Increased expression of interleukin-6 family members and receptors in urinary bladder with cyclophosphamide-induced bladder inflammation in female rats

Abstract

Recent studies suggest that janus-activated kinases-signal transducer and activator of transcription signaling pathways contribute to increased voiding frequency and referred pain of cyclophosphamide (CYP)-induced cystitis in rats. Potential upstream chemical mediator(s) that may be activated by CYP-induced cystitis to stimulate JAK/STAT signaling are not known in detail. In these studies, members of the interleukin (IL)-6 family of cytokines including, leukemia inhibitory factor (LIF), IL-6, and ciliary neurotrophic factor (CNTF) and associated receptors, IL-6 receptor (R) α, LIFR, and gp130 were examined in the urinary bladder in control and CYP-treated rats. Cytokine and receptor transcript and protein expression and distribution were determined in urinary bladder after CYP-induced cystitis using quantitative, real-time polymerase chain reaction (Q-PCR), western blotting, and immunohistochemistry. Acute (4 h; 150 mg/kg; i.p.), intermediate (48 h; 150 mg/kg; i.p.), or chronic (75 mg/kg; i.p., once every 3 days for 10 days) cystitis was induced in adult, female Wistar rats with CYP treatment. Q-PCR analyses revealed significant (p ≤ 0.01) CYP duration- and tissue- (e.g., urothelium, detrusor) dependent increases in LIF, IL-6, IL-6Rα, LIFR, and gp130 mRNA expression. Western blotting demonstrated significant (p ≤ 0.01) increases in IL-6, LIF, and gp130 protein expression in whole urinary bladder with CYP treatment. CYP-induced cystitis significantly (p ≤ 0.01) increased LIF-immunoreactivity (IR) in urothelium, detrusor, and suburothelial plexus whereas increased gp130-IR was only observed in urothelium and detrusor. These studies suggest that IL-6 and LIF may be potential upstream chemical mediators that activate JAK/STAT signaling in urinary bladder pathways.

Keywords: LIF; Q-PCR; Western blotting; detrusor smooth muscle; gp130; urothelium.

Figure 1

Regulation of interleukin (IL)-6 type cytokines and receptors in urothelium and detrusor smooth muscle with cyclophosphamide (CYP)-induced cystitis. IL-6 (A), leukemia inhibitory factor [LIF; (B)], ciliary neurotrophic factor [CNTF; (C)], IL-6 receptor [R; (D)] α, LIFR (E), and gp130 (F) transcript level in control and after 4 h, 48 h, and chronic (8 days CYP-induced bladder inflammation. (A–F) Summary histograms of relative expression of the transcript expressed in urothelium and detrusor smooth muscle. Urothelium samples were set equal to 100% and normalized to the relative expression of the housekeeping gene, L32. Detrusor samples were expressed relative to urothelium samples and normalized to the relative expression of the housekeeping gene, L32. n = 6 for each group; *p ≤ 0.01 versus control or as indicated between groups by horizontal lines.

Figure 2

Interleukin-6, LIF, and gp130 protein expression in whole bladder using Western blotting. (A) Representative Western blot examples of IL-6, LIF, and gp130 expression in whole urinary bladders (25 μg) from control rats and those treated with cyclophosphamide (CYP) of varying duration. Actin expression was used as a loading control. (B–D) Summary histogram of relative IL-6 (B), LIF (C), gp130 (D) band density in each group normalized to total actin expressed as a percentage of control (no inflammation). CYP-induced cystitis significantly (p ≤ 0.01) increased IL-6, LIF, and gp130 expression in comparison to control urinary bladder. *p ≤ 0.01 versus control or as indicated between groups by horizontal lines. n = 6 for each group.

Figure 3

Leukemia inhibitory factor-immunoreactivity (IR) in cryostat sections of urothelium (U) after varying durations of CYP treatment. CYP treatment [4 h (B), 48 h (C,D), chronic (E,F)] significantly (p ≤ 0.05–0.01) upregulated the percent of LIF-IR in U in comparison control (A). Higher power fluorescence images of selected regions (white dashed boxes) in (C,E) are shown in (D,F). For all images, exposure times were held constant, and all tissues were processed simultaneously. In 4 h, 48 h, and chronic CYP-treated rats, LIF expression was visible in the U (B–F) and lamina propria [LP; (B–D)], whereas control (A) urinary bladder showed little or no LIF-IR. Calibration bar represents 50 μm in (A,C,E,G) and 25 μm in (B,D,F,G): Immunoabsorptions with LIF peptide (5 μg/ml) and antisera in bladder sections resulted in no staining above background. (H) Histogram of the percent of LIF expression above threshold in the urothelium of CYP-treated rats (4 h, 48 h, and chronic) expressed as a percentage of control. CYP treatment (4 h, 48 h, and chronic) significantly (#, p ≤ 0.05; *, p ≤ 0.01) upregulated LIF-IR in the urothelium. sm, smooth muscle. Data are a summary of n = 6 for each group.

Figure 4

Fluorescence photographs of LIF-immunoreactivity (IR) in urothelial cells and suburothelial plexus in the bladder neck region in whole mount preparations of the urinary bladder. In control (A) whole mount preparations, LIF-IR is weak compared to preparations from 48 h CYP-treated (B), and chronic CYP-treated (C) rats. LIF-IR in the suburothelial nerve plexus in the neck region increased in density with CYP-induced cystitis [48 h (E) and chronic (F)] compared to control (D). Arrows (D,E) and arrowheads (F) indicate small or large caliber LIF-IR nerve fibers respectively. With CYP treatment [chronic, (G–I)], LIF-IR in nerve fibers (G) in the suburothelial plexus exhibited complete overlap (I) with PGP9.5 (H), a pan neuronal marker. Calibration bar represents 80 μm.

Figure 5

Fluorescence photographs of LIF-immunoreactivity (IR) in detrusor smooth muscle in the bladder neck region in whole mount preparations of the urinary bladder in control (A), 4 h CYP-treated (B), 48 h CYP-treated (C), and chronic CYP-treated (D) rats. LIF-IR in the detrusor smooth muscle increased in density with CYP-induced cystitis (4 h, 48 h, chronic). (E) Summary histogram of percent LIF expression above threshold in detrusor smooth muscle with CYP-induced cystitis. Data are a summary of n = 6 for each group. *p ≤ 0.01 versus control. Calibration bar represents 80 μm.

Figure 6

gp130-immunoreactivity (IR) in urothelium (U) and increased expression with CYP-induced cystitis. (A,B) gp130-IR in control U is weakly expressed and is restricted to apical urothelial cells (arrows). With 4 h (C,D), 48 h (E,F), and chronic (G,H) CYP-induced cystitis, gp130-IR is increased in intensity and additional urothelial layers (i.e., intermediate) express gp130-IR. gp130-IR is absent from the lamina propria (LP) of the urinary bladder from control or CYP-treated rats. (I) Summary histogram of percent gp130 expression above threshold in U with CYP-induced cystitis. Data are a summary of n = 6 for each group. *p ≤ 0.01 versus control. L, lumen. Calibration bar represents 80 μm in (A,C,E,G) and 45 μm in (B,D,F,H).

Figure 7

gp130-Immunoreactivity (IR) in detrusor smooth muscle (sm) and increased expression with CYP-induced cystitis. (A) gp130-IR in control sm is very weakly expressed. With 4 h (B), 48 h (C–E) and chronic CYP-induced cystitis, gp130-IR is increased in intensity throughout the sm. (F) Immunoabsorptions with gp130 peptide (5 μg/ml) and antisera in bladder sections resulted in no staining above background. (G) Summary histogram of percent gp130 expression above threshold in sm with CYP-induced cystitis. Data are a summary of n = 6 for each group. *p ≤ 0.01 versus control. S, serosa. Calibration bar represents 80 μm in (A–C,F), 45 μm in (D), and 25 μm in (E).