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

Vera PL, Wang X, Bucala RJ, Meyer-Siegler KL - PLoS ONE (2009)

Bottom Line: 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.GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes.

View Article: PubMed Central - PubMed

Affiliation: The Bay Pines VA Healthcare System, Research & Development, Bay Pines, Florida, United States of America. pvera@health.usf.edu

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.

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Substance P-induced MIF and GRP78 in umbrella cells.Single panels illustrating MIF (FITC labeling), GRP78 (TRITC labeling) and overlay of both (using Leica Imaging Software). Exposure times were constant for each particular fluorochrome and images were uniformly adjusted for brightness and contrast (Adobe Photoshop Elements v.2; Adobe Systems, San Jose, CA). Control sections where primary antibodies had been omitted showed no MIF (A) or GRP78 immunofluorescence (B). Consequently, overlay panel also shows no immunofluorescence (C). Arrows mark luminal edge of urothelium and slight autofluorescence can be seen. Representative sections showing urothelial immunostaining in saline-treated (D–F) and substance P-treated (G–L) bladders. Saline treated bladders showed MIF (D) immunostaining in the urothelium, whereas the urothelium appeared devoid of GRP78 immunostaining (E). Note MIF staining in basal and intermediate cells and light (or non-existent) immunostaining in umbrella cells (D). Overlay panel shows only MIF immunostaining (F). Arrowheads show location of the lamina propria, showing MIF immunostaining (D). In substance P-treated bladders, on the other hand, both MIF (G,J) and GRP78 (H,K) immunostaining was readily seen in the urothelium, particularly in umbrella cells (arrows). Overlay shows simultaneous MIF and GRP78 immunostaining as an orange color (indicated by asterisks; I,L). MIF only (panel I; green arrow) and GRP78 only (panel L; red arrow) umbrella cells can also be seen in the overlay panels. When compared to saline-treated bladders (D) MIF immunostaining in substance P-treated bladders is also increased in the fibroblast layer of the lamina propria (arrowheads, G,J). Calibration bar = 50 µm.
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pone-0005835-g001: Substance P-induced MIF and GRP78 in umbrella cells.Single panels illustrating MIF (FITC labeling), GRP78 (TRITC labeling) and overlay of both (using Leica Imaging Software). Exposure times were constant for each particular fluorochrome and images were uniformly adjusted for brightness and contrast (Adobe Photoshop Elements v.2; Adobe Systems, San Jose, CA). Control sections where primary antibodies had been omitted showed no MIF (A) or GRP78 immunofluorescence (B). Consequently, overlay panel also shows no immunofluorescence (C). Arrows mark luminal edge of urothelium and slight autofluorescence can be seen. Representative sections showing urothelial immunostaining in saline-treated (D–F) and substance P-treated (G–L) bladders. Saline treated bladders showed MIF (D) immunostaining in the urothelium, whereas the urothelium appeared devoid of GRP78 immunostaining (E). Note MIF staining in basal and intermediate cells and light (or non-existent) immunostaining in umbrella cells (D). Overlay panel shows only MIF immunostaining (F). Arrowheads show location of the lamina propria, showing MIF immunostaining (D). In substance P-treated bladders, on the other hand, both MIF (G,J) and GRP78 (H,K) immunostaining was readily seen in the urothelium, particularly in umbrella cells (arrows). Overlay shows simultaneous MIF and GRP78 immunostaining as an orange color (indicated by asterisks; I,L). MIF only (panel I; green arrow) and GRP78 only (panel L; red arrow) umbrella cells can also be seen in the overlay panels. When compared to saline-treated bladders (D) MIF immunostaining in substance P-treated bladders is also increased in the fibroblast layer of the lamina propria (arrowheads, G,J). Calibration bar = 50 µm.

Mentions: Bladder sections from saline and SP-treated rats were simultaneously exposed to MIF and GRP78 antibodies, visualized using dual immunofluorescence and single fluorophore images were overlaid. Sections where both primary antibodies had been omitted showed no immunofluorescence (Fig. 1A–C; arrows indicate luminal edge of urothelium). Similarly, omission of either one of the secondary antibodies resulted in only the appropriate fluorescence being observed (not shown). In saline treated rats, MIF immunostaining was observed in basal and intermediate layers of the urothelium as previously described (Fig 1D)[10]; while no GRP78 immunostaining was observed in the urothelium (Fig. 1E) and thus the overlay (Fig. 1F) showed only MIF immunostaining.


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

Vera PL, Wang X, Bucala RJ, Meyer-Siegler KL - PLoS ONE (2009)

Substance P-induced MIF and GRP78 in umbrella cells.Single panels illustrating MIF (FITC labeling), GRP78 (TRITC labeling) and overlay of both (using Leica Imaging Software). Exposure times were constant for each particular fluorochrome and images were uniformly adjusted for brightness and contrast (Adobe Photoshop Elements v.2; Adobe Systems, San Jose, CA). Control sections where primary antibodies had been omitted showed no MIF (A) or GRP78 immunofluorescence (B). Consequently, overlay panel also shows no immunofluorescence (C). Arrows mark luminal edge of urothelium and slight autofluorescence can be seen. Representative sections showing urothelial immunostaining in saline-treated (D–F) and substance P-treated (G–L) bladders. Saline treated bladders showed MIF (D) immunostaining in the urothelium, whereas the urothelium appeared devoid of GRP78 immunostaining (E). Note MIF staining in basal and intermediate cells and light (or non-existent) immunostaining in umbrella cells (D). Overlay panel shows only MIF immunostaining (F). Arrowheads show location of the lamina propria, showing MIF immunostaining (D). In substance P-treated bladders, on the other hand, both MIF (G,J) and GRP78 (H,K) immunostaining was readily seen in the urothelium, particularly in umbrella cells (arrows). Overlay shows simultaneous MIF and GRP78 immunostaining as an orange color (indicated by asterisks; I,L). MIF only (panel I; green arrow) and GRP78 only (panel L; red arrow) umbrella cells can also be seen in the overlay panels. When compared to saline-treated bladders (D) MIF immunostaining in substance P-treated bladders is also increased in the fibroblast layer of the lamina propria (arrowheads, G,J). Calibration bar = 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690654&req=5

pone-0005835-g001: Substance P-induced MIF and GRP78 in umbrella cells.Single panels illustrating MIF (FITC labeling), GRP78 (TRITC labeling) and overlay of both (using Leica Imaging Software). Exposure times were constant for each particular fluorochrome and images were uniformly adjusted for brightness and contrast (Adobe Photoshop Elements v.2; Adobe Systems, San Jose, CA). Control sections where primary antibodies had been omitted showed no MIF (A) or GRP78 immunofluorescence (B). Consequently, overlay panel also shows no immunofluorescence (C). Arrows mark luminal edge of urothelium and slight autofluorescence can be seen. Representative sections showing urothelial immunostaining in saline-treated (D–F) and substance P-treated (G–L) bladders. Saline treated bladders showed MIF (D) immunostaining in the urothelium, whereas the urothelium appeared devoid of GRP78 immunostaining (E). Note MIF staining in basal and intermediate cells and light (or non-existent) immunostaining in umbrella cells (D). Overlay panel shows only MIF immunostaining (F). Arrowheads show location of the lamina propria, showing MIF immunostaining (D). In substance P-treated bladders, on the other hand, both MIF (G,J) and GRP78 (H,K) immunostaining was readily seen in the urothelium, particularly in umbrella cells (arrows). Overlay shows simultaneous MIF and GRP78 immunostaining as an orange color (indicated by asterisks; I,L). MIF only (panel I; green arrow) and GRP78 only (panel L; red arrow) umbrella cells can also be seen in the overlay panels. When compared to saline-treated bladders (D) MIF immunostaining in substance P-treated bladders is also increased in the fibroblast layer of the lamina propria (arrowheads, G,J). Calibration bar = 50 µm.
Mentions: Bladder sections from saline and SP-treated rats were simultaneously exposed to MIF and GRP78 antibodies, visualized using dual immunofluorescence and single fluorophore images were overlaid. Sections where both primary antibodies had been omitted showed no immunofluorescence (Fig. 1A–C; arrows indicate luminal edge of urothelium). Similarly, omission of either one of the secondary antibodies resulted in only the appropriate fluorescence being observed (not shown). In saline treated rats, MIF immunostaining was observed in basal and intermediate layers of the urothelium as previously described (Fig 1D)[10]; while no GRP78 immunostaining was observed in the urothelium (Fig. 1E) and thus the overlay (Fig. 1F) showed only MIF immunostaining.

Bottom Line: 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.GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes.

View Article: PubMed Central - PubMed

Affiliation: The Bay Pines VA Healthcare System, Research & Development, Bay Pines, Florida, United States of America. pvera@health.usf.edu

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.

Show MeSH
Related in: MedlinePlus