Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor a release by murine macrophages
Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor a release by murine macrophages
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DOI：
10.1074/jbc.m208742200
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发表时间：
2003-01-03
影响因子：
4.8
通讯作者：
Tsan, MF
中科院分区：
文献类型：
--
作者：
Gao, BC;Tsan, MF
利用市售的重组人热休克蛋白70（rhHsp 70），最近的研究表明rhHsp 70可以以类似于脂多糖（LPS）的方式诱导巨噬细胞和单核细胞产生肿瘤坏死因子α（TNF α），例如通过CD 14和Toll样受体4介导的信号转导途径。在目前的研究中，我们证明了LPS含量为1.4 μ g/mug的高度纯化的rhHsp 70制剂（命名为rhHsp 70 -1）在高达5 μ g/ml的浓度下不能诱导RAW 264.7小鼠巨噬细胞释放TNF α。相反，在LPS含量为0.2ng/μ g的1 μ g/ml的较低纯度的rhHsp 70制剂（命名为rhHsp 70 -2）能够诱导TNF α释放，其程度与0.2ng/ml LPS诱导的程度相同。rhHsp 70 -1未能诱导TNF α释放不是因为物理性质缺陷，因为rhHsp 70 -1和rhHsp 70 -2含有相同的hsp 70含量，如通过用考马斯蓝染色的SDS凝胶和用抗rhHsp 70抗体探测的蛋白质印迹所测定的。两种rhHsp 70制剂也具有相似的酶活性，如通过其从网格蛋白包被的囊泡中去除网格蛋白的能力所判断的。用多粘菌素B-琼脂糖柱去除rhHsp 70 -2中的LPS或直接向培养液中加入多粘菌素B基本上消除了rhHsp 70 -2的TNF α诱导活性。以rhHsp 70 - 2中发现的浓度向rhHsp 70 -1中添加LPS导致与用rhHsp 70 -2观察到的相同的TNF α诱导活性。rhHsp-2、单独LPS和LPS + rhHsp 70 -1的TNF α诱导活性对热灭活都同样敏感。这些结果表明rhHsp-70不诱导TNF α从鼠巨噬细胞释放，并且在rhHsp 70 -2制剂中观察到的TNF α诱导活性完全是由于污染的LPS。
Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor alpha (TNFalpha) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70-1) with a LPS content of 1.4 pg/mug was unable to induce TNFalpha release by RAW264.7 murine macrophages at concentrations up to 5 mug/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70-2) at 1 mug/ml with a LPS content of 0.2 ng/mug was able to induce TNFalpha release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70-1 to induce TNFalpha release was not because of defective physical properties since rhHsp70-1 and rhHsp70-2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70-2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFalpha-inducing activity of rhHsp70-2. The addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same TNFalpha-inducing activity as observed with rhHsp70-2. The TNFalpha-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70-1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFa release from murine macrophages and that the observed TNFa-inducing activity in the rhHsp70-2 preparation is entirely due to the contaminating LPS.