Suppression Of Contact Hypersensitivity Response Research Articles

Triggering receptor expressed on myeloid cells (Trem)-1 plays important roles in UVB induced immune suppression and cutaneous carcinogenesis

Abstract American Cancer Society acknowledges that most skin cancers result from exposure to ultraviolet (UV) rays in sunlight. UVB radiation induces mutant cells as well as immune suppression. Tumors only occur when mutant cells are in an immune suppressive environment. The mechanisms for UVB induced immune suppression are yet to be fully elucidated. The goal of this work is to explore new mechanisms for UVB induced immune suppression and skin carcinogenesis by using advanced technology. Analysis of UVB exposed mouse (Nanostring) and human (RNAseq) skin shows that triggering receptor expressed on myeloid cells (TREM)-1 signal pathway is the top canonic pathway increased by UVB. TREM-1 has roles in inflammatory diseases and cancers. However, its role in UVB induced immune suppression and skin cancers is not understood. We have found by using multi-color flow cytometry that UVB induces TREM-1 expression in a subset of conventional dendritic cell type 2 (cDC2) in the draining lymph nodes (LN). TREM-1+ cDC2 cells are barely detected in normal LN and express a high level of the co-inhibitory molecule PD-L1. In functional assays, treatment of mice with a specific TREM-1 blocking peptide LP-17 significantly diminishes UVB-induced suppression of contact hypersensitivity responses that are mediated by T cells. Further, mice treated with this peptide show decreased incidence of photocarcinogenesis and reduced number of tumors. In summary, our findings demonstrate that UVB induces the development of a novel TREM-1+ cDC2 cell subset and that TREM-1 has an important role in UVB induced immune suppression and skin carcinogenesis. Importantly, our study implies new strategies for targeting TREM-1 in the prevention and treatment of UVB induced skin cancers. Supported by grants from NIH (R01AR072213 and R01AI154842)

768 Type I interferons modulate ultraviolet radiation induced suppression of immune responses via Stimulator of Interferon Genes (STING)

UV (ultraviolet) B induced immune suppression contributes in a major way to the growth and development of UVB-induced skin cancers. Type I interferons (IFNs) are cytokines that play a role in regulation of proliferation, differentiation, and immune function. All characterized type I IFNs transmit their signals through the type I IFN receptor (IFNAR) composed of IFNAR1 and IFNAR2 components. Loss of either subunit results in complete abrogation of receptor function. There is limited information on the role for type I IFNs (IFN-α/β) signaling in UVB induced immune responses. Stimulator of IFN genes (STING) is an adaptor that responds to intracytoplasmic DNA by stimulating the production of type I IFN. The purpose of this study was to determine whether type I IFNs contributed to UV-induced DNA damage and immunosuppression via Stimulator of Interferon genes (STING) pathway. IFNAR1 deficient (IFNAR1-/-) and STING deficient (STING-/-) on C57BL/6 background and wild type (WT) mice were subjected to a local UVB regimen consisting of 100 mJ/cm2 UVB radiation for 4 days followed by sensitization with the hapten 2, 4-dinitrofluorobenzene (DNFB). UVB radiation in wild type mice induced significant (p<0.001) suppression of contact hypersensitivity response which was greatly enhanced (p<0.001) in IFNAR1-/- and STING-/- mice. The enhanced immune suppression was associated with significant decrease (p<0.001) in CD8+ T-cells and increase (p<0.001) in CD4+CD25+Foxp3+ populations in IFNAR1-/- and STING-/- mice when compared with WT mice. These findings can be used to develop type I IFN agonists for prevention against UVB induced immune suppression.

Regulatory B cells induced by ultraviolet B through toll-like receptor 4 signalling contribute to the suppression of contact hypersensitivity responses in mice.

Ultraviolet (UV) B irradiation is known to suppress contact hypersensitivity (CHS) responses in mouse models by suppressing immune responses. However, the cellular mechanisms responsible for UVB-induced systemic suppression remain unclear. Regulatory B cells have been reported to play an inhibitory role during CHS. It is presently unknown whether regulatory B cells contribute to the effect of UVB phototherapy. To investigate the inductive effect of UVB on regulatory B cells and the underlying mechanisms by using a CHS mouse model. CHS was induced with oxazolone, and evaluated by histopathology, flow cytometry, and quantitative real-time polymerase chain reaction. We found that UVB irradiation induced regulatory B cell expansion and ameliorated CHS. UVB-induced regulatory B cells contribute to systemic immunosuppression by inhibiting the proliferation of T cells. Moreover, we determined that toll-like receptor (TLR) 4, the expression of which was upregulated in B cells after UVB exposure, played an essential role in the induction of regulatory B cells. Our data identified regulatory B cells as regulators of UVB-induced immunosuppression in CHS, and suggest the importance of the UVB-TLR4 axis in the generation of regulatory B cells.

Abstract 2221: Honokiol stimulates immune reactivity in UV-irradiated skin through DNA demethylation-dependent functional activation of dendritic cells in mice

Abstract Solar ultraviolet radiation (UVR), and in particular UVB spectrum (290-320 nm), are considered as a complete carcinogen for cutaneous malignancies. The over-exposure of UVR suppresses the development of allergic contact hypersensitivity (CHS) response in both laboratory animals as well as in humans. CHS response is considered to be a prototypic T-cell mediated immune response. UVR-induced suppression of immune sensitivity has been implicated in skin cancer risk. Therefore, the treatment options which can inhibit UVB-induced suppression of immune sensitivity may be useful in the management of skin cancers, including both melanoma and non-melanoma. Previously, we have shown that topical treatment of honokiol inhibits UVB induced immune suppression, which was associated with the reduction in UVB-induced inflammatory mediators in the mouse skin. Here, we have further determined the underlying mechanism of action of honokiol on UVR-induced immunosuppression including its effect on epigenetic regulators and their relationship with immune sensitivity in mouse skin. Topical treatment of C3H/HeN mice with honokiol (0.5 and 1.0 mg/cm2 skin area) in hydrophilic-cream based topical formulation significantly inhibits UVB-induced suppression of CHS response (P&amp;lt;0.01-0.005), which was associated with reduced levels of DNA methylation as well as Dnmt activity in the mouse skin compared with non-honokiol-treated and UVB-exposed control mice. To characterize the cell population responsible for the honokiol mediated inhibition of UVB-induced immunosuppression, we used an adoptive transfer approach. DNA-methylated (most of cells) dendritic cells (DCs), isolated from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) with and without honokiol (0.5 and 1.0 mg/cm2 skin area) treatment, were transferred into naïve recipient mice. The CHS response of the recipient mice to DNFB was then measured. Honokiol treatment of UVB-exposed donor mice relieved this suppression of the CHS response in naïve mice, while the recipient mice, obtained DNA-methylated DCs from UVB-exposed donor mice that were not treated with honokiol, showed significant suppression in the CHS response. The restoration of CHS response in mice receiving DCs from honokiol treated, UV-exposed donor mice was also associated with enhanced secretion of Th1-type cytokines compared with the Th1-type cytokines from the DCs obtained from non-honokiol-treated and UVB-exposed donor mice. These data suggest that honokiol mediated inhibition of UVB induced suppression of CHS response is associated with functional activation of dendritic cells and that is dependent on DNA demethylation in DCs. Note: This abstract was not presented at the meeting. Citation Format: Santosh K. Katiyar, Tripti Singh, Harish C. Pal, Ram Prasad. Honokiol stimulates immune reactivity in UV-irradiated skin through DNA demethylation-dependent functional activation of dendritic cells in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2017-2221

Abstract 2607: Honokiol, a phytochemical from magnolia plant, prevents UV radiation-induced immune suppression in mice through inhibition of PGE2 and DNA methylation

Abstract The exposure of ultraviolet radiation (UVR) to murine skin suppresses the development of allergic contact hypersensitivity (CHS) response which is considered to be a prototypic T-cell mediated immune response. UVR-induced suppression of immune system has been implicated in skin cancer risk. Honokiol has been shown to have anti-skin carcinogenic activity, and here we have assessed the effect of honokiol on UVR-induced immunosuppression and its possible mechanism of action. Our CHS experiments revealed that exposure of C3H/HeN mouse skin with UVB radiation (150 mJ/cm2) for 4 consecutive days resulted in significant suppression of CHS response (75%, P&amp;lt;0.001) to the skin contact sensitizer, 2,4-dinitrofluorobenzene (DNFB), which was associated with the enhancement in the levels of cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. Topical treatment of mice with honokiol (0.5 and 1.0 mg/cm2 skin area) in hydrophilic-cream based topical formulation or indomethacin (50 μg/mouse), a COX-2 inhibitor, significantly inhibits UV radiation-induced suppression of CHS response (P&amp;lt;0.01-0.005). The exposure of mice with UVB resulted in DNA hypermethylation, increase in DNA methyltransferase (Dnmt) activity, and elevated levels of Dnmt proteins in mouse skin, while treatment of mice with honokiol before each UVB exposure reduced the levels of DNA methylation as well as Dnmt activity compared with non-honokiol-treated control mice. The COX-2 deficient mice are resistant to UV-induced suppression of CHS response. Treatment of UVB exposed COX-2-deficient mice with PGE2 (50 μg/mouse/day) results in UV-induced suppression of CHS, increase in DNA methylation and Dnmt activity in the skin. Further, treatment of honokiol reversed the effect of PGE2 on UV-induced suppression of CHS response in COX-2-deficient mice. Treatment of C3H/HeN mice with 5-aza-2’-deoxycytidine (5-Aza-dc, 1.0 mg/kg body weight, i.p.), a DNA demethylating agent, after each UVB exposure restored the CHS response which was associated with a reduction in Dnmt activity and global DNA methylation levels compared to the UV-exposed mice which were not treated with 5-Aza-dc. We also compared the effect of honokiol with commercially available anti-cancer drugs, such as imiquimod and 5-fluorouracil, on CHS response in UV-exposed mice. Topical treatment of mice with equi-molar concentrations of honokiol and cancer drugs did not show significant difference in terms of inhibition of UVB induced immune suppression and thus suggests honokiol as a substitute for these drugs. These findings provide evidence that honokiol acts through inhibition of inflammatory mediators and subsequently DNA demethylation in UVR-exposed mouse skin. Citation Format: Santosh K. Katiyar, Tripti Singh, Ram Prasad. Honokiol, a phytochemical from magnolia plant, prevents UV radiation-induced immune suppression in mice through inhibition of PGE2 and DNA methylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2607.

Severity of polymorphic light eruption in pre‐ and post‐menopausal women: a comparative study

Polymorphic light eruption (PLE) is approximately four times more common in women than in men and often begins in young adult life. It is hypothesized that patients with PLE have an inherent resistance to UVL-induced immunosuppression, which is a physiological phenomenon in normal healthy individuals. Consequently, in PLE there is a delayed-type hypersensitivity reaction to a UVL-modified skin antigen, which results in an inflammatory reaction and a variable rash. The female hormone, 17β-oestradiol, has been shown to inhibit UVL-induced physiological suppression of contact hypersensitivity responses. This has been postulated to account for the female preponderance of PLE. If 17β-oestradiol plays a significant part in the disease, one might hypothesize that the severity of PLE might reduce in women after menopause. To compare the severity of PLE in pre-menopausal women with that in post-menopausal women. Eighteen pre-menopausal and 18 post-menopausal women with PLE had their Polymorphic Light Eruption Severity Index (PLESI) scored by a single investigator. Pre-menopausal women (mean age 40years; range 25-50) had a mean PLESI of 54.8 (range 0-86, SD 20.2). Post-menopausal women (mean age 63years; range 53-78) had a mean PLESI of 36.8 (range 0-74, SD 18.2). A significant difference in mean PLESI values between pre- and post-menopausal women was noted (18.0; 95% CI 4.9-31.0; P = 0.008). At the time of the study, three subjects in the pre-menopausal group and one subject in the post-menopausal group were on oestrogen preparations. Even after excluding the four patients on oral oestrogens, there remained a statistically significant difference in the mean PLESI scores between the pre-menopausal and post-menopausal groups (55.10 vs. 36.64; difference of 18.46, 95% CI: 4.0-32.91; P = 0.014). The severity of PLE was significantly less in post-menopausal women as compared with pre-menopausal women.

Abstract 1275: Prostaglandin E2 promotes ultraviolet radiation-induced suppression of immune system in mice via DNA hypermethylation.

Abstract Excessive exposure of skin to solar ultraviolet (UV) radiation is known to suppress the immune system and UV-induced immunosuppression has been implicated in the promotion of the risk of skin cancer. This notion is further verified by the fact that chronically immune-suppressed patients who live in regions of intense sun exposure have an exceptionally high rate of skin cancer. To identify the molecular targets responsible for UV-induced immunosuppression, further studies were conducted. We have found that exposure of the C3H/HeN mouse skin with UV radiation (150 mJ/cm2) for 4 consecutive days resulted in suppression of contact hypersensitivity (CHS) response, which is considered to be a prototypic of T-cell mediated immune response, and suppression of CHS was associated with the enhancement in the expression levels of cyclooxygenase-2 (COX-2) and prostaglandin (PG) E2 production. Topical treatment of mice with celecoxib (500 μg) or indomethacin (50 μg), inhibitors of COX-2, or AH6809 (25 μg), an antagonist of PGE2, inhibits UV radiation-induced suppression of CHS. The mice deficient in COX-2 on C3H/HeN background are resistant to UV-induced suppression of CHS response to a contact sensitizer. Exposure of C3H/HeN mice with UVB radiation (150 mJ/cm2) for 4 consecutive days resulted in DNA hypermethylation, increase in DNA methyltransferase (Dnmt) activity, and elevated levels of Dnmt1, Dnmt3a and Dnmt3b in epidermal skin samples, while treatment of mice with PGE2 antagonist or indomethacin before and during UVB exposure down-regulated the levels of DNA methylation as well as Dnmt activity compared to the mice which were not treated with PGE2 antagonist or indomethacin. Treatment of UV exposed COX-2-deficient mice with PGE2 (150 μg in 100μl/mouse/day) results in UV-induced suppression of CHS, increase in DNA methylation, Dnmt activity and the levels of Dnmt1, Dnmt3a and Dnmt3b were elevated in the skin. Treatment of C3H/HeN mice with 5-aza-2’-deoxycytidine (5-aza-dc, 1.0 mg/kg body weight, i.p.), a DNA demethylating agent or an inhibitor of DNA methylation, 1 h before each exposure of UV irradiation resulted in inhibition of UVB-induced suppression of CHS which was associated with the reduction in Dnmt activity, global DNA methylation and reduced levels of Dnmt1, Dnmt3a and Dnmt3b proteins compared to the mice which were not treated with 5-aza-dc but exposed to UV. Further, treatment of 5-aza-dc reversed the effect of PGE2 on UV-induced suppression of CHS response in COX-2-deficient mice. These findings uncover a previously unrecognized role of PGE2 in the promotion of UVB-induced suppression of CHS response and that it is mediated through DNA methylation. Citation Format: Ram Prasad, Santosh K. Katiyar. Prostaglandin E2 promotes ultraviolet radiation-induced suppression of immune system in mice via DNA hypermethylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1275. doi:10.1158/1538-7445.AM2013-1275

Regulation of ultraviolet radiation induced cutaneous photoimmunosuppression by Toll-like receptor-4

UVB radiation is a potent immunosuppressive agent that inhibits cell-mediated immune responses. The mechanisms by which UVB radiation influences cell-mediated immune responses have been the subject of extensive investigation. However, the role of innate immunity on photoimmunological processes has received little attention. The purpose of this study was to determine whether Toll-like receptor-4 (TLR4) contributed to UV-induced suppression of contact hypersensitivity (CHS) responses. TLR4 −/− and wild type C57BL/6 (TLR4 +/+) mice were subjected to a local UVB immunosuppression regimen consisting of 100 mJ/cm 2 UVB radiation followed by sensitization with the hapten DNFB. Wild type TLR4 +/+ mice exhibited significant suppression of contact hypersensitivity response, whereas TLR4 −/− developed significantly less suppression. The suppression in wild type TLR4 +/+ mice could be adoptively transferred to naïve syngeneic recipients. Moreover, there were significantly fewer Foxp3 expressing CD4 +CD25 + regulatory T-cells in the draining lymph nodes of UV-irradiated TLR4 −/− mice than TLR4 +/+ mice. When cytokine levels were compared in these two strains after UVB exposure, T-cells from TLR4 +/+ mice produced higher levels of IL-10 and TGF-β and lower levels of IFN-γ and IL-17. Strategies to inhibit TLR4 may allow us to develop immunopreventive and immunotherapeutic approaches for management of UVB induced cutaneous immunosuppression.

Wavelength dependency for UVA-induced suppression of recall immunity in humans

Ultraviolet (UV) A radiation, which has both mutagenic and immune suppressive effects on the skin, is increasingly recognised as a key contributor to cutaneous carcinogenesis. Whilst short wavelength UVB (290-320 nm) is well-recognised as an environmental health hazard, the dangers of UVA (320-400 nm) are relatively unexplored. Using the nickel model of recall immunity in healthy human volunteers, we determined the wavelength dependency for UV-induced immunosuppression across the UVA spectrum. Dose-response curves were established for local suppression of contact hypersensitivity responses to nickel at 7 wavelengths between 331 and 392 nm. We found a broad peak of UVA immune suppressive effectiveness at 364-385 nm. Whilst we had previously found linear dose-responses for UVB-induced immunosuppression in this model, long wavelength UVA caused bell-shaped, Gaussian dose-responses, suggesting different chromophores and mechanisms of immunosuppression for UVB and UVA. The immunosuppressive peak induced by longwave UVA has not been described previously for any species and being close to the border with visible light indicates an unexpected role for these long wavebands in human health and disease.

Abstract 1875: Dietary grape seed proanthocyanidins induce rapid repair of DNA damage via nucleotide excision repair genes in preventing UV-induced immunosuppression

Abstract Ultraviolet (UV) radiation-induced immunosuppression has been implicated in the development of skin cancers, including melanoma and non-melanoma. 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 skin carcinogenesis. We have shown earlier that administration of dietary grape (Vitis vinifera) seed proanthocyanidins (GSPs) supplemented with AIN76A control diet inhibit photocarcinogenesis in mice; however, the molecular mechanism of chemopreventive effects of GSPs are not clearly understood. To investigate the possible mechanism of action of GSPs we used genetically modified human cells and animal model. We observed that administration of dietary GSPs (0.2 and 0.5%, w/w) with supplementation of AIN76A control diet inhibited (52-65%, P&amp;lt;0.001) UV-induced suppression of contact hypersensitivity response to a contact sensitizer, 2,4-dinitrofluorobenzene, in a contact hypersensitivity mouse (C3H/HeN) model. Dietary GSPs repaired UV-induced DNA damage (CPD-positive cells) faster in the skin of mice as demonstrated by reduced number of CPD-positive cells (59%, p&amp;lt;0.001), and reduced the migration of CPD-positive cells from the skin to draining lymph nodes compared to non-GSPs-fed control mice, which was associated with the elevated levels of nucleotide excision repair (NER) genes. GSPs did not prevent UV-induced immunosuppression in NER-deficient mice but significantly prevented in NER-proficient mice (p&amp;lt;0.001) concomitantly repaired UV-induced DNA damage in NER-proficient mice (p&amp;lt;0.01) but did not repair in NER-deficient mice as indicated by immunohistochemical analysis of CPD-positive cells. Further, southwestern dot-blot analysis revealed that GSPs repaired UV-induced CPDs 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 xeroderma pigmentosum disease, indicating that NER mechanism is involved in DNA repair. Together, these data identify a novel mechanism by which dietary GSPs prevent UV-induced immunosuppression which is mediated through rapid repair of damaged DNA, and that these effects lead to the prevention of UV radiation-induced skin tumors in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1875.

Immune-modifying properties of topical vitamin D: Focus on dendritic cells and T cells

Topical creams containing the active form of vitamin D (1,25-dihydroxyvitamin D 3; 1,25(OH) 2D 3) or analogues of this compound are currently used with some success to treat skin conditions including psoriasis and vitiligo. As well as targeting inflammatory processes in the skin, topical application of 1,25(OH) 2D 3 also affects the function of immune cells in the skin and draining lymph nodes. Topically applied 1,25(OH) 2D 3 reduces the number of dendritic cells in the skin, resulting in suppressed immunity and in particular reduced contact hypersensitivity (CHS) responses. Topical 1,25(OH) 2D 3 may also promote the migration of dendritic cells from the skin to the draining lymph nodes. Skin application of 1,25(OH) 2D 3 prevented the inflammatory effects of UVB irradiation on lymph node hypertrophy, when cell numbers were examined 4 days after skin treatment. In contrast, when 1,25(OH) 2D 3 was applied to UVB irradiated skin, there was no reversal in the suppression of CHS responses caused by UVB irradiation. Instead, 1,25(OH) 2D 3 had an additive effect with UVB to suppress CHS responses to a greater degree than UVB alone. In these studies, 1,25(OH) 2D 3 was applied to the treated skin of BALB/c mice immediately following UVB irradiation. Finally, topical 1,25(OH) 2D 3 also enhanced the number and suppressive activity of CD4+CD25+ regulatory T cells in the lymphatic tissue draining skin.

Immune protective effect of a moisturizer with DNA repair ingredients

Ultraviolet (UV) light damages DNA and impairs immune surveillance. The faulty repair of DNA after UV exposure is associated with immune suppression and facilitates photodamage that leads to photoaged skin and the growth of skin cancer. Sunscreens have been developed to filter UV light from entering the skin, but are not beneficial once DNA damage has occurred. Enhancing DNA repair after UV radiation may provide added advantage and prevent UV immunosuppression. This study was performed to determine whether a product with DNA repair ingredients prevents UV-induced suppression of contact hypersensitivity responses in vivo. Solar simulated radiation was delivered on skin with and without topical treatment with a moisturizer containing DNA repair enzymes (Advanced Night Repair Concentrate). Subjects were then sensitized to the hapten dinitrochlorobenzene, and the level of resultant contact hypersensitivity response was elicited 2 weeks later. Contact hypersensitivity response measured by skin fold thickness was significantly suppressed in untreated UV-irradiated subjects but not in subjects treated with DNA repair moisturizer after solar simulated radiation. Our results indicate that DNA repair ingredients significantly prevent UV-induced immune suppression.

Silymarin inhibits UV radiation-induced immunosuppression through augmentation of interleukin-12 in mice

We have shown previously that silymarin, a plant flavonoid, inhibits UVB-induced photocarcinogenesis in mice. As UVB-induced immunosuppression has been implicated in the development of skin cancer, we investigated whether silymarin can modulate the effects of UVB radiation on the immune system. Treatment of C3H/HeN mice with topically applied silymarin (0.5 or 1.0 mg/cm(2)) or silibinin, a major component of silymarin, markedly inhibited UVB (180 mJ/cm(2))-induced suppression of contact hypersensitivity response in a local model of immunosuppression and had a moderate inhibitory effect in a systemic model of contact hypersensitivity. Silymarin reduced the UVB-induced enhancement of the levels of the immunosuppressive cytokine, interleukin (IL)-10, in the skin and draining lymph nodes and enhanced the levels of the immunostimulatory cytokine, IL-12. Intraperitoneal injection of mice treated with silymarin with an endotoxin-free neutralizing anti-IL-12 antibody abrogated the protective effects of the silymarin against UVB-induced suppression of the contact hypersensitivity response. Furthermore, the treatment of silymarin did not prevent UVB-induced suppression of the contact hypersensitivity response in IL-12 knockout mice but prevented it in their wild-type mice. Moreover, i.p. injection of IL-12 to silymarin-treated or non-silymarin-treated IL-12 knockout mice resulted in an enhanced response to contact hypersensitivity compared with the response in mice that were exposed to either UVB alone or silymarin plus UVB. These data indicate for the first time that silymarin has the ability to protect mice from UVB-induced immunosuppression and that this protective effect is mediated, at least in part, through IL-12.

Induction of Regulatory T Cells by Leflunomide in a Murine Model of Contact Allergen Sensitivity

Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-specific activation of CD8+ and CD4+ T cells and the production of cytokines resulting in an inflammatory response and tissue damage. We show here that the immunosuppressive compound leflunomide (N-[4-trifluoro-methylphenyl]-5-methylisoxazol-4 carboxamide, HWA 486) (LF) inhibited the contact allergic response induced in mice by epicutaneous application of the haptens dinitrofluorobenzene (DNFB) and oxazolone. The extent of ear swelling remained significantly reduced following repeated challenge with DNFB for up to 18 weeks. LF and DNFB had to be applied simultaneously for inhibition to occur. The loss of CHS responses was shown to be antigen-specific. Adoptive transfer of leukocytes from LF-treated mice into naïve mice resulted in a loss of CHS responsiveness. Transfer of both CD4+ and CD8+ cells was required for maximal loss of CHS responses, with CD8+ cells playing a major role. Significantly enhanced levels of IL-10 mRNA were detected in CD8+ T cells, but not in CD4+ T cells, following LF treatment of mice. LF also suppressed CHS responses in mice previously sensitized and challenged with hapten, when administered together with the hapten. Our data suggest that LF induces a long-lived tolerance in mice by inducing CD8+ and CD4+ regulatory T cells.

Ultraviolet radiation-induced non-melanoma skin cancer in the Crl:SKH1:hr-BR hairless mouse: augmentation of tumor multiplicity by chlorophyllin and protection by indole-3-carbinol

Over 1 million new cases of ultraviolet radiation-induced non-melanoma skin cancers (NMSC) per year now occur in the USA and the incidence of these diseases continues to increase. New preventative strategies are required. The hypothesis tested was that dietary administration of the putative cancer chemopreventatives sodium-copper-chlorophyllin (Chlor) or indole-3-carbinol (I3C) would inhibit UV-induced skin carcinogenesis in the Crl:SKH1:hr-BR hairless mouse. Groups of 20 mice were pre-fed isocaloric/isonutritive 20% corn-oil AIN-76a based diets that contained either Chlor (1.52 g%), I3C (5.08 g%) or no chemopreventative (control) for 2 weeks followed by exposure of their dorsal skin to a 10 week incremental, sub-erythemal, carcinogenic simulated solar UV exposure regime. Feeding was continued for the duration of the experiment. Matched non-UV exposed dietary groups were also included in the experimental design. The diets had no significant (p > 0.05) effect on body weight, feed consumption, cutaneous methanol-extractable UV photoprotective substances or on cutaneous UV-reflective characteristics. By day 180, UV-irradiated mice fed the Chlor had a significantly (p < 0.05) higher tumor multiplicity (33.6 +/- 4.72; mean +/- SEM) than UV-irradiated control animals (22.8 +/- 4.25). UV-irradiated mice fed I3C had a significantly (p < 0.001) lower tumor multiplicity (13.0 +/- 2.42) than that of both the UV-irradiated control and UV-irradiated Chlor-fed mice. The Chlor or I3C diets did not significantly (p > 0.05) affect UV-induced systemic suppression of contact hypersensitivity responses. These results demonstrate augmentation of the UV-induced cutaneous carcinogenic process by dietary chlorophyllin and protection from this carcinogenic process by indole-3-carbinol via mechanisms that do not involve changes in skin optical properties, modulation of photoimmunosuppression or caloric/nutrient effects.

Primary Defect in UVB-Induced Systemic Immunomodulation Does Not Relate to Immature or Functionally Impaired APCs in Regional Lymph Nodes

UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m(2)) to the shaved dorsum of BALB/c mice, CD11c(+)/FITC(+) cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE(2) were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c(+) lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA(323-339)) to CD4(+) cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN-gamma levels were increased in the cultures containing CD11c(+) cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c(+) APCs that directly or by production of soluble mediators (IL-12, PGE(2)) affect cellular responses in the nodes of UVB-irradiated mice.

Dietary grape-seed proanthocyanidin inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12

We have shown previously that dietary grape seed proanthocyanidins (GSPs) inhibit UVB-induced photocarcinogenesis in mice. As UVB-induced immune suppression has been implicated in the development of skin cancer risk, we investigated whether dietary GSPs can modulate the effects of UVB on the immune system. We found that the UVB-induced (180 mJ/cm2) ear swelling response (inflammatory reaction) was significantly lower in mice fed with a GSP-supplemented (0.5 and 1.0%, w/w) diet than mice fed with the standard AIN76A diet. Dietary GSPs markedly inhibited UVB-induced (180 mJ/cm2) suppression of contact hypersensitivity responses in a local model of immunosuppression but had only moderate inhibitory effect in a systemic model of immunosuppression. Dietary GSPs reduced the UVB-induced increase in immunosuppressive cytokine interleukin (IL)-10 in skin and draining lymph nodes compared with mice that did not receive GSPs. In contrast, GSPs enhanced the production of immunostimulatory cytokine IL-12 in the draining lymph nodes. Intraperitoneal injection of GSPs-fed mice with a neutralizing anti-IL-12 antibody abrogated the protective effects of the GSPs against UVB-induced suppression of the contact hypersensitivity response. These data indicate for the first time that GSPs modulate UVB-induced immunosuppression and suggest that this may be one of the possible mechanisms by which they prevent photocarcinogenesis in mice.

Association between melanoma and dermal mast cell prevalence in sun-unexposed skin.

Both exposure to intermittent intense sunlight during childhood and ultraviolet (UV) radiation-induced immunomodulation have been directly associated with melanoma development. In mice, the prevalence of dermal mast cells determines susceptibility to UVB-induced systemic suppression of contact hypersensitivity responses and thus may affect immunological responses to melanoma antigens. To determine the relevance of murine studies of dermal mast cell prevalence to human melanoma pathogenesis. The prevalence of mast cells was examined in sun-unexposed buttock skin of 45 melanoma patients and 68 control volunteers who had no history of skin cancer development. Buttock skin was studied because mast cell prevalence is stable with ageing and the confounding effects of environmental UV exposure are minimized. Using tissue immunostaining, the buttock skin from melanoma patients had a significantly higher dermal mast cell prevalence (mean +/- SEM 38 +/- 2 mast cells mm(-2)) than controls (32 +/- 2 mast cells mm(-2)) (P = 0.02). Analysis by binary logistic regression showed that the association between mast cell prevalence and melanoma outcome was not significantly altered by skin phototype. The immunomodulatory effects of mast cell products in UV-irradiated skin may contribute significantly to the initiation and development of human cutaneous malignant melanoma.

Neuronal repellent Slit2 inhibits dendritic cell migration and the development of immune responses.

One of the essential functions of dendritic cells is to take up Ags in peripheral tissues and migrate into secondary lymphoid organs to present Ags to lymphocytes for the induction of immune responses. Although many studies have demonstrated that the migration of dendritic cells is closely associated with the development of immune responses, little is known about factors that inhibit dendritic cell migration and control the extent of immune responses to Ag stimulation. We show that Slit2, a neuronal repellent factor, is up-regulated in the skin by allergen sensitization and down-regulates the migration of Langerhans cells. The effect is mediated by direct interaction of Slit2 with cells that express a Slit-specific receptor, Robo1. Slit2-mediated inhibition of Langerhans cell migration results in suppression of contact hypersensitivity responses. These findings provide insights into a novel mechanism by which Slit2 functions as an anti-inflammatory factor for the initiation of immune responses.

Protection against UV-induced suppression of contact hypersensitivity responses by sunscreens in humans.

Both in vivo skin immune responses and the skin's reaction to sun exposure integrate a complex interplay of biologic responses. The complexity and multiplicity of events that occur in the skin during an immune response make it a sensitive indication of both UVB and UVA-induced changes in the skin by sun damage, as well as those changes that are prevented by various sunscreens. Sunscreens are the most effective and widely available intervention for sun damage, other than sun avoidance or clothing. However, sunscreens vary widely in their relative ability to screen various UV waveband components, and their testing has been variably applied to outcomes other than for erythema to determine the sunburn protection factor (SPF), a measure primarily of UVB filtration only. Determination of an immune protection factor (IPF) has been proposed as an alternative or adjunctive measure to SPF, and recent studies show IPF can indeed detect added in vivo functionality of sunscreens, such as high levels of UVA protection, that SPF cannot. Clarification of the definition of IPF, however, is required. Excellent data are available on quantification of the IPF for restoring the afferent or induction arm of contact sensitivity, but other immune parameters have also been measured. Proposed here is nomenclature for whether the IPF is measured using contact sensitivity induction (IPF-CS-I), contact sensitivity elicitation (IPF-CS-E), delayed-type hypersensitivity elicitation (IPF-DTH-E), antigen-presenting cell function (IPF-APC-FXN) or numbers (IPF-APC-#), and cytokine modification such as IL-10 (i.e. IPF-cyto-IL-10). Similar nomenclatures could be used for other measures of skin function protection (i.e. DNA damage, p53 induction, oxidation products, etc.). A review of in vivo human studies, in which sunscreens are used to intervene in a UV-induced modulation of immune response, cells or cytokines, highlights the technical variables and statistical approaches which must also be standardized in the context of an IPF for regulatory or product claim purposes. Development of such IPF standards would allow the integration of both UVB and nonUVB (UVA, blue and possible IR) solar waveband effect-reversals, could be applied to integrate effects of other ingredients with protective function (i.e. antioxidants, retinoids, or other novel products), and would spur development of more advanced and complete protection products.