Isolation of rodent airway epithelial cell proteins facilitates in vivo proteomics studies of lung toxicity

Åsa M. Wheelock,1 Lu Zhang,2 Mai-Uyen Tran,3 Dexter Morin,1 Sharron Penn,2 Alan R. Buckpitt,1 and Charles G. Plopper3

Departments of 1Molecular Biosciences and 3Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California Davis, Davis 95616; and 2Advanced Research Team, Amersham Biosciences Corporation, Sunnyvale, California 94085-4520

Submitted 13 March 2003 ; accepted in final form 27 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent developments in genomics, proteomics, and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins that facilitates in vivo proteomics studies of two target-cell pheno-types of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell-specific proteins in fivefold higher concentrations and reduces the yield of nonepithelial cell proteins 13-fold compared with homogenates from microdissected airways. In addition, 36% more protein spots were detectable by two-dimensional gel electrophoresis.

two-dimensional gel electrophoresis; cytochrome P-450 monooxygenase; actin; airway epithelium; proteomics


THE LUNG IS A TARGET TISSUE for numerous environmental and industrial toxicants (12). Although substantial progress has been made in understanding the early events critical in chemically induced injury to the lung, much less is known about the cellular response that follows the initial injury or is evident during repair. The availability of methods for investigating the levels of mRNA and protein on a global scale has tremendous potential to enhance our understanding of the cellular events that occur in response to cytotoxic chemicals. However, in vivo studies of alterations in the genome and proteome in organ systems as heterogeneous as the lungs raise methodological challenges. Among the more than 40 different cell types that make up the lung, only six of the phenotypes have been reported to be pneumotoxicant targets: pulmonary alveolar macrophages, capillary endothelial cells, type I and type II alveolar epithelial cells, ciliated bronchiolar epithelial cells, and nonciliated epithelial cells (Clara cells) (12). Thus to avoid masking the alterations in one specific target cell phenotype by other resistant phenotypes, attention to the choice of isolation methods is essential.

Robust methods have made the measurement of mRNA levels for thousands of genes in a tissue routine procedure, and laser capture techniques offer specific isolation of individual cell phenotypes. However, numerous studies demonstrate poor correlations between mRNA and protein expression (5, 13, 34). Investigations of lung adenocarcinoma tissue showed correlation coefficients between mRNA and protein abundance ranging from -0.467 to 0.442 (5). The data in this and several other studies suggest that protein expression is not necessarily well predicted by the abundance of mRNA in an organ (5) and underscores the need for complementary proteomic evaluations.

Despite considerable effort to develop new separation methods for proteomics studies, two-dimensional electrophoresis (2DE), introduced more than 25 years ago, remains one of the most efficient methods for separating the thousands of proteins expressed in a eukaryotic cell (15, 18). Recent refinement of this technology, including introduction of narrow-range immobilized pH gradient strips (IPG) and fluorescent protein labels with high sensitivity and broad dynamic ranges (Cy3, Cy5, Sypro Ruby), has significantly improved the reproducibility and sensitivity of the technique (9, 15, 22, 31). However, limitations in detection of low-abundance proteins still prevail. In the investigation of the proteome of a heterogeneous organ like the lung, the limited amount of protein that can be separated in the first dimension (isoelectric focusing) becomes a major factor in restricting the sensitivity of detection. Thus the development of selective isolation methods for obtaining proteins from cells of interest is essential when applying 2DE to in vivo proteomics studies of the lung.

The limited number of investigations of the pulmonary proteome published to date have mainly utilized cultured cell lines (16, 19, 20, 30) or nasal/bronchoalveolar lavage (11, 25, 26, 33) for protein isolation. However, cultured cells do not maintain their phenotypic protein expression pattern in part because all of the airway cell populations are vital for the maintenance of differentiation and function of the epithelium (35). Collection of bronchoalveolar/nasal lavage fluid provides access to proteins in macrophages, as well as those elaborated by epithelial cells, but may not reflect changes that occur intracellularly in the airway epithelium or alveoli in response to stressors. Consequently, the lack of phenotype- and compartment-specific isolation methods compatible with proteomics is currently limiting the application of this approach to in vivo lung toxicology. In this study, we present a method for selective isolation of airway epithelial cell proteins from the lung, thus sampling two of the six target cell phenotypes of the lung, Clara cells and ciliated cells. The method involves minimizing access to the parenchyma with agarose, followed by lavage with a lysis buffer that selectively and rapidly lyses the airway epithelial cells and solubilizes the proteins of these cells (lysis-lavage). Lysis-lavage was evaluated compared with the two sampling methods available in the literature that maintain phenotypic protein expression the best: homogenates of whole lung and microdissected airways. These new approaches resulted in significant improvement in 2DE separation patterns and detection of low-abundance proteins.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. All animal use was approved in advance by the Animal Use and Care Committee at the University of California, Davis (UC Davis). Male Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN), and male Swiss Webster mice were from Charles River (Wilmington, MA). Animals were fed ad libitum and housed in an American Association for Accreditation of Laboratory Animal Care-accredited facility in HEPA-filtered cage racks at the UC Davis, for at least 5 days before use in an experiment. Twelve rats (281–318 g, 8–10 wk) were randomly assigned to four groups (n = 3), and proteins were obtained by homogenization of whole lung for group 1, homogenization of microdissected airways for group 2 (29), or lavage of the airways with lysis solution (hereafter labeled as lysis-lavage) for groups 3 and 4. The end points for group 3 were light microscopy only, and the end points for group 4 were all methods described below. Six mice (28–33 g, 5–8 wk) were randomly assigned to two groups (n = 3), and proteins were obtained by homogenization of microdissected airways or lysis-lavage, respectively. Furthermore, nine rats (139–394 g) and 13 mice (25–37 g) were used for lung volume measurements, and five rats (254–315 g) were used to evaluate the experimental variability of the lysis-lavage method.

Chemicals. Dextrose was obtained from Fisher Chemicals (Pittsburgh, PA), SeaPlaque low-melting-point agarose from FMC Bio-products (Rockland, ME), and ultrapure urea from US Biological (Cleveland, OH). Omnipure Tris, glycine, and SDS were purchased from EM Sciences (Gibbstown, NJ). 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), protease inhibitor cocktail III, and Triton X-100 were acquired from Calbiochem (La Jolla, CA). Bradford protein assay reagent, Sequi-Blot polyvinylidene difluoride (PVDF) membrane, and Tween 20 were obtained from Bio-Rad (Hercules, CA). Thiourea and dithiothreitol (DTT) were ordered from Sigma-Aldrich (St. Louis, MO). All other electrophoresis materials were obtained from Amersham Biosciences (Piscataway, NJ). All solutions were prepared with deionized water (resistivity 18.1 M{Omega}/cm).

Determination of lung volume. Nine adult male rats (weights 139–394 g, age 40–75 days) and 13 adult male mice (weight 25–37 g, age 43–89 days) were euthanized with an overdose of pentobarbital. The trachea was cannulated, and the lungs were removed from the thorax. The lung volume (LV) was measured by inflation of the lungs to a pressure of 30 cmH2O with an airtight Hamilton syringe coupled to a manometer. The procedure was repeated three times with each animal, and the latter two were used for analysis. The correlation between animal weight and LV was evaluated by linear regression using Excel software (Microsoft, Redmond, WA).

Recovery of epithelial cell proteins by lysis-lavage. Animals were killed with an overdose of pentobarbital, and the trachea was exposed and cannulated. The lungs were removed from the thorax and inflated with agarose solution [0.75% low-melting-point agarose, 5% dextrose, volume = (0.5·LV) + valve void volume], immediately followed by dextrose solution [1% dextrose, 2% protease inhibitor cocktail III, 330 mosmol/kgH2O, volume = (0.5·LV) + valve void volume] through a three-way valve. Both solutions were preheated to 37°C to prevent the agarose from solidifying during inflation. The inflated lungs were incubated in 5% dextrose at 25°C to allow the agarose to solidify (15 min). The dextrose solution was then removed through simultaneous inversion of the lungs and gentle suction with a syringe. The procedure was repeated until no more solution could be recovered (4–5 times). The airways were then lavaged with 25°C lysis buffer containing 2 M thiourea, 7 M urea, 4% wt/vol CHAPS, 0.5% wt/vol Triton X-100, 1% wt/vol DTT, and 2% vol/vol protease inhibitor cocktail III (rat volume = 0.30·LV, mouse volume = 0.40·LV). The lavage was performed with a Hamilton syringe connected directly to the cannula. The syringe was briefly removed from the cannula upon inflation with the lysis buffer to release backpressure and allow the lysis buffer to reach the distal airways. The lysis buffer, now containing the proteins, was recovered immediately through repeated simultaneous inversion of the lungs, gentle massage of the lung lobes, and suction (4–5 times). The protein sample obtained by the lysis-lavage procedure was flash frozen on dry ice and stored at -80°C until use. Controls were prepared in an identical manner, with the exception that lysis buffer was substituted for dextrose solution (1% wt/vol dextrose, 2% vol/vol protease inhibitor cocktail III, 330 mosmol/kgH2O). Protein concentration was determined according to Bradford (2), with bovine serum albumin as the standard.

In preparation for the histopathological studies, the airways were lavaged once with 5% dextrose to flush out any remaining lysis buffer. The lungs were fixed for >2 h with 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the airway tree of the left lobe was exposed by microdissection.

Preparation of homogenates of microdissected airways and whole lung. Animals were killed with an overdose of pentobarbital, and the trachea was exposed and cannulated. The lungs were removed from the thorax and inflated with 1% agarose and incubated in 5% dextrose on ice for 10 min to allow the agarose to solidify. For samples of microdissected airways, the conducting airways of the left lobe were isolated as previously described (29). For whole lung samples, the entire left lobe was collected. Both whole lung and microdissected airway samples were homogenized with a Polytron homogenizer in lysis buffer (same as for lysis-lavage). Samples were kept at room temperature for 1 h to allow complete solubilization of the proteins. The homogenate was centrifuged for 75 min at 100,000 g, 15°C. The high concentration of chaotropes and detergents in the lysis buffer causes solubilization of intracellular organelles and its proteins, and the centrifugation step only serves to pellet agarose and insoluble cell debris such as collagen. Protein concentration was determined according to Bradford (2), and samples were flash frozen on dry ice and stored at -80°C until use.

Immunoblot analysis. Protein samples were separated with SDS-PAGE using 10% Nupage minigels (Invitrogen, Carlsbad, CA). Proteins were electroblotted to a PVDF membrane. The primary antibodies used were monoclonal anti-{alpha} smooth muscle actin (product no. A-2547, diluted 1:10,000; Sigma-Aldrich) and a rabbit polyclonal anti-cytochrome P-450 2F2 diluted 1:20,000 [donated by Dr. H. Sasame (24)]. For the {alpha}-actin antibody, the NH2-terminal decapeptide of the mouse isoform (TrEMBL accession number O88990 [GenBank] ) was used as the antigen, which is identical to the NH2-terminal decapeptide of the rat isoform (TrEMBL accession number Q8R4I6). Cross-reactivity of the P-450 2F2 antibody to the rat isoform (CYP2F4) has been established in prior studies (3). Additional experiments to verify the cross-reactivity of the two antibodies to the rat isoforms were conducted in our laboratory. The primary antibodies were detected using anti-mouse and anti-rabbit secondary antibodies and chemiluminescence using an ECL-plus kit according to the protocol provided by the manufacturer (Amersham Biosciences). Film exposures were scanned using an Umax III flat bed scanner and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Differential gel electrophoresis. We prepared samples obtained from microdissected airways, lysis-lavage, and whole rat lung by pooling triplicate samples obtained by each isolation method respectively to reduce interanimal variation. In addition, aliquots containing the same amount of protein from each of these three pooled samples were combined to create an internal standard. The internal standard was labeled with Cy3 dye, whereas the other samples were labeled with Cy5 dye according to the manufacturer's protocol (Amersham Biosciences). Samples were centrifuged at 4°C, 14,000 rpm for 10 min. The supernatant was collected, and the pH was adjusted to ~8.5 by dropwise addition of 1 M Tris·HCl (pH 9.5). Protein concentrations were determined according to Bradford (2). Cy dye stock solution (1 mM) was diluted 1:5 with fresh, dry dimethylformamide, which was added to each protein sample to achieve a ratio of 50 µg of protein to 200 pmol of Cy dye. The reaction mixture was incubated in the dark at 0°C for 30 min, and 10 nmol of lysine were added (0°C, 10 min) to stop the reaction. An equal volume of sample buffer (8 M urea, 4% CHAPS, 20 mg/ml DTT, and 0.5% IPG, pH 4–7 buffer) was added, and the sample was placed at 0°C for 15 min. The samples were stored at -70°C until use.

Cy5-labeled sample (50 µg, obtained from microdissected airways, lysis-lavage, or whole lung) was mixed with 50 µg of Cy3-labeled internal standard, and proteins were separated by 2DE according to the manufacturer's protocols (Amersham Biosciences). Samples were run in duplicate. Briefly, samples were diluted in sample buffer (same as above, except the DTT concentration was 2 mg/ml) to 450 µl and rehydrated on a 24-cm Immobiline DryStrip, pH 4–7. Isoelectric focusing (IEF) was performed with the IPGphor IEF System (Amersham Biosciences) for 135 kVh. The proteins were separated in the second dimension on 10% polyacrylamide gels, 255 x 205 x 1 mm, with an Ettan DALT 12 SDS-PAGE System (Amersham Biosciences) with 2.5 W/gel for 30 min followed by 20 W/gel for 220 min at 20–22°C. The Cy dyes were visualized on a Typhoon 9410 laser scanner (Molecular Dynamics) with excitation 532 nm, emission 580BP30 (Cy3) and excitation 633 nm, emission 670BP30 (Cy5), respectively. We performed matching spot analysis with DeCyder Biological Variation Analysis Software (Amersham Biosciences) by relating the spot volume of the Cy5-labeled sample proteins to the Cy3-labeled internal standard. Total protein spot detection analysis was performed with PDQuest software 6.0 (Bio-Rad) by standard protocols without normalization (sensitivity, 12; size scale, 5; minimum peak, 1,000; vertical radius, 53; and horizontal radius, 49).

Samples collected though lysis-lavage from five adult rats (254–315 g) were analyzed with differential gel electrophoresis (DIGE) to evaluate the experimental variability. An aliquot of each sample was labeled with Cy5 dye as described above. An internal standard was created through pooling of all five samples and labeled with Cy3 dye. Each of the five Cy5-labeled samples was coseparated with an equal amount of Cy3-labeled internal standard by 2DE and visualized as described. Eight representative high-abundance protein spots [range 749,000–25,200,000 relative fluorescence units (rfu)] and eight representative low-abundance protein spots (range 28,600–205,000 rfu), respectively, were selected for analysis. The 16 spots were quantified on each of the five gels, and we calculated the coefficient of variance (CV) for each of them using Excel.

High-resolution light microscopy. The airway tree of the left lobe was exposed though microdissection and incubated for 15 min with the nuclear probe 4,6-diamidino-2-phenylindole (DAPI, 2 µg/ml, catalogue no. D-1306; Molecular Probes, Eugene, OR) followed by incubation for 20 min with 0.2 µM of the smooth muscle probe Alexa Fluor 568 phalloidin (catalogue no. A-12380, Molecular Probes). Control specimens were permeabilized with 0.3% Triton X-100 before staining. Images of four different airway levels (Fig. 1) were collected under an Olympus BH2-RFCA microscope coupled to a Cool Snap digital camera (Roper Scientific, Tucson, AZ).



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Fig. 1. Light micrograph of the left lobe of rat with the airway tree exposed through microdissection. The frames illustrate the 4 different airway levels analyzed by light and scanning electron microscopy (A, trachea; B, midlevel airway; C, distal bronchioles; D, terminal bronchioles). Methacrylate sections were also obtained from the same 4 airway levels.

 

Scanning electron microscopy. After high-resolution light microscopy, the left lobe was treated with osmium and dehydrated through a graded ethanol series. The dehydrated lobes were then bathed in hexamethyldisilizane (Electron Microscopy Sciences, Fort Washington, PA) for 5 min at room temperature. The lung lobes were glued to scanning electron microscopy (SEM) chucks with Nexaband and allowed to air-dry overnight. The lungs were sputter-coated for six cycles with gold using a Polaron II ES100 sputter-coater (acceleration voltage 2.5 kV, 10 mA current in argon; Energy Beam Sciences, Agawan, MA). The exposed airway tree was viewed under a Philips SEM 501 microscope (FEI, Hillsboro, OR), and images of four different airway levels (Fig. 1) were collected.

Methacrylate sectioning. After light microscopy, a representative left lobe of each sample set was embedded in glycol methacrylate using Immunobed (Electron Microscopy Sciences) as described elsewhere (7). Sections of 0.2 µm were cut with a Leica RM 2155 Microtome (Leica, Nussloch, Germany), and fluorescent images of four different airway levels (Fig. 1) and parenchyma were collected under an Olympus BH2-RFCA microscope coupled to a Cool Snap digital camera (Roper Scientific). Subsequently, the sections were stained with a solution of 1% toluidine blue and 1% sodium borate, and additional images of the parenchyma were collected.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Determination of LV. The great variation in size over the adult life span of a rat results in sufficiently large variations in LV to cause inconsistent lysis of the distal and terminal bronchiolar epithelial cells when a constant LV is assumed. Thus the LV of nine adult male rats between 40 and 75 days old and weighing between 139 and 394 g was determined. The LV measurement was highly reproducible (CV <5%), and a linear relationship between body weight and the LV was observed {LV = [0.034·(g body wt) +1.09] ml, r2 = 0.91} (data not shown). In contrast, the LV of adult mice was found to be constant over their life span. For the 13 mice in the study (weight 25–37 g, age 43–89 days), the average LV was determined to be 0.90 ml (CV = 10.8%).

Lysis-lavage. The essential steps in the lysis-lavage procedure are inflation of the lungs with an agarose solution, followed by inflation with a dextrose solution that forces the agarose into the parenchyma. The dextrose solution is then recovered, and the open airways are lavaged with the lysis buffer. The three aspects optimized for the inflation steps were 1) the volume of agarose solution required (as percentage of the LV), 2) the density of the agarose solution, and 3) the volume of dextrose solution required (as a percentage of the LV). The optimal conditions were determined for minimizing access to the alveolar air spaces with agarose without any coverage of the terminal bronchioles. This was achieved by using a volume of agarose solution (concentration 0.75% wt/vol) corresponding to 50% of the animal's LV followed by an equal volume of dextrose solution. The optimal ratio of agarose to dextrose solution was determined to be 1:1. The optimal recovery of the dextrose solution was obtained by inversion of the lungs, via a syringe to apply "gentle" suction. Alternatively, the dextrose solution was poured out to avoid collapse of the distal airways. This was found to be particularly useful in mice, where the softer structure of the terminal airways increases the risk of collapse. The recovery of dextrose solution from rats (n = 27) averaged 61% (CV = 6.2%).

The components of the lysis buffer were based on the composition required for optimal separation of airway protein samples in IEF. Concentrations of Triton X-100 >0.5% vol/vol caused excessive foaming during lysis-lavage. Use of a Hamilton syringe was required to form a tight seal during the lavage step with lysis buffer due to the high content of detergents. We found that release of the back pressure by quickly disconnecting the Hamilton syringe from the cannula after inflation with lysis buffer, but before recovery, improved the lysis of the epithelium of the terminal bronchioles. In the rat, the volume of the lysis buffer used for the lavage was optimized to 0.3·LV. This volume is identical to the average recovered volume of the dextrose solution [61% recovery of a (0.5·LV) infused = 0.3·LV]. The recovery of the lysis buffer solutions in rat (n = 27) averaged 68% (CV = 7.6%). The protein concentration of the recovered lysis-lavage was 5.8 ± 1.0 mg/ml (mean ± SD) with an average protein recovery of 11.6 ± 0.8 mg/rat. In the mouse, the optimal relative volume of lysis buffer was higher than for rat (0.4·LV). Protein concentrations in recovered lysis-lavage from mouse were 7.7 ± 1.2 mg/ml, with the total protein recovered averaging 1.7 ± 0.4 mg/mouse.

Immunoblot analysis. To evaluate the reproducibility and selectivity of this new method for isolation of proteins from the airway epithelium, we performed immunoblot analysis. An antibody selective for a protein mainly localized to smooth muscle ({alpha}-actin) was used as a marker of airway wall cell proteins, and an antibody selective for a Clara cell-specific cytochrome P-450 monooxygenase (CYP2F) was used as a marker of airway epithelial cell proteins. The content of {alpha}-actin was on average 17.6-fold lower for mouse (CVmicrodissected = 11%, CVlysis-lavage = 34%) and 12.7-fold lower for rat (CVmicrodissected = 45%, CVlysis-lavage = 56%) in samples obtained by lysis-lavage compared with microdissected airway homogenates (Fig. 2). In contrast, the content of CYP2F present in samples obtained by lysis-lavage was 6.4-fold higher in mouse (CVmicrodissected = 44%, CVlysis-lavage = 33%) and 4.9-fold higher in rat (CVmicrodissected = 47%, CVlysis-lavage = 11%) compared with microdissected airway homogenates. {alpha}-Actin levels were similar in homogenates of whole lung and microdissected airways. CYP2F was not detectable in whole lung homogenates.



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Fig. 2. Comparison of the content of {alpha}-actin and CYP2F in protein samples obtained by microdissection of airways (n = 3) or lysis-lavage (n = 3) in rats and mice. Identical amounts of protein were applied to 10% SDS-PAGE gels, immunoblotting was performed using antibodies against {alpha}-actin (A, left) and against CYP2F (A, right), and proteins were visualized by ECL-plus and X-ray film. The immunoblot of the rat samples is shown in A; each lane represents an individual animal. The staining intensity was quantified with ImageQuant software, and the relative amounts of protein are presented in rat (B) and mouse (C). Data are presented as means ± SD (n = 3).

 

DIGE. To evaluate the experimental variability of the lysislavage method, we analyzed samples collected from five rats using DIGE. For the eight low-abundance protein spots (spot volume range 28,600–205,000 rfu) and eight high-abundance protein spots (spot volume range 749,000–25,200,000 rfu) selected (Table 1), the maximum CV detected was 50%, the mean CV was 24%, and the median CV was 20%.


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Table 1. Experimental variability of five rat lysis-lavage samples according to DIGE

 

In addition, samples from rat obtained by the three different isolation methods, lysis-lavage, homogenization of microdissected airways, and homogenization of whole lung, were analyzed by DIGE. To correct for the variability between different 2DE gels, we created a standard by combining equal aliquots from each of the samples. The standard was labeled with Cy3 and was coseparated with Cy5-labeled samples obtained from lysis-lavage, airway homogenates, or whole lung homogenates on individual 2DE gels. The abundance of the various proteins in samples obtained by lysis-lavage vs. microdissected airway homogenates showed significant differences. With a twofold threshold and 95% confidence limit, 168 of the 2,112 matched protein spots (8%) had altered abundance (Table 2). Ninety-six of the protein spots were more abundant in lysis-lavage samples compared with microdissected airway homogenates, whereas 72 were less abundant. When the threshold was increased to fourfold, 22 spots belonging almost exclusively to five protein groups (Fig. 3) were observed at lower levels in lysis-lavage compared with microdissected airway homogenates. Those protein groups have similar molecular weight and isoelectric point (pI), suggesting that they might come from the same family but have different posttranslational modifications (glycosylation, phosphorylation, etc.). In addition to the matched spot analysis performed with the DeCyder software, the total number of detectable protein spots was evaluated with PDQuest software. The microdissected airway homogenates yielded 36% fewer detectable protein spots (1,734) than did protein samples obtained by lysis-lavage (total of 2,365).


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Table 2. Altered abundance of protein spots in lysis-lavage vs. airway homogenate samples

 


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Fig. 3. Two-dimensional electrophoresis (2DE) gel image of proteins obtained by lysis-lavage and analyzed by differential gel electrophoresis (DIGE). Protein samples obtained by lysis-lavage or homogenates of microdissected airways from rat were labeled with Cy3 and coseparated with an internal standard labeled with Cy5. DeCyder software was used to indicate proteins present in lysis-lavage samples in at least 4-fold higher levels than microdissected airways (U) and in at least 4-fold lower concentrations compared with homogenates obtained from microdissected airways (D).

 

Even larger differences in protein abundances were observed when lysis-lavage samples were compared with those from whole lung homogenates. At a twofold threshold, 95% confi-dence limit, 163 of the matched protein spots had altered abundance, in a range from 51-fold increase to 30-fold decrease (data not shown).

Light microscopy. After the final lavage and fixation, the airway tree of the left lobe of each replicate was exposed through microdissection and stained for smooth muscle (Alexa 568-phalloidin) and nuclei (DAPI) to allow assessment of the depth of penetration of the lysis solution in various airway compartments. High-resolution light micrograph images are shown from the trachea, midlevel airways, distal bronchioles, and terminal bronchioles of the rat (Fig. 4) and from distal and terminal bronchioles of mouse (Fig. 5). The observations for trachea and midlevel airways in the mouse are very similar to those for the rat and are not shown here. The spherically shaped, DAPI-labeled nuclei of intact epithelial cells observed in the control micrographs (Fig. 4, A1, C1, E1, and G1) are not present in the specimens obtained from lysis-lavaged airways of the rat (Fig. 4, B1, D1, F1, and H1). In the mouse, spherical nuclei are observed only in the terminal bronchioles of lysislavaged specimens (Fig. 5D1). Micrographs of the lysis-lavaged specimens show nuclei with an elliptical shape, which is consistent with the shape of nuclei of the basement membrane. In control specimens stained with Alexa 568-phalloidin, the actin mesh of the cytoskeleton is visible, whereas the structure of the underlying smooth muscle is barely discernible. In comparison, the bundle structure of the smooth muscle (perpendicular to the airway direction) is clearly visible in the lysis-lavaged specimens. Residues of the cytoskeleton were observed only in the terminal bronchioles of the mouse (Fig. 5D2).



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Fig. 4. High-resolution light micrographs of control (left columns) and lysis lavage-treated (right columns) specimens from four airway levels of rat (as defined in Fig. 1); A and B: trachea, C and D: midlevel, E and F: distal bronchioles, and G and H: terminal bronchioles. The blue images were obtained from samples stained with 4,6-diamidino-2-phenylindole (DAPI), which labels nuclei (X1). The control images show the spherical nuclei of the intact epithelium, whereas in lysis lavage-treated specimens, the elliptical nuclei of the basement membrane are visible. The red images are stained with Alexa 568-phalloidin, which labels actin (X2). The meshwork of the cytoskeleton is visible in the control images, whereas in the lysis lavage-treated specimens, the epithelial cell layer is removed, and the underlying smooth muscle is visible. The inset in top left of each image is a magnification of the boxed area.

 


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Fig. 5. High-resolution light micrographs of control (left columns) and lysis lavage-treated (right columns) specimens of distal (A and B) and terminal (C and D) bronchioles of mouse. Blue images (X1) display staining with DAPI (nuclei) and red images (X2) display staining with Alexa 568-phalloidin (actin). In control images, the spherical nuclei (A1 and C1) and the meshwork of the cytoskeleton (A2 and C2) of the epithelial cells are visible. In lysis lavage-treated specimens of distal bronchioles (B), the epithelial cell layer is removed and the underlying smooth muscle is visible, whereas images of terminal bronchioles (D) indicate that some of the epithelial cells remained. The inset in top left of each image is a magnification of the boxed area.

 

SEM. One specimen from each sample group (n = 3) was analyzed by SEM. Electron micrographs were obtained at all four airway levels as defined in Fig. 1. The results were similar at all four airway levels, and only micrographs of the distal airways are displayed (Fig. 6). The dome-shaped Clara cells and the cilia of the ciliated epithelial cells are easily recognized in the images of the control specimens from both rat and mouse. These cell types are absent in the lysis lavage-treated specimens. The basement membrane is completely denuded, and its fibrillar structures are clearly discernible.



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Fig. 6. Scanning electron micrographs of lysis lavage-treated (right) vs. control (left) distal airways of rat (top) and mouse (bottom). In the control specimens (left), the intact epithelium, consisting of dome-shaped Clara cells and ciliated cells, is visible. In the lysis lavage-treated specimens, all of the epithelial cells have been removed, exposing the underlying fibrillar structure of the basement membrane. The inset in top left of each image is a magnification of the boxed area.

 

Methacrylate sections. After light microscopy, one to two specimens from each set of samples (n = 3) were embedded in methacrylate, and sections from all four airway levels defined in Fig. 1 and parenchyma were prepared. The Alexa 568-phalloidin and DAPI stains retained their fluorescence following the embedding procedure, and these are displayed for images of distal and terminal airways in Figs. 7 (rat) and 8 (mouse). In rat, the airway epithelium is not visible at either airway generation in the lysis lavage-treated specimens (Fig. 7, B and D). However, traces of epithelial cells are observed in the terminal bronchiole in the corresponding images of the mouse specimens (Fig. 8D), indicating that the terminal airways of mouse are only partially lysed by the procedure. This is further supported by the micrographs of the parenchyma of mouse (Fig. 9), where intact epithelial cells can be seen in the terminal airway (Fig. 9B10x, top left). This is not the case in the images collected from rat parenchyma (Fig. 10), where all bronchial epithelial cells appear to be removed (Fig. 10B10x). The parenchymal tissue of lysis lavage-treated mouse (Fig. 9B) and rat (Fig. 10B) shows that the alveolar epithelium is intact. However, the volume of alveolar epithelial cells appears to be slightly reduced in the lysis lavage-treated specimens of both species (Figs. 9B and 10B) compared with control specimens (Figs. 9A and 10A).



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Fig. 7. Methacrylate sections of control (left columns) and lysis lavage-treated (right columns) specimens of distal (A and B) and terminal (C and D) bronchioles of rat. Blue images (X1) display staining with DAPI (nuclei) and red images (X2) display staining with Alexa 568-phalloidin (actin). In control images the cuboidal epithelium is visible (A and C), whereas in the lysis lavage-treated specimens the basement membrane appears denuded (B and D). The inset in top left of each image is a magnification of the boxed area.

 


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Fig. 8. Methacrylate sections of control (left columns) and lysis lavage-treated (right columns) specimens of distal and terminal bronchioles of mouse. Blue images (X1) display staining with DAPI (nuclei) and red images (X2) display staining with Alexa 568-phalloidin (actin). In the images of distal bronchioles, the cuboidal epithelium is visible in the control specimen (A), whereas the basement membrane appears denuded in the lysis lavage-treated specimens (B). In the terminal bronchioles, the epithelial cells are still attached to the basement membrane in the lysis-lavage treated specimen but are blebbing (D). The inset in top left of each image is a magnification of the boxed area.

 


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Fig. 9. Methacrylate sections of control (left column) and lysis lavage-treated (right column) parenchyma of mouse. The top 4 panels are stained with DAPI (nuclei), and the 2 bottom panels are stained with toluidine blue (all tissue). The boxed areas each indicate the part of the image that has been enlarged in the panel shown below the respective image. The terminal bronchiolar epithelium of the lysis lavage-treated specimen (B10x, top left) is partially lysed. The alveolar region has intact epithelium, but the volume of these cells appears to be slightly reduced in the lysis lavage-treated specimen (B) compared with the control specimen (A).

 


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Fig. 10. Methacrylate sections of control (left column) and lysis lavage-treated (right column) parenchyma of rat. Top 4 panels are stained with DAPI (nuclei), and the 2 bottom panels are stained with toluene blue (all tissue). The boxed areas each indicate the part of the image that has been enlarged in the panel shown below the respective image. The terminal bronchiolar epithelium of the lysis lavage-treated specimen (B10x, top left) is completely lysed. The alveolar region has intact epithelium, but the volume of these cells appears to be slightly reduced in the lysis lavage-treated specimen (B) compared with the control specimen (A).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Investigating global changes in protein expression in response to toxicants, which produce site-selective cellular injury to heterogeneous organs like the lung, presents significant methodological challenges. Of the >40 cellular phenotypes present in the lung, only six have been reported as targets of pneumotoxicants (12). Thus the choice of the protein isolation method becomes a central issue for successful proteomics studies. Conducting proteomics studies on homogenates of whole lung is time efficient, and the protein yield is high. However, none of the cell types targeted by pulmonary toxicants comprise >10% of the total cell mass in the lung, and alterations of the proteome in a responsive cell type will be confounded by proteins contributed to the sample by other resistant cell types. Techniques introduced more than 10 years ago, which involve preparation of pulmonary subcompartments (airways) by microdissection, have been useful for assessing xenobiotic metabolism (29). However, because the airway epithelial cells constitute only ~20% of the total airway structure (23, 32), this approach would not be ideal for work designed to examine alterations of airway epithelial cell proteins in response to toxicants. The goal of this study was to develop a method for selective protein extraction from cells of the tracheobronchial airway epithelium. The method presented here involves minimizing access to the parenchyma with agarose followed by introduction of a lysis buffer directly to the airway epithelial cells by lavage of the airways, which solubilizes the proteins of epithelial cells (lysis-lavage). Application of the method to two species routinely used in toxicological and carcinogenesis studies, mouse and rat, was evaluated.

In the rat, airway cells comprise ~26% of the total lung cells (32), whereas the airway epithelial cells comprise only ~5% of the total lung cells (23). Thus the five- to sixfold enhancement of CYP2F protein in lysis-lavage samples compared with microdissected airway homogenates observed in this study (Fig. 2) is consistent with the relative cell composition reported in earlier morphometric work. CYP2F, a protein that is expressed in high abundance in airway epithelial Clara cells, was not even detectable in whole lung homogenate. The total P-450 content in the lung of both humans and animals is <10% of that in the liver (1). However, these proteins are concentrated 5- to 10-fold in the Clara cells compared with other lung cell types (12), which explains the masking of its presence when suboptimal isolation methods are used.

In addition to high recovery of an epithelial cell-specific protein like CYP2F, samples obtained by lysis-lavage contain significantly lower amounts of {alpha}-actin, a protein primarily associated with nonepithelial cells of the airway wall. In microdissected airway homogenates, the extremely high abundance of some nonepithelial proteins (such as {alpha}-actin) dilutes the proteins of interest, thus decreasing the loading capacity and ability to detect changes in low-abundance proteins in the epithelial cells. In addition, high concentrations of single proteins produce streaking and precipitation during IEF (data not shown), which may lead to coprecipitation of other low-abundance proteins and ultimately cause them to focus at the wrong pI during IEF separation (36). Thus the exclusion of high-abundance nonepithelial cell proteins is essential for improved 2DE separation and greatly enhances the limit of detection (Fig. 3). As indicated by the data in Table 2 and Fig. 3, there are striking differences between the abundance of several dozen proteins when lysis-lavage and microdissected airway techniques are compared.

A potential problem that can arise when using samples from intact airways for proteomics is carbamylation of the proteins. Urea is an essential constituent of the lysis buffer for IEF. At room temperature, urea can degrade to ammonium cyanate (). The alkaline conditions created by the slightly basic urea can cause deprotonation of the amines, with subsequent carbamylation of protein amino residues (14). In addition to causing an obscured separation pattern in the 2DE gel, this can also block the NH2 terminus for subsequent sequencing of the protein. In our hands, the microdissected explant has to be left in urea-containing lysis buffer for at least 1 h at room temperature following homogenization to allow complete lysis of the airway epithelial cells. In combination with the long centrifugation time required at temperatures >15°C, this could increase the risk of carbamylation. Although recent reports from McCarthy et al. (21) suggest that a much longer time in room temperature is needed to cause substantial carbamylation, the rapid solubilization of airway epithelial cells during lysis-lavage theoretically minimizes the risk of carbamylation.

The lysis-lavage method maintains one of the major advantages of the use of microdissected airways, namely that proteins are isolated directly from cells maintained in their natural microenvironment (29). Collapse or rupture of the integrity of the airway wall, which is unavoidable in other isolation methods of airway epithelial cells, results in transformation of the airway epithelial cell population (28). In addition, all cell populations in the airway appear to respond to stress even when only one subpopulation is targeted, and this further emphasizes the importance of maintaining the epithelial cells in their natural microenvironment during exposure (28).

Sampling of epithelium from distal and terminal bronchioles is of special interest, since this area is specifically targeted by several toxicants (27). Light micrographs of airways, both in whole mounts and in methacrylate sections, indicate that the lysis-lavage method samples this region in a reproducible manner. The smooth muscle layer and the basement membrane were left intact, while epithelial cells were completely removed throughout the entire airway tree in the rat and from all airways proximal to the terminal bronchioles in mice. The difference between the species may depend on the less-rigid structure of the mouse terminal bronchiole, which allows it to collapse more easily than that of the rat. The terminal bronchiole is also the hardest and most time-consuming lung subcompartment to dissect, and the problem with partial recovery of the terminal airway persists when microdissection is used for protein isolation. The fact that the terminal airway is only partly lysed by the lysis-lavage procedure also indicates that minimal amounts of lysis buffer reach the parenchymal tissue, which is supported by the light micrographs of this region (Fig. 9). This observation explains the difference in protein patterns between whole lung homogenates and lysis-lavaged airways.

Although the nonalveolar tissues of the lung represent ~13% of the total lung volume (32), a 1:1 ratio of the agarose and dextrose solutions used for inflation of the lungs was optimal for achieving efficient lysis of the distal and terminal bronchioles. For sampling of proteins from target cells of the alveoli, lysis-lavage without prior agarose inflation could potentially be used. To restrict protein extraction to proximal airways only, one could use a higher ratio of agarose to dextrose solution during inflation of the lungs to deliberately block access of the lysis buffer to the distal or terminal bronchioles. By subtraction of the information obtained using various approaches, the subpopulations of cells can be evaluated on an airway generation level without masking by alterations in other airway levels.

Although the technique described in this paper has many advantages for obtaining proteins from target cell populations in the lung for proteomic studies, the method has its limitations. Ciliated and nonciliated cells of the airway epithelium are two distinct populations of cells that line the airways, and these cells respond very differently to both reactive gases and metabolically activated cytotoxicants. Both cell types are effi-ciently removed from the airway epithelium during lysislavage, and alterations in the proteome of one cell type could be diluted by a lack of response of the other cell type. New saturation labeling techniques for fluorescent protein derivatization (17) combined with selective cell isolations by laser capture offer the promise of being able to differentiate changes that occur in select cell populations. In addition, although the contribution of proteins in lining fluid would be expected to be small in normal lung, these could be considerable in lungs from animals exposed to toxicants that either cause massive release of proteins into the airway lumen or cause a breach in the barrier between capillary blood supply and the airway. Finally, although the high-resolution light micrographs of alveolar tissue of both mouse and rat (Figs. 9 and 10) show that both type I and type II alveolar epithelial cells are intact after lysis-lavage treatment, the volume of these cells appear to be slightly reduced. This may simply be due to dehydration of these cells by the agarose solution, but the possibility that some lysis occurs also of the alveolar epithelium during lysis-lavage cannot be excluded.

The ultimate goal for in vivo proteomic studies of cellularly heterogeneous organs like the lung is to enable isolation of proteins from the individual cell phenotypes of the lung. Laser capture techniques have proven successful in examining changes in gene expression of specific cell types, but identification of the small amounts of protein obtained by laser capture remains a daunting analytical challenge. There are significant drawbacks to the use of isolated primary cells of specific phenotypes such as type II cells and Clara cells (6, 8, 10) for global proteome studies. Specifi-cally, the use of proteases in isolation procedures to recover the cells degrades both cell surface and intracellular proteins (8). Furthermore, methods for isolation of some airway epithelial cell phenotypes such as ciliated cells have not been developed (4), although this cell type is specifically targeted by inhaled oxidizing agents such as ozone (27). The development of an isolation method with high selectivity for airway epithelial cell proteins represents a significant step toward the goal of facilitating investigations of changes in the proteome in response to toxicants in the individual target cell phenotypes of the airway epithelium. Although some proteins from the tracheobronchial lining fluid or the alveolar epithelium may be sampled simultaneously, the more than fivefold increase in the sensitivity of detection for proteins specific to epithelial cells, and the >36% increase in detection of the number of low-abundance proteins in 2DE analysis makes lysis-lavage superior to other isolation methods for in vivo proteomics studies of the airway epithelium. We are currently utilizing this isolation method to examine alterations in the proteome of the airway epithelium in response to both inhaled and parenterally administered lung toxicants.


    ACKNOWLEDGMENTS
 
Special thanks to Dr. Ed Schelegle for help in designing the lung volume measurements, Kimberly Day for advice regarding the methacrylate sectioning procedures, and Dr. Craig Wheelock for kind support and advice.

GRANTS

These studies were supported in part by National Institute of Environmental Health Sciences Grants ES-04311, P42 ES-04699, ES-09681, and ES-06700. UC Davis is a Center for Environmental Health Sciences, and support for core facilities used in this work is gratefully acknowledged (ES-05711).


    FOOTNOTES
 

Address for reprint requests and other correspondence: Å. M. Wheelock, Dept. of Molecular Biosciences, School of Veterinary Medicine, Univ. of California, Davis, CA 95616 (E-mail: amkarlsson{at}ucdavis.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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