Functional tight junction barrier localizes in the second layer of the stratum granulosum of human epidermis

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Abstract

Background

Mammalian epidermis has two diffusion barriers, the stratum corneum (SC) and tight junctions (TJs). We reported previously that a single living cell layer exists between the SC and TJ-forming keratinocytes in mice; however, the exact location of the TJ barrier in human epidermis has not been defined.

Objective

To investigate the precise distribution of epidermal TJs in relation to various cell–cell junction proteins and the SC and to clarify the barrier function of TJs against macromolecules in human skin.

Methods

The localization of various junctional proteins was investigated in human skin sections and in the roofs of bullae formed by ex vivo exfoliative toxin (ET) treatment in three dimensions. ET and single-chain variable fragments (scFv) against desmoglein 1 were used as large diffusion probes.

Results

Human stratum granulosum (SG) cells have a distinct distribution of TJ, adherens junction, and desmosome proteins in the uppermost three layers (SG1–SG3 from the surface inward). Ex vivo injection of ET or scFv demonstrated that only SG2–SG2 junctions function as a TJ barrier, limiting the inside-out diffusion of these proteins. The roofs of bullae formed by ex vivo ET treatment consisted of SC, SG1 cells, and TJ-forming SG2 cells, probably mimicking bulla formation in bullous impetigo.

Conclusion

Human epidermis has three SG cell layers with distinct properties just beneath the SC, of which only SG2 cells have functional TJs. Our results suggest that human epidermal TJs between SG2 cells form a paracellular diffusion barrier against soluble proteins, including immunoglobulins and bacterial toxins.

Introduction

Skin prohibits the entry of microbial pathogens and allergens, as well as the leakage of water, solutes, and nutrients. These outside-in and inside-out barrier functions are dependent on the epidermis, a stratified epithelial cellular sheet. In mammals, cornified cellular sheets called the stratum corneum (SC) constitute the outermost epidermal barrier [1]. Beneath the SC, apical paracellular spaces between the outer layer of living cells are sealed by tight junctions (TJs) in amphibians [2], reptiles [3], mice [4], [5], and humans [6], [7], [8], suggesting that TJs constitute another fundamental skin barrier system.

Early morphological studies demonstrated vertical alignment of corneocytes in rodent ear epidermis [9], [10], [11], [12]. Underneath the corneocytes, stratum granulosum (SG) cells are also aligned vertically [5], [9], [10]. The uppermost three SG layers are named (from the surface inward) SG1, SG2, and SG3 in mice [4], [5]. These cells have distinct claudin-1 expression patterns: SG1 cells do not express claudin-1, SG2 cells have TJs at their apical cell–cell contacts and express claudin-1 on their basolateral membranes but not on their apical membranes, and SG3 cells express claudin-1 over their entire surface but do not form TJs in between [4], [5]. As keratinocytes undergo continuous turnover, these observations suggest that the distinct distribution patterns of claudin-1 in SG cells are dependent on the differentiation of SG cells from SG3 to SG1. In contrast to claudin-1, occludin or zonula occludens-1 (ZO-1) proteins were shown to exclusively localize to TJs of SG2 cells [4], [5]. The en face observation of the epidermal cellular sheets of the mouse ear demonstrated that a single layer of ZO-1 honeycombs between SG2 cells demarcates the entire surface of the epidermis [5].

In contrast to rodent ear epidermis, human corneocytes and SG cells have been shown to be more disordered [13], [14]. When observed en face, occludin-positive junctions appeared to be multiplied [15]. This complexity of the human epidermis make it difficult to determine whether human epidermis has a counterpart to SG1–3 of mouse epidermis, particularly the SG1 cells that form a single viable cell layer between the SC and TJ-forming keratinocytes (SG2 cells). As we have demonstrated in mice that the SG1 layer is important for Langerhans cells to take up foreign antigens outside the TJ barrier [5], [16], we have focused much attention on whether the SG1 layer also exists outside the TJ barrier in human epidermis. Previous immunofluorescence observations of human skin sections showed that the uppermost living cells have TJs beneath the SC; no counterparts of the mouse SG1 layer existing outside the TJ barrier were described [6], [7], [15]. In electron microscopic analysis, viable nucleated cells have been occasionally observed on the apical side of cells that form a permeability barrier against lanthanum [15], [17], suggesting the existence of a counterpart to the SG1 layer. As both methods visualize a single section of the epidermis at a single time point, it is difficult to determine whether the single viable cell layer is constantly present outside the TJ barrier in human epidermis.

Here, we demonstrated that the SG1–3 cells identified in mice are well conserved in human skin, and that the TJ barrier is single-layered and exists between SG2 cells. En face three-dimensional imaging showed that a single layer of nucleated SG1 cells, partly cornified, is constantly present between the SC and TJ-forming SG2 cells in human epidermis, as in mice.

Section snippets

Study participants

Healthy skin samples from seven men and nine women patients with a mean age of 52 years (24–79 years) were obtained from leftover skin collected during excisional surgery. Sample collection was approved by the institutional review board of Keio University School of Medicine (ethics committee reference number, 20090052), and written informed consent was obtained from patients prior to surgery. This study was conducted according to the principles of the Declaration of Helsinki. The diagnosis of

Identification of SG1–3 cells in human epidermis with TJs between SG2 cells

Human corneocytes and SG cells are more disordered compared with those in rodent ear epidermis and seem to pile up on one another (Supplementary Fig. S1), as previously described [13], [14]. First, we investigated whether SG1–3 cells with distinct claudin-1 expression patterns are found in human epidermis. We stained human skin sections using anti-occludin, -claudin-1, and -desmoplakin antibodies. SG cells can be identified by their flat and disk-shaped morphology (Fig. 1A). The vertical

Discussion

The ordered, columnar alignment of SG cells and corneocytes has been reported in rodent ear epidermis [9], [10], [11], [12]. Within this ordered structure, we previously demonstrated that the outermost three SG cell layers have distinct morphological features, particularly with regard to the location of TJ-related molecules [4], [5]. In this study, we demonstrated that the three distinct types of SG cells, SG1–3 cells, are conserved in human epidermis and further characterized the distribution

Acknowledgments

We thank Motoyuki Sugai, Mikio Furuse, Yoshimi Takai, Yumi Aoyama, Showbu Sato and Minae Suzuki for helpful discussions and technical support.

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    This work was partly supported by Grants-in-Aid for Scientific Research, the “Promotion of Environmental Improvement for Independence of Young Researchers” program of the Ministry of Education, Culture, Sports, Science and Technology of Japan, Health Labour Sciences Research Grants for Research on Allergic Diseases and Immunology from the Ministry of Health, Labour and Welfare of Japan, a Keio University Grant-in-Aid for Encouragement of Young Medical Scientists, a Research Grant from the Cosmetology Research Foundation, and The Mochida Memorial Foundation for Medical and Pharmaceutical Research.

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