Elsevier

Experimental Eye Research

Volume 76, Issue 1, January 2003, Pages 107-114
Experimental Eye Research

IL-4-induced cell proliferation and production of extracellular matrix proteins in human conjunctival fibroblasts

https://doi.org/10.1016/S0014-4835(02)00248-8Get rights and content

Abstract

Giant papillae, characteristic lesions of vernal keratoconjunctivitis, are formed as a result of the proliferation of conjunctival fibroblasts, the deposition of extracellular matrix, and the infiltration of inflammatory cells. The concentration of interleukin (IL)-4 is also increased in the tear fluid of individuals with ocular allergic diseases. The possible role of IL-4 in the development of giant papillae was investigated by examining the effects of this cytokine on cultured human conjunctival fibroblasts. Reverse transcription and polymerase chain reaction analysis revealed the presence of transcripts encoding the IL-4 receptor α chain in these cells, and flow cytometry demonstrated the expression of this protein on the cell surface. IL-4 induced the proliferation of conjunctival fibroblasts in a concentration-dependent manner, and this effect was inhibited by neutralizing antibodies to the IL-4 receptor. Enzyme immunoassays revealed that IL-4 also increased in a concentration-dependent manner the amounts of procollagen type I C-peptide and fibronectin released into the culture supernatant by conjunctival fibroblasts. A whole-cell enzyme-linked immunosorbent assay showed that IL-4 increased the deposition of collagen type III by conjunctival fibroblasts. Furthermore, reverse transcription combined with real-time polymerase chain reaction analysis revealed that IL-4 increased the abundance of collagen type III mRNA in these cells. These results demonstrate that human conjunctival fibroblasts express receptors for IL-4, and that IL-4 stimulates both the proliferation of and the production of extracellular matrix proteins by these cells. These effects of IL-4 might contribute to the formation of giant papillae in individuals with vernal keratoconjunctivitis.

Introduction

Vernal keratoconjunctivitis (VKC) is a chronic allergic inflammatory disease characterized by the formation of conjunctival giant papillae at the upper tarsus. Histological analysis has revealed that the giant papillae are composed of infiltrated inflammatory cells, including eosinophils, mast cells, and T helper type 2 (Th2) cells, as well as conjunctival fibroblasts and extracellular matrix (ECM) proteins such as fibronectin and collagen types I and III (Leonardi et al., 1995). However, the mediators responsible for the proliferation of conjunctival fibroblasts and deposition of ECM that contribute to these structures remain unknown.

Interleukin (IL)-4 is a Th2 cell-derived cytokine that induces immunoglobulin isotype switching in B cells and maintains the production of IgE (Gauchat et al., 1990). This cytokine plays important roles in the development of both ocular and systemic allergic reactions. The concentration of IL-4 has been shown to be increased in the tear fluid of individuals with ocular allergic diseases and is especially high in individuals with VKC (Fujishima et al., 1995). We and others have recently shown that IL-4 acts not only on inflammatory cells but also on tissue-resident cells such as epithelial cells and fibroblasts (Doucet et al., 1998, Kumagai et al., 2000a, Fukuda et al., 2002). It is, thus, possible that IL-4 contributes to the formation of conjunctival giant papillae.

Characterization of the regulation by inflammatory mediators of the proliferation of and ECM production by conjunctival fibroblasts should provide insight into the pathogenesis of VKC. We have, therefore, now investigated whether cultured human conjunctival fibroblasts express IL-4 receptors as well as the possible effects of IL-4 on the proliferation of these cells and their synthesis of ECM proteins.

Section snippets

Materials

Minimum essential medium (MEM), fetal bovine serum (FBS), trypsin (0·05%)–EDTA (0·53%), and nonenzymatic cell dissociation solution were obtained from GIBCO BRL (Grand Island, NY, USA). Collagenase of Clostridium histolyticum was from Sigma (St Louis, MO, USA), bovine serum albumin (BSA) was from Nacalai Tesque (Kyoto, Japan), and l-ascorbic acid phosphate (Mg2+ salt) was from Wako (Osaka, Japan). Tissue culture dishes (Falcon) were from Becton-Dickinson (Franklin Lakes, NJ, USA). Recombinant

Culture of conjunctival fibroblasts

The purity of the conjunctival fibroblast cultures was judged on the basis of both cell morphology and reactivity with antibodies to vimentin and to cytokeratin (Fig. 1). All the cells were positive for vimentin and negative for cytokeratin, suggesting the absence of contamination of the cultures by epithelial cells. No changes in cell morphology or immunoreactivity were apparent during culture for up to three to eight passages. Identical results were obtained with cells derived from the two

Discussion

We have shown that IL-4 stimulates both the proliferation of and the production of ECM proteins by cultured human conjunctival fibroblasts. These effects of IL-4 appear to be mediated by IL-4 receptors expressed on the surface of the cells. Thus, the conjunctival fibroblasts were shown to express IL-4Rα at both the transcript and protein levels, and neutralizing antibodies to IL-4 receptors inhibited the IL-4-induced proliferation of these cells. IL-4 also increased the production of collagen

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