Technical Advance: Langerhans cells derived from a human cell line in a full-thickness skin equivalent undergo allergen-induced maturation and migration

Abstract

In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a(+)Langerin(+)HLA-DR(+) MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO(4) and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

MUTZ-3 Langerhans Cell maturation and CXCL12 independent migration in reconstructed human gingiva

“…MUTZ-3 LC closely resemble their native counterparts, both phenotypically and functionally (Kosten et al, 2015b;Masterson et al, 2002;Santegoets et al, 2008). Indeed, in the past we have shown in skin equivalents with integrated Langerhans Cells (SE-LC) that upon allergen exposure MUTZ-3 LC mature and migrate in a CXCL12 dependent manner from the epidermis to the dermis, whereas upon irritant exposure MUTZ-3 LC migrate in a CCL5 dependent manner and undergo an IL-10 dependent phenotypic switch into a macrophage-like cell in the dermis, closely mimicking our observations in excised skin (Kosten et al, 2015b;Ouwehand et al, 2008Ouwehand et al, , 2011b). Recently, we described a full thickness oral gingiva equivalent (GE) consisting of a reconstructed epithelium on a fibroblast populated collagen hydrogel (lamina propria) and shown that the GE secretes negligible amounts of key chemokines involved in LC migration in skin (Kosten et al, 2015a).…”

“…The number and phenotype of migrated MUTZ-3 LC was determined by flow cytometry in the SE-LC and GE-LC after disrupting the collagen gels with collagenase as described previously (Ouwehand et al, 2011b;Kosten et al, 2015b). Cell staining was performed using mouse anti-human CD1a-PE (IgG1, BD Pharmingen, San Diego, CA, USA).…”

“…We have previously described a human immune-competent skin equivalent that consists of a fully differentiated reconstructed epidermis containing LC derived from the MUTZ-3 cell line, on a fibroblast populated collagen hydrogel (Ouwehand et al, 2011b;Kosten et al, 2015b). MUTZ-3 is an acute myeloid leukemicderived human cell line with CD34 + proliferating progenitor cells that can be differentiated into LC (MUTZ-3 LC) in a cytokine dependent fashion.…”

In Vitro Approaches for Detection of Chemical Sensitization

“…4) [42]. Using a full-thickness tissue-engineered skin model containing fully functional MUTZ-3-derived LCs (MUTZ-LC), the MUTZ-LCs were demonstrated to mature and to acquire the ability to migrate towards C-X-C motif ligand (CXCL)12 and C-C motif ligand (CCL)19/21 in a comparable manner with primary LCs in skin explants [43].…”

Advanced tests for skin and respiratory sensitization assessment

“…An immunocompetent skin equivalent model (by Sue Gibbs) the ideal future will have an immunocompetent equivalent model that may replace one-to-one the in vivo method with the construction of a functional, immunocompetent, full thickness skin equivalent model (Ouwehand et al, 2011).…”