CYCLOSPORINE-H IS A POTENT AND SELECTIVE COMPETITIVE ANTAGONIST OF HUMAN BASOPHIL ACTIVATION BY N-FORMYL-METHIONYL-LEUCYL-PHENYLALANINE

Background: Cyclosporin A (CsA) binds with high affinity to cyclophili n, a critical step in the molecular mechanism of action of cyclosporin s, whereas cyclosporin H (CsH) has extremely low affinity for cyclophi lin. CsH differs from CsA by the substitution of the L-methyl valine a t position 11 with its D-isomer.Methods: We compared the effects of Cs A and CsH on the release of preformed (histamine) and de novo synthesi zed inflammatory mediators (peptide leukotriene C-4) from peripheral b lood basophils activated by N-formyl-methionyl-leucyl-phenylalanine (F MLP). Results: CsH (8 to 800 nmol/L) concentration-dependently inhibit ed 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 F MLP 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 re lease of histamine caused by C5a, platelet activating factor; monocyte chemotactic activating factor, RANTES, IL-8, bryostatin 1, and phorbo l myristate. Preincubation of basophils with granulocyte-macrophage co lony-stimulating factor (30 and 100 pmol/L), but not IL-1 beta (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 dissociati on constant (K-d) for the CsH-FMLP receptor complex was approximately 9 x 10(-8) mol/L, more than 10-fold lower than that (congruent to 1.3 x 10(-6) mol/L, of N-t-BOC-methionyl-L-leucyl-phenylalanine (BocMLP), a known formyl peptide, receptor antagonist. CsH inhibited tritiated F MLP binding to human polymorphonuclear leukocytes with a concentration required to inhibit binding by 50% of approximately lately 5.4 x 10(- 7) mol/L, whereas BocMLP was less potent with a concentration required to inhibit binding by 50% of approximately 9.1 x 10(-5) mol/L. Scatch ard 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 x 1 0(6) cells vs 0.09 +/- 0.01 nmol/L/5 x 10(6) 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 inhi bitor of mediator release from basophils induced by activation of the formyl peptide receptor; if acts by interfering with agonist binding t o FMLP receptors.