A role for NF-κB–dependent gene transactivation in sunburn

Exposure of skin to ultraviolet (UV) radiation is known to induce NF-κB activation, but the functional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be prevented by blocking UV-induced, NF-κB–dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-κB cis element (NF-κB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-α, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-κB decoy ODNs also inhibited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proinflammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Importantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irritant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-κB target genes, rather than from nonspecific changes associated with tissue damage.

Ultraviolet (UV) radiation causes sunburn reactions, immunosuppression, accelerated skin aging, and skin cancer, and it is considered to be an important environmental hazard for humans (1). At cellular levels, UV radiation has been shown to trigger cytokine production (2), regulate surface expression of adhesion molecules (3), affect cellular mitosis (4), and induce apoptotic cell death (5, 6). With respect to molecular mechanisms, UV radiation is known to alter cellular function via DNA damage (7–10), generation of reactive oxygen intermediates (ROIs) (11, 12), ligand-independent signaling through cell surface receptors, such as EGF, TNF, and IL-1 receptors (13–15), phosphorylation of receptor-associated tyrosin kinases and protein kinase C (15–18), and activation of selected transcription factors, including NF-κB, AP-1, and AP-2 (13, 19–21). These mechanisms are by no means mutually exclusive; rather, they are likely to operate in an interdependent manner. For example, NF-κB activation can be induced experimentally by damaging DNA chemically (6), exposure to hydrogen peroxide (22), clustering of surface receptors (13), or phosphorylation of tyrosin kinases or protein kinase C (17, 23, 24). NF-κB activation, in turn, leads to the expression of many genes involved in immunological and inflammatory responses, including genes that encode cytokines, adhesion molecules, and regulators of cell growth and death (24, 25). Thus, we sought to determine the causative relationship between UV-dependent NF-κB activation and the development of sunburn reactions.

For this aim, we used the recently developed technology of decoy ODNs. The original concept of using synthetic double-stranded ODN as “decoy” cis elements to block the binding of nuclear factors to promoter regions of target genes was introduced in 1990 by Sullenger et al. and Bielinska et al. (26, 27). In 1997, Morishita et al. reported the first in vivo application of this technology; they prevented myocardial infarction after reperfusion in rats by direct infusion of synthetic double-stranded 20-bp ODNs containing the NF-κB cis element into cannulated coronary arteries (28). Subsequently, Ono et al. prevented cerebral angiopathy after subarachnoid hemorrhage in rabbits by delivering NF-κB decoy ODNs into the subarachnoid space (29), and Kawamura et al. showed that direct injection of NF-κB decoy ODNs into implanted tumors in mice inhibited cachexia, without affecting the tumor growth (30). Here we report the first application to our knowledge of the decoy ODN technology to the skin (as a target organ) and to prevent inflammatory responses to an environmental hazard (as a target disease).

In vitro effect of NF-κB decoy ODN on UV-induced secretion of proinflammatory cytokines by skin-derived cell lines. In the first set of experiments, we irradiated three skin-derived cell lines with conventional FS20 sunlamps and examined NF-κB activation by the gel shift assays. A single exposure to a sublethal dose (50 J/m²) resulted in significant NF-κB activation in all cell lines (Pam 212 keratinocyte, NS47 fibroblast, and XS106 Langerhans cell lines), and in vitro binding of radiolabeled NF-κB probe to the nuclear extracts isolated from UV-irradiated cells was competed with “cold” NF-κB probe, as well as with phosphorothioated, double-stranded 20-bp NF-κB decoy ODN (data not shown). To determine the function of the NF-κB decoy ODN at cellular levels, we next transfected each cell line with the NF-κB reporter construct (3× κB luc), in which the Luc gene was inserted downstream from the NF-κB–binding motifs. As shown in Figure 1a, UV radiation at 50 J/m² (FS20 sunlamps) elevated Luc activity significantly in each cell line. Likewise, each cell line transfected with the AP-1 reporter construct (4× AP-1 luc) also responded to UV radiation by elevating Luc activity. Importantly, addition of the NF-κB decoy ODN blocked UV-induced Luc elevation with the NF-κB reporter construct almost completely, whereas scrambled ODN had no significant effect. Moreover, NF-κB decoy ODN showed minimal, if any, effects on UV-induced Luc elevation with the AP-1 reporter construct, confirming the specificity. Thus, UV radiation triggers activation of the NF-κB and AP-1 pathways in each of three skin-derived cell lines, and NF-κB decoy ODNs selectively block the NF-κB–dependent gene transactivation pathway alone.

Figure 1

Impact of NF-κB decoy ODNs on UV-triggered cytokine production. (a) Pam 212 keratinocytes, NS47 fibroblasts, and XS106 Langerhans cells were transfected with the NF-κB reporter construct (3× κB luc) or the AP-1 reporter construct (4× AP-1 luc). After 6 hours of preincubation with 10 μM NF-κB decoy or scrambled ODNs, cells were washed and exposed to 50 J/m² UVB radiation (FS20 sunlamp) in PBS, cultured for an additional 24 hours in the continuous presence of fresh ODNs, and then examined for Luc activity. Data shown are representative of four independent experiments, showing the mean ± SD from triplicate samples. (b and c) Cells (1 × 10⁶ cells/mL) were incubated for 6 hours with 10 μM NF-κB decoy or scrambled ODNs, washed, and then exposed to the indicated fluences of UV radiation using either FS20 sunlamps (b) or a Xenon arc solar simulator (c). Subsequently, cells were cultured for an additional 24 hours in complete RPMI 1640 in the presence of fresh ODNs, and culture supernatants were examined for the indicated cytokines by ELISA. Data shown are representative of three independent experiments, showing the mean ± SD from triplicate samples. ^AStatistically significant differences (P < 0.05) compared with the UV-irradiated group without added ODNs. ^BStatistically significant differences (P < 0.01) compared with the UV-irradiated group without added ODNs. ^CStatistically significant differences compared with the UV plus scrambled ODN group (P < 0.05). ^DStatistically significant differences compared with the UV plus scrambled ODN group (P < 0.01).

A single exposure of the Pam 212 keratinocyte line to UV radiation at sublethal doses (50 J/m² with FS20 sunlamps or 100 J/m² with a Xenon arc solar simulator) induced the secretion of VEGF in a dose-dependent manner (data not shown). Likewise, the NS47 fibroblast line secreted primarily IL-6 and VEGF in response to radiation. The XS106 Langerhans cell line secreted TNF-α, IL-1β, and IL-6 in response to UV irradiation. Thus, three cell lines derived from skin all responded to radiation by secreting proinflammatory cytokines (albeit in different profiles), thus, providing an in vitro experimental system to study the impact of NF-κB decoy ODN on UV-induced changes in cellular function. As noted in Figure 1b, NF-κB decoy ODNs (10 μM) prevented the secretion of VEGF by Pam 212 keratinocytes, IL-6 and VEGF by NS47 fibroblasts, and TNF-α, IL-1β, and IL-6 by XS106 Langerhans cells after exposure to FS20 sunlamps (50 J/m²). Similar inhibition was also observed for cytokine production triggered by exposure to the solar simulator (100 J/m²) (Figure 1c). By contrast, scrambled control ODNs showed no inhibitory effect on any tested cytokine.

Consistent with the functional data described earlier here, the FITC-conjugated NF-κB decoy ODNs were incorporated into the XS106 cells in a time- and temperature-dependent fashion (Figure 2a). Importantly, the FITC-conjugated scrambled ODNs were also incorporated with similar kinetics and efficiency, thus, validating the use of the scrambled ODNs as a control for the NF-κB decoy ODNs. Confocal microscopic analysis revealed FITC-conjugated NF-κB decoy ODN within the cytoplasm of XS106 cells after 4 hours of incubation at 37°C (Figure 2b). NF-κB decoy ODN (≤ 30 μM) showed no significant cytotoxicity for XS106 cells even when combined with sublethal irradiation (Figure 2c). Likewise, NF-κB decoy ODN had no cytotoxic effect on UV-irradiated Pam 212 cells or NS47 cells (data not shown). In dose-dependency experiments, NF-κB decoy ODNs exhibited optimal inhibition of UV-induced cytokine production at 10-30 μM (Figure 2d). Taken together, these in vitro observations indicate that UV-induced NF-κB activation is critical to UV-dependent secretion of proinflammatory cytokines, with the implication that NF-κB decoy ODN may provide a useful tool to study pathogenic mechanisms of sunburn reactions in skin.

Figure 2

Pharmacokinetics of NF-κB decoy ODNs. (a) XS106 cells were incubated for the indicated periods with 10 μM FITC-conjugated NF-κB decoy ODNs (open symbols) or scrambled ODNs (closed symbols) at 4°C (triangles) or 37°C (circles) and then analyzed by FACS. Data shown are the mean ± SD (n = 3) of the mean fluorescence intensities (MFI). (b) After 4 hours of incubation with FITC-conjugated NF-κB decoy ODNs at 37°C, XS106 cells were examined by confocal microscopy. (c) XS106 cells were exposed to FS20 sunlamps at 100 J/m² (reversed triangles), 50 J/m² (triangles), 25 J/m² (squares), or 0 J/m² (circles), cultured in the presence of the indicated concentrations of NF-κB decoy ODNs (open symbols) or scrambled ODNs (closed symbols), and then tested for cell viability by FACS. Data shown are representative of two independent experiments. (d) XS106 cells were exposed to FS20 sunlamps at 50 J/m² (triangles) or sham-irradiated (circles), cultured for 24 hours in the presence of the indicated concentrations of NF-κB decoy ODNs (open symbols) or scrambled ODNs (closed symbols), and then examined for IL-1β secretion. Data shown are the mean ± SD from triplicate samples.

In vivo impact of NF-κB decoy ODNs on UV-induced skin inflammation. In the preliminary experiments, we observed that a single dorsal exposure of BALB/c mouse skin to UV radiation (FS20 sunlamps) caused marked cutaneous inflammation, most remarkably on their ears, and that the extent of inflammation could be examined by measuring the ear swelling responses. We also found that a relatively large dose (3,000 J/m²) of irradiation was required to induce experimental sunburn reactions in a reproducible and quantitative fashion. Under these conditions, ear swelling became detectable within 24 hours after irradiation, and these swelling responses progressed over the next 2–3 days (Figure 3a). Mice receiving two intraperitoneal injections of NF-κB decoy ODN 24 hours and 1 hour before UV exposure showed significantly reduced ear swelling responses at each time point tested, compared with control mice that received PBS alone or scrambled ODNs (Figure 3a). Having observed the efficacy of NF-κB decoy ODNs to prevent UV-induced skin inflammation, we next determined whether other UV-induced changes would be also affected by systemic administration of NF-κB decoy ODNs. Consistent with a previous report (43), UV radiation reduced the surface densities of epidermal Langerhans cells significantly with 60–70% reduction observed at 24 hours (Figure 3b). Two intraperitoneal injections of NF-κB decoy ODNs failed to prevent UV-induced reduction in Langerhans cell numbers compared with PBS or scrambled ODN controls, suggesting that the NF-κB pathway was not directly involved in UV-dependent Langerhans cell death and/or migration (Figure 3b). As shown in Figure 3c, UV radiation caused rapid and marked DNA damage mainly in the epidermis, as revealed by immunofluorescence staining with anti-CPD mAb. Injected NF-κB decoy ODNs had minimal effect on UV-induced CPD formation, indicating that DNA damage was not a direct consequence of UV-dependent NF-κB activation. At the same time, this implied further that DNA damage played little, if any, causative role in the development of UV-induced skin inflammation.

Figure 3

Inhibition of UV-induced ear skin swelling by systemic application of NF-κB decoy ODN. BALB/c mice received two intraperitoneal injections of NF-κB decoy ODN (open circles), scrambled ODNs (closed triangles), or PBS alone (closed circles) at 24 hours and 1 hour before irradiation. These animals were exposed to UV radiation and then examined for ear swelling responses at the indicated time points (a), surface density of epidermal Langerhans cells at 24 hours after irradiation (b), and CPD formation at 5 minutes after irradiation (c). Data shown in a are representative of three independent experiments, showing the mean ± SD (n = 10) of ear swelling responses (compared with ear thickness before irradiation). ^AStatistically significant differences (P < 0.05) compared with the UV plus PBS group. ^BStatistically significant differences (P < 0.01) compared with the UV plus PBS group. ^CStatistically significant differences compared with the UV plus scrambled ODN group (P < 0.05). ^DStatistically significant differences compared with the UV plus scrambled ODN group (P < 0.01). Data shown in b are the mean ± SD (n = 10) of numbers of IA⁺ epidermal cells/mm² in ear skin samples. Immunofluorescence pictures shown in c are representative staining profiles with anti-CPD mAb H3 or with an isotype-matched control IgG. ×400.

UV-induced ear swelling was also inhibited significantly by a single intraperitoneal injection (data not shown) or a single subcutaneous injection of NF-κB decoy ODNs into ear skin 6 hours before irradiation (Figure 4a). When mouse ear skin was first treated with acetone (to remove the barrier effect of surface stratum corneum) and then painted with FITC-conjugated NF-κB decoy ODNs, a fluorescence signal became detectable histologically in the epidermis and underlying reticular dermis in ear skin sections (data not shown). This protocol enabled us to deliver NF-κB decoy ODNs directly into the target site of UV irradiation. Topical application of NF-κB decoy ODNs was found to be at least as effective as either the systemic or local injection protocol in reducing ear swelling responses. Importantly, topically applied NF-κB decoy ODNs had no inhibitory effect on ear swelling induced by skin painting with a skin irritant chemical, croton oil, or by skin challenge with the contact sensitizer oxazolone in previously sensitized animals (Figure 4a). These observations excluded the possibility that NF-κB decoy ODN had acted simply as a nonspecific anti-inflammatory agent. In the next set of experiments, mice were exposed to UV radiation 1 hour after topical application of NF-κB decoy ODNs to their right ears. As shown in Figure 4b, ear swelling was diminished significantly in the right ears compared with PBS-treated left ears in the same UV-exposed animals, indicating that topically applied NF-κB decoy ODNs had exerted pharmacologic activity locally. In summary, skin-swelling responses to UV radiation, but not to irritant or allergic compounds, were inhibited significantly (50–70%) by systemic, local, or even topical application of NF-κB decoy ODN.

Figure 4

Impact of topically applied NF-κB decoy ODN on UV-induced inflammation. (a) BALB/c mice received two intraperitoneal (ip) injections, a single subcutaneous (sc) injection, or topical application of NF-κB decoy ODNs, scrambled ODNs, or PBS alone. Three groups of animals were exposed to UV irradiation, whereas two additional groups received topical application of croton oil on the ear skin or skin challenge with oxazolone (7 days after sensitization to the same hapten). Data shown are the mean ± SD (n = 10) of ear swelling responses (compared with ear thickness before treatment) at 4 days after irradiation or skin painting with croton oil (CO) or oxazolone (OX). (b) BALB/c mice received topical application of NF-κB decoy ODN (on right ears; open circles) or PBS alone (left ears; closed circles) 1 hour before UV irradiation. Data shown are the mean ± SD (n = 10) of the ear swelling responses at the indicated time points after irradiation. ^AStatistically significant differences (P < 0.05) compared with the UV plus PBS group. ^BStatistically significant differences (P < 0.01) compared with the UV plus PBS group. ^CStatistically significant differences compared with the UV plus scrambled ODN group (P < 0.05). ^DStatistically significant differences compared with the UV plus scrambled ODN group (P < 0.01). (c and d) BALB/c mice received topical application of NF-κB decoy or scrambled ODNs as already described here. Half of these mice were exposed to UV radiation, whereas the other half were sham-irradiated. Ear samples were harvested 4 days after irradiation and subjected to histological examination. Data shown are representative fields after H&E staining (c). All specimens (10 ear samples per group) were examined microscopically for ear skin thickness, the number of skin-infiltrating leukocytes, and the number of keratinocyte layers in the epidermis (d). In a different set of experiments, ear specimens were harvested 24 hours after irradiation to determine the impact of NF-κB decoy on sunburn cell formation. Data shown are the mean ± SD (n = 10) of each histological parameter. NT, not tested.

Histologically, UV-induced edema and leukocyte infiltration were both inhibited by topical application of NF-κB decoy ODN (Figure 4c). In fact, quantitative analyses of ear skin sections revealed significant reductions in ear swelling, numbers of skin infiltrating leukocytes, and extent of epidermal hyperplasia (Figure 4d). By contrast, the scrambled ODN had no inhibitory effects on any of these inflammatory parameters. Interestingly, NF-κB decoy ODN had no significant effects on the formation of sunburn cells in UV-exposed skin (Figure 4d). Thus, some, but not all, histological changes induced by UV radiation were preventable by topical application of NF-κB decoy ODNs.

To determine the impact of NF-κB decoy ODN on UV-induced accumulation of proinflammatory cytokines in vivo, we measured cytokines in whole ear skin extracts by ELISA. As observed for skin-derived cell lines in vitro (Figure 1), VEGF, TNF-α, IL-1β, and IL-6 were all elevated significantly in skin within 24 hours after UV irradiation (Figure 5a), and they remained elevated even 4 days after irradiation, when maximal ear swelling occurred (Figure 5b). Importantly, intraperitoneal injection of NF-κB decoy ODNs diminished almost completely UV-induced accumulation of these cytokines at both time points (Figure 5, a and b). Similar inhibition was also achieved by topical application of NF-κB decoy ODNs (Figure 5c). Again, scrambled ODNs had no significant effect. Moreover, NF-κB decoy ODNs failed to affect the accumulation of the same cytokines in irritant contact dermatitis, documenting the specificity of their action (Figure 5d). These results indicate that UV-induced NF-κB activation is the critical event required for local accumulation of proinflammatory cytokines in irradiated skin.

Figure 5

Prevention of UV-induced alteration in local cytokine profiles by NF-κB decoy ODN. (a and b) BALB/c mice received two intraperitoneal injections of NF-κB decoy or scrambled ODNs. (c and d) BALB/c mice received topical application of NF-κB decoy or scrambled ODNs. These animals were treated with UV irradiation (a–c) or topical application of croton oil (CO) onto ear skin (d). Ear skin samples harvested 1 day (a and d) or 4 days (b and c) after treatment were then examined for the indicated cytokines. Data shown are the mean ± SD (n = 10) of cytokine concentrations, normalized by total protein concentrations in each sample. ^AStatistically significant differences (P < 0.05) compared with the UV plus PBS group. ^BStatistically significant differences (P < 0.01) compared with the UV plus PBS group. ^CStatistically significant differences compared with the UV plus scrambled ODN group (P < 0.05). ^DStatistically significant differences compared with the UV plus scrambled ODN group (P < 0.01).

The present study demonstrates that UV-induced activation of NF-κB–dependent gene transactivation pathways is a critical event for the subsequent development of sunburn reactions in skin. This conclusion is supported by several lines of experimental evidence. First, UV radiation with conventional FS20 sunlamps or with a solar simulator–induced NF-κB activation in cells of three lineages that were derived from skin, and irradiation concurrently triggered the secretion of VEGF, TNF-α, IL-1β, and IL-6. Second, NF-κB decoy ODNs inhibited significantly UV-induced secretion of each proinflammatory cytokine. Third, systemic, local, or topical administration of NF-κB decoy ODN reduced the severity of experimentally induced sunburn reactions, as assessed by ear swelling, histological changes, and the accumulation of proinflammatory cytokines. On the other hand, NF-κB decoy ODN did not affect noninflammatory changes (Langerhans cell depletion, CPD formation, and sunburn cell formation) that were also observed after radiation, thus, documenting specificity of the pharmacologic activity of NF-κB decoy and the physiological role played by NF-κB–dependent gene transactivation pathways. Finally, topically applied NF-κB decoy ODN resulted in local inhibition of sunburn reactions, without affecting inflammation in nontreated skin sites, with the implication that NF-κB decoy ODN acted primarily on cells that reside in skin. Our conclusion is further supported, albeit indirectly, by the clinical observation that sunburn reactions can be treated successfully by nonsteroidal anti-inflammatory agents (44), which are now known to block NF-κB activation by interfering with IkB kinase-2 (IKK2) activity (45). Moreover, two groups reported independently that mice lacking the IkBα gene developed spontaneously widespread severe dermatitis with leukocyte infiltration, suggesting a pathway through which NF-κB activation directly leads to skin inflammation (46, 47).

In addition to the recent progress in antisense ODN and decoy ODN technologies, an entirely new field of immunomodulatory ODNs has attracted significant attention (48). Several nucleotide sequences or motifs that exert nonspecific immunostimulatory or inhibitory effects have been identified. For example, the 27-bp ODN complementary to a portion of the rev region of the HIV gene have been shown to induce polyclonal activation of B cells in vitro (49, 50) and to cause massive splenomegaly and hyper-γ-globulinemia upon in vivo administration (49). The CpG motif, originally identified in bacterial DNA (51), is now known to stimulate both humoral and cellular immune responses via acting on B cells, macrophages, dendritic cells, natural killer cells, and T cells and to control the balance between the Th1- and Th2-type immune responses (52–55). An important question then concerned whether the NF-κB decoy ODNs, indeed, prevented sunburn reactions by blocking NF-κB–dependent gene transactivation pathway. Three lines of evidence support the specificity of our ODN-based approach. First, neither the NF-κB decoy ODNs nor the scrambled control ODNs used in this study contained any of the currently recognized immunomodulatory sequences, including the antisense ODN for the HIV rev gene, the conventional CpG motif (TCG)_n or (CG)_n repeats, or palindrome sequences (49–53). Second, the NF-κB decoy ODNs, but not the scrambled control ODNs, blocked NF-κB–mediated transactivation of the Luc gene, without affecting the AP-1–mediated Luc gene expression. Finally, topical application of the NF-κB decoy ODN prevented UV-induced skin inflammation, without affecting inflammatory responses to skin irritant or allergic compounds. We believe that these observations have excluded the possibility that the NF-κB decoy ODNs prevented sunburn reactions by simply acting as a nonspecific, anti-inflammatory reagent or as a “generic” transcriptional inhibitor.

UV radiation was found, more than a decade ago, to induce the production of TNF-α, IL-1β, and IL-6 by keratinocytes (56–58), and these cytokines are thought to play pathogenic roles in the development of UV-induced cutaneous inflammation (2). More recently, VEGF production by UV-irradiated keratinocytes was proposed to serve as an additional pathogenic event (59, 60). In fact, working with VEGF transgenic mice (keratin 14 promoter), Detmar et al. demonstrated recently that overproduction of VEGF by keratinocytes is sufficient for the development of pathophysiological features characteristic of cutaneous inflammation, including increased vascular density and permeability and enhanced leukocyte rolling and adhesion (61). Thus, it is reasonable to state that UV-induced alterations in the local cytokine milieu are essential to the development of sunburn reactions. Our finding that NF-κB decoy ODNs inhibited both cytokine production and sunburn reactions supports this concept.

Although NF-κB is one of the key transcription factors driving the expression of VEGF, TNF-α, IL-1β, and IL-6 genes (24, 62), it has remained unclear whether UV-induced NF-κB activation indeed plays any functional role in the production of these pro-inflammatory cytokines after UV irradiation. We have observed that local accumulation of these cytokines in sunburn reactions, but not in contact dermatitis reactions, is inhibited by NF-κB decoy ODNs almost completely. These results indicate that the NF-κB pathway plays an essential role in transducing UV-dependent signals. As described earlier here, UV radiation may lead to NF-κB activation by different mechanisms (e.g., DNA damage, ROI generation, kinase phosphorylation, and ligand-independent receptor signaling), and the present study was not designed to determine the relative contribution of each mechanism. Likewise, it was beyond the scope of this study to determine the role of NF-κB pathway in the development of other UV-induced skin changes (e.g., immunosuppression, accelerated aging, and cancer development). In this regard, topical application of T4 endonuclease immediately after radiation was found to prevent sunburn cell formation and Langerhans cell depletion significantly, without affecting UV-induced skin inflammation (63). Thus, it is conceivable that UV irradiation may cause different types of skin manifestation via different pathways/mechanisms.

In conclusion, the present study has provided the first experimental evidence for the function of NF-κB pathway in the pathogenesis of sunburn reactions. Our data also introduce the concept that sunburn reactions are the culmination of specific inflammatory events transcriptionally triggered by UV-dependent NF-κB activation, instead of a sum of inflammatory changes associated with tissue damage.

We thank A. Weiss for providing two reporter gene constructs, and S. Hayashi and M. Mummert for thoughtful comments. We also thank J. Loftus, B. Liu, and L. Ellinger for their technical support, and P. Adcock for her administrative assistance. This study was supported by NIH grants (RO1-AR40042, RO1-AR35068, RO1-AR43777, and RO1-AI43262) and by a Centre de Recherches et Investigations Epidermiques et Sensorielles (CE.R.I.E.S.) Award (A. Takashima).

1. Kripke, ML. Photoimmunology. Photochem Photobiol 1990. 52:919-924.
   View this article via: PubMed CrossRef Google Scholar
2. Takashima, A, Bergstresser, PR. Impact of UVB radiation on the epidermal cytokine network. Photochem Photobiol 1996. 63:397-400.
   View this article via: PubMed CrossRef Google Scholar
3. Krutmann, J, Grewe, M. Involvement of cytokines, DNA damage, and reactive oxygen intermediates in ultraviolet radiation-induced modulation of intercellular adhesion molecule-1 expression. J Invest Dermatol 1995. 105(Suppl. 1):67S-70S.
   View this article via: PubMed CrossRef Google Scholar
4. Bielenberg, DR, et al. Molecular regulation of UVB-induced cutaneous angiogenesis. J Invest Dermatol 1998. 111:864-872.
   View this article via: PubMed CrossRef Google Scholar
5. Aragane, Y, et al. Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L. J Cell Biol 1998. 140:171-182.
   View this article via: PubMed CrossRef Google Scholar
6. Kasibhatia, S, et al. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-κB and AP-1. Mol Cell 1998. 1:543-551.
   View this article via: PubMed CrossRef Google Scholar
7. Eller, MS, Ostrom, K, Gilchrest, BA. DNA damage enhances melanogenesis. Proc Natl Acad Sci USA 1996. 93:1087-1092.
   View this article via: PubMed CrossRef Google Scholar
8. Eller, MS, Maeda, T, Magnoni, C, Atwal, D, Gilchrest, BA. Enhancement of DNA repair in human skin cells by thymidine dinucleotides: evidence for a p53-mediated mammalian SOS response. Proc Natl Acad Sci USA 1997. 94:12627-12632.
   View this article via: PubMed CrossRef Google Scholar
9. Vink, AA, et al. The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers. Proc Natl Acad Sci USA 1997. 94:5255-5260.
   View this article via: PubMed CrossRef Google Scholar
10. O’Connor, A, et al. DNA double strand breaks in epidermal cells cause immune suppression in vivo and cytokine production in vitro. J Immunol 1996. 157:271-278.
    View this article via: PubMed Google Scholar
11. Morita, A, et al. Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of ultraviolet-A radiation phototherapy. J Exp Med 1997. 186:1763-1768.
    View this article via: PubMed CrossRef Google Scholar
12. Scharffetter-Kochanek, K, et al. UV-induced reactive oxygen species in photocarcinogenesis and photoaging. Biol Chem 1997. 378:1247-1257.
    View this article via: PubMed Google Scholar
13. Rosette, C, Karin, M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science 1996. 274:1194-1197.
    View this article via: PubMed CrossRef Google Scholar
14. Simon, MM, Aragane, Y, Schwarz, A, Luger, TA, Schwarz, T. UVB light induces nuclear factor κB (NFκB) activity independently from chromosomal DNA damage in cell-free cytosolic extracts. J Invest Dermatol 1994. 102:422-427.
    View this article via: PubMed CrossRef Google Scholar
15. Knebel, A, Rahmsdorf, HJ, Herrlich, P. Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents. EMBO J 1996. 15:5314-5325.
    View this article via: PubMed Google Scholar
16. Derijard, B, et al. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 1994. 76:1025-1037.
    View this article via: PubMed CrossRef Google Scholar
17. Lee, FS, Hagler, J, Zhijian, JC, Maniatis, T. Activation of the IκB kinase complex by MEKK1, a kinase of the JNK pathway. Cell 1997. 88:213-222.
    View this article via: PubMed CrossRef Google Scholar
18. Gilchrest, BA, Park, H-Y, Eller, MS, Yaar, M. Mechanisms of ultraviolet light-induced pigmentation. Photochem Photobiol 1996. 63:1-10.
    View this article via: PubMed CrossRef Google Scholar
19. van Dam, H, et al. ATF-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents. EMBO J 1995. 14:1798-1811.
    View this article via: PubMed Google Scholar
20. Grether-Beck, S, et al. Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene. Proc Natl Acad Sci USA 1996. 93:14586-14591.
    View this article via: PubMed CrossRef Google Scholar
21. Iordanov, M, et al. CREB is activated by UVC through a p38-HOG-1–dependent protein kinase. EMBO J 1997. 16:1009-1022.
    View this article via: PubMed CrossRef Google Scholar
22. Schmidt, KN, Amstad, P, Cerutti, P, Baeuerle, PA. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-κB. Chem Biol 1995. 2:13-22.
    View this article via: PubMed CrossRef Google Scholar
23. Nemoto, S, DiDonato, JA, Lin, A. Coordinate regulation of IκB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-κB-inducing kinase. Mol Cell Biol 1998. 18:7336-7343.
    View this article via: PubMed Google Scholar
24. Barnes, PJ, Karin, M. Nuclear factor-κB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997. 336:1066-1071.
    View this article via: PubMed CrossRef Google Scholar
25. Baeuerle, PA, Baltimore, D. NF-κB: ten years after. Cell 1996. 87:13-20.
    View this article via: PubMed CrossRef Google Scholar
26. Sullenger, BA, Gallardo, HF, Gilboa, E. Overexpression of TAR sequence renders cells resistant to human immunodeficiency virus replication. Cell 1990. 63:601-608.
    View this article via: PubMed CrossRef Google Scholar
27. Bielinska, A, Shivdasani, RA, Zhang, L, Nabel, GJ. Regulation of gene expression with double-stranded phosphorothioate oligonucleotides. Science 1990. 250:997-1000.
    View this article via: PubMed CrossRef Google Scholar
28. Morishita, R, et al. In vivo transfection of cis element “decoy” against nuclear factor-κB binding site prevents myocardial infarction. Nat Med 1997. 3:894-899.
    View this article via: PubMed CrossRef Google Scholar
29. Ono, S, et al. Decoy administration of NF-κB into the subarachnoid space for cerebral angiopathy. Hum Gene Ther 1998. 9:1003-1011.
    View this article via: PubMed CrossRef Google Scholar
30. Kawamura, I, et al. Intratumoral injection of oligonucleotides to the NFκB binding site inhibits cachexia in a mouse tumor model. Gene Ther 1999. 6:91-97.
    View this article via: PubMed CrossRef Google Scholar
31. Yuspa, SH, Hawley-Nelson, P, Koehler, B, Stanley, JR. A survey of transformation markers in differentiating epidermal cell lines in culture. Cancer Res 1980. 40:4694-4703.
    View this article via: PubMed Google Scholar
32. Xu, S, et al. Successive generation of antigen-presenting, dendritic cell lines from murine epidermis. J Immunol 1995. 154:2697-2705.
    View this article via: PubMed Google Scholar
33. Timares, L, Takashima, A, Johnston, SA. Quantitative analysis of the immunopotency of genetically transfected dendritic cells. Proc Natl Acad Sci USA 1998. 95:13147-13152.
    View this article via: PubMed CrossRef Google Scholar
34. Matsue, H, et al. Induction of antigen-specific immunosuppression by CD95L cDNA-transfected “killer” dendritic cells. Nat Med 1999. 5:930-937.
    View this article via: PubMed CrossRef Google Scholar
35. Kitajima, T, Ariizumi, K, Bergstresser, PR, Takashima, A. UVB radiation sensitizes a murine epidermal dendritic cell line (XS52) to undergo apoptosis upon antigen presentation to T cells. J Immunol 1996. 157:3312-3316.
    View this article via: PubMed Google Scholar
36. Kaminski, MJ, Bergstresser, PR, Takashima, A. In vivo activation of mouse dendritic epidermal T cells in sites of contact dermatitis. Eur J Immunol 1993. 23:1715-1718.
    View this article via: PubMed CrossRef Google Scholar
37. Kitajima, T, et al. T cell–mediated terminal maturation of dendritic cells: loss of adhesive and phagocytotic capacities. J Immunol 1996. 157:2340-2347.
    View this article via: PubMed Google Scholar
38. Roza, L, van der Wulp, KJM, MacFarlane, SJ, Lohman, PHM, Baan, RA. Detection of cyclobutane thymine dimers in DNA of human cells with monoclonal antibodies raised against a thymine dimer-containing tetranucleotide. Photochem Photobiol 1988. 48:627-633.
    View this article via: PubMed CrossRef Google Scholar
39. Vink, AA, et al. Localization of DNA damage and its role in altered antigen-presenting cell function in ultraviolet- irradiated mice. J Exp Med 1996. 183:1491-1500.
    View this article via: PubMed CrossRef Google Scholar
40. Elmets, CA, Bergstresser, PR, Tigelaar, RE, Wood, PJ, Streilein, JW. 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.
    View this article via: PubMed CrossRef Google Scholar
41. Shapiro, VS, Mollenauer, MN, Greene, WC, Weiss, A. c-rel regulation of IL-2 gene expression may be mediated through activation of AP-1. J Exp Med 1996. 184:1663-1669.
    View this article via: PubMed CrossRef Google Scholar
42. Yablonski, D, Kuhne, MR, Kadlecek, T, Weiss, A. Uncoupling of nonreceptor tyrosine kinases from PLC-γ1 in an SLP-76–deficient T cell. Science 1998. 281:413-416.
    View this article via: PubMed CrossRef Google Scholar
43. Aberer, W, Romani, N, Elbe, A, Stingl, G. Effects of physicochemical agents on murine epidermal Langerhans cells and Thy-1–positive dendritic epidermal cells. J Immunol 1986. 136:1210-1216.
    View this article via: PubMed Google Scholar
44. Stern, RS, Dobson, TB. Ibuprofen in the treatment of UV-B–induced inflammation. Arch Dermatol 1985. 121:508-512.
    View this article via: PubMed CrossRef Google Scholar
45. Yin, M-J, Yamamoto, Y, Gaynor, RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of IκB kinase-β. Nature 1998. 396:77-80.
    View this article via: PubMed CrossRef Google Scholar
46. Beg, AA, Sha, WC, Bronson, RT, Baltimore, D. Constitutive NF-κB activation, enhanced granulopoiesis, and neonatal lethality in IκBα-deficient mice. Genes Dev 1995. 9:2736-2746.
    View this article via: PubMed CrossRef Google Scholar
47. Klement, JF, et al. I κBα deficiency results in sustained NF-kB response and severe widespread dermatitis in mice. Mol Cell Biol 1996. 16:2341-2349.
    View this article via: PubMed Google Scholar
48. Klinman, DM, Verthelyi, D, Takeshita, F, Ishii, KJ. Immune recognition of foreign DNA: a cure for bioterrorism. Immunity 1999. 11:123-129.
    View this article via: PubMed CrossRef Google Scholar
49. Branda, RF, Moore, AL, Mathews, L, McCormarck, JJ, Zon, G. Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1. Biochem Pharmacol 1993. 45:2037-2043.
    View this article via: PubMed CrossRef Google Scholar
50. Liang, H, Nishioka, Y, Reich, CF, Pisetsky, DS, Lipsky, PE. Activation of human B cells by phosphorothioate oligodeoxynucleotides. J Clin Invest 1996. 98:1119-1129.
    View this article via: JCI PubMed CrossRef Google Scholar
51. Kreig, AM, et al. CpG motifs in bacterial DNA trigger direct B cell activation. Nature 1995. 371:546-549.
    View this article via: PubMed Google Scholar
52. Iho, S, Yamamoto, T, Takahashi, T, Yamamoto, S. Oligodeoxynucleotides containing palindrome sequences with internal 5′-CpG-3′ act directly on human NK and activated T cells to induce IFN-γ production in vitro. J Immunol 1999. 163:3642-3652.
    View this article via: PubMed Google Scholar
53. Carson, DA, Raz, E. Oligonucleotide adjuvants for T helper 1 (Th1)-specific vaccination. J Exp Med 1997. 186:1621-1622.
    View this article via: PubMed CrossRef Google Scholar
54. Hartmann, G, Weiner, GJ, Kreig, AM. CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc Natl Acad Sci USA 1999. 96:9305-9310.
    View this article via: PubMed CrossRef Google Scholar
55. Jakob, T, Walker, PS, Kreig, AM, Udey, MC, Vogel, JC. Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J Immunol 1998. 161:3042-3049.
    View this article via: PubMed Google Scholar
56. Köck, A, et al. Human keratinocytes are a source of tumor necrosis factor α: evidence for synthesis and release upon stimulation and endotoxin or ultraviolet light. J Exp Med 1990. 172:1609-1614.
    View this article via: PubMed CrossRef Google Scholar
57. Urbanski, A, et al. Ultraviolet light induces increased circulating interleukin-6 in humans. J Invest Dermatol 1990. 94:808-811.
    View this article via: PubMed CrossRef Google Scholar
58. Ansel, JC, Luger, TA, Green, I. The effect of in vitro and in vivo UV irradiation on the production of ETAF activity by human and murine keratinocytes. J Invest Dermatol 1983. 81:519-523.
    View this article via: PubMed CrossRef Google Scholar
59. Brown, LF, et al. Overexpression of vascular permeability factor (VPF/VEGF) and its endothelial cell receptors in delayed hypersensitivity skin reactions. J Immunol 1995. 154:2801-2807.
    View this article via: PubMed Google Scholar
60. Brauchle, M, Funk, JO, Kind, P, Werner, S. Ultraviolet B and H₂O₂ are potent inducers of vascular endothelial growth factor expression in cultured keratinocytes. J Biol Chem 1996. 271:21793-21797.
    View this article via: PubMed CrossRef Google Scholar
61. Detmar, M, et al. Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 1998. 111:1-6.
    View this article via: PubMed CrossRef Google Scholar
62. Chilov, D, et al. Genomic organization of human and mouse genes for vascular endothelial growth factor C. J Biol Chem 1997. 272:25176-25183.
    View this article via: PubMed CrossRef Google Scholar
63. Wolf, P, Cox, P, Yarosh, DB, Kripke, ML. Sunscreens and T4N5 liposomes differ in their ability to protect against ultraviolet-induced sunburn cell formation, alterations of dendritic epidermal cells, and local suppression of contact hypersensitivity. J Invest Dermatol 1995. 104:287-292.
    View this article via: PubMed CrossRef Google Scholar