IL-17 enhances the net angiogenic activity and in vivo growth of human non-small cell lung cancer in SCID mice through promoting CXCR-2-dependent angiogenesis.
ABSTRACT In this study, we examined the biological action of IL-17 on human non-small cell lung cancer (NSCLC). Although IL-17 had no direct effect on the in vitro growth rate of NSCLC, IL-17 selectively augmented the secretion of an array of angiogenic CXC chemokines, including CXCL1, CXCL5, CXCL6, and CXCL8 but not angiostatic chemokines, by three different NSCLC lines. Endothelial cell chemotactic activity (as a measure of net angiogenic potential) was increased in response to conditioned medium from NSCLC stimulated with IL-17 compared with those from unstimulated NSCLC. Enhanced chemotactic activity was suppressed by neutralizing mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2 but not to vascular endothelial growth factor-A. Transfection with IL-17 into NSCLC had no effect on the in vitro growth, whereas IL-17 transfectants grew more rapidly compared with controls when transplanted in SCID mice. This IL-17-elicited enhancement of NSCLC growth was associated with increased tumor vascularity. Moreover, treatment with anti-mouse CXCR-2-neutralizing Ab significantly attenuated the growth of both neomycin phosphotransferase gene-transfected and IL-17-transfected NSCLC tumors in SCID mice. A potential role for IL-17 in modulation of the human NSCLC phenotype was supported by the findings that, in primary NSCLC tissues, IL-17 expression was frequently detected in accumulating and infiltrating inflammatory cells and that high levels of IL-17 expression were associated with increased tumor vascularity. These results demonstrate that IL-17 increases the net angiogenic activity and in vivo growth of NSCLC via promoting CXCR-2-dependent angiogenesis and suggest that targeting CXCR-2 signaling may be a novel promising strategy to treat patients with NSCLC.
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ABSTRACT: Although numerous recent studies have implicated a role for interleukin 17(IL17) in tumor development, the mechanisms of IL17 involvement are still uncharacterized. The aims of this study were to determine whether single nucleotide polymorphisms (SNPs) in IL17 and IL17R contribute to the development of papillary thyroid cancer (PTC) and to assess the relationship between IL17 and IL17R SNPs and the clinicopathologic characteristics of PTC. Eight SNPs located within the IL17A, IL17RA, and IL17RB genes were genotyped using direct sequencing in 94 patients with PTC and 260 patients without PTC (controls). Genetic data were analyzed using commercially available software. Statistical analyses were then performed to examine the relationships between these SNPs and the clinicopathologic characteristics of PTC. Genotyping analysis demonstrated that the IL17RA SNP rs4819554 (codominant model 1, odds ratio (OR)=0.39, P=0.001) and the IL17RB SNP rs1025689 (dominant model, OR=0.59, P=0.043) were significantly associated with lack of PTC. Interestingly, the IL17A SNP rs2275913 (codominant model 2, OR=0.19, P=0.034) was significantly associated with lack of multifocality. Furthermore, the IL17RA SNP rs4819554 (dominant model, OR=0.25, P=0.010) was significantly associated with lack of cancer bilaterality. In this study of SNPs in the IL17 and IL17R genes in patients with PTC, we demonstrated that IL17RA polymorphisms can influence both the development and the bilaterality of PTC. Copyright © 2014 Elsevier Ltd. All rights reserved.Cytokine 12/2014; 71(2):283-288. · 2.87 Impact Factor
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ABSTRACT: To investigate the expression of transcriptional factors (TFs) T-bet, GATA-3, RORγt and FOXP in peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) and to evaluate the correlation between the imbalances of Th1/Th2, Th17/Treg at the expression levels and liver cancer Methods: The peripheral venous blood was drawn from 20 HCC-patients (HCC-group) and 20 health participants (C-group). The expression levels of Th1, Th2 and Th17 and the major Treg-specific TFs T-bet, GATA-3, RORγt and FOXP3 in the PBMC were measured with quantitative real-time PCR(RT-qPCR). The mRNA level of Th1-specific TF T-bet in HCC-group was significantly lower than that of C-group (52.34±34.07 VS 104.01±56.00, P<0.01); the mRNA level of Th2-specifc TF, GATA-3, in HCC group was significantly higher than that in C-group (1.38±1.15 VS 0.58±0.65, P<0.05) and T-bet mRNA/GATA-3 mRNA ratio was significantly lower in HCC-group than in C-group (86.01±116.71 VS 461.88±708.81, P<0.05). The mRNA level of Th17-specific TF RORγt in HCC-group was significantly higher than that of C-group (72.32±32.82 VS 33.07±22.86, P<0.01). Treg-specific TF FOXP3 mRNA level was significant higher in HCC-group than in C-group (3.17±1.59 VS 1.39±1.13, P<0.01) CONCLUSION: T-bet mRNA level was reduced whereas GATA-3 mRNA level was increased and T-bet/GATA-3 ratio was significantly reduced in PBMC, indicating that Th1/Th2 ratio was of imbalance at TF levels in PBMC of HCC, displaying Th2 thrift phenomena. The mRNA levels of RORγt and FOXP3 in PBMC of HCC were significantly increased, indicating the existence of a predominant phenomenon of Th17- and Treg-expressing PBMC in HCC.International journal of medical sciences 01/2015; 12(1):7-16. · 1.55 Impact Factor
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ABSTRACT: The prognostic value of Interleukin 17 (IL-17) in cancer patients is currently under debate and remains inconclusive. We performed a systematic review and meta-analysis to evaluate the role of IL-17 as a prognostic marker in cancer. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were combined to measure the effective value of IL-17 expression on prognosis. Nineteen eligible studies enrolling 2390 patients were identified. We found expression of IL-17 was not significantly correlated with overall survival (OS) in cancer (HR=1.29, 95% Cl: 0.94-1.76; P=0.12). Furthermore, compared to the data from our analysis that high expression of IL-17 predicted poor OS in both non-small cell lung carcinoma (NSCLC) (HR=2.30; 95% CI: 1.45-3.64; P<0.001; I(2)=0%) and hepatocellular carcinoma (HCC) (HR=2.02; 95% CI: 1.44-2.83; P<0.001; I(2)=0%), high expression of IL-17 was associated with favorable OS in esophageal squamous cell carcinoma (ESCC) (HR=0.63; 95% CI: 0.51-0.79; P<0.001; I(2)=0%). This meta-analysis showed that IL-17 has the potential to become a novel prognostic marker in HCC, NSCLC and ESCC. It could potentially help to monitor patients' prognosis and assess therapeutic efficacy in clinical treatment.International Journal of Clinical and Experimental Medicine 01/2014; 7(10):3258-3269. · 1.42 Impact Factor
IL-17 Enhances the Net Angiogenic Activity and In Vivo
Growth of Human Non-Small Cell Lung Cancer in SCID Mice
through Promoting CXCR-2-Dependent Angiogenesis1
Muneo Numasaki,2* Mika Watanabe,‡Takashi Suzuki,†Hidenori Takahashi,*
Akira Nakamura,¶Florencia McAllister,?Takanori Hishinuma,§Junichi Goto,§
Michael T. Lotze,#Jay K. Kolls,?and Hidetada Sasaki*
In this study, we examined the biological action of IL-17 on human non-small cell lung cancer (NSCLC). Although IL-17 had no
direct effect on the in vitro growth rate of NSCLC, IL-17 selectively augmented the secretion of an array of angiogenic CXC
chemokines, including CXCL1, CXCL5, CXCL6, and CXCL8 but not angiostatic chemokines, by three different NSCLC lines.
Endothelial cell chemotactic activity (as a measure of net angiogenic potential) was increased in response to conditioned medium
from NSCLC stimulated with IL-17 compared with those from unstimulated NSCLC. Enhanced chemotactic activity was sup-
pressed by neutralizing mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2 but not to vascular endothelial growth factor-A.
Transfection with IL-17 into NSCLC had no effect on the in vitro growth, whereas IL-17 transfectants grew more rapidly
compared with controls when transplanted in SCID mice. This IL-17-elicited enhancement of NSCLC growth was associated with
increased tumor vascularity. Moreover, treatment with anti-mouse CXCR-2-neutralizing Ab significantly attenuated the growth
of both neomycin phosphotransferase gene-transfected and IL-17-transfected NSCLC tumors in SCID mice. A potential role for
IL-17 in modulation of the human NSCLC phenotype was supported by the findings that, in primary NSCLC tissues, IL-17
expression was frequently detected in accumulating and infiltrating inflammatory cells and that high levels of IL-17 expression
were associated with increased tumor vascularity. These results demonstrate that IL-17 increases the net angiogenic activity and
in vivo growth of NSCLC via promoting CXCR-2-dependent angiogenesis and suggest that targeting CXCR-2 signaling may be
a novel promising strategy to treat patients with NSCLC. The Journal of Immunology, 2005, 175: 6177–6189.
tropic Herpesvirus saimiri (2). Several recently discovered homol-
ogous proteins with similar or different biological profiles
including IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F form a
novel cytokine family (3–5). IL-17 has pleiotropic biological ac-
tivities including induction of IL-6, CXCL8/IL-8 and vascular en-
dothelial growth factor-A (VEGF-A),3as well as enhancement of
nterleukin 17, initially termed CTLA-8 (1), is produced
mainly by activated CD4 T cells (2) and has sequence ho-
mology with the open reading frame 13 of the T lympho-
ICAM-1 expression (2–11). In addition, IL-17 induces the secre-
tion of TNF-? 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
(14–18). IL-17 has been also implicated in the tumors. Ciree et al.
(19) reported that cutaneous T cell lymphomas spontaneously se-
crete IL-17, which is associated with infiltration of polymorpho-
nuclear neutrophils into inflamed tissues. Kato et al. (20) reported
that a considerable proportion of ovarian carcinomas naturally ex-
press IL-17 and its expression significantly correlates with the in-
creased vascularity. However, until now, the possible role for
IL-17 in non-small cell lung cancer (NSCLC) has not been
The salient feature of solid tumor growth such as NSCLC is the
strict dependence on the sustained new vessel growth (21). Hana-
han 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 sup-
pressors. Although there are a wide variety of putative mediators
of angiogenesis, certain tumors have been found to produce factors
*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 Immu-
nology and Host Defense, Department of Pediatrics, University of Pittsburgh, Pitts-
burgh, PA 15213; and#Translational Research, Molecular Medicine Institute, Uni-
versity of Pittsburgh School of Medicine, Pittsburgh, PA 15219
Received for publication May 20, 2004. Accepted for publication August 3, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This 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.).
2Address 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:
3Abbreviations 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?-diaminobenzi-
dine; ELR, glutamic acid-leucine-arginine; aFGF, acidic FGF.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
that are directly angiogenic while others may depend upon vascu-
larization induced by products of infiltrating inflammatory cells
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,
VEGF-A, fibroblast growth factors (FGFs), hepatocyte growth fac-
tor (HGF), platelet-derived growth factor (PDGF)-BB, TGF-?,
CXCL12/stromal derived factor-1?, angiopoietin-2, and PGE2
over the course of their growth (24–32). Especially, multiple stud-
ies have established that angiogenic CXC chemokines are criti-
cally 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 neutral-
ization of CXCL5 in vivo significantly suppressed the growth of
NSCLC. In contrast, tumor-derived CXCL10/IFN-inducible pro-
tein-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. There-
fore, 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 ac-
tivities 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-de-
Materials and Methods
Mice and reagents
Male SCID mice, 5–7 wk of age, were obtained from Charles River Lab-
oratories. Human IL-17 cDNA was supplied by Schering-Plough. Recom-
binant 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
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, Ag-
ing 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 ?g/ml streptomycin (all from In-
vitrogen 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 ?g/ml hydrocortisone, 5
ng/ml basic FGF, 10 ?g/ml heparin, 39.3 ?g/ml dibutyryl cAMP, 50 ?g/ml
gentamicin, 50 ng/ml amphotericin B, and 5% FCS (all from Kurabo). CM
was generated as follows. Cells (1 ? 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.
Tumor tissues (n ? 77) were obtained from patients with NSCLC, who
underwent complete resection at surgery without any preoperative therapy.
Cells (1 ? 106), washed twice with PBS containing 2% FCS and 0.1%
NaN3, were incubated with mouse anti-human IL-17R mAb or with irrel-
evant normal mouse IgG1 (R&D Systems) at 4°C for 60 min. After wash-
ing, 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).
Cells (1 ? 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 ?g/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 ?l 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 ?l 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
Cells (1 ? 105/ml) were cultured in RPMI 1640 containing 5% FCS, 2 mM
L-glutamine, 0.1 mM NEAA, 100 IU/ml penicillin, and 100 ?g/ml strep-
tomycin with or without IL-17 or IL-17 plus 5 ?g/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 com-
mercially available ELISA kits (R&D Systems). PGE2concentration was
measured as reported previously (36).
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 ? 104cells/cm2onto
the polycarbonate filter with 5-?m pores (Kurabo) coated with 10 ?g/ml
fibronectin (Sigma-Aldrich). CM from NSCLC unstimulated or stimulated
with 50 ng/ml IL-17 (NSCLC/IL-17) was applied in the lower compart-
ments 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 mi-
grating cells was quantified by counting cells in five randomly selected
high-powered fields (HPFs) (?200) in each well. For the inhibitory assay,
neutralizing mAb(s) (5 ?g/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 Technol-
ogies) 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 ?g/ml
streptomycin, and 1000 ?g/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 ? 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 ? 106
Sq-19WT, Sq-19Neo, or Sq-19IL-17 cells or with 1 ? 107A549WT,
A549Neo, or A549IL-17 cells into the right flank. The animals were main-
tained 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 ? 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
6178ENHANCED ANGIOGENIC ACTIVITY OF HUMAN NSCLC BY IL-17
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 sec-
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 deparaf-
finization, 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 tem-
perature. 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
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 (Dako-
Cytomation). 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-conju-
gated 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 (?40),
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
?200. 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 sec-
tions of NSCLC tissues from surgery, using the streptavidin-biotin ampli-
fication 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 sec-
tions from blocks of Formalin-fixed, paraffin-embedded, IL-23-stimulated
human CD4 T cells. As a negative control, an immunohistochemical pre-
absorption 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 con-
ducted 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 (Intel-
ligent 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 Transcrip-
tase (Invitrogen Life Technologies). PCR was performed in a DNA Ther-
mal Cycler (PerkinElmer/Cetus) using Taq polymerase (Boehringer Mann-
heim). The primer sequences of the oligonucleotides used for PCR and
PCR product sizes were as follows: ?-actin, sense, 5?-TCTGGTCAATG
GAAGCCTGT-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 Se-
quence Detection SystemandTaqMan
(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 ex-
amined by using TaqMan ?-actin control reagents (PerkinElmer Biosys-
tems). The amount of amplified IL-17 mRNA was correlated to that of
PCR CoreReagent kit
Statistical analysis was performed using an unpaired two-tailed Student’s t
test with confirmation by parametric and F tests. Differences were consid-
ered to be statistically significant when the p value was ?0.05.
Expression of IL-17R on human NSCLC lines Sq-19, A549, and
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 ubiq-
uitous expression of IL-17R (2).
IL-17 has no direct effect on the in vitro growth of NSCLC cells
Because NSCLC cells expressed IL-17R, we investigated whether
IL-17 can modulate the phenotype of these cells. We first exam-
ined 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.
IL-17 selectively up-regulates the production of angiogenic CXC
chemokines by three different NSCLC lines
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 pro-
duction (Fig. 3, A–C), whereas an isotype-matched control mAb
had no effect (data not shown). In contrast, IL-17 did not signifi-
cantly 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
6179 The Journal of Immunology
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 chemo-
kines, but not angiostatic CXC chemokines, from NSCLC.
IL-17 significantly augments the net angiogenic activity of
IL-17 selectively up-regulated the production of important angio-
genic 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 chemo-
taxis 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 demon-
strated 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
Increased endothelial cell chemotaxis in response to CM from
NSCLC/IL-17 is suppressed in the presence of neutralizing
mAb(s) to CXCL1, CXCL5, and CXCL8 or to CXCR-2
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 che-
motaxis 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 en-
dothelial 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-de-
Establishment and characterization of the IL-17-producing
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). Af-
ter G418 selection, the expression of human IL-17 mRNA by sta-
ble transfectants was determined by RT-PCR (data not shown).
Sq-19IL-17 or A549IL-17 secretes 67 or 35 ng/1 ? 106cells/48 h
NSCLC cells. Cells (1 ? 103) were seeded into 96-well flat-bottom plates
and cultured in RPMI 1640 with 3% FCS in the presence or absence of
0.1–1000 ng/ml human IL-17. On day 5 or 7, cells were washed with RPMI
1640, and 100 ?l of MTT solution were added to each well. A wide range
of doses of human IL-17 has no direct effect on the in vitro growth of Sq-19
and A549 NSCLC cells. Each value represents mean ? SD (n ? 7). The
result is representative of two independent experiments.
IL-17 has no direct effect on the in vitro growth of human
anti-human IL-17R mAb (solid line) or irrelevant mouse IgG1 (dotted line), followed by PE-conjugated goat anti-mouse IgG1 Ab. The expression of IL-17R
on the surface of NSCLC cells was analyzed by flow cytometry.
IL-17R expression on human NSCLC lines. Three different human NSCLC lines, Sq-19, A549, and LK-87, were incubated with mouse
6180ENHANCED ANGIOGENIC ACTIVITY OF HUMAN NSCLC BY IL-17
concentrations of IL-17 on CXCL1,
CXCL5, CXCL8, and VEGF-A pro-
duction from human NSCLC lines.
NSCLC lines, Sq-19 (A), A549 (B),
and LK-87 (C), were stimulated with
or without 0.1, 0.5 1, 5, 10, 50, 100,
and 500 ng/ml IL-17 for 48 h.
VEGF-A concentrations in cell cul-
ELISA are shown. Data are ex-
pressed as mean ? SD (n ? 3). The
result is representative of two inde-
pendent experiments. (control vs 0.1–
500 ng/ml IL-17, ?, p ? 0.05; 50
ng/ml IL-17 vs 50 ng/ml IL-17 ? 5
?g/ml anti-human IL-17 mAb, ??,
p ? 0.003).
Effects of increasing
6181 The Journal of Immunology
A549, or A549/IL-17. A, Endothelial cell chemotaxis to 50 ng/ml IL-17,
CM from Sq-19, CM from Sq-19/IL-17, or CM from Sq-19/IL-17 ? anti-
IL-17 mAb. Bars represent the mean number of migrated LMVECs ? SD
per five HPFs (?200) (n ? 5). (Control vs IL-17, ?, p ? 0.05; IL-17 vs
IL-17 ? anti-IL-17 mAb, ??, p ? 0.05; control vs Sq-19, ???, p ? 0.01;
Sq-19 vs Sq-19/IL-17 or Sq-19/IL-17 ? anti-IL-17 mAb; ????, p ?
0.003.) B, Endothelial cell chemotaxis to 50 ng/ml IL-17, CM from A549,
CM from A549/IL-17, or CM from A549/IL-17 ? anti-IL-17 mAb. Bars
represent the mean number of migrated LMVECs ? SD per five HPFs
(?200) (n ? 5). The result is representative of two independent experi-
ments. (Control vs IL-17, ?, p ? 0.05; IL-17 vs IL-17 ? anti-IL-17 mAb,
??, p ? 0.05; control vs A549, ???, p ? 0.02; A549 vs A549/IL-17 or
A549/IL-17 ? anti-IL-17 mAb, ????, p ? 0.005.)
LMVEC chemotaxis to CM from Sq-19, Sq-19/IL-17,
ence of mAb(s) to CXCL1, CXCL5 and CXCL8, VEGF-A, HGF, IFN-?,
CXCR-1 or CXCR-2. A, Endothelial cell chemotaxis to CM from Sq-19/
IL-17 in the presence of neutralizing mAb(s) to CXCL1, CXCL5 and
CXCL8, VEGF-A, HGF, IFN-?, or CXCR-2. Bars represent the mean
number of migrated LMVECs ? SD per five HPFs (?200) (n ? 5). The
result is representative of two independent experiments. (Control IgG vs
anti-CXCL1, CXCL5 and CXCL8 mAbs, or anti-CXCR-2, ?, p ? 0.003).
B, Endothelial cell chemotaxis to CM from A549/IL-17 in the presence of
neutralizing mAb(s) to CXCL1, CXCL5 and CXCL8, VEGF-A, HGF,
IFN-?, or CXCR-2. Bars represent the mean number of migrated LM-
VECs ? SD per five HPFs (?200) (n ? 5). The result is representative of
two independent experiments. (Control IgG vs anti-CXCL1, CXCL5, and
CXCL8 mAbs or anti-CXCR-2, ?, p ? 0.007.)
LMVEC chemotaxis to CM from NSCLC/IL-17 in the pres-
Table I. IL-17 selectively enhances the production of angiogenic CXC chemokines by human NSCLC cellsa
21.6 ? 0.5
132 ? 4
8.9 ? 1.4
150 ? 9
0.9 ? 0.1
1.7 ? 0.1
3.6 ? 0.7
2.1 ? 0.1
10.7 ? 1.1
79 ? 12
367 ? 21
4 ? 1
1.4 ? 0.1
22.4 ? 3.6
140 ? 21
9.3 ? 2.1
147 ? 14
2.1 ? 0.1*
7.3 ? 0.6*
74.7 ? 10.5*
4.2 ? 0.1*
11.1 ? 0.7
77 ? 37
360 ? 8
5 ? 1
1.3 ? 0.1
20.8 ? 0.9
47 ? 3
157 ? 7
13.9 ? 0.7
178 ? 10
0.6 ? 0.1
10.6 ? 0.7
50.8 ? 7.0
0.15 ? 0.02
37.7 ? 2.7
93 ? 30
337 ? 5
65 ? 4
0.65 ? 0.13
17.7 ? 0.5
50 ? 3
147 ? 11
13.3 ? 0.7
162 ? 17
2.5 ? 0.1*
14.8 ? 1.1*
91.7 ? 22.6*
0.52 ? 0.05*
40.7 ? 3.7
91 ? 5
334 ? 7
63 ? 7
0.66 ? 0.15
20 ? 2.0
17 ? 1
133 ? 18
23.7 ? 3.9
135 ? 8
0.9 ? 0.1
13.7 ? 0.5
23.0 ? 7.7
0.23 ? 0.03
17.1 ? 0.5
64 ? 3
330 ? 23
108 ? 5
0.9 ? 0.1
21.9 ? 2.4
18 ? 1
123 ? 11
25.5 ? 4.3
137 ? 6
2.5 ? 0.3*
19.2 ? 1.9*
60.2 ? 6.0*
0.43 ? 0.03*
17.7 ? 1.5
67 ? 5
335 ? 7
111 ? 7
1.0 ? 0.1
aCells were cultured in RPMI 1640 containing 5% FCS, 2 mM L-glutamine, 0.1 mM NEAA, 100 IU/ml penicillin, and 100 ?g/ml streptomycin with or without 50 ng/ml
IL-17 for 48 h. Cell-free supernatants were collected and stored at ?70°C. Concentrations of cytokines were measured using commercially available ELISA kits. PGE2
concentration was measured as reported previously (36). (pg/ml for aFGF, bFGF, angiopoietin-2, TGF-?, HGF, CXCL6, CXCL10, CXCL11, CXCL12, PDGF-BB, and PGE2;
ng/ml for CXCL1, CXCL5, CXCL8, and VEGF-A).
?, p ? 0.05.
6182 ENHANCED ANGIOGENIC ACTIVITY OF HUMAN NSCLC BY IL-17
IL-17, respectively, determined by a commercially available
ELISA kit (R&D Systems).
IL-17 significantly promotes the in vivo growth of NSCLC in
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 strik-
ingly 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.
IL-17 significantly increases the intratumoral microvessel
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 pro-
liferation 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 stimu-
lating the proliferation of NSCLC cells. To examine this possibil-
ity, 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 vas-
cularized 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 tu-
mors 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 stud-
ies, which showed that angiogenesis stimulators or inhibitors can
modulate the in vivo tumor growth by increasing or decreasing
apoptosis of tumor cells (40).
The elevated levels of circulating serum angiogenic chemokines
during tumorigenesis of NSCLC cells transfected with IL-17 in
SCID mice correlate with increased growth
Because IL-17 selectively augments the secretion of angiogenic
CXC chemokines by NSCLC and promotes tumor angiogenesis,
promotes the in vivo NSCLC growth in SCID
mice. A, Transduction with the IL-17 gene has
no direct effect on the in vitro growth of NSCLC
cells. Each value represents mean ? SD (n ? 3).
The result is representative of two independent
experiments. B, The time course of the in vivo
growth for Sq-19WT, Sq-19Neo, and Sq-
19IL-17 in SCID mice. Data are mean tumor
volume ? SD for seven mice per group. The
result is representative of two independent ex-
periments. C, The time course of the in vivo
growth for A549WT, A549Neo, and A549IL-17
in SCID mice. Data are mean tumor volume ?
SD for seven mice per group. The result is rep-
resentative of two independent experiments. D,
These are representative photographs of the
gross NSCLC tumors in SCID mice.
Expression of IL-17 markedly
6183The Journal of Immunology
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 angio-
genic 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.
Administration of anti-mouse CXCR-2-neutralizing Ab into
tumor-bearing SCID mice markedly abrogates the IL-17-induced
increased growth and vascularity
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 an-
giogenic 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 inocula-
tion 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 ad-
dition, 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 re-
sults indicate that the increased growth of NSCLC elicited by
IL-17 is mediated by CXCR-2 signaling and that NSCLC growth
ination of NSCLC tissues. Tumor tissues
were excised from SCID mice, fixed in 4%
buffered paraformaldehyde, embedded in
paraffin or immediately soaked in OCT com-
pound, and frozen in liquid nitrogen. Repre-
sentative photomicrographs show a typical
immunochemical appearance in tumor tis-
sues from Neo transfectants (A, C, E, G, I,
and K) and IL-17 transfectants (B, D, F, H, J,
and I). Proliferation, apoptosis and angiogen-
esis were detected using anti-Ki-67 Ab (A–
D), the TUNEL method (E–H), and anti-
CD31 mAb (I–L), respectively. M, Graphs
show the change in Ki-67-positive cells,
TUNEL-positive cells, and blood vessel
number in the tumor tissues. Data represent
the mean ? SD (n ? 5).
Table II. Markedly elevated serum concentrations of angiogenic CXC
chemokines in SCID mice bearing tumors transfected with IL-17a
475.5 ? 117.7
1577.2 ? 317.1?
175.7 ? 48.7
575.7 ? 138.7?
717.5 ? 171.7
1877.8 ? 517.5?
1657.7 ? 177.5
2146.7 ? 217.7?
117.5 ? 37.1
233.8 ? 51.7?
51.7 ? 13.7
205.2 ? 34.3?
aOn day 25 for Sq-19 or on day 40 for A549, peripheral blood was collected from
SCID mice, kept over night at 4°C, and centrifuged. The serum was stored at ?70°C
until use. The circulating levels of angiogenic CXC chemokines were measured using
commercially available ELISA kits. Data are the mean ? SD (pg/ml) (n ? 3). The
result is representative of two independent experiments. ?, p ? 0.05.
6184ENHANCED ANGIOGENIC ACTIVITY OF HUMAN NSCLC BY IL-17
is partly dependent on angiogenic CXC chemokines, which com-
monly bind to CXCR-2. These findings also suggest that the block-
ing of CXCR-2 signaling might be a promising strategy to treat
patients with NSCLC.
IL-17 production and vascularity in primary NSCLC tissues
To further explore the possible role for IL-17 in promoting angio-
genic activity of NSCLC, we investigated the IL-17 mRNA ex-
pression 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 classi-
fied 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 im-
munohistochemical 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 in-
flammatory 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 or-
igin 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).
We also examined the expression of IL-17R in NSCLC tissues
from surgery by immunohistochemical staining. The strong immu-
noreactivity for IL-17R on NSCLC cells was detected (Fig. 9, I
and J). We further examined the vascular density of primary
NSCLC tissues by immunostaining using anti-human CD34 mAb.
A statistically significant association was found between high lev-
els of IL-17 expression and tumor vascularity (Fig. 9, K and L).
The vascular density of NSCLC tissues with high levels of IL-17
expression was significantly higher than that of NSCLC tissues
with undetectable levels of IL-17 (Table IV).
In the present study, we demonstrate that IL-17, a T cell cytokine,
markedly increases the net angiogenic activity and promotes the in
vivo growth of NSCLC transplanted in SCID mice through a
CXCR-2-dependent mechanism. We found that IL-17 stimulates
NSCLC to selectively up-regulate the production of an array of
angiogenic, but not angiostatic, CXC chemokines and strikingly
enhances the NSCLC-derived net angiogenic activity. Although
two NSCLC lines transfected with the IL-17 gene grew more rap-
idly when compared with controls in SCID mice, administration of
anti-mouse CXCR-2-neutralizing Ab into mice largely abolished
into SCID mice significantly attenuates the in vivo growth of NSCLC.
SCID mice were inoculated s.c. with NSCLC cells and treated with either
control Ab or anti-mouse CXCR-2 Ab at 4-day intervals. A, The time
course of in vivo growth for Sq-19Neo and Sq-19IL-17 in SCID mice
treated with either control Ab or anti-mouse CXCR-2 Ab. Data are mean
tumor volume ? SD for five mice per group. The result is representative
of two independent experiments. B, The time course of in vivo growth for
A549Neo and A549IL-17 in SCID mice treated with either control Abs or
anti-mouse CXCR-2 Ab. Data are mean tumor volume ? SD for five mice
per group. The result is representative of two independent experiments.
Administration of anti-mouse CXCR-2-neutralizing Ab
Table III. Treatment with anti-mouse CXCR-2-neutralizing Ab
significantly reduced the intratumoral microvessel densitya
TreatmentNo. of Tumor Vessels/10 HPFs
Sq-19Neo ? control Ab
Sq-19Neo ? anti-CXCR-2 Ab
Sq-19IL-17 ? control Ab
Sq-19IL-17 ? anti-CXCR-2 Ab
A549Neo ? control Ab
A549Neo ? anti-CXCR-2 Ab
A549IL-17 ? control Ab
A549IL-17 ? anti-CXCR-2 Ab
81 ? 11
57 ? 7?
171 ? 31
64 ? 8??
75 ? 10
47 ? 5???
130 ? 17
56 ? 6????
aTumor tissues were harvested from SCID mice on day 25 for Sq-19 or on day
40 for A549, immediately soaked in OCT compound, and frozen in liquid nitrogen.
Sections were stained for CD31. Specimens (n ? 4) were evaluated by quantifying the
number of stained blood vessels in 10 selected most vascularized HPFs per tumor
section. The mean number of microvessels/10 HPFs/tumor section (?200) from mice
treated with anti-mouse CXCR-2-neutralizing Ab was much less than that from mice
treated with control Ab. Data represent the mean number of vessels ? SD. (Sq-
19Neo ? control Ab versus Sq-19Neo ? anti-CXCR-2 Ab, ?, p ? 0.01; Sq-19IL-
17 ? control Ab vs Sq-19IL-17 ? anti-CXCR-2 Ab, ??, p ? 0.002; A549Neo ?
control Ab vs A549Neo ? anti-CXCR-2 Ab, ???, p ? 0.01; A549IL-17 ? control Ab
vs A549IL-17 ? anti-CXCR-2 Ab, ????, p ? 0.005).
6185The Journal of Immunology
this enhanced NSCLC growth. A direct effect of IL-17 on the in
vivo growth of NSCLC cells seems unlikely because a wide range
of doses of IL-17 did not affect the in vitro growth rate and wild-
type, Neo-, or IL-17-transfected tumors exhibited the same in vitro
proliferation rate. Immunostaining for CD31 revealed that the vas-
cular elements within tumor tissues of IL-17 transfectants signif-
icantly increased when compared with those of controls. Because
angiogenesis is an essential process in the development and pro-
gression of malignant solid tumors, our findings strongly suggest
that IL-17 might accelerate the in vivo NSCLC growth via en-
hancing the net angiogenic activity.
The CXC chemokines can be divided into two groups on the
basis of the presence or absence of the glutamic acid-leucine-argi-
nine (ELR) motif. CXC chemokines with the ELR motif are potent
angiogenic factors that directly promote endothelial cell prolifer-
ation, 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 im-
portant 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 determi-
nant of the net angiogenic activity in NSCLC (24, 46). The IFNs
such as IFN-?, IFN-?, and IFN-? are potent agonists for the ex-
pression of CXCL10 from a variety of cells, including keratino-
cytes, fibroblasts, endothelial cells, mononuclear cells, and tumor
cells (47–49). The IFNs are also potent inhibitors of the production
of CXCL8 by monocytes, fibroblasts, and endothelial cells (50–
NSCLC tissues. A, Total cellular RNA was extracted from human NSCLC
tissues. Five micrograms of total RNA were applied for the synthesis of
cDNAs. IL-17 mRNA expression was detected in five of seven NSCLC
IL-17 and IL-17R expression and vascularity in human
Table IV. Relation between vascular density and IL-17 mRNA
expression in NSCLC tissuesa
IL-17 mRNA Expression Vascular Density
Undetectable (n ? 33)
High (n ? 17)
175.4 ? 15.7
217.7 ? 27.4?
aNSCLC sections were stained for CD34. Specimens were evaluated by quanti-
fying the number of stained blood vessels/10 HPFs/section (?200). Data represent the
mean number of vessels ? SD. (Undetectable levels of IL-17 mRNA expression
versus high levels of IL-17 mRNA expression; ?, p ? 0.03).
cases by RT-PCR. Lanes 1, 3, 4, 5, and 7 are IL-17 mRNA positive. B and
C, As a positive control, we stained the sections from blocks of formalin-
fixed and paraffin-embedded, IL-23-stimulated CD4 T cells with anti-
human IL-17 Ab. As a negative control, immunohistochemical preabsorp-
tion test was performed in these sections. D, Anti-IL-17 reactivity in
NSCLC tissues with undetectable levels of IL-17 mRNA expression. The
immunoreactivity for IL-17 is not observed. E, Anti-IL-17 reactivity in
NSCLC tissues with high levels of IL-17 mRNA expression. The strong
immunoreactivity for IL-17 is observed in infiltrating inflammatory cells
but not in NSCLC cells. F and G, The serial sections of NSCLC tissues
were stained with Ab against IL-17 (F) or CD3 (G). Not all cells, which are
stained positively with anti-IL-17 Ab, are stained positively with anti-CD3
Ab. H, Some of the cells stained positively with anti-IL-17 Ab have poly-
morphonuclear morphology. I and J, IL-17R expression in NSCLC tissues
from surgery was examined by immunohistochemical staining using goat
anti-human IL-17R polyclonal Ab. The strong immunoreactivity for IL-
17R is detected on the surface of NSCLC cells. K and L, NSCLC tissues
from surgery were fixed in 4% buffered paraformaldehyde and embedded
in paraffin. NSCLC sections were stained with anti-CD34 mAb and coun-
terstained with hematoxylin. NSCLC tissues with high levels of IL-17
mRNA expression are more markedly vascularized than those with unde-
tectable levels of IL-17 expression.
6186ENHANCED ANGIOGENIC ACTIVITY OF HUMAN NSCLC BY IL-17
53). Conversely, IL-17 is a potent inducer of angiogenic chemo-
kines from a number of cells, including keratinocytes, fibroblasts,
epithelial cells, and tumor cells (2–8). 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 predomi-
nance of angiostatic chemokines to reduce the net angiogenic ac-
tivity, whereas IL-17 may promote the angiogenic activity of
NSCLC by causing a biological imbalance to increase the produc-
tion 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 an-
giogenic CXC chemokines, we performed an endothelial cell che-
motaxis assay on CM from NSCLC/IL-17. IL-17 markedly pro-
moted NSCLC-induced endothelial chemotactic activity. To assess
whether this biological property of IL-17 could be caused by an-
giogenic chemokines, we tested the inhibitory effects of neutral-
izing 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
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 signifi-
cantly reduced in CXCR-2?/?mice as compared with that in con-
trol 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 monothera-
pies, which target a single mediator such as CXCL8, to treat pa-
tients with NSCLC. Moreover, combination antiangiogenesis pro-
tocols, which target both CXCR-2 and VEGF-A signalings, could
greatly improve the therapeutic efficacy of NSCLC. Taken collec-
tively, 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 fre-
quently detected in NSCLC tissues. Moreover, our immunohisto-
chemical 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, poly-
morphonuclear 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 bio-
logical action on NSCLC cells in a paracrine fashion. Immuno-
staining for CD34 revealed that NSCLC tissues with high levels of
IL-17 mRNA expression had significantly higher microvessel den-
sity than those in which IL-17 mRNA expression was not detected.
These findings raised the possibility that the infiltrating inflamma-
tory cells such as T cells and polymorphonuclear neutrophils may
occasionally stimulate the production of angiogenic CXC chemo-
kines 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 endothe-
lial cells in vitro and elicits neovessel formation in vivo (37, 58).
Thus, it is a little strange that the enhanced NSCLC growth me-
diated 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 pro-
mote the in vivo NSCLC growth through enhancing CXCR-2-de-
pendent 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 depend-
ing on the stimuli.
In conclusion, our findings illustrate the biological action of
IL-17 on human NSCLC. IL-17 markedly enhances the net angio-
genic 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 microenvi-
ronment. Our results also demonstrate that targeting CXCR-2 sig-
naling 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.
We thank Eri Takahashi and Toshie Suzuki for their excellent technical
assistance in carrying out this study.
The authors have no financial conflict of interest.
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