Intravesical CD74 and CXCR4, macrophage migration inhibitory factor (MIF) receptors, mediate bladder pain

Abstract

Background: Activation of intravesical protease activated receptor 4 (PAR4) leads to release of urothelial macrophage migration inhibitory factor (MIF). MIF then binds to urothelial MIF receptors to release urothelial high mobility group box-1 (HMGB1) and elicit bladder hyperalgesia. Since MIF binds to multiple receptors, we investigated the contribution of individual urothelial MIF receptors to PAR4-induced HMGB1 release in vivo and in vitro and bladder pain in vivo.

Methodology/principal findings: We tested the effect of intravesical pre-treatment with individual MIF or MIF receptor (CD74, CXCR4, CXCR2) antagonists on PAR4-induced HMGB1 release in vivo (female C57/BL6 mice) and in vitro (primary human urothelial cells) and on PAR4-induced bladder hyperalgesia in vivo (mice). In mice, PAR4 induced HMGB1 release and bladder hyperalgesia through activation of intravesical MIF receptors, CD74 and CXCR4. CXCR2 was not involved in these effects. In primary urothelial cells, PAR4-induced HMGB1 release through activation of CD74 receptors. Micturition parameters in mice were not changed by any of the treatments.

Conclusions/significance: Urothelial MIF receptors CD74 and CXCR4 mediate bladder pain through release of urothelial HMGB1. This mechanism may set up persistent pain loops in the bladder and warrants further investigation. Urothelial CD74 and CXCR4 may provide novel targets for interrupting bladder pain.

Fig 1. Experimental protocol for inducing and antagonizing PAR4 bladder hyperalgesia (BHA).

Mice were administered a baseline abdominal von Frey (VF) test and then anesthetized with isoflurane. Bladder was emptied through a transurethral catheter and MIF or MIF-receptor antagonists were instilled for 10 min prior to instillation of PAR4 activating peptide (PAR4-AP; to elicit BHA) or a scrambled peptide (Scramb; as control). After 1 hour the intravesical fluid was collected and mice were allowed to recover. Next day, mice were again tested for VF and micturition volume and frequency were recorded in awake mice using the Voided Stain on Paper Method (VSOP). Alternatively, mice were anesthetized and instrumented for cystometry (as described). At the end of the study, bladders were collected for histology and further analysis.

Fig 2. Intravesical MIF and MIF receptors mediate PAR4-induced bladder hyperalgesia.

Bladder hyperalgesia (BHA; decreases in 50% VF threshold) at 24 hr post treatment were compared across all groups. A) In mice pre-treated with intravesical PBS, intravesical PAR4 peptide resulted in a marked and significant decrease in 50% threshold score when compared to scrambled peptide treatment. B) Pre-treatment with anti-MIF mAb prevented PAR4-induced change in 50% threshold while pre-treatment with isotype mIgG1 had no effect. C) Pre-treatment with anti-CD74 monoclonal antibody also blocked PAR4-induced changes in 50% threshold while pre-treatment with isotype control (rIgG2b) was not effective. D) Pre-treatment with MIF098 (MIF antagonist that prevents binding to CD74) had a significant effect on 50% threshold so that it was still significantly different from the vehicle treated group. E) Pre-treatment with CXCR4 antagonist/AMD3100 followed by PAR4-AP also blocked PAR4-induced changes in 50% threshold. F) Pre-treatment with CXCR2 antagonist/SB225002 followed by PAR4-AP treatment had no effect on 50% threshold and similar results were obtained from mice pre-treated with vehicle, 0.1% DMSO in PBS followed by PAR4 treatment. **** = p < 0.0001, *** = p < 0.001 when compared to appropriate control group; (###) = p < 0.001 when compared to PBS-PAR4 (24 hr).

Fig 3. Awake micturition volume in treatment groups.

Micturition volume (μl) observed using awake Voided Stain on Paper (VSOP) for all treatment groups and analyzed using ANOVA followed by post-hoc Dunnett tests. There were no significant differences between the groups when compared to control (no pain; PBS-pretreatment, scrambled peptide-treatment; PBS-Scramb). A) Micturition volume in mice pre-treated with intravesical PBS followed by intravesical scrambled PAR4 peptide or PAR4-AP. B) Pre-treatment with isotype mIgG1 (IgG) antibody or anti-MIF mAb or anti-MIF mAb, both groups treated with PAR4 to elicit bladder pain. C) Pre-treatment with isotype control (rIgG2b; IgG) or anti-CD74 monoclonal antibody and then treated with intravesical PAR4. D) Pre-treatment with PBS or CXCR4 antagonist AMD3100 (AMD) followed by PAR4-AP.

Fig 4. Awake micturition frequency in treatment groups.

Micturition frequency (number of voids in 3 hour observation period) recorded using awake Voided Stain on Paper (VSOP) for all treatment groups and analyzed using ANOVA followed by post-hoc Dunnett tests. There were no significant differences between the groups when compared to control (no pain; PBS-Scramb). A) Micturition frequency in mice pre-treated with intravesical PBS followed by intravesical scrambled PAR4 peptide or PAR4-AP. B) Pre-treatment with isotype mIgG1 antibody (IgG) or anti-MIF mAb, both groups treated with PAR4 to elicit bladder pain. C) Pre-treatment with isotype control (rIgG2b; IgG) or anti-CD74 monoclonal antibody and then treated with intravesical PAR4. D) Pre-treatment with PBS or CXCR4 antagonist AMD3100 (AMD) followed by PAR4-AP.

Fig 5. Anesthetized cystometry showed no effect from PAR4 treatment.

Mice were instrumented with a transvesical bladder catheter as described that allowed for filling of the bladder and recording of bladder pressure. Single cystometrograms (A; B) showed very similar profiles between PBS-Scrambled treated and PBS-PAR4-AP treated mice. Continuous cystometry (C; D) also showed no difference in profiles between the two groups. MT = micturition threshold; PP = peak pressure; ICI = intercontraction interval.

Fig 6. PAR4 treatment does not induce significant changes in bladder edema or inflammation.

Representative sections of H&E bladder sections from all groups. A) PBS-Scrambled peptide treatment caused mild edema likely due to catheter insertion. B) No significant difference in edema or inflammation seen in PBS-PAR4 treatment. Pre-treatment with isotype IgG (mIgG1; C or rIgG2b; E) caused a small but statistically significant increase in inflammation when compared to PBS-Scrambled group. No significant changes in edema or inflammation were noted for pre-treatment with anti-MIF mAb (D), anti-CD74 mAb (F), vehicle for MIF098 (G), MIF098 (H), AMD3100 (CXCR4 antagonist; I) or SB225002 (CXCR2 antagonist: J). Calibration bar = 200 μm.

Fig 7. Diagram of possible mechanism for MIF-CD74-HMGB1 mediated bladder pain.

PAR4-activating peptide (PAR4-AP), and more likely, urine proteases activate PAR4 receptors found in the urothelium. This event triggers a release of urothelial MIF to the extracellular (and even intravesical) space where MIF can bind CD74 and/or CXCR4 (MIF receptors) to mediate urothelial HMGB1 release. Once released, HMGB1 can bind HMGB1 receptors (most likely intravesical TLR4) to mediate bladder pain. Exact, detailed mechanisms are not known as this point and indicated by question marks.