Ultraviolet (UV) irradiation from the sun is the primary environmental cause of skin damage such as thickening, wrinkle formation, inflammation and carcinogenesis. It is well established that nuclear factor κB (NF-κB) is activated upon UV irradiation and induces various genes including interleukin-1 (IL-1), tumor necrosis factor α (TNFα), matrix metalloprotease-1 (MMP-1) and bFGF in keratinocytes and skin fibroblasts. In this study, we synthesized TNFR-peptide as an antagonist of TNFR which is based on the sequence of DNA binding site of the TNFR and TRAF2 and confirmed that TNFR-peptide block to NF-κB activation through the observation of TNFR-peptide of inhibitory effect on TNF-α induced IκB degradation and nuclear translocalization of NF-κB dimer. We examined if TNFR-peptide, an NF-κB pathway inhibitor, could block the UVB-mediated skin changes. We found that TNFR-pep could effectively inhibit MMP expression in TNF-α-stimulated fibroblasts and block TNF-α-induced inhibition of collagen synthesis in human dermal fibroblast. We also found that TNFR-peptide inhibits the UVB-induced proliferation of keratinocytes and melanocytes in the mouse skin and observed that TNFR-peptide inhibits inflammatory response which is known to be considered that COX2 expression and neutrophils infiltration. These results suggest that TNFR-peptide should be useful for the prevention of skin photoaging.
UV, Photoaging, NF-κB, TNF-α
When skin is repeat exposed to UV, this leads to photoaging. The photoaging process can lead to skin changes such as coarse wrinkles, laxity, dryness, roughness and pigmentation, leathery appearance and formation of wrinkles. These changes can be explained by keratinocyte hyperproliferation and degradation of collagen fibers (1) causing skin wrinkling and laxity, and melanocyte proliferation that leads to pigmentation characterized by dysregulation of melanocyte homeostasis and increase in the melanocyte density (2). Also the UV-induced production of proinflammatory cytokines, such as interleukin- 1 (IL-1) and tumor necrosis factor α (TNF-α), has been considered attributable to these changes by inducing inflammatory processes in the skin (3, 4, 5).
It is well known that NF-κB activation by various stimuli, such as UV, leading to induce proinflammatory cytokines, such as IL-1 and TNF-α, which subsequently stimulate the signal transduction pathway to activate NF-κB, thus conforming a vicious cycle (8, 9). UV-induced TNF-α modulate expression of MMP and bFGF causing skin aging through the NFκB activation pathway. MMP-9 is responsible for the degradation of collagen fibers that causes skin aging (3, 6). In addition, TNF-α is known to affect the stimulation of both keratinocytes and fibroblasts by basic fibroblast growth factor (bFGF) that is responsible for the proliferations (7). Thus, inhibition of the TNF-α and NF-κB activation pathway would block the vicious cycle induced by UV irradiation and effectively prevent the photoaging.
In this study, TNFR-peptidomimetics was synthesized based on the sequence of DNA binding site of the TNFR and TRAF2. The TNF receptor-associated factor (TRAF) family is a group of structurally similar scaffold protein that link members of the TNF receptor superfamily to signaling cascades. (10, 11, 12) Downstream signaling components of these cascades include the IκB kinase and mitogen-activated protein kinases, which in turn control gene expression through transcription factors such as NF-κB. Tumor-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-κB signaling triggered by TNF-α 1, 2. We synthesized based on the sequence of DNA binding site of the TNFR and TRAF1 to interfere that TRAF1 bind to TNFR, and expect to block NF-κB activation pathway.
In this study, we examined the inhibitory effect of TNFR-peptidomimetics in the processes of UVB-mediated cutaneous alterations through suppressing TNF-α-NF-κB signaling cascade using cultured cells and mouse models.
Material and methods
2.1 Cell culture
HaCat keratinocyte and NIH3T3 fibroblast were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium containing 10% FBS, 5% penicillin streptomycin. HDFa(human dermal fibroblast) were purchased from the Lifetechnoglies and in medium106 supplemented with LSGS. All cells were maintained at 37°C, 5% CO2.
2.2 Western blotting
Cells were lysed in lysis buffer [50 mM Tris-HCl, pH 7.4,150 mM NaCl, 1 mM EDTA, 5 mM sodiumorthovanadate, 1% NP-40 and proteaseinhibitors cocktail (Quatte)] for 30min on ice, and centrifuged at 13,000 rpm for 20 min at 4°C. After centrifugation, supernatants were collected and total protein concentration was compensated using Bradford assay kit (BIO-RAD, USA). Equal amounts of protein were loaded onto 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane and probed with proper antibodies. Primary antibodies, FGF-2, MMP1, COX-2, Actin (Santa cruz), MMP9 (Millipore) and IkB (cell signaling) (diluted 1:1000 in 0.5 % bovine serum albumin), were used and detected by incubation with HRP-conjugated secondary antibodies (1:10000 in TTBS), using an ECL system.
2.3 Gelatin Zymography
MMP-9 activity was assessed by gelatin zymography. Briefly, cells were exposed to TNFα (25ng/ml) and TNFR-pep indicated does for 24h. Culture supernatants were mixed with non-reducing sample buffer containing 125mM Tris–HCl (pH 6.8), 20% glycerol, 0.005% BPB and 4% SDS was added to aliquots of culture medium at a ratio of 1:4 and proteins were subjected to SDS-PAGE in 10% polyacrylamide gels containing 0.5% gelatin (v/v). After electrophoresis, the gels were washed twice in 10 mM Tris-Cl (pH 7.5) and 2.5% Triton X-100 for 1 h at room temperature to remove SDS, followed by incubation for 24 h at 37oC in developing buffer containing 50 mM Tris-Cl (pH 7.5), 10 mM CaCl2, and 0.01% NaCl. The gels were then stained with Coomassie Brilliant Blue R250 (Bio-Rad, CA) (0.25%) for 30 min and destained for 1 h in a solution of acetic acid and methanol. Clear bands (zone of gelatin degradation) against the blue background of stained gelatin indicate proteolytic activity.
2.4 UVB irradiation and treatment
Cells were seeded at a density of 3×105 cells/well in 6 well plates and cultured in DMEM for 24 h. Cells were then starved in FBS free DMEM for 24 h and rinsed with phosphate-buffered saline (PBS). UVB irradiation was performed through a thin film of PBS via exposure to 15mJ/cm2 at 312 nm, as measured using a VLX-3W research radiometer (VILVER, France). After irradiation, cells were cultured in serum free DMEM containing TNFR-pep for 24h.
2.5 Chromatin immunoprecipitation assay
To detect the in vivo association of NF-κB (p65) with human MMP-9 promoter, chromatin immunoprecipitation (ChIP) analysis was performed. HaCaT in 100-mm dishes were grown to confluence and serum starved for 24 h. After stimulation with TNF-α or UV, TNFR-pep for 4h, protein-DNA complexes were fixed by 1% formaldehyde in PBS. The fixed cells were washed and lysed in SDS-lysis buffer (1% SDS, 10 mM EDTA, 1 mM PMSF, 50 mM Tris -HCl, pH 8.1), and sonicated on ice until the DNA size became 200–1,000 base pairs. The samples were centrifuged, and the soluble chromatin was precleared by incubation with sheared salmon sperm DNAprotein agarose A slurry for 20 min at 4°C with agitation. After centrifugation at 14000 rpm for 5 min, one portion of the precleared supernatant was used as DNA input control, and the remains were subdivided into aliquots and then incubated with a nonimmune rabbit immunoglobulin G, IgG Ab to NF-κB p65 (Cell signaling Technology), overnight at 4°C. The immunoprecipitated complexes of Ab-protein-DNA were collected and washed with Dialysis buffer (2mM EDTA, 50mM Tris-Cl pH8.0) and then eluted with elution buffer (1% SDS, 100 mM NaHCO3). To reverse cross linking add 5 M NaCl to the combined eluates and incubated at 65°C for 4 h and then eluates was digested with 10 mg of proteinase K (Sigma)/ml for 1 h at 45°C. The DNA was then extracted with phenol-chloroform, and the purified DNA pellet was precipitated with ethanol. After washing, the DNA pellet was resuspended in H2O and subjected to PCR amplification with the forward (5-TGTCCCTTTACTGCCCTGA-3) and reverse (5-ACTCCAGGCTCTGTCCTCCTCTT-3), which were specifically designed from the MMP-9 promoter region (657 to 484) . PCR products were analyzed on agarose gels.
2.6 In situ PLA
To determine the nuclear translocation of NFκB dimer, we performed in situ PLA assay. The “in situ” PLA studies on fixed NIH3T3 fibroblast cells were performed as follows (O-LINK Bioscience, Upsalla, Sweden). Cells were grown on slide glass for at least 16 h, and treated with TNFR-pep or dexamethasone for 3h and then exposed to TNF-α of 25ng/ml for 1hour or After UVB irradiation of 1.5J/cm2 at 356nm, treated with TNFR-pep or dexamethasone or ascorbic acid for 4h hr in 5% C02 at 37°C and washed by PBS twice. In situ PLA detection was carried out using the appropriate DUOLINK II In Situ kit components according to the protocol of the manufacturer.
In brief, cells washed once with PBS, and then subjected to blocking using the DUOLINK blocking solution (1 drop) at 37°C in a wet chamber for 30 min. after tapping off the Blocking solution from the slides, antibodies were added at a dilution of 1 100 in 40 µl DUOLINK antibody diluent and incubated in a wet chamber at 37°C for 30 min. The slides were washed two times with Wash buffer A for 5 min each, and then secondary antibodies (DUOLINK anti-rabbit PLA-plus probe, DUOLINK anti-goat PLA-minus probe) were added and incubated at 37°C for 1 h. Two washes with Wash buffer A were then followed by addition of the ligation mix and incubation at 37°C for 30 min, followed by another two washes. Thereafter, the amplification reaction was carried out at 37°C for 100 min. Subsequently, the slides were washed twice with Wash buffer B, and once with 0.1×Wash buffer B. Mounting was done with mounting solution containing DAPI. Antibodies used for PLA were rabbit anti-NF-κB p65 Ab (Cell Signaling Technology) combined with mouse anti- NF-κB p50 Ab (Santa Cruz Biotechnology).
2.7 Mouse model for the UVB-irradiated skin
Male 6-week-old DBA/2 mice were purchased from Dooyeol Biotech (Seoul, Korea) and were maintained for 1 week before starting the experiment. All mice were randomly divided into 3 groups of 5 animals: UV+TNFR-pep treatment, UV treatment and control. TNFR-pep was administered 10mg/kg TNFR-pep using i.p. injections for 12 days. And for the group of UV+TNFR-pep and UV treatments, the heads of mice were locally exposed to at 180 mJ/cm2 of UVB irradiation using a VLX-3W research radiometer (VILVER, France). The same amount of saline was injected to UV treatment and control groups.
After 12 days, ears were excised from all subjects. One of the ear specimens of each animal was stored in -80°C for staining of melanocytes, and the other ear specimen was paraffin-embedded for the immunohistochemical analysis of the determination of skin thickness by H&E staining and neutrophils staining by using neutrophil marker monoclonal antibody (Santa Cruz Biotechnology).
For melanocyte counting, L-DOPA staining performed as following method of Hiramoto et al. (2003). Briefly, for separation of the epidermal and basal layers from the rest, the skin tissues were soaked in 2 N NaBr solution at 37°C for 2 h. Melanocytes in epidermal layer were stained by immersing in 0.1 M phosphate-buffered saline (pH 7.2) containing 0.14% L-DOPA at room temperature for 3h and observed under a microscope.
3.1 TNFR-peptide inhibits TNFR signaling by interrupting interaction of TNFR and TRAF2
In this study, TNFR-peptidomimetics was synthesized based on the sequence of DNA binding site of the TNFR and TRAF2 for expecting of blocking to TNF-α-NF-κB pathway. To evaluate capacity of TNFR-pep for blocking in TNFR and TRAF2 interaction directly, we performed in situ PLA assay using specific antibody of TNFR and TRAF2.
We observed increase interaction of TNFR and TRAF2 as a red dot in the cells was stimulated with TNF-α and treatment with TNFR-pep decreased interaction in dose dependent manner (Fig. 1).
3.2 TNFR-peptide prevents activation of NF-κB
It is well established that a nuclear transcription factor, NF-κB, is activated upon UV irradiation. (Nfkb-ref2) Activation of NF-κB by TNF-α is essential to elicit an effective response, since many of the TNF-a-regulated genes contain binding sites for NF-κB. To understand how TNFR-peptide regulates gene transcription and expression, we conducted western blot, in situ PLA and ChIP assay for transcription factor NF-κB (Fig. 2).
Using western blot, TNF-α treating on mouse fibroblast degraded Iκ-B which physically holds NF-κB subunit p65 and p50 complex in cytosol to prevent nuclear translocation (Fig. 2A). On in situ PLA assay, TNF-α promote p65-p50 complex moved from cytoplasm to nucleus (Fig. 2B). Finally nuclear p65 Binding to MMP-9 promoter region was enhanced in ChIP assay (Fig. 2D). UVB irradiation also increased p65-p50 complex nuclear translocation and p65 binding to MMP-9 promoter (Fig. 2C, D). But in TNFR-peptide co-stimulation, it prevented Iκb degradation as dose dependent manner and shown similar capacity compared with DHA used as an anti-inflammatory positive control (Fig. 2A). Also p65-p50 complex translocation (Fig. 2B, C) and p65 binding to MMP-9 promoter are prevented (Fig 2D). Dexamethasone was used as well-known anti-inflammatory agent.
3.3 TNFR-peptide has a TNF-α antagonistic effect on the protein expression of bFGF, MMP-1, MMP-9
The UV-induced cutaneous alterations are concerned with bFGF and MMP-1,-9. It was previously shown that production of bFGF and MMP-1 was induced by UVB and that UVB irradiation induced production of IL-1 and TNF-α in keratinocytes and fibroblasts (Nfkb-ref1).
To find out the effect of TNFR-peptide on TNF-α stimulated human keratinocyte, bFGF expression level was evaluated by western blotting (Fig. 3A). Stimulation of TNF-α increased bFGF expression level but co-treatment with TNFR-peptide decreased the bFGF expression in dose dependent manner. Similar to Fig. 3A data, MMP-1 and-9 expression was also reduced by TNFR-peptide on TNF-α activated mouse fibroblast (Fig. 3B).
3.4 TNFR-peptide block type 1 procollagen decrease and elevation of COX2 protein expression level in TNF-α stimulated mouse fibroblast.
Decrease of procollagen expression caused by repeated UV irradiation has been considered to be a cause of photo-aging. UV irradiation induced TNF-α which reduces ECM deposition either by inducing the production of stromal collagenases or by inhibiting the synthesis of structural components such as the type I collagen, the major structural component of connective tissue. We investigated the effect of TNFR-peptide on TNF-α-induced decrease of procollagen expression by western blot. TNF-α caused a decrease in type I procollagen expression but co treatment with TNFR-peptide blocked that (Fig. 3C).
Skin Chronic inflammation by UV leads to the up regulation of cyclo-oxygenase-2 (COX-2), which is known as prostaglandin-endoperoxide synthase 2. TNF-α is known to activate several cellular signaling pathways, which mediate the expression of COX-2 by facilitating the recruiting of various transcription factors to the COX-2 promoter (Cox2-ref1). To verify the change of COX2 protein, we used western blot. TNF-α stimulation induced Cox2 protein expression on mouse fibroblast. Similar to former data, in co-stimulation with TNFR-peptide prevented increase of COX2 as much as positive control DHA (Fig. 3D).
3.5 Effects of the TNFR-pep on epidermal hyperproliferation, melanocyte growth and neutrophil infiltration in UVB-induced animal model.
It is well known that UV irradiation stimulate both keratinocyte and fibroblast to induce basic fibroblast growth factor bFGF) which is resulted in proliferations of melanocytes and keratinocytes. (Brenneisen et al., 2002; Chung, 2003; Hirobe et al., 2003 Pittelkow and Shipley, 1989; Bielenberg et al., 1998) nfkb-ref1 Thus, we examined the effects of TNFR-pep on proliferations of keratinocytes and melanocytes in UVB irradiation mouse model. As shown in Fig. 4A, although the thickness of epidermis was significantly increased by UVB exposure compared to control untreated skin, treatment with TNFR-pep markedly decreased the epidermal hyperproliferation. The melanocyte growth was analyzed by L-DOPA staining which is used the epidermal and basal layers were separated from the skin tissue. It was significantly reduced by the treatment with TNFR-pep to 2.1-fold compared with the untreated skin (Fig. 4B). Inflammatory response that includes increased blood flow and vascular permeability, the infiltration of neutrophils into the dermis and production of pro-inflammatory cytokine occur in the skin exposed to repeated UV irradiation. To further confirmation of TNFR-pep inhibitory effect on UV-induced mouse model, we detected neutrophil infiltration in dermis by immunochemical staining (Fig. 4C). We observed a significant increase infiltrated neutrophils in the skin was exposed to UVB and treatment with TNFR-pep decreased the neurophils in dermis.
Ultraviolet irradiation leads to skin damages through the condition changes such as epidermal hyperplasia, sunburn, immunosuppression, inflammation and photoaging (10). UV radiation induce activation of several signal transduction pathways related to growth, differentiation, senescence and connective tissue degradation by the activation of several cell surface receptors. This includes cytokines or growth factors receptors as the receptors for epidermal growth factor (EGF), tumor necrosis factor (TNF), interleukin-1 (IL-1)15 and keratinocyte growth factor (KGF). Tumor necrosis factor-alpha (TNF-alpha) is a central regulator of inflammation. Several studies have revealed that UV-induced TNF-α is involved in the formation of sunburn cells, suppression of contact hypersensitivity, Langerhans cell migration from the skin, diminished antigen presentation and loss of immunosurveillance [12, 22, 31]. And It has found that significant amount of TNF-α is produced in human keratinocytes following low dose UVB (0.1 kJ/m2) exposure. Kock et al.  In the dermis, monocyte, macrophages and fibroblasts release varying quantities of TNF-α depending on the UV type and dose [89, 100, 107]. TNF NFkb pathway ref1 Upon UV irradiation, several signal transduction pathways involved in cutaneous alterations are activated through the NF-κB pathway. Activation of NF-κB by UV induced-TNF-α is essential to elicit an effective response, since many of the TNF-a-regulated genes contain binding sites for NF-κB (reviewed in reference 6). TNF NFkb pathway ref2 Thus, inhibition of TNF-α evoked outside in signaling is considered to prevent inflammatory responses and photoaging process induced by UVB.
In this study, we synthesized peptide based on the sequence of DNA binding site of the TNFR and TRAF2 to interfere that TRAF2 bind to TNFR and evaluated to the effect of peptide to block UV-induced TNF-α and NF-κB pathway. Engagement of TNF receptor 1 (TNFR1) by tumour necrosis factor (TNF) leads to the recruitment of a signaling complex containing TNFR1 associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), receptor-interacting protein 1 (RIP1; also known as RIPK1), cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2. Interaction of TRAF2 with sphingosine kinase 1 (SPHK1) stimulates it and brings it to the plasma membrane where its substrate, sphingosine, resides. Intracellular sphingosine 1 phosphate (S1P) is a required cofactor for the K63 linked polyubiquitylation of RIP1 by TRAF2, and the ubiquitin chain then acts as a scaffold to recruit and activate TGFβ-activated kinase 1 (TAK1; also known as MAP3K7) and IκB kinase (IKK) complexes. The IKK complex phosphorylates NF κB inhibitor-α (IκB-α), leading to the activation of nuclear factor-κB (NF-κB). Thus we expect that peptide we synthesized bind to TRAF2 and TNFR binding site and block TNF and NF-κB pathway.
Peptidomimetics are compounds which mimic essential elements of a natural peptide or protein in 3D space and retain the ability to interact with the biological target and produce the same biological effect ( Peptidomimetics, a synthetic tool of drug discovery Current Opinion in Chemical Biology 2008, 12:292–296). Peptides and their homologous compounds can be used in multiple pathologies. We confirmed that TNFR-peptide block to NF-κB activation through the observation of TNFR-peptide of inhibitory effect on TNF-α induced IκB degradation and nuclear translocalization of NF-κB dimer and assumed that it is due to shut off the TNF-α signal transduction by blocking TRAF2 bind to TNFR (Fig. 2).
UV-induced TNF-α modulate expression of MMP through the NF-κB activation pathway. MMPs are responsible for the degradation or synthesis inhibition of collagenous extracellular matrix in connective tissues (3). Collagen represents the main component of the extracellular matrix of dermal connective tissue, and its concentration decreases in chrono- and photoaging. We found a potential of TNFR-peptide as an anti-aging material by confirming that inhibitory effect of TNFR-peptide on MMP expression by TNF-α-stimulated NIH3T3 cells using Western blot. And TNFR-peptide blocked TNF-α inhibits collagen synthesis in human dermal fibroblast (Fig. 3). Also, skin photoaging is characterized by increase in skin thickness and melanin pigmentation consisting of hyperproliferative keratinocytes and melnocytes is due to the expression of bFGF. Shown in Fig. 3A, when treat with TNFR-peptide, TNF-α-induced bFGF expression was significantly decreased in HaCaT cells and these in vitro results were confirmed through showing that application of TNFR-peptide prevented the increase in epidermal thickness and melanocyte proliferation in the UV-irradiated mouse model (Fig. 4). We observed that TNFR-peptide inhibit inflammatory response which is known to be considered that COX2 expression and neutrophils infiltration.
These results support an idea that inhibition of TNF-α induced NF-κB by using TNFR-peptide should be effective in preventing the process of UV-mediated photoaging. Thus we suggested that TNFR-peptide could be used as an anti-photoaging cosmetic ingredient.
Fig. 1. TNFR-peptide inhibits TNFR signaling by interrupting interaction of TNFR and TRAF2
(A) Mouse fibroblast were treated with TNFR-peptide 1hour and then exposed to TNF-α of 25ng/ml for 10min. After fixation, in situ PLA for TNFR and TRAF2 was performed with specific antibodies. The detected interaction of TNFR and TRAF2 is represented by the fluorescent rolling circle products (red dot). (B) Interaction of TNFR and TRAF2 (in situ PLA signals) in the cell population (n=5) was assessed and normalized against the total number of cells counted. The average number of RCPs per cell is expressed as mean±SD (*p<0.01, ****p<0.0001).
Fig. 2. Inhibitory effect of TNFR-peptide on NF-κB activation by TNF-α stimulation and UV irradiation in NIH3T3.
(A) Mouse fibroblast were treated with TNF-α or TNFR-peptide or ascorbic acid (“DHA”, 1mM) alone and in combination for 10min and expression of IκB analyzed by western blot. (B) Mouse fibroblast were treated with TNFR-peptide or dexamethasone (“Dex”, 10μM) for 3h and then exposed to TNF-α of 25ng/ml for 1hour. After fixation, in situ PLA for NF-κB p65 and p50 dimer was performed with p65-, p50- specific antibodies. The detected location of dimer is represented by the fluorescent rolling circle products. p65/p50 dimer (in situ PLA signals) localization in the cell population (n=5) was assessed and normalized against the total number of cells counted. The average number of RCPs per cell is expressed as mean±SD (*p<0.01). (C) After UVB irradiation of 15mJ/cm2 at 312nm, NIH3T3 fibroblasts were treated with TNFR-peptide or dexamethasone for 4h and then performed in situ PLA same as (B). (D) After UVB irradiation of 15mJ/cm2 at 312nm, NIH3T3 fibroblasts were treated with TNFR-peptide or dexamethasone or ascorbic acid for 4h. ChIP assay was performed using lysate from NIH3T3 fibroblasts and anti- NF-κB p65 antibody. The primer set the _657 to _484 region in the MMP-9 gene promoter.
Fig. 3. The Effect of TNFR-peptide on expression of proteins which are related with photoaging.
(A) Human keratinocyte were treated with TNF-α or TNFR-peptide or ascorbic acid alone and in combination for 24h and expression of bFGF was analyzed by western blot. (B) Mouse fibroblast were treated with as (A) and MMP-1 and -9 expression assessed by western blot. (C, D) Human dermal fibroblasts were stimulated with TNF-α, TNFR-peptide and ascorbic acid for 24h or 6h and procollagen type I and COX2 expression was analyzed by western blot.
Fig. 4. Effect of the TNFR-peptide on epidermal hyperproliferation, melanocyte growth and neutrophil infiltration in UVB-induced animal model.
TNFR-pep or PBS as control was administered to DBA/2 mice (n= 5 for each group) by repeated i.p. injections every day for 12 days. The groups of UV+TNFR-peptide and UV treatments were exposed to UVB irradiation at 180mJ/cm2 every other day. (A) Ear samples were prepared from the anesthetized mice, paraffin-embedded, and cut with a sliding microtome to 5-μm thickness. Tissue sections were subjected to histological examination (H&E staining). Data shown are the mean ±S.D. (n=5) of the average epidermal thickness measured using software for image analysis (NIS-elements).*, p < 0.05; **, scale p < 0.01 bar=20μm.(B) To stain melanocyte, the ears obtained from each mouse were soaked in 2N NaBr solution for exfoliating epidermis and immersed in 0.14% L-DOPA solution for 3 h at room temperature. Data shown are the mean ±S.D. of the number of melanocytes per frame. Data shown are the mean ±S.D. (n=5) of the average number of melanocyte measured using software for image analysis (NIS-elements). **, p < 0.01; ****, p < 0.0001, scale; scale bar=80μm. (C) Immunohistochemical staining for infiltrated neutrophils with neutrophil marker monoclonal Ab. Data shown are the mean ±S.D. of the number of neutrophils per frame. Data shown are the mean ±S.D. (n=5) of the average number of neutrophils measured using software for image analysis (NIS-elements). *, p < 0.05; scale bar=40 μm.