Huang S J, Schatz F, Masch R, Rahman M, Buchwalder L, Niven-Fairchild T, Tang C, Abrahams V M, Krikun G, Lockwood C J
Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University, School of Medicine, New Haven, CT 06510, USA.
J Reprod Immunol. 2006 Dec;72(1-2):60-73. doi: 10.1016/j.jri.2006.03.002. Epub 2006 Jun 27.
Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1beta (IL-1beta), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity.
Leukocyte-free first trimester decidual cells were treated for 6h with estradiol (E(2)) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E(2) and MPA and IL-1beta (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture.
Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1beta in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1beta-treated decidual cells.
Treatment of first trimester decidual cells with IL-1beta induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1beta could mediate excessive macrophage infiltration of the decidua.
趋化因子通过控制白细胞迁移和淋巴细胞发育来启动免疫反应。蜕膜中巨噬细胞浸润与复发性流产和先兆子痫的发生有关。因此,我们确定培养的人蜕膜细胞是否会响应强效促炎细胞因子白细胞介素-1β(IL-1β)而产生募集单核细胞/巨噬细胞的趋化因子,以及蜕膜细胞条件培养基是否含有单核细胞和巨噬细胞趋化活性。
用雌二醇(E₂)和醋酸甲羟孕酮(MPA)处理孕早期无白细胞的蜕膜细胞6小时,以模拟妊娠的甾体环境,或用E₂、MPA和IL-1β(1 ng/ml)处理以模拟炎症蜕膜。总RNA用于cDNA合成。生成生物素化的cRNA并进行化学片段化,用于在Affymetrix HG_U133 Plus 2.0芯片上杂交,随后进行荧光标记和光学扫描。由Affymetrix GCOS 1.2(基因芯片操作软件)生成的原始数据通过GeneSpring 7.2软件进行分析。随后通过实时RT-PCR和蛋白质印迹法验证微阵列结果。还使用培养的条件培养基进行了单核细胞迁移的功能研究。
在培养的孕早期蜕膜细胞中,暴露于IL-1β后,包括C-C基序配体2(CCL2)、CCL5、C-X-C基序配体2(CXCL2)、CXCL3和CXCL8在内的五种负责单核细胞/巨噬细胞趋化和激活的趋化因子显著升高了29至975倍。实时RT-PCR(上调43至3069倍)和蛋白质印迹法(上调15至300倍)的结果证实了微阵列研究结果。IL-1β处理的蜕膜细胞条件培养基显著诱导了单核细胞迁移。
用IL-1β处理孕早期蜕膜细胞可诱导单核细胞/巨噬细胞募集趋化因子的分泌并促进单核细胞迁移。将这些体外结果外推至着床部位环境提示了一种机制,即IL-1β可介导蜕膜中巨噬细胞的过度浸润。