Potent immunosuppression by oxidized cholesterol.
Potent immunosuppression by oxidized cholesterol.
复制标题
氧化胆固醇产生有效的免疫抑制作用。
DOI：
10.4049/jimmunol.122.1.121
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发表时间：
1979
影响因子：
4.4
通讯作者：
H. Mcconnell
中科院分区：
文献类型：
--
作者：
G. M. Humphries;H. Mcconnell
当与小鼠脾细胞一起培养4天时，与ε-二硝基苯基赖氨酸共价连接的Sepharose 4 B表现为体外T非依赖性抗原。当由二肉豆蔻酰磷脂酰胆碱（DMPC）和0.05 μ mol重结晶但老化胆固醇的特定样品组成的脂质体与107个活脾细胞培养时，几乎完全抑制空斑形成细胞（PFC）反应;当使用仅由DMPC组成的脂质体时，无明显抑制作用。用二氯甲烷在SephadexLH 20上纯化后，胆固醇不再具有抑制作用。通过各种形式的色谱分离，连同生物测定，已导致的结论是，在不纯的胆固醇的主要抑制成分是25-氢过氧胆固醇，胆固醇的氧化产物，其在硅胶上的Rf，在几种溶剂系统中，是非常接近的胆固醇。过氧化物可以通过代谢或NaBH 4的作用被还原，产生25-羟基胆固醇，这是一种至少与其前体一样具有免疫抑制作用的化合物。据估计，25-氢过氧胆固醇以略低于1%的水平存在于不纯胆固醇样品中。
由DMPC：纯化胆固醇：25-羟基胆固醇（摩尔比100：100：1）组成的脂质体已被用于在更好定义的系统中研究该现象。这种脂质体的生物活性与含有DMPC：不纯胆固醇（摩尔比1：1）的脂质体的生物活性基本相同。当在培养的第一个24小时加入脂质体时，脂质体具有高度的抑制作用，但在接下来的24小时内，系统变得对抑制作用不那么敏感。到培养的第3天，即当可测量的PFC的快速发生变得明显时，细胞不再对25-羟基胆固醇的抑制作用敏感。
在25-羟基胆固醇的浓度下，PFC反应几乎完全被抑制，如果化合物是单分子分散体（即，0.5纳摩尔/1.0 ml培养体积）。在该浓度下，每个最初培养的活细胞中有3 × 107个25-羟基胆固醇分子，并且在测定时细胞的活力与不含脂质体的对照品无显著差异。氢化可的松对绵羊红细胞（SRBC）的体外主要反应的抑制发生在该类固醇的最低浓度大致相同的情况下。
众所周知，25-羟基胆固醇是3-羟基-3-甲基戊二酰辅酶A（HMG CoA）还原酶活性的有效抑制剂，因此抑制生物合成途径中醋酸盐和甲羟戊酸其他前体的胆固醇合成;然而，甲羟戊酸并不能消除含有25-羟基胆固醇的脂质体对PFC反应的抑制作用。在不存在脂质体的情况下，PFC对二硝基苯化珠粒的反应在存在甲羟戊酸的情况下比在不存在甲羟戊酸的情况下显著且可再现地更大。这表明细胞能够同化外源甲羟戊酸。如果这是真的，25-羟基胆固醇对PFC的抑制作用是通过其对HMG CoA还原酶活性本身的作用介导的假设是站不住脚的。
Sepharose 4B to which ε-dinitrophenyl lysine has been covalently attached behaves as an in vitro T-independent antigen when cultured for 4 days with mouse spleen cells. Almost total inhibition of the plaque-forming cell (PFC) response is obtained when liposomes composed of dimyristoylphosphatidylcholine (DMPC) and 0.05 μmole of a particular sample of recrystallized but aged cholesterol are cultured with 107 viable spleen cells; no inhibition is apparent when liposomes composed only of DMPC are used. After purification on Sephadex LH 20 with methylene dichloride, the cholesterol is no longer inhibitory. Fractionation by various forms of chromatography, together with biologic assays, has led to the conclusion that the major inhibitory component in the impure cholesterol is 25-hydroperoxycholesterol, an oxidation product of cholesterol whose Rf on silica gel, in several solvent systems, is very close to that of cholesterol. The peroxide can be reduced metabolically, or by the action of NaBH4, to give 25-hydroxycholesterol, a compound that is at least as immunosuppressive as its precursor. It is estimated that 25-hydroperoxycholesterol is present in the impure cholesterol sample at a level slightly less than 1%.
Liposomes composed of DMPC:purified cholesterol:25-hydroxycholesterol (molor ratio 100:100:1) have been used to study the phenomenon in a better defined system. The biologic activity of such liposomes is essentially identical to that of liposomes containing DMPC:impure cholesterol (molar ratio 1:1). The liposomes are highly inhibitory when added in the first 24 hr of culture, but the system becomes rapidly less susceptible to inhibition during the next 24 hr. By the 3rd day of culture, which is when a rapid onset of measurable PFC becomes apparent, the cells are no longer susceptible to inhibition by 25-hydroxycholesterol.
Almost total inhibition of the PFC response is obtained at a concentration of 25-hydroxycholesterol, which would be expressed as 5 × 10-7 M if the compound were in monomolecular dispersion (i.e., 0.5 nanomoles/1.0 ml culture volume). At this concentration there are 3 × 107 molecules of 25-hydroxycholesterol per viable cell originally cultured and the viability of the cells at the time of assay is not significantly different from that of controls lacking liposomes. Suppression of the in vitro primary response to sheep red blood cells (SRBC) by hydrocortisone occurs at about the same minimum concentration of that steroid.
It is well known that 25-hydroxycholesterol is a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and hence inhibits cholesterol synthesis from acetate and other precursors of mevalonate in the biosynthetic pathway; however, mevalonate does not abrogate the inhibition of the PFC response by liposomes containing 25-hydroxycholesterol. In the absence of liposomes, the PFC response to dinitrophenylated beads is significantly and reproductibly greater in the presence of mevalonate than it is in its absence. This suggests that the cells are capable of assimilating the exogenous mevalonate. If this is true, a hypothesis that PFC suppression by 25-hydroxycholesterol is mediated by its action on HMG CoA reductase activity per se is not tenable.