In situ depletion of CD4+ T cells in human skin by Zanolimumab.

CD4(+) T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease-driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4(+) T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, kappa) against CD4, was studied in a human psoriasis xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4(+), but not CD8(+) CD3(+) T cells. The capacity of Zanolimumab to deplete the CD4(+) T cells in the skin may be of importance in diseases where CD4(+) T cells play a central role. Indeed, in a phase II clinical trial Zanolimumab has shown a dose-dependent clinical response in patients with CTCL and the antibody is currently in a phase III clinical trial for CTCL, a disease for which there is no safe and effective treatment available today.

Levels of enzymatic antioxidants activities in mononuclear cells and skin reactivity to sodium dodecyl sulphate.

Chemical irritants are able to produce several biological modifications of the skin, including the direct or indirect production of cytokines and reactive oxygen species leading to an inflammatory reaction. This report examines the existence of a possible correlation between the skin sensitivity to the irritant sodium dodecyl sulphate (SDS) and the activity of the enzymatic antioxidants. In twenty-three healthy subjects the evaluation of the epidermal and peripheral blood mononuclear cells (PBMCs) activities of Superoxide Dismutase (SOD) and Catalase (Cat) demonstrate a significant correlation (r= 0,85 and p< 0,005 for SOD, and r= 0,87 and p< 0,0001 for Cat). Based on this result, on a further group of normal subjects (n=13) we studied the link between the threshold dose of skin reactivity to SDS and the activities of the enzymatic antioxidants in PBMCs. The degree of skin modification induced by SDS, applied at different concentrations for 24 hrs, was determined by means of Trans Epidermal Water Loss (TEWL), Erythemal Index or by Visual Score (VS). The minimal dose of the irritant capable of inducing skin modifications, was significantly correlated with SOD (r=0,77) and Cat (r=0,81) activities in PBMCs, and the modification of TEWL or EI were inversely correlated with levels of antioxidants in PBMCs (r=-0,62 for SOD and r=-0,66 for Cat). Our results indicate that the skin reactivity to irritants can be modulated by the levels of antioxidants, and suggest a possible therapeutical approach in preventing irritant contact dermatitis.

Increased expression of Fas on human epidermal cells after in vivo exposure to single-dose ultraviolet (UV) B or long-wave UVA radiation.

BACKGROUND: Apoptosis has been proposed to act as an important mechanism for eliminating keratinocytes that have been irreversibly damaged by ultraviolet (UV) irradiation. One way to induce apoptosis in keratinocytes is through activation of the cell surface receptor Fas (CD95), either with the ligand (FasL) or directly with UV radiation. OBJECTIVES: To investigate the regulation of Fas and FasL expression in human skin and the formation of apoptotic cells after in vivo exposure to UVB or long-wave UVA radiation. METHODS: Volunteers were irradiated with either 3 minimal erythema doses (MED) of UVB (n = 6) or 3 MED of long-wave UVA (n = 6) on buttock skin 12, 24 and 72 h before skin punch biopsies were taken. Expression of Fas and FasL was demonstrated by immunohistochemistry on cryostat sections. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labelling reaction. RESULTS: In five of six subjects, exposure to UVB radiation resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 24 and 72 h after irradiation. In all subjects, exposure to long-wave UVA resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 12 h after irradiation. In five of six subjects, exposure to UVB radiation resulted in temporarily decreased expression of FasL, but after 72 h the expression of FasL had returned to the preirradiation level. The expression of FasL on epidermal cells after exposure to long-wave UVA showed considerable variation. UVB irradiation was a stronger inducer of epidermal apoptosis than was UVA irradiation. The number of apoptotic epidermal cells did not correlate with expression of Fas or FasL. CONCLUSIONS: In human skin the expression of Fas on epidermal cells increases after in vivo exposure to UVB or long-wave UVA. Exposure to UVB causes a temporary decrease in the expression of FasL on epidermal cells.

Decrease in nickel sensitization in a Danish schoolgirl population with ears pierced after implementation of a nickel-exposure regulation.

BACKGROUND: To reduce the skin nickel exposure of the population, the Danish Ministry of Environment issued a regulation that was implemented in 1992, and the European Union countries have recently adopted an expanded regulation. OBJECTIVES: The aim of our combined patch testing and questionnaire investigation of girls in public schools and high schools/production schools was to evaluate whether the regulation has had an impact on the prevalence of nickel sensitization. METHODS: To find a group of girls with ears pierced mainly after implementation of the nickel-exposure regulation in Denmark, girls were recruited from the fifth and sixth grade in 12 public schools (the public school group). After the public school level almost all girls from a public school population continue their education in high schools or other schools such as production schools or technical schools. Therefore, to find girls demographically similar to the public school girls but older, and with ears pierced before implementation of the regulation, girls from seven high schools and two production schools were recruited (the high school group). Four hundred and twenty-seven girls in the public school group (mean age 12.4 years, range 10-14) and 534 in the high school group (mean age 18.8 years, range 17-22) participated. All participants filled out a questionnaire concerning ear piercing, use of oral braces and former patch testing for nickel sensitivity. Three hundred and five girls (71.4%) in the public school group and 275 (51.5%) in the high school group were patch tested or had been tested previously and the results of these tests were included in the study. The relation between the frequency of nickel sensitization and the various factors that might influence the prevalence of nickel sensitization was evaluated by multivariate logistic regression analysis. The investigation was conducted from March 1999 to March 2000. RESULTS: The study showed that both increasing age and having ears pierced before 1992 enhanced the prevalence of nickel sensitization. We found that 17.1% of the girls in the high school group demonstrated a positive patch test reaction to nickel. In contrast, the prevalence of nickel sensitization in the public school group was only 3.9%. Comparing girls with and without pierced ears, the prevalence of nickel sensitization was significantly higher in girls with ears pierced before, but not after, 1992 (odds ratio 3.34 and 1.20, respectively). Only in the high school group was there a tendency that wearing oral braces before ear piercing had a protective effect on nickel sensitization, but this did not reach statistical significance. CONCLUSIONS: As we found an effect of ear piercing before but not after 1992, this study strongly suggests that implementation of the nickel-exposure regulation in 1992 in Denmark has had the intended effect of protecting the female population from becoming allergic to nickel.

Communications: high dermal mast cell prevalence is a predisposing factor for basal cell carcinoma in humans.

Ultraviolet B radiation (280-320 nm) can initiate skin cancer as well as suppress the immune system, thereby preventing the rejection of ultraviolet-B-induced tumors. Recently we reported that there was not only a correlation but also a functional link between dermal mast cell prevalence and susceptibility to ultraviolet-B-induced systemic immunosuppression in multiple strains of mice. In this study, we investigated whether increased dermal mast cell prevalence is a significant predisposing factor for basal cell carcinoma development in humans. In 21 Danes with a history of basal cell carcinoma and 20 control subjects of similar age, sex, skin phototype, and recreational sun exposure over the past 12 mo, dermal mast cell prevalence was quantified on non-sun-exposed buttock skin. We investigated this skin site in order to avoid any changes in mast cell prevalence caused by sun exposure and assumed that the prevalence of mast cells in buttock skin correlated with that at sun-exposed sites at critical times in the development of basal cell carcinomas. Patients with a history of basal cell carcinoma had a significantly higher median dermal mast cell prevalence than control subjects (p = 0.01, Mann-Whitney U ). No correlation was observed between dermal mast cell prevalence and age of basal cell carcinoma patients and control subjects. These results suggest that increased dermal mast cell prevalence is a predisposing factor for basal cell carcinoma development in humans. We hypothesize that mast cells function in humans, as in mice, by initiating immunosuppression and thereby allowing a permissive environment for basal cell carcinoma development.

Primary cutaneous B-cell lymphoma: a clinical, histological, phenotypic and genotypic study of 21 cases.

The clinical, histological, phenotypic and genotypic features of 21 primary cutaneous B-cell lymphomas (CBCLs) have been investigated. The patients were 13 men and eight women aged 34-91 years (median 67) at diagnosis. Eighteen patients had localized disease, and three had multiple skin lesions at diagnosis. Twelve patients developed cutaneous or extracutaneous recurrences, and five died from malignant lymphoma 7-84 months (median 36) after diagnosis. Histological examination showed features of marginal zone/mucosa-associated lymphoid tissue (MALT)-type lymphoma in 12 cases. Three of these had transformed to diffuse large B-cell lymphoma (DLBCL) in relapse biopsies. The remaining cases were seven primary DLBCLs and two cases tentatively classified as follicle centre cell (FCC) lymphoma. The neoplastic B cells showed similar phenotypes and genotypes in most cases (CD20+, CD79+, CD5-, CD10-, cyclin D1-, bcl-2+, bcl-x-, bax-, t(14;18)-negative). p53 protein was expressed in five cases, and four harboured mis-sense or loss-of-function mutations in the p53 gene. Deletion or promoter region hypermethylation of the p16INK4a gene was detected in two patients with DLBCL. The level of retinoblastoma protein expression and the proliferative fraction were significantly higher in DLBCL (> 50%) than in MALT- or FCC-type lymphomas (< 10%). Features associated with an unfavourable prognosis were the presence of multiple skin lesions at diagnosis, transformation from MALT-type lymphoma to DLBCL, and possibly p16INK4a aberrations. It is concluded that most CBCLs are dissimilar from FCC lymphomas and seem to be more closely related to marginal zone/MALT-type lymphomas. It is also suggested that there are fundamental differences between DLBCL and other histological categories of CBCL, indicating that cutaneous DLBCL is a separate entity with an increased growth potential and genetic features similar to DLBCL originating in other anatomical sites.

Application of Staphylococcal enterotoxin B on normal and atopic skin induces up-regulation of T cells by a superantigen-mediated mechanism.

BACKGROUND: The skin of patients with inflammatory skin diseases such as atopic dermatitis is frequently colonized with Staphylococcus aureus. Colonization with S aureus has been reported to exacerbate atopic dermatitis. Recent studies have demonstrated that S aureus isolated from the skin of patients with atopic dermatitis releases bacterial toxins that act as superantigens. We have previously applied the staphylococcal superantigen staphylococcal enterotoxin B (SEB) on intact human skin and found that the application led to induction of dermatitis. OBJECTIVE: The purpose of the study was to determine whether superantigen-induced dermatitis is primarily due to a T cell-superantigen-mediated reaction or represents nonspecific cytokine-driven inflammation. METHODS: We applied SEB, vehicle, and sodium lauryl sulfate on normal skin in healthy (n = 6) and atopic subjects (n = 6) and biopsy specimens were taken from all treated areas. The biopsy specimens from all subjects and peripheral blood from the atopic subjects were analyzed for the T-cell receptor (TCR) Vbeta repertoire with mAbs against TCR Vbeta 2, 3, 8.1, 12, 14, and 17. RESULTS: From all subjects, both healthy and patients with atopic dermatitis, skin biopsy specimens from SEB-treated areas demonstrated selective accumulation of T cells expressing SEB-reactive TCR Vbeta 12 and 17 (P <.05). This selective up-regulation was not found in the sodium lauryl sulfate-treated areas. CONCLUSION: Our data strongly support that superantigen-induced T-cell activation is involved in the dermatitis seen after experimental application of SEB on intact skin.

Superantigen Staphylococcal enterotoxin B induces release of IL-1beta in human epidermis.

Lesional skin in patients with inflammatory skin diseases is often colonized with Staphylococcus aureus, which is capable of releasing superantigens. We therefore studied whether application of superantigen on the skin led to release of cytokines, especially IL-1beta. Suction blisters were raised on vehicle- and superantigen-treated skin and IL-1beta protein levels measured in suction blister fluid and supernatant from blister roofs. In all volunteers studied, application of the superantigen Staphylococcal enterotoxin B led to increased release of IL-1beta protein from suction blister roofs (n=7). In contrast, we did not detect any difference in IL-1beta in the blister fluid (n=5). IL-1beta is known as a mediator of inflammation, and the increase in IL-1beta may be involved in the aggravation of inflammatory skin diseases seen following Staphylococcus aureus colonization.

Bacterial superantigens and inflammatory skin diseases.

Bacteria seem to play an important role in the induction and maintenance of inflammatory skin diseases such as psoriasis and atopic dermatitis. Toxins from bacteria including Streptococcus and Staphylococcus aureus, have been shown to function as a new type of allergen termed 'superantigen'. Superantigens bypass the normal control of T-cell activation and activate all T-cell clones bearing certain types of variable chain on the T-cell receptor: this leads to vigorous T-cell activation and cytokine release. These bacterial superantigens may be involved in induction and aggravation of inflammatory skin diseases. Guttate psoriasis is often preceded by a streptococcal throat infection and T cells specific for streptococcal superantigens have been identified in the skin of patients. The skin of patients with atopic dermatitis is often colonized with superantigen-releasing Staph. aureus, and application of a staphylococcal superantigen to human skin induces an eczematoid reaction.

Association of TNFA gene polymorphism at position -308 with susceptibility to irritant contact dermatitis.

Mechanisms underlying susceptibility to skin irritants are not clearly understood. Cytokines play a key role in inflammation, and functional polymorphisms in cytokine genes may affect responses to irritants. We investigated the relationship between polymorphism in the tumor necrosis factor (TNF) alpha-chain gene and responses to irritants. Volunteers (n=221) tested with sodium dodecyl sulfate (SDS) and benzalkonium chloride (BKC) were divided into responders and nonresponders and high and low irritant-threshold groups. DNA was assayed for the TNF-308 polymorphism by a polymerase chain reaction-restriction fragment length polymorphism method. There was a significant increase in the A allele (P=0.030) and AA genotype (P=0.023) in both the SDS low irritant-threshold group and in SDS responders (A allele P=0.022, AA genotype P=0.048). In the BKC low irritant-threshold group, we found a significant increase in the A allele (P=0.002) and AA genotype (P=0.016). Individuals with a low threshold to both irritants demonstrated a significant increase (P=0.002) in the A allele. This is the first description of a nonatopic genetic marker for irritant susceptibility in normal individuals. Genotyping for the TNF-308 polymorphism may thus contribute to screening of individuals deemed at risk of developing irritant contact dermatitis.

Long-wave UVA offers partial protection against UVB-induced immune suppression in human skin.

Ultraviolet-B (UVB, 280-320 nm) interferes with the generation of cell-mediated immunity to contact allergens applied epicutaneously on the irradiated site. To investigate whether pretreatment with UVA-1 (340-400 nm) protects against the UVB-induced immune suppression we sensitized human volunteers with diphenylcyclopropenone (DPCP) on normal buttock skin (n= 12), on UVB-irradiated buttock skin (n=21), on buttock skin pretreated with UVA-1 (n= 12), and on buttock skin pretreated with UVA-1 and thereafter irradiated with UVB (n=22). Sensitization on UVB-irradiated skin reduced the immunization rate to DPCP compared with sensitization on non-irradiated skin (p<0.01) and skin pretreated with UVA-1 (p<0.01). In contrast, the immunization rate in the group of volunteers sensitized on skin pretreated with UVA-1 before UVB irradiation was significantly higher than the immunization rate in the group of volunteers sensitized on UVB-irradiated skin alone (p<0.05). These results indicate that pretreatment with UVA-1 under certain conditions offers partial protection against the UVB-induced reduction in the immunization rates to epicutaneous allergens.

[Biotechnological pharmaceuticals and their potential use in inflammatory skin diseases]. / Bioteknologiske farmaka. Og deres mulige anvendelse ved inflammatoriske hudsygdomme.

In recent years, a more detailed understanding of the pathogenesis of several inflammatory skin diseases, and developments in biotechnology, have made it possible to design specific response modifiers, which hold a potential for greater effectiveness and fewer side effects than do the systemic therapies in current use in cases of severe psoriasis, contact dermatitis and atopic dermatitis. The immune system plays a pivotal role in the pathogenesis of inflammatory skin diseases, and this is where such biological response modifiers as monoclonal antibodies, recombinant cytokines, or fusion proteins may be effective. Several biological response modifiers have already shown positive results in phase II clinical trials; phase III trials are being carried out.

[Superantigens and their importance for inflammatory skin diseases]. / Superantigener. Og deres betydning for inflammatoriske hudsygdomme.

Superantigens are a group of bacterial and viral proteins that are characterized by their capacity to stimulate a large number of T-cells simultaneously. Superantigens bind directly to the MHC class II molecule on the antigen-presenting cell and crosslink the cell with T-cells expressing certain V beta-chains on their T-cell receptor which leads to a vigorous polyclonal T-cell activation. Staphylococcal superantigens seem to be involved in the pathogenesis of systemic diseases such as toxic shock syndrome and Kawasaki syndrome. Furthermore, superantigens seem to play an important role in the induction and maintenance of inflammatory skin diseases such as atopic dermatitis and psoriasis. The skin of patients with atopic dermatitis is often colonized with superantigen-releasing Staphylococcus aureus, and application of staphylococcal superantigen on intact human skin induces a local dermatitis reaction. Guttate psoriasis is often preceded by a streptococcal throat infection, and T-cells specific for streptococcal superantigens have been identified in fresh guttate psoriasis lesions.

Nickel-induced proliferation of both memory and naive T cells in patch test-negative individuals.

Lymphocyte transformation test has often been used as an in vitro test for nickel allergy. We have previously demonstrated the presence of nickel-reactive T cells in individuals with no history of allergic disease and with a negative patch test towards NiSO4. In this study, we show that this proliferative response was mainly confined to T cells within the CD4+ subset. In contrast to conventional recall antigens such as tetanus toxoid, in vitro stimulation using NiSO4 activated both FACS-purified CD4+CD45RA+ 'naive' and CD4+CD45RO+ 'memory' T cells. To determine which cell population reacted with nickel to induce T cell activation, peripheral blood mononuclear cells were separated into macrophages and non-adherent, HLA-DR-depleted T cells. We found that preincubation of monocytes/macrophages but not T cells with NiSO4 resulted in subsequent T cell proliferation. This result demonstrated that nickel did not exhibit any direct effect on the T cell. Furthermore, the NiSO4-induced T cell proliferation could be blocked by antibodies towards MHC class II (HLA-DR) molecules. Our results substantiate the concept that individuals with a negative patch test towards NiSO4 contain in their peripheral blood T cells capable of recognizing nickel or nickel-modified peptides. In contrast to conventional recall antigens, both memory and naive T cells were activated. Thus, when compared with data obtained from nickel-allergic individuals, this study shows a comparable nickel-inducible T cell activation in non-allergic individuals.

Nickel-induced activation of T cells in individuals with negative patch test to nickel sulphate.

Contact hypersensitivity to nickel is the most common form of allergic contact dermatitis. To gain insight into the induction of this frequent disease, T cell reactivity towards nickel was investigated in "nonallergic" individuals defined as those with no skin manifestations and a negative patch test towards NiSO4. Surprisingly, we found that nickel induced proliferation of peripheral blood mononuclear cells (PBMC) from 16 of 18 adult individuals tested. This activation was specific, and no stimulation of PBMC was observed using control stimulants at equimolar concentrations. Furthermore, the NiSO4-induced activation required the presence of professional antigen-presenting cells. To describe the functional capacity of the nickel-inducible T cells, cytokine release was investigated in both nickel-allergic and nonallergic individuals. The T cells from both groups released interferon-gamma but no interleukin-4 upon stimulation with nickel, suggesting that the functional capacities of these cell populations were similar in nickel-allergic and nonallergic individuals. Thus, at this level, no qualitative differences could be demonstrated between T cells obtained from nickel-allergic and nonallergic individuals.

In vivo UVA-1 and UVB irradiation differentially perturbs the antigen-presenting function of human epidermal Langerhans cells.

Ultraviolet B (UVB, 290-320 nm) radiation is known to suppress the immune function of epidermal Langerhans cells. We have recently described that in vitro UVB irradiation perturbs the antigen-presenting cell function of Langerhans cells by inhibiting their expression of functional B7 costimulatory molecules (B7-1, B7-2). The aim of this study was to determine wavelength-specific UV effects on Langerhans cells function in vivo, specifically UVB and UVA-1. To address this issue, volunteers were irradiated on the sun protected volar aspects of their forearms with 3 x minimal erythema dose of UVB (Philips TL-12) and UVA-1 (UVASUN 5000 Mutzhaas). Langerhans cells were isolated from suction blister roofs immediately following irradiation. Langerhans cells isolated from UVB- but not from UVA-1-irradiated skin failed to activate naïve resting allogeneic T cells (mixed epidermal cell leukocyte reaction) or primed tetanus toxoid reactive autologous T cells. Langerhans cells isolated from sham-irradiated or UVA-1-irradiated skin strongly upregulated B7-2 molecules during short-term tissue culture. By contrast, Langerhans cells from UVB-irradiated skin did not upregulate B7-2 molecules. Furthermore, exogenous stimulation of the B7 pathway by anti-CD28 stimulatory monoclonal antibodies restored the capacity of UVB-irradiated Langerhans cells to activate both alloreactive and tetanus toxoid-reactive T cells, implying suppressed antigen-presenting cell activities and perturbed B7 expression of Langerhans cells isolated from UVB-irradiated skin are related. Those studies demonstrate that in vivo UVB, but not UVA-1, interferes with the activation-dependent upregulation of B7 molecules on Langerhans cells, which in turn is of functional relevance for the perturbed antigen-presenting cell function of Langerhans cells within UVB- but not UVA-1-irradiated skin.

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) alpha and interleukin (IL) 10 in conjunction with other factors. In humans a transition polymorphism in the TNF-alpha gene may affect TNF-alpha 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-alpha 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 chi2-test. For both the TNF-alpha 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-alpha-308 transition or IL-10 CA repeat polymorphisms, although other as yet undetected DNA sequence variants of these genes may be involved.

Susceptibility to effects of UVB irradiation on induction of contact sensitivity, relevance of number and function of Langerhans cells and epidermal macrophages.

Sensitization on skin exposed to acute low-dose UVB irradiation separates normal humans into two phenotypically distinct groups: One group, following sensitization on UVB-irradiated skin, develops contact sensitivity, designated UVB resistant (UVB-R) and the second group, following sensitization on UVB-irradiated skin, fails to develop contact sensitivity, designated UVB susceptible (UVB-S). To investigate whether UVB susceptibility in humans in related to antigen-presenting activity in the skin we studied the effect of UVB irradiation on the number and function of the epidermal antigen-presenting cells in volunteers identified as UVB-R and UVB-S. Single cell suspensions of epidermal cells from control skin and skin exposed to 3 minimal erythema doses (MED) of UVB 3 days previously were stained for Langerhans cells (CD1a+HLA-DR+) and epidermal macrophages (CD1a-HLA-DR+). The UVB exposure of the skin significantly decreased the percentage of Langerhans cells (UVB-R: n = 7, P < 0.02, UVB-S: n = 6, P < 0.03) and increased the percentage of epidermal macrophages (UVB-R: n = 7, P < 0.03, UVB-S: n = 6, P < 0.03) however to the same degree in both the UVB-R and the UVB-S group. To study the effect on Langerhans cell alloreactivity, epidermal cells were harvested immediately after UVB irradiation. However, in both UVB-R and UVB-S subjects the Langerhans cell alloreactivity was blocked to the same degree immediately after UVB irradiation compared to nonirradiated epidermal cells. To determine the effect of UVB irradiation on epidermal macrophages, epidermal cells were harvested 3 days after UVB irradiation. Irradiated epidermal cells from both UVB-R and UVB-S subjects demonstrated a strong antigen-presenting capacity compared to epidermal cells from control skin leading to activation of T cells that mainly secrete interferon (IFN)-gamma and not interleukin (IL)-4. In conclusion we found that UVB susceptibility was not correlated with the number of Langerhans cells or epidermal macrophages in the skin at the same time of sensitization. Neither was it correlated with the capacity of Langerhans cells nor UVB-induced epidermal macrophages to activate T cells in vitro.

Contrasting effects of ultraviolet A1 and ultraviolet B exposure on the induction of tumour necrosis factor-alpha in human skin.

Ultraviolet B (UVB) irradiation of the skin causes immunosuppression which is relevant to the induction of skin cancer. The mechanism of this immunomodulation is unclear but various regulatory molecules have been implicated, including cis-urocanic acid (cis-UCA) and the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL-10). Whether ultraviolet A (UVA) induces similar changes has not been investigated fully. We studied the effect of in vivo UVB and long-wave UVA (UVA1) exposure on the induction of TNF-alpha, IL-10 and cis-UCA in human skin. Volunteers were irradiated with three minimal erythema doses (MED) of UVB or UVA1. At different times after irradiation, suction blisters were raised from irradiated and from non-irradiated (control) skin. The TNF-alpha and IL-10 protein concentration, and the percentage of cis-UCA in the blister fluid, were then determined. UVB irradiation of human skin led to a rapid and significant increase in TNF-alpha concentration in suction-blister fluid, with maximal values 6 h after irradiation (n = 6, P < 0.05). In contrast, UVA1 irradiation led to a decrease in TNF-alpha concentration in the suction-blister fluid compared with non-irradiated skin, with the lowest values 6 h after irradiation (n = 6, P < 0.05). Both UVB and UVA1 exposure of the skin induced a slight increase in IL-10 concentration. However, the increase in IL-10 was only significant after UVB irradiation (UVB, n = 6, P < 0.05; UVA, n = 7, P < 0.1). As previously shown, both UVB and UVA1 result in the photo-isomerization of trans-UCA and an increased percentage of cis-UCA was found in the suction-blister fluid. Thus the results show differential effects of UVB and UVA1 irradiation on the induction of immunoregulatory molecules, which may help to explain the variation in immune responses after UVB and UVA1 exposure of human skin.

Calcipotriol inhibits the proliferation of hyperproliferative CD29 positive keratinocytes in psoriatic epidermis in the absence of an effect on the function and number of antigen-presenting cells.

The aim of this study was to elucidate some of the possible mechanisms of action of the vitamin D analogue calcipotriol in vivo. Calcipotriol is finding increasing use in the treatment of psoriasis, but the primary target cell in vivo has not yet been identified. We treated psoriatic patients and healthy volunteers with calcipotriol and placebo ointment for 4 and 7 days, and obtained epidermal cell suspensions from treated areas. Epidermal cells were cocultured with autologous T cells, isolated from peripheral blood, in the absence or the presence of a classical antigen or a superantigen. In both psoriatic and normal skin, calcipotriol treatment did not alter the capacity of epidermal antigen-presenting cells to stimulate the proliferation of autologous T cells, either in the absence or in the presence of exogenous antigen. Epidermal cell suspensions were analysed further by staining for infiltrating leucocytes (CD45+) and Langerhans cells (CD1a+). Flow cytometric analysis showed that calcipotriol did not alter the number of CD45+ cells or Langerhans cells in psoriatic skin. These results indicate that calcipotriol does not alter either the number of the function of epidermal antigen-presenting cells in psoriatic epidermis. In contrast, we found that calcipotriol significantly inhibited the proliferation of epidermal cells isolated from psoriatic skin after in vivo treatment, as determined by propidium iodide staining and flow cytometry. More specifically, we stained for CD29+ keratinocytes and found an even more significant reduction in proliferative capacity. This cell type contains the population of hyperproliferative keratinocytes in psoriatic epidermis. In conclusion, calcipotriol seems to act via an inhibitory effect on hyperproliferative basal keratinocytes of psoriatic epidermis, rather than via an effect on infiltrating leucocytes, including antigen-presenting cells.