Biochemical and functional characterization of soluble form of IL-5 receptor alpha (sIL-5R alpha). Development of ELISA system for detection of sIL-5R alpha.
Interleukin-5 (IL-5) mediates pleiotropic functions in various types of cells through its specific receptor (IL-5R) which is composed of two distinct subunits, alpha and beta. In mice, the alpha subunit (IL-5R alpha) specifically binds IL-5 with low affinity. The beta subunit (IL-5R beta) does not bind IL-5 by itself, but constructs the high affinity receptor with IL-5R alpha. We have isolated cDNA encoding the soluble form of IL-5R alpha (sIL-5R alpha). To elucidate the biochemical and functional properties of sIL-5R alpha, we developed an expression system for sIL-5R alpha cDNA in insect cell line Sf21 using baculovirus expression vector and obtained conditioned medium containing large quantities of mouse sIL-5R alpha. Mouse sIL-5R alpha was purified from the conditioned medium by using anti-IL-5R alpha mAb-coupled beads. Immunoaffinity-purified sIL-5R alpha with an approximate molecular mass of 42 kDa inhibited the binding of 125I-labeled IL-5 to IL-5R. By using purified sIL-5R alpha, we prepared rabbit anti-sIL-5R alpha antibody and developed a sandwich ELISA for detection of sIL-5R alpha. Significant amounts of sIL-5R alpha were detected in sera and ascitic fluids of mice bearing tumors (BCL1 and MOPC104E) that responded to IL-5 for DNA synthesis, but not in sera of normal mice. Interestingly, elevated levels of serum sIL-5R alpha were observed in NZB and NZW mice. The sIL-5R alpha may, therefore, have an immunoregulatory role in vivo.
Kikuchi
Y
Y
Department of Immunology, University of Tokyo, Japan.
Migita
M
M
Takaki
S
S
Tominaga
A
A
Takatsu
K
K
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015848:Interleukin-5 / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009036:Moths; D012333:RNA, Messenger / Q000032:analysis; D018123:Receptors, Interleukin / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D014018:Tissue Distribution
D015848:Interleukin-5; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins