Department of Dermatology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106
Correspondence: Santosh K. Katiyar, Ph.D., Department of Dermatology, The University of Alabama at Birmingham, 1530 3rd Ave. South, Birmingham, AL 35294.. E-mail: skatiyar{at}uab.edu
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Key Words: Key words: • Green tea • (-)-epigallocatechin-3-gallate (EGCG) • ultraviolet radiation • immune suppression • oxidative stress • antigen-presenting cell
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Solar ultraviolet (UV) radiation, particularly in the UV-B (290–320 nm) spectrum, is the primary cause of the vast majority of cutaneous malignancies each year in the United States [8 ]. Experimental studies have shown that solar UV radiation, particularly UV-B, has suppressive effects on the immune system [9 ], and exposure of UV-B to skin induces generation of reactive oxygen species (ROS), which can act as tumor initiators [10 ] as well as tumor promoters [11 ] by damaging critical cellular macromolecules such as DNA, proteins, and lipids. UV-B-induced immune suppression is also considered as a risk factor for skin cancer development [12 13 ]. Exposure of UV-B radiation causes a number of changes within the skin, which have been linked to immunosuppression; for example, UV-B irradiation reduces the density of epidermal Langerhans cells (LCs) [14 15 16 ] either by induced migration 17, 18] or by cell death [19 ], and UV-B exposure can cause infiltration of macrophages and granulocytes into the epidermis [20 ]. Furthermore, LCs and macrophages from UV-irradiated epidermis can induce hapten-specific immunosuppression and tolerance [21 22 ], suggesting that UV radiation causes functional alterations in these cells. Earlier, it was also shown that blocking of UV-induced infiltrating leukocytes using anti-CD11b antibody (specific for activated monocytes/macrophages) [23 ] or murine-specific soluble complement receptor type 1 [24 ] reversed UV-induced immunosuppression and tolerance induction. Studies have also shown that the growth of UV-induced skin tumors in mice is stimulated by the production of paracrine growth factors from infiltrating leukocytes [25 26 27 ]. These observations strongly suggest an important role of UV-B-induced infiltrating leukocytes in immunosuppression and cancer induction.
Regular intake of natural antioxidants has been suggested as a useful preventive strategy against the mutagenic and carcinogenic effects of solar UV radiation. Consistent with the idea of cancer chemoprevention, in earlier studies, we and others have shown the preventive effects of polyphenols isolated from green tea against UV radiation-induced skin inflammation, immunosuppression, and tumorigenesis in mouse models [reviewed in ref. 7 28 ]. Based on several in vitro and in vivo studies, it has become clear that EGCG is the major and most effective chemopreventive polyphenolic compound present in green tea, which is also a most potent antioxidant among many other naturally occurring antioxidants [7 28 ]. We have also shown that topical application of a green tea polyphenolic fraction (GTP) or EGCG before a single exposure of mouse skin to UV-B affords protection against UV-B-induced immunosuppression [29 30 ]. Because of the high antioxidant activity of polyphenols present in green tea, in recent years green tea extract increasingly has been used in skin care products [reviewed in ref. 28 ]. Thus, it is important to define the role of a polyphenolic constituent, specifically EGCG, on UV-B-induced markers of immunosuppression and skin cancer induction. Therefore, in the present study, we determined the possible mechanism of prevention of UV-B-induced immunosuppression and skin cancer induction by EGCG, using a C3H/HeN mouse model. EGCG (3 mg/mouse/3 cm2 of skin area) was topically applied to mouse skin before a single UV-B (90 mJ/cm2) exposure to determine whether EGCG will prevent UV-B-induced (1) infiltration of leukocytes, (2) infiltrating-leukocyte-caused induction of oxidative stress, and (3) migration or depletion of antigen-presenting cells (APCs) in the skin.
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Chemicals and antibodies
Diaminobenzidine (DAB) reagent set was purchased from Kirkgaard
and Perry (Gaithersburg, MD). Dihydrorhodamine 123 (DHR) was obtained
from Molecular Probes (Eugene, OR). Monoclonal antibodies to inducible
NO synthase (iNOS) [anti-macNOS; rat immunoglobulin (Ig) G2a] was
purchased from Transduction Laboratories (Lexington, KY), and CD11b
(anti-mac, M1/70; rat IgG2b) and Ia
k (mouse IgG2b) antibodies were
purchased from PharMingen (San Diego, CA). Purified EGCG was obtained
as a gift from Yukihiko Hara (Mitsui Norin Co., Shizuoka, Japan).
For all experiments, EGCG was dissolved in acetone, and the required concentration in 0.2 mL of acetone was topically applied to the mouse skin 20 min before UV-B exposure. Treatment of EGCG 20 min before UV-B exposure of the skin allows sufficient time for it to be absorbed by the skin cells.
UV-B-irradiation of mice and skin punch biopsies
Forty-eight hours before UV-B exposure, mice were shaved with
electric clippers and Nair depilatory lotion (Carter Products, New
York, NY) was applied for 2 to 3 min. The time period of Nair lotion
treatment was kept constant in each group of animals as well as in each
set of experiments. Non-UV-B-exposed groups of mice also were shaved
and received depilatory lotion for the same time period to maintain a
same-treatment protocol. UV-B-irradiation was performed as described
earlier [20
30
]. Briefly, the razor-shaved and
chemically depilated dorsal skin was exposed to UV irradiation from a
band of six FS-40 fluorescent lamps from which short-wavelength UV-B
(280–290 nm) and UV-C wavelengths not normally present in natural
solar radiation were filtered out using Kodacel cellulose film (Eastman
Kodak Co., Rochester, NY) [31
]. After filtration with a
Kodacel film, the majority of the resulting wavelengths of UV radiation
were in the UV-B (290–320 nm) and UV-A range, which mimics the type of
UV to which humans are generally exposed. The UV-B emission was
monitored with an IL-443 phototherapy radiometer equipped with an IL
SED 240 detector fitted with a W side angle quartz diffuser and a SC5
280 filter (all from International Light, Newburyport, MA) . During
UV-B irradiation mice were housed in specially designed cages where
they were held in dividers separated by Plexiglas. A single dose of 90
mJ/cm2 of UV-B was applied to the skin surface of each
mouse.
Mice were sacrificed 48 h after UV-B irradiation by a cervical dislocation procedure. Skin punch biopsies of 5–6 mm in size were collected for immunostaining purposes and were frozen in optimal-cutting-temperature compound under liquid nitrogen immediately after removal. These skin biopsies were stored at -80°C for further use. All immunohistochemical and analytical assays were performed in skin samples collected at 48 h after UV-B irradiation of the mouse skin, because this is the peak time point for UV-B-induced infiltration of leukocytes [20 30 ].
Myeloperoxidase activity
Myeloperoxidase (MPO) activity in epidermal and dermal skin
cytosols was determined by the procedure of Bradley et al.
[32
] with some modification [33
]. In
brief, the skin samples were taken from non-UV-B-exposed mouse skin as
well as mouse skin at 48 h after UV-B exposure. Epidermis and
dermis were separated using dispase enzyme as described earlier
[30
] after removing subcutaneous fat. The epidermis and
dermis separately were homogenized in 50 mM potassium phosphate buffer,
pH 6.0, containing 0.5% hexadecyltrimethyl-ammonium bromide, followed
by sonication of the homogenate at 4° C for three 10-s bursts with a
heat system sonicator equipped with a microprobe. For complete
extraction of MPO from infiltrating neutrophils, the tissue homogenates
were frozen and thawed three times, and each time sonication was
repeated. The tissue homogenate thus obtained was centrifuged at 40,000
g for 15 min at 4°C, and the resulting supernatants from
epidermis and dermis were used for MPO estimation.
MPO activity in supernatant (0.1 mL) was assayed by mixing with 50 mM phosphate buffer (2.9 mL), pH 6.0, containing 0.167 mg/mL of orthodianisidine dihydrochloride and 0.0005% hydrogen peroxide. The change in absorbance resulting from decomposition of H2O2 in the presence of orthodianisidine was measured at 460 nm using a Beckman DU 640 spectrophotometer. The results are expressed as the mean optical density/min/assay at 25°C, as previously described [33 ].
Immunohistochemical detection of CD11b+ cells
Immunostaining of CD11b was used as a cell surface marker of
monocytes/macrophages and neutrophils. For immunohistochemical
detection of CD11b+ cells, 6-µm-thick sections of frozen skin
biopsies were used. After blocking nonspecific staining by normal goat
serum [10% in phosphate-buffered saline (PBS)], sections were
incubated with either rat anti-mouse CD11b monoclonal antibody or
RIgG2b isotype control. After washing in Tris-HCl buffer, pH 7.5,
endogenous peroxidase was blocked using 0.5%
H2O2 in PBS buffer for 30 min. Sections were
incubated with biotinylated rabbit anti-rat IgG (Vector) and thereafter
with peroxidase-labeled streptavidin. After washing in PBS buffer,
sections were incubated with DAB substrate with peroxidase enzyme.
Sections were counterstained with methyl green for 1 h. The
DAB-peroxidase reaction gave a brown reaction product, and the methyl
green gave a blue nuclear counterstain.
Immunohistochemical detection of Ia+ cells (class II MHC+ cells)
Immunostaining of class II MHC+ Ia+ cells was used as a cell
surface marker of APCs in C3H/HeN mouse skin. Immunostaining was
performed to detect and localize Ia+ cells in UV-B-exposed and
non-UV-B-exposed skin. Briefly, 6-µm-thick sections from frozen skin
biopsies were fixed in cold acetone. After blocking nonspecific
staining by using normal goat serum (10% in PBS), sections were
incubated for 1 h with mouse anti-mouse Iak antibody or its
isotype control mouse IgG2b. After washing in PBS buffer, sections were
incubated with peroxidase-labeled secondary antibody (anti-mouse IgG2b)
in PBS buffer containing 0.2% bovine serum albumin and 10% goat
serum. After washing in PBS buffer, sections were incubated with DAB
substrate solution with peroxidase enzyme. Sections were counterstained
with methyl green for 1 h.
Immunohistochemical detection of H2O2
producing cells
Immunohistochemical detection of H2O2 in
normal as well as in UV-irradiated skin was performed as described
earlier [34
]. Briefly, 6-µm-thick frozen skin sections
were incubated with 0.1 M Tris-HCl buffer, pH 7.5, containing 1 mg/mL
of glucose and 1 mg/mL of DAB for 6 h at 37°C. Sections were
then washed in distilled water and counterstained with methyl green.
Quantitative determination of intracellular release of
H2O2 by cytometric analysis
Intracellular levels of H2O2 in
epidermal and dermal cells were determined using DHR 123 as a specific
fluorescent dye probe as described by Peus et al. [35
]
with some modification. Epidermal and dermal single-cell suspensions
were prepared as described earlier [30
]. Briefly,
106 cells each from different treatment groups were placed
in separate wells of a 24-well tissue culture plate in duplicate. These
cells were treated with DHR (5 µM) for 45 min. Reduced DHR is
irreversibly oxidized and converted to the red fluorescent compound
rhodamine 123 [36
] by UV-B-induced intracellular release
of H2O2. Plates were read on a Cytofluor II
fluorescence plate reader with an excitation wavelength of 485 nm and
an emission wavelength of 530 nm.
Immunohistochemical detection of iNOS expression
Immunohistochemical detection of iNOS expression was performed
using frozen skin biopsies from different treatment groups. Briefly,
6-µm-thick skin sections were fixed in cold acetone for 10 min. After
blocking nonspecific staining in 10% goat serum in PBS buffer,
sections were incubated with anti-mouse iNOS (IgG2a) for 1 h at
room temperature. After blocking endogenous peroxidase activity in
0.5% H2O2 in PBS buffer, sections were
incubated with secondary-antibody anti-mouse IgG2a labeled with
horseradish peroxidase. After washing in PBS buffer, sections were then
incubated with DAB and peroxidase enzyme and counterstained with
hematoxylene. The DAB-peroxidase reaction gave a brown reaction
product, and the hematoxylene, a violet nuclear counterstain.
Quantitative determination of nitric oxide production
Nitric oxide levels were determined by measuring their stable
degradation products, nitrate and nitrite, separately in epidermal and
dermal cytosolic fraction. For accurate assessment of the total nitric
oxide generated, we must monitor both nitrate and nitrite. In this
procedure nitrate is enzymatically converted into nitrite by the enzyme
nitrate reductase, followed by quantitation of nitrite using Griess
reagent. Thus nitric oxide is estimated in the form of total nitrite
formed, using a colorimetric nitric oxide assay kit (Oxford Biomedical
Research, Inc., Oxford, MI) and by following the manufacturer’s
protocol.
Analysis of CD11b+, Ia+,
H2O2+, and iNOS+ cells
To compare marker-specific positively stained cells in different
treatment groups, these cells were counted in the epidermis or dermis
compartment of the skin at six–eight places in each section using an
ocular micrometer grid with x200 magnification under Zeiss Axiophot
microscope and Zeiss Plan-Neofluar objective. The ocular micrometer
grid corresponds to 0.0625 mm2. CD11b-, Ia-,
H2O2-, or iNOS-positive cells were counted at
least in three sections per biopsy per animal. Immunostaining
experiments were repeated at least in three animals per treatment
group.
Microscopy and photography
Images from immunostaining experiments were obtained using a
Zeiss Axiophot microscope (Thornwood, NY) and Kodak Ektachrome 160T
film (Rochester, NJ). These images were scanned (SprintScan; Polaroid,
Cambridge, MA) and formatted as Tag Image File Format (TIFF) images in
Adobe Photoshop 5.0 software and Microsoft PowerPoint to make the
composite figures.
Statistical analysis
The results were expressed as the mean plus or minus standard
deviation. Statistical analysis of all the data between groups
receiving UV-B exposure alone and those receiving EGCG treatment plus
UV-B exposure was performed by Student’s t-test. The
P value < 0.05 was considered statistically
significant.
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 View larger version (106K): [in a new window] |
Figure 1. Effect of topical treatment of mouse skin with EGCG on
UV-B-induced infiltration of leukocytes. Topical treatment of mouse
skin with EGCG before UV-B exposure inhibited UV-B-induced infiltration
of leukocytes, CD11b+ cells, and MPO activity measured at 48 h
after UV-B exposure. (A) Pretreatment with EGCG inhibited UV-B-induced
infiltration of leukocytes. Skin biopsies were processed by hematoxylin
and eosin staining by a routine procedure. Black nuclei indicate the
presence of cells. (B) Pretreatment of EGCG inhibited UV-B-induced
infiltration of CD11b+ cells. Immunohistochemical detection of CD11b+
cells, a marker of monocytes/macrophages and neutrophils, was performed
in frozen skin biopsies. Sections were stained with monoclonal
antibodies to CD11b as described in Materials and Methods. CD11b+ cells
are shown in brown, and a representative section of immunostaining from
three independent experiments is shown. (C) Pretreatment of EGCG
inhibited (P < 0.001) UV-B-induced activity of MPO in
both epidermis and dermis. MPO activity was determined as a marker of
tissue infiltration of leukocytes and assayed in both epidermal and
dermal cytosolic fractions separately as described in Materials and
Methods._art>
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Furthermore, to confirm that EGCG treatment before UV-B exposure of the skin inhibits UV-B-induced infiltration of leukocytes in the UV-B irradiated sites, we determined the MPO activity in cytosolic fractions of epidermis and dermis separately in the different treatment groups. MPO was commonly used as a marker of infiltrating leukocytes (monocytes/macrophages and neutrophils) in skin after UV-B exposure. We found an increase in MPO activity both in epidermis and dermis 48 h after UV-B exposure of the skin when compared to that of the normal, non-UV-B-exposed skin (Fig. 1C) . The increase in MPO activity after UV-B exposure indicates an influx of leukocytes to the inflamed skin. Topical treatment with EGCG before UV-B exposure was found to inhibit MPO activity both in epidermis and dermis by 67 and 65%, respectively (P < 0.001). The decrease in MPO activity suggests the inhibition of UV-B-induced infiltration of leukocytes by EGCG treatment. This fact is also evident from Figure 1A and 1B , where we found that EGCG treatment inhibited UV-B-induced infiltration of leukocytes and specifically CD11b+ cell types.
Skin application of EGCG before UV-B exposure prevented
UV-B-induced depletion of APCs or Ia+ cells
In C3H/HeN mice, detection of Ia+ cells was used as a marker of
class II MHC+ cells, which represent APCs in the skin. In epidermis,
these cells are also called LCs. UV exposure of the skin reduces the
density of epidermal LCs [15
16
37
] either by induced
migration [17
18
] or by cell death [19
].
As shown by immunostaining (Fig. 2
), UV-B exposure depleted the number of Ia+ cells both in the
epidermis and the dermis (Fig. 2
, middle panel) when compared with
normal, non-UV-B-exposed skin (Fig. 2
, left panel). Depletion of Ia+
cells was critical in the induction of immune suppression after UV-B
exposure. We found that EGCG treatment before UV-B exposure inhibited
the migration, depletion, or death of APCs (Ia+ cells) (Fig. 2
, right
panel) when compared with skin exposed to UV-B alone (Fig. 2 , middle
panel). This observation indicated that EGCG treatment protects APCs
from the adverse effect of UV-B irradiation, and this may be one
reason for protection by EGCG against UV-B-induced immunosuppression in
mice [30
].
 View larger version (61K): [in a new window] |
Figure 2. Effect of topical treatment with EGCG applied to mouse skin before UV-B
exposure on UV-B-induced migration or depletion of APCs. Topical
treatment with EGCG applied to mouse skin before UV-B exposure
prevented UV-B-induced migration or depletion of APCs. Class II MHC+
Ia+ cells were immunohistochemically detected as a marker of APCs,
using anti-mouse Iak antibody. Frozen skin sections were processed
for immunoperoxidase staining as detailed in Materials and Methods. Ia+
cells are dark brown. A few distinct Ia+ cells (APCs) are indicated by
arrowheads in the epidermis. A representative cross-section of
immunostaining from three independent experiments is
shown._art>
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 View larger version (70K): [in a new window] |
Figure 3. Effect of topical treatment of mouse skin with EGCG on UV-B-induced
production of H2O2. Topical treatment of mouse
skin with EGCG inhibited UV-B-induced production of
H2O2. Immunohistochemical detection and
quantitative assay of H2O2 was
performed 48 h after UV-B exposure and used as a marker of
UV-B-induced oxidative stress in the skin. (A). Immunohistochemical
detection of H2O2-producing cells is shown as
dark-brown staining, using DAB as a substrate. The detailed staining
procedure is described in Materials and Methods. A representative
section of immunostained cells from three independent experiments is
shown. (B). Quantitative analysis of UV-B-induced intracellular release
of H2O2 was performed in single-cell
suspensions from epidermis and dermis separately, using
dihydrorhodamine 123 as a fluorescence dye probe as described in
Materials and Methods. _art>
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 View larger version (71K): [in a new window] |
Figure 4. Effect of EGCG before UV-B exposure on expression of iNOS and NO
production. Topical treatment of mouse skin with EGCG before UV-B
exposure inhibited UV-B-induced expression of iNOS and NO production
measured at 48 h after UV-B exposure. (A) Immunohistochemical
detection of iNOS expression in activated macrophages was performed
using rat anti-mouse iNOS antibody (anti-Mac) specific to activated
macrophages as detailed in Materials and Methods. iNOS+ cells are shown
in dark brown. A representative section of immunostained cells from
three independent experiments is shown. (B) A quantitative assay of NO
production in epidermal and dermal cytosols was performed using Griess
reagents. _art>
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UV-B-induced suppression of the murine immune response to a contact sensitizer is mediated through alterations of APCs [38 39 ]. The initial effect of UV is on the constitutive APCs within the skin, epidermal LCs, and dermal LC-like APCs, such that cell death [19 ] or functional alteration [40 ] of these cells occurs. As a result of in vivo UV-B exposure, the constitutive skin APCs are no longer capable of initiating in vivo contact sensitivity responses [20 21 ] or stimulating in vitro Th1-type cells [41 ]. In our study, we found that EGCG treatment before a single UV-B exposure prevented migration or depletion of APCs from the UV-irradiated skin (Fig. 2) . It appears that the prevention of UV-B-induced migration or depletion of class II MHC+ Ia+ cells by EGCG may be associated with the prevention of UV-B-induced suppression of the immune responses to a contact sensitizer [30 ]. In addition to a prominent role played by APCs in immune responses, it has been shown that the induction of a state of tolerance to a contact sensitizer applied to UV-irradiated skin receiving a single UV dose is due to the appearance of tolerance-inducing infiltrating class II MHC+CD11b+ monocytes/macrophages in both mice [20 21 ] and humans [42 43 44 ]. Our observation that treatment with EGCG prevented UV-B-induced immunosuppression and tolerance induction [30 ] is probably related, therefore, to the prevention of UV-B-induced infiltration of CD11b+ cells in the UV-B-irradiated skin. Application of EGCG alone to normal, non-UV-B exposed, mouse skin sites did not induce infiltration of leukocytes or affect the constitutive cell structure of the skin and APCs (data not shown). Moreover, it is important to mention that EGCG showed a UV absorption peak at 270–273 nm, and, therefore, it is likely that EGCG treatment may absorb or block penetration of some short UV-B wavelengths (e.g., 280–290 nm) into the skin. It is certain that EGCG did not absorb or block the penetration of the whole UV-B wavelength range into the skin.
The UV-B-induced infiltrating leukocytes were found to be a major source of H2O2 (Fig. 3A and 3B) , iNOS expression (Fig. 4A) , and NO production (Fig. 4B) . Production of H2O2 and NO may thus create a state of oxidative stress in the skin and act as a tumor initiator [10 ] and tumor promoter [11 ]. Recently, Iwai et al. [45 ] suggested that UV-induced immune suppression might be mediated through ROS, at least in part. To confirm their observations, they found that application of glutathione as an antioxidant to the skin during irradiation significantly reversed UV-induced immunosuppression. Similar observations were made with the use of EGCG on mouse skin, in which EGCG treatment before a single dose of UV exposure inhibited the induction of oxidative stress in UV-irradiated skin. Thus the reduction in UV-B-induced oxidative stress by pretreatment with EGCG may be associated with the prevention of UV-induced suppression of immune responses.
UV-induced immune suppression is also considered a risk factor for skin cancer development [12 13 ]. Clinical observations have long documented an association between chronic inflammation and increased incidence of tumor formation at the inflammatory site [46 ]. The mediating agent(s) between inflammation and the development of the tumor at the inflammatory site has been proposed to be oxidative products produced by the inflammatory leukocytes [47 48 ]. As we found in our study that UV-induced infiltrating leukocytes are the potential source of H2O2 and NO production in the skin, these ROS may play an important role as a tumor initiator [10 ] and tumor promoter [11 ] by damaging macromolecules such as DNA, proteins, and lipids. The prevention of UV-induced infiltrating leukocytes and their ROS metabolites by skin treatment with EGCG may help in reducing oxidative stress in the skin. Therefore, reduction in UV-induced oxidative stress by EGCG may help in the reduction of mutagenic and carcinogenic effects of UV radiation.
In summary, our data demonstrated the potent preventive effects of EGCG in mouse skin against UV radiation-induced infiltration of activated leukocytes, which are the potential mediating agents of UV-induced suppression of immune responses and oxidative stress in the UV-irradiated skin. These factors all together make the skin more susceptible to various skin disorders including photoaging and skin cancer development. Together, on the basis of the data obtained in our animal model, we recommend an in-depth study to translate the preventive effects of GTPs in human skin.
Received October 16, 2000; revised December 20, 2000; accepted December 21, 2000.
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