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Cyclophosphamide-induced cystitis increases bladder CXCR4 expression and CXCR4-macrophage migration inhibitory factor association.

Vera PL, Iczkowski KA, Wang X, Meyer-Siegler KL - PLoS ONE (2008)

Bottom Line: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro.We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels.

View Article: PubMed Central - PubMed

Affiliation: Bay Pines VA Healthcare System, Research & Development (151), Bay Pines, Florida, USA. pvera@health.usf.edu

ABSTRACT

Background: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro. We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.

Methods and findings: Twenty male rats received saline or cyclophosphamide (40 mg/kg; i.p.; every 3(rd) day) to induce persistent cystitis. After eight days, urine was collected and bladders excised under anesthesia. Bladder CXCR4 and CXCR4-MIF co-localization were examined with immunhistochemistry. ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels. Bladder CXCR4 expression (real-time RTC-PCR) and protein levels (Western blotting) were examined. Co-immunoprecipitations studied MIF-CXCR4 associations.Urothelial basal and intermediate (but not superficial) cells in saline-treated rats contained CXCR4, co-localized with MIF. Cyclophosphamide treatment caused: 1) significant redistribution of CXCR4 immunostaining to all urothelial layers (especially apical surface of superficial cells) and increased bladder CXCR4 expression; 2) increased urine MIF with decreased bladder MIF; 3) increased bladder SDF-1; 4) increased CXCR4-MIF associations.

Conclusions: These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated signal transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate signal transduction mediated by CXCR4.

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Co-localization of CXCR4 and MIF in urothelium.Representative sections from rats treated with saline (A–C) or CYP (D–I) are shown. The figure shows MIF immunostaining (green immunofluorescence), CXCR4 immunostaining (red immunofluorescence) and an overlay panel combining both immunostaining and a DAPI nuclear stain. MIF immunostaining is seen in basal and intermediate cells and in fibroblasts in the lamina propria of saline treated rats (A), while superficial cells do not stain for MIF. Arrows show luminal edge of urothelium. CXCR4 is restricted to basal and intermediate cells of urothelium (B) and lamina propria is not stained. Overlay of these panels (C) demonstrate co-localization of MIF and CXCR4 as orange coloring of cells. CYP treatment resulted in superficial cell staining for MIF (D,G) and CXCR4 (E,H) and overlay panels (F,I) demonstrate co-localization as orange color in urothelial cells. Arrows point to superficial cells showing MIF-CXCR4 co-localization. Calibration bar = 50 µm.
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pone-0003898-g003: Co-localization of CXCR4 and MIF in urothelium.Representative sections from rats treated with saline (A–C) or CYP (D–I) are shown. The figure shows MIF immunostaining (green immunofluorescence), CXCR4 immunostaining (red immunofluorescence) and an overlay panel combining both immunostaining and a DAPI nuclear stain. MIF immunostaining is seen in basal and intermediate cells and in fibroblasts in the lamina propria of saline treated rats (A), while superficial cells do not stain for MIF. Arrows show luminal edge of urothelium. CXCR4 is restricted to basal and intermediate cells of urothelium (B) and lamina propria is not stained. Overlay of these panels (C) demonstrate co-localization of MIF and CXCR4 as orange coloring of cells. CYP treatment resulted in superficial cell staining for MIF (D,G) and CXCR4 (E,H) and overlay panels (F,I) demonstrate co-localization as orange color in urothelial cells. Arrows point to superficial cells showing MIF-CXCR4 co-localization. Calibration bar = 50 µm.

Mentions: We examined the co-localization of CXCR4 and MIF in the urothelium using dual-immunofluorescence. Figure 3 shows representative bladder sections from each group immunostained for MIF (FITC color), CXCR4 (TRITC color) and an overlay of those two panels (co-localization indicated by orange color; nuclear staining by DAPI shown in blue). In saline-treated rats, both MIF and CXCR4 could be localized in the basal and intermediate layers of the urothelium (but not in superficial cells) (Fig 4A–C). After CYP-treatment, MIF and CXCR4 are readily localized throughout the urothelium (Fig 4D–I), even on superficial cells previously devoid of MIF or CXCR4 (arrows in Fig 4F,I).


Cyclophosphamide-induced cystitis increases bladder CXCR4 expression and CXCR4-macrophage migration inhibitory factor association.

Vera PL, Iczkowski KA, Wang X, Meyer-Siegler KL - PLoS ONE (2008)

Co-localization of CXCR4 and MIF in urothelium.Representative sections from rats treated with saline (A–C) or CYP (D–I) are shown. The figure shows MIF immunostaining (green immunofluorescence), CXCR4 immunostaining (red immunofluorescence) and an overlay panel combining both immunostaining and a DAPI nuclear stain. MIF immunostaining is seen in basal and intermediate cells and in fibroblasts in the lamina propria of saline treated rats (A), while superficial cells do not stain for MIF. Arrows show luminal edge of urothelium. CXCR4 is restricted to basal and intermediate cells of urothelium (B) and lamina propria is not stained. Overlay of these panels (C) demonstrate co-localization of MIF and CXCR4 as orange coloring of cells. CYP treatment resulted in superficial cell staining for MIF (D,G) and CXCR4 (E,H) and overlay panels (F,I) demonstrate co-localization as orange color in urothelial cells. Arrows point to superficial cells showing MIF-CXCR4 co-localization. Calibration bar = 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2588654&req=5

pone-0003898-g003: Co-localization of CXCR4 and MIF in urothelium.Representative sections from rats treated with saline (A–C) or CYP (D–I) are shown. The figure shows MIF immunostaining (green immunofluorescence), CXCR4 immunostaining (red immunofluorescence) and an overlay panel combining both immunostaining and a DAPI nuclear stain. MIF immunostaining is seen in basal and intermediate cells and in fibroblasts in the lamina propria of saline treated rats (A), while superficial cells do not stain for MIF. Arrows show luminal edge of urothelium. CXCR4 is restricted to basal and intermediate cells of urothelium (B) and lamina propria is not stained. Overlay of these panels (C) demonstrate co-localization of MIF and CXCR4 as orange coloring of cells. CYP treatment resulted in superficial cell staining for MIF (D,G) and CXCR4 (E,H) and overlay panels (F,I) demonstrate co-localization as orange color in urothelial cells. Arrows point to superficial cells showing MIF-CXCR4 co-localization. Calibration bar = 50 µm.
Mentions: We examined the co-localization of CXCR4 and MIF in the urothelium using dual-immunofluorescence. Figure 3 shows representative bladder sections from each group immunostained for MIF (FITC color), CXCR4 (TRITC color) and an overlay of those two panels (co-localization indicated by orange color; nuclear staining by DAPI shown in blue). In saline-treated rats, both MIF and CXCR4 could be localized in the basal and intermediate layers of the urothelium (but not in superficial cells) (Fig 4A–C). After CYP-treatment, MIF and CXCR4 are readily localized throughout the urothelium (Fig 4D–I), even on superficial cells previously devoid of MIF or CXCR4 (arrows in Fig 4F,I).

Bottom Line: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro.We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels.

View Article: PubMed Central - PubMed

Affiliation: Bay Pines VA Healthcare System, Research & Development (151), Bay Pines, Florida, USA. pvera@health.usf.edu

ABSTRACT

Background: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro. We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.

Methods and findings: Twenty male rats received saline or cyclophosphamide (40 mg/kg; i.p.; every 3(rd) day) to induce persistent cystitis. After eight days, urine was collected and bladders excised under anesthesia. Bladder CXCR4 and CXCR4-MIF co-localization were examined with immunhistochemistry. ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels. Bladder CXCR4 expression (real-time RTC-PCR) and protein levels (Western blotting) were examined. Co-immunoprecipitations studied MIF-CXCR4 associations.Urothelial basal and intermediate (but not superficial) cells in saline-treated rats contained CXCR4, co-localized with MIF. Cyclophosphamide treatment caused: 1) significant redistribution of CXCR4 immunostaining to all urothelial layers (especially apical surface of superficial cells) and increased bladder CXCR4 expression; 2) increased urine MIF with decreased bladder MIF; 3) increased bladder SDF-1; 4) increased CXCR4-MIF associations.

Conclusions: These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated signal transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate signal transduction mediated by CXCR4.

Show MeSH
Related in: MedlinePlus