Ken Fukuda1, Youichiro Fujitsu, Naoki Kumagai, Teruo Nishida. 1. Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, Ube City, Yamaguchi, Japan. k.fukuda@po.cc.yamaguchi-u.ac.jp
Abstract
PURPOSE: To characterize the interleukin (IL)-4 receptor (IL-4R) complex in human corneal fibroblasts. METHODS: The presence of IL-4R subunit mRNAs and proteins in cultured human corneal fibroblasts was examined by reverse transcription-polymerase chain reaction and flow cytometry, respectively. The interaction of 125I-labeled IL-4 with specific cell surface receptors was characterized by saturation binding and Scatchard analysis. The effects of IL-4 on the tyrosine phosphorylation and subcellular localization of signal transducer and activator of transcription 6 (STAT6) were evaluated by immunoblot and indirect immunofluorescence analyses, respectively. The concentration of eotaxin in cell culture supernatant was measured by enzyme-linked immunosorbent assay. RESULTS: Transcripts encoding the IL-4R components IL-4Ralpha, IL-2Rgammac, IL-13Ralpha1, and IL-13Ralpha2 were detected in human corneal fibroblasts; IL-4Ralpha and IL-2Rgammac proteins were also expressed on the cell surface. The maximum number of IL-4 binding sites was 2.3 x 10(4) per cell, and the dissociation constant for the interaction of IL-4 with these sites was 10.1 +/- 0.3 pM. IL-4 induced tyrosine phosphorylation of STAT6 as well as translocation of this protein to the nucleus. Eotaxin release from corneal fibroblasts stimulated by the combination of IL-4 and tumor necrosis factor-alpha was inhibited by pretreatment of the cells with neutralizing antibodies to IL-4R. CONCLUSIONS: Cultured human corneal fibroblasts express high-affinity functional IL-4Rs on the cell surface, suggesting that these cells may contribute to the role of IL-4 as a key mediator of allergic reactions in the cornea.
PURPOSE: To characterize the interleukin (IL)-4 receptor (IL-4R) complex in human corneal fibroblasts. METHODS: The presence of IL-4R subunit mRNAs and proteins in cultured human corneal fibroblasts was examined by reverse transcription-polymerase chain reaction and flow cytometry, respectively. The interaction of 125I-labeled IL-4 with specific cell surface receptors was characterized by saturation binding and Scatchard analysis. The effects of IL-4 on the tyrosine phosphorylation and subcellular localization of signal transducer and activator of transcription 6 (STAT6) were evaluated by immunoblot and indirect immunofluorescence analyses, respectively. The concentration of eotaxin in cell culture supernatant was measured by enzyme-linked immunosorbent assay. RESULTS: Transcripts encoding the IL-4R components IL-4Ralpha, IL-2Rgammac, IL-13Ralpha1, and IL-13Ralpha2 were detected in human corneal fibroblasts; IL-4Ralpha and IL-2Rgammac proteins were also expressed on the cell surface. The maximum number of IL-4 binding sites was 2.3 x 10(4) per cell, and the dissociation constant for the interaction of IL-4 with these sites was 10.1 +/- 0.3 pM. IL-4 induced tyrosine phosphorylation of STAT6 as well as translocation of this protein to the nucleus. Eotaxin release from corneal fibroblasts stimulated by the combination of IL-4 and tumor necrosis factor-alpha was inhibited by pretreatment of the cells with neutralizing antibodies to IL-4R. CONCLUSIONS: Cultured human corneal fibroblasts express high-affinity functional IL-4Rs on the cell surface, suggesting that these cells may contribute to the role of IL-4 as a key mediator of allergic reactions in the cornea.