Differential production of cytokines and activation of NF-kappaB in HPV-transformed keratinocytes.

We have proposed that chronic infection of keratinocytes by HPV modifies the expression of potentially important cytokines by interfering with the NF-kappaB signal pathway. We evaluated the constitutive and IL-1beta-induced expression of GM-CSF and TNF-alpha and the expression/activity of NF-kappaB in HPV+ and HPV- cell lines. Despite the enhanced expression of the functional components of the NF-kappaB signaling pathway in HPV+ cell lines by a mechanism implicating the HPV oncoprotein E6, the constitutive activity of NF-kappaB and the expression of GM-CSF/TNF-alpha were significantly reduced relative to the HPV- cell line and normal keratinocytes. In contrast, we observed a superactivation of NF-kappaB activity after IL-1beta stimulation, a strong and transient induction of GM-CSF/TNF-alpha mRNA, but undetectable levels of secreted proteins in HPV+ cell lines. Our data demonstrate that E6 modulates the NF-kappaB signaling pathway and suggest that other HPV proteins also interfere with GM-CSF/TNF-alpha expression by transcriptional and/or posttranscriptional mechanisms.

Dendritic cells induce the death of human papillomavirus-transformed keratinocytes.

Although human papillomavirus (HPV) antigens are expressed in a majority of (pre)neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix, progression to invasive cancer may occur, which suggests that the presentation of viral antigens to the immune system is deficient in some SILs. To determine whether professional antigen-presenting cells die in SILs, we assayed for the apoptosis of immature dendritic cells (DC) in organotypic cultures of HPV-transformed keratinocytes, which reproduce many features of in vivo observed SILs. Unexpectedly, the infiltration of organotypic cultures by DC specifically induced the apoptosis of HPV+ tumor cells, whereas DC were not affected. In the same conditions and in coculture experiments, apoptosis was not observed in normal keratinocytes. The induction of apoptosis required membrane contacts between DC and HPV-transformed keratinocytes. Although the HPV+cell lines were sensitive to the effects of TRAIL, soluble TRAILR2-Fc did not block the DC-induced apoptosis. Furthermore, although FasL and Fas were detected on DC and HPV+ cell lines, respectively, functional analysis revealed that this pathway is not responsible for the apoptosis induced by the DC. All together these results suggest that DC may be at the interface between innate and adaptive immunity by inducing the apoptosis of (pre)neoplastic cells.

The organotypic culture of HPV-transformed keratinocytes: an effective in vitro model for the development of new immunotherapeutic approaches for mucosal (pre)neoplastic lesions.

The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous cell carcinoma that develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (interferon-gamma and tumor necrosis factor-alpha) and by integrating activated lymphocytes or dendritic cells into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium that develops on a mucosal surface.

Colonization of in vitro-formed cervical human papillomavirus- associated (pre)neoplastic lesions with dendritic cells: role of granulocyte/macrophage colony-stimulating factor.

The purpose of this study was to investigate the ability of CD1a+ Langerhans/dendritic cells (LCs/DCs) to infiltrate human papillomavirus (HPV)-associated (pre)neoplastic lesions of the uterine cervix. Migration of LCs/DCs in the presence of keratinocytes derived from normal cervix and HPV-transformed cell lines was evaluated in Boyden chambers and in organotypic cultures and correlated with granulocyte/macrophage colony-stimulating factor (GM-CSF) production by the cells, as determined by ELISA. Conditioned media of HPV-transformed keratinocytes contained lower amounts of GM-CSF and induced a decreased motile response of LCs/DCs in the Boyden chamber assay compared with those of normal cervical keratinocytes. The migration of LCs/DCs in the presence of conditioned media from normal keratinocytes could be blocked by an anti-GM-CSF antibody, and the migration of LCs/DCs in the presence of conditioned media from HPV-transformed keratinocytes could be increased by supplementing the media with recombinant GM-CSF. GM-CSF was also a potent factor in enhancing the colonization of LCs/DCs into organotypic cultures of HPV-transformed keratinocytes, as the infiltration of LCs/DCs in the in vitro-formed (pre)neoplastic epithelium was minimal under basal conditions and dramatically increased after the addition of GM-CSF to the cultures. These results suggest that GM-CSF could play an important role in the recruitment of LCs/DCs into the HPV-transformed (pre)neoplastic cervical epithelium and be useful as a new immunotherapeutic approach for cervical (pre)cancers.

Generation of T lymphocytes from the epithelium and stroma of squamous pre-neoplastic lesions of the uterine cervix.

In this study, we have developed a simple and efficient technique for the isolation of viable lymphocytes from the epithelium and stroma of small pre-neoplastic squamous intraepithelial lesions (SIL) of the uterine cervix. Following the separation of the epithelium from the stroma using dispase II, both biopsy fragments were used to generate T lymphocytes. The stroma-derived lymphocytes were obtained by collecting and culturing the cells migrating out of the biopsy in the presence of IL2 (50 U/ml). An average of 0.7 x 10(6) and 1.4 x 10(6) lymphocytes could be obtained after 20 and 30 days of culture, respectively. For the expansion of lymphocytes derived from the pre-neoplastic epithelium (SIL) it was necessary to use a combination of irradiated peripheral blood mononuclear cells (PBMC) as a feeder layer with PHA (0.1%), in addition to IL2 (50 U/ml). Interestingly, these lymphocytes could be obtained using either allogeneic or syngeneic PBMCs. With this protocol, we were able to generate up to 100 x 10(6) lymphocytes from the epithelium, the majority of which were T lymphocytes.

Cytokine expression in squamous intraepithelial lesions of the uterine cervix: implications for the generation of local immunosuppression.

We have addressed the notion that the progression of cancer of the uterine cervix is associated with a preferential constraint on the development of a type 1 cellular mediated response, which is necessary to efficiently eliminate (pre)neoplastic cells. Based on the importance of cytokines in the regulation of an appropriate immune response, we have evaluated the expression of IL-12p40, IL-10 and transforming growth factor-beta 1 (TGF-beta1). Using reverse transcriptase-polymerase chain reaction (RT-PCR), the expression of these three cytokines was evaluated in both low-grade (LG) and high-grade (HG) cervical squamous intraepithelial lesions (SIL) and in normal exocervix and transformation zone biopsies. Our results show that the average level of IL-12 increases within both the LG and HG SIL, compared with both control groups. Interestingly, the percentage of HG SIL expressing IL-12p40 was lower compared with LG SIL. In contrast, the expression of IL-10 increased in parallel with the severity of the lesion to a maximal level in HG SIL. Using immunohistochemistry, we ascertained the presence of IL-12 protein in SIL and IL-10 protein in the transformation zone and SIL biopsies. Both IL-12- and IL-10-producing cells were localized in the stroma, not within the SIL. Furthermore, in this study we also observed that the region of the cervix the most sensitive to lesion development, the transformation zone, was associated with higher average levels of the immunosuppressive cytokines IL-10 and TGF-beta1.

Correlation of T-helper secretory differentiation and types of antigen-presenting cells in squamous intraepithelial lesions of the uterine cervix.

This study addressed the notion that the progression of cervical cancer is associated with a T-helper 2 (TH2) immunodeviation by analysing cytokine expression in 60 cervical biopsy specimens, spanning the spectrum from normal cervical tissue to high-grade squamous intraepithelial lesions (SILs). The biopsies were analysed by immunohistochemistry for the expression of TH1 [interleukin-2 (IL2), interferon gamma (IFN gamma)] and of TH2-type cytokines (IL4, IL6). Positive cells were usually observed in the subepithelial connective tissue, where most CD4+ cells were also detected. The density of IL2+ cells was significantly lower in high-grade SILs than in normal tissues taken either from the ectocervix or from the transformation zone. In contrast, significantly higher densities of IL4+ cells and, to a lesser degree, IL6+ cells were found in SIL biopsies compared with histologically normal tissues taken from the adjacent ectocervical region. A significantly higher IL4+/CD4+ cell ratio was also found in high-grade SILs (82 per cent) than in normal cervical biopsies taken from the transformation zone of healthy women showing squamous metaplasia (27 per cent). The elevated density of TH2+ cells in SIL biopsies was associated with both the expression of HLA-DR by keratinocytes and a diminished number of intraepithelial Langerhans' cells (CD1a+). In conclusion, the increased TH2+/CD4+ cell ratio in SIL biopsies suggest the presence, during cervical carcinogenesis, of a TH2 immunodeviation that could participate in the immunoescape of preneoplastic cervical keratinocytes.

Inverse modulation of IL-10 and IL-12 in the blood of women with preneoplastic lesions of the uterine cervix.

It has been postulated that an impaired immune response may contribute to the progression of human papillomavirus (HPV)-associated preneoplastic lesions. Based on this hypothesis, we evaluated the cytokine production in the blood of patients with squamous intraepithelial lesions (SIL) of the uterine cervix. The levels of type-1 (interferon-gamma (IFN-gamma) and IL-12) and type-2(IL-4 and IL-10) cytokines were measured in whole blood culture supernatants of patients with low- and high-grade SIL and control women. There was no difference in IL-4 and IFN-gamma levels between patients with SIL and the control group. In contrast, the ratio of IL-12/IL-10 levels was significantly lower in patients with SIL compared with the control group. A lower IL-12/IL-10 ratio in women with SIL was also observed when peripheral blood mononuclear cell (PBMC) culture supernatants and plasma samples were analysed. In patients, neither the lower expression of the CD3epsilon chain nor the higher frequency of HLA-DRB1*1501 expression could be correlated with abnormal cytokine production. These results suggest that a part of the cytokine network, namely IL-10 and IL-12, is perturbed in patients with SIL. A better knowledge of the role of these cytokines in regulating the growth of HPV-associated SIL might have practical implications for the development of vaccines or immunomodulatory strategies in the treatment of cervical cancers.

Interleukin-7 in the skin of Schistosoma mansoni-infected mice is associated with a decrease in interferon-gamma production and leads to an aggravation of the disease.

The effect of recombinant interleukin-7 (rIL-7) on the course of murine schistosomiasis and the development of the accompanying immune response were investigated. We demonstrated that IL-7 expression could be detected in the skin of infected mice from 1 to 21 days following infection. We here report that intradermal injection of exogenous human IL-7, prior to the penetration of the parasite into the skin, leads to a more severe liver pathology and an increased number of surviving adult parasites. In addition, injection of rIL-7 alters parasite migration (estimation of burdens of young larvae in lungs and liver). Administration of rIL-7 led to a decrease of IL-12 and interferon-gamma-(IFN-gamma) specific messengers RNA in skin and, more markedly, in skin-draining lymph nodes. The number of B220 expressing cells was increased, and T-cell number was reduced, in IL-7-treated infected mice. In addition, levels of IFN-gamma and IL-4 in sera were significantly reduced, whereas there was a shift from a Th1 to a Th2 type associated humoral response towards the egg antigens. Our experimental observation illustrate that the exogenous administration of rIL-7 affects both the development of the host's immune response and the behaviour of the parasite within the infected host. The early and specific production of IL-7 in the host skin, following infection with Schistosoma mansoni, raises fascinating questions concerning the relationships between the parasite and its host at the very beginning of their interaction.

Dyad symmetry within the mouse 3' IgH regulatory region includes two virtually identical enhancers (C alpha3'E and hs3).

The transcription of the murine Ig heavy chain locus is regulated not only by the intronic enhancer, E mu, but also by a 3' regulatory region located downstream of the C alpha membrane exon. Several DNase I-hypersensitive sites (hs1-4) and enhancer elements (e.g., C alpha3'E) have been identified in this 3' regulatory region, and some of these were suggested to comprise a locus control region. However, little is known about the coordinate regulation or function of these individual elements. Here we provide evidence that C alpha3'E and hs3 are virtually mirror images of each other and demarcate the edges of an approximately 25-kb region of quasi-dyad symmetry with 3'alphaE(hs1,2) at its center. Flanking 3'alphaE(hs1,2) are inverted repeats and families of repetitive sequences uniquely located in this region. We have observed that, like 3'alphaE(hs1,2) and hs3, C alpha3'E is DNase I hypersensitive in plasma cell lines, but not in a pre-B cell line. Additionally, we found that C alpha3'E and hs3 show significant transcriptional synergy in transfection assays only in a plasma cell line. The DNA topology of the 3' regulatory region coupled with new and existing data on the activity of its individual enhancers during B cell differentiation lead us to propose a biphasic model for the activity of this region. According to our model, one unit, consisting of the 3'-most enhancer, hs4, is active early and throughout B cell development. The second unit, which comprises C alpha3'E, 3'alphaE(hs1,2), and hs3, becomes active later in development, when it contributes to such processes as class switching and increased levels of Ig heavy chain gene transcription in plasma cells.

Identification of 3' alpha-hs4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation.

In addition to E mu, several elements downstream of the IgH cluster, i.e. 3' of the C alpha gene, are involved in regulating IgH gene rearrangement and expression. This entire downstream regulatory region was shown to be deleted in the mutant myeloma cell line, LP1.2. The deletion encompasses approximately 34 kb and is presumably responsible for the reduced levels of IgH expression in this cell line. An additional regulatory element, included in the LP1.2 deletion, was identified by investigation of a DNase I hypersensitivity site located approximately 33 kb downstream of the alpha gene and present in pre-B and plasma cells. This novel IgH gene enhancer element, termed 3' alpha-hs4, is capable of activity throughout B cell development. Transient transfection of 3' alpha-hs4 in a CAT reporter gene construct shows transcriptional enhancement activity approximating that of E mu in S194 plasmacytoma and M12.4.1 and A-20 B cell lines; while in a pre-B cell line, 18-81, the average activity is 25% that of E mu. Enhancer activity was localized to an 800 bp fragment. The activity of 3' alpha-hs4 is orientation independent and appears to be B cell specific. Tight regulation of 3' alpha-hs4 is inferred from its variable activity in different plasmacytoma cell lines and within the pre B cell line, 18-81.

DNA regions flanking the mouse Ig 3' alpha enhancer are differentially methylated and DNAase I hypersensitive during B cell differentiation.

Two B cell-specific enhancer elements are associated with the IgH gene cluster. One enhancer is located within the J-C mu intron (E mu), whereas a second enhancer (3' alpha E) is approximately 12.5 kb 3' of the C alpha membrane exon. In an attempt to understand the function of 3' alpha E, we have characterized its surrounding structural milieu during various stages of B cell differentiation through analysis of methylation patterns and the identification of DNAse I-hypersensitive sites. We observed a correlation between the chromatin structure of this region and the differentiation state of the cell. Compared to liver and brain, the region 3' of alpha was hypermethylated in pre-B and T cell lines and became progressively demethylated as B cell differentiation continued. A DNAse I-hypersensitive site was present in pre-B cell lines about 17 kb 3' of 3' alpha E. In fully differentiated myeloma cell lines, a second cluster of DNAse I-hypersensitive sites was present immediately 5' of 3' alpha E. Our data indicate that the 3' alpha enhancer is relatively sequestered during early stages of B cell differentiation and becomes increasingly accessible at later stages.

A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.

DNA sequences 3' of the Ig H chain cluster rearrange in mouse B cell lines.

A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to study DNA rearrangements that occur at the Ig H chain locus. One variant, F5.5, has acquired both VH gene and C epsilon gene rearrangements. Through genomic Southern blot analysis initially directed to mapping the C epsilon gene rearrangement, we observed that the VH region rearrangement was linked, through an inversion event, to sequences that originate 3' of the CH cluster, i.e., 3' of the C alpha gene. Subsequent studies have shown that DNA rearrangements within the region 3' of the C alpha gene are detected in several other mouse myeloma and hybridoma cell lines and are not associated with the expression of specific isotypes.

An enhancer at the 3' end of the mouse immunoglobulin heavy chain locus.

A tissue-specific enhancer (E mu) lies between the joining (JH) and mu constant region (C mu) gene segments of the immunoglobulin heavy chain (IgH) locus. Since mouse endogenous IgH genes are efficiently transcribed in its absence, the normal function of this enhancer remains ill-defined. Recently, another lymphoid-specific enhancer of equal strength has been identified 3' of the rat IgH locus. We have isolated an analogous sequence from mouse and have mapped it 12.5 kb 3' of the 3'-most constant region gene (C alpha-membrane) of the BALB/c mouse locus. The mouse and rat sequences are 82% homologous and share with other enhancers several DNA sequence motifs capable of binding protein. However, in transient transfection assays, the mouse sequence behaves as a weaker enhancer. The role of this distant element in the expression of endogenous IgH genes, both in E mu-deficient, Ig-producing cell lines and during normal B cell development, is discussed.