RRC ID 52185
Author Nagata N, Iwanari H, Kumagai H, Kusano-Arai O, Ikeda Y, Aritake K, Hamakubo T, Urade Y.
Title Generation and characterization of an antagonistic monoclonal antibody against an extracellular domain of mouse DP2 (CRTH2/GPR44) receptors for prostaglandin D2.
Journal PLoS One
Abstract Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.
Volume 12(4)
Pages e0175452
Published 2017-4-10
DOI 10.1371/journal.pone.0175452
PII PONE-D-16-33637
PMID 28394950
PMC PMC5386288
MeSH Animals Antibodies, Monoclonal / biosynthesis* Antibodies, Monoclonal / chemistry Antibodies, Monoclonal / immunology* Antibody Specificity CD4-Positive T-Lymphocytes / metabolism CHO Cells COS Cells Cricetulus Cyclic AMP / metabolism Disease Models, Animal Epitope Mapping HEK293 Cells Humans Hybridomas / metabolism Immunization Immunohistochemistry Kidney / metabolism Kidney / pathology Mice Mice, Inbred BALB C Mice, Inbred C57BL Mice, Knockout Precursor Cells, B-Lymphoid / immunology Prostaglandin D2 / analogs & derivatives Prostaglandin D2 / antagonists & inhibitors Receptors, Immunologic / genetics Receptors, Immunologic / immunology* Receptors, Prostaglandin / genetics Receptors, Prostaglandin / immunology* Ureteral Obstruction / immunology Ureteral Obstruction / metabolism Ureteral Obstruction / pathology beta-Arrestins / metabolism
IF 2.776
Times Cited 4
Human and Animal Cells Ba/F3(RCB0805)