Effects of UVA on TNF-α, IL-1β, and IL-10 expression levels in human keratinocytes and intervention studies with an antioxidant and a JNK inhibitor

Objective: To understand the expressions and transduction pathways of cytokines in ultraviolet (UV)A-irradiated keratinocytes.
Methods: We cultured human keratinocytes of the HaCaT cell line and investigated both mRNA and protein expressions of cytokines in cells that were not irradiated or were exposed to 2.4 J/cm² UVA, with or without an antioxidant (β-carotene) or a c-Jun N-terminal kinase (JNK) inhibitor (SP600125).
Results: We demonstrated that the expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were up-regulated in irradiated cells. IL-10 was not detected in non-irradiated cells, but was observed in irradiated cells. JNK was activated in irradiated cells and this could be antagonized by β-carotene. The UVA-induced up-regulation of these cytokines was also antagonized by β-carotene. SP600125 inhibited the UVA-induced increase in the expression of TNF-α mRNA and protein and in the expression of IL-1β mRNA.
Conclusions: The results suggest that oxidative stress may be an early intermediate effect in JNK-dependent UVA induction of cytokine expression in human keratinocytes in vitro .     

α-melanocyte stimulating hormone inhibits tumour necrosis factor-α stimulated intercellular adhesion molecule-1 expression in normal cutaneous human melanocytes and in melanoma cell lines

α-Melanocyte stimulating hormone (α-MSH) was found significantly to reduce tumour necrosis factor-α (TNF-α) stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in normal adult cutaneous melanocytes. The maximum inhibitory response to α-MSH was obtained at around 10⁻¹⁰ mol/L α-MSH when cells were coincubated with α-MSH and TNF-α for 24 h. α-MSH had little or no effect on basal ICAM-1 expression in melanocytes and the effects of α-MSH could be mimicked with 3-isobutyl-1-methylxanthine (IBMX). Preliminary data in three human melanoma cell lines also showed α-MSH and forskolin to be effective in significantly reducing TNF-α stimulated ICAM-1 expression over 24 h. The extent of the inhibition varied from cell line to cell line and was greatest in those cells with the highest number of α-MSH receptors. These data suggest that α-MSH has the ability to oppose the action of the pro-inflammatory cytokine TNF-α on melanocytes and melanoma cells.     

Reduced IL-1Ra/IL-1 ratio in ultraviolet B-exposed skin of patients with polymorphic light eruption

Abstract:  Polymorphic light eruption (PLE) is a putative delayed-type allergic reaction to (solar) ultraviolet (UV) exposure. Inadequate immune suppression after UVB-induced sunburn appears to be associated with reduced trafficking of Langerhans cells (LCs) out of and neutrophils into the epidermis of patients sensitive to UVB provocation of PLE. Therefore, we investigated whether pro-inflammatory and chemotactic cytokines are differentially expressed in UVB-irradiated skin of UVB-provocable PLE patients ( n  = 6) and age- and gender-matched healthy controls ( n  = 6). Interstitial interleukin-1α (IL-1α), IL-1β, IL-1Ra, IL-4, IL-8, tumor necrosis factor-α (TNF-α), macrophage inflammatory protein 1-α (MIP-1α), MIP-1β and monocyte chemotactic protein-1 (MCP-1) were measured in suction blister fluid raised 16 h after exposure to 0, three and six minimal erythemal UVB doses. In unirradiated skin, the IL-1Ra levels were significantly lower in the PLE patients than in controls ( P  < 0.05). IL-8 and TNF-α levels increased strongly upon UVB irradiation in both groups. No differential shifts in cytokine profiles were found that could explain a reduced trafficking of Langerhans cells and neutrophils in PLE patients. Dose-trend analyses showed that UVB irradiation caused significant increases in IL-1α in both groups, and that the levels of IL-1α and IL-1β were on average twofold higher in the PLE group ( P  = 0.03 and P  = 0.004, respectively.). Accordingly, the ratios of IL-1Ra over IL-1α and over IL-1β were overall lower in the skin of PLE patients ( P  = 0.015 and P  < 0.001, respectively.). This shift in cytokines in UVB-irradiated skin of PLE patients reveals an amplified early pro-inflammatory cytokine response, which may contribute to the allergic reaction to UVB radiation.     

Immunoreactivity of α-melanocyte-stimulating hormone, adrenocorticotrophic hormone and β-endorphin in cutaneous malignant melanoma and benign melanocytic naevi

Melanocyte-stimulating hormone (MSH) has been reported to enhance the experimental metastatic behaviour of melanoma cells in the mouse model. α-MSH production and MSH receptor (melanocortin 1 receptor gene) expression have been detected in cultured normal human melanocytes and metastasized melanomas. The exact role of MSH in the metastatic behaviour of human melanoma cells is, however, not yet known. To clarify a possible role of proopiomelanocortin (POMC)-derived peptides, including α-MSH, in melanoma development and progression, we analysed immunohistochemically the localization of α-MSH, adrenocorticotrophic hormone (ACTH) and β-endorphin in various kinds of benign pigmented naevocytic lesions and malignant melanomas.   Three of 21 samples of common and dysplastic naevi showed detectable α-MSH staining in naevus cells, and five and six of 15 samples were weakly positive for ACTH and β-endorphin staining, respectively. In melanoma samples, 24 of 45, 23 of 39 and 30 of 42 samples showed positive staining with α-MSH, ACTH and β-endorphin antibodies, respectively. Furthermore, staining for all three antibodies was noted to be more intense and diffuse in samples of nodular melanoma, vertically growing acral lentiginous melanoma and superficial spreading melanoma as well as metastatic lesions compared with those of naevi. Although it is yet to be determined whether or not this strong staining for POMC-derived peptides in advanced melanoma cells indicates a role of autocrine or paracrine regulation, our results suggest a possible involvement of POMC gene products in melanoma progression.     

Gpnmb is a melanosome-associated glycoprotein that contributes to melanocyte/keratinocyte adhesion in a RGD-dependent fashion

Abstract:  Gpnmb is a glycosylated transmembrane protein implicated in the development of glaucoma in mice and melanoma in humans. It shares significant amino acid sequence homology with the melanosome protein Pmel-17. Its extracellular domain contains a RGD motif for binding to integrin and its intracellular domain has a putative endosomal and/or melanosomal-sorting motif. These features led us to posit that Gpnmb is associated with melanosomes and involved in cell adhesion. We showed that human Gpnmb is expressed constitutively by melanoma cell lines, primary-cultured melanocytes and epidermal melanocytes in situ , with most of it found intracellularly within melanosomes and to a lesser degree in lysosomes. Our newly developed monoclonal antibody revealed surface expression of Gpnmb on these pigment cells, albeit to a lesser degree than the intracellular fraction. Gpnmb expression was upregulated by UVA (but not UVB) irradiation and by α-melanocyte-stimulating hormone (MSH) (but not β-MSH); its cell surface expression on melanocytes (but not on melanoma cells) was increased markedly by IFN-γ and TNF-α. PAM212 keratinocytes adhered to immobilized Gpnmb in a RGD-dependent manner. These results indicate that Gpnmb is a melanosome-associated glycoprotein that contributes to the adhesion of melanocytes with keratinocytes.     

Suprabasal expression of epidermal α2β1 and α3β1 integrins in skin treated with topical retinoic acid

In normal adult human skin, expression of epidermal integrins is confined to keratinocytes in the basal layer. However, suprabasal expression of α2, α3 and β1 integrin subunits is noted in hyperproliferative epidermis in wound repair and psoriasis. In this study, we examined the effect of topical all- trans -retinoic acid (RA), known to induce epidermal hyperplasia, on expression of integrins in human epidermis. Immunostaining of vehicle-treated skin revealed expression of α2, α3 and β1, as well as α6 and β4 integrin subunits entirely on basal keratinocytes. Topical application of RA (0.1%) for 2 weeks resulted in marked suprabasal expression of α2, α3 and β1 integrin subunits, whereas α6 and β4 staining remained on basal keratinocytes. Staining for putative ligands of α2β1 and α3β1 integrins, i.e. type IV collagen, laminin-5 and fibronectin, was not detected in the epidermal layer in RA- or vehicle-treated skin. Treatment of HaCaT keratinocytes in culture with RA (1 μmol/L) enhanced α2 and β1 mRNA abundance. Furthermore, RA slightly up-regulated the expression of α2, α3 and β1 integrin subunits on primary epidermal keratinocytes and HaCaT cells in culture with no effect on cell proliferation. These results provide evidence that RA-elicited epidermal hyperplasia is associated with aberrant suprabasal expression of α2β1 and α3β1 integrins, and that this also involves direct stimulation of keratinocyte integrin expression by RA.     

An increased ratio of interleukin-1 receptor antagonist to interleukin-1α in inflammatory skin diseases

Abstract: IL-1 receptor antagonist (IL-1ra) is a cytokine that competitively binds the IL-1 receptor to antagonize IL-1 activity without any agonist function. Previous experiments indicated that the ratio of IL-1ra to IL-1α in the normal stratum corneum (SC) was much higher in the sunexposed face than in the sun-protected area, upper arms. It was also reported by another laboratory that IL-1ra is increased in the lesional skin of psoriatic patients. This study was designed to measure the contents of IL-1α and IL-1ra in non-lesional and pathological SC obtained from inflammatory skin diseases including psoriasis and non-psoriatic dermatoses such as atopic dermatitis. The SC materials were obtained with a noninvasive tape-stripping method. Their soluble fractions were prepared and assayed for IL-1α and IL-1ra by enzyme-linked immunosorbent assays. As a result we confirmed the previous findings that the ratio of IL-1ra to IL-1α in the normal SC was much higher in the face than in the sun-protected sites, the trunk as well as extremities. Next, we found that IL-1α contents were significantly reduced in the SC samples obtained from inflammatory skin regardless of whether their IL-1ra contents increased or unchanged. Moreover, we noted that an increased ratio of IL-1ra to IL-1α in the SC was not specific to psoriasis, but was also found in other inflammatory skin diseases including atopic dermatitis. This ratio was found to become lower after successful treatment of these skin lesions with topical glucocorticoids. We conclude from these observations that the increased ratio of IL-1ra to IL-1α in the SC is a non-specific phenomenon that can occur in any inflammatory skin diseases regardless of the inflammatory pattern, probably reflecting a skin regulation process against various kinds of inflammation.     

Interleukin-1 but not tumour necrosis factor α synergistically upregulates the granulocyte—macrophage colony-stimulating factor-induced B7-1 expression of murine Langerhans cells

Epidermal Langerhans cells (LC) express several co-stimulatory molecules such as B7/BB1. which has been Implicated as one of the important determinants for potent antigen-presenting function of LC. Recent studies have shown that B7/BB1 antigens comprise three distinct molecules termed B7-1, B7-2 and B7-3. Previous studies have revealed that the phenotypic and functional properties of murine LC are enormously affected by various cytokines including granulocyle-macrophage colony stimulating factor (GM-CSF), interleukin-l (IL-1), and tumour necrosis factor α (TNF-α)derived from surrounding keratinocytes. We have already demonstrated that the expression of B7-1 of murine LC is significantly enhanced by GM-CSF, IL-1 or TNF-α. In this paper, we present that IL-I, hut not TNF-α, synergistically up-regulates the GM-CSF-induced B7-1 expression of murine LC.     

Ultraviolet B induced suppression of induction of contact sensitivity in human skin is not associated with tumour necrosis factor-alpha-308 or interleukin-10 genetic polymorphisms

Low doses of ultraviolet B (UVB) can induce localized immunosuppression in skin. This effect may be important in the induction of skin cancers and is thought to be mediated by tumour necrosis factor (TNF) α and interleukin (IL) 10 in conjunction with other factors. In humans a transition polymorphism in the TNF-α gene may affect TNF-α secretion and the promoter region of the IL-10 gene contains a CA repeat polymorphism which may affect gene function. We have therefore investigated the association of these polymorphisms with UVB-induced immunosuppression in humans. Volunteers ( n  = 42) were irradiated with UVB then sensitized on irradiated skin with diphenylcyclopropanone (DPCP) and subsequently antigen challenged with DPCP. DNA was extracted from blood samples and volunteers genotyped for the TNF-α polymorphism by polymerase chain reaction (PCR) and restriction digestion. The CA repeat polymorphism was amplified by PCR and sized by gel electrophoresis. Twenty-four volunteers were susceptible to UVB-induced immunosuppression and 18 were resistant. The association of allele frequencies and phenotype was statistically tested using a χ²-test. For both the TNF-α and IL-10 polymorphisms, there was no statistically significant association between allele types and response to UVB. These results indicate that variation in the immune response to UVB in humans is not associated with the TNF-α-308 transition or IL-10 CA repeat polymorphisms, although other as yet undetected DNA sequence variants of these genes may be involved.     

UVB-induced melanogenesis may be mediated through the MSH-receptor system

Ultraviolet B radiation (UVB) elicits an increase in melanin production in mammalian skin. The mechanisms regulating this process are not understood, although it is well documented that there is an increase in the number of melanin-producing melanocytes. The melanotropins (MSH) are a family of peptides that increase the melanin content of melanocytes through an interaction with high affinity receptors. We have obtained evidence that the effects of UVB on melanogenesis may be mediated through an increase in MSH receptor activity on melanocytes. First, exposure of Cloudman S91 mouse melanoma cells to UVB resulted in increased binding of 125I-MSH to cells within 24 h. In five separate experiments, UVB-irradiated cultures displayed 2-10-fold increases in MSH binding capacity over that of unirradiated control cultures (optimum doses 10-20 mJ/cm2). Second, UVB and MSH potentiated one another in promoting cutaneous melanogenesis in both mice and guinea pigs. In the areas of guinea pig skin that received both UVB and MSH, there was a fivefold increase in active melanocytes/mm2 over the sum of active melanocytes/mm2 in areas receiving either MSH or UVB separately. Our results suggest that UVB light causes an increase in MSH receptor activity on cutaneous melanocytes, thus increasing cellular responsiveness to MSH. Implicit in this mechanism is a transduction of radiant energy into chemical energy during the process of UVB-induced melanogenesis.     

Opposing effects of ultraviolet B irradiation on interleukin-1 receptor antagonist and interleukin-1α messenger RNA expression in a human epidermoid carcinoma cell line

Interleukin-1 receptor antagonist (IL-1RA) is a cytokine that acts to antagonize IL-1 activity without agonist function. The expression of IL-1RA has been reported in many cell types, including the keratinocyte that covers the outer most part of the skin. However the modulation of IL-1RA by ultraviolet B (UVB), which is the most biologically active UV, has not been reported yet. We therefore selected a keratinocyte cell line with a cytokine-producing profile similar to that of keratinocytes and tested the effect of UVB on its ability to produce IL-1RA mRNA. IL-1RA mRNA was constitutively expressed in the cell line and began to be suppressed by 3 h after the UVB irradiation with 100 mJ/cm². The level of IL-1RA expression became lowest by 16 h after the irradiation with 100 mJ/cm². Simultaneously, IL-1α mRNA started to increase by 1 h and peaked by 3–16 h after the irradiation with 10–100 mJ/cm². The differential expression of IL-1α and IL-1RA mRNA following exposure to a high dose (100 mJ/cm²) of UVB may markedly potentiate the role of IL-1 in UV-induced inflammation.     

TNF-α, RANTES, and MCP-1 are major chemoattractants of murine Langerhans cells to the regional lymph nodes

We have previously reported that lymph node cells generated chemotactic factors for Langerhans cells (LCs) in the induction phase of contact dermatitis. In order to clarify the chemotactic factors involved in migration to the regional lymph nodes, we investigated the migratory activity of murine LCs toward several cytokines and chemokines in vitro . One-day cultured LC-enriched epidermal cells were added to the upper compartment of a modified Boyden chamber and cytokines were added to the lower compartment. We counted dendritic cells migrated to the lower chamber as LCs under phase contrast microscopy. About 99% of migrated dendritic cells were positively reacted with anti-Ia^d and NLDC145 antibodies and considered to be LCs. We could detect LC migration more accurately by this direct examination than by counting the migrated cells in the filter membrane of a Boyden chamber. In our system, migration of murine epidermal LCs was stimulated by TNF-β, RANTES and MCP-1, but not by GM-CSF, IL-1β, IL-4, and IL-6. TNF-α induced LC migration at concentrations from 4X10³ U/ml to 5X10⁴ U/ml. RANTES at concentrations from 10 to 100 ng/ml and MCP-1 a concentration of 100 ng/ml induced LC migration in a dose-dependent manner. These data confirmed that TNF-α, RANTES, and MCP-1 induced LC migration from epidermis during contact sensitization.     

Solar-simulated, ultraviolet radiation-induced, upregulation of the melanocortin-1 receptor, pro-opiomelanocortin, and α-melanocyte- stimulating hormone in human epidermis in vivo

Ultraviolet (UV) light is one of the most crucial environmental factors with regard to its capacity to induce skin cancer, premature aging of the skin, and immunosuppression. Although UV directly affects the function of epidermal cells, many of these effects are mediated by the induction of cytokines, growth factors, and neuropeptides such as α-melanocyte-stimulating hormone (α-MSH). Recently, in addition to its well-known pigmentation inducing activity, a strong anti-inflammatory as well as an immunomodulatory potential of α-MSH has been recognized. The aim of this study was to determine whether UV irradiation affects the expression of both α-MSH and the melanocortin-1 receptor (MC-1R) in human epidermis in vivo . The volar aspects of the forearms were exposed to twice the minimal erythema dose of solar simulating radiation (SSR). Three, 6, and 24 h after irradiation, the pro-opiomelanocortin (POMC) and interleukin-10 (IL-10) mRNA levels in suction blister-induced epidermal sheets were considerably upregulated as detected by semiquantitative RT-PCR. Furthermore, α-MSH and IL-10 protein levels in blister fluids were significantly increased 24 h after UV irradiation, an effect which could be abolished by the application of the broadspectrum sunscreen AnthéliosXL^® prior to UV (SSR) exposure. In addition, enhanced MC-1R mRNA and receptor protein expression upon SSR was ascertained by RT-PCR and immunohistochemistry of the epidermal sheets, respectively. POMC-derived neuropeptides such as α-MSH may therefore play an important role in modulating UV-induced inflammation.     

Re-epithelialization of normal human excisional wounds is associated with a switch from αvβ5 to αvβ6 integrins

Summary During cutaneous wound repair, keratinocytes move laterally across the wound surface. For this lateral movement epidermal cells must disassemble their tenacious connections to the basement membrane and their neighbouring cells, and express surface receptors that permit translocation over the wound surface extracellular matrix. If the basement membrane is disrupted, the epidermis migrates over a provisional matrix that contains fibrinogen, fibronectin, vitronectin and tenascin. Although α5β1 integrin, a fibronectin receptor, is expressed by human epidermis during reepithelialization of excisional and incisional wounds, the spatial and temporal patterns of vitronectin, tenascin, and other fibronectin receptors are less clear. Other potential receptors include αvβ5 for vitronectin and αvβ6 for fibronectin and tenascin. To study provisional matrix integrin expression during human wound healing, full-thickness 4-mm punch biopsies were performed on the inner surface of the upper arm in adult volunteers. At 3, 7 and 14 days after injury wound sites were excised, bisected, quick frozen in liquid nitrogen, and examined for the expression of α5, β1, αv, β5 and β6. At 3 days, α5β1 and αvβ5, but not αvβ6, appeared around the basal and suprabasalar cells of the migrating epidermis. At 7 days, α5β1, αvβ5, and αvβ6 appeared around the perimeter of the basal cells of the migrating epidermis. By 14 days, when re-epithelialization was complete, all basal and suprabasalar cells overlying the wound expressed α5β1 and αvβ6, but not αvβ5. Thus, αv appeared to switch its heterodimeric association from β5 to β6 subunit during re-epithelialization.     

Red hair, fair skin and melanoma – melanocortin 1 receptor

POMC processing in human melanocytes has been widely documented, and the α-MSH/MC1R/cAMP cascade has been implicated in the control of pigmentation. Only very recently, a role of β-endorphin, one cleavage product of β-LPH, has been demonstrated to influence melanocyte growth, dendricity and melanin biosynthesis via the µ-opiate receptor. However, much earlier, it was shown that β-MSH, the other cleavage product of β-LPH, controls melanogenesis and melanin transfer in amphibians. To date, a specific receptor for β-MSH has not been identified. Earlier POMC processing has been found in melanosomes. Therefore, an MC1R-independent role of α-MSH was postulated and demonstrated in control of 6-tetrahydrobiopterin (6BH₄)-inhibited tyrosinase. Utilizing the depigmentation disorder vitiligo, we were now able to follow the fate of epidermal POMC processing in the presence of mM levels of hydrogen peroxide (H₂O₂). In vitiligo epidermal PC2 and 7B2 protein expression is increased, whereas α-MSH, β-MSH and β-endorphin are significantly decreased. Analysis of the peptide sequences revealed in all three cases H₂O₂ oxidation targets such as methionine and tryptophan yielding significant structural alterations. Moreover, we have identified a new function of β-MSH due to its capacity to bind the important cofactor 6BH₄ as well as its isomer 7BH₄. Hence, we propose for the first time that β-MSH can control both the supply of l -tyrosine from l -phenylalanine via phenylalanine hydroxylase and l -Dopa synthesis via tyrosinase hydroxylase in melanocytes and keratinocytes. Therefore, both melanogenesis and catecholamine synthesis could be regulated by this peptide.     

Increase in dopa‐positive melanocytes in the mouse intestine in response to ultraviolet B rays via the eyes

Background. Irradiation by ultraviolet (UV) initiates pigmentation of skin; however, it is not known whether changes in intestinal pigmentation are also induced by UVB irradiation of the eye. Aim. To examine the influence of UVB irradiation of the eye or ear on the pigmentation of mouse epidermis and intestine. Methods. DBA/2 male mice were locally exposed to UVB (280–320 nm) using a 20SE sunlamp directed at the eye or ear. The irradiation was given over 3 days, at a dosage of 2.5 kJ/m² per day. Five days after irradiation, samples were taken from the skin and intestine. Melanocytes in both epidermis and intestine were stained for dopa and expression of melanocortin‐1 receptor (MC1R). Levels of plasma α‐melanocyte‐stimulating hormone (α‐MSH) were measured using ELISA. Results. Ultraviolet B irradiation of either the eye or ear in increased the number of dopa‐positive melanocytes in the skin and the intestine (jejunum and colon). Irradiation of the eye caused a much greater increase in dopa than did irradiation of the ear. Both eye and ear irradiation increased blood α‐MSH level to a similar extent, but only irradiation to the eye increased MC1R expression in the intestine. Conclusions. These results suggest that the UVB‐induced pigmentation in the epidermis and the intestine is related to increased levels of α‐MSH and MC1R.     

Polymorphisms in the interleukin-1 gene influence the stratum corneum interleukin-1 alpha concentration in uninvolved skin of patients with chronic irritant contact dermatitis

Background:  Interleukin (IL)-1α and its receptor antagonist IL-1ra play a role in skin inflammation. Several polymorphisms in the IL1 gene cluster, coding for IL-1α, IL-1ra, and IL-1β, influence their protein expression. Within this cluster, strong linkage disequilibrium has been shown.
Objective:  We studied the association between the polymorphisms IL1A -889 (C→T) and IL1B -31 (T→C) and the concentration of IL-1α and IL-1ra in the stratum corneum (SC).
Method:  In 124 patients with chronic irritant contact dermatitis, we genotyped the IL1A -889 and IL1B -31 polymorphisms and determined the amount of IL-1α and IL-1ra on tape strips obtained from uninvolved skin of the volar forearm.
Results:  The SC IL-1α concentration was 23% and 47% lower in subjects with IL1A -889 C/T genotype and T/T genotype, respectively, compared with wild-type genotype. In subjects with IL1B -31 C/C genotype, the IL-1α concentration was 51% lower compared with C/T and T/T genotypes. The ratio IL-1ra/IL-1α increased twofold in IL1A -889 C/T genotype and threefold in T/T genotype compared with wild type.
Conclusions:  We have shown a clear effect of IL1 genotype on protein expression in the SC. This altered expression may be responsible for the interindividual differences in the inflammatory response of the skin.     

The Expression of Endothelin-1 and Its Binding Sites in Mouse Skin Increased after Ultraviolet B Irradiation or Local Injection of Tumor Necrosis Factor α

Endothelin (ET)-1 is a 21-amino acid peptide which has vasoconstrictor and growth regulatory activity. Recently, cultured keratinocytes have been reported to express ET-1 and its receptor when irradiated by ultraviolet (UV) B. In order to further understand the role of ET-1 in vivo during UVB-induced inflammation, we examined the localization, intensity and time course of the expression levels of ET-1 and its binding sites in UVB-exposed BALB/c mouse skin. Frozen and paraffin sections prepared from mouse skin 48 h after treatment with UVB irradiation (0.36 or 0.72 J/cm²) or after injection with tumor necrosis factor (TNF)-α (1.0 μg) or interleukin (IL)-1α (0.05 μg) were incubated with monoclonal anti-ET-1 IgG and then visualized by peroxidase staining. In normal skin, faint ET-1 immunoreactivity was observed in the epidermis, pilosebaceous structures and blood vessels. Upon exposure to UVB irradiation or administration of TNF-α injection or IL-1α injection, such immunoreactivity was found to be significantly enhanced. Subsequently, the frozen sections were incubated with ¹²⁵I ET-1 for 30 min, and visualized by autoradiographic technique. In normal skin, ET-1 weakly bound to the skin, while UVB irradiation and TNF-α injection significantly enhanced ET-1 binding in the epidermis, pilosebaceous structures and blood vessels. Time course experiments (1, 2, 4 and 7 days) indicated that ET-1 immunoreactivity and ET-1 binding peaked 1 or 2 days after UVB irradiation or TNF-α injection. These results suggest that the up-regulated expression of ET-1 and its binding sites in the epidermis and pilosebaceous structures may act as an autocrine/paracrine factor during UVB-induced inflammation.