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Published as doi: 10.1096/fj.06-7646com.
 (The FASEB Journal. 2007;21:2765-2775.)
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Defensins induce the recruitment of dendritic cells in cervical human papillomavirus-associated (pre)neoplastic lesions formed in vitro and transplanted in vivo

Pascale Hubert*,1, Ludivine Herman*, Catherine Maillard{dagger}, Jean-Hubert Caberg*, Arjen Nikkels{ddagger}, Gerald Pierard{ddagger}, Jean-Marie Foidart{dagger}, Agnès Noel{dagger}, Jacques Boniver* and Philippe Delvenne*

* Department of Pathology,

{dagger} Laboratory of Tumor and Development Biology,

{ddagger} Department of Dermatopathology, University Hospital of Liège, CHU Sart Tilman, Liège, Belgium

1Correspondence: Department of Pathology B35, University of Liège, Sart Tilman, 4000 Liège, Belgium, E-mail: P.hubert{at}ulg.ac.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to their direct antimicrobial activity, defensins might also influence adaptive immunity by attracting immature dendritic cells (DC). As these cells have been shown to be deficient in uterine cervix carcinogenesis, we evaluated the ability of {alpha}-defensin (HNP-2, human neutrophil defensin 2) and ß-defensin (HßD2, human beta defensin 2) to stimulate their migration in human papillomavirus (HPV)-associated (pre)cancers. We first observed, using RT-PCR and immunohistology, that HßD2 is absent in HPV-transformed keratinocytes and that it is weakly expressed in cervical (pre)neoplastic lesions in comparison with normal keratinocytes. We next demonstrated that defensins exert a chemotactic activity for DC in a Boyden Chamber assay and stimulate their infiltration in an in vitro-formed (pre)neoplastic epithelium (organotypic culture of HPV-transformed keratinocytes). To evaluate the ability of defensins also to recruit DC in vivo, we developed a model of immunodeficient mice grafted with organotypic cultures of HPV+ keratinocytes, which form an epithelium similar to a high-grade neoplastic lesion, with tumoral invasion and neovascularization. Intravenously injected human DC were able to infiltrate grafts of HPV+ keratinocytes after administration of HNP-2 in the transplantation chamber. Taken together, these results suggest that defensins could reverse a frequent immune alteration observed in cancer development.—Hubert, P., Herman, L., Maillard, C., Caberg, J-H., Nikkels, A., Pierard, G., Foidart, J-M., Noel, A., Boniver, J., Delvenne, P. Defensins induce the recruitment of dendritic cells in cervical human papillomavirus-associated (pre)neoplastic lesions formed in vitro and transplanted in vivo.


Key Words: alpha and beta defensins • antigen presenting cells • HPV • cervical lesions • migration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SOME TYPES OF HUMAN PAPILLOMAVIRUS (HPV) have been established as the central cause of cervical cancer and its precursors (squamous intraepithelial lesions, SILs) (1) . High-risk HPVs are necessary but not sufficient for the development of cervical cancer. Additional environmental and/or host factors act in concert with HPV to promote the neoplastic process (2) . Among these cofactors, host immune surveillance is likely to play an important role in the control of cervical lesions caused by HPV infection (3) . Although the nature of an effective immune response against HPV is not completely understood, there is accumulating evidence that cell-mediated immune response is more important than humoral immunity in controlling tumor progression (4) . Indeed, several studies have described a localized immune dysfunction accompanying cervical HPV infection (5 , 6) . Most SILs are associated with a lower density and/or function of Langerhans cells (LC), which constitute a subpopulation of immature dendritic cells (DC) present in the mucosal squamous epithelium (2 , 7) . As the presence of DC precursors in peripheral blood mononuclear cells (PBMC) is identical for patients with SILs and for normal donors, the decreased LC/DC density observed in SILs could be related to local mechanisms rather than to systemic alterations affecting DC precursors (8) . LC/DC form a network in suprabasal epithelial layers and initiate, maintain, and regulate adaptive immunity by taking up antigens, emigrating into the regional draining lymph nodes, and presenting the processed antigens to T cells. Importantly, keratinocytes are capable of producing soluble factors such as cytokines (e.g., GM-CSF, IL1ß, and IL10), chemokines (e.g., MIP3{alpha} and MCP-1), and defensins, all of which have an important influence on the migration or activation of LC/DC (9 , 10) .

Defensins are a family of small (2–6 kDa) cationic, microbicidal peptides. On the basis of their size and pattern of disulfide bonding, mammalian defensins are classified into different categories that are either constitutively produced or induced by inflammatory mediators. {alpha} defensins (HNP-1 to 4) were first found in human polymorphonuclear leukocytes and intestinal Paneth cells (11) . They have also been recently described in the walls of coronary vessels and in specific lymphocyte and monocyte subpopulations (12) . To date, six human ß defensins (HßD1 to 6) have been identified. They are produced by epithelial cells lining various organs (e.g., epidermis, bronchial tree, and genitourinary tract) (13) . HßD1 is constitutively produced by keratinocytes of various epithelial tissues. In contrast, the expression of HßD2 and HßD3 is induced predominantly in inflamed skin and lung tissues on treatment with LPS or cytokines such as TNF-{alpha} and IL-1ß (14) . All defensins identified to date show the capacity to kill and/or activate different gram-positive and gram-negative bacteria, fungi, some enveloped viruses, and parasites. This direct antimicrobial activity is presumably operative in vivo, and therefore defensins are generally considered to function as effectors of innate immunity. However, there are also clues suggesting that defensins might play a role in adaptive immunity, such as the observation that HNP-2 and HßD2 are chemotactic for T cells and immature DC (10 , 15) .

The current study was designed to evaluate the capacity of {alpha} (HNP-2) and ß (HßD2) defensins to influence the migratory capacity of in vitro generated CD1a+ DC in Boyden Chambers and in organotypic cultures of HPV-transformed keratinocytes formed in vitro or maintained in vivo in immunodeficient mice. The organotypic culture of keratinocytes has been used previously to examine the effects of therapeutic agents on a variety of malignant keratinocytes (16) or as a model for immuno-pharmaco-toxicologic studies (17) . 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, while HPV-transformed and established squamous carcinoma cell lines exhibit morphologies similar to those of high-grade lesions seen in vivo (16) . We have also shown that these organotypic cultures, when grafted into mice, are useful models for studying tumor angiogenesis (18) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture of normal cervical keratinocytes and keratinocyte cell lines
Human exocervical epithelial cells were obtained from hysterectomy specimens of healthy women. Cell cultures were established following a previously reported method (19) . This study protocol was approved by the Ethics Committee of the University Hospital of Liège.

SiHa, CasKi and KT1 are cervical HPV-transformed keratinocyte cell lines (20 21 22) . These tumor cell lines were grown and maintained in the same culture medium as that used for normal keratinocyte cultures.

Cervical biopsy specimens
Forty frozen cervical biopsy specimens were retrieved from the Tumor Bank of the University of Liege. These biopsies included 13 normal exocervical tissues, 27 squamous intraepithelial lesions (SILs) (8 low-grade and 10 high-grade) and 9 invasive squamous cell carcinoma (SCCs). The histological diagnosis was confirmed after hematoxylin and eosin staining.

RT-PCR
Total RNA from normal keratinocytes, CasKi, and SiHa cells and biopsies was extracted using the High Pure RNA isolation kit (Roche Diagnostics, Mannheim, Germany). RT-PCR amplification was performed using the Superscript first-strand synthesis system kit (Invitrogen, Life Technologies, Paisley, UK). The following reverse (R) and sense (S) primers were used: HBDR 5'-CTCTGTAACAGGTGCCTTGA-3', HBDS 5'-CCAGTTCCTGAAATCCTGAG-3', HPRTR 5'-CAGATGTTTCCAAACTCAAC-TTGAA-3', HPRTS 5'-GTTGGATATAAGCCAGACTTTGTTG-3'. PCR amplification was run as follows: 30 cycles for HBD and 35 cycles for HPRT at 94°C 1 min, 60°C 1 min, 72°C 1 min followed by a final 5 min extension step at 72°C. RT-PCR products were resolved on 1.8% agarose gels and analyzed using a Fluor-S MultiImager (Bio-Rad, Hercules, CA, USA) after staining with ethidium bromide.

The following primers were also used: MMP-2R 5'-GGCTGGTCAGTGGCTTGGGGTA-3', MMP-2S 5'-AGATC-TTCTTCTTCAAGGACCGGTT-3', MMP-9R 5'-GACGCGCCTGTGTACACCCACA-3', MMP-9S 5'-GCGGAGATTGGGAACCAGCTGTA-3', 28SR 5'-GATTCTGACT-TAGAGGCGTTCAGT-3' and 28SS 5'-GTTCACCCACTAATAGGGAACGTGA-3'. PCR amplification was run as follows: 30 cycles for MMP-2, 35 cycles for MMP-9 and 17 cycles for 28S at 94°C 20 s, 68°C 20 s, 72°C 10 s followed by a final 2 min extension step at 72°C. RT-PCR products were resolved on 10% polyacrylamide gels and analyzed using a Fluor-S MultiImager after staining with Gelstar dye (FMC BioProducts, Heidelberg, Germany).

Immunostaining
Briefly, 5 µm biopsy sections were blocked using rabbit nonimmune serum supplemented with 3% BSA. Incubation with anti- HßD2 (HBD21-A, 1/100, Rabbit polyclonal antibody, Alpha Diagnostic Int, San Antonio, TX, USA) and anti-HNP-2 (DEF3, BMA Biomedicals AG, Augst, Switzerland) antibodies was performed during 1 h and overnight at 4°C, respectively. Subsequently, the slides were incubated with secondary antibody (biotinylated swine anti-rabbit, 1:300, Dakopatts, Glostrup, Denmark for HßD2 and avidin-biotin-peroxidase kit for HNP-2; Vectastain ABC kit, Vector laboratories, Burlingame, CA). For HßD2 detection, slides were rinsed and covered by a polymer-based revelation system EnVision (Dakopatts). After washing, Fast Red (Dakopatts) was used as chromogen substrate for 5 min. For HNP-2 detection, positive cells were visualized by a 3,3'-diaminobenzidine substrate (DAB). The last steps consisted of counterstaining with Mayer's hemalun and mounting in Glycergel mounting medium (Dakopatts). The following control staining conditions were employed: 1) absence of the primary (specific) antibody, as a negative control and 2) neutralization of the anti-defensin antibody with recombinant defensins.

The HßD2 immunostaining was evaluated in cervical biopsy specimens by using a semiquantitative score, as described previously (23) . Scoring of the intensity and extent of the staining was performed according to an arbitrary scale. For staining intensity, 0 represented samples in which HßD2 expression was undetectable whereas 1+, 2+, and 3+ denoted samples with a low, moderate and strong staining, respectively. For staining extent, 1+ represented samples in which HßD2 expression was detectable in up to 33% of the epithelium, 2+ denoted samples in which 33–66% of the epithelium exhibited a detectable HßD2 expression and 3+ represented those in which more than 66% of the epithelial cells were stained. To provide a global score for each case, the results obtained with the two scales were multiplied, yielding a single scale with steps from 0 to 9. The HNP-2 expression, which was detected predominantly in the stroma was evaluated only by the staining intensity.

Dendritic cell cultures and labeling with lipophilic fluorescent cell tracer
DC were generated from the adherent fraction of human PBMC with 800 U/ml GM-CSF (Amoytop Biotech, Xiamen, China) and 40 U/ml IL4 (ImmunoTools, Friesthe, Germany), as described previously (8) . DC generated for this study constituted a 90% pure cell population based on several criteria, including morphology, forward-scatter and side-scatter values observed by FACS analysis and surface phenotype (CD1a+, HLA-DR+, MHC-Class I+, CD4+, CD54+, CD86dim, CD3–, CD20–, and CD14–). The DC were labeled with a lipophilic fluorescent marker (CM-DiL, Molecular Probes, Leiden, The Netherlands), according to a previously described procedure (24) . To induce the cell surface expression of CCR6 necessary for the chemoattractant activity of HßD2 (15) , dendritic cells were also generated by culturing CD34+ cord blood mononuclear cells in the presence of tumor growth factor-ß1 (TGF-ß1) (25) . Briefly, 15 x 104 CD34+ cells were seeded in T25 flasks (Sarstedt, Newton, NC) in 10 ml of RPMI 1640 medium containing 10% FCS, antibiotics and 50 µM mercaptoethanol (all from GIBCO-Life Technologies). Cultures were supplemented with optimized concentrations of the following human molecules: stem cell factor (SCF), thrombopoietin (TPO), fetal liver tyrosine kinase 3 ligand (Flt3L), GM-CSF, tumor necrosis factor {alpha} (TNF-{alpha}), interleukin 4 (IL4) and TGF-ß1. All these agents were purchased from PeproTech (Rockey Hill, NJ, USA), except for GM-CSF and IL4, which were obtained from Amoytop and ImmunoTools, respectively. The cells produced with this protocol were CD1a+, CD207+, E-cadherin+, CLA+, CCR6+ and constituted a 80% pure population.

Boyden chamber assay
Chemotactic migration of DC was tested as described previously (19) . Briefly, DC were resuspended in serum-free growth medium containing 0.1% BSA. Polyvinylpyrolidone-free polycarbonate membrane 8 µm-pore filters (Poretics Corp., Livermore, CA, USA) coated with 100 µg/ml gelatin was placed in a chemotactic Boyden microchamber (Neuroprobe, Cabin John, MD, USA). The lower compartment was filled up with 27 µl of a GM-CSF (1 µg/ml), HNP-2 (Sigma, St. Louis, MO, USA) (0.5 to 1 µg/ml), or HßD2 solution (PeproTech, Rocky Hill, NJ, USA) (0.5 to 1 µg/ml), containing 0.1% BSA. Culture medium was used as a control for random migration. Six wells were used for each experimental condition. Fifty-five microliters of DC suspension (2x106 cells/ml) were added to the upper compartment of the chamber. The chambers were incubated for 5 h at 37 C in a 5% CO2/95% air atmosphere. Two random fields were counted per well using an eyepiece with a calibrated grid, to evaluate the number of fully migrated cells.

Organotypic cultures
Organotypic cultures were prepared as previously reported (19) . After stratification of keratinocytes, DC were seeded on top of the in vitro formed epithelium at a concentration of 2 x 105 cells/50 µl of growth medium in the presence or in the absence of GM-CSF (800 U/ml), HNP-2 (0.75 µg/ml) or HßD2 (0.75 µg/ml) in the medium of organotypic culture. After 48 h at 37°C, the collagen rafts were harvested. The cultures were then embedded in O.C.T. compound (Tissue Tek, Sakura, The Netherlands) at –70°C and sectioned with a cryostat microtome for immunohistochemical analysis.

Assessment of DC infiltration in organotypic cultures
The density of DC migrating into the epithelial layer was assessed by the avidin-biotin-peroxidase detection (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) with an anti-CD1a monoclonal antibody (clone NA1/34 from Dako), as described previously (19) . 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, Erembodegem, Belgium) following a method previously described (19) . The entire surface of each culture section was analyzed, and five sections of each culture were stained with the anti-CD1a antibody. The results were expressed as percentages of immunoreactive areas to the total surface analyzed.

Xenograft assay in NOD/SCID mice
HPV-transformed keratinocytes (2x105) were plated on a collagen gel (2 mg/ml of type I collagen) inserted in Teflon rings (Renner, Dannstadt, Germany) and maintained in culture for 1 day before transplantation onto mice. Twelve-week-old NOD-scid/scid mice were used for this study. The cell-coated collagen gels were then covered with a silicone transplantation chamber (Renner) and implanted entirely onto the dorsal muscle fascia of mice, as described previously (18) . Two million fluorescent-labeled DC were intravenously injected at day 21, at the time of the first application of chemoattractant molecules. Three groups of 10 mice were treated by injecting 200 µl of HNP-2 (1.5 µg/ml), GM-CSF (4x105 U/ml) or PBS into the transplantation chamber every day, for a period of 2 days before the killing of the mice. The mice were killed 24 days later. The transplants were excised and embedded in O.C.T. compound (Tissue Tek). Infiltration of fluorescent DC was visualized with a fluorescent microscope (Leica DMLB microscope, Heidelberg GmBH, Germany) equipped with a x40 objective. Five perpendicular sections in the central part of each transplant were analyzed. Fluorescent DC were counted in the epithelium and, after antikeratin immunolabeling, the epithelial surface was evaluated with a computerized system of image analysis (CAS, Becton Dickinson). DC infiltration results were expressed as numbers of DC per square millimeter of epithelium.

Immunofluorescent labeling of xenografts
Graft cryosections (5 µm in thickness) were fixed in acetone at –20°C and in 80% methanol at 4°C. They were then incubated for 60 min at room temperature with the primary antibodies: anti-mouse type IV collagen (SIF105 rabbit anti-mouse (18) ) and anti-human keratin (KL1, mouse anti-human, Immunotech, Marseille, France). The appropriate secondary antibodies were then applied for 30 min: swine anti-rabbit conjugated to Texas red (Dako) and sheep anti-mouse conjugated to fluorescein-isothiocyanate (Sigma). After washing, we analyzed the labeling under a microscope equipped with epifluorescence optics.

Statistical analysis
Statistical analysis was performed by using the Student's t test (Instat Mac 2.01 software; Graph-Pad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Decreased expression of HßD2 mRNA in high-grade SILs and SCC biopsies and HPV-transformed cultures
HßD2 and HNP-2 defensin expression was investigated by RT-PCR in normal exocervix, SIL, and SCC biopsies. Variable levels of HßD2 mRNA were detected in all exocervical biopsies analyzed. There was no difference in HßD2 expression between normal and low-grade SIL biopsies (Fig. 1 A, B). In high-grade SILs, HßD2 expression was significantly lower in comparison with exocervical or low-grade SIL biopsies. HßD2 mRNA was not detected in SCC except in one biopsy specimen. Similar results were obtained with biopsies consisting only of epithelium or including a small amount of stroma (data not shown).


Figure 1
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  		Figure 1. A) HßD2 defensin mRNA expression in biopsies of normal and (pre)neoplastic epithelium. HPRT represents a housekeeping gene, used as a positive control for RNA integrity and concentration. EXO, exocervical epithelium; LSIL, low-grade SILs; HSIL, high-grade SILs; SCC, squamous cell carcinoma. These tissues were obtained from 4 different donors in each diagnostic category. B) Graphical representation of the expression of HßD2 mRNA levels in biopsies normalized to HPRT mRNA. The results are expressed as the means ± SD. Asterisks indicate statistically significant differences (*P<0.05, ***P<0.001).

In agreement with the in vivo data and as observed in the native epidermis (26) , variable levels of HßD2 mRNA were detected in normal keratinocyte cultures from different donors.

Concerning HNP-2, the RT-PCR showed a weak expression in a limited number of biopsies, which is in agreement with the nonepithelial expression of HNP-2. There was no significant difference in the proportion of normal, SIL, and SCC biopsies positive for HNP-2. The positivity was mainly related to inflammatory cell infiltration (data not shown).

HßD2 immunoreactivity is lower in high-grade SILs and SCC biopsies
HßD2 expression was studied in normal exocervix (n=8), low-grade SILs (n=3), high-grade SILs (n=5) and invasive SCCs (n=4). As shown in Fig. 2 A, the normal exocervical epithelium was found to be generally strongly positive for HßD2. Immunohistochemical localization of HßD2 revealed a cytoplasmic staining predominantly in the (para)basal and intermediate cell layers of the epithelial compartment. In contrast, high-grade SILs and SCCs showed a lower anti-HßD2 immunoreactivity. Macrophages present in the stroma were strongly positive and served as an internal positive control (27) . Preincubation of the anti-HßD2 antibody with HßD2 peptide inhibited the positive signals, thus confirming the specificity of the staining (Fig. 2C ). Semiquantitative evaluation of HßD2 intraepithelial expression is shown in Fig. 2D . The HßD2 score was statistically higher in the normal exocervical epithelium in comparison with high-grade SILs and SCCs (P<0.05). As for the RT-PCR analysis, there was no difference in the proportion of normal, SIL, and SCC biopsies positive for HNP-2.


Figure 2
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  		Figure 2. HßD2 immunostaining in cervical biopsy specimens. A, B) HßD2 exocervix immunoreactivity in normal squamous epithelium and in high-grade SIL. A) Normal squamous epithelium showing a diffuse staining. Arrows mark positive macrophages, which serve as positive internal controls. B) HSIL demonstrating a diminished HßD2 immunoreactivity. C) The specificity of the staining is demonstrated by the absence of immunoreactivity after neutralization of anti-HßD2 antibody with the recombinant peptide. D) HßD2 staining scores in normal exocervical epithelium (EXO), low-grade SILs (LSIL), high-grade SILs (HSIL), and invasive squamous cell carcinoma (SCC). Asterisks indicate statistically significant differences (*P<0.05).

Defensins induce the recruitment of DC in the Boyden chamber assay
To determine whether defensins could influence the migration of antigen-presenting cells, a migration assay was conducted. DC generated for this study were judged to be 90% pure based on various criteria, including morphology, forward-scatter and sidescatter values obtained by FACS analysis and surface phenotype (data not shown).

As shown in Fig. 3 , a chemotactic activity of HßD2 was observed for CCR6+ DC at a concentration as low as 0.5 µg/ml, and it reached a level similar to the chemotaxis induced by GM-CSF (1 µg/ml), the positive control (19 , 28 ). The chemotactic activity of HNP-2 (already at 0.5 µg/ml) on CCR6– DC was similar to that detected when using GM-CSF (1 µg/ml).


Figure 3
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  		Figure 3. Chemotactic activity of HßD2 or HNP-2 DC generated in vitro. Results are expressed as the incremental percentages of migrating CCR6+ DC (HßD2) and CCR6– DC (HNP-2) in comparison with control nonconditioned medium (represented as 0%) (mean percentages ± SD of 4 experiments). Asterisks indicate statistically significant differences (**P<0.01; ***P<0.001).

Defensins stimulate the infiltration of DC into organotypic cultures of HPV-transformed keratinocytes
We investigated whether the addition of {alpha} and ß defensins could modulate the ability of DC to infiltrate an in vitro-formed (pre)neoplastic epithelium, reminiscent of a cervical high-grade lesion observed in vivo.

After 1–2 wk of culture, the SiHa cell line grown on a collagen gel at the air-liquid interface formed an epithelial layer of more than 10 cell layers in thickness, which closely resembled a high-grade cervical lesion. DC were layered on top of the epithelial sheets in the presence or not of HNP-2 or HßD2 in the culture medium. In all experiments, recombinant human GM-CSF was used as a positive control for DC recruitment (19) . The effect of defensins on DC infiltration was assessed by examining immunohistological sections of organotypic cultures at 48 h following the addition of chemoattractant. The ability of defensins to influence DC infiltration was determined by the detection of CD1a+ cells. Figure 4 (A–D) illustrates representative experiments showing the density of CD1a-labeled DC in HPV-transformed keratinocyte organotypic cultures incubated or not with GM-CSF or defensins.


Figure 4
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  		Figure 4. A–D) Effect of defensins on the infiltration of CD1a+ cells into HPV+ keratinocyte organotypic cultures. CD1a+ immunolabeled sections of organotypic culture of SiHa cells in the absence of chemoattractant (A), in the presence of GM-CSF (B), HßD2 (C), and HNP-2 (D). Quantitative evaluation of CCR6+ and CCR6– DC infiltration into organotypic cultures of HPV-transformed keratinocytes in the presence of HßD2 (E) or HNP-2 (F). The infiltration of DC under basal conditions and in the presence of GM-CSF, HNP-2, or HßD2 was assessed by immunolabeling with an anti-CD1a antibody. Results are expressed as percentages of the surface labeled with anti-CD1a in comparison with unlabeled surface of the epithelial sheet ± SD (n=6 for each HPV-transformed cell line). Asterisks indicate statistically significant differences (*P<0.05; **P<0.01).

DC layered onto organotypic cultures of SiHa infiltrated poorly the epithelial layer in the absence of chemoattractant molecules (Fig. 4A ), whereas the addition of defensins caused a significant increase in the intraepithelial migration of DC (Fig. 4C,D ). The CCR6+ DC recruitment induced by HßD2 (Fig. 4C ) was similar to that observed with recombinant GM-CSF, used as positive control (Fig. 4B ). Similar observations were made when HNP-2 was used to recruit CCR6– DC (Fig. 4D ).

Quantitative analysis of DC infiltration was performed by evaluating the surface labeled with the anti-CD1a antibody throughout the full thickness of organotypic cultures (Fig. 4E,F ). Under basal conditions, the level of DC infiltration in HPV+ cell line cultures was very low. Interestingly, when the medium of organotypic culture was supplemented with HßD2, the infiltration of DC increased in comparison with that observed in the absence of chemoattractant, but the DC recruitment was significantly lower (P<0.05) than the migration induced by GM-CSF (Fig. 4E ). When the medium of organotypic cultures of HPV+ cell line was supplemented with GM-CSF, the infiltration of DC increased in the epithelial sheets and the addition of HNP-2 induced a DC recruitment similar to that observed with GM-CSF (Fig. 4F ). HNP-2 also increased the density of DC detected in CasKi cell line-derived organotypic cultures, which are refractory to the effect of GM-CSF (19) (data not shown). This observation suggests that {alpha} defensins could bypass, more efficiently than GM-CSF, the possible inhibition of DC migration induced by HPV+ keratinocytes, and this may be related to the fact that defensins mobilize cells more rapidly than chemokines (29) .

HNP-2 stimulates the migration of human DC in xenografts of HPV-transformed keratinocytes
To address the in vivo efficacy of defensins, HPV-transformed keratinocytes cultured on a collagen gel were grafted onto the back of NOD-SCID mice (Fig. 5 A). Twenty-four days after transplantation, the mice were euthanized and the grafts were removed for morphological analysis. Histological examination of thin sections revealed that the growth pattern of the grafts reproduced a high-grade squamous lesion and that the collagen gel was replaced by a granulation tissue. Although SiHa cells proliferated to form a multilayered surface epithelium, these cells failed to invade host tissues and remained as an irregular stratified epithelium on top of the collagen gel. Concomitantly, no angiogenic response was observed since capillaries failed to pierce through the collagen gel and remained confined to the area beneath it (Fig. 5D-E ). In contrast, the CasKi cell line induced an angiogenic response in the host tissue starting from vessels of the dorsal muscle and subsequently extending far up into the collagen gel. The vessels sprouted into the tumor epithelium, which started to invade the newly formed granulation tissue (Fig. 5B ,C).


Figure 5
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  		Figure 5. Schematic cross section through the organotypic culture of HPV-transformed keratinocytes grafted onto NOD-SCID mice and protected by a silicone chamber (A). Epithelial grafts of CasKi (B and C) and SiHa cells (D and E) in NOD-SCID mice. H&E staining (B and D) (e, epithelial tissue; h, host connective tissue; c, collagen gel). Immunofluorescent labeling of keratinocytes and vessels (C and E). Human keratinocytes and murine vessels are detected with anti-keratin (in green) and anti-type IV collagen (in red) antibodies, respectively.

As invasive growth and angiogenesis depend on the coordinated action of an array of proteolytic enzymes such as matrix-metalloproteinases (MMPs) and their inhibitors (30) , we analyzed the expression pattern of several MMPs (MMP-1, -2, -9, -12, -13) in SiHa and CasKi cell lines. Our qualitative results showed that SiHa cells express MMP-9 and MMP-2 at lower levels than CasKi cells, suggesting the role of these proteases during tumor invasion (data not shown). Preliminary experiments evaluating the effect of chemoattractant molecules on DC recruitment in organotypic cultures of SiHa cells did not show any DC infiltration, suggesting the need for a neoangiogenesis in tumor tissues for the migration of human DC injected into the blood (data not shown).

On the basis of these results, 30 mice were grafted with CasKi cell-derived organotypic cultures and were intravenously injected with fluorescent CCR6– DC (CM-DiL labeled cells). The level of DC infiltration into the epithelial transplants was then assessed by fluorescence microscopy (Fig. 6 ). As CCR6+ DC were not maintained in vivo under our experimental conditions, the effect of HßD2 could not be tested in this model. Interestingly, the inoculation of HNP-2 in the transplantation chamber induced a recruitment of DC in the tumor grafts (Fig. 6, C1,C2 ). The density of DC was similar to that observed when GM-CSF was used as a chemoattractant (Fig. 6 B1, B2 ), whereas DC were rarely detected in the epithelium of control nontreated mice (Fig. 6 A1, A2 ). Quantitative analysis of DC infiltration was performed by determining the number of DC per mm2 of epithelial tissue (Fig. 6D ). The level of DC infiltration in the CasKi grafts was very low (1.1±1.4 DC/mm2) in the absence of chemoattractant and increased drastically after treatment with GM-CSF (16.6±14.6 DC/mm2) or HNP-2 (30.6±18.2 DC/mm2). HNP-2 was significantly more efficient at inducing the DC migration than GM-CSF (P<0.05). Similar data were obtained with the KT1 cell line (Fig. 6 A3, B3, C3 ). No significant difference in the level of DC recruitment was observed, however, between HNP-2 and GM-CSF.


Figure 6
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  		Figure 6. Detection of DC prelabeled with a fluorescent red probe in transplanted organotypic cultures of CasKi or KT1 cells in the absence (A) and in the presence of GM-CSF (B) or HNP-2 (C). Detection of fluorescent DC in organotypic cultures of CasKi cells without counterstaining (A1, B1, C1). Localization of fluorescent DC (in red) in the grafted HPV+ epithelium formed by CasKi cells (A2, B2, C2) or KT1 cells (A3, B3, C3) and immunostained with anti-human keratin antibody (in green). Quantitative evaluation of DC infiltration in transplanted organotypic cultures in the presence or not of GM-CSF or HNP-2. (e, epithelial tissue; h, host connective tissue). The insets show higher magnifications of fluorescent DC in the tumor epithelium (scale bar: 50 µm). (D). The results are expressed as numbers of DC per mm2 of HPV+ epithelium ± SD (n=10 for each condition). Asterisks indicate statistically significant differences (***P<0.001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Defensins are peptides for which the pharmacological potential cannot be ignored, especially considering their strong antimicrobial but also antiviral activity. Indeed, HßD2 and HßD3 are able to inhibit HIV infection by both viral strains due to a direct interaction with virions and through modulation of the CXCR4 coreceptor (31) . Alpha defensins also have an anti-HIV activity (32) , bringing new hope for a cure for this worldwide disease. Moreover, a recent report showed that {alpha} defensins 1–3 (HNP 1–3) and {alpha} defensin 5(HD-5) are also potent antagonists of infection by both cutaneous and mucosal types of HPV by blocking virion escape from endocytic vesicles (33) . These data suggest that defensins could not only be a natural barrier to the sexual transmission of HPV or HIV but could also serve as a model for the development of broad-spectrum topical microbicides. In addition to antimicrobial properties, the anticancer effects of defensins are becoming increasingly documented. Indeed, despite evidence that immune effectors can play a significant role in controlling tumor growth under natural conditions or in response to therapeutic manipulations, cancer cells usually evade immunosurveillance. Interestingly, defensins could overcome these limitations by using a unique mechanism of cancer cell killing that involves membrane lysis (34) . This cytotoxic effect of defensins on cancer cells, including both solid tumors and leukemias, has stimulated interest in these peptides as possible candidates for new anticancer therapies (35) .

In this study, we analyzed the potential impact of defensins on cancer development, not in terms of lytic function but with reference to their capacity to recruit immune cells (15) . Indeed, previous studies have demonstrated quantitative and qualitative alterations of Langerhans/dendritic cells (LC/DC) in most cervical SILs in comparison with normal exocervical mucosa (2) . Because LC/DC play a central role in the generation of an effective cellular immune response, such a defect may contribute to the persistence of HPV and indirectly to the development of cervical cancer. We postulated that HPV-transformed keratinocytes could influence directly the number and/or function of LC by an altered production of factors necessary for their migration and maturation. In agreement with this hypothesis, GM-CSF, an essential factor not only for the differentiation and maturation of DC but also for their motility (36) , is poorly produced in HPV+ keratinocytes (19) and HPV16 E6 and E7 proteins have been shown to modulate the regulation of this gene in part by interfering with the NF-{kappa}B signaling pathway (37) . As LC/DC express a large array of receptors, other molecules with chemotactic activity could also be involved in the recruitment of LC/DC into (pre)neoplastic lesions.

In the current report, we demonstrated, using immunostaining and RT-PCR techniques, a diminished expression of HßD2 in high-grade SILs and in SCC. This observation is in agreement with the absence of HßD2 transcripts in HPV-transformed cells in comparison with normal exocervical keratinocytes. HßD2 has been previously detected in normal keratinocytes with, however, interindividual and site-specific differences in the intensity of immunostaining on normal skin samples from various body sites (38) . Although the mechanisms of reduced defensin expression in preneoplastic lesions are still unclear, a recent study demonstrated, in a model of atopic dermatitis, a down-regulatory role of IL10 on the expression of HßD2 (39) . Interestingly, this immunosuppressive cytokine has also been shown to be overexpressed in cervical lesions in comparison with normal exocervix (40) .

Using Boyden Chamber and organotypic culture assays, we showed that HNP-2 and HßD2 induce the migration of DC.

Preliminary evidence also suggests that DC recruited in the HPV+ epithelium remain active at least for their innate function. Indeed, we observed that the infiltration of organotypic cultures by DC under the action of defensins induces the apoptosis of HPV+ tumor cells to a similar extent as that detected when GM-CSF is used as chemoattractant (24) (data not shown).

To approximate the in vivo situation with a LC/DC colonization from the basal layers of the epithelium, we used a mouse xenograft model for the in vivo testing of chemoattractant molecules. The dorsal muscle of NOD/SCID mice as the transplantation site and the use of a silicone chamber were shown to be useful in evaluating the effects of chemoattractant molecules on DC recruitment in vivo, since the organotypic cultures of HPV-transformed CasKi cells were invaded by blood vessels of the murine host, allowing intravenously injected human DC to migrate directly from the blood circulation. Tumor vascularization and invasion were probably related, in our model, to the expression of MMP-2 and -9, as demonstrated by the high level of expression of these enzymes in CasKi cells and previous reports showing that organotypic cultures grafted in double MMP-2/MMP-9 deficient mice are not invasive (41) . By using this in vivo model, we showed that HNP-2 is able to stimulate the migration of human DC into the epithelium and that it is more efficient than GM-CSF for recruiting DC in vivo. Interestingly, the density of DC obtained after recruitment with HNP-2 was similar to that observed in the normal exocervix (42) , suggesting that HNP-2 could restore a local antigen presentation function similar to the nontransformed squamous epithelium.

The mechanisms by which defensins increase the infiltration of DC in HPV+ epithelium are currently under investigation. The specificity of DC trafficking is controlled by the interactions of defensins with their specific receptors. CC chemokines receptor 6 (CCR6) has been shown to be the receptor used by ß defensins to attract CCR6+ DC (10) . Although these receptors are still unidentified for HNP-2, previous studies have demonstrated that {alpha} defensin-induced chemotaxis may be inhibited by preincubation of target cells with pertussis toxin, suggesting that {alpha} defensin receptors are related to Gi{alpha} proteins and are different from CCR6 (15) . In addition to a direct chemotactic action on DC, HNP-2 could also down-regulate the expression of cytokine/chemokine receptors on DC and make them less susceptible to the repulsive effect of molecules produced by HPV+ keratinocytes such as TNF-{alpha}. Indeed, a previous study demonstrated that {alpha} defensins down-regulate CXCR1 expression on polymorphonuclear cell membrane (43) . Moreover, expression of molecules important for DC migration, such as MCP-1 or GM-CSF, might be also modulated by {alpha} defensins as already observed for bronchial cells (44) .

Although a few studies have detected defensins in epithelial tumors (45 , 46) , their role in the regression or progression of (pre)neoplastic lesions is still unknown. However, since defensins act as chemotactic gradients for immature DC and since DC are crucial for the induction of adaptive immune responses, it is likely that the greater the recruitment of DC to the site of antigen deposition, the higher the induction of antigen-specific immune responses. Congruently, defensins have been shown to activate murine T-helper cell responses in vitro, suggesting that they provide signals for linking innate and acquired immunity (47) .

It has also recently been reported that ß defensins are proangiogenic (48) . This effect is mediated by the chemotactic recruitment of DC precursors to tumors where they may promote neovascularization by undergoing endothelial-like transdifferentiation. However, {alpha} defensins are important regulators of neovascularization by their inhibitory effects on capillary-like tube formation in three-dimensional fibrin matrices (49) and on pathological retinal neovascularization in vivo (50) . The activity of {alpha} defensins in human tumors (51) may therefore help to control tumor angiogenesis and, indirectly, tumor growth.

In addition to the recruitment of DC to the epithelium, defensins have been shown to display cytotoxic effects toward cancer cells, whereas other investigators have suggested that defensins may exert mitogenic activity on renal cell carcinomas (51) . In the current study, defensins were not associated with any effect on cell proliferation at the concentrations that elicited DC migration (data not shown).

In conclusion, we demonstrated that defensins are able to recruit DC in organotypic cultures of HPV-transformed keratinocytes maintained in vitro or grafted in vivo, suggesting that these molecules may restore some immune functions, which have been shown to be altered during cervical carcinogenesis.

These findings, associated with previous results demonstrating the inhibitory effects of defensins on HPV infection and angiogenesis, could have important implications for the development of new antitumor approaches.


   ACKNOWLEDGMENTS
 
This work was supported by the Centre de Recherche Interuniversitaire en Vaccinologie with a grant from the Walloon Region and GlaxoSmithKline, the Marshall Programme of the Walloon Region (Neoangio 616476), the Belgian Fund for Medical Scientific Research, the L. Fredericq Fund, the BIO4-CT98–0097 contract of the EU, the IAP (Interuniversity Attraction Pole Network P5/31), the Centre Anti-Cancereux près l'Université de Liège and the CRCE. P. Delvenne is a senior Research Associate of the Belgian National Fund for Scientific Research (FNRS).

We thank Elisabeth Franzen-Detrooz for her excellent technical assistance.

Received for publication October 30, 2007. Accepted for publication March 8, 2007.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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