Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses.
Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.
Ishida
H
H
DNAX Research Institute of Molecular and Cellular Biology, Incorporated, Palo Alto, California 94304.
Yang
G
G
Harada
N
N
Hastings
R L
RL
Castle
B E
BE
Kastelein
R
R
Miyajima
A
A
Howard
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D002460:Cell Line; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011917:Rats, Inbred Lew; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D019948:Receptors, Interleukin-4; D011975:Receptors, Mitogen; D015719:Single-Strand Specific DNA and RNA Endonucleases