| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Department of Clinical and Experimental Medicine, Medical School of Novara, Via Solaroli 17, 28100 Novara, Italy
2 Department of Medical Sciences, Medical School of Novara, Via Solaroli 17, 28100 Novara, Italy
3 Department of Public Health and Microbiology, Medical School of Torino, Via Santena 9, 10126 Torino, Italy
4 International Agency for Research on Cancer–World Health Organization, 150 Cours Albert Thomas, 69372 Lyon, France
5 NoToPharm s.r.l., Bioindustry Park del Canavese, Via Ribes 5, 10010 Colleretto Giacosa (TO), Italy
Correspondence
Marisa Gariglio
gariglio{at}med.unipmn.it
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
was downregulated in HPV-immortalized cells. These data support the functional similarity between HPV16 and 38, and suggest an active role of these viruses in modulation of the inflammatory process. |
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). Persistent expression of pro-inflammatory cytokines at tumour sites may exert both protective and detrimental effects, ranging from an increase in malignant cell invasiveness to promotion of an anti-tumour immune surveillance that may counteract malignant cell growth (de Visser et al., 2006).
Keratinocytes can be induced to produce cytokines by several exogenous stimuli and dysregulation of this production has been described in several skin diseases, including cancer (van Kempen et al., 2003). One of the main exogenous inducers seems to be UVB irradiation. It stimulates keratinocyte cultures to secrete pro-inflammatory cytokines such as tumour necrosis factor-
(TNF-), interleukin-1β (IL-1β), IL-6, IL-10 and IL-8. The release of these cytokines can trigger a cutaneous inflammatory response that develops in skin exposed to sunlight and sunburn (Kupper et al., 1987; Kondo et al., 1993; Strickland et al., 1997). The preferential localization of skin squamous cell carcinoma (SCC) and human papillomavirus (HPV) infection to chronically sun-exposed body sites strongly suggests that UV and HPV may synergistically cooperate in the development of skin lesions (Pfister 2003; Akgül et al., 2006). One possibility is that this synergism may partly act at the level of the skin inflammatory response, but the effects of UVB irradiation on cytokine production by HPV-infected cells has not been studied yet (Ruhland & De Villiers, 2001).
The differential expression profile of pro-inflammatory molecules comparing the conditions of mucosal (alpha genus) and cutaneous (beta genus) HPV genotypes (de Villiers et al., 2004) has been reported (Woodworth & Simpson, 1993; Smola-Hess et al., 2001; De Andrea et al., 2007), but much less is known about the synergistic effect of E6/E7 oncoproteins and UVB on induction of inflammation (Ruhland & De Villiers, 2001; Akgül et al., 2005).
The aim of this study was to investigate the effect of UVB irradiation on secretion of the cytokines TNF-, IL-1β, IL-6, IL-8 and transforming growth factor-β (TGF-β) involved in the inflammatory process by human keratinocytes immortalized with HPV16 or HPV38 (Kupper et al., 1987; Kondo et al., 1993; Strickland et al., 1997). In addition, expression of the pro-inflammatory molecules S100A8, S100A9 and keratinocyte-specific interferon- (IFN-) was also evaluated.
Keratinocyte cell lines, grown in KGM-2 serum-free medium (Lonza), were immortalized by infection of pooled primary human keratinocytes derived from neonatal foreskin (HFK) with an amphotropic LXSN retrovirus (Pear et al., 1993) expressing the open reading frames of the E6 and E7 genes from HPV16 (HPV16-HFK) or HPV38 (HPV38-HFK) (Caldeira et al., 2003). As negative control, HFK (passage 2) were infected with the empty viral vector (LXSN-HFK), selected with G418, pooled and used at the very early passages (De Andrea et al., 2007). To exclude variation due to the specific genetic background of donors, HFK from two different pools were used independently. RT-PCR analysis was performed to confirm that the transgenes were expressed at similar levels (data not shown).
Since it has been previously reported that the immortalization process subsequent to the expression of HPV16 E6 and E7 may modify UVB sensitivity (Jackson et al., 2000; Simbulan-Rosenthal et al., 2002), and to determine a UVB dose that would stimulate the cells, but not be so toxic that mRNA would be degraded, the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide dye] assay was used to assess viability of HFK cell lines 24 h after exposure to increasing doses of UVB. As shown in Table 1, viability was lower in HPV38-HFK than in HPV16-HFK and LXSN-HFK at the highest UVB doses (i.e. 400 and 500 J m–2) (P<0.05, one-way ANOVA test). In contrast, survival of both HPV16-HFK (62 %) and HPV38-HFK (57 %) was not significantly different from that of LXSN-HFK (68 %) at the 300 J m–2 UVB dose (P>0.05, one-way ANOVA test). Cellular morphology of HFKs at 24 h post-irradiation showed that more than 50 % of the keratinocytes irradiated at 300 J m–2 survived the UVB exposure (Supplementary Fig. S1, available in JGV Online). As the effect of 300 J m–2 appeared to be similar in respect to cell viability in all three cell lines, together with the observation that lower UVB doses did not substantially induce cytokine production (data not shown), it was decided to use this dose for all subsequent experiments.
|
. The housekeeping gene hypoxanthine phosphoribosyltransferase 1 was used to normalize for variations in cDNA and PCR was performed in triplicate for each sample. The results were analysed with a relative expression software tool (REST©) (Pfaffl et al., 2002). To verify expression at the protein level, supernatants from the same cultures were collected at 24 and 48 h after stimulation and analysed by the Human Inflammation kit (BD Biosciences) or ELISA assay (TGF-β1) (R&D Systems). For the latter, the supernatants were treated by acidification before processing. Statistical significance was determined by one-way ANOVA, followed by Bonferroni's multiple comparison post-hoc test.
|
. UVB irradiation strongly upregulated IL-6 mRNA in both HPV16-HFK and HPV38-HFK, especially at 24 h post-irradiation (4.7- and 3.1-fold, respectively; P<0.05), whereas its induction in LXSN-HFK was minimal. Consistent with the mRNA expression analysis, IL-6 secretion was increased in both HPV-immortalized cell lines, but not in the normal counterpart, although maximal levels were observed with HPV16 48 h after UVB irradiation. According to our previous report (De Andrea et al., 2007), the basal level of IL-6 was higher in both HPV-immortalized cell lines compared with LXSN-HFK. A similar induction pattern was detected with IL-8; its mRNA was strongly upregulated by UVB in both HPV16-HFK and HPV38-HFK, especially at 24 h after irradiation (22.5- and 3.1-fold, respectively; P<0.01), whereas its induction was weak in LXSN-HFK. According to our previous report (De Andrea et al., 2007), the basal level of IL-8 mRNA was higher in HPV38-transduced cells and lower in HPV16-transduced cells compared with the normal counterpart. When the levels of secreted IL-8 were assessed in cell supernatants, there was a consistent increase in its production in all cell lines 48 h after UVB irradiation. Analysis of IL-1β mRNA expression levels revealed no significant (P>0.05) variation in the basal condition. Interestingly, after UVB exposure, there was a significant induction (P<0.01) of its mRNA in control cells, but no relevant upregulation in HPV-immortalized cell lines. Production of IL-1β in the cell supernatant, measured as total cytokine, was induced by UVB only in control cells, as already observed at the mRNA level. In the case of TNF- mRNA, we observed a significant (P<0.05) upregulation of its mRNA in both control cells (24-fold) and HPV38-transduced cells (31-fold), but not in HPV16-transduced cells, at 6 h post-irradiation. Its release into the cell supernatant was very low in all samples, below 4 pg ml–1, and no variation was detected after UVB treatment. TGF-β mRNA and secretion were substantially upregulated by UVB irradiation in both HPV16-HFK and HPV38-HFK, but not in control cells. Basal levels of both mRNA and protein were higher in HPV38-HFK than in HPV16-HFK and control cells. The Human Inflammation kit also includes evaluation of IL-10 and IL-12 protein levels, but their levels were always low, close to the detection limit of the assay, with no significant variation in either basal or stimulated condition (data not shown).
Several reports have suggested a direct link between the expression of two small calcium-binding proteins, S100A8 and S100A9, and inflammation in cancer. Both genes are highly upregulated in keratinocytes and infiltrating cells upon TPA (12-O-tetradecanoylphorbol-13-acetate) treatment, as well as during tumour promotion in chemically induced skin carcinogenesis (Gebhardt et al., 2006). Therefore, we evaluated S100A8 and S100A9 by real-time RT-PCR in our HFK cell lines, with and without UVB irradiation. In parallel, the same technique was used to analyse the expression of IFN-, a cytokine belonging to the type I IFN family that is specifically expressed by keratinocytes (Scarponi et al., 2006; Buontempo et al., 2006). Results show that expression of S100A8, S100A9 and IFN- mRNA was high in LXSN-HFK, but low in both HPV16-HFK and HPV38-HFK. Moreover, UVB irradiation upregulated their mRNA in LXSN-HFK, but not in HPV16-HFK or HPV38-HFK (Fig. 2).
|
; Stanley 2006). While some studies show a lack of association between inflammation and epithelial skin cancer, predominant evidence clearly supports a tumour-promoting effect of inflammation on the development and progression of these cancers in both human and animal models (Coussens et al., 1996; van Kempen et al., 2003; Mueller 2006). Despite these findings, few reports are available on the modulation of cytokine expression by UV irradiation in HPV-immortalized keratinocytes (Ruhland & De Villiers, 2001). In this study, we show that immortalization by E6/E7 oncoproteins influences not only the basal cytokines secretion profile of keratinocytes, but also its modulation upon UVB irradiation. The influence on the cell response to UVB was detectable at the relatively toxic dose of 300 J m–2, and not at 200 J m–2 (data not shown), but it was not simply ascribable to differential cell death because toxicity was similar in all cell lines and the effect was heterogeneous on different cytokines. We demonstrate that UVB upregulates IL-6, IL-8 and TGF-β in HPV-immortalized cells to a significantly higher extent than in control keratinocytes. Since these cytokines display heterogeneous functions during the inflammatory process, including promotion of angiogenesis, neutrophil chemotaxis, keratinocyte proliferation and matrix metalloproteinase (MMP) production (Apte et al., 2006; Gabay 2006; Kishimoto 2006; Li et al., 2006), they may play a key role in micro-environmental promotion of skin cancer development via the paracrine pathway. They may synergistically promote keratinocyte proliferation and inflammation by recruiting mast cells, neutrophils and monocytes/macrophages. In line with this hypothesis, a mouse model of HPV16-induced squamous epithelial carcinogenesis showed that both pre-malignant skin and cervix lesions display chronic infiltration of innate immune cells contributing to cancer development (Coussens et al., 1996). Furthermore, in our experimental system, the UVB-mediated effect was somehow selective, since it did not induce the release of other related cytokines, such as IL-10 and IL-12, for which involvement in the inflammatory process has also been demonstrated.
Inhibition of S100A8/9 expression in HPV16-immortalized or transformed cells has been previously reported (Tugizov et al., 2005), but this is the first report of S100A inhibition by the cutaneous beta-genotype HPV38. This inhibition has been associated with upregulation of cellular casein kinase II (CKII)-mediated E7 phosphorylation, and enhancement of its growth-promoting and oncogenic activity. Our finding of S100A8/9 inhibition in HPV38-immortalized cells indicates that both genotypes may exploit the same molecular pathway to escape the anti-proliferative activity of secreted S100A8/9, increase E7 oncogenic activity and possibly favour the progression of HPV-associated neoplasia.
IFN- has been shown to signal via the type I IFN-stimulated transcription pathway that produces an antiviral state in cells exposed to the recombinant protein (Buontempo et al., 2006). Several in vitro studies demonstrated the ability of HPV oncoproteins to control signalling pathways that lead to the expression of IFNs and IFN-inducible genes (Barnard et al., 2000; Koromilas et al., 2001; O'Brien & Saveria Campo, 2002). Expression of HPV16 E6 in human keratinocytes was able to diminish the induction of IFN-β gene expression by Sendai virus and, consequently, the expression of IFN-inducible genes (Ronco et al., 1998). In line with these findings, we observed that IFN- mRNA is significantly (P<0.01) downregulated in cells expressing E6/E7 from both HPV16 and 38. Thus, the capability of HPVs to limit IFN- production during infection may be responsible for reducing the host's ability to mount an effective antiviral state.
It has previously been shown that HPV38 E6 and E7, similar to HPV16 E6 and E7, deregulate cell cycle control, increase the life span of primary human keratinocytes and induce cellular transformation (Caldeira et al., 2003; Accardi et al., 2006). Here, we show novel findings that further confirm the functional similarity between the two HPV types and suggest an active role of these viruses in modulation of the inflammatory process.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Np73. EMBO Rep 7, 334–340.[CrossRef][Medline]Akgül, B., Lemme, W., Garcìa-Escudero, R., Storey, A. & Pfister, H. J. (2005). UV-B irradiation stimulates the promoter activity of the high-risk, cutaneous human papillomavirus 5 and 8 in primary keratinocytes. Arch Virol 150, 145–151.[CrossRef][Medline]
Akgül, B., Cooke, J. C. & Storey, A. (2006). HPV-associated skin disease. J Pathol 208, 165–175.[CrossRef][Medline]
Apte, R. N., Krelin, Y., Song, Y., Dotan, S., Recih, E., Elkabets, M., Carmi, Y., Dvorkin, T., White, R. M. & other authors (2006). Effects of micro-environment- and malignant cell-derived interleukin-1 in carcinogenesis, tumour invasiveness and tumour-host interactions. Eur J Cancer 42, 751–759.[CrossRef][Medline]
Barnard, P., Payne, E. & McMillan, N. A. J. (2000). The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-. Virology 277, 411–419.[CrossRef][Medline]
Buontempo, P. J., Jubin, R. G., Buotempo, C. A., Wagner, N. E., Reyes, G. R. & Baroudy, B. M. (2006). Antiviral activity of transiently expressed IFN- is cell-associated. J Interferon Cytokine Res 26, 40–52.[CrossRef][Medline]
Caldeira, S., Zehbe, I., Accardi, R., Malanchi, I., Dong, W., Giarrè, M., de Villiers, E. M., Filotico, R., Boukamp, P. & other authors (2003). The E6 and E7 proteins of the cutaneous human papillomavirus type 38 display transforming properties. J Virol 77, 2195–2206.
Coussens, L. M., Hanahan, D. & Arbeit, J. M. (1996). Genetic predisposition and parameters of malignant progression in K14–HPV16 transgenic mice. Am J Pathol 149, 1899–1917.[Abstract]
De Andrea, M., Mondini, M., Azzimonti, B., Dell'Oste, V., Germano, S., Gaudino, G., Musso, T., Landolfo, S. & Gariglio, M. (2007). Alpha- and betapapillomavirus E6/E7 genes differentially modulate pro-inflammatory gene expression. Virus Res 124, 220–225.[CrossRef][Medline]
de Villiers, E. M., Fauquet, C., Broker, T. R., Bernard, H. U. & zur Hausen, H. (2004). Classification of papillomaviruses. Virology 324, 17–27.[CrossRef][Medline]
de Visser, K. E., Eichten, A. & Coussens, L. M. (2006). Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 6, 24–36.[CrossRef][Medline]
Gabay, C. (2006). Interleukin-6 and chronic inflammation. Arthritis Res Ther 8 (Suppl. 2), S3
Gebhardt, C., Nemeth, J., Angel, P. & Hess, J. (2006). S100A8 and S100A9 in inflammation and cancer. Biochem Pharmacol 72, 1622–1631.[CrossRef][Medline]
Jackson, S., Harwood, C., Thomas, M., Banks, L. & Storey, A. (2000). Role of Bak in UV-induced apoptosis in skin cancer and abrogation by HPV E6 proteins. Genes Dev 14, 3065–3073.
Kishimoto, T. (2006). Interleukin-6: discovery of a pleiotropic cytokine. Arthritis Res Ther 8 (Suppl. 2), S2
Kondo, S., Kono, T., Sauder, D. N. & McKenzie, R. C. (1993). IL-8 gene expression and production in human keratinocytes and their modulation by UVB. J Invest Dermatol 101, 690–694.[CrossRef][Medline]
Koromilas, A. E., Li, S. & Matlashewski, G. (2001). Control of interferon signaling in human papillomavirus infection. Cytokine Growth Factor Rev 12, 157–170.[CrossRef][Medline]
Kupper, T. S., Chua, A. O., Flood, P., McGuire, J. & Gubler, U. (1987). Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation. J Clin Invest 80, 430–436.[Medline]
Li, A. G., Lu, S. L., Han, G., Hoot, K. E. & Wang, X. J. (2006). Role of TGFβ in skin inflammation and carcinogenesis. Mol Carcinog 45, 389–396.[CrossRef][Medline]
Mueller, M. M. (2006). Inflammation in epithelial skin tumours: old stories and new ideas. Eur J Cancer 42, 735–744.[CrossRef][Medline]
O'Brien, P. M. & Saveria Campo, M. (2002). Evasion of host immunity directed by papillomavirus-encoded proteins. Virus Res 88, 103–117.[CrossRef][Medline]
Pear, W. S., Nolan, G. P., Scott, M. L. & Baltimore, D. (1993). Production of high titre helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90, 8392–8396.
Pfaffl, M. W., Horgan, G. W. & Dempfle, L. (2002). Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30, e36
Pfister, H. (2003). Chapter 8: Human Papillomavirus and skin cancer. J Natl Cancer Inst Monogr 31, 52–56.
Ronco, L. V., Karpova, A. Y., Vidal, M. & Howley, P. M. (1998). Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes Dev 12, 2061–2072.
Ruhland, A. & De Villiers, E. M. (2001). Opposite regulation of the HPV 20-URR and HPV 27-URR promoters by ultraviolet irradiation and cytokines. Int J Cancer 91, 828–834.[CrossRef][Medline]
Scarponi, C., Nardelli, B., Lafleur, D. W., Moore, P. A., Madonna, S., De Pita, O., Girolamoni, G. & Albanesi, C. (2006). Analysis of IFN- expression in pathologic skin conditions: downregulation in psoriasis and atopic dermatitis. J Interferon Cytokine Res 26, 133–140.[CrossRef][Medline]
Simbulan-Rosenthal, C. M., Velena, A., Veldman, T., Schlegel, R. & Rosenthal, D. S. (2002). HPV-16 E6/7 immortalization sensitizes human keratinocytes to ultraviolet B by altering the pathway from caspase-8 to caspase-9-dependent apoptosis. J Biol Chem 277, 24709–24716.
Smola-Hess, S., de Silva, U. S., Hadaschik, D. & Pfister, H. (2001). Soluble IL-6 receptor activates the human papillomavirus type 18 long control region in SW756 cervical carcinoma cells in a STAT3-dependent manner. J Gen Virol 82, 2335–2339.
Stanley, M. (2006). Immune responses to human papillomavirus. Vaccine 24, S16–S22.[CrossRef][Medline]
Strickland, I., Rhodes, L. E., Flanagran, B. F. & Friedmann, P. (1997). TNF and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression. J Invest Dermatol 108, 763–768.[CrossRef][Medline]
Tindle, R. W. (2002). Immune evasion in human papillomavirus-associated cervical cancer. Nat Rev Cancer 2, 59–65.[CrossRef][Medline]
Tugizov, S., Berline, J., Herrera, R., Penaranda, M. E., Nakagawa, M. & Palefsky, J. (2005). Inhibition of human papillomavirus type 16 E7 phosphorylation by the S100 MRP-8/14 protein complex. J Virol 79, 1099–1112.
van Kempen, L. C. L., Ruiter, D. J., van Muijen, G. N. P. & Coussens, L. M. (2003). The tumour microenvironment: a critical determinant of neoplastic evolution. Eur J Cell Biol 82, 539–548.[CrossRef][Medline]
Woodworth, C. D. & Simpson, S. (1993). Comparative lymphokine secretion by cultured normal human cervical keratinocytes, papillomavirus-immortalized and carcinoma cell lines. Am J Pathol 142, 1544–1555.[Abstract]
Received 13 November 2007;
accepted 23 June 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
