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Intraluminal blockade of cell-surface CD74 and glucose regulated protein 78 prevents substance P-induced bladder
Pedro L Vera1, Xihai Wang, Richard J Bucala
1The Bay Pines VA Healthcare System, Research & Development, Bay Pines, Florida, United States of America. pvera@health.usf.edu
Background:
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.
Insights
Substance P triggers urothelial cell-surface expression of glucose regulated protein 78 (GRP78), which binds macrophage migration inhibitory factor (MIF) complexes. Blocking GRP78 or CD74 receptors prevents inflammation, offering therapeutic targets for bladder conditions.
Area of Science:
- Urology
- Immunology
- Cell Biology
Background:
- Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine in urothelial cells.
- MIF complexes with other proteins, including alpha1-inhibitor III (A1-I3), and binds urothelial cell receptors.
- Glucose regulated protein 78 (GRP78) is a known receptor for A1-I3.
Purpose of the Study:
- To investigate if substance P induces urothelial cell-surface expression of GRP78.
- To determine the role of CD74 (MIF receptor) and GRP78 in substance P-induced bladder inflammation.
Main Methods:
- Rats received substance P or saline; bladders were analyzed for histology, protein, and mRNA.
- In vivo biotinylation assessed urothelial cell-surface GRP78 expression.
- Intraluminal antibodies blocked CD74, GRP78, or both to evaluate effects on inflammation and MIF release.
Main Results:
- Substance P treatment increased GRP78 and MIF co-localization in urothelial umbrella cells.
- Immunoprecipitation confirmed increased GRP78-MIF complexes post-substance P.
- In vivo biotinylation verified substance P-induced GRP78 cell-surface expression.
- Blocking CD74 and/or GRP78 inhibited substance P-induced bladder edema, MIF release, and inflammatory cytokine production.
Conclusions:
- GRP78 is upregulated on urothelial cell surfaces by substance P, facilitating MIF complex binding.
- Blocking CD74 and/or GRP78 effectively mitigates substance P-induced bladder inflammation.
- These receptors represent promising therapeutic targets for MIF-mediated bladder inflammation.
