Gröndahl G, Johannisson A, Jensen-Waern M, Nilsson Ekdahl K
Department of Large Animal Clinical Sciences, Swedish University of Agricultural Sciences, P.O. Box 7018, S-750 07 Uppsala, Sweden.
Vet Immunol Immunopathol. 2001 Aug 10;80(3-4):209-23. doi: 10.1016/s0165-2427(01)00262-8.
The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells (Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS-PAGE and Western blot analyses. Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3-6-fold, indicating spatial competition between C3 and IgG at binding. Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With > or =6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents. It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.
血清中的主要调理素是抗体和补体因子C3。包括补体激活和沉积在内的调理机制在吞噬作用和微生物免疫逃逸机制的研究中很重要。本研究的目的是使用流式细胞术免疫测定法监测马血清中的补体C3和IgG在酵母细胞（酿酒酵母）上的沉积情况。使用马血中性粒细胞对调理包被与吞噬能力之间进行相关性分析。此外，通过SDS-PAGE和蛋白质印迹分析对结合的C3片段进行表征。马C3和IgG对酵母的调理包被反应迅速，血清浓度低至0.75%时即可检测到。C3沉积是补体激活的结果，未观察到被动吸附现象。当补体失活时，指示IgG沉积的荧光增加3至6倍，表明C3和IgG在结合时存在空间竞争。用1.5%的血清进行调理导致酵母细胞的马中性粒细胞吞噬作用欠佳，这取决于经典途径的补体激活。血清浓度≥6.25%时，IgG有助于调理和吞噬作用。血清浓度为50%及更高时，C3也可通过替代途径沉积。血清浓度为3%时吞噬率达到最佳，血清浓度更高时吞噬率不再进一步增加。酵母细胞上C3的主要形式是iC3b，其余为C3b，未检测到任何裂解产物（C3c或C3dg）。马补体成分的大小与人补体成分相似。可以得出结论，导致吞噬作用的酵母颗粒调理作用在非常低的血清浓度（1.5%）下即可发生，并且在这种低调理水平下依赖于经典补体途径的激活。这对于有效的宿主防御，例如感染部位的血管外吞噬作用，是一项重要发现。
