Elevated epithelial expression of interleukin-8 correlates with myofibroblast reactive stroma in benign prostatic hyperplasia

Abstract

Objectives: Numerous inflammatory diseases display elevated interleukin (IL)-8, and most are associated with a reactive stroma. IL-8 expression is also elevated in benign prostatic hyperplasia (BPH), yet little is known about reactive stroma in BPH. Whether a reactive stroma response exists in BPH, whether this correlates with elevated IL-8, and whether IL-8 can induce a reactive stroma phenotype have not been determined. This study was designed to specifically address these issues.

Methods: Normal prostate transition zone tissue and BPH specimens, as identified by the Baylor College of Medicine pathology department, were examined by quantitative immunohistochemistry to correlate IL-8, smooth muscle alpha-actin, vimentin, calponin, and tenascin-C. In addition, human prostate stromal cell cultures were used to evaluate the effect of IL-8 on the expression of reactive stroma biomarkers.

Results: BPH nodules exhibited elevated epithelial IL-8 immunoreactivity, and this correlated with elevated smooth muscle alpha-actin, reduced calponin, and altered deposition of tenascin-C, relative to the normal prostate transition zone tissue (P <0.05). Multiple vimentin-positive prostate stromal fibroblast cultures were induced by IL-8 to also co-express smooth muscle alpha-actin and tenascin-C, typical of a reactive stroma myofibroblast phenotype.

Conclusions: These data show that BPH reactive stroma is fundamentally different from normal prostate fibromuscular stroma and is typified by the emergence of a reactive stroma myofibroblast phenotype. This reactive stroma pattern correlated spatially with IL-8 elevation in adjacent epithelium. Additionally, IL-8 induced expression of myofibroblast markers in human prostate fibroblasts in vitro. These results suggest that IL-8 acts as a regulator of BPH reactive stroma and is therefore a potential therapeutic target.

Fig. 1. Elevated IL-8 immunoreactivity in the epithelium associates with reactive stroma in BPH, but not with smooth muscle stroma in normal transition zone tissue

Serial NPTZ (A-C & G-I) and BPH (D-F & J-L) tissue sections were IF dual-labeled for: IL-8 (B,C,E,F; red) and tenascin-C (A,C,D,F; green), or α-SMA (H,I,K,L; red) and calponin (G,I,J,L; green) with overlap-expression in orange. Nuclei are labeled with DAPI (blue). Images are representative of typical staining patterns seen in BPH (N=5) and NPTZ (N=5) tissues. D-F & J-L at X400 in order to capture entire periacinar stroma; A-C & G-I at X600. All scale bars = 50 μm.

Fig. 2. Quantitative IHC for IL-8 and reactive stroma biomarkers

Serial NPTZ (A,B) and BPH (C,D) tissue sections were IHC stained for: IL-8 (A&C) or calponin (B&D) and sections were counterstained with hematoxylin. Images are representative of typical IHC staining patterns seen in all BPH (N=50) and NPTZ (N=28) specimens. Images acquired at X600, all scale bars = 50 μm. For quantitation (E-H), boxes in gray represent significant differences (p<0.05) between tissue types. Data are represented as a distribution illustrated using a box-and-whisker plot, where the whiskers represent all data analyzed, excluding outliers (represented by black dots), the black line indicates the median, and the box represents 25% of data above and below the median. E, IL-8 is significantly elevated in all BPH nodules, except S nodules, when compared to NPTZ (p<0.05). The summed score for quantitation of IL-8 is shown for each tissue type listed. Data represent N = 28 NPTZ, 47 E1, 13 E2, 22 E1/E2, 12 S and 5 S/E specimens, respectively. F, Calponin is significantly lower in stromal cells in all BPH nodule types when compared to NPTZ (p<0.05). Data represent N = 28 NPTZ, 45 E1, 14 E2, 23 E1/E2, 12 S and 5 S/E specimens, respectively. G, α-SMA is significantly different in stromal cells, when compared to NPTZ, in all BPH nodules except S/E nodules (p<0.05). Data represent N = 28 NPTZ, 48 E1, 14 E2, 24 E1/E2, 11 S and 5 S/E specimens, respectively. H, Tenascin-C is significantly lower in S BPH nodules when compared to NPTZ (p<0.05). Data represent N = 28 NPTZ, 47 E1, 14 E2, 23 E1/E2, 9 S and 5 S/E specimens, respectively. Vimentin exhibited no significant fluctuation between NPTZ and all epithelial BPH nodules (data not shown).

Fig. 3. IL-8 induces a myofibroblast phenotype in human prostate fibroblasts

PrSC cells (A-F), HPS-19I cells (G-L) and HTS-2T cells (M-R), human prostate stromal cells, were treated with IL-8 (38pM, 4nM & 28nM; 38pM shown) or vehicle (PBS + 1 % BSA) in M₀ and analyzed by IF ICC: vimentin (red) A,D,G,J,M&P and α-SMA (green) B,E,H,K,N&Q. Vehicle treated PrSC and HPS-19I cells displayed a fibroblast phenotype, as evidenced by expression of vimentin (A & G, respectively), and no expression of α-SMA (B & H, respectively). In contrast, IL-8 induced the myofibroblast phenotype, as evidenced by expression of α-SMA in PrSC (E, green) and HPS-19I (K, green) cells. HTS-2T cells, whether treated with vehicle (M-O) or IL-8 (P-R) were strongly positive for vimentin (red, M&P) and α-SMA (green, Q&R). Nuclei are stained with DAPI (blue). Data are representative of N = 9 experiments (N = 3 coverslips/condition, per independent experiment, with N = 3 independent experiments). A-L at X200, M-R at X400; Scale bars = 50 μm. Western blot (S) for changes in α-SMA levels after 48hr or 72hr of treatment with IL-8 (38 pM, 4 nM, 28 nM) or vehicle (PBS + 1 % BSA) in M₀. For all 3 cell cultures, α-SMA immunoreactive bands are shown above the same lanes re-probed for GAPDH. Each pair of bands, at 48hr and 72hr, displays vehicle-treated sample on the left and IL8-treated sample on the right. Band intensity (normalized to GAPDH) was quantitated using NIH Image J software.

Fig. 4. IL-8 induces tenascin-C, a marker of reactive stroma, in human prostate fibroblasts

PrSC cells (A-B), HPS-19I cells (C-D) and HTS-2T cells (E-F), human prostate stromal cells, were treated with vehicle (PBS + 1 % BSA) or IL-8 (38pM, 4nM & 28nM; 38pM is shown) in M₀ and analyzed by IF ICC for tenascin-C (green). Vehicle treated PrSC and HPS-19I cells displayed a non-reactive phenotype, as evidenced by lack of tenascin-C expression (A & C, respectively), In contrast, IL-8 induced the reactive stroma phenotype, as evidenced by altered and more focal immunoreactivity for tenascin-C deposition in PrSC (B, green) and HPS-19I (D, green) cells. HTS-2T cells, whether treated with vehicle (E) or IL-8 (E), displayed a similar, more diffuse deposition of tenascin-C. Nuclei are stained with DAPI (blue). Data are representative of N = 9 experiments (N = 3 coverslips/condition, per independent experiment, with N = 3 independent experiments). Images acquired at X200; Scale bars = 50 μm.