Colonization of in Vitro-Formed Cervical Human Papillomavirus-Associated (Pre)Neoplastic Lesions with Dendritic Cells
1999; Elsevier BV; Volume: 154; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)65324-2
ISSN1525-2191
AutoresPascale Hubert, Frédéric van den Brûle, Sandra L. Giannini, Elizabeth Franzen‐Detrooz, Jacques Boniver, Philippe Delvenne,
Tópico(s)Immune Response and Inflammation
ResumoThe 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. 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. Cervical cancer is the second leading cause of cancer in women worldwide.1Parkin D Läärä E Muir CS Estimates of the worldwide frequency of sixteen major cancers in 1980.Int J Cancer. 1988; 41: 184-197Crossref PubMed Scopus (1037) Google Scholar It is now well established that human papillomavirus (HPV) plays a major role in the development of cervical cancer and its precursors (squamous intraepithelial lesions (SILs)).2Bosch FX Manos MM Munoz N Sherman M Jansen AM Peto J Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International Biological Study on Cervical Cancer (IBSCC) Study Group.J Natl Cancer Inst. 1995; 87: 796-802Crossref PubMed Scopus (2992) Google Scholar Additional environmental and/or host factors are probably involved in malignant progression as suggested by the isolation of HPV DNA from normal cervical cell samples,3Cox MF Meanwell CA Maitland NJ Blackledge G Scully C Jordan JA Human papillomavirus type 16 homologous DNA in normal human ectocervix.Lancet. 1986; ii: 157-158Abstract Scopus (61) Google Scholar, 4Toon PG Arrand JR Wilson LP Sharp DS Human papillomavirus infection of the uterine cervix of women without cytological signs of neoplasia.Br Med J. 1986; 239: 1261-1264Crossref Scopus (84) Google Scholar the small number of infected individuals developing cervical cancer, and the relatively long latency period before cancer emergence.5Al-Saleh W Giannini SL Jacobs N Moutschen M Doyen J Boniver J Delvenne P Correlation of T helper secretory differentiation and types of antigen-presenting cells in squamous intraepithelial lesions of the uterine cervix.J Pathol. 1998; 184: 283-290Crossref PubMed Scopus (91) Google Scholar Although the nature of an effective immune response to HPV infection is not well understood, humoral immunity to HPV seems to be a reflection of tumor progression, not a protective immune response.6Thivolet J Viac J Staquet MJ Cell mediated immunity in wart virus infection.Int J Dermatol. 1982; 21: 94-98Crossref PubMed Scopus (31) Google Scholar In contrast, several lines of evidence suggest that cellular immunity is important in controlling HPV-associated SILs.7Wu TC Immunology of the human papillomavirus in relation to cancer.Curr Opin Immunol. 1994; 6: 746-754Crossref PubMed Scopus (74) Google Scholar Indeed, several studies have described a localized immune dysfunction accompanying cervical HPV infections.8Hughes RG Norval M Howie SEM Expression of major histocompatibility class II antigens by Langerhans' cells in cervical intraepithelial neoplasia.J Clin Pathol. 1988; 41: 253-259Crossref PubMed Scopus (56) Google Scholar, 9Memar OM Arany I Tyring SK Skin-associated lymphoid tissue in human immunodeficiency virus-1, human papillomavirus, and herpes simplex virus infections.J Invest Dermatol. 1995; 105: 99S-104SCrossref PubMed Scopus (42) Google Scholar Quantitative and qualitative alterations of CD4+ T lymphocytes have been reported.5Al-Saleh W Giannini SL Jacobs N Moutschen M Doyen J Boniver J Delvenne P Correlation of T helper secretory differentiation and types of antigen-presenting cells in squamous intraepithelial lesions of the uterine cervix.J Pathol. 1998; 184: 283-290Crossref PubMed Scopus (91) Google Scholar, 10Nickoloff JB Turka LA Immunological functions of non-professional antigen-presenting cells: new insights from studies of T-cell interactions with keratinocytes.Immunol Today. 1994; 15: 464-469Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 11Coleman N Birley HDL Renton AM Hanna NF Ryait BK Byrne M Taylor-Robinson D Stanley MA Immunological events in regressing genital warts.Am J Clin Pathol. 1994; 102: 768-774Crossref PubMed Scopus (406) Google Scholar Moreover, the number, distribution, and morphology of Langerhans cells (LCs) have been shown to be altered in HPV-infected (pre)neoplastic cervical epithelium.12Morelli AE Sananes C Di Paola G Paredes A Fainboim L Relationship between types of human papillomavirus and Langerhans' cells in cervical condyloma and intraepithelial neoplasia.Am J Clin Pathol. 1993; 99: 200-207Crossref PubMed Scopus (47) Google Scholar LCs belong to the lineage of dendritic cells (DCs) that constitute a network of sentinels considered to be the most important professional antigen-presenting cells in the immune system. Currently, DCs can be obtained from monocytic precursors (CD14+).13Sallusto F Lanzavecchia A Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor α.J Exp Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4479) Google Scholar They can be induced to proliferate and to differentiate into DCs (CD1a+/CD14−) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. LCs/DCs are known to play key roles in the initiation and regulation of cutaneous, mucosal, and systemic immunity.14Knight SC Stagg AJ Antigen-presenting cell types.Curr Opin Immunol. 1993; 5: 374-380Crossref PubMed Scopus (141) Google Scholar LCs infiltrate different tumors, including cervical cancer, and have been shown to be associated with a better prognosis.15Tazi A Bouchonnet F Grandsaigne M Boumsell L Hance AJ Soler P Evidence that granulocyte macrophage-colony-stimulating factor regulates the distribution and differentiated state of dendritic cells/Langerhans cells in human lung and lung cancers.J Clin Invest. 1993; 91: 566-576Crossref PubMed Scopus (187) Google Scholar, 16Tsujitani S Oka A Kondo A Ikeguchi M Maeta M Kaibara N Infiltration of dendritic cells into regional lymph nodes in gastric cancer.Cancer. 1995; 75: 1478-1483Crossref PubMed Scopus (17) Google Scholar, 17Nakano T Oka K Hanba K Morita S Intratumoral administration of Sizofiran activates Langerhans cell and T-cell infiltration in cervical cancer.Clin Immunol Immunopathol. 1996; 79: 79-86Crossref PubMed Scopus (37) Google Scholar This observation, associated with the decreased density of LCs in most cervical SILs, suggests that a quantitative and/or qualitative disturbance of LCs could interfere with the immune surveillance of HPV-associated cervical (pre)neoplastic lesions. The epithelial microenvironment is known to play an important role in determining the density and/or function of LCs.18Wu TC Kurman RJ Analysis of cytokine profiles in patients with human papillomavirus-associated neoplasms.J Natl Cancer Inst. 1997; 87: 185-187Crossref Scopus (40) Google Scholar, 19Barker JNW Mitra RS Griffiths CEM Dixit VM Nickoloff BJ Keratinocytes as initiators of inflammation.Lancet. 1991; 337: 211-214Abstract PubMed Scopus (637) Google Scholar However, little is known about the factors controlling the number, distribution, and functional activity of LCs in HPV-associated (pre)neoplastic lesions of the cervix. Several hypotheses have been proposed to explain the local deficiency of LCs observed in most SILs. Some authors have suggested a cytopathic effect of the virus on LCs.20Rosini S Caltagirone S Tallini G Lattanzio G Demopoulos R Piantelli M Musiani P Depletion of stromal and intraepithelial antigen-presenting cells in cervical neoplasia in human immunodeficiency virus infection.Hum Pathol. 1996; 27: 834-839Abstract Full Text PDF PubMed Scopus (35) Google Scholar Others have hypothesized that the altered expression of E-cadherin, as observed on HPV-infected keratinocytes, contributes to the reduction in number and function of LCs.21Tang A Amagai M Granger LG Stanley JR Udey MC Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin.Nature. 1993; 361: 82-85Crossref PubMed Scopus (420) Google Scholar, 22Vessey CJ Wilding J Folarin N Hirano S Takeichi M Soutter P Stamp GWH Pigntelli M Altered expression and function of E-cadherin in cervical intraepithelial neoplasia and invasive squamous cell carcinoma.J Pathol. 1995; 176: 151-159Crossref PubMed Scopus (138) Google Scholar Another possibility is that the immunosuppressive cytokine IL-10, which has been shown to be highly expressed in SILs,23Giannini SL Al-Saleh W Piron H Jacobs N Doyen J Boniver J Delvenne P Cytokine expression in squamous intraepithelial lesions of the uterine cervix: implications for the generation of local immunosuppression.Clin Exp Immunol. 1998; 113: 183-189Crossref PubMed Scopus (83) Google Scholar could play a role in the functional inhibition of LCs.24Qin Z Noffz G Mohaupt M Blankenstein T Interleukin-10 prevents dendritic cell accumulation and vaccination with granulocyte-macrophage colony-stimulating factor gene-modified tumor cells.J Immunol. 1997; 159: 770-776PubMed Google Scholar An additional interesting hypothesis is based on the findings that keratinocytes can produce immunoregulatory cytokines that are likely to have a stimulatory effect on LCs, such as tumor necrosis factor (TNF)-α or GM-CSF.25Katz S The skin as an immunological organ: allergic contact dermatitis as a paradigm.J Dermatol. 1993; 20: 593-597Crossref PubMed Scopus (17) Google Scholar Transformation of keratinocytes with HPV has been shown to alter this function,26Woodworth CD Simpson S Comparative lymphokine secretion by cultured normal human cervical keratinocytes, papillomavirus-immortalized, and carcinoma cell lines.Am J Pathol. 1993; 142: 1544-1555PubMed Google Scholar causing a reduction of cytokine production that may influence the distribution and differentiation of LCs. GM-CSF is an important factor not only for the maturation and differentiation of LCs/DCs but also for their motility.27Kaplan G Walsh G Guido LS Meyn P Burkhardt RA Abalos RM Barker J Frindt PA Fajardo TT Celona R Cohn ZA Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth and enhanced wound healing.J Exp Med. 1992; 175: 1717-1728Crossref PubMed Scopus (253) Google Scholar, 28Pastore S Fanales-Belasio E Albanesi C Chinni LM Giannetti A Girolomoni G Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis.J Clin Invest. 1997; 99: 3009-3017Crossref PubMed Scopus (179) Google Scholar GM-CSF produced by keratinocytes acts as a selective chemoattractive stimulus for the continual migration of LCs/DCs into the epithelium. Exposure of LCs/DCs to GM-CSF in vitro prolongs their survival and increases their capacity to present antigens to lymphocytes.29Koch F Heufler C Kampgen E Schneeweiss D Bock G Schuler G Tumor necrosis factor α maintains the viability of murine epidermal Langerhans cells in culture, but in contrast to granulocyte/macrophage colony-stimulating factor, without inducing their functional maturation.J Exp Med. 1990; 171: 159-165Crossref PubMed Scopus (193) Google Scholar In vivo, a correlation has been observed between the amount of GM-CSF produced by some carcinomas and the distribution/differentiation of tumor-associated DCs.30Tazi A Bonay M Bergeron A Grandsaigne M Hance AJ Soler P Role of granulocyte-macrophage colony stimulating factor (GM-CSF) in the pathogenesis of adult pulmonary histiocytosis X.Thorax. 1996; 51: 611-614Crossref PubMed Scopus (59) Google Scholar, 31Stoppacciaro A Paglia P Lombardi L Parmiani G Baroni C Colombo MP Genetic modification of carcinoma with the IL-4 gene increase the influx of dendritic cells relative to other cytokines.Eur J Immunol. 1997; 27: 2375-2382Crossref PubMed Scopus (44) Google Scholar The current study was designed to evaluate the capacity of GM-CSF to influence the migratory capacity of in vitro generated DCs in Boyden chambers and in organotypic cultures of HPV-transformed keratinocytes. The organotypic culture of keratinocytes has been used previously to examine the effects of therapeutic agents on a variety of malignant keratinocytes32Delvenne P Al-Saleh W Gilles C Thiry A Boniver J Inhibition of growth of normal and human papillomavirus-transformed keratinocytes in monolayer and organotypic cultures by interferon-γ and tumor necrosis factor-α.Am J Pathol. 1995; 146: 589-598PubMed Google Scholar, 33Aebischer F McDougall JK Organotypic raft cultures for the in vitro evaluation of vaginal microbicidal agents. Microbicides have epithelium-specific effects when repeatedly added onto organotypic human keratinocyte cultures.Sex Transm Dis. 1997; 24: 69-76Crossref PubMed Scopus (9) Google Scholar, 34Eicher SA Clayman GL Liu TJ Shillitoe EJ Strothz KA Roth JA Lotan R Evaluation of tropical gene therapy for head and neck squamous cell carcinoma in organotypic model.Clin Cancer Res. 1996; 2: 1659-1664PubMed Google Scholar, 35Shindoh M Sun Q Pater A Pater MM Prevention of carcinoma in situ of human papillomavirus type 16 immortalized human endocervical cells by retinoic acid in organotypic raft culture.Obstet Gynecol. 1995; 85: 721-728Crossref PubMed Scopus (23) Google Scholar or as a model for immuno-pharmaco-toxicological studies.36Regnier M Staquet MJ Schmitt D Schmidt R Integration of Langerhans cells into a pigmented reconstructed human epidermis.J Invest Dermatol. 1997; 109: 510-512Crossref PubMed Scopus (107) Google Scholar In this system, keratinocytes are grown at the air-liquid interface on top of a dermal equivalent support. The normal keratinocytes stratify and exhibit a typical pattern of differentiated squamous epithelium, whereas HPV-transformed and established squamous carcinoma cell lines exhibit morphologies similar to those of high-grade lesions seen in vivo.37Rader JS Golub TR Hudson JB Patel D Bedell MA Laimins LA In vitro differentiation of epithelial cells from cervical neoplasias resembles in vivo lesions.Oncogene. 1990; 5: 571-576PubMed Google Scholar, 38Blanton RA Perez-Reyes N Merrick DT McDougall JK Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic cultures.Am J Pathol. 1991; 138: 673-685PubMed Google Scholar So, the organotypic culture system of HPV-transformed keratinocytes might be particularly suited to examine the effects of new therapeutic strategies and particularly to investigate the interactions between cervical (pre)neoplastic lesions and various types of immunocompetent cells, among them LCs/DCs. Because three-dimensional cultures are obtained under these conditions, the ability of LCs/DCs to infiltrate HPV-transformed (pre)neoplastic epithelium may be assessed by a quantitative image analysis of immunohistochemical markers (CD1a). Human exocervical epithelial cells were obtained from hysterectomy specimens of healthy women. Cell cultures were established and maintained following a previously reported method.39Gilles C Piette J Rombouts S Laurent C Foidart JM Immortalization of human cervical keratinocytes by human papillomavirus 33.Int J Cancer. 1993; 53: 872-879Crossref PubMed Scopus (27) Google Scholar Briefly, tissue fragments were incubated with 0.25% trypsin (GIBCO BRL, Gaithersburg, MD) for 24 hours at 4°C. The epithelial cells were detached and cultured with irradiated 3T3 mouse fibroblasts as a feeder. The growth medium was a 1/3 mixture of HAM F12 (GIBCO BRL)/Dulbecco's modified Eagle's medium (GIBCO BRL), supplemented with 0.5 μg/ml hydrocortisone (Sigma Chemical Co., St Louis, MO), 10 ng/ml epidermal growth factor (Sigma), 10% fetal calf serum (Life Sciences International, Zelik, Belgium), 2 mmol/Ll-glutamine (GIBCO BRL), 10 mmol/L Hepes (GIBCO BRL), 1 μg/ml fungizone (GIBCO BRL), 1 mmol/L sodium pyruvate (GIBCO BRL), 3000 U/ml penicillin-streptomycin (GIBCO BRL), 10−10 mol/L cholera toxin (Sigma), 5 μg/ml insulin (Sigma), 20 μg/ml adenine (Sigma), 5 μg/ml human transferrin (Sigma), and 15 × 10−4 mg/ml 3,3′,5-triiodo-l-thyronine (Sigma). Subconfluent cultures were dispersed with 0.0025% trypsin and 0.02% EDTA. These primary cultures of keratinocytes were used for the organotypic cultures. This study protocol was approved by the Ethics Committee of the University Hospital of Liège. SiHa, CasKi, and C4-II are tumorigenic cervical carcinoma-derived keratinocyte cell lines.40Friedl F Kimura I Osato T Ito Y Studies on a new human cell line (SiHa) derived from carcinoma of uterus. I. Its establishment and morphology.Proc Soc Exp Biol Med. 1970; 135: 543-545Crossref PubMed Scopus (184) Google Scholar, 41Pater M Pater A Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix.Virology. 1985; 145: 313-318Crossref PubMed Scopus (208) Google Scholar, 42Auersperg N Hawryluk AF Chromosome observations on three epithelial cell cultures derived from carcinomas of the uterine cervix.J Natl Cancer Inst. 1962; 28: 605-627PubMed Google Scholar The SiHa cell line contains one copy of integrated HPV-16 DNA. Three different passages of this cell line were used (SiHa I, SiHa II, and SiHa III), which differed in their production of GM-CSF, as measured by ELISA. The CasKi cell line contains approximately 600 copies of integrated HPV-16 DNA, whereas the C4-II cell line contains HPV-18 DNA sequences. The CK2 cell line was established by transfection of human cervical keratinocytes with HPV-33 DNA and is not tumorigenic in nude mice.39Gilles C Piette J Rombouts S Laurent C Foidart JM Immortalization of human cervical keratinocytes by human papillomavirus 33.Int J Cancer. 1993; 53: 872-879Crossref PubMed Scopus (27) Google Scholar All of these HPV-transformed keratinocyte cell lines were grown and maintained in a mixture of HAM F12 and DMEM supplemented with the same additives as those used for normal keratinocyte cultures. DCs were generated by culturing adherent fractions of human peripheral blood mononuclear cells as previously described.13Sallusto F Lanzavecchia A Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor α.J Exp Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4479) Google Scholar, 43Hubert P Greimers R Franzen-Detrooz E Doyen J Delanaye P Boniver J Delvenne P In vitro propagated dendritic cells from patients with human-papillomavirus-associated preneoplastic lesions of the uterine cervix: use of Flt3 ligand.Cancer Immunol Immunother. 1998; 47: 81-89Crossref PubMed Scopus (21) Google Scholar Briefly, mononuclear cells were isolated from leukocyte-enriched buffy coats by centrifugation on Ficoll-Hypaque. The plastic adherent fraction (10 × 106 cells/well, 18 hours at 37°C) was cultured with 800 U/ml human recombinant GM-CSF (kindly provided by Schering-Plough, Brussels, Belgium) and 100 U/ml IL-4 (Genzyme Diagnostics, Cambridge, MA) in 3 ml of RPMI containing 10% FCS/50 μmol/L mercaptoethanol. Cultures were fed every 3 days with fresh medium containing cytokines and harvested at day 7. Cells of each culture were analyzed by flow cytometry and displayed the previously described surface phenotype of DCs (HLA-DR+, MHC class I+, CD1a+, CD4+, CD54+, CD80+, CD86+, CD3−, and CD14−).13Sallusto F Lanzavecchia A Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor α.J Exp Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4479) Google Scholar, 43Hubert P Greimers R Franzen-Detrooz E Doyen J Delanaye P Boniver J Delvenne P In vitro propagated dendritic cells from patients with human-papillomavirus-associated preneoplastic lesions of the uterine cervix: use of Flt3 ligand.Cancer Immunol Immunother. 1998; 47: 81-89Crossref PubMed Scopus (21) Google Scholar Organotypic cultures of normal cervical keratinocytes and HPV-transformed keratinocytes were prepared by procedures slightly modified from those described previously.32Delvenne P Al-Saleh W Gilles C Thiry A Boniver J Inhibition of growth of normal and human papillomavirus-transformed keratinocytes in monolayer and organotypic cultures by interferon-γ and tumor necrosis factor-α.Am J Pathol. 1995; 146: 589-598PubMed Google Scholar, 44Merrick DT Blanton RA Gow AM McDougall JK Altered expression of proliferation and differentiation markers in human papillomavirus 16 and 18 immortalized epithelial cells grown in organotypic cultures.Am J Pathol. 1992; 140: 167-177PubMed Google Scholar For the preparation of dermal equivalents, a collagen matrix solution was made with 32 mg of collagen (Cellagen solution AC-5, type I, ICN, Biomedical, Asse-Relegen, Belgium) mixed on ice with 1.6 ml of 0.1% acetic acid, 1 ml of chilled 10-fold concentrated Hanks' buffer supplemented with phenol red and 1 N NaOH to give a pH of 7.2. One milliliter of FCS containing 4 × 105 normal human fibroblasts was then added. One milliliter of the collagen/fibroblast solution was poured into 24-well plates (Nunclon Δ Multidishes, Nunc, Roskilde, Denmark) and allowed to solidify at 37°C for 1 hour. The final concentrations of collagen and fibroblasts were 3.2 mg/ml and 4 × 104 cells/ml, respectively. After gel equilibration with 1 ml of growth medium overnight at 37°C, 25 × 104 to 1 × 106 keratinocytes (normal or HPV-transformed) resuspended in 100 μl of growth medium were seeded on top of the gels and maintained submerged for 24 to 96 hours. The collagen rafts were raised in a 25-mm tissue culture insert (8-μm pore size; Nunc) and placed onto stainless-steel grids at the interface between air and liquid culture medium. Epithelial cells were then allowed to stratify over 2 weeks. After stratification of keratinocytes, DCs were seeded on top of the in vitro-formed epithelium at a concentration of 2 × 105 cells/50 μl of growth medium, and organotypic culture medium was supplemented or not with 800 U/ml GM-CSF. After 24 hours at 37°C, the collagen rafts were harvested. The cultures were then embedded in OCT compound (Tissue Tek, Sakura, The Netherlands) at −70°C and sectioned with a cryostat microtome for the immunohistochemical analysis. The density of DCs migrating into the epithelial layer was assessed by the avidin-biotin-peroxidase technique (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) with an anti-CD1a monoclonal antibody (clone NA1/34 from Dako, Glostrup, Denmark). Nine-micron frozen sections were fixed in cold acetone for 3 minutes, and endogenous peroxidases were blocked with 0.1% H2O2 for 30 minutes. Sections were then incubated sequentially with anti-CD1a antibody (at a 1/40 dilution in PBS containing 2% bovine serum albumin (BSA) and 0.01 mol/L NaN3) or with an isotype-matched control antibody for 1 hour, with a biotinylated mouse anti-Ig antibody for 30 minutes, and with streptavidin/horseradish peroxidase/avidin/biotin complex for another 30 minutes. Positive cells were visualized by a 3,3′-diaminobenzidine substrate (DAB). The sections were counterstained with hematoxylin. Direct double-immunofluorescence staining was performed to evaluate the relative proportion of macrophages (CD14+) and DCs (CD1a+) in the epithelial layer. Phycoerythrin-conjugated anti-CD14 (Leu-M3, IgG2b; Becton Dickinson, Erembodegem, Belgium) and fluorescein-isothiocyanate-conjugated anti-CD1a (OKT6; Ortho, Raritan, NJ) were used at a 1/10 dilution. The DC infiltration in organotypic cultures was evaluated by measuring the surface of immunostained cells with a computerized system of image analysis (CAS, Becton Dickinson) following a method previously described.32Delvenne P Al-Saleh W Gilles C Thiry A Boniver J Inhibition of growth of normal and human papillomavirus-transformed keratinocytes in monolayer and organotypic cultures by interferon-γ and tumor necrosis factor-α.Am J Pathol. 1995; 146: 589-598PubMed Google Scholar The entire surface of each culture section was analyzed. To determine the number of CD1a+ cells within a given culture, three sections stained with anti-CD1a were counted. The positive surface was expressed as the percentage of the total surface analyzed. Statistical analysis was performed by using the Student t-test (Instat Mac 2.01 software; Graph-Pad Software, San Diego, CA) for each separate cell line or normal keratinocyte culture. Chemotactic migration of the DCs was tested as previously described.45Castronovo V Taraboletti G Liotta LA Sobel ME Modulation of laminin receptor expression by estrogen and progestins in human breast cancer cell lines.J Natl Cancer Inst. 1989; 81: 781-788Crossref PubMed Scopus (93) Google Scholar Briefly, after phenotypic characterization, DCs were resuspended in serum-free growth medium containing 0.1% BSA. Polyvinylpyrollidone-free polycarbonate membrane 8-μm pore filters (Poretics Corp., Livermore, CA) were coated by incubation with 100 μg/ml gelatin in 0.1% acetic acid solution and placed in a chemotactic Boyden microchamber (Neuroprobe, Cabin John, MD). The lower compartment was filled with 27 to 28 μl of keratinocyte-derived cell-conditioned medium, obtained from subconfluent keratinocyte cultures, containing 0.1% BSA. Nonconditioned medium was used as a control for random migration. Each condition was repeated six times, and 55 μl of DC suspension (2 × 106 cells/ml) was added to the upper compartment of the chamber. The chambers were incubated for 5 hours at 37°C in a 5% CO2/95% air atmosphere. The filters were removed, fixed, and stained using the Diff-Quick stain set (Baxter Diagnostics, Düdingen, Switzerland), and the upper side of the filter was scraped to remove residual nonmigrating cells. Two random fields were counted per well using an eyepiece with a calibrated grid to evaluate the number of fully migrated cells. The amount of GM-CSF in keratinocyte cultures was measured by ELISA (Medgenix, Fleurus, Belgium) using supernatants from normal keratinocytes and HPV-transformed cell lines collected from subconfluent cultures after 48 hours. GM-CSF was quantified by ELISA using supernatants collected from subconfluent cultures after 48 hours. Normal keratinocyte cultures, derived from four different individuals, were tested at the second passage to avoid any possible contamination with feeder cells. Figure 1 shows that the spontaneous production of GM-CSF by HPV-transformed cell lines was uniformly low. In contrast, keratinocytes from the normal exocervix released substantial amounts of GM-CSF, up to 95-fold higher than cervical carcinoma-derived cell lines. Similar results were obtained when using cultures maintained in medium without FCS, although the secreted quantities of GM-CSF were systematically lower in the absence of serum (data not shown). To determine whether conditioned media of HPV-transformed keratinocytes could influence the migration of DCs, we conducted a migration assay using a Boyden microchemotaxis chamber. DCs generated for this study were judged to be 90% pure based on several criteria, including morphology, forward-scatter a
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