Agatha Schwarz

Proopiomelanocortin-derived peptides are synthesized and released by human keratinocytes.

Proopiomelanocortin (POMC), the precursor for melanotropic, corticotropic, and opioid peptides such as alpha-melanocyte-stimulating hormone (alpha MSH), ACTH, and other related peptides, was originally identified as a product of the pituitary gland. However, recent evidence shows that POMC products can also be produced by nonpituitary tissues. Because keratinocytes, the major constituent of the epidermis exhibit the capacity to release a variety of proinflammatory and immunomodulatory mediators, the present study was performed to investigate whether human keratinocytes are able to produce POMC-derived peptides. Supernatants of human normal keratinocytes and an epidermal carcinoma cell line (A431) contained significant levels of immunoreactive alpha MSH and ACTH. Upon immuneprecipitation and size-exclusion chromatography, keratinocyte-derived alpha MSH exhibited a molecular mass of approximately 1 kD and was biologically active as demonstrated in a tyrosinase bioassay. Northern blot analysis revealed the expression of POMC-specific transcripts (1.3 kb) in both normal keratinocytes and A431 cells. The production of alpha MSH and ACTH could be significantly upregulated both at the protein and mRNA level upon treatment with phorbol myristate acetate, ultraviolet light, or interleukin 1. These data provide first evidence that human keratinocytes produce POMC-derived peptides such as alpha MSH and ACTH. Because POMC-derived peptides recently have been recognized as potent immunomodulatory mediators, their presence in the epidermis may have a major impact on the skin immune system.

UVA‐INDUCED AUTOCRINE STIMULATION OF FIBROBLAST‐DERIVED COLLAGENASE/MMP‐1 BY INTERRELATED LOOPS OFINTERLEUKIN–1 andINTERLEUKIN–6

Abstract— Previous work has shown that fibroblast‐derived collagenase/matrix‐metalloproteinase‐1(MMP–1), responsible for the breakdown of dermal interstitial collagen, was dose‐dependently induced in vitro and in vivo by UVA irradiation and this induction was at least partly mediated byinterleukin–6(IL–6). We here provide evidence that UVA‐inducedIL–1α andIL–1β play a central role in the induction of the synthesis both ofIL–6 and collagenase/MMP–1. In contrast to the late increase ofIL–1α andIL–1β mRNA levels at 6 h postirradiation, bioactivity ofIL–1 is already detectable at 1 h postirradiation. This early peak ofIL–1 bioactivity appears to be responsible for the induction ofIL–6 synthesis and together withIL–6 lead to an increase of the steady‐state mRNA level of collagenase/MMP–1 as deduced from studies usingIL–1α andIL–1β antisense oligonucleotides or neutralizing antibodies againstIL–1α andIL–1β Besides the early posttranslationally controlled release of intracellularIL–1, a latter pretranslationally controlled synthesis and release ofIL–1 perpetuates the UV response. From these data we suggest a UV‐induced cytokine network consisting ofIL–1α,IL–1β andIL–6, which via interrelated autocrine loops induce collagenase/MMP–1 and thus may contribute to the loss of interstitial collagen in cutaneous photoaging.

Interleukin-12 suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair

Induction of apoptosis of keratinocytes by ultraviolet (UV) radiation is a protective phenomenon relevant in limiting the survival of cells with irreparable DNA damage. Changes in UV-induced apoptosis may therefore have significant impact on photocarcinogenesis. We have found that the immunomodulatory cytokine IL-12 suppresses UV-mediated apoptosis of keratinocytes both in vitro and in vivo. IL-12 caused a remarkable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In accordance with this, IL-12 induced the expression of particular components of the nucleotide-excision repair complex. Our results show that cytokines can protect cells from apoptosis induced by DNA-damaging UV radiation by inducing DNA repair, and that nucleotide-excision repair can be manipulated by cytokines.

Heat shock protein 70 overexpression affects the response to ultraviolet light in murine fibroblasts. Evidence for increased cell viability and suppression of cytokine release.

To elucidate cellular concepts for protection against ultraviolet (UV) light we investigated the effect of heat shock protein 70 (hsp70) overexpression on cell viability and on the secretion of UV-inducible immunological cytokines. Transfected murine fibrosarcoma cells (WEHI-S), overexpressing hsp70 or a sham transfected control were used. Overexpression of hsp70 was sufficient to markedly increase cell viability upon treatment with UVB (290-320 nm). Since long wave UV (UVA, 320-400 nm) as well as UVB turned out to stimulate the release of O2- radicals we studied the cell viability upon oxidative stress. Hsp70 overexpression increased viability upon treatment with hydrogen peroxide or menadione, but had no influence on UV-induced O2- release. UV-light is known to upregulate immunologic and proinflammatory cytokines such as IL-1 and IL-6. Oxidative stress appeared to exert a similar effect. Hsp70 overexpression markedly decreased the release of IL-6 induced by UVA, UVB and oxidative stress. To test whether the hsp70 mediated suppression is confined to events caused by UV-light we determined IL-1-mediated effects. IL-1-induced IL-6 release was reduced by hsp70 overexpression, whereas the IL-1 mediated activation of nuclear factor kappa B was not affected. Our data suggests that hsp70 plays a central role not only in cell protection against UV-light, but also in the regulation of proinflammatory cytokine release induced by UV-exposure.

Intravenous Infusion of Syngeneic Apoptotic Cells by Photopheresis Induces Antigen-Specific Regulatory T Cells

The basis of extracorporeal photopheresis is the reinfusion of leukocytes previously exposed to 8-methoxypsoralen (8-MOP) and UVA radiation. It has been approved for the palliative treatment of cutaneous T cell lymphoma and has reported benefits in autoimmune diseases, transplant rejection, and graft-vs-host disease. However, the underlying mechanism of photopheresis remains unresolved. Because UVB radiation can cause immune tolerance via induction of regulatory T cells, we studied whether photopheresis exerts a similar effect extracorporeally. Therefore, we established a model of photopheresis using a murine model of contact hypersensitivity. Splenocytes and lymph node cells of mice that were sensitized with dinitrofluorobenzene were exposed to 8-MOP plus UVA in vitro. Intravenous injection of these cells into naive mice caused inhibition of a hapten immune response, which was lost upon depletion of CD11c+ cells but not T cells. Mice that received untreated cells or cells exposed to UVA or 8-MOP alone were not affected. Inhibition was cell-mediated and Ag-specific as demonstrated by transfer of tolerance from the primary recipients into naive animals, which could, however, properly respond to the unrelated hapten oxazolone. Transfer activity was lost when cells were depleted of CD4+ or CD25+ subpopulations. These data suggest that photopheresis exerts its immunomodulatory effects via the induction of Ag-specific regulatory T cells.

Ultraviolet Radiation-Induced Regulatory T Cells Not Only Inhibit the Induction but Can Suppress the Effector Phase of Contact Hypersensitivity

Epicutaneous application of haptens to UV-exposed skin induces hapten-specific tolerance. This is mediated via regulatory T cells (Tr), as i.v. injection of T cells from UV-tolerized mice into naive animals renders the recipients unresponsive to the respective hapten. However, when UV-induced Tr are injected i.v. into sensitized mice, contact hypersensitivity (CHS) is not suppressed, suggesting that Tr inhibit the induction, but not the elicitation, of CHS and are inferior to T effector cells. As sensitization takes place in the lymph nodes, but elicitation occurs in the area of challenge, we postulated that Tr injected i.v. locate to the lymph nodes and not to the periphery and therefore only suppress the induction, not the elicitation, of CHS. Indeed, i.v. injection of Tr into sensitized mice did not inhibit CHS, although injection of Tr into the ears of sensitized mice suppressed the challenge. Inhibition was hapten specific, as injection of dinitrofluorobenzene (DNFB)-specific Tr into the ears of oxazolone (OXA)-sensitized mice did not affect challenge with OXA. However, when ears of OXA-sensitized mice were injected with DNFB-specific Tr and painted with DNFB before OXA challenge, CHS was suppressed. Inhibition correlated with the local expression of IL-10. Depletion studies and FACS analysis revealed that Tr express the lymph node-homing receptor L-selectin, but not the ligands for the skin-homing receptors E- and P-selectin, suggesting that UV-induced Tr, although able to inhibit T effector cells, do not suppress the elicitation of CHS upon i.v. injection, because they obviously do not migrate into the skin.

Prevention of UV radiation-induced immunosuppression by IL-12 is dependent on DNA repair.

The immunostimulatory cytokine IL-12 is able to antagonize immunosuppression induced by solar/ultraviolet (UV) radiation via yet unknown mechanisms. IL-12 was recently found to induce deoxyribonucleic acid (DNA) repair. UV-induced DNA damage is an important molecular trigger for UV-mediated immunosuppression. Thus, we initiated studies into immune restoration by IL-12 to discern whether its effects are linked to DNA repair. IL-12 prevented both UV-induced suppression of the induction of contact hypersensitivity and the depletion of Langerhans cells, the primary APC of the skin, in wild-type but not in DNA repair-deficient mice. IL-12 did not prevent the development of UV-induced regulatory T cells in DNA repair-deficient mice. In contrast, IL-12 was able to break established UV-induced tolerance and inhibited the activity of regulatory T cells independent of DNA repair. These data identify a new mechanism by which IL-12 can restore immune responses and also demonstrate a link between DNA repair and the prevention of UV-induced immunosuppression by IL-12.

Evidence for functional relevance of CTLA-4 in ultraviolet-radiation-induced tolerance.

Hapten sensitization through UV-exposed skin induces hapten-specific tolerance that can be adoptively transferred by injecting T lymphocytes into naive recipients. The exact phenotype of T cells responsible for inhibiting the immune response and their mode of action remain unclear. Evidence exists that CTLA-4 negatively regulates T cell activation. We addressed whether CTLA-4 is involved in the transfer of UV-induced tolerance. Injection of lymph node cells from mice that were sensitized with dinitrofluo-robenzene (DNFB) through UV-irradiated skin inhibited induction of contact hypersensitivity against DNFB in the recipient animals. When CTLA-4+ cells were depleted, transfer of suppression was lost. Likewise, significantly fewer lymphocytes enriched for CTLA-4+ cells were necessary to transfer suppression than unfractionated cells. Expression of CTLA-4 appears to be functionally relevant, since in vivo injection of a blocking anti-CTLA-4 Ab was able to break UV-induced tolerance and inhibited transfer of suppression. Upon stimulation with dendritic cells in the presence of the water-soluble DNFB analogue, DNBS, CTLA-4+ T cells from DNFB-tolerized mice secreted high levels of IL-10, TGF-β, and IFN-γ; low levels of IL-2; and no IL-4, resembling the cytokine pattern of T regulatory 1 cells. Ab blocking of CTLA-4 resulted in inhibition of IL-10 release. Accordingly, transfer of tolerance was not observed when recipients were treated with an anti-IL-10 Ab. Hence we propose that T cells, possibly of the T regulatory 1 type, transfer UV-mediated suppression through the release of IL-10. Activation of CTLA-4 appears to be important in this process.

Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro

Ultraviolet (UV) A irradiation of human dermal fibroblasts elicits an increase in specific mRNA amounts and bioactivities of the cytokines IL‐1α, IL‐1β, and IL‐6. These effects are enhanced in deuterium oxide‐based medium and are diminished in the presence of non‐toxic concentrations of sodium azide. Furthermore, generating singlet oxygen outside the cells by irradiation of rose bengal‐coated resin particles with visible light (λ>450 nm) results in the induction of interstitial collagenase, IL‐1 and IL‐6, similar to the response observed with UVA irradiation. These observations suggest that singlet oxygen is an early intermediate in the signaling pathway of IL‐1 and IL‐6 mediating UVA induction of interstitial collagenase (E.C. 3.4.24.7). Furthermore, singlet oxygen appears to initiate this complex UV response at the cell membrane.

Langerhans cells are required for UVR-induced immunosuppression

Painting of haptens onto UVR-exposed skin does not result in sensitization but induces regulatory T cells (Treg). This was explained by UVR-mediated depletion of Langerhans cells (LCs). Furthermore, migration of UVR-damaged but still viable LCs into lymph nodes appears to be essential to induce Treg. Accordingly, the steroid mometasone, which kills LCs, inhibited sensitization but did not induce Treg. In Langerin-diphtheria toxin receptor knock-in (DTR) mice, LCs can be depleted by injection of diphtheria toxin (DT). LC-depleted mice could be sensitized though less pronounced than wild-type mice, but sensitization was not suppressed by UVR. Similarly, Treg did not develop. Langerin is not only expressed in LCs but also in some dermal dendritic cells (dDCs). Langerin-positive dDCs repopulate within 10 days after depletion, whereas LCs are still absent. Langerin-DTR mice treated with DT 10 days before UVR and sensitization were still resistant to UVR-induced inhibition of contact hypersensitivity (CHS). Similarly, Treg did not arise. As in this setting only LCs but not Langerin-positive dDCs are absent, LCs appear to be essential for both the suppression of CHS and the induction of Treg by UVR. This supports the concept that LCs are more important for the downregulation than the induction of immune responses in the skin.