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Published ahead of print on March 3, 2005, doi:10.1165/rcmb.2004-0223OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 540-547, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0223OC

Autocrine and Paracrine Regulation of Interleukin-8 Expression in Lung Cancer Cells

Pei-Li Yao, Yi-Chen Lin, Chien-Hsun Wang, Ya-Chen Huang, Wei-Yu Liao, Shan-Shue Wang, Jeremy J. W. Chen* and Pan-Chyr Yang*

Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine; NTU Center for Genomic Medicine, National Taiwan University College of Medicine; Institute of Biomedical Sciences, Academia Sinica; National Health Research Institute, Taipei; Institutes of Biomedical Sciences and Molecular Biology, National Chung Hsing University, Taichung; and Department of Biochemical Engineering, Kao Yuan Institute of Technology, Kaohsiung, Taiwan

Correspondence and requests for reprints should be addressed to Pan-Chyr Yang, M.D., Ph.D., Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Rd, Taipei, Taiwan 100, R.O.C. E-mail: pcyang{at}ha.mc.ntu.edu.tw


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We had previously demonstrated that lung cancer cells, upon contact with macrophages, could be induced to secrete angiogenic factors to promote tumor angiogenesis. In this study, we focused on the paracrine and autocrine regulation of interleukin (IL)-8 expression in sensitized lung cancer cells after interacting with macrophages. We found that the IL-8 mRNA expression in lung cancer cells significantly increased after coculture with phorbol myristate acetate–treated THP-1 cells and human primary lung macrophages. Fresh lung cancer CL1-5 cells cocultured with macrophage-sensitized lung cancer cells still had a 35% of increase in IL-8 mRNA expression. The addition of anti-inflammatory agents pyrrolidine dithiocarbamate, pentoxifylline, aspirin, and dexamethasone could completely suppress the expression of IL-8 mRNA in fresh/sensitized lung cancer cell cocultures. Human recombinant tumor necrosis factor (TNF)-{alpha} and IL-1{alpha} could induce IL-8 expression in lung cancer cells in a dose-dependent manner. Neutralization with TNF-{alpha} and IL-1{alpha} antibodies in cocultures decreased the levels of IL-8 expression in sensitized lung cancer cells. Nuclear factor-{kappa}B transcriptional activity was also suppressed by the same antibodies, as confirmed by a reporter gene assay and the electrophoretic mobility shift assay. Our results highly suggest that both autocrine and paracrine regulation are involved in IL-8 expression of lung cancer cells cocultured with macrophage. Also, the regulations of IL-8 expression in lung cancer cells were through the nuclear factor-{kappa}B pathway and modulated by TNF-{alpha} and IL-1{alpha}.

Key Words: autocrine • inflammation • lung cancer • macrophages • NF-{kappa}B


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Lung cancer is a serious problem worldwide (1). The mechanisms underlying the rapid increase and progression of lung cancer are still not known. We have previously demonstrated that angiogenesis plays a critical role in lung cancer progression (2–4). Angiogenesis is an important process in tumor development and is closely associated with the growth, progression, and metastasis of tumors (2, 5, 6). This process is believed to be regulated by many angiogenic factors, including interleukin (IL)-8, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (7–10). We have demonstrated that VEGF expression in lung cancer tumor specimen is correlated with histologic type, tumor angiogenesis, patient survival, and time to relapse in non–small cell lung cancer (NSCLC) (4). The expression of VEGF is correlated with the expression of IL-8, tumor angiogenesis, and aberrant p53 expression (3).

The interaction between the tumor and the surrounding stromal cells is complex. Normal surrounding stroma cells may promote the growth and dissemination of tumors by modulating the release of critical angiogenic peptides (11, 12). Cocultures of fibroblasts and NSCLC cells show increased expression of IL-8 mRNA and protein in both cancer cells and fibroblasts (11), demonstrating that cancer cells stimulate stroma cells to express larger amounts of angiogenic factors. The increased IL-8 expression seen in monocytes after coculture with NSCLC cells (13) indicates that cancer cells can also stimulate inflammatory cells to express increased amounts of angiogenic factors.

Our previous study showed that the presence of infiltrating macrophages in sections from patients with lung cancer is accompanied by increased expression of IL-8 mRNA and that increased tumor cell IL-8 expression is also seen in cancer cell/macrophage cocultures and that this correlates positively with tumor angiogenesis and negatively with patient survival (14). We also showed that, in cocultures, this effect is blocked by anti-inflammatory agents acting through the nuclear factor-{kappa}B (NF-{kappa}B) pathway (14). Paracrine regulation of angiogenesis between various cell types and surrounding stroma cells was observed and may play an important role in tumor angiogenesis (14, 15).

Tumor necrosis factor (TNF)-{alpha} activates angiogenic factors in several human tumor cell types and in vascular endothelial cells (9, 16), and a previous report also hinted that TNF-{alpha} and IL-1{alpha} produced by activated macrophages are involved in tumor progression and angiogenesis in human malignant melanoma (16).

In this study, we investigated the autocrine and paracrine regulation of tumor angiogenesis in lung cancer cells. The lung cancer cells were first sensitized by coculture with macrophages, then these sensitized lung cancer cells were used to sensitize naïve cancer cells, which were, in turn, used to sensitize more naïve cells. Levels of the IL-8 mRNA or protein were measured in both cancer cells and macrophages at each step of sensitization. We demonstrated autocrine and paracrine effects on tumor angiogenesis, and also showed that these might play a crucial role in tumor progression. This autocrine effect on tumor angiogenesis is mediated, in part, through the NF-{kappa}B pathway and can be modulated by anti–TNF-{alpha} and anti–IL-1{alpha}.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Lines, Alveolar Macrophage Isolation, and PMA Treatment
The human monocyte cell line THP-1 (ATCC TIB 202; American Type Culture Collection, Manassas, VA), the human NSCLC cell line A549 (ATCC CCL-185), the human bronchial epithelial cell line BEAS2B (ATCC), and the human lung adenocarcinoma cell lines CL1-0, CL1-5, and PC14 (2), were grown in RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) supplemented with 1.5 g/liter of Na2HCO3, 4.5 g/liter of glucose, and 10% fetal bovine serum (FBS; GIBCO-BRL). The THP-1 cells were pre-treated with 3.2 x 10–7M phorbol myristate acetate (PMA; SIGMA Chemical Co., St. Louis, MO) for 24 h and then applied to the following experiments. PMA-pretreated THP-1 cells were washed three times with phosphate-buffered saline (PBS) and incubated for another 24 h to eliminate the effect of PMA.

Alveolar macrophages were obtained by bronchoalveolar lavage (BAL) procedures from normal lung during routine bronchoscopic examination for patients with solitary pulmonary nodule with written informed consent. BAL was performed from the right middle lobe or lingula using three to five successive aliquots of 20 ml of 0.9% sterile NaCl. The BAL fluid was centrifuged at 800 x g for 10 min at 4°C. After washing twice, the cells were plated on plastic Petri dishes in serum-free RPMI 1640 media and allowed to adhere for 2 h at 37°C. Nonadherent cells were removed by washings with PBS. Adherent cells contained more than 95% alveolar macrophages (17, 18).

Effect of PMA-Treated THP-1 Cells Conditioned Medium or Cocultures on Lung Cancer Cells
PMA-treated THP-1 cells were incubated in serum-free RPMI 1640 medium (SF-RPMI) for 24 h, then the culture supernatants were collected as conditioned medium (CM). The lung cancer cell line CL1-5 was cultured for 24 h in SF-RPMI containing various percentages of CM, then the cells were washed three times with PBS and total RNA extracted using RNAzolB (Tel-Test, Friendswood, TX).

The detailed method of coculture system was described in our previous study (14). Briefly, 5 x 105 lung cancer cells in 2.5 ml of SF-RPMI were seeded into the lower chamber of a six-well plate transwell apparatus (Costar, Cambridge, MA). After allowing 24 h for the cells to reach confluence, they were washed three times with PBS, then 2.5 ml of SF-RPMI was added and the upper chamber immediately placed on top of the lower chamber. PMA-treated THP-1 cells or isolated alveolar macrophages (5 x 105 cells in 2 ml of SF-RPMI) were then seeded into the upper chamber.

To determine the time of maximal IL-8 mRNA expression, the lung cancer cells were harvested at various times after the start of coculture, washed three times with PBS, and total RNA isolated using RNAzolB. All experiments were performed in triplicate. To measure the decay kinetic curve of IL-8 mRNA expression in CL1-5 cells cocultured with PMA-treated THP-1 cells, CL1-5 cells were cocultured with macrophages for 24 h, then the macrophages were removed and the CL1-5 cells harvested after various times.

Sensitized Lung Cancer Cell/Fresh Lung Cancer Cell Cocultures Treated by Agents
Cancer cells cocultured with PMA-treated THP-1 cells or isolated alveolar macrophages for 24 h were designated as sensitized cells (sen-1'). These were detached using trypsin-EDTA (Gibco), washed three times with SF-RPMI, then seeded (5 x 105 cells in 2 ml of SF-RPMI per well) into the upper chamber of a new transwell apparatus and cocultured with fresh lung cancer cells (5 x 105 cells in 2.5 ml of SF-RPMI per well) that had been incubated in the lower chamber for 24 h. The lung cancer cells in the lower chambers became sensitized and were designated as sen-2' cells; these were then added to the upper chamber of a fresh transwell apparatus and used to sensitize fresh cancer cells to generate sen-3' cells. Cancer cells cocultured with untreated THP-1 cells were used as nonsensitized control. In each instance, the cells in the lower chamber after sensitization were assayed for IL-8 mRNA. Recombinant human IL-1{alpha} and TNF-{alpha} were added to nonsensitized cells; sensitized cells were treated with anti-inflammatory agents and antibodies for 24 h.

Fibroblast or Bronchial Epithelial Cell/PMA-Treated THP-1 Cell Cocultures
Primary cultured fibroblasts or bronchial epithelial cells (5 x 105 cells in 2.5 ml of SF-RPMI) were seeded into the lower chamber of the transwell apparatus. After allowing 24 h to reach confluence, the fibroblasts or bronchial epithelial cells were washed three times with PBS, and 2.5 ml of SF-RPMI was added. The upper chamber was placed into the lower chamber and seeded with PMA-treated THP-1 cells (5 x 105 cells in 2 ml of SF-RPMI). After 24 h of coculture, the fibroblasts or bronchial epithelial cells were washed three times with PBS and total RNA isolated using RNAzolB. All experiments were performed in triplicate.

Anti-Inflammatory Drug Treatment
Four anti-inflammatory agents, pyrrolidine dithiocarbamate (PDTC; 50 µM), pentoxifyllline (10 mM), aspirin (100 µM), and dexamethasone (1 µM) (all from Sigma) were added to the lower chamber of sen-1'/sen-2' and sen-2'/sen-3' CL1-5 cell cocultures. The drug concentrations used were determined in a previous dose–response and cytotoxicity study (14). After incubation for 24 h, the cells in the lower chamber were harvested and total RNA isolated using RNAzolB.

Stimulation by Recombinant IL-1{alpha} and TNF-{alpha}
To determine the effect of IL-1{alpha} and TNF-{alpha} on IL-8 mRNA levels, CL1-5 cells in SF-RPMI were incubated for 24 h with various concentrations of recombinant human IL-1{alpha} and TNF-{alpha} (Peprotech EC, London, UK). In addition, various concentrations of recombinant human IL-1 receptor antagonist (IL1-RA) were added to cocultures for 24 h, then the cancer cells were harvested and total RNA isolated with RNAzolB.

Antibody Neutralization Experiments
Various concentrations of anti–IL-1{alpha} and/or anti–TNF-{alpha} primary antibodies (IL-1{alpha}, H159; TNF-{alpha}, H156; Santa Cruz Biotech, Santa Cruz, CA) were added to the lower chamber of CL1–5/macrophage co-cultures for 24 h, then the cancer cells were harvested and total RNA isolated with RNAzolB.

In addition, 1 µg/ml of anti–IL-1{alpha} or anti–TNF-{alpha} primary antibodies, alone or in combination, was added to the lower chamber of CL1–5/macrophage, sen-2'/sen-1', or sen-3'/sen-2' CL1-5 cocultures for 24 h, then the cells in the lower chamber were harvested and total RNA isolated with RNAzolB. Anti-rabbit IgG or anti-goat IgG antibodies (Santa Cruz Biotech) were used as negative controls.

Real-Time Quantitative RT-PCR
IL-8 mRNA expression was quantified by real-time quantitative RT-PCR. The TATA box binding protein (TBP) was used as an internal control. The primers, probes, and detailed procedures have been described previously (10, 14). Briefly, each amplification mixture containing 10 ng of total RNA was subjected to one cycle of reverse transcription and 40 cycles of the polymerase chain reaction. All experiments were performed in triplicate. The relative expression level of IL-8 against that of TBP was defined as –{Delta}CT = –[CTIL-8 – CTTBP]. The IL-8 mRNA/TBP mRNA ratio was calculated as 2–{Delta}CT x K (K being a constant) (10, 14).

Protein Preparation and Western Blot Analysis
The details of nuclear extract preparation and Western blot analysis have been described previously (14). IL-1{alpha} and TNF-{alpha} were detected by rabbit polyclonal anti–IL-1{alpha} and anti–TNF-{alpha} primary antibodies (1:200) and alkaline phosphatase–conjugated anti-rabbit IgG secondary antibody (1:500). The CDP-Star chemiluminescent substrate (Tropix, Bedford, MA) was used as detection reagent and ß-actin was as the control for gel loading.

Transcriptional Regulation Assay
Luciferase activity assay for NF-{kappa}B. NF-{kappa}B transcriptional activity was determined using a reporter gene assay. IL-8 proximal promoter, as well as site-directed mutagenesis of the IL-8 AP-1, NF–IL-6, and NF-{kappa}B sites, was constructed into pGL3 basic vector (Promega, Madison, WI) according to the previous report (19). A quantity of 5 x 105 cells of CL1-5 was cotransfected with pGL3–IL-8–luciferase encoding wild-type or mutant IL-8–specific NF-{kappa}B binding domain-luciferase and pSV–ß-galactosidase (Promega) by the lipofectamine method (14, 20). The pSV–ß-galactosidase construct was used to normalize transfection efficiency. After 4 h of incubation, the transfected cells were washed three times with RPMI 1640 and incubated for 24 h in 3 ml of RPMI-FBS. They were then cocultured for 24 h with macrophages or sensitized CL1-5 cells in a 6-well transwell apparatus in the presence or absence of anti–TNF-{alpha} or anti–IL1-{alpha} antibodies. A cell lysate was prepared from the cancer cells in the lower chamber by adding 250 µl of lysis buffer (Tropix) and scraping the cells off the plate and centrifugation (3,000 x g for 10 min at 4°C), and was assayed for luciferase activity using a Tropix Luciferase Assay kit (Tropix). All experiments, including nontransfected and vector-treated cells as negative controls, were performed in triplicate; the results were normalized for both transfection efficiency and amount of protein.

Electrophoretic mobility shift assay. Cancer cells cocultured with macrophages for 24 h were designated as sensitized cells (sen-1'). These were then seeded into the upper chamber of a new transwell apparatus and cocultured with fresh lung cancer cells in the lower chamber for 24 h. The lung cancer cells in the lower chambers became sensitized and were designated as sen-2' cells. In addition, 1 µg/ml of anti–IL-1{alpha} or anti–TNF-{alpha} primary antibodies was added to the lower chamber of CL1–5/macrophage or sen-2'/sen-1' CL1-5 cocultures for 24 h. A nuclear extract was prepared from the cancer cells in the lower chamber as described previously (14). Double-stranded oligonucleotides containing two sequences coding the IL-8–specific NF-{kappa}B binding site (5'-AAAAATCGTGGAATTTCCCCCGAATCGTGGAATTTCCCCCGA-3' annealed with 5'-AAATCGGGGGAAATTCCACGATTCGGGGGAAATTCCACGATT-3') was designed for electrophoretic mobility shift assay (EMSA). Oligonucleotides were labeled using [{gamma}-32P]ATP (3,000 Ci/mmol) and T4 polynucleotide kinase. Labeled probes were purified from unincorporated [{gamma}-32P]ATP using MicroSpin G-25 columns. Nuclear extract (5 µg of protein) was incubated for 20 min at room temperature in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl [pH 7.5], and 0.1 µg of poly[d(I-C)]) containing {gamma}-32P-end-labeled, double-stranded oligonucleotide in a final volume of 10 µl. Samples were then resolved by electrophoresis on 4% polyacrylamide gels at 110 V in 1x Tris/borate/EDTA buffer for 150 min at 4°C. Gels were dried and placed on a phosphoimage screen overnight.

Statistical Analysis
All experiments were performed in triplicate and repeated at least three times. The results are shown as the mean ± SD, and differences were analyzed by ANOVA (Excel, Microsoft; Taipei, Taiwan, R.O.C). A P value < 0.05 was taken as statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
PMA-Treated THP-1 Cell CM Causes Dose-Dependent Induction of IL-8 mRNA Expression in the Lung Cancer Cell Line, CL1–5
PMA-treated THP-1 cell CM was added to CL1-5 cells, and IL-8 mRNA expression in the CL1-5 cells was measured by real-time quantitative RT-PCR. We found that IL-8 expression increased in a dose-dependent fashion. With 100% CM, the IL-8 mRNA levels were 16-fold higher than in the absence of CM (Figure 1A) ({alpha} = 0.05, P < 0.0014 compared with levels in the absence of CM).



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  		Figure 1. Effect of macrophage conditioned medium or macrophage cocultures on lung cancer cells. (A) Dose-dependent induction of IL-8 mRNA expression in CL1-5 cells by macrophage-conditioned medium (CM). CL1-5 cells were incubated for 24 h in SF-RPMI containing 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% macrophage CM, and IL-8 mRNA expression quantified by RTQ-RT-PCR. The asterisks indicate a P value of 0.00014 compared with the zero CM control. (B) Time-course of IL-8 mRNA expression in cocultured CL1-5 cells. CL1-5 cells were harvested after coculture with macrophages for the indicated time, and IL-8 mRNA was quantified by RTQ-RT-PCR. *{alpha} = 0.05, P = 0.00072 compared with the zero time control; **{alpha} = 0.05, P > 0.358 compared with the longer incubation times (>= 36 h). (C) Decay kinetics of induced IL-8 mRNA expression in cocultured CL1-5 cells. After coculture for 24 h, the macrophages were removed, and total RNAs prepared from the CL1-5 cells after a further 0, 2, 4, 8, 24, 36, 48, and 72 h of incubation and used to measure IL-8 mRNA expression. Non-Co (arrowhead) indicates non-cocultured control cells. All the results are shown as the mean ± SD of relative expression level of IL-8 against to that of TBP.

 
Optimal Induction Time for IL-8 mRNA Expression in CL1–5/PMA-Treated THP-1 Cell Cocultures
In CL1–5/PMA-treated THP-1 cell cocultures, IL-8 mRNA expression in CL1-5 cells increased in a time-dependent manner (Figure 1B). After coculture for 8 h, there was a significant increase in IL-8 mRNA expression ({alpha} = 0.05, P = 0.00072). Maximal expression was detected at 24 h, with no further difference at longer incubation times ({alpha} = 0.05, P = 0.358).

Decay Time of IL-8 mRNA Expression in Sensitized CL1-5 Cells
To investigate the decay kinetics of IL-8 mRNA levels, we cocultured the cells for 24 h, then removed the PMA-treated THP-1 cells and measured the change in IL-8 mRNA expression in the cancer cells after 0, 2, 4, 8, 24, 36, 48, and 72 h (Figure 1C). IL-8 mRNA expression was reduced to less than 20% within 4 h, but was still significantly higher than in nonsensitized cells (arrowhead) at 72 h ({alpha} = 0.05, P = 0.02).

Autocrine Regulation of Induction of IL-8 mRNA Expression in Sensitized Lung Cancer Cell Cocultures
Expression of IL-8 mRNA in CL1-5 sen-1' cells was significantly increased, as compared with nonsensitized cells (Non-Sen) ({alpha} = 0.05, P = 0.0003). IL-8 expression in sen-2' cells was lower than in sen-1' cells (Figure 2A, left panel), but was still higher than in Non-Sen ({alpha} = 0.05, P = 0.00018). There was no difference between sen-3' cells and Non-Sen ({alpha} = 0.05, P = 0.192). The results of IL-8 mRNA induction in cancer cells cocultured with primary macrophages is similar to that of cocultures with PMA-treated THP-1 cells (Figure 2A, right panel).



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  		Figure 2. Autocrine regulation of IL-8 mRNA expression in lung cancer cells CL1-5. (A) A series of sensitizations was performed as described in MATERIALS AND METHODS to generate sen-1', sen-2', and sen-3', which were then tested for IL-8 mRNA levels. Non-Sen (nonsensitized): cancer cells cocultured with untreated THP-1 cells; Sen-1': cancer cells cocultured with PMA-treated THP-1 cells; P-Sen-1': cancer cells cocultured with primary macrophages. {alpha} = 0.05, *P = 0.00018, **P = 0.192 compared with Non-Sen; #P = 0.00028 compared with CL1-5. (B) IL-8 mRNA expression level in CL1-0, CL1-5, A549, and PC14 sen-1' cells was significantly increased ({alpha} = 0.05, P = 0.000002, 0.0003, 0.0006, and 0.00006, respectively, compared with Non-Sen). A significant increase was also measured in sen-2' cells ({alpha} = 0.05, P = 0.0000006, 0.000176, 0.0061, and 0.0013). The results are shown as the mean ± SD of relative expression level of IL-8 against to that of TBP.

 
Meanwhile, other sen-1' cells of the NSCLC cell lines, CL1-0, A549, and PC14, also resulted in a significant increase in IL-8 mRNA expression (Figure 2B). IL-8 mRNA expression in CL1-0, A549, and PC14 sen-2' cells was lower, but still higher than in Non-Sen ({alpha} = 0.05, P = 0.0000006, 0.0061, and 0.0013). These results indicate that autocrine regulation of IL-8 expression is rather a general phenomenon in lung cancer cells.

Anti-Inflammatory Drugs Markedly Suppress IL-8 mRNA Expression in Cocultures
When 50 µM PDTC, 10 mM pentoxifyllline, 100 µM aspirin, or 1 µM dexamethasone was added to cocultures, IL-8 mRNA expression in sen-1' cells was significantly reduced, as compared with cocultures without drug treatment ({alpha} = 0.05, P = 0.00076, 0.00043, 0.00001, and 0.00195). When serially sensitized cocultures were treated with the same agents, IL-8 mRNA expression in sen-2' cells was further reduced as compared with sen-1', but was still significantly different from the drug-treated non-coculture control (Figure 3). However, the four drugs tested could more or less suppress intrinsic level of IL-8 mRNA expression in CL1-5 cells.



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  		Figure 3. Suppression of IL-8 mRNA induction in CL1-5 cocultures by anti-inflammatory agents. Pyrrolidine dithiocarbamate (PDTC; 50 µM), pentoxifylline (10 mM), aspirin (100 µM), or dexamethasone (1 µM) was added to cocultures for 24 h . Nonsensitized (Non-Sen) and sensitized CL1-5 (sen-1' and sen-2') not treated with drugs were used as controls. The results were shown as the mean ± SD of relative expression level of IL-8 against to that of TBP. *{alpha} = 0.05, P = 0.000002 compared with sen-1' cells treated with drugs.

 
Induction of IL-8 Expression in Cocultured CL1-5 Cells Is Modulated by IL-1{alpha} and TNF-{alpha} Derived from Macrophages
When recombinant TNF-{alpha} and IL-1{alpha} was added to CL1-5 cells only, a dose-dependent increase in IL-8 mRNA expression was shown (maximal increase of 14.6- and 16.8-fold, respectively) (Figure 4A). In addition, IL-8 mRNA expression in cocultured CL1-5 cells declined with treatment by 10 µg/ml anti–TNF-{alpha} or anti–IL-1{alpha} antibodies (to 10.1% and 11.8%, respectively) (Figure 4B). Human recombinant IL1-RA also almost completely suppressed IL-8 mRNA expression in cocultured CL1-5 cells ({alpha} = 0.05, P = 0.21, compared with Non-Co) (Figure 4C).



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  		Figure 4. Effect of TNF-{alpha} and IL-1{alpha} on IL-8 mRNA induction. (A) CL1–5 cells were incubated for 24 h with the indicated final concentration of human recombinant TNF-{alpha} or IL-1{alpha}, then IL-8 mRNA levels were measured. *{alpha} = 0.05, P = 0.009 compared with CL1–5 cells with recombinant proteins. (B) The designated concentration of anti–TNF-{alpha} or anti–IL-1{alpha} antibodies was added to CL1–5/macrophage cocultures for 24 h, and then IL-8 mRNA levels of CL1-5 cells were measured. *{alpha} = 0.05, P = 0.008 compared with cocultured CL1-5 cells treated with antibodies. (C) The indicated final concentration of human recombinant IL1 receptor antagonist (IL1-RA) was added to CL1–5/macrophage cocultures for 24 h, then IL-8 mRNA levels of CL1-5 cells were measured. *{alpha} = 0.05, P = 0.002 compared with cocultured CL1-5 cells treated with IL1-RA. The results are shown as the mean ± SD of relative expression level of IL-8 against that of TBP.

 
Anti–IL-1{alpha} and/or anti–TNF-{alpha} antibodies were also added to sen-1' and sen-2' cells to neutralize IL-1{alpha}– and TNF-{alpha}–mediated regulation. Significantly, anti–IL-1{alpha} or anti–TNF-{alpha} antibodies blocked induction of IL-8 mRNA expression in sen-1' cells (reduced to 9.25% or 28.17%, respectively) (Figure 5A). Sen-2' cells showed a further reduction (reduced to 5.05% and 3.58%, respectively, compared with the sen-2' no-antibody control). There was no significant difference between sen-3' cells and Non-Co after antibody neutralization (data not shown) ({alpha} = 0.05, P = 0.124 and 0.053, respectively). Moreover, IL-8 mRNA levels were reduced to 8.87% when both antibodies were added simultaneously to sen-1' cells. Anti-rabbit IgG or anti-goat IgG antibodies were used as negative controls ({alpha} = 0.05, P = 0.117 and 0.158, respectively).



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  		Figure 5. Effect of IL-1{alpha} and TNF-{alpha} on induction of IL-8 expression in sensitized cancer cells. (A) Anti–IL-1{alpha} and/or anti–TNF-{alpha} antibodies (each 1 µg/ml) were added to cocultures for 24 h, then IL-8 mRNA levels were measured. Non-cocultured CL1-5 cells and cocultured CL1-5 cells without antibodies were used as the negative and positive controls, respectively. The results are shown as the mean ± SD of relative expression level of IL-8 against that of TBP. *{alpha} = 0.05, P = 0.0066 compared with sen-1' cells without antibody neutralization; **{alpha} = 0.05, P = 0.255 compared with sen-1' cells without antibody neutralization. #{alpha} = 0.05, P = 0.0024 compared with sen-2' cells without antibody neutralization; ##{alpha} = 0.05, P = 0.382 compared with sen-2' cells without antibody neutralization. (B) Nuclear extract (60 µg protein) from non-cocultures (Non-Co) or cocultures (Co) were analyzed by Western blotting using antibodies against IL-1{alpha} or TNF-{alpha}. ß-Actin was used as the loading control. The number below each lane represents the relative percentage of IL-1{alpha} and TNF-{alpha} protein level compared with Non-Co macrophages (left part) or Non-Co CL1-5 cells (right part). (C) Reporter gene assay for NF-{kappa}B transcriptional activity in cocultures was performed. The results are shown as the mean ± SD of luciferase activity in arbitrary units. *{alpha} = 0.05, P = 0.0014 compared with sen-1' cells with antibody neutralization; **{alpha} = 0.05, P = 0.0091 compared with sen-2' cells with antibody neutralization (upper panel). The site-directed mutagenesis was also performed to evaluate the transcriptional activity (lower panel). m: mutation of indicated binding sites. (D) EMSA for NF-{kappa}B–binding domain interactions in CL1-5 cocultures was detected. A double-stranded oligonucleotide containing NF-{kappa}B binding sites was reacted with the nuclear extract. Unlabeled oligonucleotide was added as competitor (+ competitor).

 
Interestingly, IL-1{alpha} and TNF-{alpha} protein levels in co-cultured macrophages were much higher than in non-cocultured macrophages (2.70- and 2.94-fold, respectively) (Figure 5B). Similar results were seen for the sen-1' CL1-5 cells, showing a 3.93- and 3.06-fold increase in IL-1{alpha} and TNF-{alpha}, respectively, compared with Non-co. In sen-2' CL1-5 cells, IL-1{alpha} and TNF-{alpha} protein were still higher (2.54- and 2.78-fold, respectively). However, in sen-3' CL1-5 cells, IL-1{alpha} and TNF-{alpha} expression was not significantly different from nonsensitized cells.

NF-{kappa}B transcriptional activity was assessed by luciferase reporter gene assay and EMSA. Luciferase activity in sen-1' CL1-5 cells increased 2.8-fold, and this increase was significantly suppressed by addition of anti–TNF-{alpha} or anti–IL-1{alpha} antibody (down to 23% and 25.4%, respectively) (Figure 5C, upper panel). In sen-2' cells, the antibodies almost reduced expression to basal levels. The mock transfectant and the nontransfected control cells showed essentially no luciferase activity. The result of site-directed mutagenesis indicated that the mutation of NF-{kappa}B binding site could lead to no significant response to coculture with PMA-treated THP-1 cells (Figure 5C, lower panel; {alpha} = 0.05, P = 0.11). Similar results were obtained by EMSA (Figure 5D). A strong band shift pattern in sen-1' CL1-5 cells was reduced by either anti–TNF-{alpha} or anti–IL-1{alpha} antibody. The lanes containing extract, probe, and cold competitor showed no band. Similar results were obtained in sen-2' cells.

TNF-{alpha} and IL-1{alpha} Antibodies Do Not Suppress Induction of IL-8 mRNA Expression in Fibroblasts and Bronchial Epithelial Cells Cocultured with Macrophages
Interestingly, fibroblasts and bronchial epithelial cells sensitized by macrophages showed a significant increase in IL-8 mRNA levels (Figure 6). However, in contrast to the results obtained from lung cancer cell/PMA-treated THP-1 cell cocultures, induction of IL-8 mRNA expression in normal lung fibroblasts and bronchial epithelial cells was not suppressed by addition of anti–TNF-{alpha} or anti–IL-1{alpha} antibodies.



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  		Figure 6. Induction of IL-8 mRNA expression in non-tumoral cells. Fibroblasts or BEAS2B were cultured for 24 h in the presence or absence of 1 µg/ml of anti–TNF-{alpha} or anti–IL-1{alpha} antibodies. The results are shown as the mean ± SD of relative expression level of IL-8 against to that of TBP. (A) Normal lung fibroblasts. F indicates fibroblasts alone and F/M indicates fibroblasts in fibroblast/PMA-treated THP-1 cocultures. *{alpha} = 0.05, P = 0.00002 compared with cocultures with antibody treatment; **{alpha} = 0.05, P = 0.207 compared with cocultures with antibody treatment. (B) Bronchial epithelial cells. B: BEAS2B alone; T/B: indicates BEAS2B in BEAS2B/untreated THP-1 cocultures; PT/B: indicates BEAS2B in BEAS2B/PMA-treated THP-1 cocultures. #{alpha} = 0.05, P = 0.00061 compared with cocultures with antibody treatment; ##{alpha} = 0.05, P = 0.845 compared with cocultures with antibody treatment.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Tumor angiogenesis is induced by a progressive increase in production of angiogenic factors and a decrease in expression of angiogenic inhibitors by cancer cells, vascular endothelial cells, and other stromal cells (12, 15, 21). We previously demonstrated that IL-8 mRNA expression is increased in lung cancer cell/macrophage cocultures and correlates positively with tumor angiogenesis and negatively with patients' survival (14). In this report, we focused on paracrine and autocrine regulation of the angiogenic chemokine IL-8 expression in lung cancer CL1-5 cells interacting with either PMA-treated THP-1 cells or sensitized CL1-5 cells. In CL1-5 cells cultured alone, induction of IL-8 mRNA expression was proportional to the concentration of PMA-treated THP-1 cell–derived CM added (Figure 1A), whereas in the cancer cell/PMA-treated THP-1 cell coculture system it was maximal after coculture for 24 h (Figure 1B). We showed further that IL-8 mRNA expression in sensitized CL1-5 cells decayed to basal levels within 72 h of macrophage removal (Figure 1C). Tumor angiogenesis in lung cancer cells was found to be subject to autocrine and paracrine regulation. The autocrine effect of tumor angiogenesis was mediated, in part, through the NF-{kappa}B pathway and could be blocked by anti-inflammatory agents (Figure 3). Anti–IL-1{alpha} or anti–TNF-{alpha} antibodies partially blocked this autocrine regulation in terms of IL-8 mRNA production in a dose-dependent manner (Figure 4B).

Macrophages are attracted by monocyte chemotactic protein-1 and transforming growth factor (TGF)-ß1 secreted by tumor cells, and tumor production of TGF-ß1 is responsible for activating macrophages (15). In this study, sen-2' CL1-5 cells still showed 8-fold higher IL-8 mRNA levels than nonsensitized cells (Figure 2A). The level of IL-8 mRNA expression decreased in successively sensitized cancer cells, reaching basal levels in sen-3' cells. However, the effect of continuous sensitization on cancer cells cocultured with primary macrophages was not observed, reaching basal levels in sen-2' cells. These results still show that macrophages play an important initiator role in the regulatory pathway of IL-8 expression in lung cancer cells. Whether other factors are involved in this regulatory process is still unknown. In addition, this autocrine effect was also seen using cocultures of PMA-treated THP-1 cells and other lung cancer cell lines, such as CL1-0, A549, and PC14 (Figure 2B), suggesting that autocrine regulation of IL-8 expression is rather a general phenomenon in lung cancer cells. In the previous study, by using a Transwell invasion chamber, we selected a series of cell sublines with varying degrees of invasiveness from a clonal cell line (CL1-0) of a human male patient with lung cancer with a poorly differentiated adenocarcinoma (22). The invasiveness of the six cell lines followed a trend of: CL1-5 > CL1-4 > CL1-3 > CL1-2 > CL1-1 > CL1-0. Although the CL1-5 cell line was derived from the CL1-0 cell line, they showed different anchorage-independent growth and invasive/metastatic potential in vitro and in vivo, CL1-5 having a greater potential than CL1-0 (2, 22). Our results showed that IL-8 mRNA expression was increased to a lesser degree in cancer cells with low invasive/metastatic capacity (CL1-0) than in those with a higher invasive metastatic capacity (CL1-5).

The anti-inflammatory agents tested in this study could significantly suppress IL-8 mRNA induction in sensitized CL1-5 cells. Although their possible mechanisms of action might be quite different, these drugs have the common property of NF-{kappa}B inhibition, which might account for the similar suppressive effects. PDTC, an NF-{kappa}B inhibitor, suppresses the lipopolysaccharide-induced expression of proinflammatory genes, such as those coding for TNF-{alpha} and intercellular adhesion molecule-1 (23). Pentoxifylline is a phosphodiesterase inhibitor that inhibits protein kinase C–dependent activation of NF-{kappa}B (24). Aspirin, a nonsteroidal cyclooxygenase inhibitor, blocks TNF-{alpha}–induced IL-8 expression and inhibits NF-{kappa}B activation (25). Dexamethasone, a glucocorticoid analog, suppresses IL-8 expression by impairment of NF-{kappa}B activation in human glioblastoma cells (26). These results also confirm those of our previous study showing that IL-8 mRNA expression in CL1–5/macrophage cocultures is mediated through the NF-{kappa}B pathway (14).

Block of NF-{kappa}B expression using an antisense oligonucleotide almost completely inhibits TNF-{alpha}–dependent IL-8 production in microvascular endothelial cells (9). Moreover, TNF-{alpha} and IL-1{alpha}, secreted by activated monocytes and macrophages, enhanced the production of IL-8 and VEGF in tumor cells in vitro (11, 15, 16, 21). A previous report also suggested that angiogenesis stimulated by TNF-{alpha} could be modulated by various angiogenic factors, and that this pathway might be controlled through paracrine and autocrine mechanisms (21). The present study showed that recombinant human TNF-{alpha} and IL-1{alpha} could cause a more than 15-fold increase in IL-8 mRNA expression in CL1-5 cells (Figure 4A). We also observed that IL-1ß–induced 10-fold increase in IL-8 mRNA expression (data not shown). We therefore tested the effect of anti–TNF-{alpha} and/or anti–IL-1{alpha} antibodies on the induction of IL-8 mRNA expression in cocultures and found that each of the antibodies caused a dose-dependent decrease in IL-8 mRNA expression (Figure 4B). However, both antibodies together were no more effective than anti–IL-1{alpha} antibody alone (Figure 5A), showing that the two antibodies did not have an additive effect between TNF-{alpha} and IL-1{alpha} in suppressing IL-8 expression. Moreover, more than 1 ng/ml of IL1-RA almost completely suppressed IL-8 mRNA induction in cocultured CL1-5 cells (Figure 4C). These results suggest that IL-1{alpha} may have the main effect on IL-8 induction.

Similar results were also seen in PMA-treated THP-1 cells. Western blotting revealed that both CL1-5 cells and macrophages in cocultures showed an ~ 3-fold increase in TNF-{alpha} protein levels and a 4-fold increase in IL-1{alpha} protein levels, and increased levels of both were still observed in sen-2' CL1-5 cells (Figure 5B). The reporter gene assay and EMSA revealed that the transcriptional activity of NF-{kappa}B was also increased in sensitized CL1-5 cells (Figures 5C and 5D). These findings show that TNF-{alpha} and IL-1{alpha} are involved in IL-8 mRNA induction in CL1–5/macrophage cocultures through autocrine and paracrine regulation. In addition, our results also revealed that both NF-{kappa}B and AP-1 might participate in the multiple control of IL-8 gene expression of lung cancer cells cocultured with PMA-treated THP-1 cells, which may explain why NF-{kappa}B–specific inhibitor, PDTC, and other anti-inflammatory drugs cannot completely suppress the induction of IL-8 expression (Figure 3). This observation also demonstrated the multiple control of IL-8 gene expression in cocultures and is consistent with the previous study (27).

In addition, we investigated the interaction between PMA-treated THP-1 cells and nontumoral cells, such as fibroblasts and bronchial epithelial cells. In contrast to the result in CL1–5/PMA-treated THP-1 cocultures, the induction of IL-8 was not blocked by anti–TNF-{alpha} or anti-IL-1{alpha} antibodies, suggesting that the regulation of IL-8 mRNA induction in CL1-5 cells differs from that in normal lung fibroblasts or bronchial epithelial cells (Figure 6). It is reasonable to speculate that other pathways regulate IL-8 induction in fibroblasts or bronchial epithelial cells.

As shown in Figure 7, lung cancer cells attract macrophages and it is possible to produce certain inflammatory cytokines, such as TNF-{alpha} and IL-1{alpha}, and then secrete into the surrounding environment. Increased TNF-{alpha} and IL-1{alpha} levels might induce lung cancer cells to activate NF-{kappa}B and increase IL-8 expression in lung cancer cells further. Interestingly, lung cancer cells might regulate IL-8 production by themselves or by adjacent lung cancer cells to induce more IL-8 production with an autocrine fashion, which would induce more angiogenesis in lung cancer cells.



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  		Figure 7. A model for the possible interaction between macrophage and CL1-5 cells during angiogenesis. In this model, macrophages are attracted by chemokines derived from NSCLC cells (CL1-5). Activated macrophages produce angiogenic factors, such as TNF-{alpha} and IL-1{alpha}, and stimulate the cancer cells to produce more IL-8 and other soluble factors. Meanwhile, sensitized cancer cells, by providing TNF-{alpha}, IL-1{alpha}, or other factors, stimulate neighboring cancer cells to produce IL-8 and other soluble factors. In summary, there is a complex interaction between chemokines and angiogenic factors produced by macrophages and cancer cells in inducing angiogenesis.

 
In conclusion, this study demonstrated the paracrine regulation of IL-8 expression is present in lung cancer cells by macrophages, and there also exists an autocrine regulation of IL-8 expression in lung cancer cells. The inhibition of IL-8 mRNA induction in lung cancer cells by anti-inflammatory agents suggests a means for restraining local autocrine regulation of angiogenesis in cancer cells. Finally, we suggest that IL-8 mRNA induction in lung cancer cell cocultures occurs, at least in part, through the NF-{kappa}B pathway and is modulated by IL-1{alpha} and TNF-{alpha}. Further studies to clarify the effect of the interaction of lung cancer cells, macrophages, fibroblasts, and stromal cells in angiogenesis will be undertaken.


   Footnotes
 
This work was supported by the National Science Council and National Health Research Institutes of the Republic of China through National Research Program for Genomic Medicine grant (NSC 91-3112-P-005-008–Y, NHRI92A1-NSCLC09-5, and NHRI93A1-NSCLC09-5).

Conflict of Interest Statement: P.-L.Y. has no declared conflicts of interest; Y.-C.L. has no declared conflicts of interest; C.-H.W. has no declared conflicts of interest; Y.-C.H. has no declared conflicts of interest; W.-Y.L. has no declared conflicts of interest; S.-S.W. has no declared conflicts of interest; J.J.W.C. has no declared conflicts of interest; and P.-C.Y. has no declared conflicts of interest.

* J.J.W.C. and P.-C.Y. contributed equally to this work and are joint corresponding authors. Back

Received in original form July 14, 2004

Received in final form February 2, 2005


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 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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