Lorton D
Hoover Arthritis Research Center, Sun Health Research Institute, Sun City, AZ 85372, USA.
Mech Ageing Dev. 1997 Mar;94(1-3):199-211. doi: 10.1016/s0047-6374(96)01847-7.
The proinflammatory cytokine, interleukin-1 (IL-1) is elevated in the Alzheimer's disease (AD) brain. Studies from our laboratory have demonstrated that beta-amyloid (A beta) 1-42, fibrillar A beta 1-40 and A beta 25-35 induce the release of IL-1 beta from activated THP-1 cells, a human monocyte cell line. A beta also is chemotactic for primary rodent microglia and peritoneal macrophages. We hypothesize that A beta is a chemokine and induces these responses by interaction with chemotactic receptors. If this is true, then these A beta-induced responses should be calcium-dependent and require activation of pertussis toxin-sensitive G-proteins. To test this hypothesis, THP-1 cells were grown in culture with lipopolysaccharide (LPS) and incubated with A beta 1-42 (5 muM) in the presence and absence of a calcium chelator, an inhibitor of intracellular calcium mobilization, a calcium channel blocker, or pertussis toxin, a bacterial endotoxin which uncouples G proteins from receptors by catalyzing the ADP ribosylation of cysteine near the carboxy-terminus of the alpha subunit. The media was collected and IL-1 beta present in the media was measured using an ELISA. Treatment of LPS-activated THP-1 cells with A beta 1-42 significantly elevated IL-1 beta released into the media as previously shown. Addition or ethylene glycol-bis (beta-aminothyl ether) N,N,N'N'-tetraacetic acid (EGTA) (0.5 mM), a calcium chelator, to the media blocked A beta-induced IL-1 beta release, but had no effect on LPS-activated THP-1 cell release of IL-1 beta. The presence of 3,4,5-trimethoxybenzoic acid 8-(diethyl amino)-octyl ester (TMB-8), an inhibitor of intracellular calcium mobilization, as well as nickel chloride, a non-specific calcium channel blocker, in the media also inhibited A beta-induced IL-1 release from LPS-activated THP-1 cells. IL- 1 beta release from activated THP-1 monocytes incubated with TMB-8 and nickel chloride without A beta remained at baseline values. Pretreatment of THP-1 monocytes with pertussis toxin for 4 h, followed by LPS activation and incubation with A beta, antagonized the release of IL-1 beta from these cells, but did not alter IL-1 beta release from activated THP-1 monocytes. These data suggest that A beta-induced IL-1 beta release from these cells is calcium-dependent and requires the activation of specific G-proteins. These findings are consistent with known second messengers that are activated following stimulation of chemotactic receptors.
促炎细胞因子白细胞介素-1（IL-1）在阿尔茨海默病（AD）脑内水平升高。我们实验室的研究表明，β-淀粉样蛋白（Aβ）1-42、纤维状Aβ1-40和Aβ25-35可诱导人单核细胞系活化的THP-1细胞释放IL-1β。Aβ对原代啮齿动物小胶质细胞和腹腔巨噬细胞也具有趋化作用。我们推测Aβ是一种趋化因子，并通过与趋化受体相互作用诱导这些反应。如果这一推测正确，那么这些Aβ诱导的反应应依赖于钙，并需要百日咳毒素敏感的G蛋白激活。为验证这一假设，将THP-1细胞与脂多糖（LPS）共同培养，并在存在和不存在钙螯合剂、细胞内钙动员抑制剂、钙通道阻滞剂或百日咳毒素（一种细菌内毒素，通过催化α亚基羧基末端附近半胱氨酸的ADP核糖基化使G蛋白与受体解偶联）的情况下，与Aβ1-42（5μM）孵育。收集培养基，使用酶联免疫吸附测定法（ELISA）检测培养基中存在的IL-1β。如先前所示，用Aβ1-42处理LPS激活的THP-1细胞可显著提高释放到培养基中的IL-1β水平。向培养基中添加乙二醇双（β-氨基乙基醚）N，N，N'，N'-四乙酸（EGTA）（0.5 mM），一种钙螯合剂，可阻断Aβ诱导的IL-1β释放，但对LPS激活的THP-1细胞释放IL-1β没有影响。培养基中存在细胞内钙动员抑制剂3,4,5-三甲氧基苯甲酸8-（二乙氨基）辛酯（TMB-8）以及非特异性钙通道阻滞剂氯化镍，也可抑制LPS激活的THP-1细胞中Aβ诱导的IL-1释放。在没有Aβ的情况下，用TMB-8和氯化镍孵育的活化THP-1单核细胞释放的IL-1β保持在基线水平。用百日咳毒素预处理THP-1单核细胞4小时，随后进行LPS激活并与Aβ孵育，可拮抗这些细胞释放IL-1β，但不改变活化的THP-1单核细胞释放IL-1β。这些数据表明，Aβ诱导这些细胞释放IL-1β依赖于钙，并需要特定G蛋白的激活。这些发现与趋化受体刺激后激活的已知第二信使一致。
