Tryptase-PAR2 axis in experimental autoimmune prostatitis, a model for chronic pelvic pain syndrome

Abstract

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) affects up to 15% of the male population and is characterized by pelvic pain. Mast cells are implicated in the murine experimental autoimmune prostatitis (EAP) model as key to chronic pelvic pain development. The mast cell mediator tryptase-β and its cognate receptor protease-activated receptor 2 (PAR2) are involved in mediating pain in other visceral disease models. Prostatic secretions and urines from CP/CPPS patients were examined for the presence of mast cell degranulation products. Tryptase-β and PAR2 expression were examined in murine EAP. Pelvic pain and inflammation were assessed in the presence or absence of PAR2 expression and upon PAR2 neutralization. Tryptase-β and carboxypeptidase A3 were elevated in CP/CPPS compared to healthy volunteers. Tryptase-β was capable of inducing pelvic pain and was increased in EAP along with its receptor PAR2. PAR2 was required for the development of chronic pelvic pain in EAP. PAR2 signaling in dorsal root ganglia led to extracellular signal-regulated kinase (ERK)1/2 phosphorylation and calcium influx. PAR2 neutralization using antibodies attenuated chronic pelvic pain in EAP. The tryptase-PAR2 axis is an important mediator of pelvic pain in EAP and may play a role in the pathogenesis of CP/CPPS.

Keywords: CPPS; Mast cells; Pelvic pain; Prostatitis.

Conflict of interest statement

The authors do not have any conflict of interest to report with regard to the study reported in this manuscript.

Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1. Tryptase and carboxypeptidase A are increased in CP/CPPS patients

(A) Mast cell tryptase was significantly increased in EPS from CP/CPPS (n=16) patients compared to healthy volunteers (n=6). (B) Urine samples (V3 and V2) collected from CP/CPPS (n=29) patients showed increased presence of carboxypeptidase A (CPA3) compared to control (n=12); (C) however, NGF levels did not change compared to control group. (D) Western blots were performed with 20µg/sample of mouse prostate from NOD mice (n=4) with EAP. (E) Densitometry analysis of immunoblot demonstrated higher molecular weight forms of mMCP-6 in the prostate of EAP-NOD mice compared to naive. Data represent the mean ± SEM. (*) denotes p<0.05, ** p<0.01 and ***p<0.001 compared with control.

Fig. 2. PAR2 is elevated in the prostate of EAP mice

(A) Representative immunofluorescence images of the prostate showed that NOD mice with EAP have elevated PAR2 expression (green) in the stroma (insets) compared to naive cohorts. DAPI is labeled blue and bars represent 50 microns. (B) Immunoblot and (C) densitometry data confirmed that the prostate from EAP mice (n=4) up-regulate PAR2 expression, compared to naive (n=4). (D) Representative H&E stained section of a prostate from a NOD mouse with EAP were used to microdissect epithelium (arrows) and stroma (asterisks) separately. (E) Real-time PCR analysis showed increased in mRNA for PAR2 in the stroma and epithelial layers of mice with EAP (pooled). Data was normalized to L-19 and expressed as fold change respective to naive. (*) denotes p<0.05. Real-time PCR experiments were performed three times.

Fig. 3. Tryptase induces calcium influx located in prostate and neuronal cell lines

(A) Calcium elevation [Ca2+]i in RWPE-1 (p<0.001; n=4), BPH (stromal) (p=0.119; n=3), and B35 cells (p<0.01; n=4) was recorded at baseline and after the addition of recombinant human-tryptase before or after 1-hour ENMD (PAR2 antagonist) incubation. (B) Real-time PCR was conducted to determine the expression of proinflammatory markers CCL2, CCL3, CXCL2, TNF-α, and IL-1β in cell cultures. Data represent the mean ± SEM. **p<0.01 and ***p<0.001 compared with respective control. Real-time PCR experiments were performed three or more times.

Fig. 4. Tryptase/mMCP-6 induced pelvic pain via PAR2

NOD mice were assessed for pelvic tactile allodynia/hyperalgesia with von Frey filaments before (baseline) and after intraurethral instillation of (A) mMCP-6 (n= 5) or (B) saline (n=5) at 1hr and 24hrs. (C) The pain increase (%) was generated from the total response frequency at 1hr or 24 hrs divided by respective baseline x100. Pelvic pain response was evaluated in (D) C57BL/6J (B6) (n=5) and (E) PAR2 deficient mice (n=4) with EAP (PAR2 KO-EAP) at day 0, 20, and 30. The following von Frey filaments were used to evaluate each mouse: 2.44, 3.22, 3.61, 4.08 and 4.56. Data represent the mean ± SEM. (*) denotes p<0.05. In vivo experiments were performed two or more independent times.

Fig. 5. PAR2 is involved in ERK signaling and calcium influx

(A) Western blot shows DRG (S1–S4) p-ERK1/2 expression at day 30 from mice with EAP (n=3) compared to naive (n=3) and PAR2 deficient mice with EAP (PAR2 KO-EAP; n=3) compared to control (PAR2 KO-Naive; n=3). (B) Densitometry data showed a significant increased in p-ERK1/2 expression in mice (B6) with EAP compared to naive cohorts and (C) no difference in p-ERK1/2 expression was observed between PAR2 KO-Naive and PAR2 KO-EAP. (D) Representative immunofluorescence of p-ERK 1/2 (green) in DRG of mice with EAP and naive at day 30 showed increased p-ERK1/2. (E and G) Changes in [Ca2+]i were recorded before (45 seconds) and after (150 seconds) delivery of 1nm capsaicin to DRG extracted from mice with EAP and compared to naive cohorts. (G) EAP treated animals showed a significant increase in [Ca2+]i compared to control. (F and H) However, [Ca2+]i remained the same in DRG from PAR2 KO-EAP compared to PAR2 KO-Naive at day 30. Calcium experiments were repeated independently at least three times. Scale bars represent 50µm and (*) denotes p<0.05.

Fig. 6. PAR2 loss does not inhibit inflammation and mast cell activation in EAP mice

(A) Representative H&E staining of the prostate excised from (panel a) EAP mice and (panel b) PAR2 KO with EAP at day 30 show leukocytic influx at day 30. Also, mouse prostate sections were stained with acidified toluidine blue and showed the same number of mast cells in (panel c) B6 mice with EAP and (panel d) PAR2 KO with EAP. (B) Inflammation score was assessed from H&E staining sections of the prostates obtained from B6 EAP and PAR2 KO with EAP at day 30 and were quantified by a blinded observer. (C) Resting mast cells, partially activated, and activated mast cells were also quantified and show no difference between groups. Data reflect mean ± SEM for 3 non-serial sections from 3 animals.

Fig. 7. Anti-PAR2 antibody (SAM11) reduced pelvic pain

NOD mice (5 per group) were induced with EAP for 20 days and pain development was confirmed at day 20 followed by intraperitoneal administration of saline or a PAR2 neutralizing antibody (SAM11). Mice assessed for tactile hyperalgesia/allodynia with von Frey filaments at day 0 (baseline) day 20 and after injection with (A) saline or (B) SAM11 at day 3, 7, and 10. (C) Graph depicts percent change in total responses from day 20 EAP at day 3, 7, and 10. Data represent the mean ± SEM. (*) and (**) denotes p<0.05 and p<0.01, respectively. Behavioral experiments were performed independently two times.