Entrapment of conjunctival goblet cells by desiccation-induced cornification

Abstract

Purpose: To evaluate the effects of desiccating stress on conjunctival goblet cell density and morphology and the expression of cornified envelope precursors by the ocular surface epithelia.

Methods: Experimental dry eye (EDE) was created in C57BL/6 mice. Real-time PCR evaluated the expression of cornified envelope (CE) precursor proteins (involucrin and small proline-rich [Sprr] -1a, -1b, -2a, -2b, -2f, and -2g proteins), the cross-linking transglutaminase 1 enzyme (Tg-1) and Muc5AC mRNA transcripts by the ocular surface epithelia. Laser scanning confocal microscopy evaluated the expression of the CE precursor proteins Tg-1 and Muc5AC in cryosections. Tg-1 activity was measured by a fluorescein cadaverine assay. Muc5AC concentration was measured by ELISA.

Results: Levels of involucrin; Sprr-1a, -1b, -2a, -2b, -2f, and -2g; and Tg1-1 mRNA transcripts in ocular surface tissues increased in response to desiccating stress. Expression and activity of Tg in the conjunctiva markedly increased after EDE. Desiccating stress caused progressive loss of mucin-filled goblet cells. The apical portion of the remaining conjunctival goblet cells became entrapped by adjacent stratified apical epithelia expressing increased levels of cornified envelope precursors.

Conclusions: Exposure to desiccating stress stimulates ocular surface epithelia to produce cornified envelope precursors and the tissue transglutaminase enzyme that cross-links them. This effect is accompanied by loss of mucin-filled goblet cells and entrapment of mucin contents in the remaining ones by cornifying cells that block the egress of mucin contents to the ocular surface. This mechanism may contribute to the conjunctival mucin deficiency that develops in dry eye.

Figure 1.

Mean ± SD of goblet cell density in UT mice and after 5 and 10 days (D) of EDE.

Figure 2.

Laser scanning confocal immunofluorescence microscopy of cornea cryosections stained for involucrin (A), Sprr-1a (B) and Sprr-2 (C) (all markers in green) with propidium iodide (red) nuclear counterstaining in UT control mice and after 5 and 10 days (D) of EDE. Scale bar, 50 μm.

Figure 3.

Laser scanning confocal immunofluorescence microscopy of conjunctiva cryosections stained for involucrin (A), small proline-rich protein [Sprr]-1a (B) and Sprr-2 (C) (all markers in green) with propidium iodide (red) nuclear counterstaining in UT control mice and after 5 and 10 days (D) of EDE. Insets: higher magnification of areas boxed in blue. Scale bar, 50 μm.

Figure 4.

Laser scanning confocal immunofluorescence microscopy of conjunctiva cryosections dual-stained for Muc5AC (blue) and Sprr-1a (green) with propidium iodide (red) nuclear counterstaining in UT control and after 5 and 10 days (D) of EDE. Solid arrows: SPRR-1a staining around the goblet cells. Scale bar, 50 μm.

Figure 5.

Laser scanning confocal immunofluorescence microscopy of tissue sections stained for Tg-1 protein in cornea (A) and conjunctiva (B) and with a Tg activity assay in cornea (C) and conjunctival tissue (D) with propidium iodide (red) nuclear counterstaining in UT control mice and after 5 and 10 days (D) of EDE. Scale bar, 50 μm.

Figure 6.

Transmission electron microscopy of conjunctiva. The unstressed control (UT) conjunctiva consistently showed areas with contiguous goblet cells that opened to the conjunctival surface (arrows). The goblet cells were packed with secretory vesicles of uniform electron density. The epithelium is two to three cells deep in this view. After 5 and 10 days (D) of EDE fewer goblet cells reached the conjunctival surface. Frequently, mature and maturing goblet cells were covered by one or two layers of thin epithelial cells (). Secretory granules in these goblet cells were reduced and varied greatly in electron density compared with those in UT control eyes.

Figure 7.

Mean ± SD of MUC5AC concentration measured by ELISA in UT mice and after 5 and 10 days (D) of EDE in conjunctiva (A) and corneal epithelial (B) samples. Concentrations are reported per nanogram of total protein.