Amniotic membrane induces apoptosis of interferon-γ activated macrophages in vitro
Amniotic membrane induces apoptosis of interferon-γ activated macrophages in vitro
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DOI：
10.1016/j.exer.2005.06.022
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发表时间：
2006-02-01
影响因子：
3.4
通讯作者：
Tseng, SCG
中科院分区：
文献类型：
--
作者：
Li, W;He, H;Tseng, SCG
羊膜（AM）作为临时或永久移植用于眼表重建具有有效的抗炎作用。我们想研究AM诱导巨噬细胞体外凋亡的机制。小鼠巨噬细胞，Raw 264.7细胞，在无血清培养基中分别在塑料、I型胶原、角膜基质片或AM基质上培养，含或不含干扰素- γ (ifn - γ)。采用LIVE/DEAD法、Hoechst-33342法和TUNEL法对细胞死亡和凋亡进行染色。采用细胞死亡检测ELISA法对细胞裂解液和条件培养基进行定量分析。条件培养基中亚硝酸盐浓度采用Griess法测定，肿瘤坏死因子α (tnf - α)浓度采用ELISA法测定。细胞裂解液对ikk - α、ikk - β、核因子κ B (nf - κ B) p65 （RelA）亚基、总Akt、phospho-Akt （Ser473）和phospho-FKHR (Thr24)/phospho - fkhrl1 （Thr32）进行Western blot分析。培养48小时后，细胞在含或不含ifn - γ的塑料、I型胶原和角膜基质片上以及不含IFN-y的AM上均表现出低水平的凋亡。然而，在活化ifn - γ的AM上培养时，细胞凋亡明显增加，这种现象仅在48小时后才显现出来。ifn - γ激活的巨噬细胞在72小时的培养过程中持续产生一氧化氮（NO）和tnf - α。相比之下，AM培养48小时后没有no和TNF-a的产生。NO抑制剂L-NMMA和L-NIL可减弱AM上ifn - γ活化巨噬细胞NO的产生，但细胞凋亡未相应减少。AM上培养的ifn - γ活化巨噬细胞中ikk - α、ikk - β、NF-omicron B的p65 （ReIA）亚基、磷酸化Akt （Ser473）和磷酸化fkhr (Thr24)/FKHRL1 （Thr32）的表达均下调。综上所述，AM基质诱导IFN-y激活而非激活的巨噬细胞凋亡，不是通过产生NO，而是通过下调抗凋亡的NF-kappa B和Akt-FKHR信号通路。(c) 2005 Elsevier Ltd版权所有。
Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has a potent anti-inflammatory effect. We would like to investigate the mechanism whereby AM induces macrophage apoptosis in vitro. Mouse macrophages, Raw 264.7 cells, were cultured on plastic, type I collagen, corneal stromal slice or AM stromal matrix in serum-free medium with or without interferon-gamma (IFN-gamma). Cells were stained by LIVE/DEAD assay, Hoechst-33342, and TUNEL assay for cell death and apoptosis. Cell lysates and conditioned media were analysed by Cell Death Detection ELISA assay for quantitation of apoptosis. Conditioned media were also analysed by Griess assay for the nitrite concentration and ELISA assay for tumour necrosis factor alpha (TNF-alpha) concentration. Lysates of cells were subjected to Western blot analyses of IKK-alpha, IKK-beta, p65 (RelA) subunit of nuclear factor kappa B (NF-kappa B), total Akt, phospho-Akt (Ser473), and phospho-FKHR (Thr24)/phosphor-FKHRL1 (Thr32). At 48 hr after cultivation, cells showed a low level of apoptosis when cultured on plastic, type I collagen and corneal stromal slice with or without IFN-gamma and on AM without IFN-y. Nevertheless, cells showed a significant increase of apoptosis when cultured on AM with IFN-gamma activation, and this phenomenon became apparent only after 48 hr. IFN-gamma-activated macrophages on plastic continuously produced nitric oxide (NO) and TNF-alpha during 72 hr culturing. In contrast, there was no NO and TNF-a production after 48 hr culture on AM. NO inhibitors, L-NMMA and L-NIL, attenuated NO production of IFN-gamma-activated macrophages on AM, while apoptosis was not decreased accordingly. Expression of IKK-alpha, IKK-beta, p65 (ReIA) subunit of NF-omicron B total Akt, phosopho-Akt (Ser473), and phospho-FKHR (Thr24)/FKHRL1.(Thr32) was all down-regulated in IFN-gamma-activated macrophages cultured on AM. In conclusion, AM stromal matrix induces apoptosis of IFN-y activated, but not non-activated macrophages, not through the generation of NO, but instead by down-regulating anti-apoptotic NF-kappa B and Akt-FKHR signalling pathways. (c) 2005 Elsevier Ltd. All rights reserved.