Weidemann B, Schletter J, Dziarski R, Kusumoto S, Stelter F, Rietschel E T, Flad H D, Ulmer A J
Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany.
Infect Immun. 1997 Mar;65(3):858-64. doi: 10.1128/IAI.65.3.858-864.1997.
Previously, we were able to show that soluble peptidoglycan (sPG)-induced monokine production in human peripheral monocytes is inhibited by anti-CD14 monoclonal antibodies and by lipid A partial structures. This suggested but did not prove that monocytic surface protein CD14 is involved in the activation of human monocytes not only by cell wall components of gram-negative bacteria such as lipopolysaccharide (LPS) but also by cell wall components of gram-positive bacteria such as sPG. In the present study, we provide experimental evidence that CD14 indeed constitutes a binding site for sPG recognition and activation of human monocytes. The results show that fluorescein isothiocyanate-sPG (FITC-sPG) binds to human monocytes in a saturable, dose-dependent, and specific manner. For maximal binding, 2 to 3 microg of FITC-sPG per ml was sufficient, and this binding is completed within 90 min; about 40% of the binding is completed within the first 3 min. The FITC-sPG binding is considered specific because unlabeled sPG and also muramyldipeptide (MDP), the minimal bioactive structure of sPG, inhibit the binding of sPG to monocytes in a dose-dependent manner. This specific binding was also inhibited by an anti-CD14 monoclonal antibody, LPS, and lipid A partial structure compound 406. Direct evidence for an interaction of sPG with CD14 is provided by experiments involving native polyacrylamide gel electrophoresis that showed a shift of the electrophoretic mobility of CD14 by LPS as well as by sPG. These results allow the conclusion that sPG binds directly to CD14, that MDP represents the active substructure of sPG, and that CD14 may be a lectin-like receptor which plays a key role in cellular stimulation by bioactive components of not only gram-negative but also gram-positive bacteria.
此前，我们已经能够证明，可溶性肽聚糖（sPG）诱导的人外周血单核细胞中的单核因子产生受到抗CD14单克隆抗体和脂多糖A部分结构的抑制。这表明但并未证明单核细胞表面蛋白CD14不仅参与革兰氏阴性菌细胞壁成分如脂多糖（LPS）对人单核细胞的激活，还参与革兰氏阳性菌细胞壁成分如sPG对人单核细胞的激活。在本研究中，我们提供了实验证据，证明CD14确实构成了sPG识别和激活人单核细胞的结合位点。结果表明，异硫氰酸荧光素标记的sPG（FITC-sPG）以饱和、剂量依赖性和特异性的方式与人单核细胞结合。为实现最大结合，每毫升2至3微克的FITC-sPG就足够了，且这种结合在90分钟内完成；约40%的结合在前3分钟内完成。FITC-sPG的结合被认为具有特异性，因为未标记的sPG以及sPG的最小生物活性结构胞壁酰二肽（MDP）均以剂量依赖性方式抑制sPG与单核细胞的结合。这种特异性结合也受到抗CD14单克隆抗体、LPS和脂多糖A部分结构化合物406的抑制。涉及天然聚丙烯酰胺凝胶电泳的实验提供了sPG与CD14相互作用的直接证据，该实验表明LPS以及sPG会使CD14的电泳迁移率发生改变。这些结果可以得出结论：sPG直接与CD14结合，MDP代表sPG的活性亚结构，并且CD14可能是一种凝集素样受体，它在不仅由革兰氏阴性菌而且由革兰氏阳性菌的生物活性成分引起的细胞刺激中起关键作用。
