Claassen I J, Osterhaus A D, Claassen E
Laboratory for Control of biological products, National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands.
Eur J Immunol. 1995 May;25(5):1446-52. doi: 10.1002/eji.1830250547.
Several mechanisms have been postulated to explain the relatively high immunogenicity of antigens presented in immune-stimulating complexes (iscom). Their potency can in part be explained by the specific targeting of these structures to cells presenting antigens to the immune system. However, until now no method for the subcellular detection of iscom in situ was available. In the present study, a novel, fast and simple method for the detection of iscoms in situ is demonstrated. By making use of the lipophilic fluorescent carbocyanine dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), rabies virus antigen and iscom prepared with this antigen were visualized with fluorescence microscopy. The labeled antigen and iscoms were observed in macrophages of spleen and liver of mice within 1-2 h after intravenous administration. When administered intramuscularly or in the footpad, uptake in macrophages of draining lymph nodes could be demonstrated. In the spleen, labeled inactivated virus antigen localized preferentially in the marginal zone macrophages and to a lesser extent in the red pulp macrophages. In contrast, antigen presented in iscom was taken up mainly by the marginal metallophilic macrophages and to a much lesser extend by marginal zone macrophages or follicular-dendritic and -B cells. This method enables the detection of iscom and membrane viruses and allows the analysis of their relation to antigen-presenting cells in situ. Here, we demonstrate that iscom containing rabies virus antigen are taken up by a subset of macrophages in the spleen distinct from those that take up inactivated rabies virus antigen not presented in iscom, thereby possibly explaining the observed difference in immunogenicity of these antigen preparations. Furthermore, we show a lower efficiency on the induction of humoral and cellular responses after intravenous immunization for both types of antigen when compared with subcutaneous immunization.
人们已经提出了几种机制来解释免疫刺激复合物（iscom）中呈现的抗原具有相对较高免疫原性的原因。这些结构的效力部分可以通过其对向免疫系统呈递抗原的细胞的特异性靶向作用来解释。然而，到目前为止，还没有一种用于原位亚细胞检测iscom的方法。在本研究中，展示了一种用于原位检测iscom的新颖、快速且简单的方法。通过利用亲脂性荧光碳菁染料，即1,1'-二辛基-3,3,3',3'-四甲基吲哚碳菁高氯酸盐（DiI）和3,3'-二辛基氧杂碳菁高氯酸盐（DiO），用荧光显微镜观察了狂犬病病毒抗原以及用该抗原制备的iscom。静脉注射后1 - 2小时内在小鼠脾脏和肝脏的巨噬细胞中观察到了标记的抗原和iscom。当进行肌肉注射或足垫注射时，可以证明引流淋巴结的巨噬细胞有摄取。在脾脏中，标记的灭活病毒抗原优先定位于边缘区巨噬细胞，在红髓巨噬细胞中的定位程度较低。相比之下，iscom中呈现的抗原主要被边缘金属嗜性巨噬细胞摄取，而被边缘区巨噬细胞或滤泡树突状细胞及B细胞摄取的程度要小得多。该方法能够检测iscom和膜病毒，并允许原位分析它们与抗原呈递细胞的关系。在此，我们证明含有狂犬病病毒抗原的iscom被脾脏中一部分巨噬细胞摄取，这部分巨噬细胞与摄取未在iscom中呈现的灭活狂犬病病毒抗原的巨噬细胞不同，从而可能解释了这些抗原制剂在免疫原性上观察到的差异。此外，我们还表明，与皮下免疫相比，静脉免疫这两种抗原后诱导体液和细胞反应的效率较低。
