Role of mast cells in male chronic pelvic pain

Abstract

Purpose: Chronic pelvic pain syndrome accounts for 90% of all chronic prostatitis but it has an unknown pathogenesis. We sought to understand the role of mast cells and nerve growth factor in chronic pelvic pain.

Materials and methods: Expressed prostatic secretions in men with chronic pelvic pain syndrome and controls were tested for mast cell tryptase and nerve growth factor. Mast cell number, activation status and nerve growth factor expression were examined in the NOD/ShiLtJ experimental autoimmune prostatitis model and in mast cell deficient KitW-sh/KitW-sh mice. Tactile allodynia was quantified using von Frey filaments as a measure of pelvic pain behavior. Inhibitors of mast cell degranulation, histamine receptor antagonists and anti-nerve growth factor neutralizing antibodies were tested to decrease pelvic pain behavior.

Results: Men with chronic pelvic pain syndrome showed increased mast cell tryptase and nerve growth factor in expressed prostatic secretions. In the experimental autoimmune prostatitis model increased total and activated mast cells were observed in the prostate. Mast cell deficient KitW-sh/KitW-sh mice showed attenuated pelvic pain behavior but no difference in inflammatory infiltrates in the prostate from controls. Mice with experimental autoimmune prostatitis also demonstrated increased intraprostatic nerve growth factor compared to that of KitW-sh/KitW-sh mice. Treatment of experimental autoimmune prostatitis with a mast cell stabilizer combined with a histamine 1 receptor antagonist resulted in a synergistic decrease in chronic pelvic pain. In contrast, neutralization of nerve growth factor in vivo did not result in pain relief.

Conclusions: Results suggest that mast cells are important mediators of chronic pelvic pain in experimental autoimmune prostatitis cases. They may be potential targets for therapeutic intervention in men with chronic prostatitis/chronic pelvic pain syndrome.

Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Figures

FIGURE 1. Tryptase and NGF levels in EPS are higher in patients with CPPS

(A) EPS samples from CPPS patients (n=7) and controls (n=5) were normalized with regard to protein concentration (200 µg) and analyzed for mast cell tryptase levels. Results are expressed relative to activity of a tryptase positive standard (B) EPS (50µg protein) from CPPS patients (CP1-4) and control subjects (control 1–3) as well as recombinant NGF as positive control were subjected to SDS-PAGE separation followed by detection with an anti-NGF polyclonal antibody. Immunoblot images were analyzed using ImageJ and intensity of NGF protein bands relative to the NGF standard is displayed. The immunoblot image shown is representative of two separate experiments but the densitometric analysis represents data from both experiments with CPPS (n=6) and control (n=5) subjects. Significance is indicated by absolute p values.

FIGURE 2. Mast cell numbers and NGF expression increase in EAP

(A) Mast cell in toluidine blue stained sections of mouse prostate harvested at 0, 10, 20, and 30 days after EAP induction. Note increased degranulation with time denoted by less intracytoplasmic granular staining of mast cells. The arrows point to mast cells in different stages of activation. (B) Fluorescent stained sections of mouse prostates harvested at 0, 10, 20, and 30 days after EAP induction. The sections were stained with anti-NGF antibody (red) and DAPI (blue). The arrows point to areas of increased NGF expression. (C) Mast cell numbers were determined in non-serial sections of the mouse prostate stained with toludine blue. The total number of mast cells in EAP were categorized as activated, partially activated, and resting. (I) Shows the total amount mast cells during the time course. (II, III, IV) Show the amount of activated, partially activated, and resting mast cells respectively. Scale bar represents 50µm.

FIGURE 3. Chronic pelvic pain is dependant on mast cells

Male B6 (n=10) (A) or KitW-sh/KitW-sh mice (n=8) (B) were tested for tactile allodynia using Von Frey filaments at baseline and 5, 10, 20 and 30 days after EAP induction. (C) Serial sections of the prostate of B6 and KitW-sh/KitW-sh mice following EAP induction were stained using hematoxylin and eosin and scored in a blinded manner for inflammation using standardized criteria. (D) B6 and KitW-sh/KitW-sh prostate sections show foci of inflammation but only B6 mice show the presence of mast cells. Arrows in upper panel indicate inflammatory infiltrates and in lower panels, the presence of mast cells in B6 and its absence in KitW-sh/KitW-sh prostate sections. Scale bar represent 50µm.

FIGURE 4. NGF mediates chronic pelvic pain

(A) Prostate sections of B6 and KitW-sh/KitW-sh mice with EAP were stained with anti-NGF antibody (green) or anti-NGF antibody (green) with blocking peptide and DAPI (blue) as nuclear stain. Arrows indicate areas of strong NGF staining appreciable in B6 prostates and less so in the KitW-sh/KitW-sh prostate. Male B6 (B, n=7) or KitW-sh/KitW-sh mice (C, n=7) were tested for tactile allodynia using Von Frey filaments at baseline and at day 20 prior to initiating treatment with an anti-NGF neutralizing antibody or an isotype control antibody. Pain behavior was tested 1, 2, 3, 4 and 5 days following injection of antibody. (D) NOD mice with EAP were treated at PID 20 with 100 µg of anti-NGF neutralizing antibody (Anti-NGF) or control IgG (Control) followed by daily testing for tactile allodynia.

FIGURE 5. Simultaneous stabilization of mast cells and inhibition of H1 receptor attenuates chronic pelvic pain

EAP was induced in NOD mice (n=5 in all groups) for 10 days followed by treatment with saline (A), cetirizine (10mg/kg, H1 receptor antagonist) (B), ranitidine (10mg/kg, H2 receptor antagonist) (C), cromolyn sodium (10mg/kg, mast cell stabilizer) (D) or a combination of all three drugs (E) administered intraperitoneally. Mice were tested for tactile allodynia with Von Frey filaments at baseline, day 10, and 1 hour after treatment. (F) Pain behavior expressed as percent decrease from pretreatment levels. Combination therapy was statistically significant from control (p

FIGURE 6. A mast cell stabilizer and H1 receptor inhibitor show synergistic inhibition of chronic pelvic pain

Dose response of cromolyn sodium (CS) (A) and cetirizine (H1R) (B) in the reduction of chronic pelvic pain at 1 hour and 24 hours following intraperitoneal therapy in EAP (n=5 in all groups). Administration of cromolyn sodium at 0.5mg/kg (C and D) in combination with cetirizine at 2.5 mg/kg (C and D) shows significant synergism for reduction in pain behavior when administered orally (n=6, p