Prostate secretions from men with chronic pelvic pain syndrome inhibit proinflammatory mediators

Abstract

Purpose: In the past numerous chemokines have been noted in the expressed prostatic secretions of patients with chronic prostatitis/chronic pelvic pain syndrome. We examined the functional effects of chemokines in expressed prostatic secretions of patients with chronic pelvic pain syndrome.

Materials and methods: We studied the functional effects of expressed prostatic secretions on human monocytes by examining monocyte chemotaxis in response to monocyte chemoattractant protein-1, a major chemoattractant previously identified in chronic prostatitis/chronic pelvic pain syndrome cases. We determined effects on cellular signaling by quantifying intracellular calcium increase in monocytes and nuclear factor-κB activation in normal prostate epithelial cells.

Results: Results show that the monocyte chemoattractant protein-1 in expressed prostatic secretions is nonfunctional with an inability to mediate human monocyte chemotaxis, or mediate signaling in monocytes or prostate epithelial cells. This lack of functionality could be extended to other proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, when incubated with expressed prostatic secretions from patients with chronic pelvic pain syndrome. The mechanism underlying this apparent ability to modulate proinflammatory cytokines involves heat labile extracellular proteases that mediate the inhibition of immune and prostate epithelial cell function.

Conclusions: These results may have implications for the design of specific diagnostic and therapeutic methods targeted toward the complete resolution of prostate inflammatory insults.

Figure 1. EPS from CPPS patients inhibit MCP-1 induced chemotaxis of human monocytes

Pooled EPS from CPPS or BPH patients with 0, 1–10 or 10–25 leukocytes per high power field (HPF) were compared with EPS from control patients with non-prostate disease (penile pain). Chemotaxis was measured with phosphate buffered saline alone (negative control) and FMLP (positive control). EPS from CPPS, BPH were assayed initially by ELISA for MCP-1 levels followed by use in the chemotaxis assay. EPS diluted 1:10 in PBS, EPS spiked with MCP-1 and PBS spiked with MCP-1 were used to stimulate chemotaxis of human monocytes. The presence of EPS from CPPS patients significantly inhibited MCP-1 induced monocyte chemotaxis compared to MCP-1 in PBS (A). No such inhibition was observed upon incubation with prostatic fluid from BPH patients (B). Data is representative of at least three separate experiments with similar results. Statistical significance was examined by ANOVA and is indicated by *p<0.05 or **p<0.001.

Figure 2. EPS from CPPS patients inhibit MCP-1 induced intracellular calcium elevation in human monocytes

Human monocytes were loaded with fura-2 AM dye and intracellular calcium elevation was imaged using ratiometric methods. Monocytes treated with 10ng/ml of recombinant MCP-1 (arrow) demonstrated a rapid increase in intracellular calcium levels (A) that was however attenuated if the treatment was done simultaneously with EPS from CPPS patients (A). When the MCP-1 was preincubated with EPS for 30 minutes before stimulation, calcium elevation was completely abolished (B). Repeated administration of MCP-1 (2nd stimulation) did not elicit any further calcium increase in EPS treated monocytes (B). Data shown is representative of at least three separate experiments with similar results.

Figure 3. EPS from CPPS patients modulates NF-κB activation in benign prostate epithelial cells

The proinflammatory cytokines IL-1β (1ng/ml), MCP-1(50ng/ml) and TNFα (1ng/ml) were used to stimulate NF-κB activation in RWPE-1 cells stably transformed with an NF-κB luciferase reporter construct. Cell cultures were treated for 4 hours either with the cytokine alone in PBS or in the presence of three different dilutions (1:10, 1:100 and 1:1000) of pooled EPS from CPPS patients. EPS significantly inhibited IL-1β (A), MCP-1 (B) and TNFα mediated NF-κB activation in a concentration-dependent manner. In contrast, pretreatment of EPS with heat (56 °C for 30 minutes) abrogated the ability of EPS to inhibit IL-1β mediated NF-κB activation (D). Data shown is the mean +/− SEM from three separate experiments. Statistical significance is indicated at *p<0.05 or **p<0.001.

Figure 4. EPS modulation of MCP-1 signaling is sensitive to protease inhibition

THP-1 cells loaded with 5 μM Fluo-4 AM were treated with 10ng/ml of recombinant human MCP-1 and imaged at 488 nm using real-time video fluorescence microscopy and maximal fluorescence after MCP-1 treatment was subtracted from baseline. Preincubation of MCP-1 with CPPS EPS for 30 minutes prior to stimulation of THP-1 cells significantly (p<0.001) inhibited intracellular calcium elevation (A). Pretreatment of EPS with a protease inhibitor or fetal bovine serum (FBS) removed the inhibitory effect of EPS pretreatment on MCP-1 induced intracellular calcium elevation (B). Pretreatment of EPS with an MMP inhibitor (GM6001, 10μM) retained the inhibition of MCP-1 induced intracellular calcium elevation. Data shown is the mean +/− SEM from three separate experiments. Statistical significance is indicated at *p<0.05 or **p<0.001.