Kobzik L, Godleski J J, Brain J D
Respiratory Biology Program, Harvard School of Public Health, Boston, MA.
J Immunol. 1990 Jun 1;144(11):4312-9.
We have compared the oxidative response of alveolar macrophages (AM) during opsonin-dependent and independent phagocytosis by using multiparameter flow cytometry. The respiratory burst of AM during phagocytosis was quantitated by the intracellular oxidation of the nonfluorescent precursors dichlorofluorescin diacetate (DCFH) or hydroethidine (HE, a reduced precursor of ethidium) to their fluorescent (oxidized) counterparts. After loading freshly isolated normal hamster AM with DCFH or HE, red or green fluorescent beads, respectively, were added to the shaking cell suspensions. Ingestion of opsonized particles by AM caused a marked increase in oxidation of both DCFH and HE proportional to the number of beads ingested. In contrast, uptake of one to three unopsonized particles per cell led to inhibition of oxidative activity compared to control cells incubated without particles. AM ingesting four or more unopsonized particles showed some increase in oxidative metabolism, but far less than that with identical numbers of particles in opsonin-dependent ingestion. Similar results were obtained using fluorescent labeled staphylococcal bacteria. Using three-color flow cytometry to study cells ingesting both types of particles, cells first ingesting unopsonized beads were also found to have an inhibited oxidative response to subsequently ingested opsonized particles. The mitochondrial poison antimycin inhibited most of the intracellular oxidative response to either type of phagocytosis. The remaining antimycin-insensitive, membrane derived respiratory burst of AM was also substantially diminished after phagocytosis of unopsonized particles vs similar numbers of opsonized particles. The greatly increased mitochondrial respiration in AM during phagocytosis of opsonized particles may be related to bactericidal mechanisms. Killing of ingested Staphylococcus by AM was markedly impaired in the presence of antimycin. The results suggest that AM may ingest the numerous, unopsonized inert particles that are inhaled without generation of potentially toxic oxygen metabolites, while retaining the capacity to undergo a respiratory burst after ingesting opsonized particles and bacteria. The mechanism(s) for this distinct response may include generation of an inhibitor of intracellular oxidative metabolism.
我们通过多参数流式细胞术比较了肺泡巨噬细胞（AM）在调理素依赖性和非依赖性吞噬过程中的氧化反应。吞噬过程中AM的呼吸爆发通过非荧光前体二氯荧光素二乙酸酯（DCFH）或氢乙锭（HE，乙锭的还原前体）在细胞内氧化为其荧光（氧化）对应物来定量。用DCFH或HE分别加载新鲜分离的正常仓鼠AM后，将红色或绿色荧光珠添加到振荡的细胞悬液中。AM摄取调理过的颗粒会导致DCFH和HE的氧化显著增加，与摄取的珠子数量成比例。相比之下，与未与颗粒一起孵育的对照细胞相比，每个细胞摄取一到三个未调理的颗粒会导致氧化活性受到抑制。摄取四个或更多未调理颗粒的AM显示氧化代谢有一些增加，但远低于调理素依赖性摄取中相同数量颗粒的情况。使用荧光标记的葡萄球菌也获得了类似的结果。使用三色流式细胞术研究摄取两种类型颗粒的细胞时，还发现首先摄取未调理珠子的细胞对随后摄取的调理过的颗粒的氧化反应受到抑制。线粒体毒物抗霉素抑制了对任何一种吞噬类型的大部分细胞内氧化反应。与摄取类似数量的调理过的颗粒相比，摄取未调理的颗粒后，AM剩余的对抗霉素不敏感的、源自膜的呼吸爆发也显著减少。在摄取调理过的颗粒过程中，AM中线粒体呼吸的大幅增加可能与杀菌机制有关。在抗霉素存在的情况下，AM对摄取的葡萄球菌的杀伤明显受损。结果表明，AM可能摄取吸入的大量未调理的惰性颗粒而不产生潜在有毒的氧代谢物，同时保留摄取调理过的颗粒和细菌后发生呼吸爆发的能力。这种不同反应的机制可能包括产生细胞内氧化代谢的抑制剂。
