环孢菌素H是一种强效且具有选择性的竞争性拮抗剂，可抑制N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸对人嗜碱性粒细胞的激活作用。

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

作者信息

de Paulis A, Ciccarelli A, de Crescenzo G, Cirillo R, Patella V, Marone G

机构信息

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

出版信息

J Allergy Clin Immunol. 1996 Jul;98(1):152-64. doi: 10.1016/s0091-6749(96)70237-3.

DOI:10.1016/s0091-6749(96)70237-3

PMID:8765829

Abstract

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).

CONCLUSION

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.

摘要

背景

环孢素A（CsA）与亲环蛋白具有高亲和力结合，这是环孢素分子作用机制中的关键步骤，而环孢素H（CsH）对亲环蛋白的亲和力极低。CsH与CsA的不同之处在于其11位的L-甲基缬氨酸被D-异构体取代。

方法

我们比较了CsA和CsH对经N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（FMLP）激活的外周血嗜碱性粒细胞释放已生成的（组胺）和新合成的炎症介质（肽白三烯C4）的影响。

结果

CsH（8至800 nmol/L）浓度依赖性地抑制FMLP激活的纯化和未纯化嗜碱性粒细胞释放组胺和白三烯C4，而CsA（8至800 nmol/L）对FMLP刺激的嗜碱性粒细胞释放组胺几乎没有抑制作用。FMLP刺激的嗜碱性粒细胞释放组胺的抑制作用极其迅速，且在刺激前洗涤细胞（三次）可消除该抑制作用。CsH（8至800 nmol/L）对C5a、血小板活化因子、单核细胞趋化激活因子、RANTES、IL-8、苔藓抑素1和佛波酯肉豆蔻酸酯引起的组胺释放无影响。嗜碱性粒细胞与粒细胞-巨噬细胞集落刺激因子（30和100 pmol/L）预孵育，但与IL-1β（30和100 ng/ml）预孵育则不能，可浓度依赖性地逆转CsH对FMLP诱导的组胺释放的抑制作用。CsH竞争性抑制FMLP对嗜碱性粒细胞组胺释放的作用。CsH-FMLP受体复合物的解离常数（Kd）约为9×10⁻⁸mol/L，比已知的甲酰肽受体拮抗剂N-叔丁氧羰基-甲硫氨酰-L-亮氨酰-苯丙氨酸（BocMLP）的解离常数（约等于1.3×10⁻⁶mol/L）低10倍以上。CsH抑制氚标记的FMLP与人多形核白细胞的结合，抑制50%结合所需的浓度约为5.4×10⁻⁷mol/L，而BocMLP的效力较低，抑制50%结合所需的浓度约为9.1×10⁻⁵mol/L。Scatchard分析显示，CsH引起的氚标记FMLP结合减少是由于Bmax降低（0.22±0.04 nmol/L/5×10⁶细胞对0.09±0.01 nmol/L/5×10⁶细胞；p<0.05），而Kd无显著差异（5.16±1.22 nmol/L对6.32±2.42 nmol/L；p=NS）。