Skip to main content
NCBI home page
Carcinogenesis logoLink to Carcinogenesis
. 2012 Apr 30;33(7):1360–1367. doi: 10.1093/carcin/bgs152

The environmental stressor ultraviolet B radiation inhibits murine antitumor immunity through its ability to generate platelet-activating factor agonists

Ravi P Sahu 1,2, Matthew J Turner 1, Sonia C DaSilva 1, Badri M Rashid 2, Jesus A Ocana 1, Susan M Perkins 3, Raymond L Konger 1,2, Christopher E Touloukian 4,5, Mark H Kaplan 5,6, Jeffrey B Travers 1,6,7,8,*
PMCID: PMC3405652  PMID: 22542595

Abstract

Ubiquitous pro-oxidative stressor ultraviolet B radiation (UVB) to human or mouse skin generates platelet-activating factor (PAF) and novel oxidatively modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell; PAF-R-expression as UVB or the PAF-R agonist, carbamoyl PAF (CPAF), both promote B16F10 tumor growth in wild-type (WT) mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in Pafr − /− mice. UVB-mediated augmentation of experimental murine tumor growth was inhibited with antioxidants, demonstrating the importance of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth which is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice or depletion of CD25-positive cells in FoxP3EGFP transgenic mice block UVB and/or CPAF-induced tumor growth supporting a requirement for IL-10 and Tregs in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression.

Introduction

Malignant melanoma is the seventh most common type of cancer (1,2). The incidence of melanoma is increasing more than any other malignancy in the USA (1,2). The American Cancer Society estimates over 68 000 new patients diagnosed with melanoma and 8700 died from malignant melanoma in 2010 (3). Once metastatic spread has occurred, melanoma is highly lethal. Importantly, metastatic melanoma is highly resistant to chemotherapy and radiation therapy (4). In contrast, melanoma has been shown to be susceptible to immune-based therapeutic approaches (5–7). Thus, it is critical to further understand how melanoma evades immune-mediated clearance and how stromal and environmental factors influence melanoma immuno surveillance.

Exposure of human skin to the ubiquitous environmental stimulus ultraviolet B radiation (UVB, 290–320 nm) is associated with both acute and chronic consequences ranging from the induction of inflammatory responses to immunosuppression and development of malignant melanoma and non-melanoma skin cancer (8–10). It is becoming recognized that UVB-initiated effects are mediated in part through membrane phospholipids. UVB irradiation is a potent pro-oxidative stressor (11,12) and can induce reactive oxygen species (ROS), generating lipid mediators as a direct result of lipid peroxidation. Among these biologically active oxidized lipids, the proinflammatory phospholipid platelet-activating factor (PAF; 1-O-alkyl-2-acetyl glycerophosphocholine) can be produced by UVB radiation through non-enzymatic-free radical attack on the sn-2 fatty acid of parent glycerophosphocholines as well as via tightly controlled enzymatic pathways (12). In addition to native PAF, this sn-2 oxidation also results in the production of other oxidized glycerophosphocholines (Ox-GPCs) exhibiting PAF-like activity (13). Once produced, PAF stimulates diverse biological functions such as acute inflammatory responses (e.g. anaphylaxis) and delayed processes such as systemic immunosuppression (14–17). PAF agonist production via ROS contrasts with the tightly controlled enzymatic pathways of PAF synthesis (18,19).

PAF and PAF-like species exert their effects via binding to a single specific transmembrane G-protein-coupled receptor, the PAF-receptor (PAF-R) (19). The expression of the PAF-R has been identified in immune cell types as well as keratinocytes of the skin (20,21). The interaction of PAF with PAF-R has been shown to activate various intracellular signaling pathways including intracellular calcium ion mobilization, phosphatidylinositol turnover and activation of cellular phospholipases and intracellular kinases (22,23).

Several lines of evidence have implicated the PAF system in melanoma pathogenesis. First, though not expressed on primary melanocytes, PAF-Rs have been reported to be expressed on melanoma cell lines (24). Second, several studies have demonstrated that PAF directly stimulates melanoma cell growth (25–27). Injection of B16F10 murine melanomas together with PAF in matrigel results in an augmentation of tumor growth in vivo (25). Overexpression of the PAF catabolizing enzyme, PAF-acetylhydrolase, in melanoma tumor cells has been reported to decrease tumor growth in vivo (26). Finally, systemic treatment with the PAF-R antagonist WEB2170 also inhibits experimental melanoma tumor growth (27). What is not clear from these studies, however, is whether host/stromal PAF-R or tumor cell PAF-R is mediating these effects.

UVB-mediated systemic immunosuppression has been classically measured by the ability of distant UVB irradiation to inhibit contact hypersensitivity (CHS) responses to chemical antigens such as dinitrofluorobenzene or delayed type hypersensitivity responses to antigens such as Candida (15). In regards to cell types and mediators involved, studies from several laboratories have demonstrated the importance of ROS, PAF, IL-10, cyclooxygenase-2 and mast cells (12,15–17,28). UVB has been shown to induce systemic immunosuppression in humans (29). Though UVB-mediated systemic immunosuppression has been linked to photocarcinogenesis, the significance of UVB-mediated systemic immunosuppression in other processes including melanoma progression is unknown (30). The present studies were designed to test whether systemic immunosuppression induced by UVB can modulate tumor growth in a murine model of melanoma tumor progression. This investigation demonstrates that UVB irradiation augments melanoma tumor growth via the generation of PAF-R agonists that act upon the host in a process that involves IL-10 and regulatory T cells (Treg). Delineation of the ability of environmental pro-oxidative stressors such as UVB to modulate melanoma tumor growth could have significant clinical implications in terms of providing potential insights into the mechanisms by which developing melanoma lesions evade immune surveillance.

Materials and methods

Reagents and UVB irradiation source

All chemicals were obtained from Sigma–Aldrich (St Louis, MO) unless indicated otherwise. RPMI 1640 media was ordered from Mediatech (Manassas, VA) and G418, Penicillin and streptomycin were from Invitrogen (Frederick, MD). Antibody against mouse anti-IL-10 was purchased from BD Biosciences (San Jose, CA), and anti-CD25 was from BioXcell (West Lebanon, NH). Isotype control antibodies against IgG and IgM were obtained from eBiosciences (San Diego, CA) and BioXcell. Murine PAF-R, enhanced green fluorescent protein (EGFP), CD3 and glyceraldehyde 3-phosphate dehydrogenase RT2 primers, first-strand synthesis and SYBR Green quantitative reverse transcription–PCR (qRT–PCR) reagents were purchased from SuperArray Bioscience Corp. (Frederick, MD). As previously reported (16,17) UV source was a Philips F20T12/UVB lamp. The intensity of the UVB source was measured before each experiment using an IL1700 radiometer and a SED240 UVB detector (International Light) at a distance of 8 cm from the UVB source to the anesthetized mice.

Cells and mice

The mouse melanoma B16F10 cell line, originally obtained from a metastatic lesion from the lungs of C57BL/6 mice (31), was maintained in RPMI 1640 (Mediatech) supplemented with 10% fetal bovine serum (Invitrogen) and 100 μg/ml penicillin and streptomycin (Invitrogen). A B16F10-PAF-R model system stably expressing the PAF-R was created by transduction of PAF-R-negative B16F10 cells with the MSCV2.1 retrovirus encoding the human leukocyte PAF-receptor as described previously (32). B16F10 cells stably transduced with the PAF-receptor (designated as B16-PAFR cells) or with control empty vector MSCV2.1 retrovirus (defined as B16-MSCV cells) were characterized by calcium mobilization studies using the calcium-sensitive dye Fura-2 (32) and by real-time quantitative PCR (16).

Pafr− /− mice on a C57BL/6 background, generated as described previously (33), were a kind gift of Prof. Takao Shimizu (University of Tokyo Department of Biochemistry). C57BL/6-wild-type (WT) mice (PAF-R expressing; age 8–12 weeks) were purchased from the Charles River Laboratories. Immunodeficient NOD.CB17-PrkdcSCID/J (common name: NOD SCID) mice were purchased from the Indiana University Simon Cancer Center Core facility. FoxP3EGFP knock-in transgenic mice on the C57BL/6 background (age 8–12 weeks) were procured from the Jackson Laboratories (34). These mice were housed under specific pathogen-free conditions at the Indiana University School of Medicine. All procedures were approved by the Animal Care and Use Committee of Indiana University School of Medicine.

Measurement of intracellular calcium ion influx

The presence of a functional PAF-R in B16-PAFR was identified by the ability of the metabolically stable PAF-R agonist, carbamoyl PAF (CPAF), to induce an intracellular Ca2+ mobilization response in B16-PAFR cells as described previously (17). In brief, B16-PAFR and B16-MSCV cells were preloaded with the Ca2+-sensitive indicator, fura-2-AM (4 μM in Hanks' balanced salt solution) at 37°C for 90 min, washed and resuspended in Hanks' balanced salt solution at room temperature before use. CPAF dissolved in ethanol (adjusted to 1 μM) was added to an aliquot of these cells (1.0–1.5 × 106 cells/2 ml) in a cuvette at 37°C with constant stirring. Fura-2-AM fluorescence was monitored in a Hitachi F-4010 spectrophotometer with excitation and emission wavelengths of 331 and 410 nm, respectively. The Ca2+ influx in suspensions was calculated as described (17) and shown as percentage of maximal peak calcium flux induced by 1 μM CPAF.

In vivo tumor growth studies

To determine the effect of UVB-induced systemic immunosuppression in melanoma tumor growth, 0.5 × 106 B16F10 cells, which lack functional PAF-R, were implanted s.c. into the shaved right hind flank of WT and Pafr− /− mice (six to seven mice per group) distant from UVB-irradiated site. Mice were either UVB irradiated on ∼2.5 × 2.5 cm area of the back skin at 5000 J/m2 dose (on day 0, 6 and 12) under anesthesia with an area from the neck and the lower body covering tumor injection site ∼2.5 cm distant from the UVB-irradiated site of mice was shielded with light impermeable black paper. The mice left unirradiated served as sham control.

To assess the role of antioxidants on UVB-induced melanoma tumor progression, WT mice were fed with regular chow or vitamin C-enriched (10 g/kg) chow (Research Diets, New Brunswick, NJ) and 5 mM N-acetyl cysteine (NAC) in water ad libitum for 10 days before tumor cell implantation. After 10 days, WT mice on regular chow or antioxidant diet were randomly distributed and divided into two groups (sham versus UVB; 6 mice per group) and 0.5 × 106 B16F10 cells were implanted followed by treatment with either sham or UVB as mentioned above.

To delineate whether host cell PAF-R or melanoma tumor cell PAF-R is playing a significant role in CPAF-induced enhanced melanoma tumor growth, 0.5 × 106 PAF-R-expressing (B16-PAF-R) or its vector control (B16-MSCV) cells were implanted into WT mice (six to nine mice per group) and treated with vehicle [50 μl phosphate-buffered saline (PBS)] or with 250 ng of CPAF (diluted in 50 μl PBS) on day 0, 6 and 12. To further confirm the role of host PAF-R, similar experiments were performed in Pafr− /− mice (6–7 mice per group).

To evaluate the involvement of host immune cells in CPAF-mediated systemic immunosuppression and the resulting increase in melanoma tumor growth, 0.5 × 106 B16F10 cells were implanted into immunodeficient NOD SCID mice (eight mice/group) followed by treatment with either vehicle or CPAF as mentioned above.

To evaluate the involvement of IL-10 in UVB-induced augmentation of melanoma tumor growth and to determine whether the PAF agonist CPAF mimics UVB-induced effects, WT mice (six mice per group) were first implanted with 0.5 × 106 B16F10 cells and then injected either with depleting antibody against mouse IL-10 or isotype control (IgG) antibody (100 μg per mouse) i. p. These mice were then treated with CPAF (250 ng) i. p. or UVB irradiated (5000 J/m2) on days 0, 6 and 12. For control groups, WT mice were either injected with vehicle (50 μl PBS) or left unirradiated (sham).

To determine the role of Tregs in UVB-induced melanoma tumor progression, FoxP3EGFP transgenic mice on the C57BL/6 background were injected with either depleting antibodies against CD25 (clone PC61.5.3 against IgG1 and 7D4 against IgM1) or isotype controls (IgG1 and IgM1) (1 mg each) 2 days before (−2 days) UVB irradiation. After 2 days, 0.5 × 106 B16F10 tumor cells were implanted followed by either sham or UVB irradiation as described above. The depletion of CD4+CD25+ Tregs in FoxP3EGFP mice was confirmed (on day 10; including 2 days of either isotype control or anti-CD25 antibody injection) by analyzing the expression of EGFP as a surrogate for Tregs in inguinal lymph nodes and spleen by flow cytometry analysis in a separate experiment.

In all the above experiments, tumor growth (length and width) was monitored and measured daily until day 14 with digital caliper, and tumor volume was calculated (length/width2/2). At the end of the experiment (day 14), mice were euthanized by CO2 asphyxiation and cervical dislocation; tumors were excised and their weights were measured. In addition, tumors were processed for histology, stored in RNA later for real-time quantitative PCR analysis and fluorescence-activated cell sorting (FACS) analysis.

Real-time RT–PCR

Total RNA was extracted from melanoma tumors using the RNAeasy kit (QIAGEN). In brief, tissue was homogenized in a RLT buffer containing β-mercaptoethanol by bullet blender (Next Advance, Averill Park, NY) using carbide beads following the manual's protocol (QIAGEN). Purified RNA was quantitated with the Nano Drop 2000 (Thermo Fisher Scientific, Lafayette, CO). Reverse transcription of whole RNA was done using Super Script cDNA synthesis kit (Invitrogen) with random hexamers. Complementary DNA was analyzed for content using an SYBR green-based, quantitative fluorescent PCR method (SA Biosciences, Frederick, MD) for PAF-R and EGFP and normalized against glyceraldehyde 3-phosphate dehydrogenase and CD3 as endogenous controls. Each assay was performed in duplicate in a 20 μl reaction volume with 2 × SYBR green mastermix, 2 μl of complementary DNA and primers at 10 μM. The following PCR conditions were used: 95°C for 15 min, followed by 45 cycles of 94°C for 15 s, 50°C for 30 s and 72°C for 30 s, followed by 72°C for a final 10 min. Each PCR reaction was also tested to assure a single product of the predicted size. Fluorescence was detected with the Step One Real-time PCR machine (Bio-Rad Laboratories, Hercules, CA). Quantification of each PCR product was expressed relative to glyceraldehyde 3-phosphate dehydrogenase or CD3.

Flow cytometry analysis

Tissue samples (draining lymph nodes and spleens) were harvested from isotype control antibody or anti-CD25 antibody treated FoxP3EGFP mice at day 10 (including −2 days of antibodies injection) and processed for the flow analysis. In brief, these tissues were dissociated into single cell suspension in RPMI media containing 10% fetal bovine serum using gentle MACS™ (Miltenyl Biotech, Auburn, CA). Tissue samples were filtered with cell strainer (40 μm) and centrifuged at 12 000 r.p.m. for 5 min. Spleens were processed initially with red blood cell lysis buffer. Cells from each tissue samples were washed twice with FACS buffer (PBS containing 2% fetal bovine serum) and counted. 1 × 106 cells from each organ were incubated with Fc blocking (CD16/32) antibody for 10 min after which cells were washed twice with FACS buffer. Foxp3-positive cell populations were quantitated based on EGFP as surrogate by FACS. Data files were analyzed using FlowJo software (Tree Star, Ashland, OR).

Statistical analysis

In the present study, at least five mice per group were used in all murine experiments. Statistical analyses were conducted using SAS version 9.3 (Cary, NC). Shapiro–Wilk’s and Levene’s tests were used to test normality and equal variance for days 12 and 14 tumor volume, tumor weight and qRT–PCR values. Normality was a reasonable assumption in all cases. For comparing two groups, we used equal or unequal variance t-tests. For comparing more than two groups, we used analysis of variance (with the Welch approximation if the variances were unequal) and post hoc Tukey-adjusted pair-wise tests. Only the P values from the overall F-tests are presented in the text. Statistical significance was defined as a P value <0.05.

Results

UVB-induced augmentation of melanoma tumor growth is dependent on the PAF-R system

Previous studies have demonstrated the importance of PAF-R signaling in UVB-mediated systemic immunosuppression as measured by the inhibition of CHS reactions to the contact allergen dinitrofluorobenzene (DNFB) using C57BL/6 mice expressing PAF-R (WT) and PAF-R-deficient mice (Pafr− /−) (16,17). Our first studies tested whether UVB irradiation at doses 2500, 5000 and 7500 J/m2, which induce systemic immunosuppression, could augment experimental murine melanoma growth. The dose of UVB irradiation (5000 J/m2) was selected by performing a pilot study in WT mice (five mice per group) harboring B16F10 cells followed by either sham or UVB irradiation at doses 2500, 5000 and 7500 J/m2 (Supplementary Figure 1 is available at Carcinogenesis Online and data not shown). The dorsal skin of WT and Pafr− /− mice was shaved, and 5 × 105 B16F10 cells were inoculated s.c. into the right flank of each mouse. This was followed by UVB irradiation (5000 J/m2 on day 0, 6 and 12) on an ∼2.5 × 2.5 cm area of shaved dorsal skin which was ∼3 cm away from the shielded tumor injection site (Figure 1A). Tumor growth was measured daily with calipers. As shown in Figure 1B, C and D, UVB irradiation of WT mice augmented the growth of B16F10 mouse melanoma tumors significantly compared with their non-irradiated (sham) counterparts. Importantly, Pafr− /− mice were resistant to this UVB-mediated augmentation of melanoma tumor growth (Figure 1B and D).

Fig. 1.

Fig. 1.

Open in a new tab

Treatment with UVB augments B16F10 melanoma tumor growth via PAF-R-dependent system: (A) Schematic representation of the experimental protocol. (B) As outlined in Materials and methods, 0.5 × 106 B16F10 cells were injected s.c. into dorsal hind flank skin of either C57BL/6 (WT) or Pafr− /−. An ∼2.5 × 2.5 cm area of shaved upper back was then treated with UVB (5 KJ/m2) or without UVB (sham) at days 0, 6 and 12. The tumors were measured daily with calipers and volume was calculated. (C) Representative pictures of sham and UVB-irradiated mice bearing melanoma tumors. (D) At the end of the experiment (day 14), whole tumors were excised and weighed (tumor weight). The data are represented as mean ± SE of tumor volume/weight from six to seven mice each group. For tumor volume, significant differences were observed (day 12, *P value <0.05; day 14, **P value <0.01) with the WT-UVB group being different from KO-UVB and KO-Sham on day 12 and being different from all other groups on day 14. Significant differences were also seen for tumor weight (*P value <0.05), with UVB-WT being different from Sham-KO.

Effect of antioxidants on UVB-induced melanoma tumor progression

Our previous studies have demonstrated that UVB irradiation resulted in the production of PAF and PAF-like Ox-GPCs via its ability to induce ROS (12,13). Feeding WT mice a vitamin C-enriched diet resulted in less UVB-generated PAF-R agonists and blocked systemic immunosuppression induced by UVB but not that induced by PAF-R agonist CPAF (17). These studies indicate that blocking ROS production abrogates UVB-induced immunosuppression by inhibiting the formation of Ox-GPC PAF-R agonist species. To determine the effect of antioxidants on UVB-induced melanoma tumor progression, WT mice were fed a vitamin C-enriched diet (10 g/kg) and 5 mM NAC in water for 10 days followed by the treatment with UVB as outlined in Materials and methods. As shown in Figure 2, the UVB-mediated increase in B16F10 melanoma tumor growth was significantly attenuated in the group of mice, which were treated with vitamin C/NAC as compared with UVB-treated mice on a regular diet. Final tumor weights mirrored the tumor volume measurements, demonstrating increased weight of tumors from UVB-irradiated hosts fed normal diets compared with those fed with an antioxidant diet (data not shown). Of note, other studies demonstrated that systemic administration of vitamin C or NAC alone (five mice per group) exerted only partial protection against UVB-induced melanoma tumor progression (Supplementary Figure 2 is available at Carcinogenesis Online). The finding that antioxidants vitamin C and NAC could block UVB-mediated augmentation of tumor growth suggests the involvement of oxidatively produced PAF species rather than enzymatically produced ones.

Fig. 2.

Fig. 2.

Open in a new tab

Treatment with vitamin C + NAC attenuates UVB-mediated augmentation of B16F10 melanoma tumor growth: C57BL/6 (WT) mice were either fed vitamin C-enriched chow diet and 5 mM NAC in water for 10 days or standard diet/water. The mice then underwent the protocol of UVB irradiation and melanoma implantation outlined in Figure 1. The tumors were measured daily with calipers. The data are represented as mean ± SE of tumor volume from six to seven mice each group. Significant differences were observed (day 12, **P value <0.01; day 14, ***P value <0.0001) with the regular diet + UVB group being different from the two vitamin C groups on day 12 and being different from all other groups on day 14.

Importance of host PAF-R signaling in melanoma tumor progression

Since PAF-Rs have been reported to be expressed on melanoma cells (24) and PAF-R activation has been reported to enhance tumor growth (25–27), the next studies were designed to define whether the augmentation of tumor growth by PAF-R signaling was due to direct effects of PAF on either the tumor or on the host cells. We first tested B16F10 cells for PAF-R messenger RNA (mRNA) expression by RT–PCR and for PAF-R activity by functional intracellular calcium ion mobilization studies using calcium-sensitive FURA-2 dye. These studies did not find significant levels of PAF-R transcripts in B16F10 cells nor did B16F10 cells respond with a calcium mobilization response to the PAF-R agonist, CPAF (data not shown). To test the role of the melanoma PAF-R in tumor cell growth, we transduced B16F10 cells with a PAF-R or empty MSCV2.1 retroviral construct to create B16F10PAF-R (B16-PAF-R) and B16F10MSCV2.1 (B16-MSCV), respectively. PAF-R mRNA expression was present in B16-PAF-R cells as demonstrated by qRT–PCR (Supplementary Figure 3 is available at Carcinogenesis Online), and PAF-R function was confirmed by intracellular calcium mobilization studies in response to CPAF (Figure 3A and B). Using these cell lines, we next assessed whether or not PAF-R expression in B16F10 cells alone would exert a growth promoting effect on melanoma tumors. To that end, 5 × 105 B16-PAF-R or B16-MSCV cells were implanted into the flanks of WT mice (six mice per group) and the growth of melanoma tumors was recorded. We observed that the expression of PAF-R alone was not associated with increased melanoma tumor growth as no difference in the tumor volumes or tumor weights was seen with these cells when implanted into WT mice (Supplementary Figure 4 is available at Carcinogenesis Online). We then evaluated the effect of PAF-R activation on the tumor growth of B16-PAF-R and B16-MSCV cells. To that end, we implanted 5 × 105 B16-PAF-R or B16-MSCV cells into the flanks of WT mice followed by treatment with or without PAF-R agonist (CPAF; 250 ng per mouse on day 0, 6 and 12). This dose of CPAF was used as our previous studies have demonstrated that i. p. injection of 250 ng CPAF mimics UVB-induced inhibition of CHS reactions to DNFB (17). As shown in Figure 3C and D, CPAF treatment resulted in a significant increase in tumor growth regardless of the PAF-R status of the B16F10 cells (no significant differences were noted between the tumor growths of B16-MSCV and B16-PAF-R cells exposed to CPAF treatment at day 14), suggesting the role of host PAF-R signaling rather than melanoma tumor cell PAF-R in mediating CPAF-induced melanoma tumor growth.

Fig. 3.

Fig. 3.

Open in a new tab

CPAF augments B16F10 tumor growth in WT mice regardless of B6F10 PAF-R expression status: (A) B16-PAF-R and (B) B16-MSCV cells were loaded with Fura-2-AM dye and intracellular Ca2+ levels monitored in response to treatment with 1 μM CPAF. C57BL/6 (WT) mice were injected with either (C) B16-PAFR or (D) B16-MSCV melanoma cells (0.5 × 106) on the dorsal hind flank skin s.c. followed by treatment with either vehicle or CPAF (250 ng) on days 0, 6 and 12. The tumors were measured daily with calipers. Similarly, Pafr− /− mice were injected with either (E) B16-PAFR or (F) B16-MSCV melanoma cells followed by treatment with either vehicle or CPAF. The tumors were measured daily with calipers. The data are represented as mean ± SE of tumor volume from six to nine mice in each group. Significant differences were observed for WT mice with either B-16 PAFR (day 12, *P value <0.05; day 14, *P value <0.05) and B16-MSCV cells (day 12, **P value <0.01; day 14, ***P value <0.001). No differences were observed in the Pafr− /− mice with any treatments at any time points. No significant differences were seen between similarly treated B16-MSCV and B16-PAF-R cells.

To further confirm the role of the host PAF-R signaling in mediating melanoma tumor growth, we performed the same studies in Pafr− /− mice, which demonstrated that CPAF failed to augment the growth of B16F10 cells regardless of the status of their PAF-R expression (Figure 3E and F). These studies indicate that activation of the PAF system augments the growth of B16 tumors in a manner dependent on host cell expression, but not melanoma tumor cell expression, of the PAF-R.

Role of immune cells in mediating PAF-R effects on melanoma tumor growth

The next studies were designed to assess the importance of the adaptive immune system in the PAF-R-mediated augmentation in an experimental murine melanoma tumor growth model. To that end, the ability of systemic CPAF to modulate tumor growth in T- and B-cell deficient severe combined immunodeficient (NOD SCID) mice was tested. As depicted in Figure 4A and B, CPAF treatment had no appreciable effect on tumor growth in NOD SCID mice. Of note, B16F10 tumors grew approximately twice as fast in these immunodeficient mice in comparison with their WT counterparts. In fact, the rate of tumor growth of B16F10 cells in NOD SCID mice resembled WT mice treated with UVB or CPAF. These findings demonstrate the importance of the adaptive immune system in mediating PAF agonist-mediated augmentation of melanoma tumor growth and reflect the immunosuppressive effect of UVB and CPAF.

Fig. 4.

Fig. 4.

Open in a new tab

Effect of CPAF treatment on the growth of melanoma tumors in NOD SCID mice. Immunodeficient NOD SCID mice were implanted with 0.5 × 106 B16F10 cells s.c. on dorsal hind flank skin followed by treatment either with vehicle or CPAF (250 ng) at days 0, 6 and 12. The tumors were measured daily with calipers. The data are represented as mean ± SE of tumor volume/weight from seven to eight mice each group. No significance differences were observed in tumor volume or tumor weight.

Involvement of IL-10 in UVB/CPAF-induced melanoma tumor progression

The following lines of evidence indicate the immunomodulatory cytokine IL-10 mediates PAF-induced immunosuppression. First, activation of the PAF-R in the murine epithelial cell line PAM-212 (15) as well as i.d. CPAF injection induce epidermal production of IL-10 in PAF-R-expressing WT mice (16). Second, UVB irradiation increases IL-10 mRNA levels in the lymph nodes and spleens of WT mice as compared with their respective control groups (16). Finally, depleting antibodies against IL-10 block both CPAF- and UVB-induced inhibition of CHS reactions to DNFB in WT mice (16).

The following studies were designed to assess whether IL-10 is involved in UVB or CPAF-induced melanoma tumor progression. WT mice engrafted with B16F10 tumor cells were injected with either depleting antibodies against IL-10 or isotype control antibodies, followed by the treatment with either CPAF or UVB and monitoring tumor growth. As shown in Figure 5, anti-IL-10 antibodies blocked both CPAF- and UVB-mediated melanoma tumor progression compared with isotype control antibody injected CPAF or UVB-treated mice. These studies indicate that IL-10 mediates both CPAF- and UVB-induced melanoma tumor progression.

Fig. 5.

Fig. 5.

Open in a new tab

Treatment with anti-IL-10 blocks both UVB/CPAF-mediated augmentation of B16F10 melanoma tumor growth. C57BL/6 (WT) mice were treated with either depleting Ab against IL-10 (100 μg) or isotype control (IgG) on days 0, 6 and 12 and treated with either (A) Sham or UVB (5000 J/m2) or (B) vehicle or CPAF (250 ng) at days days 0, 6 and 12. B16F10 cells were implanted at day 0 and the tumors were measured daily with calipers and volume calculated. The data are represented as mean ± SE of tumor volume from six to seven mice each group. Significant differences were observed for the UVB experiment (day 12, ***P value <0.001; day 14, **P value <0.01) with the UVB + IgG1 being different from all other groups. Similarly, significant differences were observed for the CPAF experiment (day 12, *P value <0.05; day 14, *P value <0.05) with the CPAF + IgG1 being different from all other groups.

Role of regulatory T cells in UVB-induced melanoma tumor progression

Recently, CD4+CD25+ Tregs have been shown to mediate UVR-induced localized immunosuppression in a process involving IL-10 (35). We thus hypothesized that Tregs could be mediating these PAF-induced effects on experimental melanoma tumor growth.

To assess a possible role of Tregs, FoxP3EGFP transgenic mice on the C57BL/6 background were utilized (34). These mice coexpress the Treg-specific transcription factor FoxP3 and an EGFP reporter, both under control of the endogenous FoxP3 promoter. Using these mice and FACS analysis of EGFP expression as a surrogate marker for Tregs, we tested the effective of depleting antibodies against CD25 on EGFP+ cells. FoxP3EGFP mice were injected with either isotype control antibodies (IgG and IgM) or depleting antibodies against CD25 (PC61.5.3 and 7D4). EGFP expression in the lymph nodes and spleens of these mice was analyzed by FACS after the 10th day. Depleting antibodies against CD25 reduced the numbers of EGFP-positive cells in lymph nodes and spleen by 50–60% compared with mice injected with isotype control antibodies (Supplementary Figure 5 is available at Carcinogenesis Online). Next, FoxP3EGFP mice were injected either with isotype control or anti-CD25 antibodies, and 2 days later, mice were implanted with B16F10 tumor cells and treated with or without UVB irradiation according to our standard protocol. We observed that partial depletion of Tregs with anti-CD25 antibodies blocked UVB-mediated augmentation of melanoma tumor growth. In contrast, isotype control antibodies injected into UVB-treated mice had no effect on tumor growth (Figure 6A). Furthermore, EGFP transcripts, in tumors, were increased by UVB irradiation in isotype antibody-treated mice, an effect lost in UVB irradiated mice treated with anti-CD25 antibodies and not present in sham-treated mice regardless of antibody treatment (Figure 6B). These findings further support the idea that Tregs are involved in UVB-mediated augmentation of melanoma tumor growth.

Fig. 6.

Fig. 6.

Open in a new tab

Treatment with anti-CD25 blocks UVB-mediated augmentation of B16F10 melanoma tumor growth: (A). Foxp3EGFP mice on a C57BL/6 background were injected either with isotype control antibodies against IgG and IgM or depleting antibodies against two different clones of anti-CD25 (PC61.5.3 and 7D4) (1 mg each) 2 days before beginning standard sham or UVB (5000 J/m2) irradiation and melanoma implantation protocol as outlined in Figure 1. The tumors were measured daily with calipers and volume calculated. The data are represented as mean ± SE of tumor volume from 9 to 11 mice in each group. (B). RNA was extracted from the tumor tissues and analyzed for EGFP expression (marker for FoxP3-Tregs) by qRT–PCR and normalized against CD3. The data are represented as mean ± SE from four mice each group. Significant differences were observed in tumor volume (day 12, ***P value <0.0001; day 14, ***P value <0.0001) with the IgG + IgM + UVB being different from all other groups. Significant differences were also observed for qRT–PCR (*P value <0.05) with IgG + IgM − UVB being different from IgG + IgM − Sham and CD25 − Sham.

Discussion

Although the role of the PAF system in mediating UVB-induced systemic immunosuppresion (14–17) and photocarcinogenesis (14,36) has been demonstrated, it is not known whether the systemic immunosuppressive effects of UVB could modulate melanoma tumor immunity. The present study lends support to the concept that UVB-induced generation of ox-GPCs having PAF-R agonistic activity augments the growth of experimental murine melanoma tumors. Moreover, these studies indicate that IL-10 and Tregs play critical roles in this PAF-R-dependent enhanced melanoma tumor progression.

The current study demonstrates that UVB irradiation to an area of skin distant from s.c. implanted B16F10 murine melanoma tumor augments tumor growth in PAF-R-expressing C57BL/6 (WT) mice compared with non-irradiated (sham) mice. The fact that UVB did not exert these growth-promoting effects in Pafr− /− mice and the ability of exogenous CPAF to mimic this effect, both confirm that the PAF system mediates the UVB effect. These results are consistent with previous reports demonstrating that UVB irradiation of murine and human skin generates novel lipid mediators (Ox[Black and white]-GPCs) having PAF-R agonistic activity and that these PAF-R agonists mediate UVB-induced systemic immunosuppression (12,13,15–17,37). Furthermore, the ability of antioxidants to block the UVB-mediated growth enhancing effects is evident that Ox-GPC PAF-R agonists are produced non-enzymatically in this process. Of note, pro-oxidative stressors other than UVB such as tert-butyl hydroperoxide and more clinically relevant agents such as chemotherapeutic drugs and cigarette smoke have also been reported to generate Ox-GPCs having PAF-R agonistic activity (38–40). Thus, it is possible that exposure to environmental pro-oxidative stressors other than UVB could generate this pro-tumor growth effect (39,40). This process could in turn explain the beneficial effects reported for combining systemic antioxidants with chemotherapeutic agents for treating human malignancies (41,42).

Previous studies have demonstrated that PAF-R agonists augment melanoma tumor growth in vitro and in vivo, effects that have been proposed to be a direct effect on the tumor cell PAF-R (24–26). For example, Melnikova et al. (24) have demonstrated that the PAF-R agonist, CPAF, increased the in vitro growth of PAF-R-expressing human melanoma cells in a dose-dependent manner in a process involving matrix metalloproteinase 2. Similarly, these authors found that CPAF treatment augmented human melanoma growth in SCID mice (24). In contrast, the present studies, through the use of murine PAF-R-negative and -positive tumor cells and immunocompetent hosts, indicate that PAF-R agonistic (CPAF) effects on the growth of s.c. implanted B16F10 melanoma tumor cells are due to host rather than direct tumor effects. In addition, implantation of only PAF-R-negative and -positive tumor cells into WT (PAF-R expressing) host without the addition of CPAF did not result in differences of tumor growth. Differences between the findings of previous studies indicating that PAF-R activation directly augments melanoma tumor growth could be due to differences in the signaling pathways induced by the native versus ectopically expressed PAF-R used in these studies.

The present studies demonstrating the lack of effect of exogenous CPAF on tumor growth in immunodeficient NOD SCID mice indicate that the adaptive immune system mediates this PAF-R-dependent response. The ability of depleting antibodies against IL-10 and Tregs to block PAF-R-mediated augmentation of tumor growth also attests to the involvement of the adaptive immune response. Nevertheless, it is possible that systemic PAF-R agonists could contribute to melanoma growth via both effects on tumor immunity as well as direct effects on PAF-R-expressing melanoma tumors.

Studies have implicated the involvement of CD4+CD25+-expressing regulatory T cell (Treg) in mediating UVR-induced immunosuppression (35,43–45). Tregs have also been recognized as playing an active role in the prevention of autoimmunity, graft versus host disease and transplant rejection (46). The development and function of a key population of Tregs are regulated by a member of the forkhead transcription factor family protein, FoxP3 (47). Several lines of evidence suggest the possible involvement of Tregs in mediating UVB-induced local as well as systemic immune tolerance. First, Elmets et al. (43) have observed that application of DNFB onto UV-irradiated skin results in the induction of Tregs that suppress CHS. Second, UVB-induced systemic immunosuppression in mice is associated with an increase in the number of Tregs, which is suppressed by the treatment with prostaglandin E2 receptor subtype 4 (EP4) antagonist (44). Third, IL-10, which is necessary for UVB-mediated systemic immunosuppression, is an important cytokine for Treg function (35,45). Finally, the Schwarz group has characterized the phenotypic and functional aspects of UV-induced Tregs in a local immunosuppression model and demonstrated that UVB irradiation activates Tregs that secrete IL-10 which promotes suppression of CHS (45). This UVB-mediated tolerance was blocked by i. p. injection of depleting antibody against IL-10 (45). These separate lines of evidence all suggest that Tregs play an important role in UVB-mediated immunosuppression. In addition to Tregs, IL-10-producing regulatory B cells, which are generated via UV radiation, could be involved in this process (48).

The present studies demonstrate that Tregs play an important role in Ox-GPC-induced melanoma tumor growth as depleting antibodies against CD25 substantially block UVB-induced tumor growth in FoxP3EGFP mice. Furthermore, the expression of EGFP mRNA, a marker for FoxP3 expressing Tregs, was significantly increased in the tumors of UVB-irradiated mice, an effect that was lost in mice treated with depletion of CD25+ cells. Consistent with these findings, selective depletion of FoxP3-expressing Tregs has been shown to improve vaccination strategies against established melanoma (49,50).

Of interest, Donawho et al. (51) reported >20 years ago that injection of murine melanoma and other tumors into UV-irradiated skin resulted in an augmentation of tumor growth. However, UV irradiation of one ear did not augment tumor growth in the other ear, suggesting that this was a local effect. Given that there appears to be a critical threshold for the amount of UVB to allow adequate production of Ox-GPC PAF-R agonists to augment melanoma tumor growth (Supplementary Figure 1 is available at Carcinogenesis Online), it is possible that the lack of efficacy of systemic UV previously reported could be due to not enough PAF-R agonists produced in their system.

In summary, the present studies indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity target host adaptive immune cells, most notably Tregs, to mediate systemic immunosuppression thus enhancing experimental murine melanoma tumor growth. These data also suggest that host expression of PAF-R, but not melanoma tumor cell expression of PAF-R, is involved in UVB/PAF-R-induced augmentation of melanoma tumor growth. These studies describe a previously unrecognized mechanism by which an environmental stressor allows melanoma tumor cells to escape antitumor immunity. Inasmuch as other pro-oxidative stressors could potentially generate PAF-R agonists, this pathway could have clinical relevance.

Funding

This research was supported in part by grants from the Riley Memorial association; the National Institutes of Health grants (HL062996 to J.B.T. and R.L.K., CA134014 to C.E.T.); the Veteran ' s Administration Merit Award (J.B.T.) and a Postdoctoral Fellowship grant from American Institute for Cancer Research (09A062 to R.P.S.).

Supplementary Material

Supplementary Data
supp_33_7_1360__index.html (1.7KB, html)

Acknowledgments

The authors thank Drs Dan F. Spandau, Yongxue Yao, Samin Rezania, Mohammed Al-Hassani and Ms Qiaofang Yi, Deena R. Mohamed and Sarah Defitt for technical assistance and/or helpful discussions for these studies.

Conflict of Interest Statement: None declared.

Glossary

Abbreviations

CPAF

carbamoyl PAF

CHS

contact hypersensitivity

DNFB

dinitrofluorobenzene

EGFP

enhanced green fluorescent protein

FACS

fluorescence-activated cell sorting

mRNA

messenger RNA

NAC

N-acetyl cysteine

Ox-GPC

oxidatively modified glycerophosphocholine

PAF

platelet-activating factor

PAF-R

PAF-receptor

PBS

phosphate-buffered saline

qRT–PCR

quantitative reverse transcription–PCR

ROS

reactive oxygen species

Tregs

regulatory T cells

UVB

ultraviolet B radiation

WT

wild-type

References

  • 1.Miller AJ, et al. Melanoma. N. Engl. J. Med. 2004;355:51–65. doi: 10.1056/NEJMra052166. [DOI] [PubMed] [Google Scholar]
  • 2.Rigel DS, et al. The incidence of malignant melanoma in the United States: issues as we approach the 21st century. J. Am. Acad. Dermatol. 1996;34:839–847. doi: 10.1016/s0190-9622(96)90041-9. [DOI] [PubMed] [Google Scholar]
  • 3.American Cancer Society. Cancer Facts & Figures. 2010. http://www.cancer.org/downloads/STT/500809web.pdf. [Google Scholar]
  • 4.Jilaveanu LB, et al. Chemotherapy and biologic therapies for melanoma: do they work? Clin. Dermatol. 2009;27:614–625. doi: 10.1016/j.clindermatol.2008.09.020. [DOI] [PubMed] [Google Scholar]
  • 5.Agarwala SS. Novel immunotherapies as potential therapeutic partners for traditional or targeted agents: cytotoxic T-lymphocyte antigen-4 blockade in advanced melanoma. Melanoma Res. 2010;20:1–10. doi: 10.1097/CMR.0b013e328333bbc8. [DOI] [PubMed] [Google Scholar]
  • 6.Kaehler KC, et al. Pegylated interferons: prospects for the use in the adjuvant and palliative therapy of metastatic melanoma. Eur. J. Cancer. 2010;46:41–46. doi: 10.1016/j.ejca.2009.10.004. [DOI] [PubMed] [Google Scholar]
  • 7.Fang L, et al. Immunotherapy for advanced melanoma. J. Invest. Dermatol. 2008;128:2596–2605. doi: 10.1038/jid.2008.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Carroll RP, et al. Incidence and prediction of nonmelanoma skin cancer post-renal transplantation: a prospective study in Queensland, Australia. Am. J. Kidney Dis. 2003;41:676–683. doi: 10.1053/ajkd.2003.50130. [DOI] [PubMed] [Google Scholar]
  • 9.Halliday GM. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis. Mutat. Res. 2005;571:107–120. doi: 10.1016/j.mrfmmm.2004.09.013. [DOI] [PubMed] [Google Scholar]
  • 10.Fernandez AA, et al. An experimental population study of nucleotide excision repair as a risk factor for UVB-induced melanoma. Photochem. Photobiol. 2011;87:335–341. doi: 10.1111/j.1751-1097.2010.00875.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Peus D, et al. H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes. J. Invest. Dermatol. 1998;110:966–971. doi: 10.1046/j.1523-1747.1998.00210.x. [DOI] [PubMed] [Google Scholar]
  • 12.Konger RL, et al. Oxidized glycerophosphocholines as biologically active mediators for ultraviolet radiation-mediated effects. Prostaglandins Other Lipid Mediat. 2008;87:1–8. doi: 10.1016/j.prostaglandins.2008.04.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Marathe GK, et al. Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage. J. Biol. Chem. 2005;280:35448–35457. doi: 10.1074/jbc.M503811200. [DOI] [PubMed] [Google Scholar]
  • 14.Ullrich SE. Mechanisms underlying UV-induced immune suppression. Mutat. Res. 2005;571:185–205. doi: 10.1016/j.mrfmmm.2004.06.059. [DOI] [PubMed] [Google Scholar]
  • 15.Walterscheid JP. Platelet-activating factor, a molecular sensor for cellular damage, activates systemic immune suppression. J. Exp. Med. 2002;195:171–179. doi: 10.1084/jem.20011450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Zhang Q, et al. UVB radiation-mediated inhibition of contact hypersensitivity reactions is dependent on the platelet-activating factor system. J. Invest. Dermatol. 2008;128:1780–1787. doi: 10.1038/sj.jid.5701251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Yao Y, et al. Ultraviolet B radiation generated platelet-activating factor receptor agonist formation involves EGF-R-mediated reactive oxygen species. J. Immunol. 2009;182:2842–2848. doi: 10.4049/jimmunol.0802689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Blank ML, et al. Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis. Biochem. Biophys. Acta. 1990;1042:153–158. doi: 10.1016/0005-2760(90)90001-e. [DOI] [PubMed] [Google Scholar]
  • 19.Ishii S, et al. Platelet-activating factor receptor. Prostaglandins Other Lipid Mediat. 2002;68-69:599–609. doi: 10.1016/s0090-6980(02)00058-8. [DOI] [PubMed] [Google Scholar]
  • 20.Zhuang Q, et al. Activation via multiple signaling pathways induces down-regulation of platelet-activating factor receptors on human B lymphocytes. J. Immunol. 2000;165:2423–2431. doi: 10.4049/jimmunol.165.5.2423. [DOI] [PubMed] [Google Scholar]
  • 21.Travers JB, et al. Identification of functional platelet-activating factor receptors on human keratinocytes. J. Invest. Dermatol. 1995;105:816–823. doi: 10.1111/1523-1747.ep12326581. [DOI] [PubMed] [Google Scholar]
  • 22.Stafforini DM, et al. Platelet-activating factor, a pleiotrophic mediator of physiological and pathological processes. Crit. Rev. Clin. Lab. Sci. 2003;40:643–672. doi: 10.1080/714037693. [DOI] [PubMed] [Google Scholar]
  • 23.Marques SA, et al. The platelet-activating factor receptor activates the ERK MAP kinase and induces proliferation of epidermal cells through an EGF receptor-dependent pathway. J. Pharmacol. Exp. Ther. 2002;300:1026–1035. doi: 10.1124/jpet.300.3.1026. [DOI] [PubMed] [Google Scholar]
  • 24.Melnikova VO, et al. Platelet-activating factor mediates MMP-2 expression and activation via phosphorylation of cAMP-response element-binding protein and contributes to melanoma metastasis. J. Biol. Chem. 2006;281:2911–2922. doi: 10.1074/jbc.M508683200. [DOI] [PubMed] [Google Scholar]
  • 25.Heon SK, et al. Platelet-activating factor induces up-regulation of antiapoptotic factors in a melanoma cell line through nuclear factor-kappaB activation. Cancer Res. 2006;66:4681–4686. doi: 10.1158/0008-5472.CAN-05-3186. [DOI] [PubMed] [Google Scholar]
  • 26.Biancone L, et al. Platelet-activating factor inactivation by local expression of platelet-activating factor acetyl-hydrolase modifies tumor vascularization and growth. Clin. Cancer Res. 2003;9:4214–4220. [PubMed] [Google Scholar]
  • 27.de Oliveira SI, et al. Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy. BMC Cancer. 2010;10:200. doi: 10.1186/1471-2407-10-200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Hart PH, et al. Mast cells, neuropeptides, histamine, and prostaglandins in UV-induced systemic immunosuppression. Methods. 2002;28:79–89. doi: 10.1016/s1046-2023(02)00201-3. [DOI] [PubMed] [Google Scholar]
  • 29.Poon TS, et al. Sunlight-induced immunosuppression in humans is initially because of UVB, then UVA, followed by interactive effects. J. Invest. Dermatol. 2005;125:840–846. doi: 10.1111/j.0022-202X.2005.23894.x. [DOI] [PubMed] [Google Scholar]
  • 30.Beissert S, et al. Ultraviolet-induced immunosuppression: implications for photocarcinogenesis. Cancer Treat. Res. 2009;146:109–121. doi: 10.1007/978-0-387-78574-5_10. [DOI] [PubMed] [Google Scholar]
  • 31.Im SY, et al. Augmentation of tumor metastasis by platelet-activating factor. Cancer Res. 1996;56:2662–2665. [PubMed] [Google Scholar]
  • 32.Pei Y, et al. Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis. J. Immunol. 1998;161:1954–1961. [PubMed] [Google Scholar]
  • 33.Ishii S, et al. Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor. J. Exp. Med. 1998;187:1779–1788. doi: 10.1084/jem.187.11.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Bettelli E, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. doi: 10.1038/nature04753. [DOI] [PubMed] [Google Scholar]
  • 35.Schwarz T. 25 years of UV-induced immunosuppression mediated by T cells-from disregarded T suppressor cells to highly respected regulatory T cells. J. Photochem. Photobiol. 2008;84:10–18. doi: 10.1111/j.1751-1097.2007.00223.x. [DOI] [PubMed] [Google Scholar]
  • 36.Sreevidya CS, et al. Inhibition of photocarcinogenesis by platelet-activating factor or serotonin receptor antagonists. Cancer Res. 2008;68:3978–3984. doi: 10.1158/0008-5472.CAN-07-6132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Travers JB, et al. Ultraviolet B radiation of human skin generates platelet-activating factor receptor agonists. Photochem. Photobiol. 2010;86:949–954. doi: 10.1111/j.1751-1097.2010.00743.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Travers JB. Oxidative stress can activate the epidermal platelet-activating factor receptor. J. Invest. Dermatol. 1999;112:279–283. doi: 10.1046/j.1523-1747.1999.00521.x. [DOI] [PubMed] [Google Scholar]
  • 39.Darst M, et al. Augmentation of chemotherapy-induced cytokine production by expression of the platelet-activating factor receptor in a human epithelial carcinoma cell line. J. Immunol. 2004;172:6330–6335. doi: 10.4049/jimmunol.172.10.6330. [DOI] [PubMed] [Google Scholar]
  • 40.Lehr HA, et al. Vitamin C blocks inflammatory platelet-activating factor mimetics created by cigarette smoking. J. Clin. Invest. 1997;99:2358–2364. doi: 10.1172/JCI119417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Muralikrishnan G, et al. Effect of vitamin C on lipidperoxidation and antioxidant status in tamoxifen-treated breast cancer patients. Chemotherapy. 2010;56:298–302. doi: 10.1159/000320030. [DOI] [PubMed] [Google Scholar]
  • 42.An SH, et al. Vitamin C increases the apoptosis via up-regulation p53 during cisplatin treatment in human colon cancer cells. BMB Rep. 2011;44:211–216. doi: 10.5483/BMBRep.2011.44.3.211. [DOI] [PubMed] [Google Scholar]
  • 43.Elmets CA, et al. Analysis of the mechanism of unresponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation. J. Exp. Med. 1983;158:781–794. doi: 10.1084/jem.158.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Soontrapa K, et al. Prostaglandin E2-prostaglandin E receptor subtype 4 (EP4) signaling mediates UV irradiation-induced systemic immunosuppression. Proc. Natl Acad. Sci. USA. 2011;108:6668–6673. doi: 10.1073/pnas.1018625108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Maeda A, et al. Phenotypic and functional characterization of ultraviolet radiation-induced regulatory T cells. J. Immunol. 2008;180:3065–3071. doi: 10.4049/jimmunol.180.5.3065. [DOI] [PubMed] [Google Scholar]
  • 46.Raimondi G, et al. Naturally occurring regulatory T cells: recent insights in health and disease. Crit. Rev. Immunol. 2007;27:61–95. doi: 10.1615/critrevimmunol.v27.i1.50. [DOI] [PubMed] [Google Scholar]
  • 47.Hori S, et al. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003;299:1057–1061. [PubMed] [Google Scholar]
  • 48.Matsumura Y, et al. A role for inflammatory mediators in the induction of immunoregulatory B cells. J. Immunol. 2006;177:4810–4817. doi: 10.4049/jimmunol.177.7.4810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Klages K, et al. Selective depletion of Foxp3+ regulatory T cells improves effective therapeutic vaccination against established melanoma. Cancer Res. 2010;70:7788–7799. doi: 10.1158/0008-5472.CAN-10-1736. [DOI] [PubMed] [Google Scholar]
  • 50.Steitz J, et al. Depletion of CD25(+) CD4(+) T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon alpha-induced, CD8(+) T-cell-dependent immune defense of B16 melanoma. Cancer Res. 2001;61:8643–8646. [PubMed] [Google Scholar]
  • 51.Donawho CK, et al. Immunologic factors in melanoma. Clin. Dermatol. 1992;10:69–74. doi: 10.1016/0738-081x(92)90059-8. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_33_7_1360__index.html (1.7KB, html)
supp_bgs152_Fig_S-1.tif (2MB, tif)
supp_bgs152_Supplemental_Fig_2-final.tif (1.7MB, tif)
supp_bgs152_Supplemental_Fig_3-final.tif (842.3KB, tif)
supp_bgs152_Supplemental_Fig_4-final.tif (782.2KB, tif)
supp_bgs152_Supplemental_Fig_5-final.tif (1.9MB, tif)

Articles from Carcinogenesis are provided here courtesy of Oxford University Press

RESOURCES