Background
There are about 400,000 individuals with bladder cancer in the world and 150,000 patients die from the disease every year [
1,
2]. In the United States bladder cancer is the 5th most common type of cancer with 72,500 new cases and 15, 200 deaths occurring in 2013 [
3], while cystitis and polyp are considered as high-risk for bladder cancer [
4]. It has been shown that multiple bladder polyps and cystitis are easy to develop malignant disease depending on the scope and duration of those relative diseases [
5]. Although many factors may be associated with the pathogenesis and mechanisms of above diseases, some cytokines, including tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17) cytokine family and interferon (IFN) are considerably involved in the occurrence and development of cystitis, bladder polyp and bladder cancer [
6,
7].
The IL-17 cytokine family includes six ligands (IL-17A to IL-17 F) and five receptors (IL-17RA to IL-17RE). Because of their unique and distinct biological functions, most studies are focused on IL-17A and IL-17E in tumors [
8,
9]. In addition, IL-17 F has also been studied because of its high molecular homology and similar biological functions with IL-17A [
10]. Previous studies have shown that both IL-17A and IL-17E can bind to their receptors IL-17RA and IL-17RB, while IL-17 F can bind to its own receptor IL-17RC and/or IL-17RA to fulfill their biological function. It has been indicated that IL-17A and IL-17 F are key pro-inflammatory cytokines in the pathogenesis of inflammatory and autoimmune diseases [
11]. Compared with IL-17A/IL-17 F, IL-17E plays an important role in the pathogenesis of asthma and atopic diseases through binding to the heterodimeric complex of IL-17RA/IL-17RB [
12]. On the other hand, some studies have also indicated the paradoxes of the pro-tumor or anti-tumor activity of IL-17 family relative ligands [
11,
13]. Previous data have shown that macrophages secreting IL-17 family cytokines may play important roles in the pathogenesis of malignant tumors [
14]. For example, it has been shown that IL-17A transcripts in peripheral blood mononuclear cells [
15] and serum concentrations of IL-17A [
16] were significantly higher in peripheral blood mononuclear cells in subjects with bladder cancer than those of controls. In animal experiments, it has been reported that IL-17A promoted bladder cancer growth [
17], while IL-17-producing γδ T cells possibly play a key role in the Bacillus Calmette-Guéri (BCG)-induced recruitment of neutrophils to the bladder, which is essential for the antitumor activity against bladder cancer [
18]. However, the expression and location of other IL-17 family cytokines and their receptors, and their relationships to bladder relative disease progression, inflammatory cellular infiltration and structural changes are still largely unclear in cystitis, bladder polyp and bladder cancer.
In this study we expanded our previous observations in prostate cancer [
19] and hypothesized that in bladder tissues, both infiltrating inflammatory cells and structural cells can express IL-17 family cytokines and relevant receptors, and that such expressions can affect not only tissue remodelling but also angiogenesis, which are associated with disease severity and tumorigenesis. We compared expression and location of IL-17 cytokine family IL-17A, IL-17E and IL-17 F and their receptors IL-17RA, IL-17RB and IL-17RC in tissues derived from subjects with cystitis, bladder polyp and bladder cancer in parallel. We also analyzed the relationships between expression of these IL-17 family cytokines and their receptors, infiltration of inflammatory cells and changes of structural cells in these diseases.
Discussion
Although a numbers of previous studies seem to prove that IL-17 family cytokines have the capability to promote occurrence and development of cystitis, polyp and bladder cancer [
22‐
25], there is a lack of systematically comparison study of IL-17 family ligands and their corresponding receptors in these urinary system diseases. Here we analyzed the IL-17 family cytokines IL-17A, E and F and their receptors IL-17RA, RB and RC in bladder tissues from patients with cystitis, polyp and bladder cancer. We also simultaneously examined inflammatory cellular infiltration, structural changes and angiogenesis in these urinary disorders.
Accumulation of infiltrating inflammatory cells is a typical feature of cystitis, possibly in bladder cancer as well. However, proinflammatory responses are double- edged sword with protective and tumorgenesis roles. On the one hand, such accumulated phagocytes might cause further inflammation in bladder tissues, which possibly exerts an anti-tumor activity, through generating more inflammatory mediators, including IL-17 family cytokines. On the other hand, these mediators and cytokines, in turn, might promote and enhance the abnormal cellular proliferation associated with polyp and bladder cancer, through generating various mediators, including inflammatory cytokines such as interleukin-6 (IL-6) and cellular growth factor (TGF-β) [
23,
25,
26]. Our data showed that expression of IL-17 cytokine family ligands and their relevant receptors was accompanied with markedly distinct profiles of infiltration of inflammatory cells in various conditions of urinary disorders. For example, elevated expressions of IL-17A, IL-17 F and IL-17RC and increased numbers of macrophages were observed in bladder cancer. One possible explanation may be that IL-17A and IL-17 F and IL-17RC can be expressed by these macrophages in bladder cancer. However, expression of IL-17A, IL-17RA and IL-17RC also increased in polyps but with the downgrade of numbers of macrophages. This suggests that macrophages might not be major source of these members of IL-17 family in polyps.
Additionally, other changed abnormal structural cells such as endothelial cells and fibroblasts were also observed in bladder cancer, which might contribute to the increases of immunoreactivity for IL-17A, IL-17 F and IL-17RC. It has been known that IL-17A and IL-17 F can bind to both IL-17RA and IL-17RC. For example, IL-17 F exerts effects on angiogenesis, while the elevated vessels in turn contribute to elevation of IL-17A or IL-17 F production in bladder cancer [
27], which formats a positive feedback to promote malignant proliferation. In the present study, expression of IL-17A and IL-17 F increased but IL-17RA decreased in bladder cancer, suggesting that IL-17A and IL-17 F are possibly expressed by different profiles of cellular populations and mainly through binding IL-17RC to exert biological effects. Although we observed that some immunoreactivity for IL-17A and IL-17 F located in malignant cells, however, until far, there is lack of systemic study whether malignant cells in bladder cancer express IL-17A or IL-17 F. Clearly further experiments remain to be performed in this aspect.
IL-17E, through binding to its own receptor IL-17RB or IL-17RA, shows different properties from IL-17A and IL-17 F in tumor fields. Our data here showed that IL-17E and IL-17RB expression was elevated in cystitis but reduced in bladder cancer, possibly indicating that IL-17E might also be involved in the pathogenesis of benign urinary diseases. On one hand, IL-17E might participate in the pathogenesis of inflammation through acting on its receptor IL-17RB expressed on inflammatory cells and structural cells, which results in inflammation and alternation of structural cells. On the other hand, it is interesting that reduced expression of IL-17E and IL-17RB in bladder cancer. Such reduction in bladder cancer is possibly because the damaged or abnormal structural cells and malignant cells express less IL-17E and its receptor IL-17RB. This might affect anti-tumor effect of IL-17E in bladder cancer. Again, however, the details and underlying mechanisms of IL-17E in the pathogenesis of bladder cancer remain to be explored.
Our results also show that there were more vascular endothelial cells in bladder cancer compared with cystitis. It is well known that in bladder cancer, over-proliferation of cancer cells need more blood supplying, while these malignant cells and increased angiogenesis might affect other cell proliferation. On the other hand, vascular endothelial cells can express IL-17 family cytokine which might attract more inflammatory cells into malignant tissues and promote occurrence and development of bladder cancer. Apart of blood vessels, increased immunoreactivity for CD90
+ fibroblasts was observed in bladder cancer, suggesting that these fibroblasts might also contribute to expression of IL-17 family ligands and receptors and possibly to pathogenesis of bladder cancer, through enhancing tumor growth [
28,
29].
Since inflammatory microenvironment is a feature in urinary tract diseases while the relative members of IL-17 cytokine family closely link with inflammation, we also examined the status of common inflammatory cell types in the present study. The increased numbers of CD3
+ T lymphocytes and CD68
+ macrophage in bladder cancer suggest that these cells might participate in the process of bladder cancer. Macrophage is an important component of the inflammatory and tumor microenvironment and plays a key role in the progression of tumors. It has been shown that macrophage has dual function according to different tumor types [
30]. Its function is, however, extremely complex and has not been elucidated till now. Neutrophil infiltration is a typical characteristic of acute inflammation. We also observed that there were more neutrophils in cystitis tissue than polyp and cancer, accompanied with increased IL-17RA, IL-17E and IL-17RB expression. Although neutrophils might not express IL-17E, these cells do express IL-17RA/or IL-17RB.
In addition, close correlations of expression of IL-17A-IL-17RA and IL-17E-IL-17RB suggest that different signaling of IL-17A-IL-17RA and IL-17E-IL-17RB might play an important role in bladder cancer. It is known that smooth muscle can express IL-17E. Thus, it is reasonably to presume that these cells might be a major cellular source of IL-17E, which may partly explain that the decreased IL-17E expression is a part of result due to the reduction of smooth muscle in bladder cancer. At the mean time, slight but significant correlation between immunoreactivity of IL-17 F and the numbers of CD3+ T lymphocytes was observed in bladder cancer, suggesting that CD3+ T lymphocytes might also be a major cellular source expressing IL-17 F. Whether these IL-17 F+ CD3+ T cells are a subgroup of Th17 cells remains to be investigated.
Obviously our study has some limitations. Firstly, it is still unknown for certain whether the specific index assessed by immunohistochemistry has been accurately identified. Clearly other experiments need to be done to confirm the roles of IL-17A and IL-17E signals in bladder cancer occurrence and development. Secondly, there were no entirely normal bladder specimens as controls, which might affect the comparison among the groups.
Acknowledgements
We thanked Pro Hongwen Gao in the Second Affiliated Hospital, Norman Bethune Medical School of Jilin University, for his help in determination of pathological results.