Differential gene expression in human conducting airway surface epithelia and submucosal glands

Abstract

Human conducting airways contain two anatomically distinct epithelial cell compartments: surface epithelium and submucosal glands (SMG). Surface epithelial cells interface directly with the environment and function in pathogen detection, fluid and electrolyte transport, and mucus elevation. SMG secrete antimicrobial molecules and most of the airway surface fluid. Despite the unique functional roles of surface epithelia and SMG, little is known about the differences in gene expression and cellular metabolism that orchestrate the specialized functions of these epithelial compartments. To approach this problem, we performed large-scale transcript profiling using epithelial cell samples obtained by laser capture microdissection (LCM) of human bronchus specimens. We found that SMG expressed high levels of many transcripts encoding known or putative innate immune factors, including lactoferrin, zinc alpha-2 glycoprotein, and proline-rich protein 4. By contrast, surface epithelial cells expressed high levels of genes involved in basic nutrient catabolism, xenobiotic clearance, and ciliated structure assembly. Selected confirmation of differentially expressed genes in surface and SMG epithelia demonstrated the predictive power of this approach in identifying genes with localized tissue expression. To characterize metabolic differences between surface epithelial cells and SMG, immunostaining for a mitochondrial marker (isocitrate dehydrogenase) was performed. Because greater staining was observed in the surface compartment, we predict that these cells use significantly more energy than SMG cells. This study illustrates the power of LCM in defining the roles of specific anatomic features in airway biology and may be useful in examining how disease states alter transcriptional programs in the conducting airways.

Figure 1.

Laser capture microdissection (LCM) of surface epithelia and submucosal glands (SMG). Representative images from the tissue isolation process are depicted for surface epithelia (left, A–D) and submucosal glands (right, E–H). A and E represent respective tissues before dissection. Captured cells attached to the LCM cap film are demonstrated for surface epithelia (B) and submucosal glands (F). After removal of the LCM cap, unselected tissue remains behind (C and G). The LCM process yields populations of surface epithelial cells (D) and submucosal gland cells (H) on the LCM cap film.

Figure 2.

Bioinformatic analysis of gene expression. (A) Correlation of gene expression levels in surface epithelia (x axis) with submucosal glands (y axis). Expression signals were log2 transformed and normalized by the RMA procedure. Some genes selected for further analysis are displayed in red. (B) Gene set enrichment analysis (GSEA) heatmap output for the 100 most differentially expressed transcripts in human bronchus surface epithelium and submucosal glands. The columns represent unique array hybridizations using either surface epithelial RNA (SURFACE) or submucosal gland RNA (SM GLAND). The donor number is also indicated after the tissue type. Red indicates high expression and blue low expression. Probe IDs are listed on the right of the figure. (C) Metabolic pathway upregulation (mitochondrial genes) in surface epithelium assessed by gene set enrichment analysis. A total of 14,989 genes were rank ordered by relative expression in surface epithelium, and an enrichment score (ES) was calculated using these rank order data weighted by a correlation coefficient using GSEA software (7). The upward deviation of the ES curve demonstrates the positive enrichment of the gene set in surface epithelial cells. (D) The dot plot represents the ranks of individual genes belonging to the mitochondrial gene set (n = 319) within the cumulative list of genes. The distribution is skewed to the left, indicating enrichment of mitochondrial transcripts in surface epithelial cells. The solid bar represents the mean rank of this gene set.

Figure 3.

Mitochondrial staining is greater in conducting airway surface epithelial cells compared with submucosal gland cells. Immunohistochemical staining of bronchus tissue is presented for isocitrate dehydrogenase (IDH3G), a ubiquitous Krebs cycle enzyme with known localization to the mitochondrial matrix. (A) IDH3G immunostaining is highly enriched in surface epithelial cells (arrowheads), with less intense staining observed in SMG epithelial cells (arrows). (B) IDH3G staining was most intense immediately below the apical surface of ciliated epithelial cells. Representative of n = 3 human donor bronchi examined.

Figure 4.

Immunohistochemical localization of selected gene products confirms the microarray results. (A) GST-α is widely expressed in the surface epithelium. (B) Staining was observed very rarely in serous and mucus submucosal glands. (C) The most intense staining was observed at the apical surface of ciliated epithelial cells. (D) CYP4B1 was specifically observed on a subset of surface epithelial cells, with additional staining in cells lining the gland duct (arrowhead). (E) Under higher power, CYP4B1 was absent in the submucosal glands. (F) Surface CYP4B1 immunostaining was most intense near cilia, which could be consistent with the prediction that CYP4B1 is secreted. Minimal immunostaining was observed in the basal layer of surface epithelial cells. (G) Expression of CYP4B1 declined in the more distal portions of submucosal gland ducts (data not shown). NQO1 staining was widely expressed in the surface epithelium, consistent with previous studies (25). (H) NQO1 was not detectable in submucosal gland epithelia, but light staining was observed in interstitial cells. (I) The basal layer of surface airway epithelial cells was deficient in NQO1 expression. Surface epithelial staining within columnar cells appeared cytoplasmic, with a bias toward apical expression. NQO1 immunostaining was also present in vascular endothelial cells and in chondrocytes of bronchial cartilage (data not shown). (J and K) LTF is expressed highly in the serous demilunes, consistent with earlier observations (26). (L and M) ZAG expression was intense in the vast majority of acinar gland cells, and appeared to be present in both serous and mucus cell types. A representative tissue section stained with isotype control antibody is presented in panel N.