Signal transduction through CD16 in human conjunctival epithelial cells

T. Fujihara, Tsutomu Takeuchi, K. Saito, Kazuo Tsubota

Research output: Contribution to journalArticle

Abstract

Purpose. Recently epithelial cells are considered to be involved in the regulation of immunological reactions. We examined whether three types of Fc gamma receptors including Fcg RI, II, III (CD64, CD32, CD16, respectively) were expressed in human conjunctival epithelial cells. Methods. Conjunctival epithelial cells were isolated from normal human volunteers by brush cytology. Human conjunctival epithelial cell line (ChWK) was also used for the following analysis. Expression of Fc gamma receptors on thses cells were analyzed by indirect immunofluorescence using a flow cytometry. To characterize Fc gamma receptor, biotin-labeled cells were lysed and immunoprecipitated, followed by SDS-PAGE, and blotting onto PVDF membrane. The signals were detected by ECL chemiluminescent system. Furthermore, phosphorylation of intracellular proteins after crosslinking of Fc gamma receptors by monoclonal antibodies was detected by immuno-blotting. Results. Normal human conjunctival epithelial cells expressed CD16 which is a low affinity Fc gamma receptor while CD32 and CD64 were negative. Only CD16 was also positive in ChWK by flow cytometric analysis and immunoprecipitaiton. Crosslinking of the CD16 molecules by mAb resulted in the tyrosine phosphorylation of 180-190 kDa protein. Conclusions. Human conjunctival epithelial cells expressed a low affinity Fc gamma receptor, CD16, which could transfer signals, suggesting an important functional role of this molecule.

Original languageEnglish
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
Publication statusPublished - 1996 Feb 15
Externally publishedYes

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IgG Receptors
Signal Transduction
Epithelial Cells
Phosphorylation
Indirect Fluorescent Antibody Technique
Biotin
Cell Biology
Tyrosine
Polyacrylamide Gel Electrophoresis
Healthy Volunteers
Flow Cytometry
Proteins
Monoclonal Antibodies
Cell Line
Membranes

ASJC Scopus subject areas

  • Ophthalmology

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Signal transduction through CD16 in human conjunctival epithelial cells. / Fujihara, T.; Takeuchi, Tsutomu; Saito, K.; Tsubota, Kazuo.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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AB - Purpose. Recently epithelial cells are considered to be involved in the regulation of immunological reactions. We examined whether three types of Fc gamma receptors including Fcg RI, II, III (CD64, CD32, CD16, respectively) were expressed in human conjunctival epithelial cells. Methods. Conjunctival epithelial cells were isolated from normal human volunteers by brush cytology. Human conjunctival epithelial cell line (ChWK) was also used for the following analysis. Expression of Fc gamma receptors on thses cells were analyzed by indirect immunofluorescence using a flow cytometry. To characterize Fc gamma receptor, biotin-labeled cells were lysed and immunoprecipitated, followed by SDS-PAGE, and blotting onto PVDF membrane. The signals were detected by ECL chemiluminescent system. Furthermore, phosphorylation of intracellular proteins after crosslinking of Fc gamma receptors by monoclonal antibodies was detected by immuno-blotting. Results. Normal human conjunctival epithelial cells expressed CD16 which is a low affinity Fc gamma receptor while CD32 and CD64 were negative. Only CD16 was also positive in ChWK by flow cytometric analysis and immunoprecipitaiton. Crosslinking of the CD16 molecules by mAb resulted in the tyrosine phosphorylation of 180-190 kDa protein. Conclusions. Human conjunctival epithelial cells expressed a low affinity Fc gamma receptor, CD16, which could transfer signals, suggesting an important functional role of this molecule.

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