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The Journal of Immunology, 2001, 167: 497-504.
Copyright © 2001 by The American Association of Immunologists

Both E6 and E7 Oncoproteins of Human Papillomavirus 16 Inhibit IL-18-Induced IFN-{gamma} Production in Human Peripheral Blood Mononuclear and NK Cells1

Shin-Je Lee*,{dagger}, Young-Sik Cho*, Min-Chul Cho*, Jung-Hyun Shim*, Kyung-Ae Lee*, Kwang-Kjune Ko{dagger}, Yong Kyung Choe*, Sue-Nie Park{ddagger}, Tomoaki Hoshino§, SooHyun Kim, Charles A. Dinarello and Do-Young Yoon2,*

* Laboratory of Cellular Biology, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea; {dagger} Department of Microbiology, College of Medicine, Soonchunhyang University, Chungnam, Korea, {ddagger} Korean Food and Drug Administration, Seoul, Korea; § Department of Internal Medicine 1, Kurume University School of Medicine, Fukuoka, Japan; and University of Colorado Health Sciences Center, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cervical carcinoma is the predominant cancer among malignancies in women throughout the world, and human papillomavirus (HPV) 16 is the most common agent linked to human cervical carcinoma. The present study was performed to investigate the mechanisms of immune escape in HPV-induced cervical cancer cells. The presence of HPV oncoproteins E6 and E7 in the extracellular fluids of HPV-containing cervical cancer cell lines SiHa and CaSki was demonstrated by ELISA. The effect of HPV 16 oncoproteins E6 and E7 on the production of IFN-{gamma} by IL-18 was assessed. E6 and E7 proteins reduced IL-18-induced IFN-{gamma} production in both primary PBMCs and the NK0 cell line. FACS analysis revealed that the viral oncoproteins reduced the binding of IL-18 to its cellular surface receptors on NK0 cells, whereas there was no effect of oncoproteins on IL-1 binding to its surface IL-1 receptors on D10S, a subclone of the murine Th cell D10.G4.1. In vitro pull-down assays also revealed that the viral oncoproteins and IL-18 bound to IL-18R {alpha}-chain competitively. These results suggest that the extracellular HPV 16 E6 and E7 proteins may inhibit IL-18-induced IFN-{gamma} production locally in HPV lesions through inhibition of IL-18 binding to its {alpha}-chain receptor. Down-modulation of IL-18-induced immune responses by HPV oncoproteins may contribute to viral pathogenesis or carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human papillomaviruses (HPVs)3 are well known oncogenic dsDNA viruses linked to cervical cancer, and associated with the high risk group of viral oncogenes E6 and E7 (1). Among the high risk HPVs, HPV 16 and HPV 18 are associated with 70% of all cervical carcinomas, and HPV type 16 correlates to 50% of cervical carcinomas (2, 3). Both E6 and E7 proteins are necessary for immortalization of human keratinocytes (4). However, E6 or E7 alone can transform primary rodent cells in association with activated Ras (5, 6). Thus, the E6 and E7 genes of the high risk group are known to be oncogenes; first, they are selectively retained and expressed in cervical cancer cells (7); second, their encoded proteins interact with and interfere the functions of tumor suppressor proteins p53 and retinoblastoma protein (pRb), respectively (8, 9, 10); and lastly, they can transform and immortalize primary human epithelial cells (11, 12).

There is also evidence that E6 manifests its cellular response independently of p53 functions. E6 protein can bind many cellular proteins other than p53 in vitro. Using the yeast two-hybrid method, a calcium binding protein called E6BP and E6 target protein 1 were isolated in HeLa cell from a cDNA library (13, 14). Binding of E7 to pRb causes release of the transcription factor E2F, which influences expression of the genes concerned in mitosis and cell cycle control (15). E7 also functions in a pRb-independent manner. The E7 oncoprotein exhibits multiple functions, such as protein-protein interactions not only with pRb but also with other pocket proteins, the cyclin-dependent kinase-2 and transcriptional regulating proteins such as the TATA box binding protein and the AP-1 transcription factors (16, 17).

Large DNA viruses encode a variety of immune defense proteins, some of which bind to cytokines and neutralize their activities (18). For example, a homologue of the mammalian IL-18 binding protein (IL-18BP) (19) is found in poxviruses (20). Viral IL-18BP binds to IL-18, a proinflammatory cytokine (21) that plays an important role in the T lymphocyte helper type 1 response as an IFN-{gamma}-inducing factor (22). Recently, others have described IL-18BPs from other viruses (23, 24). Molluscum contagiosum virus (MCV), a human poxvirus and a causative agent of papular skin lesions, persists for a long period without signs of inflammation, similar to HPV infection. The MCV IL-18BP binds human and murine IL-18 with high affinity and inhibits IL-18-mediated IFN-{gamma} production (20). IL-18 has been found to have diverse biological functions, including stimulation of lytic activity of NK cells, induction of IFN-{gamma} and GM-CSF by activated T cells, stimulation of T cell proliferation, and induction of Th1 cell responses (21). IL-12 and IL-18 act synergistically on T cells to induce IFN-{gamma} production. IL-18, together with IL-12, may play an important role in both anti-tumor immunity and protective effects against infection of intracellular pathogens including virus (25, 26).

Infection of HPV in cervical epithelial cells and cervical intraepithelial neoplasia (CIN) can persist for decades before progression to the malignant carcinoma. In addition, the increased incidence of CIN and cervical cancer among immunosuppressed persons strongly suggests that the immune responses play an important role in disease development (27, 28). Moreover, there is accumulating evidence that the cellular immune response, but not the humoral immune response, is impaired in HPV infections compared with that in normal cervical tissues (29, 30). The constitutive expression of E6 and E7 proteins of high risk group HPV is required for maintenance of the transformed phenotype, and the corresponding open reading frames are expressed throughout the epithelium of premalignant and malignant cervical lesions (31, 32). Thus, these oncoproteins can affect tumor-specific immune responses.

Although it is generally accepted that IFN-{gamma} and cell-mediated immunity are important in determining the progression of HPV-related cervical lesions, including cervical cancer (33, 34), there is no direct evidence that the high risk HPV oncoproteins E6 and E7 can modulate the production of IFN-{gamma} by IL-18. In this study we investigated whether the high risk HPV 16 E6 and E7 oncoproteins could affect IL-18-induced IFN-{gamma} production in human PBMCs and the NK0 cell line to elucidate the possible immune escape mechanisms of HPV-infected cervical lesions including cervical cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

Monoclonal anti-human IL-18 Ab clone 8 (IgG2a) was made and characterized as previously described (35). Clone 8 recognizes both precursor and mature forms of IL-18. The following were purchased: recombinant human IL-12p70 (IL-12) and human IL-18 (PharMingen, San Diego, CA); human IL-2 (Roche, Mannheim, Germany); DMEM (Life Technologies, Grand Island, NY); penicillin-streptomycin, amphotericin B, and FBS (Life Technologies); OptEIA human IFN-{gamma} ELISA kit (PharMingen); anti-human IL-18R {alpha}-chain Ab and human IL-1{beta} (R&D Systems, Minneapolis, MN); glutathione-Sepharose beads (Amersham Pharmacia, Little Chalfont, U.K.); Ni-NTA (Qiagen, West Sussex, U.K.); Immobilon-P membrane (Millipore, Bedford, MA); goat polyclonal Abs against C-19 and N-17 of HPV 16 E6 protein and mouse monoclonal anti-HPV 16 E7 IgG2a (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-human p53 Ab (Ab-3; Oncogene, Boston, MA); 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (Bio-Rad, Hercules, CA); nitrocellulose membrane (0.45-µm pore size; Schleicher & Schuell, Dassel, Germany); Histopaque-1077, 3,3'-diaminobenzidine (DAB), isoprophyl-{beta}-D-thiogalactopyranoside; and Sephadex G-25 M, polymyxin B, OVA, RPMI 1640, PHA, imidazole, PMSF, aprotinin, FITC-conjugated anti-mouse IgG, and HRP-conjugated anti-mouse IgG (Sigma, St. Louis, MO). PCR reagents were obtained from Promega (Madison, WI) and Stratagene (La Jolla, CA). Human IL-1R antagonist (IL-1Ra) and TNF binding protein (TNFbp) were purchased from R&D Systems. IL-18BP was expressed in Chinese hamster ovary cells as a His6-tagged carboxyl-terminal recombinant protein as previously described (36). Other reagents were of analytical grade.

Cells

HPV 16-positive cervical cancer cell lines, such as SiHa and CaSki, and normal keratinocyte HaCaT were purchased from American Type Culture Collection (Manassas, VA). These cell lines were maintained in DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 25 ng/ml amphotericin B, and 10% FBS at 37°C in a humidified incubator with 5% CO2. SiHa and CaSki cell lines used in this experiment have been known to contain 1–2 and 60–600 copies of HPV 16 genome, respectively (37). NK0 cells, a subclone of the original NK92 cell line, were maintained in RPMI 1640 medium containing 10% FBS and human IL-2 (0.5 ng/ml) as previously described (38). To elucidate the effect of oncoproteins on IL-18-induced IFN-{gamma} production, the cells were suspended at 1 x 106/ml in RPMI 1640 containing 2 ng/ml IL-12 and 80 ng/ml IL-18 with or without different concentrations of E6 or E7 oncoproteins. After 20 h at 37°C in humidified air with 5% CO2, the culture supernatant was collected for IFN-{gamma} measurement. D10S, a subclone of the murine Th cell D10.G4.1 (39), was maintained at 37°C in a humidified incubator with 5% CO2 in RPMI 1640 medium supplemented with 5% FBS and 10% mouse-conditioned medium as previously described (40).

Human PBMCs

The human PBMCs used in these experiments were obtained from fresh whole blood of healthy volunteers after informed consent or from packed RBC purchased from Chung-Nam National Blood Bank (Taejon, Korea). PBMCs were isolated from buffy coat of heparinized whole blood or citrate-phosphate-dextrose-treated packed RBC by centrifugation on a density gradient of Histopaque-1077, then washed tree times with PBS. PBMCs were suspended at a final concentration of 1 x 106 cells/ml in RPMI 1640 medium supplemented with penicillin, streptomycin, amphotericin B, 10% heat-inactivated FBS, and 50 µM 2-ME, then cultured at 37°C in a humidified incubator with 5% CO2.

Plasmid constructs and expressions of proteins for E6, E7, IL-18, and IL-18R{alpha}

E6 and E7 were inserted into the BamHI-SalI site of pET28a (Invitrogen, Carlsbad, CA) and were expressed, respectively, in Escherichia coli as a His6-tagged amino-terminal recombinant protein as previously described (41, 42). IL-18 cDNA was synthesized using total RNA from a normal keratinocyte cell line, HaCaT. The primers for mature IL-18 were the following: sense, CGC GGA TCC TAC TTT GGC AAG-3'; and antisense, CCG GAA TTC AAT AGC TAG TCT TCG-3'. After subcloning the IL-18 PCR products into T vector (43) that had been prepared from pBluescript pBluescript KS(+) and sequencing of IL-18, mature IL-18 was subcloned into the BamHI-SalI site of pET28a.

IL-18R{alpha} cDNA was amplified from PBMCs by RT-PCR with the following primer pairs: sense, 5'-CGG GGT ACC ATG AATT GTA GAG AAT TAC CCT T-3'; antisense, 5'-CCG GAA TTC TTA AGA CTC GGA AAG AAC AGG CAA-3'. The PCR product was cloned into pTARGET (Promega, Madison, WI). For the construction of IL-18R{alpha}-expressing vector, IL-18R{alpha} cDNA from pTARGET/IL-18R{alpha} was subcloned into EcoRI-NotI sites of pGEX4T-1 (Amersham Pharmacia Biotech, Uppsala, Sweden). Histidine fusion proteins were expressed in E. coli BL21 (DE3) using pET28/E6/E7/IL-18 and purified with Ni-NTA as previously described (41, 42). The concentration of purified proteins was determined by the Bradford method (44). Then the purified proteins were aliquoted and stored at -70°C until use. For GST-tagged IL-18R{alpha}, pGEX/IL-18R{alpha} was expressed in E. coli DH5{alpha}, prepared in PBS containing 0.5% Triton X-100, and then sonicated. The lysates were centrifuged for 30 min at 12,000 rpm to remove pellet containing cell debris. Supernatants were used as cell lysate for the binding assay.

Production of IFN-{gamma}

Human PBMCs and NK0 cells in RPMI 1640 medium were seeded into 96-well plates in 200 µl at final concentrations of 1 x 106/ml and 5 x 105/ml, respectively, and pretreated with 10 µg/ml polymyxin B to neutralize endotoxins. Human IL-18 was used as an inducing agent, and PHA was used as a costimulator in PBMCs. PBMCs or NK0 cells treated with various recombinant proteins were incubated at 37°C in a humidified incubator with 5% CO2 for 20 h, and the amount of the IFN-{gamma} was measured with OptEIA human IFN-{gamma} ELISA kit according to the manufacturer’s instructions.

Dot ELISA

The presence of HPV 16 E6 and E7 proteins in the culture supernatants of cervical cancer cell lines SiHa and CaSki cells was detected by the dot ELISA described by Hawkes et al. (45) with some modifications. After the cells were grown to monolayers, they were washed three times with PBS and cultured overnight in 5 ml DMEM supplemented with 1% FBS. The culture supernatants were collected and centrifuged in a microcentrifuge at a speed of 10,000 rpm for 30 min at 4°C. The supernatants were concentrated to 0.5 ml using a Centricon tube (Millipore) and were dialyzed against PBS at 4°C. For a dot-blot analysis, nitrocellulose membranes were used. DMEM containing 1% FBS was used as a supernatant control. The samples and control supernatants (3 µl) were spotted on the membrane. After blocking nonspecific binding sites with PBS containing 5% skim milk, membranes were treated with goat polyclonal Abs (1/1000) against C-19 and N-17 of HPV 16 E6 protein and mouse monoclonal anti-HPV 16 E7 Ab (1/1000), respectively. Respective peroxidase-conjugated secondary Abs were treated for 45 min. After washing membranes with PBS/0.05% Tween 20 five times, DAB was used as a substrate in a solution of 0.02% Co2+, 0.02% Ni2+, and 0.03% H2O2. The specificity of the reaction was asserted by samples treated with an unrelated mouse anti-human p53 Ab.

Conjugation of IL-18 or IL-1{beta} with FITC

The FITC (isomer I) solution was prepared by dissolving 5 mg FITC in 1 ml DMSO. Ten microliters of FITC solution was added dropwise under continuous stirring to the 500 µg affinity-purified His-tagged IL-18 in 500 µl 0.1 M carbonate-bicarbonate buffer (pH 9.0), and the mixture was agitated at room temperature for 2 h. Then conjugated protein was purified by gel filtration and 1% BSA, and 0.02% sodium azide were added into conjugate. The ratio A280/A495 of IL-18-FITC was ~2. IL-1{beta} was conjugated with FITC as previously described (40) and was used in this IL-1{beta}-binding experiment.

FITC-IL-18 or IL-1{beta}-FITC binding assays using a flow cytometer

NK0 cells were pretreated with 1 ng/ml human IL-12 for 48 h and washed three times with assay buffer (1% BSA and 0.02% sodium azide in PBS). The binding reactions were performed on 1 x 106 cells for 45 min at 4°C in 100 µl assay buffer. NK0 cells were preincubated with E6 and E7 at 4°C for 15 min. Then IL-18-FITC was added to cells at 4°C for 45 min. D10S cells were washed twice with cold RPMI 1640 before resuspension at on 5 x 106 cells in 250 µl assay buffer. D10S cells were preincubated with nonlabeled IL-1{beta}, TNFbp, IL-1Ra, or E6 and E7 proteins for 30 min at 4°C, followed by addition of 128 ng IL-1{beta}-FITC with an additional rotation for 2 h. The bindings of IL-18-FITC and IL-1{beta}-FITC were quantitated by FACS analyses after washing with cold PBS/0.1% BSA containing 0.05% azide using FACSCalibur (BD Biosciences, San Jose, CA).

In vitro binding assay

Binding assays were performed by combining GST-IL-18R{alpha} immobilized on glutathione-Sepharose with increasing doses of bacterial crude lysates from E. coli expressing His-tagged IL-18 protein ranging from 5 to 800 µg in binding buffer (PBS containing 0.5% Triton X-100). The mixtures were rotated in microcentrifuge tubes at 4°C for 1 h. The beads were washed three times with binding buffer, and then boiled for 5 min in 50 µl SDS gel loading buffer. The proteins in the sample buffer were subjected to electrophoresis on a 12% polyacrylamide gel, transferred to the Immobilon-P membrane, and immunoblotted using IL-18-specific mAb 8.

The binding reactions between either E6 or E7 and IL-18R{alpha} were also performed as the IL-18 binding assay described above. E6 and E7 proteins were detected by goat polyclonal Abs against C-19 and N-17 of HPV 16 E6 and mouse monoclonal anti-HPV 16 E7 Ab, respectively. Respective secondary Abs conjugated with alkaline phosphatase were used. 5-Bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium was used as a substrate for color development.

Statistical analysis

Data are expressed as the mean ± SEM. Group means were compared by ANOVA using Fisher’s least significant difference. ANOVA and correlation analyses were performed with the statistical packages StatView 512+ (BrainPower, Calabasas, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-18-induced IFN-{gamma} production was inhibited by both E6 and E7 oncoproteins of HPV 16

Purified recombinant E6 and E7 proteins from E. coli were assessed to determine whether they could inhibit the functions of IL-18 on immune cells. We investigated whether the HPV 16 E6 or E7 oncoprotein inhibits the production of IFN-{gamma} by IL-18 in PBMCs from five different donors and in the NK0 cell line. To determine the effect of IL-18 on IFN-{gamma} production, we preincubated PBMCs in a neutralizing anti-IL-18R{alpha} Ab and treated the culture with IL-18. The treatment of anti-IL-18R{alpha} Ab inhibited IFN-{gamma} production, confirming that IL-18 ligation on the cell surface IL-18R{alpha} induced IFN-{gamma} production (Fig. 1GoA). The pretreatment of E6 or E7 proteins resulted in significant reductions in IL-18-induced IFN-{gamma} in PHA-treated PBMCs (27.1 ± 8.1 to 11.4 ± 10.6% for E6 compared with IL-18-induced IFN-{gamma} production, and 32.4 ± 16 to 11.8 ± 8.8% for E7 compared with IL-18-induced IFN-{gamma} production; Fig. 1GoB).



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  		FIGURE 1. The effect of HPV oncoproteins E6 and E7 on IL-18-induced IFN-{gamma} production in PBMCs. Polymyxin B (10 µg/ml) was added to the cells to neutralize endotoxin. The concentrations of the reagents used were as follows: IL-18, 50 ng/ml; PHA, 1 µg/ml; neutralizing anti-IL-18R{alpha} Ab, 0.5 µg/ml; and IL-18BP, 0.5 µg/ml. The IL-18BP or OVA was mixed with IL-18, preincubated at room temperature for 30 min, and treated into PBMCs for 20 h. The oncoproteins and neutralizing anti-IL-18R{alpha} Ab were preincubated with cells for 20 min. Then IL-18 was added to the cells for 20 h. OVA was added as a nonrelated protein. E6 and E7 significantly inhibited the IL-18-induced IFN-{gamma} production. *, p < 0.05. Results are the mean ± SEM of five experiments.

 
IL-18 and IL-12 synergistically activate NK0 cells to induce IFN-{gamma} production. The viral oncoproteins E6 and E7 also inhibited IL-18-induced IFN-{gamma} production in NK0 cells cotreated with IL-12 (19.6 ± 3.4, 4.7 ± 1.5, respectively; Fig. 2Go). There was no effect of OVA on IL-18-induced IFN-{gamma} production, and IL-18BP neutralized the effect of IL-18 in both PBMCs and NK0 cells.



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  		FIGURE 2. The effect of HPV oncoproteins E6 and E7 on IL-18-induced IFN-{gamma} production in NK0 cell lines coactivated with IL-12. Cells were pretreated with polymyxin B (10 µg/ml) to neutralize endotoxin. The concentrations of reagents used were as follows: IL-18, 80 ng/ml; IL-12, 2 ng/ml; E6, 50 ng/ml; E7, 50 ng/ml; or OVA, 50 ng/ml. The oncoproteins were preincubated with NK0 cells at room temperature for 20 min, and IL-18 was added to NK0 cells for 20 h. OVA was added as a nonrelated protein. *, p < 0.05 compared with coincubation of IL-12 and IL-18. Results are the mean ± SEM of three experiments.

 
HPV 16 E6 and E7 proteins were detected in culture supernatant of CaSki cells

Hence, E6 or E7 oncoproteins inhibited IL-18-induced IFN-{gamma} production in PBMCs and NK0 cells. We investigated whether those oncoproteins exist in the extracellular compartment of cervical carcinomas. The presence of E6 and E7 proteins in culture supernatant of CaSki was ascertained by dot ELISA (Fig. 3Go). In the case of SiHa, the presence of the proteins was so minute that E6 and E7 were rarely detected. Control recombinant E6 could be detected up to 10 pg (Fig. 3GoA), and control E7 up to 1 pg (Fig. 3GoB) by anti-E6 or -E7 Abs, respectively. Treatment of anti-E6 or -E7 Abs showed that there were no nonspecific binding in control culture medium (Fig. 3Go, A and B). When an unrelated Ab such as mouse anti-human p53 Ab was used, no stain was apparent in the dot (Fig. 3GoC).



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  		FIGURE 3. Dot ELISA for the presence of extracellular HPV 16 E6 and E7 proteins in the supernatants of SiHa and CaSki cells. The supernatants of cervical carcinomas were spotted on the membrane. After blocking nonspecific binding sites, membranes were treated with Abs against HPV 16 E6 and E7, respectively. Then respective peroxidase-conjugated secondary Abs were added. After washing, DAB was used as a substrate. The specificity of the reaction was asserted in samples treated with an unrelated mouse anti-human p53 Ab. A, The presence of E6 protein was detected by goat polyclonal anti-HPV16 E6 Abs. Strip 1, Purified E6 protein was deposited. Strip 2, Culture supernatant was deposited. NC, culture medium as a normal control. B, The presence of E7 protein was detected by mouse monoclonal anti-HPV16 E7 Ab. Strip 1, Purified E7 protein was deposited. C, This strip revealed nonspecific binding of E6 or E7 detected by an unrelated Ab.

 
E6 and E7 oncoproteins inhibit the binding of IL-18 to its cellular IL-18 receptors, while there is no effect of the IL-1 binding to its surface IL-1 receptors by E6 and E7

To assess the inhibitory effect of E6 and E7 on IL-18, direct binding of IL-18-FITC to NK0 cells and inhibition assays of binding by the oncoproteins were performed. The NK0 cells were pretreated with IL-12 to induce IL-18R on the cell surface for 48 h (Fig. 4GoA) as previously described (46). IL-18-FITC bound to the cell surface in a dose-dependent manner (Fig. 4GoB), while an irrelevant FITC-conjugated protein (anti-mouse IgG-FITC) did not. As shown in Fig. 5Go, we tested the specificity of the binding by displacement with unconjugated IL-18, prevention by a neutralizing anti-human IL-18R{alpha} Ab, or neutralization by IL-18BP. Neutralizing anti-human IL-18R{alpha} Ab inhibited IL-18 binding, supporting the idea that IL-18-FITC was specifically bound to its cell surface receptor. FACS analyses showed that the viral proteins E6 and E7 inhibited the staining of IL-18-FITC to its cellular IL-18 receptor; however, they did not affect the IL-1{beta} binding to its surface IL-1 receptors on D10S, and the binding of IL-18 to its cellular IL-18 receptor was not hindered with TNFbp (Fig. 5Go).



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  		FIGURE 4. A, Flow cytometric analysis of IL-18R expression on NK0 cells. NK0 cells were analyzed by flow cytometry for the expression of surface IL-18R{alpha} using mouse monoclonal anti-human IL-18R{alpha} Ab followed by FITC-conjugated anti-mouse IgG. FITC-conjugated anti-mouse IgG was used as a control Ab. NK0 cells were pretreated with IL-12 (1 ng/ml) for 48 h to induce IL-18Rs. The data show the results of one representative of three similar experiments. B, The binding of FITC-IL-18 to NK0 cells. NK0 cells (1 x 106) were incubated for 45 min at 4°C with increasing amounts of FITC-conjugated IL-18 or an unrelated FITC-conjugated anti-mouse IgG. Then the binding of FITC was assessed by FACS analysis. NK0 cells were pretreated with IL-12 (1 ng/ml) for 48 h to induce IL-18R.

 


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  		FIGURE 5. Flow cytometric analysis of competitive binding of E6 or E7 proteins with IL-18 for IL-18R on NK0 cells. A, NK0 cells were pretreated with IL-12 (1 ng/ml) for 48 h. Then, NK0 cells (1 x 106) were incubated for 45 min at 4°C with IL-18-FITC. E6, E7, or anti-IL-18R{alpha} Ab was added before 30 min, and the other cells were mixed and preincubated for 30 min at 4°C with IL-18-FITC before treatment. B, D10S cells (5 x 106) cells were preincubated with nonlabeled IL-1{beta}, TNFbp, IL-1Ra, or E6 and E7 proteins for 30 min at 4°C, followed by addition of 128 ng IL-1{beta}-FITC with an additional rotation for 2 h. Then, the binding of IL-18-FITC and IL-1{beta}-FITC was assessed by FACS analyses. *, p < 0.05 compared with IL-18-FITC binding or IL-1{beta}-FITC. Results are the mean ± SEM of three experiments.

 
Both E6 and E7 oncoproteins bound to IL-18R{alpha} and inhibited IL-18 binding to its receptor in vitro

To confirm whether E6 and/or E7 proteins could inhibit the binding of IL-18 to its receptor, IL-18R{alpha}, in vitro pull-down assays were performed with recombinant proteins. Our in vitro binding assays revealed that IL-18 bound to IL-18R{alpha} in a dose-dependent manner (Fig. 6GoA). As doses of E6 and E7 proteins were increased, E6 and E7 were gradually bound to IL-18R{alpha} (Fig. 6Go, B and C), whereas binding of IL-18 to its IL-18R{alpha} was gradually inhibited (Fig. 6GoD). The E6 and E7 proteins bound to IL-18R{alpha}, and the bindings were also inhibited by IL-18 in a dose-dependent manner (Fig. 7Go). Our previous data and FACS analyses showed that E6 protein did not bind to Rb (42), and E6 and E7 oncoproteins did not affect the IL-1{beta} binding to its surface IL-1Rs. These results suggested that E6 and E7 proteins, via binding of the viral proteins to IL-18R{alpha} and replacing bound IL-18, would inhibit the IL-18-induced cellular responses in immune cells.



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  		FIGURE 6. A, IL-18 binding to IL-18R in vitro. Binding assays were performed by combining GST-IL-18R{alpha} immobilized on glutathione-Sepharose with increasing doses of bacterial lysates from E. coli expressing His-tagged IL-18 proteins ranging from 5 to 800 µg in binding buffer (PBS containing 0.5% Triton X-100). The binding reaction was performed at 4°C for 1 h, then the unbound proteins were washed, and Western blot analysis was performed using IL-18-specific Ab. bGST, glutathione-Sepharose bead. B and C, The effect of HPV 16 E6 and E7 proteins on binding of IL-18 to IL-18R{alpha} in vitro. Competitions of E6 or E7 proteins with IL-18 protein for glutathione-Sepharose bead-bound IL-18R{alpha} were analyzed, and the proteins bound to IL-18R{alpha} were detected by Western blot analysis. A constant amount of IL-18 and bead-bound IL-18R{alpha} was incubated with an increasing amount of E6 (B) or E7 (C) proteins at 4°C for 1 h, then the unbound proteins were washed, and Western blot analysis was performed using E6- or E7-specific Ab, respectively. D, The competition assays of E6 or E7 with IL-18 protein for bead-bound IL-18R{alpha} were detected by IL-18-specific Ab.

 


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  		FIGURE 7. The effect of IL-18 on HPV 16 E6 or E7 protein binding to IL-18R{alpha} in vitro. A, Constant amounts of E6 or E7 protein and bead-bound IL-18R{alpha} were incubated with increasing amounts of His-tagged IL-18 at 4°C for 1 h, then the unbound proteins were washed, and Western blot analysis was performed using specific Ab against E6, E7, and IL-18 protein, respectively. B, Coomassie staining of polyacrylamide gel.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The link between HPV and development of squamous intraepithelial lesions as well as cervical cancer is well known (1). The pathogenesis of cervical HPV infection is affected by the immune responses of the host, which depend upon specific cytokines and recruited inflammatory cells at the site of the lesion (47). It has been reported that few inflammatory cells were infiltrated, and a reduced cytokine level was observed in HPV-related cervical lesions, including CIN (33, 47). These reports suggested that high risk group HPV-related lesions were immunologically ignored. Moreover, as a consequence of the frequent integration of the HPV genome during carcinogenesis only two viral genes, E6 and E7, are consistently expressed, always intact, and transcribed at higher levels in high grade lesions than in low grade ones (7, 48). In contrast, it was reported that IFN-{gamma} repressed the viral gene expression transcriptionally in HPV 16 immortalized cell lines and inhibited the growth of the cells (49). However, transcription of the IFN-{gamma} gene was shown to be significantly reduced in both CIN and cervical cancer tissues compared with normal cervical tissues (33).

We investigated whether HPV 16 E6 and E7 proteins can affect the host immune response. IL-18-induced IFN-{gamma} production was assessed in human PBMCs, and the results revealed that both the E6 and E7 proteins have a significant inhibitory effect on IL-18-induced IFN-{gamma} release. Similar results were also obtained from NK0 cells. Both E6 and E7 oncoproteins inhibited IL-18-induced TNF-{alpha} production in PBMCs. However, these TNF-{alpha} inhibition effects were dependent on donors (data not shown). These experiments were performed with polymyxin B pretreatment to inhibit possibly contaminated bacterial endotoxins. When we did not use polymyxin B, the results were affected by contaminated endotoxin in purified recombinant proteins from E. coli (data not shown). IFN-{gamma} was known to play important roles in reducing the HPV genome as well as tumor suppression (49). In contrast, E6 and E7 oncoproteins were known to have multiple functions not only growth regulation of the cells, but also regulation of host immune responses (33, 50). In flow cytometric analyses, HPV 16 E6 and E7 oncoproteins inhibited binding of IL-18 to its cellular IL-18R, while there was no effect of the oncoproteins on IL-1 binding to surface IL-1Rs on D10S cells (Fig. 5Go). These results suggested that the inhibition of IFN-{gamma} production by the viral oncoproteins might be mediated via blocking of IL-18 binding to IL-18R{alpha}. To confirm the inhibitory effect of oncoproteins, in vitro pull-down assays were performed with recombinant proteins from E. coli whether E6 and E7 proteins could bind to IL-18R{alpha}. These pull-down assays revealed that both E6 and E7 could bind to IL-18R{alpha} and inhibit the IL-18 binding to IL-18R{alpha}. IL-18 also inhibited the binding of E6 and E7 to IL-18R{alpha} in a dose-dependent manner, suggesting that IL-18 and the oncoproteins competed each other with IL-18R{alpha}. If E6 and E7 proteins could inhibit IL-18 function by blocking its cell surface IL-18R{alpha}, the proteins should exist in the extracellular compartment in cervical cancer cells evading host immune responses. Hence, the presence of E6 and E7 proteins in culture supernatants of cervical cancer cell lines, SiHa and CaSki, was assessed by a dot ELISA. However, the amount of the proteins was so minute that only a minimal amount of their existence could be exhibited. The E6 and E7 proteins were prominent in CaSki, but were hardly detected in SiHa. This result might be due to the copy number of the proteins in the corresponding cell lines. CaSki and SiHa cell lines have been known to contain 60–600 and 1–2 copies of HPV 16 genome, respectively (37). We used a polyclonal anti-E6 Ab and a monoclonal anti-E7 Ab. The different detection sensitivities of the two proteins might attribute to the purity and affinity of the Abs used. It was assumed that a monoclonal anti-E7 Ab could detect E7 more sensitively than a polyclonal anti-E6 Ab. We used high concentrations (5–50 ng/ml) of oncoproteins in these studies to inhibit IL-18-induced IFN-{gamma} production. Although there are minute amount of the oncoproteins in culture supernatants, the inhibition of IL-18-induced immune responses by E6 and E7 is considered to be a possible event in cervical cancer because the concentration of the proteins in microenvironment might be sufficient due to accumulation of the proteins for long periods in the HPV-infected cervical lesion. In addition, HPVs usually infect basal epithelial cells, and the lesion developed on the surface layer of the squamous epithelium. Viral proteins may be accumulated sufficiently around the infected cells during this period. According to some clinical studies, the most consistent findings have been that the presence of serum Abs to the transforming E6 and E7 proteins of the virus is associated with invasive cancer compared with the sera of controls (51, 52). These reports suggested the presence of circulating E6 and E7 proteins in cervical cancer patients. However, the Abs specific to E6 or E7 proteins did not influence the course of the disease, but, rather, were a marker for disease progression (30). The minute amounts of E6 and E7 proteins in culture supernatant of SiHa cell were previously reported using the same dot ELISA (53, 54). Although the exact mechanism of the presence of E6 and E7 protein in extracellular compartment was not known, whether actively exported or passively diffused, the circulating E6 and E7 protein might play a role in immunomodulation. HPV 16 E7 protein-induced immunosuppression by enhanced production of IFN-{alpha} and suppression of Ag-stimulated human T cells were also shown in APCs and PBMCs (54). However, it was reported that IFN-{alpha} and IL-12 enhanced IL-18R{alpha} gene expression in human NK and T cells (46). In addition, extracellular E7 inhibits the immune cell response to recall and alloantigens, and enhances the release of angiogenic cytokines, including TNF-{alpha}, IL-1{beta}, and IL-6, by macrophages and/or dendritic cells (54). The expression of HPV 16 E7 protein-abrogated IFN-{alpha}-mediated signals by direct protein-protein interaction between E7 and p48, a DNA-binding component of IFN-stimulated gene factor 3 (ISGF3), led to loss of ISGF3 transcription complex formation (55).

Our study focused on the IL-18-induced immune response in PBMCs and NK0 cells. There have been several reported biological activities of IL-18, suggesting possible roles in antiviral and anti-tumor effects (56); stimulation of IFN-{gamma}, IL-2, GM-CSF, chemokines (IL-8, macrophage inflammatory protein-1{beta}, and monocyte chemoattractant protein-1), enhancement of T and NK cell cytotoxicity, enhancement of the development of Th1 cells, and induction of Fas and FasL on some leukemic cells. In BALB/c mice, IL-18 treatment significantly suppressed pock formation on the tail of mice inoculated i.v. with vaccinia virus, while NK and CTL activities were significantly augmented (25). The infected mice showed severe deterioration of health and loss of body weight when injected with anti-IFN-{gamma} Ab. In contrast, IL-18 was also produced in response to viral infection and enhanced cellular immunity against viral infection. GM-CSF-differentiated human macrophages produce IL-18 protein against Sendai virus infection by a caspase-1-dependent pathway (57). IL-18 induces IFN-{gamma} and NK cell cytotoxicity, making it a logical target together with IFNs for viral antagonism of host defense. However, this is not so surprising, although HPV is equipped with counteracting tools against important host factors, such as IL-18 or IFNs. Many DNA viruses, rather then RNA viruses, have strategies to resist IFNs (58, 59). The viral response against IL-18 has recently been shown, especially in human poxviruses. A family of proteins encoded by several poxviruses showed high homology to IL-18BP (19), but were little homologous to IL-18R{alpha} (23, 24). The epidermal lesions including the virus, like HPV-induced lesions, frequently persist for a considerable period without signs of inflammation even in immunocompetent individuals. Some proteins were found in MCV that can bind to IL-18 with high affinity. The proteins from MCV, MC53L and MC54L, are homologues of recently discovered human and mouse IL-18BP; they bind with high affinity to human and murine IL-18 and inhibit IL-18-mediated IFN-{gamma} production (20). IL-18 is required in vivo for induction of IFN-{gamma}. Therefore, the inhibition of IL-18 activity by oncoproteins E6 and E7 of high risk HPV 16 represents a novel immune escape strategy of the virus and/or the virally induced lesions, including cervical cancer. The E6 and E7 proteins were revealed to have other immunomodulatory functions, such as induction of CTL tolerance and inhibition of IFNs. The mechanism of immune escape of HPV or HPV-induced cervical cancer needs further investigation to determine the functional biological immune responses of IL-18 such as CTL assay. Cells or tissues obtained from HPV 16-infected patients or cervical carcinomas will be further investigated to determine whether HPV infection inhibits both IL-18 expression and IL-18-induced immune responses by using immunohistochemistry and applying these experimental procedures. This study may provide new insight into the pathogenesis of HPV infection and tumorigenesis.


   Footnotes
 
1 This work was supported by Molecular Medicine Program Grant 98-J03-02-02-A-03 from the Ministry of Science and Technology, Korea (to D.Y.) and was supported in part by National Institutes of Health Grant AI15614 (to C.A.D.). Back

2 Address correspondence and reprint requests to Dr. Do-Young Yoon, Laboratory of Cellular Biology, Korea Research Institute of Bioscience and Biotechnology, Yuseong P.O. Box 115, Taejon 305-600, Korea. E-mail address: dyyoon{at}mail.kribb.re.kr Back

3 Abbreviations used in this paper: HPV, human papillomavirus; IL-18BP, IL-18 binding protein; MCV, Molluscum contagiosum virus; CIN, cervical intraepithelial neoplasia; TNFbp, TNF binding protein; Rb, retinoblastoma; DAB, 3,3'-diaminobenzidine; IL-1Ra, IL-1R antagonist. Back

Received for publication January 22, 2001. Accepted for publication April 30, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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