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Keywords:

  • γδ T cells;
  • Bladder tumor;
  • BCG;
  • IL-17;
  • Neutrophils

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Intravesical inoculation of Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used for the treatment of bladder cancer. Recent studies implied the requirement of neutrophil infiltration for the antitumor effect. In this study, we found that IL-17 was produced in the bladder after BCG treatment, preceding the infiltration of neutrophils. Neutrophils in the bladder after BCG treatment were reduced in IL-17-deficient mice, in which BCG-induced antitumor effect against intravesically inoculated bladder cancer was abolished. Notably, the level of IL-17 production and the number of neutrophils in BCG-treated bladder was reduced in γδ T-cell-deficient mice but not in CD4-depleted mice. Survival of bladder cancer-inoculated γδ T-cell-deficient mice was not improved by BCG treatment. These results suggest that IL-17-producing γδ T cells play a key role in the BCG-induced recruitment of neutrophils to the bladder, which is essential for the antitumor activity against bladder cancer.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

In 1976, Morales et al. reported intravesical inoculation of Mycobacterium bovis BCG as an effective adjuvant therapy for bladder cancers 1. Thereafter, intravesical immunotherapy with BCG has been used for 30 years, however the antitumor effector mechanisms remain elusive. Recent studies demonstrated that neutrophils infiltrated in the bladder after BCG treatment played a key role in the antitumor effect 2. Expression of TRAIL on neutrophils in voided urine following BCG therapy suggests a direct antitumor effect of neutrophils 3, 4. In addition, neutrophils isolated from BCG-treated bladder produced CC (e.g. MIP-1α) as well as CXC chemokines (e.g. IL-8 and GRO-α). The chemokines released by activated neutrophils attract monocytes, which in turn result in BCG-induced CD4 T-cell-migration 2. Th1-polarized cell-mediated immunity, which includes NK cells, and CD8+ and CD4+ T cells, was also involved in the antitumor effect of BCG immunotherapy 5–7. Thus, neutrophils might exert antitumor effect directly and indirectly. However, at present, the mechanism of neutrophil infiltration after BCG treatment is not fully understood.

IL-17 (also known as IL-17A) is a T-cell-derived proinflammatory cytokine, which is involved in various pathogenesis where neutrophils are involved. IL-17 induces mobilization of neutrophils indirectly via production of several cytokines, growth factors, and CXC chemokines 8. IL-17 is produced by a recently identified subset of helper CD4+ T cells, Th17 cells, which contribute to various inflammatory disorder as well as host defense 9. However, increasing evidence revealed that another subset of T cells, namely γδ T cells, could even play a dominant role as the source of IL-17 in vivo. We found that γδ T cells in the peritoneal cavity produced IL-17 immediately after Escherichia coli infection, which is critical to the infiltration of neutrophils 10. Furthermore, it was reported that IL-17 production in pulmonary infection with BCG was mediated by γδ T cells 11. In the present study, we found BCG treatment in murine bladder also induced IL-17 production by γδ T cells, which play essential role in local neutrophil infiltration and antitumor effect against bladder cancer.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Vesical neutrophil infiltration after BCG treatment is mediated by IL-17

Recent studies demonstrated that neutrophils infiltrated in the bladder after BCG treatment played a key role in the antitumor effect 2. In this study, we first examined the kinetics of neutrophil infiltration induced by weekly treatment with BCG. Significant infiltration of neutrophils was observed from one wk after starting BCG treatment, and it gradually increased during the observation period (Fig. 1A). We then examined intravesical IL-17 production after single BCG administration. As shown in Fig. 1B, IL-17 production was induced as early as 1 day after BCG injection, but lasted less than 5 days. During the course of repeated BCG administration, similar level of IL-17 production was induced after each injection (Fig. 1C). In order to determine the importance of IL-17 in the infiltration of neutrophils after BCG treatment, we examined the number of intravesical neutrophils in IL-17-deficient mice 22 day after starting BCG treatment. Infiltration of neutrophils was significantly reduced in IL-17-deficient mice (Fig. 2A). Therefore, IL-17 was involved in the infiltration of neutrophils into the bladder after BCG treatment.

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Figure 1. Intravesical neutrophil infiltration and IL-17 production after BCG treatment. B6 mice were intravesically injected with BCG or PBS on days 0, 7, 14, and 21. On days 0, 1, 8, 15, and 22, the number of neutrophils (A) and the level of IL-17 production (C) in the bladder were analyzed. Each group consisted of five mice. (B) Kinetics of the level of IL-17 production after single injection of BCG in the bladder (n=3). Representative data of three separate experiments are shown. *p<0.05, Student's t-test.

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Figure 2. Involvement of IL-17 in the neutrophil infiltration and antitumor effect of BCG treatment. (A) B6 and IL-17KO mice were intravesically injected with BCG or PBS on days 0, 7, 14, and 21. On day 22, the number of neutrophils in the bladder was analyzed. Each group consisted of five mice. *p<0.05, Student's t-test. (B) B6 and IL-17KO mice were intravesically inoculated with 1×105 MB49 tumor cells on day 0 and received weekly intravesical injections of BCG or PBS from day 1 to 22. Each group consisted of five mice. Representative data of three separate experiments are shown. *p<0.05 compared with other three groups, log-rank test.

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Requirement for IL-17 in the antitumor effects of intravesical BCG treatment

To examine the significance of IL-17-induced neutrophil infiltration in the antitumor effect of BCG therapy, IL-17 KO mice were inoculated with MB49 bladder cancer cells before BCG treatment (Fig. 2B). The control B6 mice treated with BCG exhibited significantly longer survival compared to PBS-treated mice. On the other hand, there was no difference in the survival between BCG- and PBS-treated IL-17-deficient mice. There was also no difference in the survival of PBS-treated B6 and IL-17-deficient mice. We confirmed that depletion of neutrophils completely abrogated the antitumor effect of BCG therapy (data not shown), as was previously demonstrated by others 2. Thus, it was revealed that IL-17-induced neutrophil infiltration was essential for the antitumor effect of intravesical treatment of BCG.

In contrast to our results, there have been reports implicating IL-17 with tumor progression. By acting on stromal cells and fibroblasts, IL-17 induces angiogenesis factors, which enhances tumor growth 12, 13. It is possible that the antitumor effect of neutrophil infiltration overwhelmed the tumor-promoting effect of IL-17 in the case of bladder tumor. Alternative explanation for the discrepancy was the short duration of IL-17 production after each injection of BCG, which might not be enough for the tumor-promoting effect (Fig. 1B). In addition, there are reports showing tumor-inhibitory effects of IL-17 14–17. Further investigation is necessary to identify factors that dictate anti- versus pro-tumor effects of IL-17 18.

γδ T cells are the major source of IL-17 production after BCG treatment

In order to identify the cell subset(s) responsible for the IL-17 production after BCG treatment, we harvested mononuclear cells in the bladder of BCG- or PBS-treated mice at day 22 and performed flow cytometric analysis of ex vivo intracellular staining for IL-17. We detected CD3+ cells producing IL-17 in BCG-treated bladder, and the IL-17+ cells were mostly TCR γδ+ (Fig. 3A). To directly address which cell population is important as the source of IL-17, we measured IL-17 production and neutrophil infiltration in the bladder of γδ T-cell-deficient mice (CδKO), and CD4 or NK1.1-depleted mice (Fig. 3B and D). We found that BCG-treated CδKO mice showed significant reduction of IL-17 production and neutrophil infiltration compared with BCG-treated control mice. On the other hand, there was no difference in either IL-17 production or neutrophil count between CD4 or NK cell-depleted mice and the control mice. These results revealed that γδ T cells significantly contributed to IL-17 production that induced recruitment of neutrophlis to the bladder after BCG treatment. Similar to our results, IL-17 production by tumor infiltrating γδ T cells was recently reported in a model of mouse sarcoma, although IL-17 supported tumor progression via angiogenesis in this case 19. In order to define the cellular source of the remaining IL-17 production in BCG-treated CδKO mice, we performed flow cytometric analysis but failed to detect cells positive for IL-17 (data not shown).

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Figure 3. Importance of γδ T cells in IL-17 production, neutrophil infiltration, and the antitumor effect of BCG treatment. (A) Flow cytometric analysis of IL-17 production by the lymphocytes in the bladder of PBS (left)- or BCG (right)-treated mice was performed after brief in vitro culture without stimulation. Representative dot plots of IL-17 and TCR Cδ expression are shown after gating on CD3+ lymphocytes. The number in upper right quadrant indicates the percent of IL-17+ cells in TCR Cδ+ cells. (B) B6 (control) and CδKO mice were intravesically injected with BCG or PBS on days 0, 7, 14, and 21. A group of PBS (control) or BCG-treated B6 mice received repeated i.p. injection of anti-CD4 mAb (C), anti-NK1.1 mAb (D), or the respective isotype control mAbs on days −1, 6, 13, and 20. Depletion of the target cell populations after mAb administration was confirmed by flow cytometry (Supporting Information Fig. 1). The level of IL-17 production (left) and the number of neutrophils (right) were measured on day 22. Each group consisted of five mice. Representative data of three separate experiments are shown. *p<0.05, Student's t-test. (E) B6 and CδKO mice were intravesically inoculated with 1×105 MB49 tumor cells on day 0 and were received weekly intravesical injection with BCG or PBS from day 1 to 22. Each group consisted of five mice. Representative data of two separate experiments are shown. *p<0.05 compared with other three groups, log-rank test.

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We lastly examined the importance of γδ T cells in the antitumor effect of BCG treatment. As shown in Fig. 3E, BCG treatment prolonged the survival of the control B6 mice inoculated with MB49 tumor cells. However, survival of CδKO mice was not improved by BCG treatment. There was also no difference in the survival of PBS-treated WT and CδKO mice, indicating that antitumor effect of γδ T cells depends on BCG treatment. Taken together, these results indicated that IL-17 produced by γδ T cells plays a key role in the recruitment of neutrophlis to the bladder after BCG treatment, which is important for the antitumor effect against bladder tumor. Although the mechanism of IL-17 production by γδ T cells is not fully elucidated yet, an involvement of IL-23-signaling has been suggested 10, 11, 20. In agreement with this, we detected a significant level of IL-23 production in the bladder after BCG treatment (data not shown). However, because the antigens recognized by γδ T cells remain unclear, possible involvement of antigenic stimulation on BCG-induced IL-17 production by γδ T cells is not excluded.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

We found in this study that γδ T cells were involved in the antitumor effect of intravesical BCG treatment via IL-17 production. Interestingly, Yuasa et al. reported that intravesical administration of γδ T cells exerted antitumor activity against bladder tumor, which is thought to be mediated by the direct cytotoxic activity to the tumor cells 21. Importantly, human γδ T cells are also known for their antitumor effect 22. Because γδ T cells exert effector function in an MHC-unrestricted manner, these findings suggest that γδ T cells could be a good target of universally applicable immunotherapy against bladder cancer.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Mice

C57BL/6 (B6) mice were purchased from Japan SLC (Hamamatsu, Japan). CδKO and IL-17KO mice (B6 background) were kindly provided by Dr. S. Itohara and Dr. Y. Iwakura, respectively. The mice were bred in specific pathogen-free conditions in our institute. 6- to 8-wk-old female mice were used for the experiments. This study was approved by the Committee of Ethics on Animal Experiment in Faculty of Medicine, Kyushu University. Experiments were conducted under the control of the Guideline for Animal Experiment.

Tumor and tumor inplantation

The murine bladder cancer cell line, MB49, was kindly provided by Dr. T. L. Ratliff. The cells were cultured in RPMI-1640 containing 10% FCS at 37°C in a humidified 5% CO2 atmosphere and passaged 2–3 times weekly.

BCG treatment protocol

We used a well-defined murine syngeneic bladder tumor model 23. Briefly, mice were catheterized to receive an intravesical inoculate of 1×105 MB49 tumor cells on day 0. On days 1, 8, 15, and 22, mice were treated intravesically with either 3×106 CFU of BCG Connaught strain (Immucyst, kindly provided by Nippon kayaku, Tokyo, Japan) or PBS. Just after BCG or PBS injection, the urethra of the mice was ligated by 3-0 silk and released 3 h later.

Isolation of neutrophils and lymphocytes for flow cytometric analysis

To harvest neutrophils and lymphocytes, the bladder was minced to yield 1–2 mm pieces and were incubated in a mixture of 1 mg/mL collagenase (Invitrogen, Carlsbad, CA, USA) and 20 μg/mL DNase (Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640 containing 10% FCS for 90 min at 37°C. The following antibodies were used for flow cytometric analysis: FITC-conjugated anti-Gr-1 (RB6-8C5), anti-TCR Cδ (GL3), and anti-CD4 (RM4-5) mAbs, PE-conjugated anti-I-A/E (M5/114.15.2), anti-NK1.1 (PK136), anti-CD8 (53-6.7) mAbs, allophycocyanin-conjugated anti-CD3e (145-2C11) mAb (BD Biosciences, San Diego, CA, USA), and PE-conjugated donkey anti-mouse IgG polyclonal antibody (eBioscience, San Diego, CA, USA). Stained cells were run on a FACS Calibur flow cytometer (BD Biosciences) after adding propidium iodide (1 μg/mL) in order to exclude the dead cells. The data were analyzed using Cell Quest software (BD Biosciences).

Intracellular cytokine staining

Freshly isolated lymphocytes from the bladder were immediately incubated with 10 μg/mL befeldin A (Sigma-Aldrich) in RPMI containing 10% FCS at 37°C for 6 h. Cells were first stained with mAbs for surface molecules and were then fixed and permeabilized using BD Perm/Wash solution (BD Biosciences) and stained with PE-conjugated anti-mIL-17 (TC11-18H10.1) mAb (BD Biosciences).

In vivo depletion of neutrophils, CD4+ cells, and NK1.1+ cells

Two hundred micrograms of anti-Gr-1 mAb (RB6-8C5), anti-CD4 mAb (GK1.5), or anti-NK mAb (PK136) or the isotype control mAb was given i.p. every 7 days. Depletion of each cell subset was confirmed by flow cytometric analysis.

ELISA for measurement of IL-17 production

Bladders were dissected from BCG-treated or PBS-treated mice and minced in 200 μL of PBS. After a centrifugation, IL-17 in the supernatant was measured by mouse IL-17 DuoSet ELISA Development System (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.

Statistical analyses

Survival of mice was evaluated using Kaplan–Meier plots and the log-rank test. Difference in the amounts of IL-17 production or neutrophil counts were analyzed by Student's t-test using GraphPad Prism 5.0 software (Prism Graphpad, San Diego, CA, USA). Differences with p values of <0.05 were considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for Promotion of Science (H. Y. and Y. Y.), and by the program of Founding Research Centers for Emerging and Reemerging Infectious Diseases launched as a project commissioned by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (Y. Y.).

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Concluding remarks
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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