Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays

Abstract

Purpose: To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression.

Methods: cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest.

Results: The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb.

Conclusions: Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that are potentially active in the function of corneal and conjunctival epithelia.

Figure 1

Biochip profiles (Agilent, Palo Alto, CA) of total RNA isolated from Dispase-released corneal and conjunctival epithelia.

Figure 2

Heat map for unsupervised hierarchical sample clustering of corneal and conjunctival transcripts with significant expression. Whole transcript sets for three conjunctival (Cnj1-3) and three corneal (Co1-3) samples were clustered by the unsupervised hierarchical sample method (Pearson correlation similarity measurement) to generate a sample tree (vertical lines). Note that the process has correctly clustered the two cell types. The 1228 statistically different transcripts (803 for the Cnj and 425 for the Co samples) are shown as individual horizontal color-coded lines within this sample tree, where color represents relative expression level in a base 10 logarithmic scale. The color code is shown in the right side bar.

Figure 3

Hierarchical tree of GO ontology processes significantly overrepresented in the Cnj epithelium.

Figure 4

Immunolocalization of sPLA₂-IIA and HLA-II antigen presenting cells in freshly isolated human conjunctival epithelium. Cryosections (5–8 mm thick) fixed in ice-cold methanol were probed with anti-human sPLA2-IIA and HLA-II antibodies. (A) Low magnification demonstrates that the sPLA2-IIA protein predominantly labeled to immature and mature Goblet cells. (B) Higher magnification of the asterisk-marked area reveals that the sPLA2-IIA protein was distributed in both Goblet and non-Goblet cells in vesicular granules structures within or in proximity to the plasma membrane. In surface goblet cells undergoing degranulation, numerous vesicle profiles were observed migrating toward the cell secretory pit, whereas vesicular density at the membrane wall appeared markedly reduced. Cells containing very high levels of HLA-II antigen distributed between the Cnj epithelial stratum (C). (A, C, insets) Results for the cornea.

Figure 5

Fontana-Mason stain of the mucocutaneous zone of human conjunctiva and immunostain for TYRP1 in the palpebral conjunctiva (inset). Note the high frequency of stained cells in the basal layer in both cases.

Figure 6

Distribution of NADPH reductase activity in the conjunctival and corneal epithelium. Activity was found in the basal stratum of the corneal epithelium (A) but not in the conjunctival epithelium (B).