Institute of Comparative Medicine, Division of Pathological Sciences, University of Glasgow, Garscube Estate, Glasgow G61 1QH, UK
Correspondence
M. S. Campo
s.campo{at}vet.gla.ac.uk
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ABSTRACT |
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MAIN TEXT |
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Papillomas that are induced by BPV develop through four well-defined stages, from stage 1 or plaque, the first clinical manifestation of infection, to stage 4, when the papilloma starts to regress (Jarrett, 1985). From infection to regression, papilloma development often takes longer than 12 months. In the case of BPV-4-induced papillomas, regression is accompanied by infiltration of immune cells, primarily CD4+ T lymphocytes in the adjacent dermis, as well as CD8+ T lymphocytes infiltrating the keratinocytes (Knowles et al., 1996
). Persistence, spread and progression of papillomas to cancer occur mainly in animals that graze on bracken fern (Pteridium spp.) (Campo et al., 1994
). These animals are immunocompromised by immunosuppressants, such as sesquiterpenes, that are present in the plant. However, papillomas can also spread and persist in cattle that are not exposed to bracken fern (Tsirimonaki et al., 2003
). Even in the absence of malignant transformation, BPV infection can persist for a significant period of time before activation of the host immune system. Lymphocytes from infected animals do not recognize early or late viral antigens until late in infection, despite the presence of numerous papillomas actively producing virus (Chandrachud et al., 1994
, 1995
; McGarvie et al., 1995
; Kirnbauer et al., 1996
). This lack of recognition suggests that the host immune system is unaware of, or disabled by, BPV infection. It is now known that papillomaviruses can subvert the immune response indirectly, as virus replication is confined to the epithelial cells above the basal membrane and therefore occurs in a site that is recognized poorly by immune cells (Frazer et al., 1999
). In addition, papillomaviruses appear to interfere directly with host antiviral immune mechanisms, including the interferon response and major histocompatibility complex class I (MHC I) antigen presentation to cytotoxic T lymphocytes (O'Brien & Campo, 2002
; Tindle, 2002
).
We have shown recently that expression of the E5 oncoprotein of BPV-1, BPV-4 and HPV-16 has a profound effect on the synthesis and transport of MHC I in cultured cells and, therefore, can potentially contribute to the ability of the virus to evade immune recognition (Ashrafi et al., 2002, 2004
; Marchetti et al., 2002
; O'Brien & Campo, 2002
). To ascertain whether the downregulation of MHC I that is observed in cultured cells takes place in tumours, we investigated the expression of BPV-4 E5 and MHC I in clinical samples of BPV-4 papillomas.
Papillomas from the palate, rumen and oesophagus, as well as samples of normal palate, tongue and buccal mucosa, were collected post-mortem from animals that were referred to the University of Glasgow Veterinary School. Tissue samples were fixed and stored in 10 % formaldehyde in PBS at pH 7·5 and embedded in paraffin wax for histological processing. Serial sections (1·5 µm) were cut and placed on microscopic slides that had been treated with VECTABOND (Vector). After deparaffinization in Histo-clear (National Diagnostics), sections were rehydrated in graded ethanol and incubated in 0·5 % H2O2/methanol for 20 min to quench endogenous peroxidase. Sections were subjected to antigen-retrieval treatment with 0·01 M sodium citrate buffer (pH 6) in a pressure cooker for 75 s at 103·4 kPa, blocked with 1 % normal unlabelled swine serum (Scottish Antibody Production Unit) in TBS containing 0·1 % Tween 20 for 30 min at room temperature and then incubated for 1·5 h at room temperature with primary antibodies for detection of E5, E7, MHC I heavy chain and the proliferation marker Ki67, as detailed below. The sections were then incubated with biotin-labelled secondary antibody (Dako) and streptavidinbiotin complex (Dako) for 45 min, following the manufacturer's instructions. Immunoreactivity was visualized with diaminobenzene (Sigma). Sections were counterstained with Gill's haematoxylin, dehydrated, cleared in Histo-clear and mounted permanently with DPX mountant under a coverslip prior to microscopic examination. A total of seven papillomas was analysed, with at least three sections and three different section fields examined per papilloma; all papillomas were classified as stage 2/3, i.e. mature papillomas producing virus, and presented the typical features of BPV-4 infection (Jarrett, 1985), including an irregular basal layer (Fig. 1a and b
), fronds of transformed cells terminating in keratinized tips (Fig. 2d
) and koilocytes, cells with highly enlarged cytoplasm that are typical of papillomavirus infection (Fig. 2d
, black arrow). In contrast, the normal bovine alimentary mucosal epithelium had a typical architecture, composed of a regular basal cell layer and suprabasal, spinous and squamous layers (Fig. 1d
).
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The E7 proteins of HPV-16 and -11 have been implicated, respectively, in the downregulation of MHC I either through inhibition of the transcriptional promoter of the MHC I heavy chain (Georgopoulos et al., 2000) or indirectly through inhibition of TAP, the transporter associated with peptide (Vambutas et al., 2001
). To ensure that the absence of MHC I in bovine papillomas was due to E5 and not to E7, we stained papilloma sections with rabbit antisera 11547 and 11823, which were raised against a
-galactosidaseE7 fusion protein (Anderson et al., 1997
). Similar results were obtained with both antisera; only results that were obtained with antiserum 11547 are shown. E5 and E7 are co-expressed in the same cells (Anderson et al., 1997
) and, accordingly, in this study, cells that expressed either E5 or E7 alone were seldom detected. Nevertheless, in cells that expressed E5 and did not express E7, or expressed it at levels below detection, there was little or no MHC I (Fig. 3ac
). Conversely, cells that expressed E7, but not E5, still had detectable MHC I (Fig. 3df and hj
). Thus, it appears that expression of E7 is not responsible for downregulation of MHC I.
Although the lack of MHC I in the uppermost layers of the papillomas (Figs 2e and 3e) was consistent with the differentiated state of the cells, its absence in the basal (Fig. 3b
) and the immediate suprabasal (transit and lower spinous) (Fig. 2e
, boxed area 1) layers could not be attributed to cell differentiation, as MHC I was present in these areas of normal mucosa (Fig. 2a
). Furthermore, MHC I was absent in similar areas of papillomas where E5 was not expressed (Fig. 2d and e
, boxed area 2).
We conclude that E5 inhibits the expression of MHC I in BPV-induced papillomas, corroborating and validating our observations on downregulation of MHC I by E5 in vitro.
HPV-16 and other high-risk HPV types induce cervical intraepithelial neoplasia (CIN), the precursor lesion of cervical cancer (zur Hausen, 2002). MHC I downregulation has been observed in CIN, but E5 expression was not investigated (Bontkes et al., 1998
). Given that HPV-16 E5 downregulates MHC I (Ashrafi et al., 2004
) and that HPV-16 E5 can be found in CIN samples (Chang et al., 2001
), it can be speculated that E5 is also responsible for MHC I downregulation in CIN.
It remains to be seen whether the E5-induced downregulation of MHC I leads to evasion of the host immune response, thus allowing the virus to establish infection and allowing the infection to persist.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 March 2004;
accepted 18 June 2004.
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