Mast Cells Populate the Corneoscleral Limbus: New Insights for Our Understanding of Limbal Microenvironment

Abstract

Purpose: Although stem cell activity represents a crucial feature in corneal and ocular surface homeostasis, other cells populating this region and the neighboring zones might participate and influence local microenvironment. Mast cells, the long-lived and tissue-sited immune cells, have been previously reported in corneoscleral specimens. Herein, mast cells were investigated in corneoscleral tissues and related to microenvironment protein expression.

Methods: Twenty-six (14 male/12 female; older than 60 years) human corneoscleral specimens were sectioned for light and fluorescent immunostaining (CD45, p63, Ck-3/7/12/19, tryptase/AA1, and chymase/CC1). Corneal, limbal, and conjunctival squares were produced for molecular and biochemical analysis. Statistical comparisons were carried out by ANOVA.

Results: Toluidine blue staining identified metachromatic intact or degranulated mast cells in the area below the palisades' Vogt (Ck-3/12-positive epithelium and underneath p63 immunoreactivity). Tryptase immunoreactivity was observed close to palisades' Vogt, whereas no specific signal was detected for chymase. Tryptase/AA1 transcripts were quantified in limbal and conjunctival RNA extracts, whereas no specific amplification was detected in corneal ones. Few mediators were overexpressed in limbal extracts with respect to corneal (Neural cell adhesion molecule (NCAM), Intercellular adhesion molecule 3 (ICAM3), Brain-derived Neurotrophic factor (BDNF), and neurotrophin 3 (NT3); P < 0.00083) and conjunctival (NCAM, ICAM3, and NT3; P < 0.05) protein extracts. A trend to an increase was observed for Nerve Growth Factor (NGF) in limbal extracts (P > 0.05).

Conclusions: The specific observation of tryptase phenotype and the interesting protein signature of microenvironment (adhesion molecules, growth factors, and neurotrophins), known to partake mast cell behavior, at least in other areas, would provide additional information to better understand this crucial zone in the framework of ocular surface healthiness.

Figure 1.

(A–C) Characterization of corneoscleral section and tissue extracts. Representative digital acquisitions defining the limbal zone between cornea and conjunctiva: (A) HE overview of a cross-sectional longitudinal tissue sample (×10/objective) and (B) Ck12-blue (cornea-limbus marker) immunoreactivity over a red nuclear staining (PI; ×40/objective). Scale bar are shown in the panels. (C) Amplicons specific for referring (18S) and target (Ck3, Ck7, and Ck12) genes amplified in corneal, limbal, and conjunctival extracts and separated in 2.5% agarose gel.

Figure 2.

(A–G) Immunohistochemical localization of mast cells at limbal zone. Representative digital acquisitions for basal histology (A–E) and immunofluorescent staining (F–H) highlighting the presence of mast cells. (A–C) Low (A, B; ×20) and high (C; ×40) optic field acquisitions from acidic (pH 3) 0.1% toluidine blue–stained sections. Note the presence of “purple-granule” stained mast cells in the limbal segment. (D, E) AA1 immunoreactive cells (brown-dark DAB staining) over a cresyl blue nuclear counterstaining. Note the presence of highly conserved secretory granules in stained cells (D; ×40) and particularly a localization close to dense blue counterstained palisades of Vogt (high nuclear affinity to cresyl violet) (E; ×40). The inset in the panel shows an internal control section from the serial section (absence of first antibody). (F, G) Representative immunofluorescent acquisitions showing AA1 (cy3/green) positive cells (×40). Note the presence of some partially degranulated mast cells (F) and mast cells with highly conserved (G) secretory granules. (H) A single MC localized in close proximity to dense nuclear palisades.

Figure 3.

(A–E) Characterization of mast cells at the limbal zone. (A, B) Double staining of AA1 (cy2/green) and Ck12 (cy5/blue) over a red nuclear counterstaining (PI; ×20/objective) and a particular AA1-positive cell close to niches (cy2/green; B). Ck12 stains part of the initial superficial limbal epithelial cells and extend over the corneal epithelium. White arrows point at AA1-positive cells. (C, D) Representative double-staining (merge) acquisition respectively for AA1 (cy2/green)–cKit (cy5/blue) and CD45 (cy2/green)–AA1 (cy3/red) over a nuclear counterstaining (DAPI/blue). Single staining is also shown close to the merge, as provided by the Nis software (Nikon). (E, G) Amplicons specific for cKit, AA1, CC1, FcεRI, and H3 are shown with respect to corneal, limbal, and conjunctival extracts. The related densitometric analysis specific for AA1 and cKit is shown in panels F and G, respectively.

Figure 4.

(A, B) NCAM-1, ICAM-3, BDNF, NT-3, and NGF are differentially expressed in the limbal zone. Adhesion molecules (NCAM1 and ICAM3; A, C) and growth factor (BDNF-NT3 [B, D, E] and NGF [F]) protein expression in corneal (C), limbal (L), and conjunctival (J) extracts, as confirmed by conventional Western blotting analysis on triplicate repeated experiments (C, D). Note the significant increase of NCAM1 and ICAM3 in limbal extracts (with respect to the other tissue extracts; P < 0.05) and BDNF and NT3 in limbal extracts with respect to corneal ones (P < 0.05). MFI stands for mean fluorescent intensity, as detected by ImageJ software (National Institutes of Health, Bethesda, MD, USA) on array chip (A, B). BDNF and NGF protein expression (mean ± SEM) quantified by ELISA and expressed as pg/µg total protein (E, F). ANOVA analysis followed by Bonferroni's correction. Asterisks (*) in the histograms point at significant differences between subgroups (P < 0.05).