Analysis of common beta chain of human GM-CSF,IL-3, and IL-5 rceptor

1994 Fiscal Year Final Research Report Summary

• Project/Area Number: 05670913
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Hematology
• Research Institution: KYUSHU UNIVERSITY
• Principal Investigator: HAYASHIDA Kazuhiro Kyushu univ., Faculty of Medicine Assistant, 医学部, 助手 (60180981)
• Co-Investigator(Kenkyū-buntansha): NIHO Yoshiyuki Kyushu univ., Faculty of Medicine Professor, 医学部, 教授 (60091287) ; OKAMURA Seiichi Kyushu univ., Faculty of Medicine Assistant Professor, 医学部, 助手 (20136435) ; OTSUKA Teruhisa Kyushu univ., Faculty of Medicine Assistant Professor, 医学部, 講師 (20185317)
• Project Period (FY): 1993 – 1994
• Keywords: GM-SCF receptor / Monoclonal antibody

Research Abstract

GM-CSF affects various cells including myeloid cells and its receptor binds to GM-CSF with specific alpha chain and shares beta chain with IL-3 and/or IL-5 receptor. This research began to study the expression level of GM-CSF receptor alpha and beta chains in vivo by using the specific antibodies.
Balb/c mice were immunized of synthetic peptides that correspond to amino acids' sequence of the extracellular domain of human GM-CSF receptor alpha chain. Then spleen cells were collected to make hybridomas with mouse myeloma cell line. After checking the elevation of the antibody titers against the synthetic peptide in the supernatant, hybridomas that produced the specific antibody ware injected to the intraperitoneal space of Balb/c mouse.
After purified the antibody, expression level of alpha chain was examined by Western blotting and FACScan sorting. By Western blotting, single band of MW 70kDa was detected for the cell membrane fractions of U937 and HL-60 that express the GM-CSF receptor alpha chain. By flowcytometry, GM-CSF receptor alpha chain was detected on the surface of TF-1, U937, neutrophil and monocyte, not on that of Raji and Jarkat. As the antibody binding to TF-1 and U937 was blocked by GM-CSF,this antibody was thought to be the blocking antibody.
The expression of alpha chain was not increased by TNF alpha and IL-1 that increase the expression of beta chain strongly. This expperiment indicated that the regulation of alpha chain is different to that of beta chain.
Although this research was aimed to produce the specific antibody for GM-CSF receptor beta chain, only the antibody for alpha chain was obtained. We think that to have the good antibody for beta chain is still the important work for analysis the GM-CSF receptor.