Monoclonal antibodies detect different distribution patterns of IL-8 receptor A and IL-8 receptor B on human peripheral blood leukocytes.
mAbs previously reported to be specific for IL-8R type A (IL-8R-A) and mAbs specific for IL-8R type B (IL-8R-B), which are described in this paper, were used to investigate the expression of each receptor on various types of cells. We generated mAbs specific for IL-8R-B, 4D1, and 10H2 by immunizing mice with 293 cells that expressed IL-8R-B and by selecting hybridoma cell lines that secreted mAbs that bind to human neutrophils. Flow cytometry showed that mAbs 4D1 and 10H2 were specific for IL-8R-B, as determined by their exclusive binding to 293-27 cells that expressed IL-8R-B, but not to 293-71 cells that expressed IL-8R-A. Epitopes recognized by these IL-8R-B-specific mAbs were shown to be within the N-terminal residues 1-18 of the IL-8R-B on the basis of their binding to various N-terminal peptides, as measured by ELISA. These IL-8R-B-specific mAbs were able to inhibit up to 90 and 50% of the 125I-labeled IL-8 binding to 293-27 cells and human neutrophils, respectively. The combination of mAb 9H1 (anti-IL-8R-A) and mAb 10H2 (anti-IL-8R-B) inhibited approximately 70% of 125I-labeled IL-8 binding to human neutrophils. Flow cytometry showed a wide range of donor variation in the expression levels of IL-8R-A and IL-8R-B on various human peripheral blood leukocytes. All neutrophils, all monocytes, and 5 to 25% of total lymphocytes (CD8+ T cells and CD56+ NK cells) expressed IL-8R. Neutrophils expressed the highest level of both IL-8R-A and IL-8R-B, at an approximately equal ratio, whereas monocytes and IL-8R+ lymphocytes expressed higher levels of IL-8R-B than IL-8R-A. Double-color flow cytometric analysis showed that 7 to 42% of CD8+ T cells and 39 to 76% of CD56+ NK cells, but no CD 20+ B cells or CD4+ T cells, expressed IL-8R.
Chuntharapai
A
A
Department of Bioanalytical Technology, Genentech, Inc., South San Francisco, CA 94080.
Lee
J
J
Hébert
C A
CA
Kim
K J
KJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007962:Leukocytes / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018123:Receptors, Interleukin / Q000096:biosynthesis* / Q000276:immunology; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D014162:Transfection / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B