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Macrophage migration inhibitory factor is released as a complex with alpha1-inhibitor-3 in the intraluminal fluid
Pedro L Vera1, Kenneth A Iczkowski, Lin Leng
1Research and Development Service, Bay Pines Veterans Affairs Medical Center, Bay Pines, Florida, USA. pvera@hsc.usf.edu
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.
Insights
Macrophage migration inhibitory factor (MIF) forms complexes with alpha1-inhibitor-3 in rat bladder inflammation. Substance P and capsaicin treatments altered these MIF complexes, suggesting a role in inflammatory responses.
Area of Science:
- Urology
- Immunology
- Biochemistry
Background:
- Macrophage migration inhibitory factor (MIF) is implicated in inflammatory processes.
- MIF is found in the intraluminal fluid (ILF) of the rat bladder during inflammation.
Purpose of the Study:
- To investigate the forms of MIF released into rat bladder ILF.
- To examine MIF release patterns following substance P (SP) or capsaicin administration.
- To identify proteins associated with MIF in ILF.
Main Methods:
- Rats received subcutaneous SP or intravesical capsaicin.
- ILF was collected and analyzed for MIF using ELISA and Western blotting.
- Mass spectrometry and immunoprecipitation identified MIF-associated proteins.
Main Results:
- Both SP and capsaicin increased total MIF levels in ILF.
- MIF existed in high molecular weight complexes, with distinct band patterns under nonreducing conditions for each treatment.
- Mass spectrometry identified MIF complexed with acute phase proteins, specifically alpha1-inhibitor-3.
Conclusions:
- MIF forms complexes with alpha1-inhibitor-3, a proteinase inhibitor, in the rat bladder.
- SP and capsaicin differentially modulate MIF complex formation during inflammation.
- These findings indicate that MIF, in complex with other proteins, is part of the inflammatory response in the bladder.
