Prevention of ultraviolet radiation-induced immunosuppression by (-)-epigallocatechin-3-gallate in mice is mediated through interleukin 12-dependent DNA repair.

PURPOSE: Solar UV radiation-induced immunosuppression is considered to be a risk factor for melanoma and nonmelanoma skin cancers. We previously have shown that topical application of (-)-epigallocatechin-3-gallate (EGCG) prevents UV-induced immunosuppression in mice. We studied whether prevention of UV-induced immunosuppression by EGCG is mediated through interleukin 12 (IL-12)-dependent DNA repair. EXPERIMENTAL DESIGN: IL-12 knockout (KO) mice on C3H/HeN background and DNA repair-deficient cells from xeroderma pigmentosum complementation group A (XPA) patients were used in this study. The effect of EGCG was determined on UV-induced suppression of contact hypersensitivity and UV-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) in mice and XPA-deficient cells using immunohistochemistry and dot-blot analysis. RESULTS: Topical treatment with EGCG prevented UV-induced suppression of the contact hypersensitivity in wild-type (WT) mice but did not prevent it in IL-12 KO mice. Injection of anti-IL-12 monoclonal antibody to WT mice blocked the preventive effect of EGCG on UV-induced immunosuppression. EGCG reduced or repaired UV-induced DNA damage in skin faster in WT mice as shown by reduced number of CPDs(+) cells and reduced the migration of CPD(+) antigen-presenting cells from the skin to draining lymph nodes. In contrast, this effect of EGCG was not seen in IL-12 KO mice. Further, EGCG was able to repair UV-induced CPDs in XPA-proficient cells obtained from healthy person but did not repair in XPA-deficient cells, indicating that nucleotide excision repair mechanism is involved in DNA repair. CONCLUSIONS: These data identify a new mechanism by which EGCG prevents UV-induced immunosuppression, and this may contribute to the chemopreventive activity of EGCG in prevention of photocarcinogenesis.

Light-Emitting Diode at 460 ± 20 nm Increases the Production of IL-12 and IL-6 in Murine Dendritic Cells.

BACKGROUND DATA: Light emitting diode (LED) therapy has been proposed as an option for the treatment of many skin inflammatory processes. Dendritic cells (DCs) are important cells of skin that participate in the initiation and activation of skin immunity. The modulation of these cells by LED could explain much of its effects. OBJECTIVE: Thus, the aim of this study was to examine the effects of LED at 460 ± 20 nm on cytokine production and the expression of surface markers on DCs. MATERIALS AND METHODS: DCs were obtained from mouse bone marrow-derived dendritic cells (BMDCs). The LED was applied giving a fluence of 3.3, 8.2, or 16.5 J/cm2 on BMDCs or lipopolysaccharide (LPS)-matured BMDCs. The production of cytokine was analyzed by enzyme linked immunosorbant assay (ELISA) and the expression of DC co- and stimulatory was analyzed markers by cytometry. RESULTS: LED increases IL-12p40 and IL-6 production in both nonstimulated BMDCs and LPS-matured BMDCs. The expression of MHC-II molecule was inhibited and the expression of the CD86 molecule was increased in nonstimulated BMDCs but not in LPS-matured BMDCs. The production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) and the expression of CD40 were not altered. CONCLUSIONS: These results demonstrate that LED stimulated cytokine production in BMDCs, suggesting a proinflammatory role in the tested conditions and maybe it can increase DC maturation.

Narrowband (312-nm) UV-B suppresses interferon gamma and interleukin (IL) 12 and increases IL-4 transcripts: differential regulation of cytokines at the single-cell level.

OBJECTIVE: To determine whether 312-nm UV-B alters production of effector and regulatory cytokines by viable T cells that remain in psoriatic lesions during UV-B phototherapy. DESIGN: Prospective study. SETTING: General clinical research center of The Rockefeller University Hospital. PATIENTS: Ten adult patients with moderate to severe psoriasis vulgaris that was difficult to manage were sequentially enrolled in our protocols, and biopsies were taken at various time points from resolving lesions. INTERVENTION: Narrowband (312-nm) UV-B was given starting at 50% of a minimum erythema dose, then increased daily 10% to 15% if no apparent erythema was induced. Patients continued with treatment until maximal benefit was noted. In some experiments, T cells were irradiated ex vivo with standard TL-01 fluorescent bulbs (Philips Lighting Co, Somerset, NJ). MAIN OUTCOME MEASURES: Intracellular cytokine staining was done using flow cytometry to quantify numbers of cytokine-producing cells from epidermal and peripheral T cells. The production of messenger RNA for interleukin (IL) 12, interferon (IFN) gamma, tumor necrosis factor alpha, IL-4, and IL-10 was measured by quantitative reverse transcription-polymerase chain reaction. RESULTS: Ultraviolet-B treatment eliminated production of IL-12 messenger RNA and decreased production of IFN-gamma messenger RNA by more than 60% in irradiated psoriasis lesions (P<.03 for both). Within 1 to 2 weeks of starting UV-B treatment, the frequency of viable T cells producing IFN-gamma decreased 40% to 65%. In contrast, mRNA for IL-4 increased by 82% (P =.05) during UV-B treatment, and the number of IL-4-producing cells increased by 228% after 1 week of treatment. In vitro experiments established that, on the single-cell level, survival and cytokine production by type 1 T cells were differentially regulated by UV-B. CONCLUSIONS: Therapeutic UV-B suppresses the type 1 (proinflammatory) axis as defined by IL-12, IFN-gamma, and IL-8, and can selectively reduce proinflammatory cytokine production by individual T cells. Knowledge of the immunomodulatory effects of UV-B will help to integrate this modality in future therapeutics for psoriasis based on deliberate blockade of inflammatory molecular pathways in the type 1 T-cell pathway.

Protective effect of epigallocatechin gallate on the immune function of dendritic cells after ultraviolet B irradiation.

AIM: To investigate the protective effect of epigallocatechin gallate (EGCG) on the immune function of dendritic cells (DCs) after ultraviolet B irradiation (UVB) and its underlying mechanisms. METHODS: The monocytes were isolated from peripheral blood and cultivated into DCs with cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. DCs were harvested after cultivation for 7 days and subjected to irradiation with different dosages of UVB. Then, 200 microg/mL of EGCG was added in certain groups 24 h before irradiation. DCs simply treated with UVB or treated with both UVB and EGCG were co-cultured with lymphocytes, and Mono-nuclear cell direct cytotoxicity (MTT) assay was used to detect the ability of DCs to stimulate proliferation of lymphocytes. Surface markers CD80, CD86, human leukocyte antigen(locus)-DR (HLA-DR), and CD40 were detected using flow cytometry, and the levels of IL-10 and IL-12 secreted from DCs 24 h after cultivation were measured using ELISA. RESULTS: UVB irradiation was able to inhibit the ability of DCs to stimulate the proliferation of lymphocytes and surface expressions of CD80, CD86, HLA-DR, and CD40 on DCs in a dose-dependent manner. The inhibition rate of DCs was improved to some extent after treatment with 200 microg/mL of EGCG. UVB showed no significant influence on the secretion of IL-10 and IL-12 from DCs, while EGCG was able to down-regulate the secretion level of IL-12 and up-regulate that of IL-10. CONCLUSIONS: EGCG can antagonize the inhibitory effect on DCs induced by UVB irradiation. This function has some relationship with its protecting effect of the expression of the costimulating molecules on the surface of DCs and the secretion level of IL-10 and IL-12.

Immunosuppressive effects of radiation on human dendritic cells: reduced IL-12 production on activation and impairment of naive T-cell priming.

Dendritic cells (DC) are professional antigen-presenting cells (APC) of the immune system, uniquely able to prime naive T-cell responses. They are the focus of a range of novel strategies for the immunotherapy of cancer, a proportion of which include treating DC with ionising radiation to high dose. The effects of radiation on DC have not, however, been fully characterised. We therefore cultured human myeloid DC from CD14+ precursors, and studied the effects of ionising radiation on their phenotype and function. Dendritic cells were remarkably resistant against radiation-induced apoptosis, showed limited changes in surface phenotype, and mostly maintained their endocytic, phagocytic and migratory capacity. However, irradiated DC were less effective in a mixed lymphocyte reaction, and on maturation produced significantly less IL-12 than unirradiated controls, while IL-10 secretion was maintained. Furthermore, peptide-pulsed irradiated mature DC were less effective at naive T-cell priming, stimulating fewer effector cells with lower cytotoxicity against antigen-specific targets. Hence irradiation of DC in vitro, and potentially in vivo, has a significant impact on their function, and may shift the balance between T-cell activation and tolerization in DC-mediated immune responses.

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.

Characterization of immunologic tolerance induced by transfusion of UV-B--irradiated allogeneic mononuclear leukocytes.

Transfusions of UV-B--irradiated peripheral blood mononuclear cells (UV-B--PBMCs) from BALB/c (H-2(d)) mice into CBA (H-2(k)) mice can induce humoral immune tolerance to H-2(d) antigens, and the induced tolerance is partially mediated by negative regulatory PBMCs. To further identify which subset of spleen mononuclear leukocytes (MNLs) in the tolerant CBA mice is responsible for the negative regulatory activity, adoptive transfer experiments were conducted using spleen MNLs from the tolerant CBA mice. Results showed that only CD4(+) T cells could transfer the negative regulatory activity in a dose-dependent manner. This negative regulatory activity was significantly reduced when CD25(+) helper T cells were removed. Further study suggested that inhibition of IL-12 production by UV-B--irradiated PBMCs played a role in the induction of immune tolerance. In vitro study of the cytokine production profile by CBA CD4(+) T cells, after stimulation with gamma-irradiated BALB/c spleen cells, revealed an enhanced production of the type 2 T-cell cytokines after tolerance induction. Induction of tolerance also prevented the development of cytotoxic T cells in CBA mice against BALB/c MNLs. Adoptive transfer study suggested that the cellular immune tolerance was also mediated by CD4(+) negative regulatory T cells. The induced immune tolerance was nullified after 400 cGy sublethal gamma irradiation. These results suggest that the ex vivo study of cytokine production by T cells may be used to monitor tolerance induction and the selection of gamma radiation dose is critical for potential clinical application of the tolerance induced by UV-B--PBMCs. (Blood. 2001;98:1239-1245)

Reduced IL-12 production by monocytes upon ultraviolet-B irradiation selectively limits activation of T helper-1 cells.

The capacity of APC to stimulate the proliferation of human peripheral blood T cells decreases upon ultraviolet-B (UVB) irradiation. The aim of this study was to investigate whether all T cell subsets are equally sensitive to this reduced APC function. Established human Th1, Th2, and Th0 clones were stimulated with monocytes in a soluble CD3 mAb-mediated assay that is dependent on the presence of APC. Monocytes were exposed to low nonlethal doses of UVB radiation before coculture with T cells. UVB irradiation inhibited the capacity of monocytes to stimulate the proliferation and IFN-gamma production of Th1 cells in a dose-related fashion. In contrast, UVB-treated monocytes induced normal proliferation and IL-4 production in Th2 cells. Stimulation of Th0 cell proliferation by UVB-irradiated monocytes was normal, but a preferential suppression of IFN-gamma production was observed, thus leading to a more Th2-like cytokine response. The loss of Th1 proliferation upon stimulation with UVB-irradiated monocytes could be overcome by rIL-2; however, IFN-gamma production remained suppressed. IFN-gamma production could be completely restored by rIL-12, whereas the addition of IL-1 beta, TNF-alpha, or indomethacin had no such effect, nor did the addition of mAb to CD28, added to compensate for the reduced B7 expression of UVB-irradiated monocytes. Monocytes exposed to UVB radiation exhibited reduced expression of mRNA for the IL-1 2 subunits p35 and p40 and suppressed production of the IL-12 p70 protein. Our results thus indicate that UVB irradiation of APC selectively impairs Th1-like responses, a phenomenon caused by the UVB-induced suppression of monocyte IL-12 production.

Exposure to ultraviolet radiation causes dendritic cells/macrophages to secrete immune-suppressive IL-12p40 homodimers.

UV-induced immune suppression is a risk factor for sunlight-induced skin cancer. Exposure to UV radiation has been shown to suppress the rejection of highly antigenic UV-induced skin cancers, suppresses delayed and contact hypersensitivity, and depress the ability of dendritic cells to present Ag to T cells. One consequence of UV exposure is altered activation of T cell subsets. APCs from UV-irradiated mice fail to present Ag to Th1 T cells; however, Ag presentation to Th2 T cells is normal. While this has been known for some time, the mechanism behind the preferential suppression of Th1 cell activation has yet to be explained. We tested the hypothesis that this selective impairment of APC function results from altered cytokine production. We found that dendritic cells/macrophages (DC/Mphi) from UV-irradiated mice failed to secrete biologically active IL-12 following in vitro stimulation with LPS. Instead, DC/Mphi isolated from the lymphoid organs of UV-irradiated mice secreted IL-12p40 homodimer, a natural antagonist of biologically active IL-12. Furthermore, when culture supernatants from UV-derived DC/Mphi were added to IL-12-activated T cells, IFN-gamma secretion was totally suppressed, indicating that the IL-12p40 homodimer found in the supernatant fluid was biologically active. We suggest that by suppressing DC/Mphi IL-12p70 secretion while promoting IL-12p40 homodimer secretion, UV exposure preferentially suppress the activation of Th1 cells, thereby suppressing Th-1 cell-driven inflammatory immune reactions.

Antigen-specific regression of established tumors induced by active immunization with irradiated IL-12- but not B7-1-transfected tumor cells.

Transfection of modestly immunogenic tumors to express B7 family co-stimulator molecules results in their rejection by syngeneic mice, suggesting a possible clinical application in cancer patients. Immunization of naive mice with irradiated B7-1-transfected P1.HTR cells is sufficient to induce specific cytolytic T lymphocytes (CTL) and to protect against tumor challenge. However, patients to be treated will have an existing tumor burden; thus, preclinical models should examine therapeutic efficacy in an established tumor setting. Contrary to expectations, immunization of mice with irradiated B7-1-transfected P1.HTR cells had no impact on the growth of pre-established control-transfected tumors. Mice bearing control-transfected P1.HTR tumors successfully rejected living B7-1 transfectants on the contralateral flank, demonstrating the ability of tumor-bearing mice to respond to B7 co-stimulation. Inasmuch as IL-12 is another important factor for CTL maturation, P1.HTR transfectants expressing B7-1 and/or IL-12 were then constructed. Remarkably, regression of pre-established tumors was achieved following immunization with irradiated IL-12 transfectants, even without co-expression of B7-1. Rejection required a shared antigen with the tumor used for immunization, could not be reproduced with rIL-12 alone, depended on host T lymphocytes and correlated with a high IFN-gamma-producing T cell phenotype. In addition, IL-12-facilitated tumor rejection required co-operation with a CTLA-4 ligand provided by the host, and correlated with up-regulation of B7-1 and B7-2 on host antigen-presenting cells. Thus, active immunization in the established tumor setting is benefitted greatly by the provision of IL-12, which may recruit participation of sufficient B7 co-stimulation from the host that it need not be provided exogenously.