Purpose: To investigate the expression of dectin-1 protein in conjunctival epithelial cells and the expression of dectin-1 and B-cell activating factor belonging to the tumor necrosis factor family (BAFF) mRNA in in vivo conjunctival epithelial cells (CECs) and in vitro cultured CECs, and its difference in topographical change and etiology of disorders.
Subjects and methods:
1. Investigation of dectin-1 and BAFF expression by cytodiagnosis of CECs.
The subjects were 12 eyes of 12 healthy volunteers (control group), 6 eyes of 6 patients with Sjögren syndrome (Sjögren group) and 10 eyes of 10 patients with vernal keratoconjuctivitis (VKC group). CECs were sampled by impression cytology using nitrocellulose membrane. The expression of dectin-1 in CECs was detected by immunofluorescence and the quantitative determination of dectin-1 mRNA and BAFF mRNA expression was performed by real-time polymerase chain reaction (real-time PCR).
2. Investigation of dectin-1 and BAFF expression using cultured CECs.
Cultured CECs which were divided into an OK-432 addition group (addition concentrations: 0.02, 0.1, 0.5 KU/mL), a lipopolysaccharide (LPS) addition group (addition concentrations: 80, 160, 320 μg/mL) and an additive-free group were cultured. Quantitative determination of dectin-1 mRNA and BAFF mRNA expression in cultured CECs was performed by real-time PCR.
Results:
1. Investigation of dectin-1 and BAFF expression by cytodiagnosis of CECs.
In the control group, there was no significant topographical difference in the expression of dectin-1 and the amount of dectin-1 mRNA among superior, inferior tarsal conjunctiva and temporal bulbar conjunctiva. The levels of dectin-1 mRNA expression were 1.5 (0.1-4.0) [median value (range)] for the control group, 2.6 (1.1-4.8) for the Sjögren group and 3.6 (1.7-16.6) for the VKC group. The VKC group showed a significantly higher level of dectin-1 mRNA than the control group (p<0.01, Kruskal-Walles H-test). The levels of BAFF mRNA expression were 2.8 (0.2-13.8) [median value (range)] for the control group, 6.3 (2.1-15.1) for the Sjögren group and 11.2 (3.5-70.8) for the VKC group. The VKC group showed a significantly higher level of dectin-1 mRNA than the control group (p<0.01, Kruskal-Walles H-test). Moreover, regarding the relationship between expression level of dectin-1 mRNA and that of BAFF mRNA in all the subjects, there was a significant correlation between them (r=0.75, p<0.001, Spearman's rank coefficient). The levels of dectin-1 mRNA expression in the moderate and severe VKC group 9.2 (2.6-16.6) [median value (range)] were significantly higher than those in mild VKC group 2.8 (1.7-3.8) (p<0.05, Mann-Whitney U-test). The levels of BAFF mRNA expression in the severe and moderate VKC groups 17.4 (9.1-70.8) [median value (range)] were significantly higher than those in the mild VKC group 4.3 (3.5-11.2) (p<0.05, Mann-Whitney U-test).
2. Investigation of dectin-1 and BAFF expression by cultured CECs.
In the OK-432 addition group, the expression levels of dectin-1 mRNA were increased dose-dependently due to the OK-432 stimulation (p<0.05, Kruskal-Wallis H-test). Moreover, regarding the relationship between the expression level of dectin-1 mRNA and that of BAFF mRNA in all the cultured conjunctival epithelial cells stimulated by OK-432, there was a significant correlation between them (r=0.85, p<0.005, Spearman's rank coefficient).
Conclusions: We concluded that dectin-1 expression in CECs was demonstrated, and expression of both dectin-1 and BAFF in CECs is thought to be involved in pathologic aggravation of allergic inflammatory in patients with VKC.
Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc) 118: 368-377, 2014.