Evaluation of eotaxin-1, -2, and -3 protein production and messenger RNA expression in patients with vernal keratoconjunctivitis

Abstract

To evaluate the in vivo expression of members of the eotaxin subfamily, eotaxin-1, -2, and -3, at the ocular surface, we analyzed the messenger RNA (mRNA) expression of the eotaxin subfamily in conjunctival epithelium and the protein expression of the eotaxin subfamily in tears of patients with vernal keratoconjunctivitis (VKC) and in those of healthy individuals. The subjects were 25 patients with VKC (25 eyes) and 11 healthy volunteers (11 eyes) as a control. Tear samples were collected using the Schirmer strip method. Tear samples were eluted, and concentrations of eotaxin-1, -2, and -3 in the tear samples were determined by enzyme-linked immunosorbent assay (ELISA). Concentration of eosinophil cationic protein (ECP) in tears was also determined by chemiluminescent enzyme immunoassay. Conjunctival epithelial cells were obtained from upper tarsal conjunctiva by impression cytology, and eotaxin-1, -2, and -3 mRNA extracted from the impression cytology membrane were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Conjunctival smears, which were obtained by tarsal conjunctival scraping, were stained for eotaxin-2 using immunohistochemical methods. In the ELISA analysis, the expression ratio of eotaxin-1 (P < 0.01) and -2 (P < 0.001) in tears was significantly higher in the VKC group than in the control group. Concentrations of eotaxin-1 and -2 in tears in the VKC group were 0.7 and 1440.5 (median values) pg/ml, respectively. In the VKC group, the concentration of eotaxin-2 in tears was higher than that of eotaxin-1. There was a significant correlation between the concentration of eotaxin-2 and that of ECP in tears in the VKC group (r = 0.53, P < 0.01). Expression of eotaxin-3 protein in tears was not detected in the VKC group or the controls. In the RT-PCR analysis, the positive ratio of eotaxin-1, -2, and -3 mRNA expression in the VKC group was significantly higher than that in the control group (P < 0.01, P < 0.01, P < 0.05, respectively). In the immunohistochemical analysis, positive staining was detected in epithelial-like cells in conjunctival smears obtained from the patients with VKC. We showed that the mRNA expression and the protein production of the eotaxin subfamily at the ocular surface are critical biomarkers when investigating the pathophysiology of eosinophilic inflammation and the effect of antiallergic treatment in patients with VKC.