Vera Pedro L, Wang Xihai, Bucala Richard J, Meyer-Siegler Katherine L
The Bay Pines VA Healthcare System, Research & Development, Bay Pines, Florida, United States of America.
PLoS One. 2009 Jun 8;4(6):e5835. doi: 10.1371/journal.pone.0005835.
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine constitutively expressed by urothelial cells. During inflammatory stimuli, MIF is released into the lumen complexed to other proteins and these complexes can bind to urothelial cell-surface receptors to activate signaling pathways. Since MIF is complexed to alpha1-inhibitor III (A1-I3; a member of the alpha2-macroglubulin family) and glucose regulated protein 78 (GRP78) is a receptor for A1-I3 the goals of this study were to determine if substance P elicits urothelial cell-surface expression of GRP78 and to assess the functional role of CD74 (receptor for MIF) or GRP78 in substance P-induced bladder inflammatory changes.
METHODOLOGY/PRINCIPAL FINDINGS: Anesthetized male Sprague-Dawley rats received either saline or substance P (s.c.), bladders were collected 1 hour after treatment and processed for histology or protein/mRNA. The expression of GRP78 at urothelial cell-surface was determined by performing in vivo biotinylation of urothelial cell-surface proteins. Finally, in order to determine the effects of receptor blockade on substance P-induced MIF release and inflammatory changes, rats received either intraluminal antibodies to CD74, GRP78, both, or non-specific IgG (as a control). GRP78 and MIF immunostaining was simultaneously visualized in umbrella cells only after substance P treatment. Immunoprecipitation studies showed GRP78-MIF complexes increased after substance P while in vivo biotinylation confirmed substance P-induced GRP78 cell-surface expression in urothelial cells. Intraluminal blockade of CD74 and/or GRP78 prevented substance P-induced changes, including bladder edema, intraluminal MIF release by urothelial cells and production of inflammatory cytokines by urothelial cells.
CONCLUSIONS/SIGNIFICANCE: GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes. Blocking CD74 (receptor for MIF) and/or GRP78 prevented substance P-induced inflammatory changes in bladder and urothelium, indicating that these urothelial receptors are effective targets for disrupting MIF-mediated bladder inflammation.
巨噬细胞移动抑制因子（MIF）是一种由尿路上皮细胞组成性表达的促炎细胞因子。在炎症刺激期间，MIF会释放到管腔中并与其他蛋白质形成复合物，这些复合物可与尿路上皮细胞表面受体结合以激活信号通路。由于MIF与α1-抑制剂III（A1-I3；α2-巨球蛋白家族的一员）形成复合物，且葡萄糖调节蛋白78（GRP78）是A1-I3的受体，因此本研究的目的是确定P物质是否能诱导GRP78在尿路上皮细胞表面表达，并评估CD74（MIF的受体）或GRP78在P物质诱导的膀胱炎症变化中的功能作用。
方法/主要发现：对雄性Sprague-Dawley大鼠进行麻醉后，分别给予生理盐水或P物质（皮下注射），治疗1小时后收集膀胱并进行组织学或蛋白质/ mRNA处理。通过对尿路上皮细胞表面蛋白进行体内生物素化来确定GRP78在尿路上皮细胞表面的表达。最后，为了确定受体阻断对P物质诱导的MIF释放和炎症变化的影响，大鼠分别接受腔内注射抗CD74、GRP78、两者的抗体或非特异性IgG（作为对照）。仅在给予P物质处理后，才在伞细胞中同时观察到GRP78和MIF免疫染色。免疫沉淀研究表明，P物质作用后GRP78-MIF复合物增加，而体内生物素化证实P物质可诱导GRP78在尿路上皮细胞表面表达。腔内阻断CD74和/或GRP78可防止P物质诱导的变化，包括膀胱水肿、尿路上皮细胞腔内MIF释放以及尿路上皮细胞产生炎性细胞因子。
结论/意义：P物质处理后，GRP78在尿路上皮细胞表面表达，可与MIF复合物结合。阻断CD74（MIF的受体）和/或GRP78可防止P物质诱导的膀胱和尿路上皮炎症变化，表明这些尿路上皮受体是破坏MIF介导的膀胱炎症的有效靶点。
