Opsonization of yeast cells with equine iC3b, C3b, and IgG

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 ≥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.     

日本血吸虫保护性免疫机制的研究——活化巨噬细胞和超敏反应测定

本文用辐照致弱日本血吸虫童虫疫苗,冷冻辐照童虫苗和速冻致死童虫苗免疫雌性C57-BL/6小鼠。免疫后对小鼠腹腔巨噬细胞(MФ)的吞噬杀伤活性和超敏反应进行动态测定。结果表明2个活苗免疫组小鼠腹腔MФ体外表现出强烈杀伤童虫活性,杀伤力显著强于死苗免疫组和对照组(P<0.01);免疫后第6周杀伤力达峰值,第8周杀伤力下降;MФ杀伤力与保护力存在显著正相关(r=0.630,r>r0.05);活化MФ在免疫血清调理后杀伤活力有所增加,而免疫血清对于未活化MФ则无调理作用;活苗和死苗免疫鼠都出现强烈的速发型超敏反应,但与保护力之间缺乏相关性;迟发型超敏反应在本次实验中出现较弱。