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Abstract |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Terminal airways are affected in many lung diseases and toxic
inhalations. To elucidate the changes in terminal airways in these diverse situations it will be helpful to profile and quantify gene expression in terminal bronchiolar epithelium. We
used laser capture microdissection (LCM) to collect terminal
bronchiolar epithelial cells from frozen sections of lungs of
mice subjected to intratracheal bleomycin. The RNA from
these cells was used for analysis of select messenger RNAs
(mRNAs) by quantitative real-time polymerase chain reaction
(PCR). In parallel, we used real-time PCR to analyze mRNAs in
whole-lung homogenates prepared from other mice given intratracheal bleomycin. We found reductions of Clara cell-specific protein and keratinocyte growth factor receptor mRNAs
in both terminal bronchiolar epithelium and whole-lung homogenates 7 d after bleomycin. In contrast, terminal bronchiolar epithelial transforming growth factor (TGF)-
mRNA was reduced but whole-lung TGF- mRNA was not changed, whereas
terminal bronchiolar epithelial epidermal growth factor (EGF)
receptor mRNA was not changed but whole-lung EGF receptor
was reduced. We conclude that LCM can isolate terminal bronchiolar epithelial cells for studies of cell-specific gene expression by quantitative real-time PCR, and that cell-specific gene
expression in terminal bronchiolar epithelium is not necessarily
reflected in analysis of whole-lung gene expression.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Currently, there is great interest in characterizing the gene expression of cells in vivo. The most frequently used methods for detection and quantification of gene expression in lung are Northern blotting and reverse transcriptase/polymerase chain reaction (RT-PCR) using total RNA derived from whole lung. Recently, oligonucleotide arrays have been used to markedly expand the number of genes that can be evaluated in samples of lung (1). However, the lung is anatomically complex, composed of over 40 different cell types, so that analysis of whole-lung messenger RNAs (mRNAs) may not reflect or reveal patterns in individual cell types. In situ hybridization can provide cell-specific information about gene expression, but the number of genes that can be studied conveniently by this method is limited and the method is not quantitative.
In the past few years, laser technology has been used to assist with the retrieval of individual cells from lung tissue. In two studies, single alveolar macrophages were retrieved from frozen sections of rat lung using a sterile needle after adjacent cells were photolysed by a nitrogen laser (2, 3). Laser capture microdissection (LCM) is another laser-based method for retrieval of single cells from tissue sections for analysis of gene expression (4, 5). To date, LCM has been applied to retrieve tumor cells (6) and cells of neuronal subtype (7), but it has not been used to obtain structural cells of the lungs.
The aim of this study was to use LCM to harvest terminal bronchiolar epithelium for quantification of mRNAs
for several genes that might be expected to change in response to intratracheal administration of bleomycin. Specifically, we determined the effects of intratracheal bleomycin upon the expression of Clara cell-specific protein
(CCSP), transforming growth factor (TGF)-, epidermal growth factor (EGF) receptor, and keratinocyte growth
factor (KGF) receptor mRNAs in terminal bronchiolar
epithelium. We compared the results from LCM samples
with measurements of mRNAs isolated from whole-lung homogenates.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mice
A total of 129 SvEv mice, 3 to 4 mo old, were housed in a pathogen-free animal facility. Bleomycin (0.05 U in 50 µL of saline) was instilled intratracheally as previously described (8). Mice were killed at 3 and 7 d. Nonmanipulated mice were used as controls. All procedures were approved by the Washington University Animal Studies Committee.
Tissue Preparation for LCM
The lungs were removed and inflated with diluted Tissue-Tek
OCT (Sakura Finetek U.S.A., Torrance, CA) (50% vol/vol) in ribonuclease (RNase)-free phosphate-buffered saline with 10% sucrose. The lungs were then cut sagitally into several pieces, placed
in cryomolds, covered with Tissue-Tek OCT, and immediately
frozen in liquid nitrogen-cold 2-methylbutane and stored at
80°C. The tissue was sectioned at 7 µm in a cryostat. The frozen
sections were placed on plain glass slides (Fisher Scientific, Pittsburgh, PA) and then immediately fixed in 100% ethanol for 1 min,
followed by rehydration through 95, 70, and 50% ethanol diluted
in RNase-free deionized H2O (5 s each); staining with 0.5% Nissl
(cresyl violet acetate) (Sigma Chemical, St. Louis, MO)/0.1 M sodium acetate buffer for 1 min; dehydration in graded ethanols
(5 s each); and cleaning in xylene for 5 min (7).
Tissue Sampling by LCM
Slides were completely air-dried and kept desiccated to prevent the activation of endogenous RNase in the tissues. A minimum of 40 sections per animal were prepared for LCM. LCM of terminal bronchiolar epithelial cells was performed in the Alvin J. Siteman Cancer Center Tissue Procurement Core Facility at Washington University Medical Center using a PixCell II System (Arcturus Engineering, Mountain View, CA) with the following parameters: laser diameter, 15 µm; pulse duration, 1.5 ms; and amplitudes, 30 to 40 mW. Bronchiolar epithelium was retrieved from the junction of the terminal bronchiole and alveolar ducts and proximally along the bronchiole ~ 250 µm. A total of 20,000 to 40,000 bursts of the laser were used to collect the cells from each mouse. After the samples were captured on transfer film (CapSure Transfer Film TF-100; Arcturus Engineering), nonspecific attached components were removed by adhesive tape (CapSure Pad; Arcturus Engineering).
RNA Extraction
Total RNA was extracted from laser-captured cells using a High Pure RNA Isolation Kit (Boehringer Mannheim, Indianapolis, IN) and eluted in 30 µL of elution buffer included in the kit. Genomic DNA was digested by incubation with 18.2 U/µL DNase I (Sigma) for 15 min at room temperature. Total RNA of whole lungs from nontreated and bleomycin-treated mice at 7 d was extracted as previously described (8).
Reverse Transcription
The quantity of 15 µL of RNA extract from laser-captured cells, containing an unknown amount of RNA, was reverse transcribed using random hexamers (Perkin-Elmer Applied Biosystems, Branchburg, NJ) and 50 units of MuLV RT (Perkin-Elmer) in 30 µL of total volume at 25°C for 10 min, at 42°C for 30 min, and at 94°C for 5 min, and the resulting first-strand complementary DNA (cDNA) was used as template for the RT-PCR. The quantity of 1 µg of total RNA from whole lungs was reverse transcribed in a total volume 20 µL, as described earlier.
5' Exonuclease-Based Fluorogenic PCR
Quantitative PCR was carried out using an ABI Prism 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, CA). Oligonucleotide PCR primer pairs and fluorogenic probes for each gene were designed from the published sequences using Primer Express software (Perkin-Elmer) (Table 1). Specific amplification using these primers was confirmed by ethidium bromide staining of the predicted size of the PCR products on an agarose gel. The probes were labeled with 6-carboxyl-fluorescein at the 5' end and 6-carboxyl-tetramethyl rhodamine at the 3' end (PE Applied Biosystems). Primers and labeled probe for rodent glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) were purchased from PE Applied Biosystems. A 1-µL aliquot of the RT product from LCM-retrieved cells or 1 µL of a 10-fold dilution of the RT product from whole lungs was placed into each tube and PCR was carried out in duplicate using a PCR reagent kit (TaqMan PCR Core Reagents Kit with AmpliTaq Gold; PE Applied Biosystems) according to the manufacturer's protocol. In brief, a master mixture including all reagents required for PCR was prepared to give final concentrations of 1× TaqMan buffer A, 5.5 µM deoxynucleotide triphosphates, 5.5 mM manganese acetate, 0.01 U/µL AmpErase UNG, and 0.025 U/µL of Taq Gold DNA polymerase. Hybridization probe and primers were added to give a final probe concentration of 100 nM-probe and 200 nM-primers, and the total reaction volume was increased to 25 µL. PCR was performed for 40 cycles, consisting of a denaturation step at 95°C for 15 s and a combined annealing and extension step at 58°C for 2 min. The PCR reactions for the target gene and GAPDH were performed in separate tubes to avoid possible competition and/or interference in a single reaction tube (9). For linear regression analysis of unknown samples in each assay, a standard curve was generated using 4-fold serial dilutions of whole-lung cDNA from a normal mouse.
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Data Presentation and Statistical Analysis
Gene expression is normalized as a ratio of the mRNA of the gene of interest to the GAPDH mRNA in the same sample. The mean value of this ratio for the bleomycin-treated animals is expressed as a percentage of the mean ± standard error of the mean (SEM) value of the control animals. Statistical analysis was performed for the samples of whole lungs but not for those of LCM samples because of the small number of animals analyzed in each group. Differences between the two groups of animals were determined using Student's unpaired t test. Statistical significance was defined at P < 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Retrieval of Terminal Bronchiolar Epithelium
In mice, the terminal bronchiole ends abruptly at alveolar ducts (10). Figure 1 shows the selective retrieval of terminal bronchiolar epithelium from nontreated mouse lung (Figures 1a-1c) and from mouse lung 7 d after intratracheal bleomycin (Figures 1d-1f) using LCM. After bleomycin, numerous inflammatory cells were evident in the interstitium and additional collagen was deposited around bronchoalveolar junctions (Figure 1d). The tissue retrieved by LCM, which was immersed in lysis buffer for RNA extraction, was confined to terminal bronchiolar epithelium (Figures 1b and 1e), and the tissue remaining after LCM further demonstrates the selectivity of the LCM procedure for removing terminal bronchiolar epithelium (Figures 1c and 1f). Efforts to do LCM 14 d after bleomycin were not successful because the terminal bronchiolar epithelial cells could not be removed without using a higher intensity of laser pulse or a longer laser pulse, either of which enlarged the field and caused retrieval of additional cell types.
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Amplification Plots and Standard Curves for Quantitative RT-PCR
Extracted RNA was reverse transcribed into cDNA and
used for quantitative RT-PCR using an ABI Prism 7700 Sequence Detector. As an example, a schematic representation of fluorescence release of the serially diluted whole-lung cDNA during signal amplification for CCSP is shown
in Figure 2a. The algorithm calculates the cycle at which
each PCR amplification reaches a pre-set threshold (Ct). The number of PCR cycles to reach the Ct signal is inversely proportional to the number of copies of the target
sequence in each sample. The linearity of dilution and the
assay range of CCSP cDNA are shown in Figure 2b. Thus,
this assay provided sufficient sensitivity and precision for
measuring the mRNA in the cells captured by LCM. The
possibility of genomic DNA contamination in the starting RNA preparation was excluded by finding no amplification of the template without inclusion of RT. Comparable
amplification plots and linear standard curves were obtained in the assays for TGF-, EGF receptor, KGF receptor, and GAPDH (data not shown).
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Rn (the fluorescence signal increase
due to template amplification) was collected during the
extension phase of each cycle of the PCR and is plotted
on a log scale against the cycle number. The bold horizontal line indicates the threshold. (b) Standard curve
shows the input cDNA amount versus the number of cycles (Ct) to the pre-set threshold (slope: CCSP mRNA in Whole Lungs and in Bronchiolar Epithelium
In whole lung there was a significant decrease in CCSP mRNA 7 d after intratracheal bleomycin (Figure 3a), consistent with findings reported in studies using oligonucleotide array (see ref. 1; the relevant data are available in the website provided in the paper: http://medicine.ucsf.edu/divisions/lbc/ ). The CCSP mRNA levels in terminal bronchiolar epithelium were also decreased by as much as 2-fold in the terminal bronchioles 7 d after bleomycin (Figure 3b and Table 2).
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TGF-, EGF Receptor, and KGF Receptor
TGF- mRNA was significantly decreased 7 d after bleomycin in whole-lung samples (Figure 4a), whereas it was
unchanged in LCM samples of untreated and bleomycin-treated terminal bronchiolar epithelium (Figure 4b). In contrast, EGF receptor mRNA showed no difference in whole-lung RNA between untreated and bleomycin-treated lung
(Figure 5a), whereas its expression tended to be decreased in
terminal bronchiolar epithelium after bleomycin (Figure 5b). KGF receptor mRNA was decreased both in whole lungs
and terminal bronchioles 7 d after bleomycin (Figure 6). The
terminal bronchiolar epithelium expression of gene expression at 3 and 7 d after intratracheal bleomycin is shown in Table 2. It is noted that there are trends of decrease at 7 d relative to 3 d for the EGF and KGF receptors.
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Typically, quantification of mRNA expression in lung in
vivo has been done using whole lung homogenates (8, 11).
To enrich the contribution of RNA from select anatomical
compartments, microdissection of trachea, lobar bronchus,
major and minor daughter airways, and parenchyma has
been described (12). However, microdissection to the level
of these structures cannot yield information about gene
expression in individual cell types. Recently, single alveolar macrophages were retrieved from hematoxylin-stained
frozen sections of rat lung using a sterile needle after adjacent cells were photolysed by a nitrogen laser (2, 3). In
those reports, tumor necrosis factor (TNF)- mRNA by
quantitative real-time PCR in the isolated alveolar macrophages matched TNF- mRNA of alveolar macrophages collected by bronchoalveolar lavage, validating the procedure of harvesting single cells from sections of lung for
analysis of mRNAs.
In this study we used LCM to obtain terminal bronchiolar epithelial cells. These cells were readily identified microscopically because of their anatomical location; and they presented an ideal situation for LCM because the airway lumen is on one side, so that the laser beam could be focused on the epithelium without including subepithelial tissue. With LCM, targeted epithelial cells were rapidly fused to the LCM capture film, which was then placed into a PCR tube. By combining LCM with 5' exonuclease-based fluorogenic PCR (13), we were able to reproducibly quantify multiple mRNAs in a small number of terminal bronchiolar epithelial cells.
Although we found that LCM worked well to harvest bronchiolar epithelium, some points of caution should be noted. Because bronchiolar injury develops heterogeneously after intratracheal bleomycin, and injured cells cannot be distinguished from normal cells by light microscopy, the cells we harvested by LCM are likely a mixture of normal and injured terminal bronchiolar epithelium. Moreover, although the cells we retrieved by LCM in terminal bronchioles are highly enriched in Clara cells, because these cells predominate in the murine terminal bronchiole we did not attempt to exclude other epithelial cell types, such as ciliated cells, which are present in this region (14, 15). Epithelial heterogeneity could be important in some situations because bronchiolar injury, such as that which occurs in response to napthalene, can be cell type-specific (16). To investigate gene expression in specific bronchiolar epithelial cells, such as stem cells which may serve as potent progenitor cells after injury (17), more precise cell isolation is required. In several reports dealing with other tissues, the specificity of LCM targeting has been improved by using immunostained frozen sections (3, 18, 19).
Intratracheal administration of bleomycin is widely used
as an experimental model of idiopathic pulmonary fibrosis
(20). Bleomycin also affects airway epithelium, causing
bronchiolar epithelial swelling (8), apoptosis (14), and the
development of bronchiolized epithelium in alveolar ducts
and alveoli, so-called "alveolar bronchiolization" (21, 22).
Recently, we found that after intratracheal bleomycin
there is expression of gelatinase B (matrix metalloproteinase-9) in terminal bronchiolar epithelial cells and in cells
comprising alveolar bronchiolization (8). Gelatinase B expression in bronchiolar epithelium after bleomycin, and
the morphologic changes of these cells, suggest that bleomycin affects the expression of many genes in bronchiolar
epithelium. In this study we focused on several genes that
we speculated would be affected: CCSP, TGF-, and the
KGF and EGF receptors.
CCSP is produced by Clara cells, which comprise ~ 90% of the cell type lining terminal airways in the mouse (10). Although the biologic role of CCSP is still unclear, growing evidence suggests that this protein operates as an anti-inflammatory agent (23, 24). Using LCM we found decreased CCSP mRNA in terminal bronchiolar epithelium within 3 d after intratracheal bleomycin that persisted at 7 d. Whole-lung homogenates also showed a significant reduction in CCSP mRNA at 7 d after bleomycin. These results support in situ hybridization data 28 d after intratracheal bleomycin (25) and the recent finding, using oligonucleotide arrays, that there is reduced CCSP mRNA in whole lung 7 and 14 d after intratracheal bleomycin (see ref. 1; the relevant data are available in the website provided: http://medicine.ucsf.edu/divisions/lbc/). Whether reduced CCSP mRNA after intratracheal bleomycin reflects a reduction in the number of Clara cells, as suggested by the in situ hybridization data at 28 d, or whether other mechanisms are involved at other times, such as reduced CCSP expression per Clara cell or accelerated turnover of CCSP mRNA in Clara cells, is not known.
Cell proliferation, as determined by bromodeoxyuridine
positivity, occurs in terminal bronchiolar epithelium after
intratracheal bleomycin in rats (22); and in mice we detected proliferation of terminal bronchiolar epithelial cells by
proliferating cell nuclear antigen immunostaining 3 d after
bleomycin (8). These findings and the occurrence of alveolar bronchiolization after intratracheal bleomycin prompted
us to investigate the expression of two growth factors that
affect bronchiolar epithelial proliferation: TGF- and KGF.
TGF- has a broad mitogenic spectrum, stimulating a variety of cell types, including epithelial cells. TGF- and its
physiologic receptor, the EGF receptor, are constitutively detectable in airway epithelium, alveolar septal cells, and
alveolar macrophages by immunohistochemistry (26). Increased expression of TGF- and EGF receptor after bleomycin-induced lung injury in rats has been reported (26),
and a critical role of TGF- in the fibrosis that ensues after
bleomycin is strongly suggested from the finding of reduced fibrosis in mice with TGF- deficiency (27).
KGF, a member of the fibroblast growth factor (FGF) family, is a mitogen for different types of epithelial cells. Epithelial cells do not express KGF, but most epithelial cells express the KGF receptor which is a splice variant of FGF receptor 2 (28). Thus, KGF acts in a paracrine manner. In the lung, KGF can have potent effects. Intratracheal administration of KGF blunts alveolar injury in various model systems (29, 30), apparently by stimulating proliferation, migration, and differentiation of alveolar epithelial cells. KGF instillation also causes proliferation of bronchiolar epithelial cells (31), indicating the presence of the KGF receptor on bronchiolar epithelium. Accordingly, we speculated that KGF receptor expression might be increased by bleomycin-induced injury and might be involved in the proliferation of terminal bronchiolar epithelium that occurs after intratracheal bleomycin.
We did not find increases in the mRNAs for TGF-,
EGF receptor, or KGF receptor in LCM-retrieved terminal bronchiolar epithelial cells in the first week after intratracheal bleomycin. On the contrary, there were decreases in TGF- and EGF receptor mRNA at 3 and 7 d
and a decrease in KGF receptor mRNA at 7 d after bleomycin. In any case, these results suggest that TGF-, EGF receptor, and KGF receptor do not contribute directly to
the early response of bronchiolar epithelium to intratracheal bleomycin, although they may have roles in the responses of alveolar epithelial and interstitial cells.
The strength of LCM is the opportunity to harvest specific cells for analysis of gene expression that might be
overlooked or even misinterpreted if those cells were only
a minor component of whole tissue samples. In this study,
the results of comparisons of gene expression patterns between bronchiolar epithelial cells retrieved by LCM and
whole-lung homogenates are intriguing. The pattern of
CCSP mRNA change after bleomycin in LCM-retrieved
terminal bronchiolar epithelium and in whole lung was
similar. This finding would be expected because Clara cells
are the only cells that express CCSP mRNA in the whole
lung. On the other hand, for factors such as KGF receptor,
EGF receptor, and TGF- that are expressed by many
lung cell types, it would not be unexpected to find discrepancies between the results with LCM-retrieved bronchiolar epithelial cells and whole lung, and discrepancies
were observed. We interpret these results as indicating
clearly that whole-lung samples should be used with caution to assess gene expression in a particular cell type for a
gene that is expressed by many cell types in the lung.
In summary, we have demonstrated the feasibility of measuring mRNA in individual structural cells in sections of lung. Combining LCM and the 5' exonuclease-based fluorogenic RT-PCR system is shown to be a method of quantifying in vivo gene expression in a small amount of terminal bronchiolar epithelium. This technique should facilitate the understanding of terminal bronchiolar responses in normal and disease states. The recent development of cDNA microarrays for monitoring thousands of genes simultaneously will further enhance the power of LCM to elucidate gene expression in vivo.
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Footnotes |
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Address correspondence to: Robert M. Senior, M.D., Dept. of Medicine, Barnes-Jewish Hospital, North Campus, 216 S. Kingshighway, St. Louis, MO 63110. E-mail: seniorr{at}msnotes.wustl.edu
(Received in original form December 8, 2000 and in revised form March 5, 2001).
Abbreviations: Clara cell-specific protein, CCSP; complementary DNA, cDNA; epidermal growth factor, EGF; glyceraldehyde-3-phosphatase dehydrogenase, GAPDH; keratinocyte growth factor, KGF; laser capture microdissection, LCM; messenger RNA, mRNA; polymerase chain reaction, PCR; reverse transcriptase, RT; transforming growth factor, TGF.Acknowledgments: This work was supported by grants HL47328 and HL29594 from the National Heart, Lung and Blood Institute of the National Institutes of Health, the Alan A. and Edith L. Wolff Charitable Trust, and a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists. The authors thank Manami Nagano, PE Applied Biosystems, for assistance in design of the optical probes and primers for 5' exonuclease-based fluorogenic RT-PCR.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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