Characterization of the interleukin-4 receptor complex in human corneal fibroblasts.
OBJECTIVE
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.
Fukuda
Ken
K
Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, Ube City, Yamaguchi, Japan. k.fukuda@po.cc.yamaguchi-u.ac.jp
Fujitsu
Youichiro
Y
Kumagai
Naoki
N
Nishida
Teruo
T
D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D054413:Chemokine CCL11; D019742:Chemokines, CC / Q000378:metabolism; D002631:Chemotactic Factors, Eosinophil / Q000378:metabolism; D002675:Child, Preschool; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015847:Interleukin-4 / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D019948:Receptors, Interleukin-4 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050800:STAT6 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
C515456:CCL11 protein, human; D054413:Chemokine CCL11; D019742:Chemokines, CC; D002631:Chemotactic Factors, Eosinophil; D012333:RNA, Messenger; D019948:Receptors, Interleukin-4; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015534:Trans-Activators; D015847:Interleukin-4; D014443:Tyrosine