Department of Biomedical Engineering, University of California Davis, Davis, California, USA.
PLoS One. 2013;8(1):e54735. doi: 10.1371/journal.pone.0054735. Epub 2013 Jan 22.
Neutrophils, in cooperation with serum, are vital gatekeepers of a host's microbiome and frontline defenders against invading microbes. Yet because human neutrophils are not amenable to many biological techniques, the mechanisms governing their immunological functions remain poorly understood. We here combine state-of-the-art single-cell experiments with flow cytometry to examine how temperature-dependent heat treatment of serum affects human neutrophil interactions with "target" particles of the fungal model zymosan. Assessing separately both the chemotactic as well as the phagocytic neutrophil responses to zymosan, we find that serum heat treatment modulates these responses in a differential manner. Whereas serum treatment at 52°C impairs almost all chemotactic activity and reduces cell-target adhesion, neutrophils still readily engulf target particles that are maneuvered into contact with the cell surface under the same conditions. Higher serum-treatment temperatures gradually suppress phagocytosis even after enforced cell-target contact. Using fluorescent staining, we correlate the observed cell behavior with the amounts of C3b and IgG deposited on the zymosan surface in sera treated at the respective temperatures. This comparison not only affirms the critical role of complement in chemotactic and adhesive neutrophil interactions with fungal surfaces, but also unmasks an important participation of IgGs in the phagocytosis of yeast-like fungal particles. In summary, this study presents new insight into fundamental immune mechanisms, including the chemotactic recruitment of immune cells, the adhesive capacity of cell-surface receptors, the role of IgGs in fungal recognition, and the opsonin-dependent phagocytosis morphology of human neutrophils. Moreover, we show how, by fine-tuning the heat treatment of serum, one can selectively study chemotaxis or phagocytosis under otherwise identical conditions. These results not only refine our understanding of a widely used laboratory method, they also establish a basis for new applications of this method.
中性粒细胞与血清协同作用，是宿主微生物组的重要守门员，也是抵御入侵微生物的一线防御者。然而，由于人类中性粒细胞不适用于许多生物学技术，因此其免疫功能的调控机制仍知之甚少。我们结合最先进的单细胞实验和流式细胞术，研究了血清的温度依赖性热处理如何影响人类中性粒细胞与真菌模型酵母聚糖“靶”颗粒的相互作用。分别评估中性粒细胞对酵母聚糖的趋化和吞噬反应，我们发现血清热处理以不同的方式调节这些反应。虽然 52°C 的血清处理几乎会损害所有趋化活性并降低细胞与靶标的黏附，但在相同条件下，中性粒细胞仍容易吞噬被操纵与细胞表面接触的靶标颗粒。即使在强制细胞-靶标接触后，较高的血清处理温度也会逐渐抑制吞噬作用。使用荧光染色，我们将观察到的细胞行为与在各自温度下处理的血清中沉积在酵母聚糖表面的 C3b 和 IgG 的量相关联。这种比较不仅证实了补体在中性粒细胞与真菌表面的趋化和黏附相互作用中的关键作用，而且还揭示了 IgG 在酵母样真菌颗粒吞噬作用中的重要参与。总之，本研究为包括免疫细胞趋化募集、细胞表面受体黏附能力、IgG 在真菌识别中的作用以及人中性粒细胞的调理素依赖性吞噬作用形态在内的基本免疫机制提供了新的见解。此外，我们展示了如何通过微调血清的热处理，可以在其他条件相同的情况下选择性地研究趋化作用或吞噬作用。这些结果不仅改进了我们对广泛使用的实验室方法的理解，还为该方法的新应用奠定了基础。
