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