Vera Pedro L, Meyer-Siegler Katherine L
The Bay Pines VA Healthcare System, Research & Development, Bay Pines, FL, USA.
BMC Urol. 2006 Sep 18;6:24. doi: 10.1186/1471-2490-6-24.
Macrophage migration inhibitory factor (MIF) is released into the intraluminal fluid during bladder inflammation in the rat complexed to alpha1-inhibitor-3 (A1-I3; a rodent proteinase inhibitor in the alpha-macroglobulin family). The location of A1-I3 in the bladder had not been investigated. Therefore, we examined the location of A1-I3 and MIF/A1-I3 complexes in the bladder and changes due to experimental inflammation.
Anesthetized male rats had bladders removed with no treatment (intact) or were injected with Substance P (SP; s.c.; saline vehicle). After one hour intraluminal fluid was removed, bladder was excised and MIF and A1-I3 levels were determined using ELISA and/or western-blotting. MIF co-immunoprecipitation determined MIF/A1-I3 complexes in the bladder. Bladder sections were immunostained for A1-I3 and MIF/A1-I3.
A1-I3 immunostaining was observed in interstitial spaces throughout the bladder (including submucosa) but not urothelium in intact and saline-treated rats. RT-PCR showed that the bladder does not synthesize A1-I3, therefore, A1-I3 in the interstitial space of the bladder must be plasma derived. In SP-treated rats, A1-I3 in the bladder increased and A1-I3 was observed traversing through the urothelium. Umbrella cells that do not show MIF and/or A1-I3 immunostaining in intact or saline-treated rats, showed co-localization of MIF and A1-I3 after SP-treatment. Western blotting demonstrated that in the bladder MIF formed non-covalent interactions and also binds covalently to A1-I3 to form high molecular weight MIF/A1-I3 complexes (170, 130 and 75-kDa, respectively, verified by co-immunoprecipitation). SP-induced inflammation selectively reduced 170-kDa MIF/A1-I3 in the bladder while increasing 170 and 130-kDa MIF/A1-I3 in the intraluminal fluid.
A1-I3 and MIF/A1-I3 complexes are resident in bladder interstitium. During SP-induced inflammation, MIF/A1-I3 complexes are released from the bladder into the lumen. Binding of MIF/A1-I3 complexes to urothelial cells during inflammation suggests these complexes participate in the inflammatory reaction through activation of receptors for MIF and/or for A1-I3.
在大鼠膀胱炎症期间，巨噬细胞移动抑制因子（MIF）与α1-抑制因子-3（A1-I3；α-巨球蛋白家族中的一种啮齿动物蛋白酶抑制剂）复合后释放到管腔内液中。尚未对A1-I3在膀胱中的定位进行研究。因此，我们研究了A1-I3和MIF/A1-I3复合物在膀胱中的定位以及实验性炎症引起的变化。
对麻醉的雄性大鼠，一组不做处理直接摘除膀胱（完整组），另一组皮下注射P物质（SP；用生理盐水作溶媒）。1小时后抽取管腔内液，然后切除膀胱，使用酶联免疫吸附测定（ELISA）和/或蛋白质印迹法测定MIF和A1-I3水平。通过MIF免疫共沉淀法测定膀胱中的MIF/A1-I3复合物。对膀胱切片进行A1-I3和MIF/A1-I3免疫染色。
在完整组和生理盐水处理组大鼠的整个膀胱（包括黏膜下层）间质中观察到A1-I3免疫染色，但尿路上皮未观察到。逆转录聚合酶链反应（RT-PCR）显示膀胱不合成A1-I3，因此，膀胱间质中的A1-I3必定来源于血浆。在SP处理组大鼠中，膀胱中的A1-I3增加，且观察到A1-I3穿过尿路上皮。在完整组或生理盐水处理组大鼠中未显示MIF和/或A1-I3免疫染色的伞细胞，在SP处理后显示MIF和A1-I3共定位。蛋白质印迹法表明，在膀胱中MIF形成非共价相互作用，并且还与A1-I3共价结合形成高分子量的MIF/A1-I3复合物（分别为170、130和75 kDa，经免疫共沉淀验证）。SP诱导的炎症选择性地降低了膀胱中170 kDa的MIF/A1-I3，同时增加了管腔内液中170和130 kDa的MIF/A1-I3。
A1-I3和MIF/A1-I3复合物存在于膀胱间质中。在SP诱导的炎症过程中，MIF/A1-I3复合物从膀胱释放到管腔中。炎症期间MIF/A1-I3复合物与尿路上皮细胞结合，提示这些复合物通过激活MIF和/或A1-I3的受体参与炎症反应。
