Cancer Res Treat.  2014 Jul;46(3):297-306.

Interleukin-17 Indirectly Promotes M2 Macrophage Differentiation through Stimulation of COX-2/PGE2 Pathway in the Cancer Cells

• Li Q
• Liu L
• Zhang Q
• Liu S
• Ge D
• You Z

Abstract

PURPOSE
Interleukin-17 (IL-17) is a proinflammatory cytokine that plays important roles in inflammation, autoimmunity, and cancer. The purpose of this study was to determine if IL-17 indirectly regulates macrophage differentiation through up-regulation of cyclooxygenase-2 (COX-2) expression in the cancer cell lines.
MATERIALS AND METHODS
Human cervical cancer HeLa, human lung cancer A549, and mouse prostate cancer Myc-CaP/CR cell lines were treated with recombinant IL-17; Western blot analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction analysis were utilized to examine the cellular responses.
RESULTS
IL-17 up-regulated expression of COX-2 mRNA and protein in HeLa, A549, and Myc-CaP/CR cell lines. IL-17's effects were mediated through nuclear factor-kappaB and ERK1/2 signaling pathways as the inhibitors of these pathways could inhibit IL-17-induced COX-2 expression. The conditional medium obtained from the cancer cells contained prostaglandin E2, the levels of which were increased by IL-17 treatment. When treated with the conditional medium, particularly with the IL-17-induced conditional medium, mouse RAW264.7 macrophages and human THP-1 monocytes expressed higher levels of IL-10 (a marker of M2 macrophages) than inducible nitric oxide synthase or tumor necrosis factor alpha (markers of M1 macrophages). In contrast, when RAW264.7 and THP-1 cells were treated directly with IL-17, expression of these marker genes was not markedly changed.
CONCLUSION
The results of this study suggest that IL-17 indirectly promotes M2 macrophage differentiation through stimulation of the COX-2/PGE2 pathway in the cancer cells, thus IL-17 plays an indirect role in regulating the tumor immune microenvironment.

Keyword

Interleukin-17; Cyclooxygenase-2; Dinoprostone; Neoplasms; Macrophages; Tumor microenvironment

MeSH Terms

Animals
Autoimmunity
Blotting, Western
Cell Line
Cyclooxygenase 2
Dinoprostone
Enzyme-Linked Immunosorbent Assay
Humans
Inflammation
Interleukin-10
Interleukin-17*
Lung Neoplasms
Macrophages*
Mice
Monocytes
Nitric Oxide Synthase Type II
Prostatic Neoplasms
Real-Time Polymerase Chain Reaction
RNA, Messenger
Tumor Microenvironment
Tumor Necrosis Factor-alpha
Up-Regulation
Uterine Cervical Neoplasms
Cyclooxygenase 2
Dinoprostone
Interleukin-10
Interleukin-17
Nitric Oxide Synthase Type II
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1. Interleukin (IL)-17 up-regulates expression of cyclooxygenase-2 (COX-2) mRNA and protein in cancer cells. (A) The cancer cells were treated without or with 20 ng/mL IL-17A for 2 hours; COX-2 mRNA levels were determined by real-time quantitative reverse transcriptase polymerase chain reaction. (B, D, F) The cancer cells were treated with 20 ng/mL IL-17A; COX-2 protein levels were determined by Western blot analysis. (C, E, G) Quantification of Western blot signals in three independent experiments. The ratio represents COX-2 signal divided by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal, where ratio=1 means that COX-2 signal is equal to GAPDH signal. Values are presented as the mean± standard deviation obtained from three independent experiments. *p < 0.05, compared to the control group.

• 

  Fig. 2. Interleukin (IL)-17 activates nuclear factor-κB and/or extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathways in cancer cells. HeLa cells (A), A549 cells (B), and Myc-CaP/CR cells (C) were treated without or with 20 ng/mL IL-17A; protein levels were determined by Western blot analysis. For protein loading control, the blots were stripped and reprobed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

• 

  Fig. 3. U0126 and pyrrolidine dithiocarbamate (PDTC) inhibit interleukin (IL)-17-induced cyclooxygenase-2 (COX-2) protein expression in cancer cells. (A, B) The cancer cells were treated without or with 100 μM nuclear factor-κB (NF-κB) inhibitor PDTC or 10 μM MEK inhibitor U0126 for 30 minutes prior to addition of recombinant IL-17A (20 ng/mL) for 12 hours treatment. COX-2 protein expression was determined by Western blot analysis. (C, D) Quantification of Western blot signals in three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Values are presented as the mean±standard deviation obtained from three independent experiments. *p < 0.05, compared to the IL-17A treatment group.

• 

  Fig. 4. Interleukin (IL)-17 induces prostaglandin E2 (PGE2) secretion in cancer cells. The cancer cells were serum starved for 15 hours and then treated without or with 20 ng/mL IL-17A for 24 hours; PGE2 levels were determined using an enzyme-linked immunosorbent assay kit. Values are presented as the mean±standard deviation obtained from three independent experiments. *p < 0.05, compared to the control group.

• 

  Fig. 5. Interleukin (IL)-17A-induced conditional medium increases expression of marker genes of M2 macrophages. The conditional medium (CM) or IL-17A-induced CM from human HeLa cancer cells (A) or human A549 cancer cells (B) was used to treat mouse RAW264.7 macrophages; the CM or IL-17A-induced CM from mouse Myc-CaP/CR cancer cells (C) was used to treat human THP-1 monocytes. The control group was treated with serum-free medium that had not been exposed to any cells. The IL-17A group was treated with 20 ng/mL IL-17A in serum-free medium. After 3 hours treatment, mRNA levels of the genes were determined by real-time quantitative reverse transcriptase polymerase chain reaction. Inducible nitric oxide synthase (iNOS) and tumor necrosis factor α (TNFα) are markers for M1 macrophages, and IL-10 and arginase I are markers for M2 macrophages. Values are presented as the mean±standard deviation obtained from three independent experiments. *p < 0.05 or **p < 0.01, compared to the groups as indicated.

Reference

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