Intraluminal blockade of cell-surface CD74 and glucose regulated protein 78 prevents substance P-induced bladder inflammatory changes in the rat.

Abstract

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.

• Authors: Pedro L Vera, Xihai Wang, Richard J Bucala, Katherine L Meyer-Siegler
• Journal: PloS one (PLoS One) Vol. 4 Issue 6 Pg. e5835 (Jun 08 2009) ISSN: 1932-6203 [Electronic] United States
• PMID: 19503733 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Antigens, Differentiation, B-Lymphocyte
• GRP78 protein, rat
• Heat-Shock Proteins
• Histocompatibility Antigens Class II
• invariant chain
• Substance P

Topics

• Animals
• Antigens, Differentiation, B-Lymphocyte (biosynthesis)
• Cell Membrane (metabolism)
• Heat-Shock Proteins (physiology)
• Histocompatibility Antigens Class II (biosynthesis)
• Inflammation
• Male
• Microscopy, Fluorescence (methods)
• Models, Biological
• Oligonucleotide Array Sequence Analysis
• Protein Transport
• Rats
• Rats, Sprague-Dawley
• Signal Transduction
• Substance P (metabolism)
• Urinary Bladder (metabolism, pathology)