Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by N-formyl-methionyl-leucyl-phenylalanine,☆☆,,★★

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Abstract

Background:Cyclosporin A (CsA) binds with high affinity to cyclophilin, a critical step in the molecular mechanism of action of cyclosporins, whereas cyclosporin H (CsH) has extremely low affinity for cyclophilin. CsH differs from CsA by the substitution of the L-methyl valine at position 11 with its D-isomer. Methods: We compared the effects of CsA and CsH on the release of preformed (histamine) and de novo synthesized inflammatory mediators (peptide leukotriene C 4) from peripheral blood basophils activated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). Results: CsH (8 to 800 nmol/L) concentration-dependently inhibited histamine and leukotriene C 4 release from purified and unpurified basophils activated by FMLP, whereas CsA (8 to 800 nmol/L) had little inhibitory effect on histamine release from basophils challenged with FMLP. Inhibition of histamine release from basophils challenged with FMLP was extremely rapid and was abolished by washing the cells (three times) before challenge. CsH (8 to 800 nmol/L) had no effect on the release of histamine caused by C5a, platelet activating factor, monocyte chemotactic activating factor, RANTES, IL-8, bryostatin 1, and phorbol myristate. Preincubation of basophils with granulocyte-macrophage colony-stimulating factor (30 and 100 pmol/L), but not IL-1β (30 and 100 ng/ml), concentration-dependently reversed the inhibitory effect of CsH on FMLP-induced histamine release. CsH competitively inhibited the effect of FMLP on histamine release from basophils. The dissociation constant (K d) for the CsH-FMLP receptor complex was approximately 9 × 10-8 mol/L, more than 10-fold lower than that (≅ 1.3 × 10-6 mol/L) of N-t-BOC-methionyl-L-leucyl-phenylalanine (BocMLP), a known formyl peptide receptor antagonist. CsH inhibited tritiated FMLP binding to human polymorphonuclear leukocytes with a concentration required to inhibit binding by 50% of approximately 5.4 × 10-7 mol/L, whereas BocMLP was less potent with a concentration required to inhibit binding by 50% of approximately 9.1 × 10-5 mol/L. Scatchard analysis revealed that the decreased tritiated FMLP binding caused by CsH was due to a decrease in the B max (0.22 ± 0.04 nmol/L/5 × 106 cells vs 0.09 ± 0.01 nmol/L/5 × 106 cells; p < 0.05), without a significant difference in the K d (5.16 ± 1.22 nmol/L vs 6.32 ± 2.42 nmol/L; p = NS). Conclusions: CsH is a potent and selective inhibitor of mediator release from basophils induced by activation of the formyl peptide receptor; it acts by interfering with agonist binding to FMLP receptors. (J ALLERGY CLIN IMMUNOL 1996;98:152-64.)

Section snippets

Reagents

The following were purchased: C5a and N-t-BOC-methionyl-L-leucyl-phenylalanine (BocMLP) (Sigma Chemical Company, St. Louis, Mo.); N-formyl-methionyl-leucyl-l-phenylalanine (FMLP) (Calbiochem- Behring Co., La Jolla, Calif.); PAF C 16:0 (Biomol, Philadelphia, Pa.); 12-0-tetradecanoyl-phorbol-13-acetate (TPA) (P-L Biochemicals Inc., Milwaukee, Wis.); dextran T 70 (Pharmacia Fine Chemicals, Uppsala, Sweden); tritiated LTC 4 (35.7 Ci/mmol; New England Nuclear, Boston, Mass.); tritiated FMLP (40.5

Effect of CsH and CsA on FMLP-induced histamine release from human basophils

In a first series of experiments we assessed the effects of increasing concentrations of CsA and CsH on histamine release from human basophils induced by FMLP. The concentrations of cyclosporins used (8 to 800 nmol/L) included those reached in vivo during CsA treatment (8 to 300 nmol/L)37 and did not induce spontaneous lactate dehydrogenase or histamine release from basophils (data not shown). Fig. 1, A, shows the results from 11 experiments in which basophils were preincubated (5 minutes at

DISCUSSION

This study demonstrates that nanomolar concentrations of CsH exert a potent antiinflammatory effect by inhibiting the release of preformed and de novo synthesized chemical mediators from human basophils activated by FMLP. Our results are compatible with the hypothesis that the inhibitory effect of CsH on the release of mediators from basophils is mediated by the competitive interaction at the FMLP receptor level. The inhibitory effect of CsH is reversible and can be reversed by preincubation

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    From the Division of Clinical Immunology, School of Medicine, University of Naples Federico II.

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    Supported in part by grants from the National Research Council (C.N.R.) (Targeted Project F.A.T.M.A.; Subproject Prevention and Control of Disease Factors SP. 2; No. 94.00607.PF41 and 95.00856.PF41) and Ministero Università Ricerca Scientifica e Technologica, Rome, Italy.

    Reprint requests: Gianni Marone, MD, Division of Clinical Immunology and Allergy, University of Naples Federico II, School of Medicine, Via S. Pansini, 5, 80131 Napoli, Italy.

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