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* Department of Geriatric and Respiratory Medicine and
Department of Anatomic Pathology, Tohoku University School of Medicine, Sendai, Japan;
Department of Pathology and
Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan;
¶ Department of Experimental Immunology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan;
|| Laboratory of Lung Immunology and Host Defense, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15213; and
# Translational Research, Molecular Medicine Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and IL-1
by activated macrophages (12). IL-17R is a type 1 transmembrane protein with an extraordinarily long intracellular domain (2, 13). Although the expression of IL-17 mRNA is restricted to activated T cells, the expression of IL-17R mRNA has been detected in virtually all cells and tissues (2, 13). In its role as a proinflammatory cytokine, IL-17 levels have been found to significantly increase in rheumatoid arthritis synovium, asthmatic airways, during allograft rejection, and in other chronic inflammatory diseases, including multiple sclerosis and psoriasis (14, 15, 16, 17, 18). IL-17 has been also implicated in the tumors. Ciree et al. (19) reported that cutaneous T cell lymphomas spontaneously secrete IL-17, which is associated with infiltration of polymorphonuclear neutrophils into inflamed tissues. Kato et al. (20) reported that a considerable proportion of ovarian carcinomas naturally express IL-17 and its expression significantly correlates with the increased vascularity. However, until now, the possible role for IL-17 in non-small cell lung cancer (NSCLC) has not been elucidated.
The salient feature of solid tumor growth such as NSCLC is the strict dependence on the sustained new vessel growth (21). Hanahan and Folkman (22) proposed that quiescent endothelium exists in a net balance of angiostatic factors over angiogenic ones, thereby maintaining homeostasis. Conversely, tumors themselves, interacting stromal cells and/or responding inflammatory cells, may cause an imbalance to increase the secretion of angiogenic inducers or decrease the production or effect of angiogenic suppressors. Although there are a wide variety of putative mediators of angiogenesis, certain tumors have been found to produce factors that are directly angiogenic while others may depend upon vascularization induced by products of infiltrating inflammatory cells (23).
NSCLC cells have been reported to produce a wide variety of angiogenic factors, including CXCL1/growth related oncogene-, CXCL5/epithelial cell-derived neutrophil-activating protein-78, CXCL6/granulocyte chemotactic protein-2, CXCL8/IL-8, VEGF-A, fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF)-BB, TGF-, CXCL12/stromal derived factor-1, angiopoietin-2, and PGE2 over the course of their growth (24, 25, 26, 27, 28, 29, 30, 31, 32). Especially, multiple studies have established that angiogenic CXC chemokines are critically involved in the angiogenic activity of NSCLC (22, 33, 34). Arenberg et al. (33) reported that inhibition of the action of CXCL8 markedly suppressed the in vivo growth of NSCLC in SCID mice model. Arenberg et al. (34) also showed that neutralization of CXCL5 in vivo significantly suppressed the growth of NSCLC. In contrast, tumor-derived CXCL10/IFN-inducible protein-10 is an important endogenous angiostatic factor in NSCLC, which negatively regulates tumor angiogenesis and growth (35).
IL-17 induces CXCL8 production from various cell types and CXCL8 is involved in the angiogenic activity of NSCLC. Therefore, we hypothesized that IL-17 might enhance the net angiogenic activity and promote the tumorigenicity of NSCLC. To address this issue, we examined the biological effect of IL-17 on NSCLC and found that IL-17 selectively enhanced the production of an array of angiogenic CXC chemokines, including CXCL1, CXCL5, CXCL6, and CXCL8 but not angiostatic chemokines, from NSCLC lines in vitro. Therefore, these characteristic biologic activities of IL-17 prompted us to further investigate the role for this cytokine in in vivo biologic action of NSCLC. In this study, we show that IL-17 significantly enhances the net angiogenic activity and in vivo growth of NSCLC through promoting CXCR-2-dependent angiogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Male SCID mice, 5–7 wk of age, were obtained from Charles River Laboratories. Human IL-17 cDNA was supplied by Schering-Plough. Recombinant human IL-17 and IL-23 proteins and anti-human IL-17, IL-17R, CXCL1, CXCL5, CXCL8, VEGF-A, HGF, CXCR-1, and CXCR-2 mAbs were purchased from R&D Systems. Anti-human IFN-
mAb was supplied by Mabtech. Goat anti-human IL-17R polyclonal Ab was from Santa Cruz Biotechnology. Rabbit anti-mouse CXCR-2-neutralizing polyclonal Ab was supplied by Dr. K. Matsushima (University of Tokyo, Tokyo, Japan).
Human NSCLC cell lines, cell cultures, and conditioned medium (CM)
Human NSCLC lines Sq-19 (squamous cell carcinoma), A549 (alveolar cell carcinoma), and LK-87 (adenocarcinoma) were supplied from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (Tohoku University). These cells were maintained in RPMI 1640 with 10% FCS, 2 mM L-glutamine, 0.1 mM nonessential amino acid (NEAA), 100 IU/ml penicillin, and 100 µg/ml streptomycin (all from Invitrogen Life Technologies). Human lung microvascular endothelial cells (LMVECs) were purchased from Cambrex and maintained in HuMedia-EB2 with 10 ng/ml epidermal growth factor, 1 µg/ml hydrocortisone, 5 ng/ml basic FGF, 10 µg/ml heparin, 39.3 µg/ml dibutyryl cAMP, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, and 5% FCS (all from Kurabo). CM was generated as follows. Cells (1 x 105/ml) were cultured in HuMedia-EB2 containing 3% FCS with or without 50 ng/ml IL-17 for 60 h. Cell-free supernatants were collected and stored at –70°C until use.
Clinical materials
Tumor tissues (n = 77) were obtained from patients with NSCLC, who underwent complete resection at surgery without any preoperative therapy.
Flow cytometry
Cells (1 x 106), washed twice with PBS containing 2% FCS and 0.1% NaN3, were incubated with mouse anti-human IL-17R mAb or with irrelevant normal mouse IgG1 (R&D Systems) at 4°C for 60 min. After washing, the cells were incubated with PE-conjugated goat anti-mouse IgG1 Ab (Caltag Laboratories) at 4°C for 30 min. The cells were analyzed with FACSCalibur (BD Biosciences).
MTT assay
Cells (1 x 103) were seeded into 96-well flat-bottom plates and cultured in RPMI 1640 containing 3% FCS, 2 mM L-glutamine, 0.1 mM NEAA, 100 IU/ml penicillin, and 100 µg/ml streptomycin with or without 0.1–1000 ng/ml human IL-17. On day 5 or 7, cells were washed with RPMI 1640, and 100 µl of MTT (Sigma-Aldrich) solution (2.5 mg/ml in RPMI 1640 with 5% FCS) were added to each well. Plates were incubated for 50 min. Next, MTT solution was removed, and 50 µl of DMSO (Sigma-Aldrich) were added to each well to solubilize formazan crystals formed in viable cells. The absorbance was read at a wavelength of 590 nm on an ELISA plate reader.
Cytokine assay
Cells (1 x 105/ml) were cultured in RPMI 1640 containing 5% FCS, 2 mM L-glutamine, 0.1 mM NEAA, 100 IU/ml penicillin, and 100 µg/ml streptomycin with or without IL-17 or IL-17 plus 5 µg/ml anti-IL-17 mAb at indicated concentrations for 48 h. Cell-free supernatants were collected and stored at –70°C. Concentrations of cytokines were measured using commercially available ELISA kits (R&D Systems). PGE2 concentration was measured as reported previously (36).
Migration assay
Migration of LMVECs was evaluated using a modified Boyden chamber assay, as described previously (37). Briefly, LMVECs were cultured in HuMedia-EB2 with 2% FCS for 8 h and plated at 12 x 104 cells/cm2 onto the polycarbonate filter with 5-µm pores (Kurabo) coated with 10 µg/ml fibronectin (Sigma-Aldrich). CM from NSCLC unstimulated or stimulated with 50 ng/ml IL-17 (NSCLC/IL-17) was applied in the lower compartments of the chamber. LMVECs were cultured for 4 h at 37°C. The filters were fixed and stained with Diff-Quick (Harleco), and the number of migrating cells was quantified by counting cells in five randomly selected high-powered fields (HPFs) (x200) in each well. For the inhibitory assay, neutralizing mAb(s) (5 µg/ml each) was added in the upper and lower compartments of the chamber.
Construction of expression vector carrying human IL-17 cDNA
A BamHI-XbaI fragment of the PCR product of IL-17 cDNA was ligated into the EcoRI site of pCRTM2.1. The insert DNA was excised from the plasmid with BamHI/XbaI and inserted into the multiple cloning site of pRc/CMV. A549 and Sq-19 cells were transfected with the human IL-17 gene expression plasmid using LipofectAMINE (Invitrogen Life Technologies) and selected in RPMI 1640 with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM NEAA, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 1000 µg/ml G418. As a control, a vector carrying only neomycin phosphotransferase gene (Neo) was used.
In vitro cell growth assay
To examine the in vitro growth, cells (5 x 104) were seeded in 10-cm culture plates on day 0, and the cell number was counted on days 2, 3, 4, 5, and 6.
Human NSCLC-SCID mouse chimeras
Eight- to 10-wk-old male SCID mice were inoculated s.c. with 7 x 106 Sq-19WT, Sq-19Neo, or Sq-19IL-17 cells or with 1 x 107 A549WT, A549Neo, or A549IL-17 cells into the right flank. The animals were maintained under sterile conditions in laminar flow rooms. In some experiments, mice were treated with either control Ab or rabbit polyclonal Ab specific for mouse CXCR-2 every 4 days. On day 25 for Sq-19 or on day 40 for A549, blood was collected from the mice, kept overnight at 4°C, and then centrifuged. The serum was stored at –70°C until use. Tumor volume (mm3) was calculated using the formula a x b2/2 (a = largest diameter; b = smallest diameter) (38).
Immunohistochemical staining for Ki-67 in human NSCLC tissues from SCID mice
Analysis of the biological effect of IL-17 on the cell proliferation of NSCLC in vivo was performed with Ki-67 immunostaining, using MIB-1 Ab (Immunotech). Sections were deparaffinized and autoclaved in acetate buffer at 120°C for 5 min. After cooling, slides were incubated overnight at 4°C with MIB-1 or with normal mouse IgG (R&D Systems). After washing, sections were then incubated with biotinylated rabbit anti-mouse IgG and HRP-conjugated streptavidin (Nichirei, Tokyo, Japan). Sections were developed with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. The proliferating cells were estimated by the percentage of Ki-67-positive staining cells in 10 randomly chosen HPFs for each section (x400).
In situ detection of apoptosis
To detect apoptotic cells, the TUNEL method was performed using the WAKO in situ apoptosis detection kit (WAKO). Briefly, after deparaffinization, sections were incubated with a protein digestion enzyme for 5 min at 37°C. TdT with biotin-11-dUTP and dATP was then applied to the slides for 10 min at 37°C in a moist chamber. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 min at room temperature. Then, anti-biotin-11-dUTP labeling was conducted for 10 min at 37°C, followed by exposure to DAB. Sections were counterstained with methyl green. The apoptotic cells were estimated by the percentage of positive staining cells visualized in 10 randomly chosen HPFs for each section (x400).
Immunohistochemical staining for vascular endothelial cells in human NSCLC tissues and quantitation of vessel density
Immunostaining for CD31 was performed on frozen sections with rat anti-mouse CD31 mAb (BD Pharmingen). Briefly, frozen sections of NSCLC tissues from SCID mice were air-dried and incubated with 1% normal goat serum for 30 min at room temperature. Endogenous peroxidase activity was blocked using DakoCytomation peroxidase blocking reagent (DakoCytomation). Then, the primary Ab was applied overnight at 4°C. After washing, the sections were incubated with biotinylated rabbit anti-rat IgG Ab (DakoCytomation) for 30 min. After washing, peroxidase-conjugated streptavidin solution (DakoCytomation) was applied. Immunoreactivity was visualized by DAB and counterstained with hematoxylin.
Immunostaining for CD34 was performed on sections of NSCLC tissues from surgery, using the streptavidin-biotin amplification method (Histofine kit). Briefly, after deparaffinization, slides were treated with 3% hydrogen peroxidase in methanol for 10 min. Sections were then incubated with 1% normal goat serum for 30 min, followed by the application of mouse anti-human CD34 mAb (Nichirei) overnight at 4°C. Sections were treated with biotinylated anti-mouse IgG for 30 min, followed by peroxidase-conjugated streptavidin for 30 min. Immunoreactivity was visualized by DAB and counterstained with hematoxylin.
Sections were examined in a blinded fashion for the presence of CD31 or CD34 immunolocalization. According to the method described by Weidner et al. (39), specimens were scanned at low magnification (x40), and the 10 most vascularized tumor areas within a section were selected for evaluation of angiogenesis. Blood vessels stained with anti-mouse CD31 mAb or anti-human CD34 mAb were counted under light microscopy at x200. Any distinct area of positive staining for CD31 or CD34 was counted as a single regardless of size. The results are shown as mean ± SD per 10 HPFs per tumor section.
Immunohistochemical staining for IL-17, IL-17R, and CD3 in human NSCLC tissues
Immunohistochemical staining for IL-17 and CD3 was performed on sections of NSCLC tissues from surgery, using the streptavidin-biotin amplification method (Histofine kit). Goat polyclonal Ab for human IL-17 and rabbit polyclonal Ab for human CD3 were used as a primary Ab. The dilution of primary Ab used in this study was 1/500 for IL-17 and 1/100 for CD3. Ag retrieval was performed by heating the slides in a microwave oven for 15 min for IL-17 or by treatment with protease K (Sigma-Aldrich) for 10 min for CD3. The Ag-Ab complex was visualized with DAB and counterstained with hematoxylin. As a positive control, we used the sections from blocks of Formalin-fixed, paraffin-embedded, IL-23-stimulated human CD4 T cells. As a negative control, an immunohistochemical preabsorption test was performed in these specimens.
For IL-17R staining, goat anti-human IL-17R polyclonal Ab (Santa Cruz Biotechnology) was used to characterize the expression of IL-17R on primary NSCLC tissues. As a control, sections were incubated with normal goat IgG (R&D Systems). The blocking of nonspecific proteins was conducted with PBS containing 2% BSA. The staining was conducted using Cy-3-conjugated rabbit anti-goat Ab as a secondary Ab (Sigma-Aldrich) and ProLong Gold antifade reagent with 4',6'-diamidino-2-phenylindole (Molecular Probes) as a mounting medium. The pictures were captured by a camera attached to an Axioplan 2 universal imaging microscope (Intelligent Imaging Innovations) and were further analyzed with SlideBook 4.1 (Intelligent Imaging Innovations).
RT-PCR and quantitative RT-PCR
Total cellular RNA was extracted using Isogen (Nippon Gene) according to the manufacturer’s instructions. Five micrograms of total RNA was used for the synthesis of cDNAs with SuperScript RNaseH-Reverse Transcriptase (Invitrogen Life Technologies). PCR was performed in a DNA Thermal Cycler (PerkinElmer/Cetus) using Taq polymerase (Boehringer Mannheim). The primer sequences of the oligonucleotides used for PCR and PCR product sizes were as follows: -actin, sense, 5'-TCTGGTCAATGGAAGCCTGT-3', and antisense, 5'-CTGTGGTGGTGAAGCTGTAC-3', 436 bp; and human IL-17, sense, 5'-ACTCCTGGGAAGACCTCATTG-3', and antisense, 5'-GGCCACATGGTGGACAATCG-3', 461 bp. Real-time quantitative PCR (TaqMan PCR) using an ABI PRISM 7700 Sequence Detection System and TaqMan PCR Core Reagent kit (PerkinElmer) was performed according to the manufacturer’s protocol. Oligonucleotides and a probe specific for human IL-17 were purchased from PerkinElmer Biosystems. Internal standard gene expression was examined by using TaqMan -actin control reagents (PerkinElmer Biosystems). The amount of amplified IL-17 mRNA was correlated to that of -actin mRNA.
Statistical analysis
Statistical analysis was performed using an unpaired two-tailed Student’s t test with confirmation by parametric and F tests. Differences were considered to be statistically significant when the p value was <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The expression of IL-17R on three NSCLC cells was examined by flow cytometry using anti-human IL-17R mAb. These NSCLC cells expressed IL-17R on the surface at the protein level (Fig. 1). No staining was observed when an isotype matched control mAb was used (Fig. 1). These results are in accordance with the ubiquitous expression of IL-17R (2).
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Because NSCLC cells expressed IL-17R, we investigated whether IL-17 can modulate the phenotype of these cells. We first examined the possibility that IL-17 might modulate the in vitro growth rate of NSCLC. To determine whether IL-17 affects the in vitro growth, cells were cultured in the presence or absence of human IL-17 for 5 or 7 days. A wide range of doses of IL-17 had no direct effect on the in vitro growth of Sq-19 and A549 cells as shown in Fig. 2, A and B. These results indicate that IL-17 has no biological action to promote or suppress the growth of NSCLC cells.
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We next examined the possibility that IL-17 might modulate the secretion of angiogenic and angiostatic factors by NSCLC, which promote or suppress tumor angiogenesis and growth. NSCLC cells have been reported to naturally produce diverse angiogenic factors. Interestingly, IL-17 selectively up-regulated the production of NSCLC-derived several angiogenic CXC chemokines, including CXCL1, CXCL5, CXCL6 and CXCL8 (Fig. 3, A–C, and Table I). Especially, IL-17 markedly augmented the production of CXCL1, CXCL5, and CXCL8 (Fig. 3, A–C). To control these results, we demonstrated that anti-IL-17 mAb inhibited the up-regulated production (Fig. 3, A–C), whereas an isotype-matched control mAb had no effect (data not shown). In contrast, IL-17 did not significantly enhance the production of acidic FGF (aFGF), bFGF, VEGF-A, PDGF-BB, HGF, TGF-, angiopoietin-2, CXCL12, and PGE2 (Fig. 3, A–C, and Table I). Moreover, we could not detect the up-regulated secretion of angiostatic CXC chemokines CXCL9/monokine induced by IFN-, CXCL10, and CXCL11/IFN-inducible T cell chemoattractant from NSCLC lines with IL-17 (Table I). These results indicate that IL-17 selectively up-regulates the production of an array of angiogenic CXC chemokines, but not angiostatic CXC chemokines, from NSCLC.
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IL-17 selectively up-regulated the production of important angiogenic CXC chemokines of NSCLC. We therefore hypothesized that IL-17 might enhance the net angiogenic activity of NSCLC. To address this question, we performed endothelial cell chemotaxis assays on CM from NSCLC or NSCLC/IL-17. As shown in Fig. 4A, in comparison to CM from Sq-19, CM from Sq-19/IL-17 demonstrated a marked increase in angiogenic activity as assessed by endothelial cell chemotaxis. Similarly, CM from A549 demonstrated less angiogenic activity than CM from A549/IL-17 (Fig. 4B). These data clearly indicate that IL-17 significantly augments the net angiogenic activity of NSCLC measured as endothelial cell chemotactic activity.
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We hypothesized that the increased endothelial cell chemotaxis to CM from NSCLC/IL-17 was attributable to the observed increased secretion of angiogenic CXC chemokines by NSCLC with IL-17. To test this postulate, endothelial cell chemotaxis was performed with CM from NSCLC/IL-17 in the presence of neutralizing mAb(s) against CXCL1, CXCL5 and CXCL8, VEGF-A, HGF, IFN-, CXCR-1, or CXCR-2. We found that endothelial cell chemotaxis to CM from Sq-19/IL-17 was reduced significantly in the presence of neutralizing mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2 as compared with control IgG (Fig. 5A). On the contrary, adding a neutralizing mAb against VEGF-A, HGF, IFN-, or CXCR-1 did not significantly inhibit the increased endothelial cell chemotaxis. Similarly, endothelial cell chemotaxis to CM from A549/IL-17 in the presence of neutralizing mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2 was significantly less than that in the presence of control mouse IgG (Fig. 5B). These data demonstrate that IL-17 increases the net angiogenic activity of NSCLC through angiogenic CXC chemokine- and CXCR-2-dependent fashion.
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To evaluate the biological effect of IL-17 on NSCLC in detail, we generated the human IL-17 gene expression vector as described in the Materials and Methods. Two NSCLC lines, Sq-19 and A549, were transfected with human IL-17 gene using LipofectAMINE. No expression of either IL-17 mRNA or protein could be detected in Sq-19 and A549 cells before transfection (data not shown). After G418 selection, the expression of human IL-17 mRNA by stable transfectants was determined by RT-PCR (data not shown). Sq-19IL-17 or A549IL-17 secretes 67 or 35 ng/1 x 106 cells/48 h IL-17, respectively, determined by a commercially available ELISA kit (R&D Systems).
IL-17 significantly promotes the in vivo growth of NSCLC in SCID mice
Significant changes were not observed in the in vitro growth of IL-17 transfectants when compared with that of parental cells or Neo transfectants (Fig. 6A). When Sq-19WT, Sq-19Neo, or Sq-19IL-17 cells were implanted s.c. in SCID mice, they all formed solid tumors. However, Sq-19IL-17 developed tumors with a strikingly increased growth rate compared with controls (Sq-19WT vs Sq-19 IL-17: p < 0.0003; Sq-19Neo vs Sq-19IL-17: p < 0.0002, on day 30) (Fig. 6, B and D). A similar increase in in vivo tumor growth was observed when A549IL-17 was implanted in SCID mice as compared with controls (A549WT vs A549 IL-17: p < 0.0007; A549Neo vs A549 IL-17: p < 0.0006, on day 45) (Fig. 6, C and D). In in vitro cultures, A549 proliferated more rapidly than Sq-19, whereas Sq-19 grew much faster in vivo than A549 when transplanted in SCID mice. The representative photographs of the gross NSCLC tumors in SCID mice were shown in Fig. 6D.
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Although IL-17 enhanced the in vivo growth of NSCLC, the result raised the question as to how IL-17 promotes the in vivo growth of these tumors, inasmuch as IL-17 has no direct effect on the proliferation of NSCLC cells in vitro. To address this question and further investigate the mechanism by which the in vivo growth of NSCLC was enhanced with IL-17, NSCLC tumors were excised, and the proliferation index of tumor cells in situ was quantified by immunohistochemical staining for Ki-67 (Fig. 7, A–D). Although the volume of NSCLC tumors transduced with IL-17 increased to 
170% of the controls, the proliferation index did not significantly change (Fig. 7M). Thus, we speculated that IL-17 increases the in vivo growth by promoting tumor angiogenesis rather than stimulating the proliferation of NSCLC cells. To examine this possibility, we evaluated the vascular density by immunohistochemical examination of NSCLC tissues. Immunostaining for CD31 showed that tumor tissues of IL-17 transfectants were more markedly vascularized when compared with those of controls (Fig. 7, I–L). To compare the vascular density, the mean number of blood vessels of CD31-stained sections obtained from five independent tumors was determined. The mean number of microvessels of Sq-19IL-17 tumors was significantly higher than that of Sq-19WT or Sq-19Neo on day 25 (p < 0.007) (Fig. 7M). Similar results were obtained in A549 tumors on day 40 (p < 0.001) (Fig. 7M). These results indicate that the enhanced in vivo growth of IL-17 transfectants closely correlated with increased vascularity. Furthermore, the TUNEL assay showed that IL-17 led to a 0.34-fold decrease for Sq-19 and 0.47-fold decrease for A549 in the number of apoptotic cells (Fig. 7M). These findings are consistent with previous studies, which showed that angiogenesis stimulators or inhibitors can modulate the in vivo tumor growth by increasing or decreasing apoptosis of tumor cells (40).
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Because IL-17 selectively augments the secretion of angiogenic CXC chemokines by NSCLC and promotes tumor angiogenesis, we postulated that the increased growth of NSCLC with IL-17 was mediated by an enhanced production of angiogenic chemokines. Thus, we initially examined the serum concentrations of angiogenic CXC chemokines in SCID mice bearing NSCLC. As shown in Table II, the serum concentrations of CXCL1, CXCL5, and CXCL8 in mice bearing IL-17 transfectants increased in direct correlation with enlarged tumor size.
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To delineate the role for up-regulated production of angiogenic chemokines during tumorigenesis of IL-17 transfectants, animals were subjected to a strategy to block the biological action of angiogenic CXC chemokines. Because the CXC chemokine receptor 2, CXCR-2, has been reported to be the receptor accounting for CXC chemokine-induced angiogenic activity on human and mouse endothelium (41), SCID mice were treated with either control Ab or neutralizing Ab against mouse CXCR-2 at the time of inoculation and every 4 days for a period of 4 or 5 wk. As illustrated in Fig. 8A, Sq-19IL-17 tumor-bearing SCID mice treated with anti-CXCR-2 Ab demonstrated a marked reduction in tumor growth as compared with animals that were treated with control irrelevant Ab (control Ab vs anti-CXCR-2 Ab; p < 0.0001, on day 30). In addition, Sq-19Neo tumors in SCID mice treated with anti-CXCR-2 Ab grew more slowly than those in animals that were treated with control Ab (control Ab vs anti-CXCR-2 Ab; p < 0.007, on day 30). Similarly, administration of anti-CXCR-2 Ab into SCID mice with A549IL-17 tumors resulted in a significant reduction of tumor growth (control Ab vs anti-CXCR-2 Ab; p < 0.0003, on day 55) (Fig. 8B). Moreover, treatment with anti-CXCR-2 Ab significantly suppressed the growth of A549Neo in SCID mice (control Ab vs anti-CXCR-2 Ab; p < 0.01, on day 55). We also investigated the microvessel density and found that the treatment with anti-CXCR-2 Ab significantly reduced the tumor vascularity of Neo transfectants as well as IL-17 transfectants (Table III). These results indicate that the increased growth of NSCLC elicited by IL-17 is mediated by CXCR-2 signaling and that NSCLC growth is partly dependent on angiogenic CXC chemokines, which commonly bind to CXCR-2. These findings also suggest that the blocking of CXCR-2 signaling might be a promising strategy to treat patients with NSCLC.
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To further explore the possible role for IL-17 in promoting angiogenic activity of NSCLC, we investigated the IL-17 mRNA expression in freshly isolated surgical specimens of NSCLC and found that its expression was detected in 57% of the cases (44 of 77 samples) (Fig. 9A and data not shown). The levels of IL-17 mRNA expression assessed by quantitative RT-PCR were classified as follows: high > 0.15, low 
0.15, and undetectable 0.001. High levels of IL-17 expression were found in 17 cases, low in 27, and undetectable in 33. To investigate what types of cells produce IL-17 in primary NSCLC tissues, we performed an immunohistochemical analysis using anti-human IL-17 Ab. The strong immunoreactivity for IL-17 was found when high levels of IL-17 expression were detected, whereas the immunoreactivity was not observed when IL-17 expression was not detected (Fig. 9, D and E). Furthermore, in NSCLC tissues, the immunoreactivity for IL-17 was found only in the accumulating and infiltrating inflammatory cells but not in tumor cells (Fig. 9E). Fossiez et al. (7) reported that IL-17 transcripts are detected only in T cells upon activation. To examine whether the infiltrating T cells are the origin of IL-17 production, we stained the serial sections of NSCLC tissues using Ab against IL-17 or CD3. Surprisingly, the cells stained positively with anti-IL-17 Ab were not necessarily stained with anti-CD3 Ab (Fig. 9, F and G). These cells, which were stained positively with anti-IL-17 Ab and negatively with anti-CD3 Ab, had polymorphonuclear morphology (Fig. 9H). These findings indicated that, in NSCLC tissues, the IL-17-producing cells are T cells and polymorphonuclear neutrophils. In addition, we found that the presence of T cells in NSCLC tissues had no direct correlation with IL-17 expression. In some NSCLC cases, infiltrating T cells were stained positively with anti-IL-17 Ab, whereas, in other cases, T cells were not stained at all (data not shown). Moreover, there was no significant relationship between IL-17 expression and any clinicopathologic parameter such as stage or histological type (data not shown).
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Discussion |
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The CXC chemokines can be divided into two groups on the basis of the presence or absence of the glutamic acid-leucine-arginine (ELR) motif. CXC chemokines with the ELR motif are potent angiogenic factors that directly promote endothelial cell proliferation, chemotaxis, and tubular morphogenesis (42, 43). ELR+ CXC chemokines are also major angiogenic factors in NSCLC and murine models of human NSCLC (33, 34, 44). In contrast, CXC chemokines without the ELR motif such as CXCL9 and CXCL10 are potent angiostatic factors (45). Especially, CXCL10 is an important endogenous angiostatic factor in NSCLC, which negatively regulates the NSCLC-derived net angiogenic activity (35). The relative expression of angiogenic, as compared with angiostatic, members of the CXC chemokine family is an important determinant of the net angiogenic activity in NSCLC (24, 46). The IFNs such as IFN-, IFN-, and IFN- are potent agonists for the expression of CXCL10 from a variety of cells, including keratinocytes, fibroblasts, endothelial cells, mononuclear cells, and tumor cells (47, 48, 49). The IFNs are also potent inhibitors of the production of CXCL8 by monocytes, fibroblasts, and endothelial cells (50, 51, 52, 53). Conversely, IL-17 is a potent inducer of angiogenic chemokines from a number of cells, including keratinocytes, fibroblasts, epithelial cells, and tumor cells (2, 3, 4, 5, 6, 7, 8). IL-17 also inhibits TNF--induced CXCL10 secretion by fibroblasts (54). In this study, we indicated that IL-17 has the capability to selectively enhance the production of angiogenic CXC chemokines from NSCLC. Taken together, the IFNs may shift the local biologic balance between angiogenic and angiostatic CXC chemokines toward a predominance of angiostatic chemokines to reduce the net angiogenic activity, whereas IL-17 may promote the angiogenic activity of NSCLC by causing a biological imbalance to increase the production of angiogenic CXC chemokines and to suppress the secretion of angiostatic CXC chemokines.
To confirm the action of IL-17 in enhancing the net angiogenic activity of NSCLC via selectively up-regulated production of angiogenic CXC chemokines, we performed an endothelial cell chemotaxis assay on CM from NSCLC/IL-17. IL-17 markedly promoted NSCLC-induced endothelial chemotactic activity. To assess whether this biological property of IL-17 could be caused by angiogenic chemokines, we tested the inhibitory effects of neutralizing mAb(s) against angiogenic molecule(s) or their receptor and found that the inhibition of CXCL1, CXCL5, and CXCL8 or CXCR-2 abolished the enhanced endothelial cell chemotaxis to CM from NSCLC/IL-17. On the contrary, neutralization of VEGF-A, HGF, IFN-, or CXCR-1 did not significantly affect the increased endothelial chemotaxis in the presence of IL-17. Thus, it is confirmed that the enhanced angiogenic activity of NSCLC stimulated with IL-17 is mediated by an array of angiogenic CXC chemokines.
Because neutralization of angiogenic CXC chemokines or their receptor CXCR-2 in vivo could constitute the direct evidence of the role for these factors as mediators of IL-17, we administered the neutralizing anti-mouse CXCR-2 Ab into mice. Treatment with anti-CXCR-2 Ab led to a marked reduction in in vivo growth of IL-17 transfectants, confirming the findings in in vitro endothelial cell chemotaxis assay. Interestingly, treatment with anti-CXCR-2 Ab significantly suppressed the neovascularization and in vivo growth of Neo transfectants as well as IL-17 transfectants. These findings are consistent with the previous report demonstrating that the in vivo growth of Lewis lung cancer primary tumors significantly reduced in CXCR-2–/– mice as compared with that in control mice (55). For antiangiogenic therapy to be successful, it must inhibit diverse angiogenic stimuli produced by the tumor and its microenvironment. Thus, the attractive feature of targeting the common receptor CXCR-2 for the angiogenic CXC chemokines is the fact that it results in the inhibition of binding of several ELR+ chemokine ligands at once. From this point of view, targeting CXCR-2 signaling may be more effective than other monotherapies, which target a single mediator such as CXCL8, to treat patients with NSCLC. Moreover, combination antiangiogenesis protocols, which target both CXCR-2 and VEGF-A signalings, could greatly improve the therapeutic efficacy of NSCLC. Taken collectively, our results indicate that CXCR-2 signaling mainly mediates the angiogenic activity of NSCLC and highlights the importance of developing the novel strategies to target CXCR-2 signaling.
In the current study, we showed that IL-17 expression is frequently detected in NSCLC tissues. Moreover, our immunohistochemical analysis provided an additional evidence that, in NSCLC tissues, the infiltrating inflammatory cells, but not tumor cells, are producing IL-17. There have been reports demonstrating that T cells infiltrating into cervical and prostate carcinoma tissues are expressing IL-17 (56, 57). Interestingly, in NSCLC tissues, polymorphonuclear neutrophils in addition to T cells are producing IL-17. In addition, IL-17R is expressed on the surface of primary NSCLC cells. Thus, it is likely that IL-17, produced by both T cells and polymorphonuclear neutrophils in situ, may have some biological action on NSCLC cells in a paracrine fashion. Immunostaining for CD34 revealed that NSCLC tissues with high levels of IL-17 mRNA expression had significantly higher microvessel density than those in which IL-17 mRNA expression was not detected. These findings raised the possibility that the infiltrating inflammatory cells such as T cells and polymorphonuclear neutrophils may occasionally stimulate the production of angiogenic CXC chemokines by NSCLC via secretion of IL-17.
We recently reported that IL-17 is an angiogenic factor, which stimulates the migration and cord formation of vascular endothelial cells in vitro and elicits neovessel formation in vivo (37, 58). Thus, it is a little strange that the enhanced NSCLC growth mediated by IL-17 was largely impaired in mice treated with anti-CXCR-2 Ab. Although the elucidation of a precise mechanism by which IL-17 mediates angiogenesis in vivo is far from complete, neovessel formation elicited by IL-17 may be mostly mediated by the angiogenic CXC chemokine family, which shares a common chemokine receptor CXCR-2.
Although our study indicates that CD4 T cell cytokine may promote the in vivo NSCLC growth through enhancing CXCR-2-dependent angiogenesis, CD4 T cells have been thought, in general, to regulate angiogenesis negatively via production of IFN-, which in turn induces the production of angiostatic CXC chemokines (59, 60). CD4 T cells stimulated by IL-12 secrete IFN- and inhibit angiogenesis (61). In contrast, physiological regulation of IL-17 production by CD4 T cells has not been fully elucidated (62, 63). Taken collectively, our results suggest that CD4 T cells may have the regulatory ability to promote or suppress angiogenesis depending on the stimuli.
In conclusion, our findings illustrate the biological action of IL-17 on human NSCLC. IL-17 markedly enhances the net angiogenic activity and promotes the in vivo growth of NSCLC via selectively up-regulated production of an array of angiogenic CXC chemokines, which lead to an imbalance between angiogenesis promoters and inhibitors present within the vascular microenvironment. Our results also demonstrate that targeting CXCR-2 signaling may represent a potential therapeutic strategy against NSCLC. Further analyses are needed to elucidate the precise role for IL-17 in tumor angiogenesis and growth of NSCLC.
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1 This work was supported in part by a grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science (Grant 15590791; to M.N.). 
2 Address correspondence and reprint requests to Dr. Muneo Numasaki, Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. E-mail address: numasaki77{at}aol.com
3 Abbreviations used in this paper: VEGF-A, vascular endothelial growth factor-A; NSCLC, non-small cell lung cancer; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; CM, conditioned medium; NEAA, nonessential amino acid; LMVEC, lung microvascular endothelial cell; HPF, high-powered field; PDGF, platelet-derived growth factor; Neo, neomycin phosphotransferase gene; DAB, 3,3'-diaminobenzidine; ELR, glutamic acid-leucine-arginine; aFGF, acidic FGF.
Received for publication May 20, 2004. Accepted for publication August 3, 2005.
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and/or TNF-. Immunol. Lett. 95:175.-184. [Medline]
and/or TNF-. Immunol. Lett. 95:97.-104. [Medline]
-induced elaboration of proangiogenic factors from fibroblasts. Immunol. Lett. 93:39.-43. [Medline]
and TNF-, by human macrophages. J. Immunol. 160:3513.-3521. synergize in the enhancement of proinflammatory cytokine production by human keratinocytes. J. Invest. Dermatol. 111:645.-649. [Medline]
is independent of ras mutations in lung adenocarcinoma. Lung Cancer 29:151.-157. [Medline]
/CXCL12-CXCR 4 axis is involved in the dissemination of NSCLC cells into pleural space. Am. J. Respir. Cell Mol. Biol. 30:671.-677. -inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J. Exp. Med. 184:981.-992. expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC receptor-2 dependent mechanism. Oncogene 19:3477.-3486. [Medline]
