A role for NF-kappaB-dependent gene transactivation in sunburn

Abstract

Exposure of skin to ultraviolet (UV) radiation is known to induce NF-kappaB 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-kappaB-dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-kappaB cis element (NF-kappaB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-alpha, 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-kappaB 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-kappaB target genes, rather than from nonspecific changes associated with tissue damage.

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).

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.

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.

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.

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).