Research and Development, The Bay Pines VA Healthcare System, Bay Pines, Florida, United States of America.
PLoS One. 2010 Dec 31;5(12):e15904. doi: 10.1371/journal.pone.0015904.
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.
MIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.
UROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.
Urothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression.
巨噬细胞移动抑制因子（MIF）是一种由尿路上皮细胞表达的促炎细胞因子，可介导膀胱炎症。我们研究了凝血酶刺激，即蛋白酶激活受体 1（PAR1）激动剂，对尿路上皮细胞中 MIF 释放和 MIF mRNA 上调的影响。
使用实时 RT-PCR、Western 印迹和双重免疫染色法检查正常人类永生化尿路上皮细胞（UROtsa）中的 MIF 和 PAR1 表达。还检查了大鼠尿路上皮中的 MIF 和 PAR1 免疫染色。在 UROtsa 细胞（体外）和大鼠（体内）中检查了凝血酶刺激（100 nM）对尿路上皮 MIF 释放的影响。用凝血酶刺激 UROtsa 细胞，在不同时间点收集培养物上清液，并通过 ELISA 测定 MIF 含量。戊巴比妥麻醉大鼠接受膀胱内生理盐水（对照）、凝血酶或凝血酶+2%利多卡因（阻断神经活动）1 小时，收集管腔内液体并通过 ELISA 测定 MIF 含量。使用实时 RT-PCR 测量膀胱或 UROtsa MIF mRNA。
UROtsa 细胞持续表达 MIF 和 PAR1，并且在这些细胞以及大鼠尿路上皮的基底和中间层中观察到两种物质的免疫染色。尿路上皮细胞的凝血酶刺激导致 MIF 释放呈浓度和时间依赖性增加，无论是在体外（UROtsa；1 小时时增加 2.8 倍）还是在体内（大鼠；增加 4.5 倍），而热失活的凝血酶则没有影响。在大鼠中，膀胱内利多卡因处理可减少但不能消除凝血酶诱导的 MIF 释放。凝血酶还上调了 UROtsa 细胞中的 MIF mRNA（增加 3.3 倍）和大鼠膀胱中的 MIF mRNA（增加 2 倍），而利多卡因处理使该作用降低（1.4 倍）。
尿路上皮细胞表达 MIF 和 PAR1。尿路上皮 PAR1 受体的激活，无论是由局部产生的凝血酶还是尿液中存在的蛋白酶引起，都可能通过诱导尿路上皮 MIF 释放和上调尿路上皮 MIF 表达来介导膀胱炎症。
