Chaka W, Verheul A F, Vaishnav V V, Cherniak R, Scharringa J, Verhoef J, Snippe H, Hoepelman A I
Division of Vaccines, University Hospital Utrecht, The Netherlands.
J Immunol. 1997 Sep 15;159(6):2979-85.
We have shown previously that specific receptors on PBMCs and a serum factor other than Ab and complement are involved in the TNF-alpha response to cryptococcal mannoprotein (MP2). To characterize the mechanism of MP2 recognition by PBMCs, 10(6) PBMCs were incubated with 25 microg of FITC-labeled MP2 in 10% normal human serum (1 h). The cells were analyzed by flow cytometry. FITC-MP2 binding was CaCl2 and temperature dependent and was enhanced by prestimulating PBMCs with unlabeled MP2. Binding to PBMCs was specific, since unlabeled MP and mannan produced dose-dependent inhibition. Beta-Glucan laminarin produced background inhibition. mAbs against CD14, CD11b, and CD18 did not prevent FITC-MP2 binding to PBMCs, implying that these receptors are not involved in MP2 recognition by PBMCs. mAb against CD14 blocked (>90%) MP2-induced TNF-alpha release by PBMCs, while mAbs against CD11b/CD18 caused no inhibition. Removal of human mannose binding protein (hMBP) by preincubation of serum with a specific mAb abrogated TNF-alpha induction by MP2 and strongly inhibited its binding to PBMCs. Recombinant hMBP enhanced TNF-alpha induction by MP2 as well as binding of FITC-MP2 to PBMCs. In addition, incubation of serum with MP2-coated beads and analysis by SDS-PAGE resulted in the detection of a protein of approximately 33/34 kDa that could be partially removed by preincubating the serum with hMBP mAb. We conclude that hMBP is involved in the binding of MP2 to PBMCs and the release of TNF-alpha.
我们之前已经表明，PBMCs上的特定受体以及除抗体和补体之外的一种血清因子参与了对隐球菌甘露糖蛋白（MP2）的TNF-α反应。为了表征PBMCs识别MP2的机制，将10⁶个PBMCs与25μg FITC标记的MP2在10%正常人血清中孵育（1小时）。通过流式细胞术分析细胞。FITC-MP2结合是CaCl₂和温度依赖性的，并且通过用未标记的MP2预刺激PBMCs而增强。与PBMCs的结合是特异性的，因为未标记的MP和甘露聚糖产生剂量依赖性抑制。β-葡聚糖海带多糖产生背景抑制。抗CD14、CD11b和CD18的单克隆抗体不能阻止FITC-MP2与PBMCs的结合，这意味着这些受体不参与PBMCs对MP2的识别。抗CD14的单克隆抗体阻断（>90%）PBMCs由MP2诱导的TNF-α释放，而抗CD11b/CD18的单克隆抗体没有抑制作用。通过用特异性单克隆抗体预孵育血清去除人甘露糖结合蛋白（hMBP）消除了MP2诱导的TNF-α，并强烈抑制其与PBMCs的结合。重组hMBP增强了MP2诱导的TNF-α以及FITC-MP2与PBMCs的结合。此外，将血清与MP2包被的珠子孵育并通过SDS-PAGE分析导致检测到一种约33/34 kDa的蛋白质，该蛋白质可以通过用hMBP单克隆抗体预孵育血清而部分去除。我们得出结论，hMBP参与了MP2与PBMCs的结合以及TNF-α的释放。
