Multiple mechanisms underlie HLA dysregulation in cervical cancer.

The consistent dysregulation of HLA expression in cervical neoplasia is likely to influence the natural history of the disease and prospects for cell-mediated vaccine therapies. We have studied the underlying mechanisms in eight new cervical cancer cell lines derived from primary tumour biopsies. At least five independent mechanisms leading to changes in HLA expression were seen: HLA class I allelic transcription but no protein; abnormal HLA class I allelic transcription; no HLA-B locus transcription; loss of heterozygosity (LOH); no gammaIFN-mediated upregulation of HLA class I expression, and/or no interferon-gamma (gammaIFN)-mediated HLA class II induction. These were evident in different combinations in 7/8 cell lines showing that multiple, mostly irreversible mechanisms not overridden by gammaIFN, are responsible for HLA dysregulation in cervical neoplasia. Point mutations were responsible for lack of HLA-A2 expression in two cases. In cell line 808, the mutation encodes a stop codon in exon 3; in cell line 778, mutation of the first intron acceptor site leads to use of an alternative AG site in exon 2, resulting in a frameshift and a stop codon after the translation of only 38 amino acids. Tumour cells showing specific HLA class I loss may have selective advantage in the face of tumour-specific cytotoxic T cells (CTL). Such immune escape mechanisms present a major obstacle for the success of CTL-mediated therapies in cervical cancer.

Integration of high-risk human papillomavirus DNA is linked to the down-regulation of class I human leukocyte antigens by steroid hormones in cervical tumor cells.

A crucial event in the malignant progression of cervical intraepithelial neoplasia appears to be the up-regulation of high-risk human papillomavirus (HPV) early gene expression. Steroid hormones have been linked to the progression from premalignant to neoplastic status in HPV positive lesions. This report demonstrates that at physiological levels, the glucocorticoid hormone hydrocortisone consistently down-regulates class I human leukocyte antigen (HLA) surface expression in HPV-positive cervical tumor cells but can up-regulate expression in HPV-negative epithelial tumor lines. Suppression of HLA expression was also seen with progesterone, another steroid hormone. The hydrocortisone-mediated modulation of HLA expression is dependent on integration and transcription of the HPV genome and can be blocked by Ru38486, an antagonist of both glucocorticoid and progesterone receptors, indicating the role of these receptors in mediating HLA suppression. The data suggest that HPV integration events in cervical epithelia correlate with hormone-dependent HLA suppression, possibly contributing to the avoidance of tumor recognition by cytotoxic T cells. These studies imply that clinical use of steroids may be contraindicated in HPV-positive individuals who have early premalignant cervical disease or neoplasia but provide evidence that the antiprogestin Ru38486 may be useful in the management of early stage cervical disease.

The association of an HPV16 oncogene variant with HLA-B7 has implications for vaccine design in cervical cancer.

HLA-restricted cytotoxic T-lymphocyte (CTL) recognition of human papillomavirus (HPV) oncogene products may be important in the control of the HPV infections associated with the development of cervical cancer. We have identified, in HLA-B7 individuals, a consistent variation in the HPV16 E6 oncoprotein sequence, which alters an HLA-B7 peptide binding epitope in a way likely to influence immune recognition by CTLs. These results illustrate a biologically relevant mechanism for escape from immune surveillance of HPV16 in HLA-B7 individuals. Thus, both HLA type and HPV16 strain variation need to be considered in the screening of at-risk individuals and for the rational design of anti-HPV vaccines.

Identification of a naturally processed HLA A0201-restricted viral peptide from cells expressing human papillomavirus type 16 E6 oncoprotein.

Human papillomavirus (HPV) DNA encoding the oncogenic proteins E6 and E7 is usually retained in cervical carcinomas, implicating these proteins as potential target antigens for immune recognition in this virally associated tumor. We have characterized endogenously processed peptides eluted from major histocompatibility complex class I molecules in cells infected with a recombinant vaccinia expressing the HPV-16 E6 oncoprotein. The reverse-phase chromatography profile of peptides eluted from isolated HLA-A0201 molecules in cells expressing the E6 oncoprotein differs from that of cells not expressing E6. Sequential Edman degradation of novel peaks found in the peptide profiles from cells expressing HPV-16 E6 led to the identification of a naturally processed HLA-A0201-restricted E6 peptide of sequence KLPQLCTEL. This approach has allowed the identification of a viral peptide which is processed and presented by cells expressing the E6 oncoprotein and is a likely target for cytotoxic T lymphocyte recognition in HLA-A0201-positive patients.

Expression of human papillomavirus type 16 E7 protein by recombinant baculovirus and use for the detection of E7 antibodies in sera from cervical carcinoma patients.

Although the presence of serum antibodies against the human papillomavirus type 16 (HPV-16) E7 protein has been linked with cervical cancer, currently available assays detect antibodies in only ca. 40% of carcinoma patients. The dependence of these serological assays on synthetic target antigens which present only linear epitopes may be a limiting factor. In order to produce a more realistic target antigen for use in serological assays, we have expressed the HPV-16 E7 protein in insect cells using a recombinant baculovirus vector. Two major E7 forms of ca. 18kDa and 16kDa were produced and characterised. The 16kDa component was shown to be truncated at the N-terminus. A radioimmunoprecipitation assay was developed for the detection of anti-E7 antibodies in human sera. This assay showed a marked increase in detection rate compared with a western blotting method based on bacterially derived E7 fusion proteins.

Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera.

Existing assays to detect antibodies to human papillomavirus type 16 (HPV-16) proteins in sera from cervical carcinoma patients rely primarily on bacterially produced recombinant proteins or synthetic peptides for use as target antigens. These methods have had limited success in the detection of antibodies against the E6 protein. To produce more authentic E6 protein for use in serological assays, we have employed a recombinant baculovirus vector to synthesize the protein in insect cells. Cells infected with the vector containing E6 gene sequences expressed a stable protein doublet comprising 18.5K and 19.1K bands. This protein reacted in Western blots with an antiserum raised against a purified E6 fusion protein produced in Escherichia coli. This antiserum, and several others raised against E. coli-derived E6 fusion proteins, were unable to recognize the baculovirus E6 protein in radioimmunoprecipitation assays (RIPAs). However, serum from a cervical carcinoma patient readily immunoprecipitated the baculovirus E6 protein, suggesting that the baculovirus-derived protein represented a realistic antigenic target. A RIPA was developed for the detection of anti-E6 protein antibodies in human sera. The assay was tested on a selected group of sera from carcinoma patients and controls, in comparison with a Western blotting method using bacterial fusion proteins. The baculovirus E6 protein-based RIPA showed a marked increase in detection rate over the Western blotting method. These findings suggest that serum antibodies to HPV-16 E6 protein may be more prevalent than has previously been shown.

Serum IgE anti-cartilage collagen antibodies in rheumatoid patients.

The presence of circulating IgG, IgA and IgM antibodies to native cartilage collagens in some patients with rheumatoid arthritis (RA) suggests that an autoimmune response to cartilage collagens may be involved in the pathogenesis of RA. However, the relevance of such antibodies to the pathological process remains unclear, and it is likely that many humoral and cellular derived factors combined to trigger events leading to the chronicity of the rheumatoid lesion. Since histological and biochemical studies have suggested the involvement of mast cells in the rheumatoid joint, we have studied the frequency of IgE antibodies directed against the cartilage collagens type II, IX and XI in patients with active rheumatoid disease. Of the 91 patients' sera tested, 32 had significant levels of IgE anti-cartilage collagen antibodies when compared with non-arthritic controls. Total serum IgE levels did not correlate with the presence of IgE anti-collagen antibodies, nor were any patients positive for IgE antibodies to fibronectin, a widely distributed extracellular matrix component. These results are consistent with an allergic type I hypersensitivity reaction to cartilage antigens in RA involving mast cell and basophil degranulation.

Plasminogen activator release from cultured murine mast cells.

Mast cells from the Furth murine mastocytoma tumour line were found to contain significant levels of plasminogen activator (PA). Cultured cells released PA activity into the culture medium in parallel with the release of histamine, and both were proportionately increased following exposure to degranulating agents. Pretreatment of the mast cells with cycloheximide did not alter their total PA content or their ability to release PA. These studies suggest that PA is a prestored granule constituent. The ability of PA to generate plasmin from plasminogen suggests an important role for mast cell PA in fibrinolysis and tissue degradation, observations that have been associated with mast cell degranulation and infiltration in vivo.

The role of serine proteinase in cartilage damage.

Arthritic cartilage from experimental animals has been shown to have a decreased proteoglycan content, a decreased rate of proteoglycan synthesis, and a marked increase in an active serine proteinase when compared with normal articular cartilage. The serine proteinase is transferred from PMN cells into cartilage during the inflammatory response where it increased the rate of proteoglycan degradation and is eventually removed by interaction with the chondrocyte surface. The interaction also results in an inhibition of proteoglycan synthesis by the chondrocytes. Both these factors contribute to the loss of proteoglycan from arthritic cartilage.

Receptor-mediated binding of leukocyte elastase by chondrocytes.

Studies on the effect of leukocyte elastase on the metabolism of chondrocytes in culture have demonstrated that these cells possess a specific cell surface receptor for leukocyte-derived elastase. Purified elastase from rabbit and human leukocytes is capable of modulating the metabolism of the cell by causing a marked decrease in both proteoglycan and protein biosynthesis. Addition of 125I-labeled elastase to chondrocytes maintained in suspension culture has shown that binding occurs, and that it is saturable and is inhibited by the addition of unlabeled enzyme. We ascertained that the active site of the enzyme was necessary for binding to the chondrocyte, since phenylmethylsulfonyl fluoride-inactivated leukocyte elastase failed to bind. Pancreatic elastase had only a slight affinity for the receptor, whereas trypsin and bovine serum albumin failed to bind to any significant extent. Autoradiographic studies and the use of inhibitors of endocytosis, such as dansyl cadaverine, confirmed that endocytosis of elastase was the secondary event after cell binding.

The effects of trypsin treatment on proteoglycan biosynthesis by bovine articular cartilage.

The effects of mild or severe trypsin treatment of bovine articular-cartilage slices in tissue culture were studied by monitoring the incorporation of [35S]sulphate into proteoglycans. Moderate trypsin treatment caused a subsequent marked inhibition of proteoglycan biosynthesis, which was reversible with time. Analysis on Sepharose CL-2B of the proteoglycan species synthesized showed that, directly after trypsin treatment, there was a 30% increase in the synthesis of the low-Mr proteoglycan (Kav. 0.71), and the total decrease in proteoglycan biosynthesis was reflected in a decrease in the synthesis of the high-Mr proteoglycan species (Kav. 0.31). The small proteoglycan was partially characterized and shown to be a true biosynthetic product and not a breakdown product. Trypsin treatment (20 micrograms/ml per 100 mg of tissue) of cartilage slices also resulted in an increase in the glycosaminoglycan chain size of the large proteoglycan, but not of the small proteoglycan.

Changes in proteoglycan biosynthesis following leukocyte elastase treatment of bovine articular cartilage in culture.

Treatment of bovine articular cartilage in culture with a low molecular weight elastase purified from rabbit polymorphonuclear leukocytes resulted in degradation and release of proteoglycans from the tissue, coupled with a prolonged inhibition of proteoglycan biosynthesis. These observations are consistent with those seen in experimental arthritis induced in rabbits. Comparison of the size of the proteoglycan degradation products extracted from elastase-treated cartilage in culture with that from arthritic cartilage showed marked similarities. The results strongly suggest that leukocyte elastase is a contributing factor in proteoglycan degradation and inhibition of synthesis in inflammatory joint disease.

Investigation of relationships between collagens, elastin and proteoglycans in bovine thoracic aorta by immunofluorescence techniques.

Types I, III and V collagens and proteoglycan were localized in the aorta by indirect immunofluorescence techniques. Type I collagen was more prominent in media and adventitia than in intima while type III collagen predominated in intima and media but appeared less significant in adventitia. Type V collagen was observed in intima and media only and was seen surrounding smooth muscle cells. Type I collagen was located between elastic fibres but type III collagen appeared to envelop the fibres, suggesting an interaction between elastic fibres and type III collagen. Pretreatment of sections with testicular hyaluronidase caused no changes in staining for type I collagen, but adventitial areas showed increased staining for type III collagen. After digestion with chondroitinase ABC, intimal and medial areas showed increased staining for type III collagen. Therefore, type III collagen forms stronger interactions with proteoglycans and hyaluronic acid than does type I collagen and type III collagen in adventitia is largely masked by hyaluronic acid, while type III collagen in intima and media is associated with proteoglycan. Thus, type III collagen is a more significant component of adventitia than previously recognized. Proteoglycan was also partly localized along elastic fibres. It is, therefore, suggested that elastic fibres are coated with type III collagen, which itself is coated with proteoglycan.

Distribution of proteoglycans and hyaluronic acid in transverse sections of bovine thoracic aorta.

The distribution of hyaluronic acid and proteoglycans in bovine thoracic aorta was studied by Alcian Blue staining of frozen tissue sections under controlled electrolyte conditions with and without prior enzymic digestion. Some sections were digested with chondroitinase ABC, testicular hyaluronidase or bacterial collagenase and subsequent staining permitted conclusions to be drawn about the distribution of specific glycosaminoglycans within the tissue. The total glycosaminoglycan content was maximal in the intima and decreased across the arterial wall to the outermost adventitial layer. The content of proteoglycan containing chondroitin sulphate and/or dermatan sulphate chains paralleled this distribution. However, other glycosaminoglycans also contributed significantly to staining, although there was no evidence for any appreciable concentration of heparin or highly sulphated heparan sulphate. Several experiments indicated that proteoglycan containing chondroitin sulphate and/or dermatan sulphate was associated with elastic laminae which were often seen stained along their periphery. Hyaluronic acid was present at significant concentrations in all locations of the aorta and there was evidence for a similar distribution of heparan sulphate which was possible also present at a high concentration in the endothelium. Staining of sections after treatment with 4 M guanidinium chloride confirmed that this extractant removed most of the proteoglycan from the tissue section.