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The Immunosuppressant Cyclosporin A Antagonizes Human Formyl Peptide Receptor through Inhibition of Cognate Ligand Binding¹

Pangke Yan^2,*, Masakatsu Nanamori^2,, Meiling Sun^*, Caihong Zhou^*, Ni Cheng, Na Li^*, Wei Zheng, Lihua Xiao, Xin Xie^*, Richard D. Ye^3, and Ming-Wei Wang^3,*

^* National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China; Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612; and Fujian Institute of Microbiology, Fuzhou, Fujian, China

Cyclosporin A (CsA) is a fungus-derived cyclic undecapeptide with potent immunosuppressive activity. Its analog, cyclosporin H (CsH), lacks immunosuppressive function but can act as an antagonist for the human formyl peptide receptor (FPR). More recent studies have shown that CsA also inhibits fMLF-induced degranulation in differentiated HL-60 promyelocytic leukemia cells. However, it is unclear whether CsA interferes with ligand-receptor interaction, G protein activation, or other downstream signaling events. In this study we used human neutrophils, differentiated HL-60 cells, and rat basophilic leukemia (RBL)-2H3 cells expressing human FPR (RBL-FPR) to identify the action site of CsA. In functional assays, CsA inhibited fMLF-stimulated degranulation, chemotaxis, calcium mobilization, and phosphorylation of the MAPKs ERK 1/2 and the serine/threonine protein kinase Akt. CsA also blocked Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm)-induced functions in RBL-FPR cells. Concentrations for half-maximal inhibition with CsA are generally 6- to 50-fold higher than that of CsH. CsA was compared with another immunosuppressant, ascomycin, relative to the inhibitory effects on FPR-mediated chemotaxis, calcium mobilization, and degranulation. In these experiments, ascomycin produced no inhibitory effects at low micromolar concentrations (1–4 µM), whereas the inhibitory effects of CsA were prominent at comparable concentrations. Finally, CsA dose-dependently inhibited the uptake of fNle-Leu-Phe-Nle-Tyr-Lys-fluoresceine and [³H]fMLF or [¹²⁵I]WKYMVm binding to FPR. However, CsA and CsH did not show any obvious inhibitory effect on FPR-like 1-mediated cellular functions. These results demonstrate that CsA is a selective antagonist of FPR and that its inhibition of fMLF-stimulated leukocyte activation is at the level of cognate ligand binding.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Ministry of Science and Technology of China Grant 2004CB518902 (to M.-W.W.), Shanghai Municipality Government Grants 30219228 and 05dz22914 (to M.-W.W.) and 2005B86 (to R.D.Y.), Chinese Academy of Sciences Grant KFRJHZ[2005]32 (to M.-W.W. and R.D.Y.), and National Institutes of Health Grant AI033503 (to R.D.Y.). M.N. is supported by a predoctoral fellowship from the American Heart Association, Midwest Affiliate.

² P.Y. and M.N. contributed equally to this study.

³ Address correspondence and reprint requests to Dr. Ming-Wei Wang, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Zhangjiang High-Tech Park, Shanghai 201203, China; E-mail address: mwwang{at}mail.shcnc.ac.cn or Dr. Richard D. Ye, Department of Pharmacology, College of Medicine, M/C 868, 835 South Wolcott Avenue, University of Illinois, Chicago, IL 60612. E-mail address: yer{at}uic.edu

⁴ The abbreviations used are: FPR, formyl peptide receptor; FPRL1, FPR-like 1; CsA, cyclosporin A; CsH, cyclosporin H; dbcAMP, N⁶-2'-O-dibutyryl-cAMP; RBL, rat basophilic leukemia; RBL-FPR, RBL-2H3 cells expressing human FPR; RBL-FPRL1, RBL-2H3 cells expressing human FPRL1; Fluo-4/AM, fluo-4-acetoxymethyl ester.

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