Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. Requirement for plasma but not for tumor necrosis factor-alpha.
Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. Requirement for plasma but not for tumor necrosis factor-alpha.
复制标题
脂多糖启动中性粒细胞以增强超氧化物的释放。
作者：
Y. Aida;M. Pabst
当人中性粒细胞与LPS孵育时，它们响应于FMLP的刺激而被激发以增强O2-的释放。我们研究了LPS引发的两个方面：1）引发是否依赖于中性粒细胞制剂中存在的单核细胞分泌TNF-α，以及2）引发是否需要血浆。使用血浆Percoll梯度，我们分离出仅含0.1%单核细胞的中性粒细胞。在37摄氏度，这些中性粒细胞显着引发LPS（100 ng/ml）在30分钟内。相反，LPS处理的单核细胞需要60分钟分泌显着的嗜中性粒细胞引发活性，其中的主要成分是TNF-α。此外，针对TNF-α的抗体在15、30和45分钟时未能抑制LPS对中性粒细胞的引发，并且在60分钟时仅抑制15%。结果表明，TNF-α或单核细胞的其他因子对于LPS对中性粒细胞的引发不是必需的。通过50%Percoll离心去除血浆的中性粒细胞对LPS的反应很弱，这与引发增强的O2释放和细胞表面碱性磷酸酶活性的表达有关。通过回加血浆（0.1 - 1.0%）可恢复洗涤中性粒细胞的预充。将等离子体加热到56摄氏度并不能阻断等离子体的这种效应，但在100摄氏度时会阻断。多粘菌素B可阻断LPS的促发作用，即使在血浆存在的情况下也是如此。因此，预充需要LPS和血浆。在不存在血浆的情况下与LPS孵育的中性粒细胞未通过随后添加血浆引发，但通过添加血浆和LPS引发。在不存在血浆的情况下，用20 ng/ml LPS孵育的中性粒细胞的培养物上清液不能引发新鲜的中性粒细胞，但在存在1%血浆的情况下，用LPS孵育的中性粒细胞的上清液能够引发新鲜的中性粒细胞。这些结果表明，中性粒细胞灭活LPS和血浆保护LPS从灭活。然而，这种灭活的LPS保留了在10 pg/ml下使鲎溶解物凝胶化的能力和在100 pg/ml下引发单核细胞的能力。因此，血浆阻止LPS的嗜中性粒细胞特异性失活。
When human neutrophils are incubated with LPS, they become primed for enhanced release of O2- in response to stimulation by FMLP. We investigated two aspects of LPS priming: 1) whether priming depends on secretion of TNF-alpha by monocytes present in neutrophil preparations, and 2) whether plasma is required for priming. Using plasma-Percoll gradients, we isolated neutrophils that contained only 0.1% monocytes. At 37 degrees C, these neutrophils were significantly primed by LPS (100 ng/ml) within 30 min. In contrast, LPS-treated monocytes required 60 min to secrete significant neutrophil-priming activity, the major component of which was TNF-alpha. Further, antibody against TNF-alpha failed to inhibit priming of neutrophils by LPS at 15, 30, and 45 min, and inhibited only 15% at 60 min. The results suggested that TNF-alpha or other factors from monocytes were not essential for priming of neutrophils by LPS. Neutrophils that had been washed free of plasma by centrifugation through 50% Percoll responded only weakly to LPS with respect to priming for enhanced O2- release and increased expression of alkaline phosphatase activity on the cell surface. Priming of washed neutrophils could be restored by adding back plasma (0.1 to 1.0%). This effect of plasma was not blocked by heating the plasma to 56 degrees C but was blocked at 100 degrees C. LPS priming could be blocked by polymyxin B, even in the presence of plasma. Thus, priming required both LPS and plasma. Neutrophils incubated with LPS in the absence of plasma were not primed by subsequent addition of plasma, but were primed by addition of plasma and LPS. Culture supernatants from neutrophils incubated with 20 ng/ml LPS in the absence of plasma failed to prime fresh neutrophils, but supernatants from neutrophils incubated with LPS in the presence of 1% plasma were able to prime fresh neutrophils. These results implied that neutrophils inactivated LPS and that plasma protected LPS from inactivation. Nevertheless, such inactivated LPS retained the ability to gel Limulus lysate at 10 pg/ml, and the ability to prime monocytes at 100 pg/ml. Thus, plasma prevented a neutrophil-specific inactivation of LPS.