Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
Anal Biochem. 2011 Jun 15;413(2):185-91. doi: 10.1016/j.ab.2011.02.031. Epub 2011 Feb 24.
Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G-fluorescein isothiocyanate (IgG-FITC) for flow cytometry or with anti-rabbit IgG-peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with (125)I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1-100ng/ml LPS; however, it was reduced by approximately 30-40% after 2 h of treatment with 1 μg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.
脂多糖（LPS）被巨噬细胞的 Toll 样受体 4（TLR4）识别，触发促炎介质的产生。决定对 LPS 反应幅度的因素之一，甚至可能导致危及生命的感染性休克，是 TLR4 在细胞表面的丰度。然而，由于受体表达水平低，TLR4 的表面定量很困难。为了开发 TLR4 评估方法，我们用兔抗体标记细胞表面上的受体，然后用抗兔免疫球蛋白 G-荧光素异硫氰酸酯（IgG-FITC）进行流式细胞术，或用抗兔 IgG-过氧化物酶进行细胞酶联免疫吸附试验（ELISA）。或者，用（125）I 标记的抗兔 IgG 检测抗-TLR4 抗体。流式细胞术不能检测 J774 细胞或人巨噬细胞表面的 TLR4。相比之下，应用细胞 ELISA 或放射性标记技术结合抗体非特异性结合的有效阻断，提供了 TLR4 特异性信号。J774 细胞表面 TLR4 的水平在用 1-100ng/ml LPS 处理时没有变化；然而，在用 1μg/ml LPS 处理 2 小时后，其水平降低了约 30-40%。这些数据表明，表面 TLR4 的下调可以作为对高剂量 LPS 细胞反应的负调节手段。
