Air exposure induced squamous metaplasia of human limbal epithelium

Abstract

Purpose: Squamous metaplasia is a pathologic process that frequently occurs in nonkeratinized stratified ocular surface epithelia. The mechanism for this occurrence is largely unknown except for vitamin A deficiency.

Methods: Human limbal explants were cultured under airlift with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or in a submerged manner for different durations up to 2 weeks. Epithelial cell proliferation, differentiation, limbal stem cell maintenance, and expansion were studied using certain markers such as Ki67, p63, K10 and K12 keratins, filaggrin, Pax6, ABCG-2, and Musashi-1. Expression of phospho-p38 MAPK and its downstream transcription factors, C/EBPalpha and C/EBPbeta, were studied by immunohistochemistry. Epithelial cells harvested from explants after 2 weeks of culturing under different conditions were seeded onto 3T3 feeder layers and cultured for 12 days. The differentiation of clonal epithelial cells was investigated by double staining to K12 and K10 keratins.

Results: The squamous metaplasia model was successfully created by culturing human limbal explants at an air-liquid interface (airlift) for 2 weeks. Increased stratification and hyperproliferation only happened in the limbal, but not the corneal, epithelium in airlift, but not submerged, cultures. Epithelial proliferation was associated with a transient increase of limbal epithelial stem cells. Abnormal epidermal differentiation-evidenced by positive expression of K10 keratin in suprabasal cells and filaggrin in superficial cells-ensued. Clones generated from epithelial cells harvested from airlift culture only expressed K12 keratin without K10. As early as 2 days in airlift cultures, p38 expression emerged in limbal basal epithelial cells and gradually extended to the cytoplasm and nuclei. Furthermore, addition of the p38 inhibitor SB203580 abolished abnormal epidermal differentiation without affecting limbal epithelial proliferation. Expression of C/EBPalpha and C/EBPbeta, downstream of the p38 MAPK signaling pathway, was strongly induced by airlift culture and partially was inhibited by SB203580.

Conclusions: Dryness resulting from exposure activates p38 MAPK signaling coupled with abnormal epidermal differentiation without intrinsic alteration of stem cells in the limbus. On the ocular surface, p38 inhibitors may have the potential to revert the pathologic process of squamous metaplasia induced by dryness.

Figure 1

Schematic drawing of human limbal explant cultures. From a human cadaveric eye, the corneoscleral tissue was retrieved and preserved in the storage medium. After the central cornea was removed for transplantation, the remaining corneoscleral rim was subdivided into six segments, of which each was cultured under airlift or submerged mode. While in submerged mode, the culture medium was added to cover the entire limbal explant. While in airlift mode, the culture medium was added to the level at which the epithelium was at the air-medium interface.

Figure 2

Limbal epithelial changes in airlift cultures. Hematoxylin staining showed stratified limbal (d0) and peripheral corneal (d0C) epithelium before culturing. Superficial limbal epithelial cells desquamated on day 2 (d2). Limbal epithelial cell layers increased dramatically from day 4 to day 14, resulting in an undulated basal epithelial plane and digital invasion. In contrast, the cell layers of the peripheral corneal epithelium decreased to one to two layers after 14 days of airlift culturing (d14C). Scale bar, 200 μm.

Figure 3

Limbal epithelial proliferation in airlift cultures. Ki67 staining (A) showed that positive nuclei were very sparse in the limbal epithelium before culturing (d0), became absent on day 2 (d2), and dramatically increased on day 4 (d4) and day 6 (d6) in mostly basal and some suprabasal epithelial cells before a gradual decline on day 10 (d10) and day 14 (d14). p63 staining (B) showed that positive nuclei were found in all basal and some suprabasal cell layers on day 0 and day 2 in airlift cultures but increased to the full-thickness cell layers on day 4 before the superficial cell layers reverted to negative from day 6 to day 14 (B). ABCG-2 staining (C, green) showed that positive staining was limited to clusters in limbal basal cells on day 0 but dramatically increased in basal and suprabasal layers on day 4 before a gradual decrease from day 6 to day 14. Musashi-1 staining (D, red) showed that positive expression was sporadic in limbal basal and very few suprabasal cells on day 0, became negligible on day 2, and dramatically increased in basal and suprabasal layers on day 4 before gradual decrease from day 6 to day 14. Scale bars, 100 μm.

Figure 4

Abnormal epidermal differentiation of limbal epithelial cells in airlift cultures. Double immunostaining to K12 keratin (A) and K10 keratin (B) and merged images (C) show that the expression of K12 keratin was limited to suprabasal cell layers of the limbal epithelium before culturing (d0). K12-negative basal cells expanded to suprabasal cell layers from day 2 to day 6 and then reverted to the basal cell layer again on day 10 and day 14. Expression of K10 keratin was negative before culturing and on day 2 in airlift cultures but became positive mostly in superficial cell layers on day 4 and gradually increased from day 6 to day 14. Merged images showed nearly all K10-positive cells maintained K12 expression. Filaggrin-positive cells were first found in superficial cells on day 6 and gradually increased in superficial cells until day 14 (D). Pax6 (E, F) was expressed by almost all epithelial cells throughout the culture duration, except some desquamated cells on the superficial limbus on day 2, day 10, and day 14 and some cells in the basal epithelium on day 0 and day 14 (arrows). Scale bars, 100 μm.

Figure 5

p38 activation of limbal epithelial cells and stromal cells in airlift cultures. Immunohistochemical staining of P-p38 at different stages of airlift cultures were performed. P-p38 staining was negative in limbal epithelial and stromal cells on day 0. On day 2, positive epithelial cells were present in limbal basal and suprabasal layers. Positive staining was increased in the basal layer on day 4, whereas stromal cells continued to stain negatively. On day 6, cytoplasmic expression of P-p38 increased without a concomitant increase in nuclear staining, whereas stromal cells started to show weak positive nuclear staining. On day 10 and day 14, cytoplasmic expression of P-p38 increased dramatically, and nuclear staining spread from the basal layer to the superficial layer. Stromal expression also increased. Scale bar, 200 μm.

Figure 6

Suppression of abnormal epidermal differentiation by p38 inhibitor SB203580. Immunostaining to K12 keratin (A), K10 keratin (B), and filaggrin (C) was performed in the normal control (before culturing), submerged cultures, and airlift cultures, of which the latter was also added with SB203580 for 2 weeks. K12 keratin was expressed by suprabasal and superficial cells in the normal control and the submerged culture, decreased to single cells and patches of cells in the airlift culture, and reverted to the normal pattern after the addition of SB203580. K10 keratin-positive cells were absent in the normal control and the submerged cultures but was found in superficial and suprabasal cell layers in airlift cultures. They disappeared after SB203580 was added. Filaggrin-positive cells were found only in superficial cell layers in airlift cultures but disappeared after SB203580 was added. Composites of micrographs at lower magnification confirmed the loss of K12 keratin expression (D). The gain of K10 keratin expression (E) was confined to the limbal epithelium in airlift cultures and was reverted by SB203580. Scale bars: (A–C) 100 μm; (D, E) 200 μm.

Figure 7

Progenitor cell status after explant cultures. Human limbal explants cultured under submerge, airlift, and airlift+SB203580 modes for 2 weeks. Single cells were harvested from the explant surface by dispase and subsequent trypsin/EDTA and were seeded onto mitomycin C-treated 3T3 fibroblast feeder layers for 12 days. The resultant epithelial clones were double stained with antibodies against K12 (top) and K10 (bottom) keratins and were counterstained to DAPI. All clones remained K12 keratin positive but K10 keratin negative, and those derived from oral mucosal epithelial cells were K12 keratin negative and K10 keratin negative. Scale bar, 200 μm.

Figure 8

Immunohistochemistry to P-p38, C/EBPα, and C/EBPβ. Immunohistochemical staining to P-p38 was negative in the normal limbal epithelium and stroma, weakly positive in the limbal basal epithelium and stromal cells in submerged cultures, and strongly positive in cytoplasms and nuclei of the limbal epithelium and in the nuclei of stromal cells in airlift cultures. P-p38 staining was decreased in epithelial and stromal cells in airlift cultures treated with SB203580. C/EBPα was weakly expressed in the normal limbal epithelium but was strongly positive in the nuclei of limbal epithelial cells in submerged and airlift cultures. Such positive nuclear staining was dramatically decreased when SB203580 was added to airlift cultures. C/EBPβ was also weakly expressed in the nuclei of limbal epithelial cells but was strongly expressed in the nuclei of almost all epithelial and stromal cells in submerged and airlift cultures. However, such nuclear staining was decreased by SB203580 in airlift cultures. Scale bar, 100 μm.