Introduction

THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Differentiated THP-1 cells acquire macrophage-like characteristics including more adherence and altered cell function. Nitric oxide (NO), an intracellular messenger, is critical in regulating cell differentiation. Here we elucidated whether NO relates to PMA-induced monocyte-to-macrophage differentiation of THP-1 cells. The mutual regulation of calcium and NO was also investigated.

Material & methods

THP-1 cells were incubated with PMA for 24 h, followed by assay of adherence, morphological change, migration or IL-1β release. L-N^G-Nitroarginine methyl ester (l-NAME, a nitric oxide synthase inhibitor) or BAPTA-AM (a calcium chelator) was added before PMA stimulation, and levels of calcium and NO were measured. Furthermore, a selective inhibitor of inducible nitric oxide synthase (iNOS) activity was employed to study the role of iNOS.

Results and Discussion

Effects of PMA on upregulation of adherence, lipopolysaccharide-triggered IL-1β, and migration ability of THP-1 cells were consistent with NO concentrations. Both l-NAME and BAPTA-AM mitigated effects of PMA on THP-1 cells differentiation. BAPTA-AM decreased levels of NO, while l-NAME had no effect on calcium levels. Of note, inhibition of iNOS activity decreased PMA-triggered upregulation of NO.

Conclusion

PMA induced differentiation of THP-1 cells partially in a NO-dependent manner. The calcium signaling may mediate PMA-triggered upregulation of NO.

中文翻译：

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佛波醇肉豆蔻酸酯以一氧化氮依赖性方式诱导 THP-1 细胞分化

介绍

THP-1 细胞是一种人类白血病单核细胞系，由醋酸佛波醇肉豆蔻酸酯 (PMA) 分化而成，被广泛用作人类巨噬细胞的替代物。分化的 THP-1 细胞获得巨噬细胞样特征，包括更多的粘附和改变的细胞功能。一氧化氮 (NO) 是一种细胞内信使，对调节细胞分化至关重要。在这里，我们阐明了 NO 是否与 PMA 诱导的 THP-1 细胞的单核细胞向巨噬细胞分化有关。还研究了钙和NO的相互调节。

材料和方法

THP-1 细胞与 PMA 一起孵育 24 小时，然后进行粘附、形态变化、迁移或 IL-1β 释放的测定。在 PMA 刺激之前加入LN ^{G -}硝基精氨酸甲酯（l -NAME，一种一氧化氮合酶抑制剂）或 BAPTA-AM（一种钙螯合剂），并测量钙和 NO 的水平。此外，采用诱导型一氧化氮合酶 (iNOS) 活性的选择性抑制剂来研究 iNOS 的作用。

结果和讨论

PMA 对上调粘附、脂多糖触发的 IL-1β 和 THP-1 细胞迁移能力的影响与 NO 浓度一致。两个升-NAME和BAPTA-AM减轻对THP-1细胞分化PMA的影响。BAPTA-AM 降低了 NO 水平，而l -NAME 对钙水平没有影响。值得注意的是，iNOS 活性的抑制降低了 PMA 触发的 NO 上调。

结论

PMA 以 NO 依赖性方式部分诱导 THP-1 细胞分化。钙信号可能介导 PMA 触发的 NO 上调。