Generation and characterization of an antagonistic monoclonal antibody against an extracellular domain of mouse DP2 (CRTH2/GPR44) receptors for prostaglandin D2.

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.

• Authors: Nanae Nagata, Hiroko Iwanari, Hidetoshi Kumagai, Osamu Kusano-Arai, Yuichi Ikeda, Kosuke Aritake, Takao Hamakubo, Yoshihiro Urade
• Journal: PloS one (PLoS One) Vol. 12 Issue 4 Pg. e0175452 ( 2017) ISSN: 1932-6203 [Electronic] United States
• PMID: 28394950 (Publication Type: Journal Article)

Chemical References

• Antibodies, Monoclonal
• Receptors, Immunologic
• Receptors, Prostaglandin
• beta-Arrestins
• 15-methylprostaglandin D2
• Cyclic AMP
• Prostaglandin D2
• prostaglandin D2 receptor

Topics

• Animals
• Antibodies, Monoclonal (biosynthesis, chemistry, 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, pathology)
• Mice
• Mice, Inbred BALB C
• Mice, Inbred C57BL
• Mice, Knockout
• Precursor Cells, B-Lymphoid (immunology)
• Prostaglandin D2 (analogs & derivatives, antagonists & inhibitors)
• Receptors, Immunologic (genetics, immunology)
• Receptors, Prostaglandin (genetics, immunology)
• Ureteral Obstruction (immunology, metabolism, pathology)
• beta-Arrestins (metabolism)