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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:B411-B415 (2004)
© 2004 The Gerontological Society of America

Aging of Human Epidermis: Reversal of Aging Changes Correlates With Reversal of Keratinocyte Fas Expression and Apoptosis

Amos Gilhar1,, Yehuda Ullmann1, Rachel Karry2, Raya Shalaginov1, Bedia Assy1, Sima Serafimovich1 and Richard S. Kalish3,

1 Skin Research Laboratory, The B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, and Flieman Medical Center, Haifa, Israel.
2 Laboratory of Psychology, Department of Psychiatry, Rambam Medical Center and Rappaport Faculty of Medicine, Technion, Israel.
3 Department of Dermatology, State University of NewYork at Stony Brook.

Additional correspondence to Dr. Amos Gilhar, Laboratory for Skin Research, Rappaport Building, Technion Faculty of Medicine, POB 9649, Bat-Galim, Haifa, 31096, Israel. E-mail: gilhar{at}techunix.technion.ac.il

Address correspondence to Dr. Richard S. Kalish, Department of Dermatology, Health Sciences Center T-16, 060, SUNY at Stony Brook, Stony Brook, NY 11794-8165. E-mail: richard.kalish{at}stonybrook.edu


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
The goal of this study was to determine the role of Fas-mediated apoptosis in human epidermal aging. Epidermal Fas expression and apoptosis are increased in aged human skin. Aging changes of human epidermis, including decreased epidermal thickness and proliferation, are reversed following grafting of human skin to SCID (severe combined immunodeficiency) mice. Skin from aged participants (n = 14; mean 70.7 years), and young participants (n = 14; mean 23.4 years) was grafted to beige SCID mice, and epidermal thickness, proliferation (Ki-67 expression), apoptosis (TUNEL [Tdt-mediated dUTP nick end labeling] reaction below granular layer), and expression of Fas and FasL were determined by histology and immunochemical staining. Aged skin was associated with thinning of the epidermis, decreased epidermal proliferation, a significant increase in apoptosis below the granular layer, and epidermal Fas expression. Engraftment significantly reversed these aging changes, including apoptosis, and Fas expression. Correlation of reversal of aging changes, with decreased epidermal Fas expression and apoptosis, supports a role for Fas-mediated apoptosis in aging of human epidermis.

AGED human skin exhibits decreased epidermal thickness, flattening of the dermal–epidermal junction, and decreased keratinocyte proliferation (1–5). Apoptosis of keratinocytes below the granular layer is also present in aged skin, along with keratinocyte expression of Fas (6,7). We proposed that the age-related decrease in epidermal thickness was mediated in part by apoptosis induced by Fas, along with decreased proliferation.

Epidermal thickness is regulated by proliferation and terminal differentiation. Keratinocyte terminal differentiation differs from apoptosis but has many features in common, such as activation of endonucleases and degradation of DNA, resulting in cells positive for the TUNEL (Tdt-mediated dUTP nick end labeling) reaction (8,9). In normal epidermis, terminal differentiation occurs at the granular layer. TUNEL-positive cells below the granular layer represent apoptosis (10–12), and are found in aging skin, in association with expression of Fas (6). Fas (CD95) belongs to the tumor necrosis factor receptor family (13,14) and plays a role in inducing apoptosis (13–15).

Grafting of aged human epidermis to immunodeficient mice results in a reversal of aging changes, including decreased proliferation index and reappearance of the convoluted dermal/epidermal junction (16–18). The aim of the present study was to test the hypothesis that Fas-mediated apoptosis has a role in age-related thinning of human epidermis. Aged skin was grafted to SCID (severe combined immunodeficiency) mice, resulting in reversal of epidermal aging changes. Increase in epidermal thickness correlated with reduction of keratinocyte Fas expression, as well as apoptosis, demonstrating that reversal of Fas-mediated apoptosis correlated with reversal of aging changes.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Human Skin Grafts
Twenty-eight participants were included in this study. These participants were included in a previous observational study that did not include skin grafting (6). All procedures were approved by the local ethics committee. Older persons (n = 14) ranged from 59 to 82 years, mean age 70.7 years ± 8.2 years. Younger donors (n = 14) ranged from 18 to 32 years, mean age 23.4 years ± 6.3 years. All donors underwent surgical procedures in the Plastic Surgery Department of the Rambam Medical Center. Remnants of normal skin were obtained from sun-protected unexposed areas of the thighs. A portion of the donor skin of each volunteer (1.2–1.5 cm in diameter) was grafted onto a "beige-SCID" mouse. The remaining skin was submitted for routine histological examination with hematoxylin eosin and immunohistochemical staining for Fas, FasL, and Ki-67. One month following transplantation, the grafts were removed from the mice and divided into portions for routine histological examination and immunochemical staining for Fas expression. The remaining portion was snap-frozen in liquid nitrogen for TUNEL staining. Comparison of values before and after engraftment was performed.

Animals
C.B-17/IcrHsd-scid-bg (beige-SCID) mice (Harlan Laboratories Ltd., Jerusalem, Israel), 2–3 months of age, were used in this study. The mice were raised in the pathogen-free animal facility of the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology. Animal care and research protocols were in accordance with institutional guidelines and approved by the institutional committee on animal use.

Skin Grafting
Graft transplantation to mice was performed as previously described (19). Split-thickness skin grafts of 0.4 mm were obtained by a Padgett Air Dermatome (Padgett Instruments, Inc., Kansas City, MO) and were transplanted onto subcutaneous tissue of a mouse's skin over the lateral thoracic cage. Graft sites were covered with petroleum impregnated gauze and covered with a standard adhesive bandage that was surgically stapled to the ventral surface of the animal.

Proliferative Index
Proliferative index (6) of epidermis was determined by percent staining of keratinocytes with monoclonal antibody to Ki-67 (Zymed Laboratories, San Francisco, CA). Immunohistochemistry was performed on OCT (Tissue Tek, Torrance, CA)-embedded specimens with a biotin-avidin system (Vectostain; Vector Laboratories, Burlingame, CA).

TUNEL Assay
Apoptotic cells below the granular layer were assayed by using the TUNEL method (20,21) as previously described (6). Frozen sections were fixed with paraformaldehyde, permeabilized, and the TUNEL reaction was performed with modified fluorescein nucleotides and a TdT (in situ cell death detection kit; Boehringer Mannheim, Indianapolis, IN). Antifluorescein antibody conjugated with horse radish peroxidase was then added to the sections, followed by substrate (aminoethyl carbazole). Slides were rinsed, counter-stained, and analyzed under a light microscope. TUNEL index (TI) (22) was determined by the formula: TUNEL positive cells x 100 per total 1000 cells. Positive control sections included in each assay were DNase treated.

Fas Immunostaining (6,23)
Immunochemistry was performed as previously described using monoclonal anti-Fas (Apo-1; Transduction Laboratories), followed by biotinylated horse antimouse IgG (Elite ABC kit; Vectastain, Vector Laboratories), and peroxidase avidin biotin mixture (Vectastain, ABC kit). Finally, the sections were stained with aminoethyl carbazole plus peroxide. Sections were counter-stained, mounted, and analyzed under light microscope. Fas index (FI) was determined by the formula: Fas positive cells x 100 per total 1000 cells.

Morphometric Analysis
Histologic assessment was performed by routine light microscope of the histological examination (6,16,18). Measurement of the epidermal thickness was carried out in the vertical plane with an ocular micrometer. Observations were taken at a minimum of 20 points along the epidermis.

Statistical Analysis
Statistical analysis was carried out using the analysis of variance (ANOVA) test for all multiple comparisons, and unpaired t test for single comparisons.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Proliferation Index
Proliferation index was determined by percent Ki-67 staining of epidermis (Figures 1 and 2). Prior to grafting, the proliferation index of young epidermis (23.5% ± 5.6%) was approximately twice that of older epidermis (12.3% ± 5.0%) (p <.05 by ANOVA). Following grafting, there was an increase in proliferation index for both (p <.001 by ANOVA), such that they became equivalent (30.0% young vs 34.0% older).



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  		Figure 1. Differences in epidermis of young (n = 14) versus aged (n = 14) skin before and after grafting to beige-SCID (severe combined immunodeficiency disorders) mice. A, Proliferation index as percentage of cells staining for Ki-67; B, epidermal thickness in microns; C, TUNEL (Tdt-mediated dUTP nick end labeling) index; and D, Fas index. The following differences are significant by analysis of variance for all four graphs: young pregraft versus old pregraft (p <.05); old pregraft versus old postgraft (p <.001)

 


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  		Figure 2. Immunohistochemistry of young and old skin, both pregraft and postgraft, stained for Ki-67, TUNEL (Tdt-mediated dUTP nick end labeling), and Fas

 
Morphometric Analysis
A significant difference (p <.001 by ANOVA) was found between the mean epidermal thickness of young skin (25.2 µm ± 5.73) versus aged skin (16.95 µm ± 4.1) prior to grafting (Figures 1 and 2). Grafting of skin onto beige-SCID mice led to an increase of 80% in the epidermal thickness of young skin (ANOVA, p <.001). Aged epidermis exhibited a 165% increase in thickness following grafting (ANOVA, p <.001), so that the thickness of young and old epidermis was equivalent (45.58 µm vs 44.97 µm).

TUNEL Index (TI)
Most of the TUNEL-positive cells of the young epidermis were located within the granular layer reflecting normal differentiation. However, in the aged epidermis, TUNEL-positive cells were detected not only within the granular layer but also below the granular layer, representing apoptosis (Figure 2). The TI of aged epidermis (2.99% ± 1.66%) was significantly greater (p <.001) than that of young epidermis (1.30% ± 0.85%) (Figure 1). Following engraftment to SCID mice, there was a dramatic change in the pattern and extent of keratinocyte apoptosis. In particular, the features of aged epidermis became closer to younger epidermis with a significant (p <.001) drop in the mean TI, both in the whole epidermis and below the granular layer. There was a smaller drop in the TI of young epidermis following engraftment, so that it was similar to that of grafted aged epidermis (0.53% young vs 0.75% old).

Fas and FasL Expression by Epidermis
Aged epidermis showed a significantly greater (p <.001 by ANOVA) Fas index than young epidermis (4.48 young vs 1.45 old) (Figure 1). Following grafting, there was a significant decrease in the Fas index of old skin (p <.001 by ANOVA), and the Fas index of old skin normalized to approximate that of young epidermis following grafting (0.99 old vs 0.73 young). In all groups, Fas antigen was not located within the granular layer but below the granular layer.

In contrast, epidermal FasL expression was increased in aged versus young epidermis (2.55 young vs 4.3 old: p <.02 by t test), but did not change significantly with grafting when determined by immunohistochemistry (2.84 young vs 4.18 old).


   DISCUSSION
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 Abstract
 Methods
 Results
 Discussion
References
 
Aging changes of human epidermis include decreased epidermal thickness, and proliferation, along with flattening of the rete ridge pattern. We have previously shown that aging of human epidermis is associated with expression of Fas and apoptosis of keratinocytes below the granular layer (6). Reversal of aging changes following grafting was associated with a decrease in both apoptosis below the granular layer and Fas expression, suggesting that Fas-induced apoptosis may have a role in the increased apoptosis associated with epidermal aging.

Fas-mediated apoptosis of keratinocytes has multiple functions (23–25), and has been implicated in such varied conditions as allergic contact dermatitis (26), lupus erythematosus (27), toxic epidermal necrolysis (28), and sunburn cell formation to ultraviolet light (29). The expression of FasL was also elevated in aged epidermis, but did not normalize along with grafting, suggesting that age-related keratinocyte apoptosis is regulated by cell surface expression of Fas.

Aged human epidermis exhibits many changes including decreased thickness, proliferation, and flattening of rete ridges. Apoptosis of keratinocytes below the granular layer was observed, along with keratinocyte expression of Fas. These changes, including the increased apoptosis, and Fas expression, were reversed by grafting to beige-SCID mice. This supports the proposal that Fas-mediated apoptosis has a role in age-related changes of the epidermis.


   Acknowledgments
 
We wish to thank Dorit Ben-Shachar, Head, Laboratory of Psychology, Department of Psychiatry, Rambam Medical Center and Rappaport Faculty of Medicine, Technion, for her valuable assistance.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received November 11, 2003

Accepted February 12, 2004


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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