In vivo and in vitro approaches in understanding the differences between Caucasian and African skin types: specific involvement of the papillary dermis

Authors


  • Conflicts of interest: The authors declare no conflicts of interest.

Sarah Girardeau-Hubert, MSc
L’Oréal Research and Innovation, 90 rue Général Roguet, 92583 Clichy cedex, France
E-mail: shubert@rd.loreal.com

Abstract

Background  Most of the identified differences between Caucasian and African skin types have been related to the superficial part of the skin, the epidermis. We investigated possible implications of the dermal compartment in cutaneous differences observed between Caucasians and Africans.

Methods  In vivo and in vitro comparative studies were carried out using normal human skin biopsies and the corresponding in vitro reconstructed skin. Skin equivalents were developed with papillary fibroblasts isolated from the superficial dermis of both Caucasian and African skin types. Expression of major components of the dermal–epidermal junction (DEJ) was examined as a function of ethnicity.

Results  Control histological examinations of skin biopsies showed that the African skin type had greater convoluted appearance of the DEJ than the Caucasian skin type. Immunostainings of type IV and VII collagens, laminin 5, and nidogen proteins at the DEJ were lower in African skin compared with Caucasian skin biopsies.

Conclusions  This study brings together new elements on involvement of the papillary dermis in differences between Caucasian and African skin types. As fibroblasts from the superficial dermis cooperate with epidermal keratinocytes in producing protein of the membrane basal zone, present in vivo results suggest that papillary fibroblasts may play a part in the distinct features observed at the DEJ. In preliminary in vitro experiments, differences in several protein expressions contributing to the DEJ framework were found in reconstructed skin models made with papillary fibroblasts from both Caucasian and African skin types. Therefore, in vitro skin equivalents may be useful for better understanding of ethnic skin differences in the future.

Introduction

The main information about ethnic skin differences is related to the superficial part of the skin. Reported studies about Caucasian and African skin types emphasize differences in the structural and functional role of the epidermis in pigmentation, photoprotective properties, and barrier function of the stratum corneum.1

So far, biological studies on ethnic differences have not focused on the dermal part of the skin. This is of importance, however, as the dermis plays a critical role in the functioning of the skin as a structural and complex tissue. Dermis can be divided into two distinct regions, namely, papillary and reticular dermis corresponding to the superficial and deeper part of the dermis, respectively. Each dermal part, and constituent fibroblasts, exhibit distinct features. The papillary dermis has been described as a thin structure with highly proliferative fibroblasts, whereas the reticular dermis, a large compartment with accumulating thick fibers populated with fibroblasts, has a slow proliferative capacity.2,3 Papillary and reticular fibroblasts differ in their constitutive expression of soluble molecules and extracellular components in the dermis as well as in their capacity to influence epidermal morphogenesis in three-dimensional cultures.4,5

A previous study showed that activities of cultured papillary fibroblasts isolated from the superficial part of the dermis of African and Caucasian skin types could differ with regard to soluble molecule secretion.6 Therefore, we focused this current comparative study on the papillary dermis. In vivo and in vitro experiments were designed to identify and understand the potential differences between dermis of Caucasian and African skin types.

Materials and methods

Skin samples and three-dimensional skin reconstruction in vitro

Normal human skin biopsies were obtained from mammary surgery (sun-protected areas) of adult women (age range 19–42 years) of Caucasian (n = 10) and African (n = 10) origins. Skin samples were used for histology and immunohistochemistry analysis.

Papillary fibroblasts were isolated from the superficial part of the dermis (up to 0.3 mm depth) of skin explants as described previously.5 Keratinocytes were obtained from normal human skin from an additional Caucasian donor under 30 years old and cultured on a feeder layer of Swiss 3T3 fibroblasts.7

Skin equivalents were produced as described previously.8 Briefly, dermal equivalents were prepared with papillary fibroblasts (1 × 106 cells per dermis) from three Caucasian skin types and three African skin types, respectively and bovine type I collagen, then kept in culture during contraction of the lattice. The epidermis was formed by seeding keratinocytes from the Caucasian donor on top of dermal equivalents. Cultures were kept submerged for 7 days for keratinocyte proliferation and then raised to the air-liquid interface to obtain epidermal differentiation.

Histology and immunohistochemistry

Samples of human and reconstructed skins were fixed in neutral formalin, embedded in paraffin and sections were stained for classical histology with hematoxylin–eosin saffron.

Samples of human skin were embedded in Tissue-tek OCT compound (Sakura Finetek, Zoeterwourde, The Netherlands) and frozen in liquid nitrogen. Immunolabelings were performed on air-dried frozen sections (5 μm thick). Mouse monoclonal primary antibodies were used to stain human type IV collagen (Dako, Glostrop, Denmark), type VII collagen (Chemicon, Millipore, Billerica, MA, USA), laminin 5 (Chemicon) proteins, and rabbit polyclonal primary antibody for human nidogen protein (Calbiochem, Nottingham, UK). Primary antibodies were revealed by fluorescein isothiocyanate-conjugated rabbit antimouse or swine antirabbit antibodies (Dako). Nuclei were counterstained using propidium iodide (Sigma, St Louis, MO, USA). Histological and immunofluorescent sections were examined using LEICA DMDR microscope (Microsystems, Wetzlar, Germany).

Results and discussion

The in vivo study on normal human skin of Caucasian and African origin revealed striking differences at the level of the dermal–epidermal junction (DEJ). The DEJ of African skin had a greater convoluted and homogeneous pattern, with numerous and evenly distributed dermal papillae and epidermal projections as opposed to that of Caucasian skin (Fig. 1a,b). As DEJ is a complex macromolecular structure,9 we focused our interest on several proteins involved in the framework of the epidermal basement membrane zone (BMZ). Our immunohistochemical investigations demonstrated that the staining of type IV collagen (Fig. 1c,d), type VII collagen (Fig. 1e,f), laminin 5 (Fig. 1g,h), and nidogen (Fig. 1i,j) proteins was higher in the biopsies from Caucasian origin than in those from African origin. Such differences may be linked to a distinct architecture or differential expression from skin cell populations of BMZ proteins between skin of Caucasian origin and skin of African origin.

Figure 1.

 Histology and basal membrane zone protein immunostaining in Caucasian and African skin types. Skin biopsies were stained with hematoxylin–eosin saffron (HES) coloration (a,b), for type IV collagen (c,d), type VII collagen (e,f), laminin 5 (g,h), and nidogen (i,j). Note more pronounced rete ridges in African skin (b) as compared with Caucasian skin (a) together with stronger staining of basement membrane components in Caucasian skin (c,e,g,i) as compared with African skin (d,f,h,j). Scale bar: 100 μm

The convoluted appearance of the DEJ area may be as a result of cumulative processes such as epidermal layer proliferation and the extracellular matrix environment. The cooperative interaction between keratinocytes and fibroblasts leads to the formation of the cutaneous basement membrane. Each cell population contributes due to a specific expression pattern of BMZ proteins10,11 and interacting effects mediated by secreted soluble factors. In previous in vitro experiments, we noticed that papillary fibroblasts of African origin cultured in monolayers had greater protein synthesis activity for keratinocyte growth factor and monocyte chemotactic protein-1 than papillary fibroblasts of Caucasian origin.6 We suggest that the molecule secretion difference of papillary fibroblasts in African skin compared with Caucasian skin may influence keratinocyte activity and modulate the protein balance of the BMZ.

The highly developed DEJ relief, the in vitro specific expression pattern of secreted proteins,6 and these new in vivo results about BMZ markers in African skin types may reflect differences in intercellular interactions between the dermis and epidermis as compared with Caucasian skin types (Fig. 2). Therefore, it is of interest to us to study these properties using in vitro comparative studies in a three-dimensional context. Using reconstructed skin in vitro containing epidermal and dermal compartments with living cells is a useful model to study cell–cell and cell–matrix interactions. Reconstructed skins were produced with skin papillary fibroblasts from either Caucasian or African skin types for the dermal equivalent and with a common source of keratinocytes from Caucasian origin for the epidermal part (Fig. 3). Our preliminary experiments with skin equivalents suggest that expression of several BMZ proteins could be higher at the DEJ when Caucasian papillary fibroblasts are included in the dermal equivalent than with African papillary fibroblasts (work in progress). These recent findings can relate to our in vivo data on BMZ protein expression at the DEJ. It appears that beside soluble mediator expression, papillary fibroblasts from Africans and papillary fibroblasts from Caucasians differ in their extracellular matrix environment protein expression and this may provide an explanation for in vivo differences observed at the DEJ level.

Figure 2.

 Diagrammatic representation of skin differences between Caucasian and African skin types. These distinct properties may result from specific differences in papillary fibroblast function. KGF, keratinocyte growth factor; MCP-1, monocyte chemotactic protein-1.

Figure 3.

 Histological appearance of reconstructed skins made with papillary fibroblasts isolated from Caucasian (a) or African skin biopsies (b). Scale bar: 100 μm

In conclusion, while recent advances have been made in the field of skin types, few of them have addressed biological studies at the cellular level. Therefore, we decided to investigate differences between skin of Caucasian and African origin using a complementary approach to in vivo and in vitro studies. Results on papillary dermis in vivo and dermal papillary fibroblasts in vitro showed that the corresponding ethnic differences are not limited to the superficial part of the skin but also extend to deeper layers. These distinct properties between African and Caucasian skin types may provide a new explanation of potential differences in their specific cutaneous physiology and physiopathologies.

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