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Ultraviolet (UV) radiation-induced immunosuppression has been implicated in the development of skin cancers. As oral administration of green tea polyphenols (GTPs) in drinking water prevents photocarcinogenesis in mice, we studied whether administration of GTPs in drinking water of mice (0.1-0.5%, w/v) prevents UV-induced immunosuppression, and determined the possible mechanism of action of GTPs. We observed that GTPs (0.2 and 0.5%, w/v) prevented UV-induced suppression of contact hypersensitivity (CHS) response to a contact sensitizer in local (58-62%, p<0.001) and systemic (51-55%, p<0.005) models of CHS. GTPs (0.2%, w/v) repaired UV-induced DNA damage faster in the skin of mice as demonstrated by reduced number of cyclobutane pyrimidine dimers (CPD)-positive cells (59%, p<0.001), and reduced the migration of CPD-positive cells (2-fold) from the skin to draining lymph nodes, which was associated with the elevated levels of nucleotide excision repair (NER) genes. GTPs did not prevent UV-induced immunosuppression in NER-deficient mice but significantly prevented in NER-proficient mice (p<0.001) concomitantly repaired UV-induced DNA damage in NER-proficient mice (p<0.001) but not in NER-deficient mice as indicated by immunohistochemical analysis of CPD-positive cells. Southwestern dot-blot analysis revealed that GTPs repaired UV-induced CPD in xeroderma pigmentosum complementation group A (XPA)-proficient cells obtained from healthy person but did not repair in XPA-deficient cells obtained from the patients suffering from XPA, indicating that NER mechanism is involved in DNA repair. These data identify a noble mechanism by which drinking GTPs prevent UV-induced immunosuppression, and this may contribute to the chemopreventive activity of GTPs in prevention of photocarcinogenesis.
Green tea is consumed as a popular beverage world-wide. Polyphenols isolated from the leaves of green tea (Camellia sinensis) have a number of beneficial health effects including anti-carcinogenic activity, which has been demonstrated in various tumor models (1,2). In previous studies, we and others have shown that oral administration of an aqueous extract of green tea or green tea polyphenols (GTPs; a mixture of polyphenols) in drinking water inhibits UV radiation-induced skin carcinogenesis in mice in terms of tumor incidence and tumor multiplicity (3,4).
The immunosuppressive effects of solar UV radiation, in particular the mid-wave range (UVB, 290-320 nm), are well established having been demonstrated most clearly by the effects of UV radiation on the inhibition of contact hypersensitivity (CHS), which is a prototypic T-cell mediated immune response (5,6). Some of the adverse effects of solar UV radiation on human health, including exacerbation of infectious diseases and initiation of skin cancer, are mediated at least in part by this ability of UV radiation to induce immune suppression (7-9). As UV-induced immunosuppression is considered to be a risk factor for the induction of skin cancer (10, 11), prevention of UV-induced immunosuppression represents a potential strategy for the management of skin cancer. Thus, to assess whether drinking green tea polyphenols (GTPs) inhibit UVB-induced immunosuppression is of considerable interest.
UV-induced DNA damage, predominantly the formation of cyclobutane pyrimidine dimers (CPDs), has been recognized as an important molecular trigger for the initiation of UVB-induced immunosuppression and carcinogenesis in the skin (12-14). Reduction of CPDs through application of DNA repair enzymes considerably reduces the risk of UV-induced skin cancer in mice and in humans (14, 15). Again, as UV-induced immunosuppression has been considered as a risk factor for the development of skin cancer, we sought to determine whether administration of GTPs in drinking water prevents UVB-induced immunosuppression, and whether the prevention of UVB-induced immunosuppression by GTPs is mediated, at least in part, through the rapid repair of DNA damage in the mouse skin exposed to UVB radiation. As green tea is commonly consumed as a beverage world-wide, we assessed the mechanism of photoprotective effect of its active ingredients (polyphenols) after mixing them in drinking water and using in vivo mouse models. We also hypothesized that the rapid repair of UVB-induced DNA damage by GTPs is mediated through the enhanced levels of nucleotide excision repair genes. If this is the case, we postulate that the treatment with GTPs in drinking water would be unable to inhibit UVB-induced immunosuppression and DNA repair in nucleotide excision repair (NER)-deficient mice.
We have used C3H/HeN mice in our experiments as these mice are inbred and therefore considered better for immunological studies compared to outbred mice, such as SKH-1 hairless mice. Also in our studies, we used xeroderma pigmentosum complementation group A-deficient mice (XPA-/-) which were generated on C3H/HeN background. Because of background of XPA-deficient mice, we also preferred to use C3H/HeN mice in the study so that resultant data can be interpreted correctly. The female C3H/HeN mice (6-7 weeks old) were purchased from Charles River Laboratory (Wilmington, MA). The XPA-/- mice which are devoid of nucleotide excision repair function were generated as described previously (16). All mice were maintained under standard conditions of a 12-hour dark/12-hour light cycle, a temperature of 24 ± 2°C, and relative humidity of 50 ± 10%. The animal protocol used in this study was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
The antibody specific for CPDs was obtained from Kamiya Biomedical Co. (Seattle, WA). The manufacturer-supplied standardized real-time PCR primers for nucleotide excision repair genes (XPA, XPC, RPA1, DDB2 and DDB1) and β-actin were obtained from the SuperArray BioScience Corp. (Frederick, MD). All other chemicals of analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO).
The purified mixture of green tea polyphenols was obtained from Mitsui Norin Co. Ltd. (Tokyo, Japan) and contains primarily 5 major epicatechin derivatives, such as, (-)-epigallocatechin-3-gallate, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epicatechin and gallocatechin gallate, as described previously (4, 17). This mixture of GTPs was given in the normal drinking water ad libitum. In all the animal experiments mice were given GTPs in drinking water at least 7 days before the start of UVB irradiation. Fresh GTPs-containing water was provided every third day. The polyphenolic constituents of samples of the three-day-old GTPs-containing drinking water were analyzed by HPLC and this confirmed that the chemical composition of the GTPs in the drinking water was not significantly altered during this time period when compared with the fresh samples (17).
The shaved backs of the mice were UVB irradiated as described earlier (4, 17) using a band of four FS20 UVB lamps (Daavlin, UVA/UVB Research Irradiation Unit, Bryan, OH) equipped with an electronic controller to regulate UV dosage. The UV lamps emit UVB (280-320 nm; ≈80% of total energy) and UVA (320-375 nm; ≈20% of total energy) with UVC emission being insignificant. This UV unit enables us to enter dose in millijoules and variations in energy output are automatically compensated so the desired UV dose can be delivered. Mice were kept under the UV lamps at a fixed distance of 24 cm. Monitoring indicated that the majority of the resulting wavelengths of UV radiation were in the UVB (290-320 nm) range with a peak emission at 314 (4, 17).
The shaved backs of the mice were exposed to UVB radiation (100 mJ per cm2) for 4 consecutive days. Twenty-four hour after the last UV exposure, the mice were sensitized by painting 25 μl of 0.5% 2,4-dinitrofluorobenzene (DNFB) in acetone: olive oil (4:1, v/v) either at the UVB-irradiated skin site (local CHS model) or at a shaved non-UVB-irradiated ventral or distant site (systemic CHS model). The CHS response was elicited 5 days later by challenging the both surfaces of the ears of each mouse with 20 μl of 0.2% DNFB in acetone: olive oil (4:1, v/v). The ear swelling was measured 24 hour after the challenge using an engineer's micrometer (Mitutoyo, Tokyo, Japan) and was compared with the ear thickness just before the challenge, as detailed previously (18). Mice that received the same dose of DNFB but were not UV irradiated served as a positive control, whereas the non-irradiated mice which received only ear challenge without sensitization with DNFB served as a negative control. To determine the chemopreventive effect of GTPs against UV-induced immunosuppression, GTPs were given in drinking water of the mice in separate groups of mice. During UV exposure of the mice, the ears of mice were protected from the UV irradiation. The mice that were not exposed to UV radiation were also shaved to maintain the identical regimen. The UV-induced suppression of CHS was determined as described previously (19). Each group consisted of five mice, and each experiment was performed at least twice.
To further determine whether GTPs-mediated prevention of UVB-induced immunosuppression leads to a long-term immunity, we extended the CHS experiment for a longer period of time. After measuring the ear swelling response to challenge with DNFB (Primary challenge), the mice were rested for 4 weeks (i.e., until the ear swelling has regressed to the basal level), and mice were not given GTPs in drinking water after the primary challenge. Mice were then re-challenged (Secondary challenge) on the ear skin with the same hapten (DNFB) and the ear thickness was measured before and 24 hour after re-challenge.
Immunohistochemical detection of CPD+ cells in the skin or draining lymph node (DLN) samples was performed using a procedure described previously (17, 20). Briefly, frozen skin or DLN sections (5 μm thick) were thawed, and kept in 70 mM NaOH in 70% ethanol for 2 min to denature nuclear DNA, followed by neutralization for 1 min in 100 mM Tris-HCl (pH 7.5) in 70% ethanol. The sections were washed with PBS buffer and incubated with 10% goat serum in PBS to prevent non-specific binding prior to incubation with a monoclonal antibody specific for CPDs, or its isotype control (IgG1). Bound anti-CPD antibody was detected by incubation with biotinylated goat-anti-mouse IgG1 followed by peroxidase-labeled streptavidin. After washing, sections were incubated with diaminobenzidine and counterstained with either H & E or methyl green.
Xeroderma pigmentosum complementation group A (XPA)-deficient (Cat. # GM02009) and XPA-proficient (Cat. # GM08399) human fibroblasts were obtained from the Coriell Institute for Medical Research (Camden, NJ). These XPA-deficient cells originally were obtained from patients suffering from xeroderma pigmentosum and the XPA-proficient cells from healthy human donors. Cells were cultured in Modified Eagle's Medium with Earle's salts (MEM) supplemented with 2mM L-glutamine, 10% heat-uninactivated fetal bovine serum (Hyclone, Logan, UT) and maintained in a incubator at 37°C in a humidified atmosphere of 5% CO2. The cells were UV irradiated using the same UV source as employed for irradiation of the mice. The cells were exposed to the UV radiation through PBS and upon UV irradiation cells were reincubated with GTPs (0.2%, w/v) for indicated time periods.
UVB-induced DNA damage and its repair by GTPs in XPA-proficient and XPA-deficient human fibroblast cells were determined using Southwestern dot-blot analysis, as described previously (17). Cells were treated with GTPs (0.2%, w/v) for 1 hour before irradiation to UVB (20 mJ/cm2). Cells were harvested 36 hours later. Genomic DNA from the cells was isolated following the standard procedures. Genomic DNA (500 ng) was transferred to a positively charged nitrocellulose membrane by vacuum dot-blotting (Bio-Dot Apparatus, Bio-Rad, Hercules, CA) and fixed by baking the membrane for 30 min at 80°C. After blocking the non-specific binding sites in blocking buffer (5% non-fat dry milk, 1% Tween 20 in 20 mM TBS, pH 7.6), the membrane was then incubated with the antibody specific to CPDs for 1 hour at room temperature. After washing, the membrane was incubated with HRP-conjugated secondary antibody. The CPDs were detected by chemiluminescence using an ECL detection system. The experiments were repeated twice.
The results of CPD+ cells in each group are expressed in terms of either percentage of CPD+ cells or number of CPD+ cells per field under microscope, and expressed as means ± SD. The statistical significance of difference between the values of control and treatment groups was determined by analysis of variance followed by post hoc test. The p value <0.05 was considered as a statistical significant.
We have shown earlier that the chemical composition of GTPs was not significantly changed in drinking water at least for three days (17).
As UVB-induced immunosuppression is considered to be a risk factor for photocarcinogenesis (10,11), and GTPs given in drinking water prevent photocarcinogenesis in mice (3, 17), we determined whether treatment of mice with GTPs in drinking water protects against UVB-induced suppression of the CHS response to DNFB in a model of local UVB-induced immune suppression in which we measure the CHS response to DNFB. We first confirmed that administration of GTPs in drinking water with various concentrations of GTPs (0.1, 0.2 and 0.5%, w/v) did not affect the ability of the mice to generate a local CHS response to DNFB in the absence of UVB irradiation (Fig 1, Compare left panel A; third to fifth bar from the top with the second bar from the top (positive control). We then confirmed that in the absence of treatment with GTPs, the local CHS response in terms of ear swelling was significantly lower (72% suppression, p<0.001; Left panel A, 6th bar from the top) in those mice that were UVB-irradiated than those mice that were not UVB-irradiated (Left panel A, 2nd bar from the top, positive control), indicating the immunosuppressive effect of the UVB radiation. The group of mice that were treated with GTPs in drinking water at a concentration of either 0.2 or 0.5%, prior to UVB irradiation exhibited a significantly less UVB-induced suppression of CHS (66% lower, p<0.001) than UV-irradiated mice that were not treated with GTPs. Administration of a lower concentration of GTPs (0.1%, w/v) failed to provide significant protection from the UVB-induced suppression of the local CHS response in mice. These data indicate that the treatment doses of 0.2 or 0.5% of GTPs are capable of protecting mice from UVB-induced immunosuppression in a local model of immunosuppression. However, it also has been observed that there was not significant difference in protection of UVB-induced immunosuppression between the doses of 0.2 and 0.5% of GTPs in drinking water.
To examine whether treatment of mice with drinking GTPs induces long-term immunity in UVB-exposed mice, the mice in local CHS model were rested for 4 weeks after primary challenge with DNFB, and were not given GTPs in drinking water during this period. As shown in Figure 1A (Right panel), again the group of mice that were given GTPs in drinking water earlier (Fig. 1A, left panel) at a concentration of 0.2 or 0.5% exhibited a significantly greater CHS response (51-55% more, p<0.001) after secondary challenge with DNFB than those UVB-irradiated mice that have not received GTPs at any stage. These data suggest that GTPs have the ability to prevent UVB-induced immune tolerance in mice and can protect for a longer period of time even after ceasing the consumption of GTPs.
We next determined whether administration of GTPs in drinking water induces inhibitory effects in a systemic model of CHS. As in the local model of CHS, treatment of GTPs did not affect the ability of the mice to generate a systemic CHS response to DNFB in the absence of UVB irradiation (Fig 1, Panel B, compare 3rd-5th bar from the top with second bar from the top). In the systemic model of CHS, treatment of the lower dose of GTPs (0.1%, w/v) did not result in a statistically significant inhibition of UVB-induced immunosuppression as compared to the positive control. Treatment at the higher doses of GTPs (0.2 and 0.5%) significantly inhibited the immunosuppressive effects of UV radiation in the systemic model of CHS with GTPs inhibiting UVB-induced immunosuppression by 58-62% (p<0.005). The prevention of UVB-induced suppression of systemic CHS response by GTPs may be due to the induction of immune response in animals against UVB-induced adverse effects.
UVB-induced DNA damage in the form of CPDs has been implicated in the UVB-induced immunosuppression (12, 13). Therefore, to determine whether GTPs prevent UV-induced immunosuppression by enhancing DNA repair, we evaluated the formation of CPDs in the UV exposed skin. As we have found that 0.2 and 0.5% of GTPs in drinking water significantly protect the mice from UVB-induced immunosuppression, and that there was no significant difference in the protection ability of 0.2 and 0.5% of GTPs, in all further experiments we used 0.2% GTPs in drinking water of mice. The shaved backs of C3H/HeN mice were exposed to UVB (60 mJ/cm2) with and without the treatment of GTPs (0.2%, w/v) in drinking water. Mice were sacrificed either immediately (≈30 min) or 72 hour later, samples of the skin were obtained and the presence of CPDs was detected and determined by immunohistochemistry using an antibody directed against CPDs. In skin samples obtained immediately after UV exposure, no differences in the staining pattern of CPDs were observed whether or not the mice were treated with GTPs (Fig 2A). This observation also eliminated the speculation that drinking GTPs might have significant filtering effect on UV radiation. In contrast, in skin samples obtained 72 hour after UVB exposure, the numbers of CPD+ cells were significantly lower (p<0.001) in the GTPs-treated mice than the mice that have not received GTPs in drinking water but were exposed to UVB. It was observed that the skin samples obtained 72 hour after UVB exposure from non-GTPs-treated mice also showed a reduction in the number of CPD+ cells indicating that some endogenous defense mechanism independent to GTPs action may be involved in repair of UV-damaged DNA. The skin samples obtained from the groups of mice that were not exposed to UV (normal skin), including those that were or were not treated with GTPs were devoid of any CPD+ cells. The numbers of CPD+ cells were counted at least 5-6 different places of the sections and are presented as percent of CPD+ cells in different treatment groups, and as means ± SD (Fig. 2B), n=5.
UV-induced DNA damage has been recognized as an important molecular trigger for the migration of antigen presenting cells (i.e., Langerhans cells in the epidermis) from the skin to the draining lymph nodes (DLN). DNA damage in antigen presenting cells impairs their capacity to present Ag, which in turn results in a lack of sensitization (21). CPD-containing antigen presenting cells have been found in the DLN of UV-exposed mice (22). These antigen presenting cells were identified to be of epidermal origin and exhibited an impaired Ag presentation capacity. As we have found that GTPs have the capacity to induce DNA repair in the UV-exposed skin (Fig. 2), we next determined whether GTPs act to reduce the migration of CPD+ cells from the skin to the DLN. For this purpose, mice were treated with GTPs and UV-irradiated. Mice were sacrificed 36 hour later, the DLN harvested, and the presence of CPDs in the DLN was detected by immunohistochemical analysis. CPD+ cells were not detectable in the DLN of mice that were not UV irradiated whether or not they were treated with GTPs (Fig 3A). The significant numbers of CPD+ cells in the DLN were found in UV-exposed mice, with the numbers of CPD+ cells in the DLN of the UV-exposed mice being more than 2-fold higher (p<0.001) than in the DLN of GTPs-treated mice. The lower number of CPD+ cell in the DLN of GTPs+ UVB-treated group of mice compared to non-GTPs-treated UVB-exposed mice was not unexpected and may be attributable to the partial removal of the damaged DNA in the migrating cells. Treatment with GTPs resulted in a 59% reduction in the numbers of CPD+ cells in the DLN of UV-exposed mice compared to non-GTPs-treated UV-exposed mice (Fig 3B, p<0.001).
Microscopic examination suggests that most of the CPD+ cells, in both GTPs-treated and non-GTPs-treated, were localized in an area extending from the subcapsular sinus to the paracortical region of the lymph nodes, including the interfollicular areas (Fig 3A). The CPD+ cells were counted in these areas and reported in terms of CPD+ cells per field. As the interfollicular areas are the sites of T-cell localization, the presence of CPD+ cells in these areas may adversely affect the induction of the sensitization response. These data suggest that the ability of GTPs to prevent UV-induced immunosuppression in mice may be due to its capacity to repair UV-induced DNA damage in epidermal antigen presenting cells.
As we have found that administration of GTPs in drinking water enhances the removal or repair of UVB-induced thymine dimers or CPDs in the skin of mice, we were interested to determine whether the rapid repair or removal of CPDs in UV-exposed skin by GTPs is mediated through the enhancement in the levels of NER genes. For this purpose, mice were exposed to acute UVB (60 mJ/cm2) with and without the treatment of GTPs (0.2%, w/v) in drinking water, as detailed in Materials and methods. Mice were sacrificed at 1 and 3 hours later, skin samples were collected, and epidermal RNA was isolated and subjected to the analysis of mRNA expression of NER genes (i.e., XPA, XPC, RPA1, DDB2, and DDB1, etc.) using real-time PCR. As shown in Fig. 4, the acute exposure of the mouse skin with UVB radiation mildly enhances the levels of NER genes (not significantly) compared to non-UVB exposed skin of the mice. However, the mRNA levels of NER genes, such as XPA, XPC and RPA1, were significantly enhanced (p<0.05-0.001) in the skin of mice treated with GTPs at both 1 and 3 hours time points after UVB exposure compared to non-GTPs-treated UVB exposed mouse skin (Fig. 4). GTPs had no significant effect on the stimulation of DDB2 and DDB1 NER genes compared with non-GTPs-treated mouse skin exposed to UV radiation. Further, the administration of GTPs in drinking water to non-UV-irradiated mice did not affect the steady levels of NER genes (data not shown).
As the enhanced repair of UVB-induced DNA damage in the form of CPDs by GTPs may be associated with the inhibition of UVB-induced immunosuppression in mice, we next determined whether GTPs prevent UVB-induced immunosuppression in XPA-deficient or XPA-/- mice which do not have the ability to repair UVB-induced DNA damage because of absence of functional NER enzymes or genes. For this purpose, XPA-/- and their wild-type counterparts (XPA+/+) were subjected to local CHS protocol/experiment with and without the treatment of GTPs in drinking water (0.2%, w/v). Following the local CHS protocol, it was observed that in the absence of treatment with GTPs, the CHS response in terms of ear swelling was significantly lower (73% suppression, p<0.001; left panel, 4th bar from the top) in XPA-/- mice that were UVB-irradiated than those XPA-/- mice that were not UVB-irradiated (Left panel, 2nd bar from the top, positive control), indicating the immunosuppressive effect of UVB radiation in XPA-/- mice. The group of mice that were treated with GTPs in drinking water (0.2%, w/v) also exhibited a significant UVB-induced suppression of CHS response (p<0.005) which was similar to non-GTPs-treated UVB-exposed mice (Fig. 5). It suggests that administration of GTPs did not prevent UVB-induced suppression of CHS response to DNFB in XPA-/- mice. In contrast, the administration of GTPs to the wild-type counterparts (XPA+/+) significantly induces contact sensitization reaction and ear swelling response to DNFB and was significantly higher (p<0.001; Right panel, bottom bar of the panel) than those mice which were not given GTPs in drinking water and exposed to UVB radiation. The ear swelling response in GTPs-treated group of wild-type mice was comparable to the mice of control group (Right panel, 2nd bar from the top). The change in ear skin thickness in XPA-/- mice in response to DNFB sensitization in GTPs +UVB was also compared to the change in ear skin thickness in XPA+/+ mice in response to GTPs +UVB. The increase in ear skin thickness after sensitization to DNFB was greater in the XPA+/+ mice treated with GTPs +UVB (53%, p<0.01) as compared to increase in ear skin thickness after sensitization to DNFB in XPA-/- mice treated with GTPs +UVB. The data from this set of experiment suggest that prevention of UVB-induced immunosuppression by GTPs requires NER genes, which have a role in repair of UVB-induced DNA damage in the form of CPDs.
It has been shown that application of DNA repair enzymes that reduce the numbers of CPD+ cells prevents UV-induced immunosuppression (14, 23). We found that drinking GTPs have the ability to prevent UVB-induced immunosuppression in XPA+/+ mice but not in XPA-/- mice (Fig. 5), that are devoid of the NER gene and that is necessary for the repair of UV-induced DNA damage in mammalian cells. Therefore, we further examined whether the GTPs-mediated repair of UV-induced DNA damage requires NER gene. For this purpose, XPA+/+ and XPA-/- mice were exposed to acute UVB exposure (60 mJ/cm2) with and without the treatment of GTPs in drinking water, and sacrificed 72 hour later. Skin samples were collected and subjected to immunohistochemical analysis of CPD+ cells. In skin samples obtained from XPA-/- mice, no significant difference in the staining pattern of CPDs were observed whether or not they were treated with GTPs (Fig. 6A). In contrast, in UVB-exposed skin samples obtained from XPA+/+ mice, the numbers of CPD+ cells were significantly lower in the GTPs-treated mice (p<0.001) than those mice which were not treated with GTPs (Figures 6A and 6B).
To further verify our observations of green tea in XPA-/- and XPA+/+ system, we used NER-deficient fibroblasts from XPA-patients and repair-proficient fibroblasts from healthy persons. The XPA gene is an essential component of the NER, thus, cells with a mutated XPA gene completely lack a functional NER. Therefore, we examined the effect of GTPs on UV-induced CPDs in XPA-proficient and XPA-deficient cells using southwestern dot blot analysis. For this purpose XPA-deficient and XPA-proficient human fibroblasts were exposed to UV radiation in the presence or absence of GTPs. Cells were harvested 48 hours later, genomic DNA was isolated and subjected to dot-blot analysis. As clearly indicated in Figure 6C, GTPs treatment of XPA-proficient cells for 48 h resulted in remarkable repair or reduction of UV-induced CPDs. However, this DNA-repairing effect of GTPs was not evident in the XPA-deficient cells 48 hours after UV irradiation. This may be due to absence of NER enzymes in these cells. The cells whether XPA-deficient or XPA-proficient and either treated with GTPs or not treated with GTPs did not show the presence of CPDs as reflected from the absence of dot blot.
Exposure of the skin to UV radiation initiates a variety of harmful effects on human health, including squamous and basal cell carcinoma and melanoma, as well as premature aging of the skin and susceptibility to infection (7-9). UVB radiation has multiple effects on the immune system (6, 11). There is ample clinical and experimental evidence to suggest that immune factors contribute to the pathogenesis of sunlight-induced skin cancer in mice and probably in humans as well (10, 11). Chronically immunosuppressed patients living in regions of intense sun exposure experience an exceptionally high rate of skin cancer (Reviewed in 24). This observation is consistent with the hypothesis that immune surveillance is an important mechanism designed to prevent the generation and maintenance of neoplastic cells. As UVB-induced immunosuppression has been implicated in the development of photocarcinogenesis, we examined the efficacy of GTPs in drinking water on UVB-induced immunosuppression using local and systemic models of CHS in C3H/HeN mice, and investigated the possible mechanism of prevention of UVB-induced immunosuppression by GTPs. The results presented here show that administration of GTPs in drinking water inhibits UVB-induced suppression of CHS response to DNFB in both local and systemic models of CHS. These data provide a first line of evidence that prevention of photocarcinogenesis by drinking GTPs may be, at least in part, due to the prevention of UVB-induced immunosuppression in mice.
In terms of the mechanisms by which GTPs mediate the inhibition of UVB-induced immunosuppression, our data demonstrate that treatment of mice with GTPs rapidly remove or repair UVB-induced DNA damage in the form of CPDs in UVB-exposed skin site, and reduces the emigration of CPD+ antigen presenting cells from the epidermis to draining lymph nodes. There is evidence that UV-induced DNA damage is the molecular trigger for the migration of Langerhans cells (antigen presenting cells in the epidermis) from the skin to the draining lymph nodes (13, 22). The UV-induced DNA damage also impairs the antigen presenting capacity of Langerhans cells which results in a lack of sensitization and the induction of tolerance to contact sensitizers (21, 22). We observed that administration of GTPs in drinking water of mice inhibited the migration of epidermal antigen presenting cells to DLN in mice, indicating that treatment of GTPs might be able to repair UV-induced CPD in the mice. We speculate that, as the migrating antigen presenting cells in the epidermis were either not damaged or were repaired in mice they were able to present Ag to T-cells in the DLN resulting in induction of sensitization to DNFB after challenge. Further, the numbers of CPD+ cells were significantly higher in the non-GTPs-fed mice in the subcapsular sinus to the paracortical region of the lymph nodes, including the interfollicular areas, which are the sites of T-cell localization. Thus, the damaged DNA in the lymph nodes of non-GTPs-fed mice may adversely affect the ability of the antigen presenting cells to present Ag to T-cells thus abrogating sensitization after DNFB challenge. In contrast, the reverse was observed in GTPs-fed mice, and that may be one of the reasons that GTPs prevent UVB-induced immunosuppression in mice.
NER is the main mechanism of repair in mammalian cells for the removal of UV radiation-induced DNA damage. Since the treatment of GTPs enhances the removal or repair of UVB-induced DNA damage, we further examined whether the removal or repair of UV-induced CPDs by GTPs is mediated via induction of NER genes. Our real-time PCR data indicate that treatment of mice with GTPs increases the levels of some NER genes (e.g., XPA, XPC and RPA1) in UVB-exposed skin sites compared to non-GTPs-fed mice and that may have contributed in the rapid repair of damaged DNA in mouse skin. However, GTPs have no effect on some other NER genes (e.g., DDB1 and DDB2). It suggests that the function of GTPs is NER gene-specific. The role of NER was further confirmed by assessing the effect of GTPs on UVB-induced immunosuppression in XPA-/- mice and data were compared with the XPA+/+ (proficient) mice. Treatment of mice with GTPs in drinking water prevents UVB-induced suppression of CHS response in XPA+/+ mice but do not prevent in XPA-/- mice further support our observations that inhibition of UVB-induced immunosuppression by GTPs require functional NER genes. This observation was important as the treatment of GTPs do not remove or repair UVB-induced DNA damage in XPA-/- mice but repair in XPA+/+ mice which were exposed to UVB. Importantly, exposure of mice to UV radiation suppresses CHS response in both XPA-/- and XPA+/+ mice. It suggests that UV-induced immunosuppression is mediated through other mechanisms in addition to DNA damage. These may include: (i) UV-induced suppression of IL-12 (19). IL-12 stimulates immune system through the development of Th1 cell types; (ii) stimulation of IL-10 in UV-irradiated skin which is considered as an immunosuppressive cytokine (24). Further to confirm our hypothesis and verify our present data, we used NER-deficient cells from XPA-patients and NER-proficient cells from healthy persons. Cells derived from patients suffering from xeroderma pigmentosum either lack or have reduced DNA repair capacity due to genetic mutations in several components of the NER. The XPA complementation type represents the most severe phenotype, because the XPA gene is the most crucial component in the repair process and, thus, cells lacking the XPA gene are completely deficient in NER (25, 26). Our dot-blot analysis indicated that GTPs were able to remove UV-induced CPDs in NER-proficient cells (XPA+/+) but was not able to remove or repair in NER-deficient (XPA-/-) human fibroblast cells. These observations indicate that repair of UV-induced DNA damage by GTPs is mediated through the NER mechanism or GTPs-induced DNA repair requires functional NER. These findings have important implications for the chemopreventive mechanism of skin cancer protection by GTPs, and identify a new mechanism by which GTPs prevent UV-induced immunosuppression. Based on the information obtained in this study, it can be suggested that the consumption of 5-6 cups (one cup=150 ml) of green tea (1 g green tea leaves/150 ml) per day by humans may provide the same level of photoprotective effect in human system. However, the magnitude of photoprotective effect or UVB-induced immunosuppression by green tea may differ person to person based on the differences in race, and intensity and exposure time of UV radiation.
Taken together, our data indicate that the prevention of UV radiation-induced immunosuppression by drinking GTPs is mediated through rapid repair of UVB-induced DNA damage and that requires NER. As UV-induced DNA damage and immunosuppression play an important role in melanoma and nonmelanoma skin cancers, it is tempting to suggest that drinking green tea should be further investigated as a chemopreventive agent for the prevention of skin cancers in humans, and its possible use in future practice of medicine.
Financial support: This work was supported by grants from the National Center for Complementary and Alternative Medicine/NIH (1 RO1 AT002536, S.K.K.) and the Veterans Administration Merit Review Award (S.K.K.).
Disclosure of Potential Conflicts of Interest: No conflict of interest.