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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 17, 2001 as doi:10.1096/fj.00-0872fje. |
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Department of Pathology, University Hospital of Liège, CHU Sart Tilman, 4000 Liège, Belgium; and
* Department of Infectious and Parasitic Diseases, B-43bis, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium
2Correspondence: Department of Pathology B35, CHU Sart Tilman, 4000 Liege, Belgium. E-mail: P.Hubert{at}ulg.ac.be.
SPECIFIC AIMS
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, suggesting that the function of antigenic presentation is deficient in some SILs. This hypothesis is reinforced by the observation that most SILs are characterized by a lower density of Langerhans cells (LC) than the normal exocervical epithelium.
To determine whether professional antigen-presenting cells die in SILs, we assessed the apoptotic status in an in vitro formed epithelium derived from HPV-transformed keratinocytes and infiltrated by immature dendritic cells (DC).
PRINCIPAL FINDINGS
1. DC induce the death of HPV-transformed keratinocytes after their infiltration in organotypic cultures
We first used a TUNEL assay and confocal microscopy to identify apoptotic cells in organotypic cultures of HPV-transformed keratinocytes infiltrated by fluorescent DC. As shown in Fig. 1
, DC infiltration into organotypic cultures of HPV+ cell lines induced an increase in the apoptosis of keratinocytes vs. cultures without DC (Fig. 1C
). In contrast, apoptotic DC were rarely observed (Fig. 1A-C
). The addition of DC into organotypic cultures of HPV- cell lines (Fig. 1A
) or normal keratinocytes (Fig. 1B
) did not affect the level of apoptosis.
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2. The cytotoxic activity of DC is specific for HPV+ cell lines and does not affect surrounding normal keratinocytes
We next wanted to ascertain whether DC induce the apoptosis of HPV+ cell lines without affecting normal surrounding keratinocytes present in the culture. We therefore performed several cocultures experiments by adding DC onto semiconfluent monolayer cultures of normal or HPV+ keratinocytes and a mixture of normal keratinocytes and HPV+ cell lines.
In triple cocultures (DC+normal keratinocytes+HPV+ cell lines), we observed an important killing of HPV+ keratinocytes (87.9±15% for SiHa in triple DC/SiHa/normal keratinocytes cultures, 95.9±2% for CasKi in triple DC/CasKi/normal keratinocytes cultures), whereas normal keratinocytes were not affected by the cytotoxicity (6.4±5.9% in triple DC/SiHa/normal keratinocytes cultures, 5.9±1.9% in triple DC/CasKi/normal keratinocytes cultures).
3. The antitumor activity of DC requires contact between DC and tumor cells
To determine the mechanism of the DC-mediated proapoptotic activity, we performed a 48 h growth inhibition assay by coculturing HPV+ cell lines, an HPV- cell line, or normal keratinocytes with DC or supernatants collected on day 7 from the DC cultures. DC exhibited a significant growth inhibitory effect on HPV+ cell lines. The DC did not inhibit the growth of HPV- cell lines and normal keratinocytes. However, supernatants had no inhibitory effect on any cells analyzed, suggesting that contact between DC and tumor cells is required to inhibit tumor cell growth. We also performed similar experiments using the TUNEL assay. The results agreed with those obtained with the growth inhibition assay. We also collected supernatants of 48 h cocultures of DC + HPV+ keratinocytes (SiHa and CasKi) or normal keratinocytes. These supernatants were tested for their killing activity on monolayer of HPV+ and HPV- keratinocytes in a 48 h growth inhibition assay. Cell proliferation in the presence of supernatants of DC + keratinocytes was similar to that obtained in the presence of supernatants derived from cultures of keratinocytes or DC alone.
4. DC do not induce apoptotic cell death of HPV+ cell lines via a TRAIL mechanism
To examine whether DC can affect the HPV+ cell lines via a TRAIL-based mechanism, we performed a 48 h growth inhibitionassay by coculturing DC pretreated with TRAILR2-Fc with different keratinocyte cell lines. However, although the HPV+ cell lines were sensitive to the effect of recombinant TRAIL, TRAILR2-Fc did not alter the cytotoxic ability of DC, which suggests that the TRAIL pathway is probably not the mechanism responsible for the apoptosis of the HPV+ keratinocytes induced by the DC.
5. Cytotoxic activity of DC is not associated with the FasL pathway
FasL was detected on the surface membrane of DC and Fas on HPV+ cell lines. Pretreatment of DC with an anti-FasL antibody (NOK2, PharMingen, San Diego, CA) did not prevent the apoptosis of HPV+ cell lines, which makes the DC cytotoxic activity mediated by Fas/FasL interactions unlikely.
DISCUSSION
Previous studies have shown a depletion of LC in most HPV-associated preneoplastic lesions (SILs) of the uterine cervix vs. normal exocervical squamous mucosa. On the other hand, the ability to generate in vitro DC from blood precursors is similar in healthy women and patients with SIL. These results suggest that local factors in the cervix play an important role in the development of LC alterations during HPV-associated cervical carcinogenesis.
Because interactions between malignant and immune cells are best studied in models approximating the tumor in vivo microenvironment, we performed organotypic cultures of HPV-transformed keratinocytes, which reproduce many features of in vivo SILs. Using this model, we recently demonstrated that GM-CSF is a potent factor for enhancing the colonization of LC/DC into organotypic cultures of HPV-transformed keratinocytes.
In this study, we demonstrated that the cross-talk between monocyte-derived DC and HPV+ keratinocytes does not induce the death of DC but causes the apoptosis of HPV-infected keratinocytes. DC-induced death was restricted to HPV+ keratinocytes, and contact between DC and tumor cells was shown to be a prerequisite for the cytotoxic activity of DC. The membrane contact did not lead to an up-regulation of cytotoxic factors (FasL, TNF-
, TRAIL) on the DC membrane or in the supernatants, suggesting that the contact between both cell types is required for a direct killing effector mechanism (data not shown).
One recent study has implicated that TRAIL (TNF-related apoptosis-inducing ligand) serves as the innate molecule on DC necessary for elimination of some tumor cells. TRAIL has been shown to be a potent mediator of tumor cell apoptosis while demonstrating minimal cytotoxicity toward normal tissues. In our study, we observed that keratinocytes, either normal or derived from the HPV-negative C33 cell line, are not sensitive to the effect of TRAIL, whereas SiHa and CasKi cells were respectively moderately and very sensitive to TRAIL. However, although 1.58–14.68% of DC expressed TRAIL on their membrane (data not shown), we did not observe an inhibition of apoptosis in cocultures with DC preincubated with the TRAILR2-Fc, which suggests that the cytotoxic activity of DC is probably not mediated in our model by this pathway.
Many other molecules have been implicated in the induction of apoptosis such as Fas/FasL or TNF/TNFR interactions. However, these molecules have not been involved in the killing of tumor cells by human DC.
FasL was detected on the surface membrane of DC and Fas on HPV+ cell lines. Pretreatment of DC with an anti-FasL antibody did not prevent the apoptosis of HPV+ cell lines, making unlikely the cytotoxic activity of DC mediated by Fas/FasL interactions.
The TNF/TNFR pathway was also considered a possible candidate to explain the mechanism of DC-mediated apoptosis. However, FACS analysis of DC did not show any significant cell membrane staining for TNF-. Another TNF receptor family member is CD40, which has been demonstrated to induce cell death in human B cell lymphoma cell lines. Preliminary results have shown that HPV+ cell lines, which die in the presence of DC, express higher levels of CD40 than the unaffected C33 HPV- cell line.
Other possible mechanisms for the cytotoxic activity of DC are the expression by the DC of the NK receptor protein 1 (NKRP1A) or the TWEAK molecule, induction of apoptosis by nitric oxide, or production of still unknown proapoptotic factors by DC in response to viral proteins. The effect(s) of HPV protein(s) on the function of DC is unknown at this time, but it is possible that contact or the nonspecific uptake of HPV proteins or viral particles influences the apoptosis-inducing abilities of DC. These data suggest that the induction of apoptosis may play an important role in the outcome of HPV-associated (pre)neoplastic lesions.
An increased number of Langerhans cells has been reported in spontaneously regressing HPV infected genital warts. Whether these observations are correlated to the improved viral antigen presentation or to the apoptosis of HPV-infected keratinocytes, however, is unknown. A recent study demonstrated that the prognosis of HPV-positive squamous cell carcinoma of the lung is better than that of cases where HPV is not detected. Moreover, in HPV-associated cancers, higher levels of apoptosis and infiltration of DC were demonstrated. In cervical carcinogenesis, apoptosis was shown to be negatively correlated with increasing histological abnormalities and the presence of high-risk HPVs. We and others have previously reported that most SILs have a lower density of Langerhans cells than nonpaired and paired normal exocervix biopsies and that SIL-derived LC exhibit a reduction in their functional activity. These observations may account for the low level of apoptosis observed in the majority of high-grade cervical lesions. This may be related to the high expression of interleukin 4 (IL-4) and IL-10 within the microenvironment of SIL. IL-10 has been shown to down-regulate several functions of DC and to protect some cells, such as macrophages or leukemic cells, from apoptotic death. In some conditions, IL-4 has been reported to protect B cells from spontaneous and dexamethasone-induced apoptosis. In addition to the direct cytotoxic effect of DC on HPV-transformed keratinocytes, apoptotic cell death induced by DC could also be an important mechanism implicated in the establishment of a long-term antitumor immune response. Recent studies have demonstrated that the cross-presentation of antigens to CD8+ or CD4+ T cells occurs after uptake of apoptotic cells by DC. Moreover, DC maturation is induced by exposure to necrotic tumor cells. DC have been shown to be able to process antigens from autologous melanoma apoptotic bodies and to stimulate Th1-directed autologous effector T cells. However, it has also been demonstrated that tumor cells undergoing apoptosis (as opposed to necrosis) are capable of rendering DC tolerogenic.
In conclusion, our findings may have important implications in host resistance to cancers. Since immature DC are widely distributed, they may participate in the growth inhibition of tumors. By exploring approaches to further enhance DC-mediated tumor apoptosis in vivo, new ways to strengthen innate and adaptive antitumor immunity could be found.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0872fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.00-0872fje.
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