Allergy, Pulmonary and Critical Care Medicine Division, Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA.
Cytokine. 2012 May;58(2):199-206. doi: 10.1016/j.cyto.2012.01.009. Epub 2012 Feb 9.
TNF (designated as TNF-α under previous nomenclature) is the preeminent activator of MMP-9 generation from a variety of cells including eosinophils. We have previously established that TNF strongly synergizes with IFN-γ and IL-4 for eosinophil synthesis of Th1- and Th2-type chemokines respectively. Thus, we sought to determine if TNF-induced synthesis of MMP-9 would be enhanced by the presence of Th1, Th2, or the eosinophil-associated common beta chain (βc) cytokines. Human blood eosinophils were cultured with TNF alone or in combination with either IFN-γ, IL-4, IL-3, IL-5, or GM-CSF. Concentrations and activities of MMP-9 in eosinophil culture supernates were measured by ELISA and gelatin zymography, mRNA transcription and stabilization by quantitative real-time PCR, and signaling events by immunoblotting and intracellular flow cytometric analysis. Individually, TNF, GM-CSF, or IL-3, but not IL-4 or IFN-γ, induced relatively small (<0.2 ng/ml) but statistically significant quantities of MMP-9. Remarkable synergistic synthesis of MMP-9 (ng/ml levels) occurred in response to TNF plus IL-3, GM-CSF or IL-5, in the order of IL-3>GM-CSF>IL-5. Zymography revealed that eosinophils release MMP-9 in its pro-form. Eosinophil stimulation with the combination of IL-3 plus TNF led to increased steady-state levels of MMP-9 mRNA, prolonged mRNA stabilization, and enhanced activation of ERK1/2 phosphorylation. Inhibition of NF-κB, MEK kinase, or p38 MAP kinase, but not JNK signaling pathways, diminished IL-3/TNF-induced MMP-9 mRNA and protein production. Thus, the synergistic regulation of eosinophil MMP-9 by IL-3 plus TNF likely involves cooperative interaction of multiple transcription factors downstream from ERK, p38, and NF-κB activation as well as post-transcriptional regulation of MMP-9 mRNA stabilization. Our data indicate that within microenvironments rich in βc-family cytokines and TNF, eosinophils are an important source of proMMP-9 and highlight a previously unrecognized role for synergistic interaction between TNF and βc-family cytokines, particularly IL-3, for proMMP-9 synthesis.
肿瘤坏死因子（在以前的命名法中指定为 TNF-α）是各种细胞（包括嗜酸性粒细胞）产生 MMP-9 的主要激活剂。我们之前已经确定，TNF 与 IFN-γ 和 IL-4 强烈协同作用，分别促进嗜酸性粒细胞合成 Th1 和 Th2 型趋化因子。因此，我们试图确定 TNF 诱导的 MMP-9 合成是否会因存在 Th1、Th2 或嗜酸性粒细胞相关的共同β链（βc）细胞因子而增强。用人血嗜酸性粒细胞单独或与 IFN-γ、IL-4、IL-3、IL-5 或 GM-CSF 一起培养 TNF。通过 ELISA 和明胶酶谱法测量嗜酸性粒细胞培养上清液中 MMP-9 的浓度和活性，通过定量实时 PCR 测量 mRNA 转录和稳定性，通过免疫印迹和细胞内流式细胞术分析测量信号事件。单独的 TNF、GM-CSF 或 IL-3，但不是 IL-4 或 IFN-γ，诱导相对较小（<0.2ng/ml）但具有统计学意义的 MMP-9 量。TNF 加 IL-3、GM-CSF 或 IL-5 的协同作用显著增加了 MMP-9 的合成（ng/ml 水平），按 IL-3>GM-CSF>IL-5 的顺序。明胶酶谱显示嗜酸性粒细胞以其前体形式释放 MMP-9。IL-3 加 TNF 刺激嗜酸性粒细胞导致 MMP-9 mRNA 的稳态水平增加，mRNA 稳定性延长，并增强 ERK1/2 磷酸化的激活。NF-κB、MEK 激酶或 p38 MAP 激酶的抑制，但不是 JNK 信号通路的抑制，减少了 IL-3/TNF 诱导的 MMP-9 mRNA 和蛋白产生。因此，IL-3 加 TNF 对嗜酸性粒细胞 MMP-9 的协同调节可能涉及 ERK、p38 和 NF-κB 激活下游的多个转录因子的协同相互作用，以及 MMP-9 mRNA 稳定性的转录后调节。我们的数据表明，在富含βc 家族细胞因子和 TNF 的微环境中，嗜酸性粒细胞是 proMMP-9 的重要来源，并强调了 TNF 和βc 家族细胞因子（特别是 IL-3）之间协同相互作用在 proMMP-9 合成中的以前未被认识到的作用。
