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Modulation of IL-8 and RANTES Release in Human Conjunctival Epithelial Cells: Primary Cells and Cell Line Compared and Contrasted

Smit, Eefke E. M.D.; Sra, Sharon K. M.D.; Grabowski, Laura R.; Ward, Sherry L. M.D., Ph.D.; Trocme, Stefan D. M.D.

Basic Investigations

Purpose. Recent research indicates that epithelial cells of the ocular surface can contribute to the allergic reaction by the release of inflammatory and/or chemotactic mediators. In this study, the role of two inflammatory mediators, previously identified in the tear film of ocular allergy subjects, TNF-α and IFN-γ, were evaluated for their effect on the release of two chemotactic mediators, IL-8 and RANTES, from cultured human conjunctival epithelial cells.

Methods. Human conjunctival epithelial cells (primary cells or HC0597 cell line) were grown to confluence and stimulated with various concentrations of TNF-α, IFN-γ, or a combination of both. Supernatants were collected at 6, 24, and 48 hours and stored frozen for subsequent ELISA analyses of RANTES and IL-8.

Results. RANTES and IL-8 release from HC0597 cells was stimulated in a dose- and time-dependent manner following treatment with TNF-α. However, only RANTES release was modulated by IFN-γ treatment. Treatment of HC0597 cells with both TNF-α and IFN-γ resulted in a synergistic increase in the release of RANTES. This synergistic effect was confirmed using primary cultures of human conjunctival epithelial cells.

Conclusions. Stimulation of conjunctival epithelium with proinflammatory mediators, TNF-α and/or IFN-γ, generated the release of the chemotactic factors IL-8 and RANTES, which could act to prolong inflammation. These two chemokines may prolong inflammation by recruiting eosinophils to the ocular surface. This is the first study to compare chemokine release in a cell line and primary cells; similar chemokine release after mediator stimulation was demonstrated, indicating that the two cell types are phenotypically similar.

Allergic conjunctivitis is a common manifestation of atopy and is characterized clinically primarily by itching and secondarily by conjunctival injection, lid swelling, chemosis, and tearing. 1,2 During an allergic reaction, the environmental allergen traverses the conjunctiva and elicits mast cell degranulation, recruitment of inflammatory cells, and release of mediators, such as proinflammatory cytokines. 3,4 Various cytokines and chemokines (including IFN-γ, TNF-α, RANTES, and IL-8) have been identified in tears of allergic patients. 5–9 One of the eosinophil granule proteins, eosinophilic cationic protein (ECP), has also been identified in tears of patients suffering from ocular allergies. 10,11 Tear ECP has been detected at minimal levels in normal, healthy, nonallergic individuals and has been shown to be increased in patients suffering from vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), and seasonal allergic conjunctivitis (SAC). 8 Many of these mediators can further stimulate the ocular surface epithelium to express intercellular adhesion molecule-1 (ICAM-1) and release chemoattractant cytokines capable of recruiting more eosinophils to the ocular surface epithelium. 12–14

Eosinophils are thought to play a significant role in the pathogenesis of ocular inflammation. 15 Some chemotactic cytokines (chemokines) are believed to be involved in the selective recruitment and functional activation of eosinophils. 16 Chemokines are divided into two subfamilies, distinguished by the presence (CXC) or the absence (CC) of an amino acid between the first two cysteine residues. 17 The chemokine RANTES belongs to the CC subfamily of chemokines and is a potent chemoattractant for eosinophils and T cells. 18,19 The chemokine IL-8 (interleukin-8) belongs to the CXC subfamily and is a potent chemotactic factor for neutrophils, T cells, 20 and eosinophils. 21

Both IL-8 and RANTES are synthesized by many cell types during an inflammatory response. In the lung, bronchial epithelium from patients with asthma has been shown to release high amounts of IL-8, in addition to other cytokines, such as GM-CSF and IL-6. 22 Wang et al. 23 have also demonstrated the stimulated release of RANTES in human bronchial epithelial cells; RANTES was released after stimulation with TNF-α. Both TNF-α and IFN-γ have been reported to synergistically stimulate the production of RANTES in bronchial epithelial cells. 24

Tumor necrosis factor α and IFN-γ are proinflammatory cytokines secreted by several cell types during an inflammatory response. In the eye, Th2-like helper T cells have been shown to be present in the conjunctiva of patients with VKC. 25 Th2 cells secrete a variety of cytokines, including TNF-α. 26 Mast cells, which have also been identified in the conjunctival cellular infiltrate of patients with VKC, 27 produce TNF-α and IFN-γ. 28 In addition, eosinophils have also been demonstrated to produce TNF-α. 29 The role of TNF-α and IFN-γ in allergic inflammation prompted our examination of these cytokines.

Primary cultures of corneal epithelium have been observed to release IL-8 and RANTES after treatment with TNF-α or IFN-γ; these cytokines produced a synergistic effect on RANTES release. 12,30,31 In addition, previous immunohistochemical investigations on conjunctival biopsies have also identified chemokines and cytokines in conjunctival epithelium. 14 Cytokine-stimulated primary cultures of human conjunctival epithelium released IL-8. 32 Although very limited research has been done on RANTES release from a neoplastically transformed conjunctival cell line (Wong-Kilbourne), 33,34 the relevance of these studies is questionable without validation in primary conjunctival epithelium. Thus, the objectives of the current study are not only to investigate chemokine release from a recently developed plasmid-transfected cell line but to also provide for the first time much needed validation studies on primary conjunctival epithelium.

From the Department of Ophthalmology and Visual Sciences (E.E.S., S.K.S., L.R.G., S.D.T.), University of Texas Medical Branch, Galveston, Texas; Gillette Medical Evaluation Laboratories (S.L.W.), Gaithersburg, Maryland.

Submitted May 8, 2002.

Revision received February 18, 2003.

Accepted February 18, 2003.

Commercial relationships: none.

This study was supported by a grant from Research to Prevent Blindness, Inc.

Address correspondence and reprint requests to Stefan D. Trocme, M.D., Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, 700 University Blvd., Galveston, TX 77555-1106. E-mail:

© 2003 Lippincott Williams & Wilkins, Inc.