animal feeding operations and prevalence of asthma in students. Chest 129: 1486–1491, 2006.[CrossRef][ISI][Medline]
  25. Slager RE, Sisson JH, Pavlik JA, Johnson JK, Nicolarsen JR, Jerrells TR, Wyatt TA. Inhibition of protein kinase C epsilon causes ciliated bovine bronchial cell detachment. Exp Lung Res 32: 349–362, 2006.[CrossRef][ISI][Medline]
  26. Spurzem JR, Romberger DJ, Von Essen SG. Agricultural lung disease. Clin Chest Med 23: 795–810, 2002.[CrossRef][ISI][Medline]
  27. Strieter RM. Interleukin-8: a very important chemokine of the human airway epithelium. Am J Physiol Lung Cell Mol Physiol 283: L688–L689, 2002.[Free Full Text]
  28. Sweeten JM, Parnell CB Jr, Shaw BW, Auvermann BW. Particle size distribution of cattle feedlot dust emission. Transactions of the ASAE 41: 1477–1481, 1998.[ISI]
  29. Veranth JM, Reilly CA, Veranth MM, Moss TA, Langelier CR, Lanza DL, Yost GS. Inflammatory cytokines and cell death in BEAS-2B lung cells treated with soil dust, lipopolysaccharide, and surface-modified particles. Toxicol Sci 82: 88–96, 2004.[Abstract/Free Full Text]
  30. Von Essen SG, Auvermann BW. Health effects from breathing air near CAFOs for feeder cattle or hogs. J Agromedicine 10: 55–64, 2005.[Medline]
  31. Wu JM, Xiao L, Cheng XK, Cui LX, Wu NH, Shen YF. PKC epsilon is a unique regulator for hsp90 beta gene in heat shock response. J Biol Chem 278: 51143–51149, 2003.[Abstract/Free Full Text]
  32. Wyatt TA, Forget MA, Sisson JH. Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells. Am J Pathol 163: 1157–1166, 2003.[Abstract/Free Full Text]
  33. Wyatt TA, Heires AJ, Sanderson SD, Floreani AA. Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells. Am J Respir Cell Mol Biol 21: 283–288, 1999.[Abstract/Free Full Text]
  34. Wyatt TA, Ito H, Veys TJ, Spurzem JR. Stimulation of protein kinase C activity by tumor necrosis factor-{alpha} in bovine bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 273: L1007–L1012, 1997.[Abstract/Free Full Text]
  35. Wyatt TA, Kharbanda KK, Tuma DJ, Sisson JH. Malondialdehyde-acetaldehyde-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells. Alcohol 25: 159–166, 2001.[CrossRef][ISI][Medline]
  36. Wyatt TA, Schmidt SC, Rennard SI, Sisson JH. Acetaldehyde-stimulated PKC activity in airway epithelial cells treated with smoke extract from normal and smokeless cigarettes. Proc Soc Exp Biol Med 225: 91–97, 2000.[Abstract/Free Full Text]
  37. Yamamoto H, Teramoto H, Uetani K, Igawa K, Shimizu E. Stretch induces a growth factor in alveolar cells via protein kinase. Respir Physiol 127: 105–111, 2001.[CrossRef][ISI][Medline]



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