Search

Human papillomaviruses (HPVs), which are contained in the alpha, beta, gamma, mu, and nu genera, differ in their oncogenic potential and their tropism for cutaneous or mucosal epidermis. Langerhans cells (LC), the only epidermal professional antigen-presenting cells, are readily detected in normal mucosal and cutaneous epithelium. The aim of this study is to determine whether LC loss, which has been reported for HPV16, occurs in other HPV genera and establish its significance in viral pathology. We found that, as for HPV16, LCs were reduced in lesions infected with high-risk mucosal (alpha7 and alpha9 species) and low-risk cutaneous (gamma and mu) types. Lesions infected with alpha10 low-risk genital types had reduced LC but contained epidermal LC patches, coincident with dermis-localized regulatory T cells (T-regs). In contrast to other genera, LCs were common in the epidermis, and T-regs occupied the dermis of the potentially high-risk cutaneous beta-HPV type infected lesions. Therefore, LC loss in the infected lesions occurred irrespective of tropism or oncogenic potential of the HPV type. LC depletion in the HPV-infected epidermis may create an environment that is permissive for viral persistence and in HPV lesions in which LCs are found, the presence of typically immunosuppressive T-regs may compensate for their continued presence.

Epithelial (E)-cadherin is a transmembrane protein that mediates calcium-dependent cell adhesion. E-cadherin has significant roles in tissue development, adhesion between keratinocytes and retention of Langerhans cells in the epidermis, and its loss on tumour cells is frequently associated with metastasis. Here we describe a simple, flow cytometric adhesion assay to measure the effects of potential regulators of cell surface E-cadherin expression on E-cadherin-mediated adhesion between cells. In this assay, cells that have been transiently transfected to express the protein of interest are enzymatically treated to remove cell surface E-cadherin. Cells are then incubated in low attachment plates, during which time the E-cadherin is re-expressed and E-cadherin-mediated aggregation occurs. The effect of the protein of interest on the percentage of E-cadherin-mediated aggregates that form during incubation is measured flow cytometrically, following staining with an E-cadherin specific antibody. A major advantage of this assay is that a potential regulatory protein of interest can be tested in a transient expression system by co-expression with green fluorescent protein and analysis of adhesion conducted on green fluorescent cells only. We have applied this assay to measure the regulatory effects of E6 protein from human papillomavirus type 16 on E-cadherin-mediated adhesion but this assay potentially has broad applicability for testing the effects of other proteins on E-cadherin-mediated adhesion in an accurate and highly reproducible manner.