Duke University Medical Center, Division of Rheumatology and Immunology, Durham, North Carolina, USA.
J Leukoc Biol. 2010 Jun;87(6):1115-23. doi: 10.1189/jlb.0709465. Epub 2010 Mar 24.
MPs are small membrane-bound particles that originate from activated and dying cells and mediate intercellular communication. Once released from cells, MPs can serve as novel signaling elements in innate immunity, with levels elevated in immune-mediated diseases. This study tested the hypothesis that TLR stimulation can induce MP release by macrophages. In these experiments, using the RAW 264.7 murine macrophage cell line as a model, LPS, a TLR4 ligand, and poly(I:C), a TLR3 ligand, induced MP release effectively, as measured by flow cytometry; in contrast, a CpG oligonucleotide, which can stimulate TLR9, induced much lower levels of particle release. To determine the role of other mediators in this response, the effects of NO were tested. Thus, MP release from RAW 264.7 cells stimulated by LPS or poly(I:C) correlated with NO production, and treatment with the iNOS inhibitor 1400W decreased particle release and NO production. Furthermore, treatment of RAW 264.7 cells with NO donors induced MP production. As TLR ligands can induce apoptosis, the effect of caspase inhibition on MP release by stimulated cells was assessed. These experiments showed that the pan-caspase inhibitor, ZVAD, although decreasing NO production, increased MP release by stimulated cells. Together, these experiments demonstrate that TLR stimulation of macrophages can lead to MP release, and NO plays a key role in this response.
MPs 是源自激活和死亡细胞的小膜结合颗粒，介导细胞间通讯。一旦从细胞中释放出来， MPs 可以作为先天免疫中的新型信号分子，其水平在免疫介导的疾病中升高。本研究检验了 TLR 刺激可以诱导巨噬细胞释放 MPs 的假设。在这些实验中，使用 RAW 264.7 鼠巨噬细胞系作为模型，LPS（TLR4 配体）和 poly(I:C)（TLR3 配体）有效地诱导 MPs 释放，如流式细胞术所测；相比之下，可刺激 TLR9 的 CpG 寡核苷酸诱导的颗粒释放水平低得多。为了确定该反应中其他介质的作用，测试了 NO 的影响。因此，LPS 或 poly(I:C)刺激的 RAW 264.7 细胞的 MPs 释放与 NO 产生相关，iNOS 抑制剂 1400W 的处理降低了颗粒释放和 NO 产生。此外，用 NO 供体处理 RAW 264.7 细胞诱导 MPs 产生。由于 TLR 配体可诱导细胞凋亡，因此评估了 caspase 抑制对刺激细胞 MPs 释放的影响。这些实验表明，泛 caspase 抑制剂 ZVAD 虽然降低了 NO 产生，但增加了刺激细胞的 MPs 释放。综上所述，这些实验表明 TLR 刺激巨噬细胞可导致 MPs 释放，而 NO 在该反应中发挥关键作用。
