Cyclosporin 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 cyclophilin, a critical step in the molecular mechanism of action of cyclosporins, where 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 it D-isomer.


METHODS
We compared the effects of CsA and CsH on the release of performed (histamine) and de novo synthesized inflammatory mediators (peptide leukotriene C4) 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 C4 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-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 dissociation constant (Kd) for the CsH-FMLP receptor complex was approximately 9 x 10(-8) mol/L, more than 10-fold lower than that (approximately equal 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 FMLP binding to human polymorphonuclear leukocytes with a concentration required to inhibit binding by 50% of approximately 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. Scatchard analysis revealed that the decreased tritiated FMLP binding caused by CsH was due to a decrease in the Bmax (0.22 +/- 0.04 nmol/L/5 x 10(6) cells vs 0.09 +/- 0.01 nmol/L/5 x 10(6) cells; p < 0.05), without a significant difference in the Kd (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.
de Paulis
A
A
Division of Clinical Immunology, School of Medicine, University of Naples Federico II, Italy.
Ciccarelli
A
A
de Crescenzo
G
G
Cirillo
R
R
Patella
V
V
Marone
G
G
D001491:Basophils / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D001667:Binding, Competitive / Q000276:immunology; D054713:Bryostatins; D018932:Chemokine CCL2 / Q000494:pharmacology; D018946:Chemokine CCL5 / Q000494:pharmacology; D015936:Complement C5a / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology*; D006636:Histamine Release / Q000187:drug effects*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000494:pharmacology; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D017997:Leukotriene C4 / Q000096:biosynthesis; D018942:Macrolides; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D054713:Bryostatins; D018932:Chemokine CCL2; D018946:Chemokine CCL5; D007375:Interleukin-1; D016209:Interleukin-8; D007783:Lactones; D018942:Macrolides; D009842:Oligopeptides; D010972:Platelet Activating Factor; D017997:Leukotriene C4; C046785:bryostatin 1; D009240:N-Formylmethionine Leucyl-Phenylalanine; C046832:t-butyloxycarbonyl-methionyl-leucyl-phenylalanine; D015936:Complement C5a; D016572:Cyclosporine; C050025:cyclosporin H; D013755:Tetradecanoylphorbol Acetate