Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes

Robert Gonzalez,1 Yee Hwa Yang,2,3 Chandi Griffin,2,4 Lennell Allen,1 Zachary Tigue,1 and Leland Dobbs1,2,5

1Cardiovascular Research Institute, Departments of 2Medicine and 5Pediatrics, 3Center of Bioinformatics and Molecular Biostatistics, and 4General Clinical Research Center, University of California San Francisco, San Francisco, California

Submitted 19 July 2004 ; accepted in final form 14 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We used microarray analysis with Affymetrix rat chips to determine gene expression profiles of freshly isolated rat type I (TI) and TII cells and cultured TII cells. Our goals were 1) to describe molecular phenotypic "fingerprints" of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about this cell type, and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. For these experiments, we used preparations of isolated TI and TII cells that contained <2% cross-contamination. With a false discovery rate of 1%, 601 genes demonstrated over twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about functions of TI and TII cells. We found similar numbers of differentially expressed genes between freshly isolated TI or TII cells and cultured TII cells (698, 637 genes) and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference including cluster dendrograms and log/log identity plots indicated major differences between the phenotypes of freshly isolated TI cell and cultured type II cell populations. The latter results suggest that experiments with TII cells cultured under these conditions should be interpreted with caution with respect to biological relevance to TI or TII cells.

gene expression profiling; transdifferentiation


THE ALVEOLAR EPITHELIUM, which covers >99% of the very large internal surface area of the lung, comprises two cell types, type I (TI) and type II (TII) cells. TI cells are very large squamous cells with calculated diameters of 50–100 µm and volumes of 3,000 µm3 (56). Two or more very thin (~50 nm) cytoplasmic sheets extend from the nucleus of the TI cell to cover the basement membrane that separates the epithelium from the interstitium. TI cells cover >98% of the internal surface area of the lungs. In contrast, TII cells are smaller, cuboidal cells (diameter ~10 µm) characterized morphologically by surfactant-containing secretory granules called lamellar bodies. TI cells have an extremely high water permeability (13) and are capable of transporting ions (4, 33), suggesting that TI cells may play a role in lung liquid homeostasis. Additional functions of TI cells have been proposed, based on the known properties of several genes expressed in this cell type (9, 49, 62). TII cells, which cover the remainder of the alveolar surface, synthesize, secrete, and recycle surfactant components (reviewed in Ref. 51); TII cells also have the capacity to transport ions (26, 45), synthesize immune effector molecules (47, 57, 60), and act as progenitor cells following injury to the alveolar epithelium (21).

Although various genes and gene products have been shown to be differentially expressed between type I and type II cells (9, 15, 18, 35, 49, 60, 62), detailed molecular phenotypes of TI and TII cells have not been described. Because TII cells cultured on tissue culture plastic lose characteristics associated with the mature TII cell phenotype and acquire some characteristics of the TI cell phenotype (2, 5, 7, 17, 18), it has been proposed that cultured TII cells are transdifferentiating into TI cells, although the similarities and differences between cultured TII cells and TI cells are largely undefined. We used microarray analysis for gene expression profiling of freshly isolated rat TI and TII cells and cultured TII cells. The goals of this study were 1) to describe molecular phenotypic "fingerprints" of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through inferred functions of differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about the functions of this cell type, and 4) to ascertain the similarities/differences in gene expression between cultured TII cells and freshly isolated TI and TII cells. For these experiments, we used only preparations of isolated TI and TII cells that contained <2% cross-contamination. Using a false discovery rate of 1%, we found 601 genes demonstrating more than twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about cellular functions of TI and TII cells. We found similar numbers of genes differentially expressed between freshly isolated TI and cultured TII cells (698 genes), freshly isolated and cultured TII cells (637 genes), and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference such as cluster dendrograms and log/log identity plots indicated major differences in phenotypes of the cultured TII cell populations compared with freshly isolated TI or TII cells. The latter results suggest that experiments with the cultured TII cell model should be interpreted with caution with respect to biological relevance to either TI or TII cells.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell Isolation

Because the purity of cell preparations is important in analyzing the results of experiments with isolated cells, we discarded any cell preparation that contained >2% cross-contamination of cell types between the TI and TII cell populations, as determined by immunostaining with antibodies specific for each cell type (16, 18). To obtain sufficiently pure cells used for the studies described herein, we found it necessary to discard ~50% of the preparations of TI cells and ~10% of the preparations of TII cells.

We used specific pathogen-free rats (Simonsen Laboratories, Gilroy, CA) as a source for isolating alveolar epithelial cells. TI cells were isolated as described previously (13), using immunoselection with magnetic particles to deplete TII cells from the preparations. The TI cell preparations used for these experiments contained 80–91% TI cells and <1.5% TII cells, as analyzed by cytocentrifuged cell preparations stained with monoclonal antibodies specific for apical membranes of each cell type. The remaining cells consisted of alveolar macrophages and lymphocytes, as identified by their typical morphology after modified Papanicolaou staining (14). A total of seven different preparations of TI cells were used in these experiments.

TII cells were isolated by previously described methods (14); TI cells were removed by negative selection with magnetic beads (28). The TII cell preparation used for these experiments contained 84–92% TII cells and <0.1% TI cells. The remaining cells consisted of alveolar macrophages and lymphocytes, as identified by their typical morphology after modified Papanicolaou staining.

The elapsed time from surgical excision of the lungs to keeping cells at 4°C (i.e., including steps of elastase digestion, mincing lungs, and filtering cells) was ~30 min for TII cells and 45 min for TI cells. The total duration from surgical excision to RNA extraction was ~2 h for TII cells and ~3 h for TI cells. We did not perform viability studies of the cells used in these experiments, but, in many parallel experiments, viability assayed by vital dye exclusion was >95%.

TII cells were cultured in the presence of 10% fetal bovine serum [University of California San Francisco (UCSF) Cell Culture Facility] as previously described (14) on tissue culture plastic previously coated with bovine fibronectin (200 µg/ml) for 7 days. A total of eight separate TII cell isolations, four of which were for the cultured TII cells, were used in these experiments.

RNA Isolation and In Vitro Transcription Labeling

Total RNA purification and biotinylated cRNA synthesis. Total RNA was purified from each individual TI, TII, or cultured TII cell preparation using Qiagen’s RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA). We obtained an average of 1.4 µg RNA/106 TI cells and 2.1 µg RNA/106 TII cells. The quantity and integrity of isolated RNA were assessed by obtaining the ratio of absorbance values at 260 and 280 nm using a Beckman spectrophotometer and by visualization of intact 28S and 18S ribosomal RNA bands on denaturing formaldehyde agarose gels after electrophoresis. For cRNA synthesis, we followed the recommended protocol supplied by Affymetrix. We used 9 µg of total RNA for each reaction. Because some of the preparations of TI cells contained less than this amount, we pooled two samples of RNA from different cell isolations to create four independent samples of TI cell RNA. We had sufficient quantities of RNA from each of the TII cell preparations to process each sample separately. RNA was reverse transcribed to cDNA using the BRL Superscript Choice System (GIBCO/BRL Life Technologies, Gaithersburg, MD). In vitro transcription and biotinylation of newly synthesized cDNA were performed using an Enzo BioArray High Yield RNA Transcript Labeling Kit (Enzo, Santa Clara, CA); biotinylated cRNA was purified on Qiagen RNeasy columns, ethanol precipitated, and fragmented by heat treatment in Tris-acetate fragmentation buffer. Samples of cDNA, cRNA, and fragmented cRNA were run on a denaturing gel to obtain an estimation of DNA and RNA size distribution.

Samples were hybridized to RGU34 Rat GeneChips (Affymetrix, Santa Clara, CA) per the manufacturer’s instructions with the following modifications. Before hybridization against the RGU34 arrays, samples were incubated for 5 min at 99°C and then for 5 min at 45°C and centrifuged for 15 min at full speed in a standard Eppendorf microcentrifuge. Posthybridization processing was performed according to the manufacturer’s instructions. To assess the quality of the resultant expression data, a number of metrics were examined for each array. Specifically, the 3':5' ratios for the signal intensities were examined for both {beta}-actin and GAPDH for each array. In addition, data quality was assessed by review of the average background, noise (RawQ), and percent present calls for each array.

Statistical Analysis of Array Data

The raw-image data were analyzed using GeneChip Expression Analysis Software (Affymetrix) to produce perfect match and mismatch values, to which we applied the robust multiarray average (RMA) algorithm (1, 31) implemented in the RMAExpress software at http://stat-www.berkeley.edu/~bolstad/RMAExpress/RMAExpress.html. This results in a matrix of log-based 2 of gene expression measures, where columns correspond to different gene chips and rows correspond to the different genes. For a typical gene (probe set), we have four replicate expression measures for day 0 type I (TId0) cells (MTId0), four replicate expression measures for day 0 type II (TIId0) cells (MTIId0), and three replicate expression measures for day 7 type II (TIId7) cells (MTIId7) in log scale.

Identification of Differentially Expressed Genes

The plot (19) provides a graphic method to identify and visualize differentially expressed genes. For example, to compare samples TIId0 and TId0, we computed the average differences between the two cell types = TIId0TId0. Genes with corresponding extreme values represent potential marker genes for either TI or TII cells. The overall expression level for a particular gene is conveniently measured by the quantity , the average of log intensities across all the slides in the experiment. Similar calculations were made for TIId7 vs. TId0 and TIId0 vs. TIId7. The provides a ranking of genes corresponding to the strength of evidence of differential expression. The main drawback of ranking based on is that large values can be driven by outliers. Smyth (55) has shown that the moderated t-statistics are more reliable than simple fold change as a ranking statistic for identifying changes in gene expression. Therefore, for each individual gene (probe set) on the array, we computed the fold change, moderated t-statistics (55). We then generated a candidate list of differentially expressed genes with a 1% false discovery rate and greater than twofold change between groups. These procedures use functions in the limma library of the Bioconductor software package (30) and Smyth (55).

Sameness/Difference Comparisons Between TId0 and TIId7 Populations

Hierarchical clustering of samples using all genes demonstrated that replicates were clustered together and that the TIId7 pattern is closer to that of TId0 than that of TIId0. To examine how similar the expression profile of TId0 is to that of TIId7, we determined whether TId0 and TIId7 share similar TI cell marker genes compared with TIId0. To this end, we used a resampling method to compare the log-fold changes between TIId0 vs. TId0 and TIId0 vs. TIId7. For each gene, we randomly selected six pairs of (TIId0*, TId0*) samples and calculated the average resampled M values for TIId0/TId0 = ave(log2TId0*/TIId0*). Similarly, we calculated the average resampled M values for TIId0/TIId7 = ave(log2TIId7*/TIId0*). Figure 7B shows a scatter plot between TIId7/TIId0 against TId0/TIId0. We compared this scatter plot to two different resampled M values for TIId0/TId0 (TId0/TIId0 vs. TId0/TIId0) (Fig. 7A).



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Fig. 7. Comparison of log-fold differences between TId0 vs. TIId0 and TIId7 vs. TIId0. A: scatter plot of resampled M values for TIId0/TId0 vs. itself; r = 1.0. B: scatter plot of resampled M values for TIId7 vs. TIId0 and TId0 vs. TIId0; r = 0.47. Marker genes previously described for TI cells (red squares) include: phospholipase A2A group II, alpha crystallin B, receptor for advanced glycosylation end products, caveolin-1, aquaporin 5, and RTI40. Marker genes for TII cells (green squares) include: growth-related oncogene, surfactant proteins A (2 probe sets), B, and C. For details, see text.

 
Groups of Differentially Expressed Genes by Functions

To investigate further the difference between the TI and TII cell markers, we compared the functional composition of probe sets differentially expressed by TId0 and TIId0. We mapped each probe set to a predefined functional group (node in a tree structure) according to the Gene Ontology (GO) annotation database. A bar plot was constructed to compare the number of genes belonging to each functional group.

Real-Time PCR

Aliquots of total RNA used for microarray hybridization were reverse transcribed using RETROscript reagents (Ambion, Austin, TX). Quantitative real-time PCR (Q-PCR) amplification of cDNA was performed using an ABI PRISM 7700HT Sequence Detector System with a 384-well block (Applied Biosystems, Foster City, CA). Reaction mixtures consisted of 9 ng cDNA, TaqMan 2X Universal PCR Master Mix (Applied Biosystems), forward primer, reverse primer, and probe (Table 1) in a reaction volume of 10 µl. Using a two-step PCR program, we heated samples to 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Differences in the amount of cDNA amplified were corrected for by normalization to endogenous levels of ribosomal RNA (TaqMan Ribosomal RNA control reagents, Applied Biosystems). Standard curves for test genes and 18S ribosomal RNA were constructed on each plate from serial log dilutions of stock whole lung cDNA or plasmid containing the cDNA insert of interest; the relative quantification in triplicate for each experimental sample was obtained by the standard curve method. Control reactions were performed without reverse transcriptase and in the absence of target DNA.


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Table 1. Quantitative real-time PCR primers and probes

 
Verification of Differential Expression of Selected Gene Products by Immunohistochemistry

We used immunohistochemistry to confirm differential expression of proteins encoded by some of the differentially expressed genes. Tissue was fixed, frozen, sectioned, and processed for immunocytochemistry as previously described (15). Cryostat sections (2 µm) were incubated overnight with the following primary antibodies: rabbit polyclonal antibody against krox-20 (Covance, Berkeley, CA); mouse anti-human osteonectin [secreted protein acidic and rich in cysteine (SPARC); US Biological, Swampscott, MA]; goat polyclonal anti-agrin (Santa Cruz Biotechnology, Santa Cruz, CA); and rabbit anti-polymeric immunoglobulin receptor [pIgR; a kind gift from Dr. Keith Mostov, UCSF, of antibody produced by Dr. Jan Kraehenbuhl (40)]. Proteins were visualized by incubating sections with goat anti-rabbit or goat anti-mouse IgG conjugated to Alexa 594 or Alexa 488 at 1:3,000 (Molecular Probes, Eugene, OR). Fluorescence and phase contrast images were captured at 2,600 x 2,060 dpi with a Leica DC500 camera on a Leica Orthoplan microscope. We tested commercially available antibodies against the protein products of other selected differentially expressed genes [IGF-specific binding protein (IGFBP)-6, bone morphogenetic protein (BMP)-3, lysyl oxidase, and {beta}-defensin 2] but found these antibodies not to be useful in tissue due to problems of high background that could not be resolved using techniques of amplification and/or blocking.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of the Cell Populations

We used previously described methods to isolate TI and TII cells from rat lungs. Cell preparations were assessed by staining with monoclonal antibodies against apical plasma membrane proteins specific to each cell type. Representative cell preparations are shown in Fig. 1. There was <2% cross-contamination between the TI and TII cell preparations.



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Fig. 1. Dual-label immunocytochemistry of cytocentrifuged preparations of freshly isolated type I (TI) and type II (TII) cells. Cytocentrifuged preparations of TI and TII cells were stained for both RTI40 (marker of TI cells in normal lung, red) and RTII70 (marker of TII cells in normal lung, green); for details, please see text. A: preparation of TI cells (red) showing one TII cell (green) in the field. B: preparation of TII cells (green) showing a fragment of a TI cell (red) in the field. Arrows indicate the contaminating TI and TII cells. Bars = 50 µm.

 
Identification of Differentially Expressed Genes

Cross-chip data were normalized by quantile normalization (1), and data were analyzed by the RMA method (32). The data have been deposited in the GEO database (accession number GSE1567). We identified genes as differentially expressed if they had greater than twofold differential expression and met a false discovery rate of <0.01.

The results demonstrate that there are substantial differences in gene expression among all three groups, freshly isolated TI cells, freshly isolated TII cells, and cultured TII cells. There were 601 probe sets with greater than twofold different expression between TI and TII cells (Fig. 2, plot) and 206 with greater than fourfold different expression. There were approximately as many differences between freshly isolated TII and cultured TII cells (689 differentially expressed probe sets greater than twofold, 233 greater than fourfold, Fig. 3) as there were between freshly isolated TI and cultured TII cells (698 probe sets greater than twofold, 163 greater than fourfold; Fig. 4).



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Fig. 2. plot (scatter plot) of comparison of expression profiles of freshly isolated TI [day 0 type I (TId0)] and TII [day 0 type II (TIId0)] cells. Each point represents the log2 (expression in TId0/expression TIId0 cells) of ~8,800 genes (y-axis) plotted against the log2 (magnitude) (x-axis). The dotted lines indicate those genes differentially expressed by >2-fold with a false discovery rate (fdr) <0.01. Red points indicate genes differentially expressed in TId0; green points indicate genes differentially expressed in TIId0. Dotted lines indicate those genes differentially expressed by >2-fold.

 


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Fig. 3. plot (scatter plot) of comparison of expression profiles of freshly isolated TII (TIId0) and type II cells cultured for 7 days (TIId7) cells. Each point represents the log2 (expression in TIId7/expression TIId0 cells) of ~8,800 genes (y-axis) plotted against the log2 (magnitude) (x-axis). The dotted lines indicate those genes differentially expressed by >2-fold with an fdr <0.01. Green points indicate differential expression in TIId0; blue points indicate differential expression in TIId7.

 


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Fig. 4. plot (scatter plot) of comparison of expression profiles of freshly isolated TI (TId0) and TII cells cultured for 7 days TII (TIId7). Each point represents the log2 (expression in TId0/expression TIId7 cells) of ~8,800 genes (y-axis) plotted against the log2 (magnitude) (x-axis). The dotted lines indicate those genes differentially expressed by >2-fold with an fdr <0.01. Red points indicate genes differentially expressed in TId0; blue points indicate genes differentially expressed in TIId7.

 
How Good Was Replication?

To assess the reproducibility of replicates, we calculated the gene-wise SD within and between the three sample groups (TId0, TIId0, and TIId7). The median of the between-group SD was 0.17; the within-group SDs were 0.12, 0.08, and 0.09 for the three samples, respectively. These results indicate good reproducibility.

Differentially Expressed Genes

TId0 and TIId0. A plot of the TId0 and TIId0 comparison is shown in Fig. 2. Of the 601 differentially expressed probe sets (190 in TId0, 411 in TIId0), 473 had locus link identifications and 322 had unique locus links. Microarray expression profiling confirmed differential expression of several genes previously known to be differentially expressed in the two cell types, including glycoprotein 38 (RTI40, T1{alpha}) (18, 50, 59), aquaporin 5 (37), caveolin-1 (46), alpha crystallin B, phospholipase A2 group IIA (9), the receptor for advanced glycosylation end products (9, 22), ICAM-1 (8) (TI cells); surfactant proteins (SP)-A, -B, and -D, GRO, a CXC-chemokine (60), and alkaline phosphatase (20) (TII cells). The genes differentially expressed in TId0 cells encompass a wide range of different functions, including cytokines, growth factors, collagen and laminin subunits, tissue inhibitors of metalloproteinases (TIMPs), signaling molecules, and matrix- and growth factor-binding proteins. Their differential expression in TI cells suggests some intriguing possible functions for this cell type. Some genes, such as TIMPs and lysyl oxidase, which cross-link collagen fibrils to form insoluble collagen (34), suggest a role for TI cells in matrix deposition and maintenance. Expression of other genes such as fibulin suggest a role in vascular growth and differentiation, which seems reasonable considering the close proximity of TI cells to capillary endothelial cells and that these two cell types often share a common basement membrane (61). Genes involved with growth repression (disabled homolog 2, cyclin-dependent kinase inhibitor 2B) suggest specific molecular mechanisms by which TI cell proliferation may be regulated in normal lungs. In keeping with the theme of TI cell/bone/brain expression of RTI40, several genes, such as neuronatin, agrin, and IGFBP-6, also are expressed in bone or nervous tissues. Differentially expressed genes in TIId0 cells include various enzymes important in both fatty acid/phospholipid metabolism (e.g., fatty acid coenzyme A ligase, fatty acid synthetase, fatty acid binding protein 5) and heme metabolism (aminolevulinic acid synthetase, heme oxygenase-1). There was differential expression of phospholipase subgroups, transcription factors, and cytokines between the two cell types.

A list of the 52 genes (26 each in TId0 and TIId0) exhibiting highest fold differential expression between the two cell types is shown in Table 2; this may be useful in identifying additional markers of each cellular phenotype and in generating additional testable hypotheses about cellular functions.


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Table 2. Fifty-two genes expressing the highest-fold differences between the TId0 and TIId0 cell populations (26 in TId0, 26 in TIId0)

 
We used Q-PCR analysis to test independently whether genes identified by microarray analysis as differentially expressed between TId0 and TIId0 were also differentially expressed by Q-PCR. We selected six genes, based on those having potential biological functions of interest. The data, shown in Table 3, confirm differential expression. In each case, the fold change measured by Q-PCR was considerably larger than that measured by microarray analysis. These findings are consistent with those reported by others (63) and probably represent both differences between solid-state and solution hybridization and the use of the RMA algorithm, which provides greater specificity and sensitivity, but may blunt the magnitude of fold change (32).


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Table 3. Comparison of fold differences by array and Q-PCR in TId0 and TIId0 cells

 
TIId0 and TIId7. There were 689 differentially expressed probe sets (Fig. 3) between the TIId0 and TIId7 cell populations (332 in TIId0, 357 in TIId7), of which 574 had locus link references and 388 unique locus link references. TII cells cultured for several days on tissue culture plastic or fibronectin lose characteristics of the TII cell phenotype and acquire some characteristics of the TI cell phenotype (2, 3, 7, 10, 17, 18, 44). There were some similarities between the expression patterns of TIId0 vs. TIId7 and TIId0 vs. TId0. For example, there were large differences in expression of previously known TII cell markers, such as SP-A, -B, -C, and -D, alkaline phosphatase, and GRO. Other proteins shared this pattern, including {beta}-defensin 2 and the pIgR. Cultured TII cells expressed some of the genes found to be differentially expressed by TId0 cells in the TId0 vs. TIId0 comparison, such as RTI40 (gp38), osteonectin (SPARC), lysyl oxidase, alpha crystallin B, and frizzled-2.

TId0 and TIId7. Somewhat surprisingly, there were similar numbers of differentially expressed genes between the TId0 and TIId7 (Fig. 4) populations as there were between the TId0 vs. TIId0 and TIId0 vs. TIId7 populations. Of the 698 differentially expressed genes between TId0 and TIId7 (195 in TId0, 503 in TIId7), 530 had locus link annotations, 383 of which were unique. Some known markers of TI cells were expressed more highly in the TId0 cells than the cultured TII cells. These included caveolin-1, phospholipase A2 group IIA, osteonectin, and aquaporin 5, all of which were expressed at >10-fold higher levels in the TId0 cell population than in the TIId7 population; in contrast, tissue inhibitory factor for metalloproteinase 3, previously shown to be expressed in TId0 cells (9), was expressed approximately fourfold higher in the TIId7 cell population, as were lysyl oxidase and frizzled-2. Q-PCR analysis of TI marker genes verified differences in the level of expression of some of these marker genes between the freshly isolated TI and cultured TII cells (Table 4).


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Table 4. Q-PCR analysis of type I cell marker genes in the TId0 and TIId7 cell populations

 
There were several differences in histocompatibilty antigens, transcription factors, and in growth factor binding proteins in the TId0 cells, but many of the differentially expressed genes were of unknown function. The TIId7 cells contained differentially expressed genes of a wide variety of functions, including cytokines, transcription factors, and enzymes important in various cellular metabolic functions.

Blunting of Fold Change Expression for Some Marker Genes

Because there was very little (<2%) cross-contamination of cell types by immunocytochemical analysis, we were initially surprised that analysis of the Affymetrix gene chip arrays did not detect differential expression of SP-C and showed only five- to eightfold changes in SP-A and -B (eightfold) expression between the TId0 and TIId0 populations. Analysis of the same RNA samples by Q-PCR showed larger fold differences in expression of these genes between these populations. For example, the ratios for TIId0/TId0 by Q-PCR were: SP-A, 22; SP-B, 41; SP-C, 22. One possible reason for this discrepancy is that the probe sets on the chip may not be uniquely specific for the gene of interest. By basic local alignment search tool (BLAST) analysis of all 16 of the 25-mer probes for SP-C, only four of these probes are specific for SP-C. Among the remaining 12 probes, there are homologies with various other genes, including topoisomerase IIA, T cell receptor loci, regulators of G proteins, and X-chromosome genes. The degree of homology of the probe set oligomers with genes other than SP-C varied from 23/23 to 17/17. There are two probe sets on the U34 chip representing SP-A; one of these (M33201 [GenBank] ) gave lower (fivefold) fold differences than the other (X13176 [GenBank] , eightfold). Other possible reasons for the observed differences in fold change between Q-PCR and array data are covered in the DISCUSSION.

Verification of Differential Expression of Selected Gene Products by Immunohistochemistry

We performed immunohistochemistry on lung tissue to confirm differential expression of proteins encoded by some of the differentially expressed genes. Differential expression in TII cells was confirmed for krox-20 and the pIgR; there was differential expression of osteonectin (SPARC) and agrin in TI cells (Fig. 5). We tested several commercially available antibodies against the protein products of other differentially expressed genes (IGFBP-6, lysyl oxidase, BMP-3, and {beta}-defensin 2) but found these antibodies not to be useful in tissue due to problems of high background that could not be resolved using techniques of amplification and/or different blocking methods.



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Fig. 5. Immunohistochemistry of 4 different gene products whose mRNAs were differentially expressed in TI or TII cells by microarray analysis. Cryostat sections (2-µm-thick) of rat lung were processed for immunohistochemistry as described in the text; paired phase contrast and immunofluorescence views are shown. Agrin (A) and osteonectin (B) staining (red) in a pattern compatible with localization in TI cells; type II cells (arrows) are negative. Polymeric immunoglobulin receptor (pIgR, C) and krox-20 (D) staining (green) in TII cells (arrows) are recognized by their lucent lamellar bodies. Krox-20 appears localized to the apical plasma membranes of TII cells; pIgR appears to be localized intracellularly.

 
Functions of Differentially Expressed Genes: the GO Database Analysis

We examined functional groups by the GO Consortium Database, in which the functions of various genes are categorized into groups. The results are shown in Fig. 6. The data should be viewed with the perspective that there were fewer differentially expressed genes in TI cells than in TII cells and many of the differentially expressed genes in TI cells do not have annotated functions. There were fewer identifiable genes in TI cells with functions in the "regulation of cell differentiation" group, and, despite the lower number of annotated genes in TI cells, the number of genes with antioxidant activity was higher in TI cells. Both TI and TII cells contained genes with other cell functions, such as transporter activity, immunoregulation, and signal transduction.



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Fig. 6. Bar graph showing functional categories of genes differentially expressed between TId0 and TIId0. We mapped each probe set to a predefined functional group (node in a tree structure) according to the Gene Ontology annotation database. A bar plot was constructed to compare the number of genes belonging to each functional group. Results should be interpreted with the information that the TId0 population contained fewer differentially expressed genes; of these, a lower proportion was annotated than in the TIId0 population. For details, see text.

 
Comparisons of Similarities and Differences Between TId0 and TIId7

An unanswered question is how close the cultured TII cell model system is to native TI cells. We compared the molecular profiles of the TId0 and TIId7 cell populations by several different methods.

First, the number of differentially expressed genes between the TId0 and TIId7 populations was similar to the number of differentially expressed genes between TId0 and TIId0 and similar to the number between TIId0 and TIId7, from which one may infer that there are substantial differences in gene expression between the TId0 and TIId7 populations.

Second, we used a resampling method to compare plots of log2 (TId0/TIId0) vs. log2 (TId0/TIId0) and log2 (TIId7/TIId0) vs. log2 (TId0/TIId0) (Fig. 7). The plot of log2 (TId0/TIId0) vs. log2 (TId0/TIId0) (Fig. 7A) is a straight line, with the individual gene intensities (the "gene cloud") centered on the line and an r = 1.0. Marker genes for the TII cell phenotype are indicated by green points, marker genes for TI cells by red points. The marker genes align on the line with a slope = 1. If the gene expression pattern were the same in TIId7 and TId0, then a plot of log2 (TIId7/TIId0) vs. log2 (TId0/TIId0) should yield similar results. In contrast, the plot of log2 (TIId7/TIId0) vs. log2 (TId0/TIId0) shows an eccentric gene cloud and an r = 0.47, indicating significant deviation from unity. Marker genes for TI and TII cells deviate significantly from unity, demonstrating differences in marker gene expression between the TId0 and TIId7 populations.

A third measure of the interrelationships between the three cell populations can be tested by hierarchical clustering, shown in a cluster dendrogram (Fig. 8). The TId0 and TIId7 populations are somewhat more closely related than either of these is to the TIId0, but the differences between the TIId0 and TIId7 are almost as great as those between both of these and TIId0.



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Fig. 8. Hierarchical clustering of samples. Hierarchical clustering of samples using all genes demonstrated that replicates were clustered together, that the TIId7 pattern is closer to that of TId0 than that of TIId0, but that the TId0 is almost as different from TIId7 as TIId0 is.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
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We have used microarray analysis to describe gene expression profiling of freshly isolated rat TI and TII cells and cultured TII cells. The goals of this study were several: 1) to establish the molecular phenotypic fingerprints of TI and TII cells; 2) to gain insight into possible functional differences between the two cell types through inferred functions of differentially expressed genes; 3) to identify genes that might indicate potential functions of TI cells, since so little is known about the functions of this cell type; and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. Because the purity of the cell preparations is critical to the interpretation of results, we used only cell preparations that had <2% cross-contamination by immunocytochemical analysis with cell-specific markers. We chose the Affymetrix Rat Gene chip system because the Affymetrix system is well characterized and because we were using rat TI and TII cells.

The data were analyzed by the RMA algorithm, which, compared with alternative methods such as MAS 5.0 and dChip, provides higher specificity and sensitivity when using fold change analysis to detect differential expression. In particular, the precision improves dramatically for probe sets with lower gene expression levels, although the fold change calculation may be somewhat blunted by this method of analysis. We used a combination of a twofold expression difference and a false discovery rate of <0.01 to determine differentially expressed genes.

The number of differentially expressed genes was relatively the same in each comparison: TId0 vs. TIId0, TIId0 vs. TIId7, and TId0 vs. TIId7. Most previously known marker genes for TI and TII cells were differentially expressed by array analysis, although the magnitude of the differences varied among probe sets and fold differences for some markers (ex. SP-A, SP-B) appeared to be blunted. Surprisingly, SP-C was not differentially expressed between the TId0 and TIId0 populations in array analysis, although SP-C was differentially expressed by Q-PCR using RNA from the same samples used for the arrays. One possible reason for these results is that the probe set for SP-C on this chip is not unique for SP-C. BLAST analysis of the 16 25-mer probes revealed significant homologies with other genes. Although the extent to which these probes cross-hybridize with genes other than SP-C is difficult to predict, this factor may be a contributing factor to our results. Even without invoking the lack of specificity of the SP-C probe, one finds precedent in the literature for marked discrepancies between expression ratios measured by Q-PCR and array analysis. In a systematic evaluation of array analysis and Q-PCR data, Yuen et al. (63) demonstrated that both oligonucleotide arrays and cDNA arrays showed "a marked tendency to underestimate the fold-change ratios of the...mRNAs." For some genes, these effects were extreme, with fold changes of 200- to 400-fold by Q-PCR and essentially no change seen on arrays. The reason for these discrepancies remains unclear, but Yuen et al. speculated that the differences may be related to nonspecific hybridization or to probe saturation effects. Because SP-A, SP-B, and SP-C are expressed in high abundance and because the signal appeared close to saturation in all samples, it seems likely that probe saturation may have occurred.

We used isolated cells for gene expression profiling, as have many other investigators. The expression of certain genes may be affected by techniques of cell isolation. However, there are technical limitations to performing gene expression profiling in alveolar epithelial cells in situ. TI and TII cells can be clearly identified at the light microscopic level only by antibody staining with specific cell markers, which requires preparation of tissue in aqueous media. Many authors have reported major problems with RNA degradation when laser-capture microdissection (LCM) is performed with tissue that has been exposed for even brief periods of time (8–10 min) to aqueous media. For example, in a series of experiments using immuno-LCM, Kohda et al. (38) reported a loss of 99% of mRNA for {beta}-actin following immunostaining; degradation probably occurred secondarily to endogenous RNases. Ideally, LCM should capture the thin cytoplasmic extensions of TI cells (50–100 nm in thickness), but this would be extremely difficult to accomplish accurately without contaminating the sample with portions of interstitial cells. Therefore, the current technology for immuno-LCM may have limited utility for studies of gene profiling of alveolar epithelium. For these reasons, we used isolated cells for gene expression profiling. During the cell isolation procedure, cells are kept at 37°C or room temperature for the period of enzymatic digestion, mincing of lungs, and filtering (30–45 min) but are otherwise kept at 4°C. Although the expression levels of certain genes may be affected by cell isolation, most of the previously identified marker genes for TI and TII cells identified by immunohistochemistry were recognized to be differentially expressed in these experiments (see RESULTS).

We also verified differential expression of four genes by immunohistochemical analysis in normal lung tissue. Agrin and osteonectin were expressed in TI cells, krox-20 and the pIgR in TII cells. Both agrin and osteonectin are multifunctional proteins that have matrix-related functions. Agrin, a heparan sulfate proteoglycan that is believed to play different important roles in neuronal function, is critical to the formation and maintenance of the neuromuscular junction. Agrin null mutant mice die either close to term or shortly after birth, presumably from lack of respiratory muscle function (23, 24), although the pulmonary parenchyma has not been carefully examined in this null mutant. It has been proposed that agrin, also a component of the basal lamina of the cerebral microvasculature, plays a role in the function of the blood-brain barrier (54). The presence of agrin in TI cells, which are in close proximity to the capillary endothelium, raises analogous questions about whether agrin may play a role in the air-blood barrier. Agrin is also concentrated in interneuronal synapses (39) and is believed to play an important role in cerebral development. Osteonectin (SPARC) is a matricellular protein expressed by a wide variety of cell types, although its name is derived from its very high expression level in bone. It has a modular structure with domains that bind to cell surfaces, matrix components, or growth factors (reviewed in Ref. 6). SPARC null mutant mice develop early cataracts (25) and are reported to have abnormal bone development and abnormal osteoblasts (11, 12). Mesenchymal cells from SPARC null mutant mice proliferate more rapidly than cells from normal mice (6). Together with the fact that TI cells differentially express other matrix proteins or inhibitors of matrix degradation, these observations suggest that TI cells may play important roles in matrix formation and maintenance.

The two genes differentially expressed in TII cells whose expression was confirmed by immunohistochemistry were krox-20 and the pIgR. Krox-20, originally identified as a serum response immediate-early gene, is a zinc-finger transcription factor essential for myelination. Krox-20 null mutant mice are deficient in rhombomeres 3 and 5 (53) and Schwann cells (58). Krox-20 inhibits Schwann cell proliferation and death (48). In addition to its effects in the nervous system, krox-20 is also essential for normal bone formation (41, 42). Although krox-20 is upregulated after wounding, its presence is not essential for wound healing (27). Its function in TII cells is a matter of speculation. The pIgR is a well-described integral membrane glycoprotein that binds IgA at the basolateral epithelial cell surface, stimulating tyrosine kinase-mediated signal transduction (43). The IgA-pIgR complexes undergo transcytosis, whereby secretory IgA is released into the mucosal lumen. Although some older studies did not find secretory IgA to be present in alveolar epithelium (52), others reported that "secretory component" was present in endoplasmic reticulum of TII cells (29). Our results (Fig. 5C) show that pIgR is detectable in TII cells, but not TI cells. Our findings and those of Haimoto et al. (29) are compatible with the concept that TII cells may play a role in transcytosis. pIgR localization in type II cells would explain observations that cultured TII cells transport secretory component (reviewed in Ref. 36).

The genes differentially expressed in the TId0 population are of particular interest, because potential functions of this cell type may be inferred from the functions of some known genes. Unfortunately, there are few analytical tools to categorize the various functions of large numbers of genes. We mapped each probe set to a predefined functional group (node in a tree structure) according to the GO annotation database. A bar plot was constructed to compare the number of genes belonging to each functional group. The results are shown in Fig. 6. Some groups of genes may be of particular interest, such as those genes involved in transport, regulation of cell growth, regulation of apoptosis, or antioxidant activity.

Although TII cells cultured on plastic or fibronectin lose various morphological and biochemical characteristics associated with the TII cell phenotype and acquire some characteristics of the TI cell phenotype, it has been uncertain how similar the cultured TII cell model system is to TI cells. By several different criteria, it appears that there are large differences between the molecular phenotypes of these two cell populations. First, there are similar numbers of differentially expressed genes between each comparison: TId0 and TIId7, TId0 and TIId0, TIId0 and TIId7. Second, the plot of log2 (TIId7/TIId0) vs. log2 (TId0/TIId0) (Fig. 7B) reveals a poor correlation (r = 0.47) between the expression patterns of the two populations. Third, hierarchical clustering analysis (Fig. 8) suggests that, although the TId0 and TIId7 cells are somewhat more closely related to each other than either is to TIId0 cells, the differences between the two cell populations is nevertheless substantial.

The reasons for these differences may be multiple, including the observations that TII cells in culture dedifferentiate rather than transdifferentiate, that the culture conditions may be insufficient to promote full transdifferentiation, and that the process of isolating cells may induce changes in gene expression. However, together, these results suggest that interpretations of experiments with the cultured TII cell model should be interpreted with caution with respect to relevance to either TI or TII cells.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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This work was supported in part by NHLBI Grant R01 HL-57426, the NHLBI Shared Microarray Facility Grant R01 HL-72301, the General Clinical Research Center M01RR00083-41, and Grant HL-24075.


    ACKNOWLEDGMENTS
 
The authors deeply appreciate the discussions with and suggestions of Drs. David Erle and Chris Barker, the facilities of the General Clinical Research Center, the National Heart, Lung, and Blood Institute (NHLBI) Shared Microarray Facility, and Michael Salazar’s help in preparation of data for submission.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. G. Dobbs, Suite 150, UCSF-LH Campus, 3333 California St., San Francisco, CA 94118 (E-mail: dobbs{at}itsa.ucsf.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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