http://onlinelibrary.wiley.com/doi/10.1 ... 215.x/full
Background. Photoageing of skin is thought to be caused by protein denaturation, which can be induced by ultraviolet radiation. Previous studies have also reported that inflammation is related to protein denaturation; however, the influence of inflammation on skin ageing has not been explored in detail.
Aim. To investigate the possible connection between inflammation and protein denaturation, which might lead to skin ageing, we focused on halogenated tyrosine as a denatured substance produced during the inflammation process.
Methods. We measured halogenated tyrosine in aged human skin. Inflammatory cells and halogenated tyrosine were detected by immunohistochemistry using antibodies to mast-cell tryptase, neutrophilic myeloperoxidase and halogenated tyrosine. Finally, using elastic van Gieson (EVG) staining, we investigated whether the sites of halogenated tyrosine coincided with the sites at which proteins were denatured.
Results. Immunohistochemical analysis indicated that both inflammatory cells and halogenated tyrosines increased with ageing in both photoexposed and photoprotected skin. EVG staining confirmed that the localization of halogenated tyrosine was close to the sites at which protein was denatured.
Conclusions. Our investigations indicate a possible connection between skin ageing and inflammation, suggesting that halogenated tyrosine could be a useful marker of ageing skin.
At an early stage of inflammation, inflammatory cells, which contain granules filled with peroxidases such as myeloperoxidase (MPO), invade the dermis. The peroxidases released from these inflammatory cells catalyse reactions between hydrogen peroxide (H2O2) and either chloride ions (Cl−) or bromide ions (Br−), producing hypochlorous acid (HOCl) and hypobromous acid (HOBr), respectively.1,2 This is an extremely effective defence system against viral pathogens or bacterial infections; however, overproduction of HOCl or HOBr results in their reaction with amino acids in peptides and proteins, and in the production of modified tyrosines, including halogenated tyrosine, nitrotyrosine and dityrosine.3
In our previous study, modified tyrosines1,2,4 were detected in mice irradiated with ultraviolet (UV) light. These mice had the typical features of ageing, such as deep wrinkles and thickening of the dermis and epidermis.5 We also found that modified tyrosine levels were increased even in nonirradiated aged mice compared with nonirradiated young mice. In addition, the results suggested that reactions to UV-induced inflammation in skin were enhanced in ageing skin. Thus, we hypothesized that inflammatory responses might increase with age in human skin.
Concerning the relationship between inflammation and the ageing process, Chung et al.6 reported that the expression of a gene cluster associated with inflammatory reactions increases with age. Hirose et al.7 reported heightened inflammatory reactions in the blood vessels of elderly subjects. However, the influence of inflammation on skin ageing has not been explored in detail.
In this study, we investigated the possible correlation between inflammation and ageing in human skin. Although the number of inflammatory cells may be increased in aged skin, this increase could be transient. Hence, this study focused on halogenated tyrosine as a specific marker of protein denaturation caused by inflammation,8 because it is produced during the inflammation process and is presumed to accumulate in the skin.4 The presence of halogenated tyrosine in aged skin would strongly suggest that inflammation is involved in skin ageing.
Using immunohistochemistry, we investigated the levels of inflammatory cells (neutrophils and mast cells) and halogenated tyrosine in aged human skin. We also investigated, using elastic van Gieson (EVG) staining, whether the sites of halogenated tyrosine coincide with the sites at which proteins are denatured.
The study was approved by the medical ethics committee of Kyoto University, and all participants gave informed consent.
Patients and sample collection
The study enrolled eight young (mean ± SD age 24 ± 6.9 years, range 7–38) and eight aged (mean ± SD age 72 ± 6.8 years, range 61–87) subjects. Photoexposed samples were obtained during surgery from the face, forearm and dorsum of the hand, and photoprotected samples were obtained from the buttock, thigh, back, abdomen, chest and shoulder. The samples were taken from skin without inflammatory lesions.
The specimens were fixed, embedded in paraffin wax, sectioned at 6 μm, and then dewaxed for EVG staining and immunohistochemistry, using an anti-MPO Ab-1 rabbit polyclonal antibody (RB-373-A0; Laboratory Vision Corp., Fremont, CA, USA) and an anti-mast-cell tryptase antibody (sc32889; Santa Cruz Biotech, Santa Cruz, CA, USA).
Staining of halogenated tyrosine
The sections were autoclaved at 120 °C for 5 min and incubated for 18 h at 4 °C with a 1 : 50 dilution of 3A5 antihalogenated tyrosine biotinylated antibody.1 After processing using a commercial kit (PK-4002; Vectastain ABC-PO Kit; Vector Laboratories, Burlingame, CA, USA), the sections were developed with diaminobenzidine (SK-4100; DAB Substrate Kit peroxidase; Vector Laboratories) for 5 min at room temperature, and then photographed and analysed.
To quantify the photographs of halogenated tyrosine and neutrophilic MPO, unnecessary structures such as hair follicles were trimmed away, and then a skin dermis area of 0.1 mm2 was automatically level corrected and binarized (Adobe Photoshop Elements version 3.0; Adobe Systems, San Jose, CA, USA), and analysed by Image J software (http://rsb.info.nih.gov/ij/) to calculate the percentage of pixels in the stained area. Similarly, an area of skin dermis 1.6-mm2 in size in immunofluorescence images of the mast-cell tryptase was binarized, and the percentage of pixels in the stained area was calculated. The Wilcoxon rank sum test was used for analysis, and significance was set at P < 0.05.
Detection of neutrophils in human skin
Neutrophils were detected in human skin. Even though samples were taken from clinically noninflammatory lesions by skin surgery, many MPO-positive sites were seen in photoprotected elderly skin (Fig. 1b). In photoexposed elderly skin, MPO-positive sites were even more abundant (Fig. 1d). By contrast, samples of young photoprotected skin and photoexposed skin had few or no MPO-positive sites (Figs 1a,c). Image analysis of the stained area showed a tendency for the MPO-stained areas to increase in elderly skin compared with the young skin, although the levels were not significant (data not shown).
Figure 1. Inflammatory cells (neutrophils and mast cells), were detected in both young and aged human skin. (a–d) Immunohistochemical detection of neutrophils using an antineutrophilic myeloperoxidase antibody. (a,c) Young skin from (a) buttock of a 7-year-old child and (c) jaw of a 27-year-old adult. (b,d) Aged skin from (b) buttock of a 76-year-old and (d) cheek of a 73-year-old. (a,b) Photoprotected and (c,d) photoexposed specimens. (e,h) Immunohistochemical detection of mast cells using an anti-mast-cell tryptase antibody. (e,g) Young skin from (e) buttock of a 22-year-old and (g) cheek of a 19-year-old. (f,h) Aged skin from (f) back of an 87-year-old and (h) dorsum of the hand of a 74-year-old. (e,f) Photoprotected and (g,h) photoexposed specimens. (i) Image analysis using binary image processing of immunofluorescence using an anti-mast-cell tryptase antibody. The vertical axis shows the ratio of positive reaction sites per 1.6 mm2 (n = 8). **P < 0.01, Wilcoxon rank sum test.
Detection of mast cells in human skin
Mast cells, which play an important role in inflammation and induce the chemotaxis of neutrophils,9 were detected in the dermis by an anti-mast-cell tryptase antibody. More mast cells were present in elderly skin than in younger skin (Figs 1e–h). Image analysis of these photographs confirmed that there was a significant increase in mast cells in elderly skin compared with young skin (Fig. 1i).
Detection of halogenated tyrosine in human skin
Immunohistochemical detection of halogenated tyrosine, using an antibody that binds dihalogenated tyrosines, such as dichlorotyrosine and dibromotyrosine, showed that there were significantly more halogenated tyrosine-positive sites in elderly skin (Fig. 2b) than in younger skin (Fig. 2a), and there was a dramatic increase in the number of halogenated tyrosine-positive sites in photoexposed elderly skin (Fig. 2d). Halogenated tyrosine was also found to be distributed in the upper dermis, which is more susceptible to denaturing with ageing. The increase in halogenated tyrosine in elderly skin was significant (P < 0.01) (Fig. 2e), and there was also a significant difference between the halogenated tyrosine of photoexposed elderly skin and that of photoprotected elderly skin (P < 0.05). These results confirm the modification of proteins in aged skin.
Figure 2. Immunohistochemical detection of halogenated tyrosine using the 3A5 antibody in young and aged human skin. (a,c) Young skin from (a) buttock of a 22-year-old and (c) jaw of a 27-year-old. Aged skin from (b) back of an 87-year-old and (d) face of a 67-year-old. (a,b) Photoprotected and (c,d) photoexposed specimens. (e) Image analysis using binary image processing of immunofluorescence using the antihalogenated tyrosine antibody. The vertical axis shows the ratio of positive reaction sites per 0.1 mm2 (n = 8). *P < 0.05, **P < 0.01, Wilcoxon rank sum test; arrows show positive reaction sites.
Elastic van Gieson staining
To investigate whether the sites of halogenated tyrosine coincided with sites at which proteins are denatured, the skin samples were exposed to EVG staining. In photoprotected young skin (Fig. 3a), collagen fibres stained bright red and elastic fibres stained black, showing that no denaturation had occurred. Correspondingly, no positive sites of halogenated tyrosine staining were seen in young skin (Fig. 3b). In photoexposed young skin (Fig. 3c), the collagen had a similar structure, but with a slight lightening of the red staining, and the elastic fibres were stained black as before. Similarly, very few positive sites of halogenated tyrosine were seen (Fig. 3d). The photoprotected skin of the older subjects (Fig. 3e) had further lightening of the red staining, probably due to denaturation with ageing. In addition, halogenated tyrosines were mainly seen in the upper dermis, suggesting that these positive sites correspond to the denatured areas (Fig. 3h). In photoexposed older subjects (Figs 3g,h), these phenomena were exaggerated, probably due to the effects of UV irradiation.
Figure 3. Adjacent photographs show the same parts of the same tissue of the skin. (a,c,e,g) Elastic van Gieson staining; (b,d,f,h) immunohistochemical detection of halogenated tyrosine. (a,b) Photoprotected young skin from buttock of a 22-year-old; (c,d) photoexposed young skin from jaw of a 27-year-old; (e,f) photoprotected elderly skin from back of an 87-year-old; and (g,h) photoexposed elderly skin from face of a 67-year-old. Orange arrows show the denatured parts of the skin dermis, and black arrows indicate the positive sites of the antihalogenated tyrosine antibody.
Our results indicate that inflammatory cells and halogenated tyrosine, which increase with ageing not only in photoexposed skin but also in photoprotected skin, occur extensively in or near areas with denatured protein in connective tissue in skin dermis. The data indicate a possible connection between skin ageing and inflammation, suggesting that halogenated tyrosine could be a useful marker of ageing skin.
A limiting factor to the study is that the samples were obtained from surgical off-cuts, preventing direct comparison of photoexposed and photoprotected specimens from the same human subject. However, our results show that the number of inflammatory cells increases with ageing even in skin without apparent inflammation. In addition, we found that halogenated tyrosine, which is one of the indicators of protein denaturation by inflammation, exists in human skin and increases with age. These findings suggest that skin inflammation, which occurs over many years, may be involved in skin ageing.
Although halogenated tyrosines can be detected in the lungs and livers of rodents treated with lipopolysaccharide or endotoxin,1,10 the existence of halogenated tyrosine in human skin has not been previously investigated. Our earlier study showed an increase in halogenated tyrosine in mouse skin exposed to moderate doses of UV irradiation.5 Taken together with the results here, the data suggest that the levels of inflammatory cells and halogenated tyrosine increase with age, and that these levels are accelerated by stimuli such as UV exposure. Our results also show that the number of mast cells increases in photoprotected aged human skin. We hypothesize that mast cells, which increase in number with age, induce the chemotaxis of neutrophils, which consequently produce halogenated tyrosines. MPO has been suggested to be involved in various diseases11–13 and in the degradation of the extracellular matrix.14 These reports suggest that inflammatory factors may affect tissue damage and induce the dysfunction of crucial proteins. Although the reason why the number of inflammatory cells increases with ageing remains unclear, this increase could have a very important influence on skin ageing.15,16
Finally, our results suggest the possible involvement of halogenated tyrosine, produced by previous skin inflammation, in skin ageing, through denaturation of the skin tissue. However, we could not clearly show a direct influence of halogenated tyrosine on elastin aggregation or collagen denaturation. We propose that the increased halogenated tyrosine levels could be useful markers of human skin ageing, reflecting denatured proteins resulting from previous skin inflammation.
Our investigations raise the possibility of a connection between skin ageing and inflammation, suggesting that halogenated tyrosine could be a useful marker of ageing skin.
• Although previous studies have suggested a possible relationship between inflammation and the ageing process, the influence of inflammation on skin ageing has not been explored.What does this study add?
• Inflammatory cells and halogenated tyrosine increase with ageing not only in photoexposed but also in photoprotected human skin, appearing extensively in or near areas with denatured protein.• Thus, halogenated tyrosine could serve as a marker of inflammatory protein damage.
We thank Dr T. J. Lin, Dr L. Anthony and Ms C. Goh for stimulating discussions and helpful advice.
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