Wurfel M M, Kunitake S T, Lichenstein H, Kane J P, Wright S D
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York 10021.
J Exp Med. 1994 Sep 1;180(3):1025-35. doi: 10.1084/jem.180.3.1025.
Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.
从正常人血浆中分离出的脂蛋白能够结合并中和细菌脂多糖（LPS），这可能是宿主抵御革兰氏阴性菌败血症休克的一种重要机制。最近的研究表明，实验性地提高循环高密度脂蛋白（HDL）水平可在动物内毒素休克模型中提供抗死亡保护。我们试图确定HDL中中和LPS所需的成分。为实现这一目标，我们使用一种快速检测方法研究了天然和重组HDL对LPS的功能中和作用，该方法可测量人中性粒细胞上CD14依赖性白细胞整合素的激活情况。我们在此报告，由纯化的载脂蛋白A-I（apoA-I）与磷脂和游离胆固醇组合制备的重组HDL颗粒（R-HDL）不足以中和LPS的生物活性。然而，添加重组LPS结合蛋白（LBP），一种已知可将LPS转移至CD14并增强细胞对LPS反应的蛋白质，可使R-HDL迅速结合并中和LPS。因此，LBP似乎不仅能够将LPS转移至CD14，还能转移至脂蛋白颗粒。与R-HDL相反，通过在抗apoA-I柱上进行选择性亲和免疫吸附（SAIS）从血浆中分离出的含apoA-I脂蛋白（LpA-I），无需添加外源性LBP即可中和LPS。多项证据表明LBP是血浆中LpA-I的一个组成部分。血浆通过抗apoA-I柱后，ELISA可检测到的LBP去除率超过99%，而通过柱洗脱可回收31%的LBP。同样，血浆使LPS激活中性粒细胞的能力（LBP/Septin活性）通过相同过程被耗尽并恢复。此外，固定化的抗LBP单克隆抗体可共沉淀apoA-I。此处描述的结果表明，除了将LPS转移至CD14的能力外，LBP还可能将LPS转移至脂蛋白。由于LBP似乎在血浆中与脂蛋白存在物理关联，它在LPS的中和过程中可能发挥重要作用。
