Lazaron V, Leslie D B, Wasiluk K R, Dunn D L
Department of Surgery, University of Minnesota, Minneapolis, USA.
Surgery. 2001 Aug;130(2):192-7. doi: 10.1067/msy.2001.115825.
Interaction between lipopolysaccharide (LPS), LPS-binding protein, and the CD14 receptor at the surface of LPS-responsive cells results in inflammatory cytokine release and internalization and detoxification of LPS. Monoclonal antibodies (mAbs) raised against the deep-core lipid A or the O-linked polysaccharide moieties of LPS accelerate internalization and detoxification of LPS without stimulating cytokine release. This study was conducted to test the hypothesis that the antibody-mediated internalization of LPS is an Fc receptor (FcR)--mediated process.
Fluoroisothiocyanate (FITC)-conjugated Escherichia coli O111:B4 LPS was incubated with RAW 264.7 cells and allowed to internalize for 2 hours in the presence and absence of anti-LPS, anti-CD14, and isotype control mAbs, and Fab fragments from the anti-CD14, anti--Fc receptor, and control mAbs. Tumor necrosis factor--alpha (TNF-alpha) release was measured by WEHI 164 cell bioassay. FITC-LPS uptake was measured by flow cytometry. Statistical analysis was by analysis of variance and Fisher exact test.
Addition of anti-LPS antibodies resulted in a 30- to 40-fold acceleration of LPS internalization (P <.01) in agreement with previous studies. This increase was blunted by anti-CD14 and also by isotype control holo-antibody (P <.01), but not by Fab fragments from anti-CD14 or isotype control antibody. Both anti-FcR antibodies and Fab fragments blocked anti-LPS antibody--stimulated uptake of FITC-LPS. Both intact anti-CD14 holo-antibody and Fab fragments blocked TNF-alpha release (P <.01).
Clearance and detoxification of LPS are thought to be essential to the host response to endotoxin. It has been shown that antibodies to LPS accelerate its internalization by monocytic cell lines without increasing the elaboration of cytokines. We found that specific blockade of CD14 by Fab fragments could block TNF-alpha release but not alter the accelerated internalization of LPS produced by anti-LPS antibodies. In contrast, a nonspecific blockade of internalization was produced by competing antibody, which suggests a mechanistic role for the FcR. Specific blockade of FcR by either holo-antibody or Fab fragments blocked accelerated internalization, which confirms a FcR mechanism. We conclude that the accelerated internalization of LPS produced by anti-LPS antibody is an Fc receptor--mediated process. These results have significance for the development of adjuvant immunotherapy for gram-negative bacterial sepsis.
脂多糖（LPS）、LPS结合蛋白与LPS反应性细胞表面的CD14受体之间的相互作用导致炎症细胞因子释放以及LPS的内化和解毒。针对LPS的深核脂质A或O-连接多糖部分产生的单克隆抗体（mAb）可加速LPS的内化和解毒，而不会刺激细胞因子释放。本研究旨在验证抗体介导的LPS内化是一种Fc受体（FcR）介导的过程这一假说。
将异硫氰酸荧光素（FITC）偶联的大肠杆菌O111:B4 LPS与RAW 264.7细胞孵育，并在存在和不存在抗LPS、抗CD14及同型对照mAb，以及抗CD14、抗Fc受体和对照mAb的Fab片段的情况下使其内化2小时。通过WEHI 164细胞生物测定法测量肿瘤坏死因子-α（TNF-α）的释放。通过流式细胞术测量FITC-LPS的摄取。采用方差分析和Fisher精确检验进行统计学分析。
与先前研究一致，添加抗LPS抗体可使LPS内化加速30至40倍（P<.01）。抗CD14以及同型对照全抗体可减弱这种增加（P<.01），但抗CD14或同型对照抗体的Fab片段则不会。抗FcR抗体及其Fab片段均能阻断抗LPS抗体刺激的FITC-LPS摄取。完整的抗CD14全抗体及其Fab片段均能阻断TNF-α的释放（P<.01）。
LPS的清除和解毒被认为是宿主对内毒素反应的关键。已表明针对LPS的抗体可加速单核细胞系对其的内化，而不会增加细胞因子的产生。我们发现Fab片段对CD14的特异性阻断可阻断TNF-α的释放，但不会改变抗LPS抗体所导致的LPS加速内化。相反，竞争性抗体产生了对内化的非特异性阻断，这提示了FcR的机制性作用。全抗体或Fab片段对FcR的特异性阻断可阻断加速内化，这证实了FcR机制。我们得出结论，抗LPS抗体所导致的LPS加速内化是一种Fc受体介导的过程。这些结果对于革兰氏阴性菌败血症辅助免疫治疗的发展具有重要意义。
