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

Abstract

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.

Figure 1.

GM-CSF secretion by cultures of cervical epithelial cells. Cultures of exocervical cells (four different donors, KN1 to KN4) and HPV-transformed cell lines were tested. Values represent the mean of three independent experiments ± SD.

Figure 2.

Chemotactic activity of conditioned media of keratinocytes on DCs. NC, nonconditioned medium; KN1 and KN2, conditioned media of normal keratinocytes from two different donors; F. Hum, conditioned medium of human fibroblasts (positive control). Other conditioned media came from HPV-transformed cell lines. Results are the mean ± SD of four experiments.

Figure 3.

A: Effect of anti-GM-CSF antibody on the chemotactic activity of conditioned media of keratinocytes. B: Effect of exogenous GM-CSF addition on the chemotactic activity of conditioned media of SiHa cell line. NC, nonconditioned medium; F. Hum, conditioned medium of human fibroblasts (positive control); KN1, KN3, and KN4, conditioned media of normal keratinocytes from three different donors. Asterisks indicate statistically significant differences: *P < 0.05; **P < 0.005; ***P < 0.0001.

Figure 4.

SiHa cell and normal keratinocyte organotypic cultures as models of high-grade cervical lesions and normal epithelium, respectively (A to D, H&E). A: Biopsy specimen of normal cervical epithelium. B: Section of an organotypic culture of normal cervical keratinocytes. C: Biopsy specimen of high-grade cervical SIL. D: Section of an organotypic culture of SiHa cells. E to H: Correlation between the penetration of CD1a⁺ cells into organotypic cultures and the addition of exogenous GM-CSF, shown by CD1a⁺ immunolabeled sections of organotypic culture of normal keratinocytes (E and F) and SiHa cells (G and H) in the absence (E and G) or in the presence (F and H) of GM-CSF.

Figure 5.

Quantitative evaluation of DC infiltration into organotypic cultures of normal and HPV-transformed keratinocytes. The penetration of DCs under basal conditions (A) and in the absence or in the presence of recombinant GM-CSF (B) is followed by immunolabeling with anti-CD1a. Results are expressed as percentages of surface labeled with anti-CD1a compared with unlabeled surface of the epithelial sheet ± SD (n = 4 for each HPV-transformed cell line and normal keratinocyte culture). Asterisks indicate statistically significant differences: *P < 0.05; **P < 0.005; ***P < 0.0001).