Gene expression in human accessory lacrimal glands of Wolfring

Abstract

Purpose: The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands.

Methods: Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry.

Results: Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring.

Conclusions: The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs.

Figure 1.

Frozen sections of human eyelid (left) and human main lacrimal gland (right) stained with toluidine blue O. Note the similarity of the structure of the gland of Wolfring to the acini of the main lacrimal gland (original magnification ×100).

Figure 2.

Integrity of RNA in eyelid samples. (A) GAPDH RT-PCR of RNA extracted from frozen sections of four eyelids (in triplicate). (B) 1% agarose gel of samples from (A), showing the GAPDH band (+ is the positive control). BP, 100 base pair ladder.

Figure 3.

Laser microdissection of a gland of Wolfring from a frozen eyelid section, showing the gland in situ (left), the eyelid section after removal of the gland (center) (original magnification ×100), and the gland of Wolfring tissue isolated from the remainder of the eyelid (right) (original magnification ×50). Tissue was stained with hematoxylin and eosin under RNAase-free conditions.

Figure 4.

Representative bioanalyzer data demonstrating good-quality RNA extracted from two glands of Wolfring isolated by laser microdissection, as indicted by strong 18S and 28S rRNA peaks. The baseline noise indicates some degradation, as expected in human surgical tissue. Samples with RNA integrity values (RIN) of 4 or greater were submitted for microarray analysis.

Figure 5.

Immunohistochemistry on frozen sections of gland of Wolfring using antibodies to several proteins known to be present in tear fluid. Nuclei were stained with DAPI. The control section was exposed to rhodamine-conjugated secondary antibody alone.

Figure 6.

Immunohistochemistry on frozen sections of gland of Wolfring for several proteins known to be involved in electrolyte and fluid secretion. Nuclei were stained with DAPI.