PURPOSE: Macrophage migration inhibitory factor (MIF) is released into intraluminal fluid (ILF) during bladder inflammation in the rat. We investigated the forms of MIF that are released. We examined MIF release after subcutaneous substance P (SP) or intravesical capsaicin and studied proteins associated with excreted MIF in ILF. MATERIALS AND METHODS: Anesthetized male rats with the bladder isolated from the kidneys were injected with SP subcutaneously (saline vehicle) or with intravesical capsaicin (vehicle, 0.1 mM and 1 mM). After 1 hour the ILF was removed and MIF levels were determined using enzyme-linked immunosorbent assay or Western blotting procedures under native, nonreducing and reducing conditions. Mass spectrometry was used to identify proteins associated with MIF in ILF and results were verified by immunoprecipitation. RESULTS: SP and intravesical capsaicin increased the total amount of MIF in ILF. MIF was found in high molecular weight complexes that resolved into 2 bands under nonreducing conditions. SP and capsaicin differentially increased the MIF bands. Mass spectrometry determined that MIF was complexed with acute phase proteins. MIF immunoprecipitation followed by Western blotting confirmed that MIF was complexed to alpha1-inhibitor-3. CONCLUSIONS: MIF is complexed with alpha1-inhibitor-3, a member of the alpha-2-macroglobulin proteinase inhibitor family, in the rat. Although SP and capsaicin increased the total amount of MIF detected by enzyme-linked immunosorbent assay in ILF, the patterns of MIF complexes elicited by these 2 treatments were different. These findings suggest that in association with other proteins MIF forms part of a complex elicited by bladder inflammation.
PURPOSE:Macrophage migration inhibitory factor (MIF) is released into intraluminal fluid (ILF) during bladder inflammation in the rat. We investigated the forms of MIF that are released. We examined MIF release after subcutaneous substance P (SP) or intravesical capsaicin and studied proteins associated with excreted MIF in ILF. MATERIALS AND METHODS: Anesthetized male rats with the bladder isolated from the kidneys were injected with SP subcutaneously (saline vehicle) or with intravesical capsaicin (vehicle, 0.1 mM and 1 mM). After 1 hour the ILF was removed and MIF levels were determined using enzyme-linked immunosorbent assay or Western blotting procedures under native, nonreducing and reducing conditions. Mass spectrometry was used to identify proteins associated with MIF in ILF and results were verified by immunoprecipitation. RESULTS: SP and intravesical capsaicin increased the total amount of MIF in ILF. MIF was found in high molecular weight complexes that resolved into 2 bands under nonreducing conditions. SP and capsaicin differentially increased the MIF bands. Mass spectrometry determined that MIF was complexed with acute phase proteins. MIF immunoprecipitation followed by Western blotting confirmed that MIF was complexed to alpha1-inhibitor-3. CONCLUSIONS:MIF is complexed with alpha1-inhibitor-3, a member of the alpha-2-macroglobulin proteinase inhibitor family, in the rat. Although SP and capsaicin increased the total amount of MIF detected by enzyme-linked immunosorbent assay in ILF, the patterns of MIF complexes elicited by these 2 treatments were different. These findings suggest that in association with other proteins MIF forms part of a complex elicited by bladder inflammation.
Authors: Dimitrios E Kouzoukas; Katherine L Meyer-Siegler; Fei Ma; Karin N Westlund; David E Hunt; Pedro L Vera Journal: PLoS One Date: 2015-05-28 Impact factor: 3.240