Abstract
Purpose
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.
Methods
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.
Results
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.
Conclusions
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.
Similar content being viewed by others
References
Friedlander MH. Conjunctivitis of allergic origin: clinical presentation and differential diagnosis. Surv Ophthalmol 1993;38Suppl:105–114.
Bonini S, Bonini S, Berruto A, et al. Conjunctival provocation test as a model for the study of allergy and inflammation in humans. Int Arch Allergy Appl Immunol 1989;88:144–148.
Pucci N, Lombardi NE, Cianferoni A, et al. Atopy and serum eosinophil cationic protein in 110 white children with vernal keratoconjunctivitis: differences between tarsal and limbal forms. Clin Exp Allergy 2003;33:325–330.
Shoji J, Kitazawa M, Inada N, et al. Efficacy of tear eosinophil cationic protein level measurement using filter paper for diagnosing allergic conjunctival disorders. Jpn J Ophthalmol 2003;47:64–68.
Leonardi A, Borghesan F, Aggian D, Secchi A, Plebani M. Eosinophil cationic protein in tears of normal subjects and patients affected by vernal keratoconjunctivitis. Allergy 1995;50:610–613.
Montan PG, van Hage-Hamsten M. Eosinophil cationic protein in tears in allergic conjunctivitis. Br J Ophthalmol 1996;80:556–560.
Humbles AA, Conroy DM, Marleau S, et al. Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo. J Exp Med 1997;186:601–612.
Forssmann UM, Uguccioni P, Loetscher CA, et al. Eotaxin-2, a novel CC chemokine that is selective for the chemokine receptor CCR3, and acts like eotaxin on human eosinophil and basophil leukocytes. J Exp Med 1997;185:2171–2176.
Shinkai A, Yoshisue H, Koike M, et al. A novel human CC chemokine, eotaxin-3, which is expressed in IL-4-stimulated vascular endothelial cells, exhibits potent activity toward eosinophils. J Immunol 1999;163:1602–1610.
Brown JR, Kleimberg J, Marini M, Sun G, Bellini A, Mattoli S. Kinetics of eotaxin expression and its relationship to eosinophil accumulation and activation in bronchial biopsies and bronchoalveolar lavage (BAL) of asthmatic patients after allergen inhalation. Clin Exp Immunol 1998;114:137–146.
Menzies-Gow A, Ying S, Phipps S, Kay AB. Interactions between eotaxin, histamine and mast cells in early microvascular events associated with eosinophil recruitment to the site of allergic skin reactions in humans. Clin Exp Allergy 2004;34:1276–1282.
Berkman N, Ohnona S, Chung FK, Breuer R. Eotaxin-3 but not eotaxin gene expression is upregulated in asthmatics 24 hours after allergen challenge. Am J Respir Cell Mol Biol 2001;24:682–687.
Fukagawa K, Nakajima T, Tsubota K, Shimmura S, Saito H, Hirai K. Presence of eotaxin in tears of atopic keratoconjunctivitis patients with severe corneal damage. J Allergy Clin Immunol 1999;103:1220–1221.
Leonardi A, Jose PJ, Zhan H, Calder VL. Tear and mucus eotaxin-1 and eotaxin-2 in allergic keratoconjunctivitis. Ophthalmology 2003;110:487–492.
Shoji J, Inada N, Sawa M. Antibody array-generated cytokine profiles of tears of patients with vernal keratoconjunctivitis or giant papillary conjunctivitis. Jpn J Ophthalmol 2006;50:195–204.
Irkec M, Bozkurt B. Epithelial cells in ocular allergy. Curr Allergy Asthma Rep 2003;3:352–357.
Japanese Ocular Allergology Study Group. Guidelines for the clinical management of allergic conjunctival disease [in Japanese with English abstract]. Nippon Ganka Gakkai Zasshi 2006;110:101–140.
Nelson JD. Impression cytology. Cornea 1988;7:71–81.
Ying S, Meng Q, Zeibecoglou K, et al. Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, monocyte chemoat tractant protein-3 (MCP-3), and MCP-4), and C-C chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic (intrinsic) asthmatics. J Immunol 1999;163:6321–6329.
Kagami S, Kakinuma T, Saeki H, et al. Significant elevation of serum levels of eotaxin-3/CCL26, but not of eotaxin-2/CCL24, in patients with atopic dermatitis: serum eotaxin-3/CCL26 levels reflect the disease activity of atopic dermatitis. Clin Exp Immunol 2003;134:309–313.
Ravensberg AJ, Ricciardolo FLM, van Schadewijk A, et al. Eotaxin-2 and eotaxin-3 expression is associated with persistent eosinophilic bronchial inflammation in patients with asthma after allergen challenge. J Allergy Clin Immunol 2005;115:779–785.
Abu El-Asrar AM, Struyf S, Al-Kharashi SA, Missotten L, Van Damme J, Geboes K. Chemokines in the limbal form of vernal keratoconjunctivitis. Br J Ophthalmol 2000;84:1360–1366.
Leonardi A, Curnow SJ, Zhan H, Calder VL. Multiple cytokines in human tear specimens in seasonal and chronic allergic eye disease and conjunctival fibroblast cultures. Clin Exp Allergy 2006;36:777–784.
Ying S, Robinson DS, Meng Q, et al. C-C chemokines in allergeninduced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (monocyte chemoattractant protein-3 and RANTES). J Immunol 1999;163:3976–3984.
Heiman AS, Abonyo BO, Darling-Reed SF, et al. Cytokinestimulated human lung alveolar epithelial cells release eotaxin-2 (CCL24) and eotaxin-3 (CCL26). J Interferon Cytokine Res 2005;25:82–91.
Komiya A, Nagase H, Yamada H, et al. Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells. Cell Immunol 2003;225:91–100.
Matsukura S, Stellato C, Plitt JR, et al. Activation of eotaxin gene transcription by NF-kappa B and STAT6 in human airway epithelial cells. J Immunol 1999;163:6876–6883.
van Wetering S, Zuyderduyn S, Ninaber DK, van Sterkenburg MA, Rabe KF, Hiemstra PS. Epithelial differentiation is a determinant in the production of eotaxin-2 and -3 by bronchial epithelial cells in response to IL-4 and IL-13. Mol Immunol 2007;44:803–811.
Author information
Authors and Affiliations
Corresponding author
About this article
Cite this article
Shoji, J., Inada, N. & Sawa, M. Evaluation of eotaxin-1, -2, and -3 protein production and messenger RNA expression in patients with vernal keratoconjunctivitis. Jpn J Ophthalmol 53, 92–99 (2009). https://doi.org/10.1007/s10384-008-0628-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10384-008-0628-5