Chronic Photodamage in Skin of Mast Cell-deficient Mice

Photochemistry and Photobiology, 1999, 70(2): 246-253

Chronic Photodamage in Skin of Mast Cell-deficient Mice*

Salvador Gonzalez, Michael Moran and Irene E. Kochevar* Wellman Laboratories of Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

Received 25 January 1999; accepted 19 May 1999

ABSTRACT Solar elastosis is a hallmark of photoaged human skin and a prominent feature in experimentally produced photoaging in murine skin. The products of mast cells have been implicated in the development of photoaged skin. We evaluated whether products from mast cells me- diate chronic UVB-induced changes in murine skin by employing a strain of mast cell-deficient mice, WWv. The responses in these mice were compared to those in BALB/c, another albino mouse strain. Mice were exposed three times per week to UVB radiation for 11 weeks; the total dose was 18.8 J/cm2. Irradiated WWv mice showed greater epidermal alterations than the irradiated BALB/ c mice. In the dermis, a 3.6-fold increase in elastin content, as measured by desmosine, was produced in the UVB-treated BALBlc mice; in contrast, no difference was observed in elastin between UVB-treated and untreated WWv mice. Collagen content was not increased by UVB treatment in either strain, and the glycosaminoglycan content increased a similar amount in UVB-treated mice in both strains. The number of mast cells increased two- fold and the number of neutrophils increased six-fold in UVB-treated BALB/c mice compared to age-matched unirradiated controls. Neutrophils, as well as mast cells, were absent in untreated and UVB-treated WWv mouse skin. These results suggest that products of mast cells are important in the development of solar elastosis in murine skin either by directly inducing elastin production by li- broblasts or indirectly by mediating the presence of other cell types that produce products that increase fibroblast elastin production.

INTRODUCTION Chronic exposure of human skin to solar UV radiation causes marked morphological, structural and biochemical changes that are collectively termed photoaging. The outward mani- festations of photoaging include fine and coarse wrinkles, mottled coloration and skin laxness. Major alterations are

*This paper is dedicated to the memory of Dr. Raymond Latarjet

*Author to whom correspondence should be addressed at: Wellman Laboratories of Photomedicine. Massachusetts General Hospital WEL-224, 37 Fruit Street, Boston, MA 02114, USA. Fax: 617- 726-3 192; e-mail: kochevar@ helix.mgh.harvard.edu

(191 1-1998).

8 1999 American Society for Photobiology 0031-8655/99 $5.00+0.00

apparent in the dermis of chronically sun-exposed skin and include increased elastin content with an amorphous appearance (solar elastosis), increased glycosaminoglycan (GAG)t content and altered collagen content. Similar responses are produced in an experimental animal model for photoaging, the chronically UVB-treated Skh-hairless- 1 mouse (1-3).

Alterations in the dermal extracellular matrix caused by years of exposure to UV radiation appear to involve both altered production of matrix macromolecules by dermal fibroblasts and increased activity of matrix-degrading en- zymes by a variety of cells (4-6). These changes have been suggested to result from a chronic inflammatory condition and inflammatory cells are present in the dermis of sun- damaged skin (7). Experiments utilizing the Skh hairless mouse model indicate that inflammation is part of the process leading to photoaging because anti-inflammatory agents inhibit chronic UVB-induced changes in mouse skin (8,9). The sources of these mediators include resident skin cells (e.g. keratinocytes, fibroblasts, mast cells, endothelial cells) and infiltrating inflammatory cells (neutrophils, macrophag- es). For example, cytokines produced by keratinocytes in response to UVB treatment that may be involved in chronic photodamage include interleukin (IL)-1. IL-6, IL-8, IL- 10, tumor necrosis factor (TNF)-a, and granulocyte-macrophage colony-stimulating factor (10-12).

Mast cells have been implicated in the development of photoaged skin. Mast cells are capable of synthesizing and releasing several mediators capable of modulating, directly or indirectly, extracellular matrix production and degradation. These mediators include TNF-a, transforming growth factor-p and prostaglandin (PG)D,. Mast cells also release proteases that degrade the extracellular matrix or activate the proenzyme form of metalloproteases. In photodamaged human skin, mast cells are present in higher numbers than in nonphotoaged skin (10) and were reported to be apposed frequently to activated fibroblasts, suggesting a functional interaction (7). The histamine increase appearing within 30 min after exposure to UVB radiation suggests degranulation of mast cells ( l l ) , although keratinocytes may also be a source of UVB-induced histamine (12). Degranulation of mast cells and release of TNF-a after a single dose of UVB radiation to human skin supports a role for products of mast cells in chronic photodamage (13). Multiple treatments of

tAbbreviurions: GAG, glycosaminoglycans; 1L. interleukin; PG, prostaglandin; TNF, tumor necrosis factor.

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Photochemistry and Photobiology, 1999, 70(2) 249

hairless mouse skin with UVB produces two- to seven-fold increases in mast cell numbers depending on the irradiation protocol and strain of mouse (9.14-17). The reported increase in mast cell growth factor expression in the skin of chronically UVB-treated Skh mice provides a molecular stimulus for the increase in mast cell numbers (16).

Our approach to evaluating whether the products of mast cells may contribute to responses in murine skin to chronic UVB treatments was to employ mice that are known to be deficient in mast cells. A strain of mice was used, WW', that has <0.5% of the normal number of cutaneous mast cells (18). Mast cell development is inhibited in these mice because the cells that normally give rise to mast cells possess mutant c-kit receptors; these receptors bind mast cell factor (stem cell factor, Steele factor) that is required for mast cell development. The lack of mast cells in these mice results in less ear swelling and lower PGD2 levels after a single UVB treatment compared to the response in other albino mice (19). In this study, chronic UVB-induced dermal and epidermal alterations in the WW' mice were measured. The wild-type littermates of the WW' mice are pigmented because normal c-kit receptors are also required for melani- zation; thus, they could not serve as controls in our experiments. Instead, the changes produced in ww' mice were compared to those produced in BALBlc mice, another albino strain that we have previously employed in a photoaging study (15).

MATERIALS AND METHODS Animals. Mast ce I I -defic ien t mice ( WB B6F, /J - Kit W/Kit w-'', referred to as WW') and BALB/c mice were obtained from the Jackson Lab- oratory (Bar Harbor, ME). Ultraviolet-B treatments were begun when the mice were 7-8 weeks old. Mice were housed in groups of four. Animals were distributed into four groups: WW' treated with UVB, WW' not exposed to UVB, BALBk treated with UVB and BALBk not exposed to UVB. Each group was identified with ear tags. Prior to UVB treatment, hair on the dorsal surface of all mice, including controls, was clipped using an Oster electric clipper A5 with a no. 40 (1-10 mm) blade. In the chronic study, this procedure was performed weekly during the first month and every 2 weeks thereafter.

Materials. Desmosine antiserum and Bolton Hunter-labeled desmosine were obtained from Elastin Products Co. (Owensville, MO). Goat anti-rabbit antiserum was from Organon Teknika Corp. (West Chester, PA). Glucuronic acid lactone, papain, sodium acetate, cys- teine, Bis-Tris buffer, polyethylene glycol, chloramine T and 4-hydroxyproline were obtained from Sigma (St. Louis. MO). Sodium tet- raborate and perchloric acid were from Fisher (Pittsburgh, PA) and 4- dimethylaminobenzaldehyde was from Aldrich (Milwaukee, WI).

Irradiation protocols. Mice were irradiated under a bank of 12 UVB-Ho-90" fluorescent tubes (Elder Pharmaceuticals Inc.) equipped with a Kodacel TA 401/407 filter (Eastman Kodak, Roch- ester, NY) to remove UVC radiation emitted by the lamp. The height of the lamps was adjusted to provide 0.5 mW/cm2 at the dorsal surface of the mice. The irradiance for each treatment was measured with an IL-1700 radiometer equipped with an SED 240 UVB detec- tor (International Light Inc.). The spectrum of the bulbs was measured periodically with a spectroradiometer (model 742, Optronix Laboratories Inc.). The spectral output was maximal at 3 14 nm with the majority of the energy emitted below 320 nm.

For the acute treatments, single doses of 25, 50 and 75 mJ/cm2 were administered. A total of 13 BALB/c and 14 WW' mice were used in groups of four or five mice per group. Contralateral skin on the dorsal surface that was covered with a thick pad and aluminum foil served as control skin on each mouse.

For the chronic treatment protocol, each group consisted of eight mice, except the UVB-treated BALB/c mouse group that only had

seven animals. Mice were irradiated on alternate days, three times a week, for I 1 weeks. The initial two treatments were 100 mJlcm2 each and the fluence deliveredltreatment was gradually increased as the mouse skin became more tolprant to the treatment. The daily fluence (mJ/cm2) and number of treatments at that fluence were: 100 (2), 200 (3). 400 (9, 600 (lo), 800 ( 5 ) and lo00 (6). The total cumulative fluence was 18.8 J/cm2. Mice were sacrificed 6 days after the last irradiation.

Histology. Paraffin sections (4 pm) of biopsies were stained with hematoxylin and eosin for evaluation of the epidermis, the cellular constituents of the dermis and the inflammatory infiltrate. Resorcin fuchsin-stained sections were utilized for evaluation of elastic fibers and mast cells. Colloidal iron stain was used to evaluate GAG content and distribution. Mast cells were counted using a 40X objective lens and an eyepiece micrometer with a 10 X 10 square grid (0.25 mm/side). The micrometer was aligned with one edge along the dermal/epidermal junction that allowed the mast cells within the papillary and midreticular dermis to be counted. Ten fields were counted per section and three sections per group were counted. Neu- trophils were counted in a similar manner on 5 pm frozen sections fixed in acetone and stained with benzidine dihydrochloride (0.3% in 50% ethanol with I % sodium acetate, 2.1 % Hz02, pH 6.0). Saf- ranin 0 was included in the primary stain and the counterstain was 1% methyl green. Sunburn cells were counted in a similar manner on hematoxylin- and eosin-stained sections; in the acute study, four sections from each animal in each group were analyzed.

Skin-fold thickness. Measurements of skin-fold thickness were made by pulling up the skin along an imaginary line from axilla to hip and using a spring-loaded micrometer to determine the thickness.

Vascular permeabiliry. Vascular permeability was quantitated by the leakage from vessels of albumin-bound Evans blue using the method previously described (9).

Biochemical assays. The procedures for preparation of skin samples and assessing skin content of collagen, elastin and GAG are identical to those described previously (9). Briefly, skin punch biopsies were hydrolyzed in 6 N HCI prior to analysis of collagen by a spectrophotometric hydroxyproline assay (20) and of elastin by radioimmunoassay for desmosine content (21). Biopsies for GAG evaluation were digested with papain before measuring uronic acid content by a spectrophotometric assay (22). All measurements were expressed per area of skin as described previously (9).

Statistics. Results are expressed as the mean t SD. The two- tailed paired Student's t-test was used to compare the intra-animal skin responses to UVB radiation and the two-tailed unpaired Stu- dent's t-test was used to compare the quantitative parameters of the chronic study.

RESULTS Responses to a single UVB treatment

The responses to a single UVB treatment were evaluated in WW' and BALBlc mice prior to initiation of the chronic protocol to determine whether the responses in these strains were similar. Groups of mice (four or five per group) were exposed to 25, 50 and 75 mJ/crn2 and measurements were made after 24 h; an unirradiated dorsal skin site was used at the control in each animal. No alteration in the skin-fold thickness was detected in any group after UVB treatment. Vascular permeability increased with UVB fluence in both strains. In WV mice, the increase became statistically significant only at 75 mJ/cm2, whereas in the BALBlc mice, the increase was significant at both 50 and 75 mJ/cm2 (Fig. IA). The number of sunburn cells per unit length of epidermis did not increase except in the BALBlc mice at the high- est dose (Fig. 1B).

Responses to chronic UVB treatments

During the irradiation protocol, two of the UVB-treated WW' mice appeared to be less active and to lose weight.

250 Salvador Gonzalez et a/.

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Figure 1. Cutaneous responses of WWv and BALBlc mice to a single UVB treatment. Measurements were made 24 h after exposure to varying fluences of UVB radiation. A. Vascular permeability evaluated by Evans blue in tissue detected at 620 nm (a, P = 0.01 vs unirradiated site; b, P = 0.007 vs unirradiated site; c, P = 0.001 vs unirradiated site). B. Sunburn cells were counted according to morphologic criteria from hematoxylin- and eosin-stained skin sections (a, P = 0.017 vs BALBlc mice receiving 75 ml/cm2). Error bars show SD.

One mouse died in week 6 after a cumulative dose of 7 J/ cm2 and the other in week 11, at the end of the protocol (skin from this mouse was not used in the analyses at the end of the protocol). The animals in all of the other groups remained healthy.

Morphology. The WWv mice but not the BALB/c mice showed strong responses to UVB treatment. Fxlema and scal- iness were clearly evident in week 4. Crusted lesions were evident on the ear skin of 3 of 8 mice at week 6 and on 5 of 7 mice at week 10. Tumors appearing as solid keratotic papules developed on the upper back and reached a size of 1-2 mm. They were present in 2 of 7 mice by week 9, in all mice by week 10 and lasted until the end of the protocol. The skin-fold thickness was measured after week 4 and at the end of the protocol. After 4 weeks, UVB-treated WWv mice showed a significant increase over unirradiated controls, but no difference was seen between the groups of BALB/c mice (Fig. 2A). After week 11, the difference between UVB-treated and nontreated WW' mice was nonsig- nificant (data not shown).

Hisrology. Unirradiated, age-matched controls of both strains showed similar histologic features; the epidermis typ- ically consisted of two cell layers. Irradiated WWv mice

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Figure 2. Effects of chronic UVB treatments on thickness of WW' and BALB/c mouse skin. Mice were treated three times per week for I1 weeks with UVB radiation as described in the Materials and Methods. A. Skin-fold thickness measured after 4 weeks of irradiation (a, P = 0.046 vs unirradiated WW"). B. Epidermal thickness (after 11 weeks of UVB) measured on hematoxylin- and eosin- stained sections (a, P = 0.007 vs unirradiated WW'; b, P = 0.013 vs unirradiated BALB/c). C. Dermal thickness (after 11 weeks of UVB) measured on hematoxylin- and eosin-stained sections (a, P = 0.039 vs unirradiated WW"). Error bars show SD.

showed greater changes in the epidermis than BALB/c mice. In two of six WW' mice, the epidermis was hyperplastic with seven or eight cell layers. Measurements of the epidermal thickness showed significant increases in both mouse strains, but the percent increase for the WWv mice was greater (273% vs 129%; Fig. 2B). Vacuolar degeneration of the basal layer was found in four of six irradiated WWv mice. All irradiated WWv mice showed mild pleiomorphic epider-


Mast cells Neutrophils

Figure 3. Effects of chronic UVB treatments on number of mast cells and neutrophils in BALBk mouse skin. Mice were treated three times per week for 11 weeks with UVB radiation as described in the Materials and Methods. Mast cells counted on resorcinol fuchsin- stained sections (a, P = 0.01 vs unirradiated mice). Neutrophils counted after staining for myeloperoxidase (b, P = O.OOO1 vs unirradiated mice). Error bars show SD.

mis focally infiltrated with lymphocytes; half showed intense vasodilation. Sunburn cells were scant; however, they were seen in skin samples from five of six of WWv mice but in only one of six from BALBk mice. The dermal thickness increased to about the same extent (20-25%) in both strains (Fig. 2C).

As expected, hypertrophy of all structures in the upper dermis, including sebaceous glands was seen. Mast cells were only present in the skin of BALBk mice. The number of mast cells in BALBk skin at the end of the chronic treatment protocol (18.8 ? 7.4 per mm) was significantly greater than the number in untreated controls (8.3 2 1.4 per mm; Fig. 3). Similarly, neutrophils were only present in the skin of the BALBk mice and the number in the UVB-treated skin (23.8 ? 5.3 per mm) was significantly greater than in the untreated controls (4.3 ? 1.7 per mm; Fig. 3).

Glycosaminoglycan staining increased after irradiation in both strains of mice. Elastic fiber hyperplasia was pro- nounced in chronically UVB-irradiated BALB/c mice show- ing foci of clear elastosis with tangled, thickened elastic fibers. Irradiated WWv mice showed fine elastic fibers without a noticeable increase compared to unirradiated WWv mice.

Macromolecule content. Treatment of BALBk mice with UVB for 11 weeks produced a 3.6-fold increase in elastin content, as measured by desmosine, compared to unirradiated controls. In contrast, no difference in elastin was observed between UVB-treated and untreated WWv mice (Fig. 4A). The UVB treatment did not alter the collagen content of skin of either mouse strain compared to age-matched controls, as measured by hydroxyproline (Fig. 4B). The GAG content of the skin in both mouse strains increased in the UVB-treated groups (Fig. 4C) and the percent increase was similar for both strains (41 and 57%).

DISCUSSION Solar elastosis is a hallmark of photoaged human skin and is a prominent feature in experimentally produced photoaging in murine skin. Development of solar elastosis may involve both increased production and degradation of elastic fibers (4-6,23). In both human and mouse skin, photoaging is associated with an increase in the number of mast cells

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Figure 4. Effects of chronic UVB treatments on dermal macromolecules in WW’ and BALB/c mouse skin. Mice were treated three times per week for 11 weeks with UVB radiation as described in the Materials and Methods. A. Desmosine measured to indicate elastin content (a, P < 0.001 vs unirradiated BALBk). B. Hydroxypro- line measured to indicate collagen content. C. Uronic acid measured to indicate glycosaminoglycan content (a, P = 0.012 vs unirradiated WW”; b, P < O.OOO1 vs unirradiated BALBk). Error bars show SD.

(7,9,15-17). The results of this study indicate that mast cell products contribute, directly or indirectly, to the increase in elastin content observed in murine skin exposed to UVB radiation over an extended period. As shown in Fig. 4A, chronic UVB treatments over 11 weeks did not increase the dermal elastin content in the mast cell-deficient mice. Chron- ic UVB treatment increased elastin content in several studies using Skh hairless mice (9,15,24,25) as well as in studies with C3WHeN mice. Our result with BALB/c mice (3.6- fold increase in elastin) is very similar to the result obtained

252 Salvador Gonzalez et a/.

in our earlier study (3.0-fold increase) using this same strain (15). Thus, the absence of a UVB-induced increase in elastin content in exposed skin of ww' mice appears to be an ex- ception to the result found in all previously examined murine strains and strongly suggests that products of mast cells are needed for the development of solar elastosis.

The lack of increased elastin content in UVB-exposed skin of WWv mice cannot be attributed to lower UVB penetration into the skin of these mice. A single UVB exposure produced similar increases in vascular permeability and sun- bum cells in both strains (Fig. l), suggesting that responses to UVB do not differ greatly between these two strains. In addition, our observation that the GAG content and dermal thickness increased to similar extents in WB-treated WWv and BALB/c mice supports similar UVB penetration into the skin of both strains.

As mentioned previously, the wild-type littermates of the WW' mice could not be used as controls because they are pigmented. Instead, BALB/c mice were chosen as a com- parable haired albino strain. Other studies employing WW' mice to investigate the impact of mast cells on tissue pathophysiology have used WWv mice reconstituted with mast cells as controls (18). This approach was not used because during chronic W B treatments mast cell progenitors mi- grate into the skin and mature into mast cells (16) and a source of normal mast cell progenitors would not be present. In addition, it was not apparent that mast cells injected into the skin would distribute throughout the area to be irradiated. Because congenic +/+ mice could not be used as controls, the possibility exists that some difference between the WW' and BALB/c strains, other than the absence or presence of mast cells, may account for appearance of solar elastosis only in the latter strain.

Chronic UVB-induced epidermal alterations were greater in the WWv strain than in the BALB/c mice, suggesting that UVB-induced mediators from the epidermis of the WW' mice might be involved in down-regulation of elastin production. However, TNF-a is the only UVB-induced mediator from keratinocytes that has been reported to down-regulate fibroblast production of elastin (26). Because mast cells are the major source of this cytokine in skin, their presence or absence should dominate any TNF-a-mediated effect. The epidermis of UVB-treated WW' mice, but not that of BALB/ c mice, showed prominent hyperplasia scaling and tumors. This response was unexpected because neutrophil infiltration after UVB exposure has been demonstrated to play a role in epidermal damage, presumably by production of reactive oxygen species (27) and the UVB-treated ww' mice lacked neutrophils as well as mast cells.

Our results do not elucidate the mechanism for mast cell involvement in UVB-induced elastin accumulation. Mast cells are the major source of preformed TNF-a in skin and release this cytokine in human skin after UVB exposure (13). In vitro TNF-a has been shown to down-regulate tropoelastin mRNA by skin fibroblasts (26) but to up-regulate tropoelastin mRNA from UVB-treated fibroblasts (28). The latter process may contribute to the development of solar elastosis in the BALB/c but not the WW' mice. The TNF-a may also act indirectly to increase elastin by stimulating P- or E-selectin expression on endothelial cells (1 3.29). Both P-selectin and E-selectin are required for neutrophil migra-

tion into skin (29). Neutrophil elastase has been implicated in UVB-induced elastin accumulation because mice that are deficient in this enzyme (beige C57BW6J-bghg) do not show solar elastosis after chronic UVB exposure (23). How- ever, neutrophils also produce other potential mediators such as reactive oxygen species that have been reported to en- hance tropoelastin mRNA levels in cultured human skin fibroblasts (30). Because the number of neutrophils did not increase in the skin of chronically UVB-treated WWv mice but did increase after similar treatment of BALBk and other mouse strains (15), some function of these cells may cause the induction of elastin synthesis by fibroblasts. Further study is necessary to identify the products of mast cells and/ or neutrophils that induce an increase of elastin content during photoaging.

If these results can be extrapolated to human skin, they suggest that mast cells may play an important role in solar elastosis. Previous studies have suggested a role for mast cells in human photoaging based on the observed close as- sociation of mast cells and activated fibroblasts (7.10). In addition, the number of mast cells has been reported to be greater in human photoaged skin than in non-sun-exposed skin (7).

Acknowledgemenr-Support of this work by NIH grant ROI AR43895 is gratefully acknowledged.

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Chronic Photodamage in Skin of Mast Cell-deficient Mice