Histamine is released following aminolevulinic acid-photodynamic therapy of human skin and mediates an aminolevulinic acid dose-related immediate inflammatory response

Acute skin inflammation occurs following topical aminolevulinic acid-photodynamic therapy (ALA-PDT), but its nature and mediation are ill defined. As we observed an urticarial response, a potential role for histamine was explored. In 13 healthy volunteers, we assessed the time course and dose-response of the acute cutaneous response(s) to ALA-PDT, the impact of H(1) antihistamine blockade, and measured dermal histamine release. An ALA dose series was iontophoresed into ventral forearm skin and exposed to red light. All participants exhibited an immediate urticarial response, both wheal and flare correlating with log ALA dose. Subsequently, a dose-related erythema developed at treatment sites by 3 hours and persisted at 24 hours. H(1) blockade with oral cetirizine doubled the median minimal urticating dose of ALA and reduced the slope of dose-response for wheal and flare, whereas at the highest ALA dose, mean wheal and flare areas reduced by 68 and 60%, respectively. In contrast, cetirizine did not influence the 24 hour minimal phototoxic dose or erythema dose-response. Histamine release after ALA-PDT mirrored the urticarial response, levels peaking within 30 minutes and returning to baseline by 24 hours. Thus, two discrete acute inflammatory responses to topical ALA-PDT occur in human skin; histamine mediates the immediate response, but does not appear involved in the delayed phototoxicity.     

Topical tretinoin replenishes CD1a-positive epidermal Langerhans cells in chronically photodamaged human skin

Excessive exposure to sunlight results in drastic degradative changes in the constitutive cells of the epidermis. Among these is a profound abrogation of Langerhans cell number and function, leading to compromised immunologic competency. Retinoids have recently been shown to restore immunologic function in the setting of iatrogenic immunosuppresion. We asked whether topical tretinoin would reverse Langerhans cell depletion in chronically photodamaged skin.
We examined the skin of 8 volunteers immunohistochemically before and after 6 months of daily applications of tretinoin. At baseline, there was a profound depletion of CD1a-positive Langerhans cells in the interfollicular epidermis, but not in the adjoining follicular epithelium. After tretinoin, all patients demonstrated replenishment of interfollicular epidermis by CD1a-positive Langerhans cells. This was associated with induction of HLA-DR expression on infundibular keratinocytes, as well as the appearance of CD1a dendritic cells in the papillary dermis. Thus, the enhancement of epidermal immunity in photodamaged skin may reflect restoration of antigen-presenting Langerhans cells. The source of this renewed dendritic cell population is likely to be follicular infundibulum and the papillary dermis.     

Topical photodynamic therapy significantly reduces epidermal Langerhans cells during clinical treatment of basal cell carcinoma

Background Topical photodynamic therapy (PDT) is a widely applied treatment for basal cell carcinoma (BCC). PDT‐induced immunosuppression leading to reduced antitumour immune responses may be a factor in treatment failure. Objectives To examine the impact of topical PDT on leucocyte trafficking following clinical treatment of BCC. Methods Superficial BCCs in eight white caucasian patients were treated with methyl aminolaevulinate (MAL)‐PDT. Biopsies for immunohistochemical assessment were taken from BCCs pre‐PDT, 1 h and 24 h post‐PDT and from untreated healthy skin. Results Treatment of BCC with MAL‐PDT produced a rapid neutrophil infiltration, commencing by 1 h and significantly increased at 24 h post‐PDT (P < 0·05 compared with baseline). An associated increase in the number of blood vessels expressing E‐selectin was observed at 1 h and 24 h post‐PDT (both P < 0·05 compared with baseline). In contrast, the number of epidermal Langerhans cells fell sharply by 1 h post‐PDT, and remained significantly reduced at 24 h post‐PDT (both P < 0·05 compared with baseline). Conclusions Reduction of Langerhans cells during clinical treatment of BCC might potentially impact negatively on antitumour responses through reduced activation of tumour‐specific effector cells. Investigation of modified PDT protocols with the aim to minimize immunosuppressive effects while maintaining antitumour efficacy is warranted.     

Langerhans cells in normal human skin capillaries

Cells with Langerhans granules are reported for the first time in normal human skin capillaries.     

Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro

It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.     

5-氨基酮戊酸乳膏光动力疗法治疗皮肤癌前病变和皮肤原位癌

目的 探讨5-氨基酮戊酸光动力疗法(ALA-PDT)治疗皮肤癌前病变和皮肤原位癌的疗效.方法 对15例光线性角化病、3例Queyrat增生性红斑和8例Bowen病患者进行ALA-PDT治疗.结果 15例光线性角化病患者的17个皮损获得完全缓解,2例复发,复发率13.33%;3例Queyrat增生性红斑除1例无效外,另2例患者的皮损获完全缓解,无复发;8例Bowen病除1例获得部分缓解外,其他7例完全缓解,1例复发.结论 ALA-PDT是一种疗效好、无明显痛苦、无瘢痕形成、复发率低、美容效果好的治疗皮肤癌前病变和皮肤原位癌的新疗法;特别适合于头面部及外生殖器部位的多发性、较大面积皮损.     

Langerhans cell density in epithelial skin tumors correlates with epithelial differentiation but not with the peritumoral infiltrate

We investigated the intraepithelial density of Langerhans cells in 17 epithelial skin tumors by immunohistologic and morphometric methods. There was a significant difference between seborrheic keratosis (Langerhans cell density 431 +/- 31/mm2; normal epidermis: 378 +/- 20/mm2), basal cell carcinoma (28 +/- 6/mm2), and squamous cell carcinoma (100 +/- 21/mm2). No correlation was found between the Langerhans cell density and the number of intraepithelial T lymphocytes or the extent of the peritumoral inflammatory infiltrate. A significant inverse correlation was demonstrated between mean nuclear area of the epithelial tissue and the Langerhans cell density (r = -0.7; p less than 0.05). These data indicate that the number of Langerhans cells does not influence the extent of the antitumoral immune response. The correlation with the level of epithelial differentiation may be due to different homing conditions.     

In situ quantification of T-lymphocyte subsets and Langerhans cells in the inflammatory infiltrate of atopic eczema

Tissue from the acute, non-infected eruption of sixteen atopic eczema subjects was subjected to an indirect immunoperoxidase technique using monoclonal antibodies recognizing T-lymphocyte subsets, Langerhans cells and natural killer cells. Over half the cells infiltrating the dermis were T lymphocytes, including a large majority of helper T cells and relatively few suppressor T cells. Langerhans cells were present in significant proportions in the dermis and probably reflected increased antigen presentation within the affected skin. There was no evidence of increased natural killer cell presence. This study suggests that type IV hypersensitivity may be implicated in the aetiology of atopic eczema.     

Topical beta-carotene is converted to retinyl esters in human skin ex vivo and mouse skin in vivo

Human epidermis contains endogenous retinoids (retinol and retinyl esters) and carotenoids (mostly beta-carotene). Previous studies have shown that the enzymes involved in retinoid metabolism are present in human epidermis. There is still a controversy about the presence in the skin of the enzymes able to convert beta-carotene into vitamin A (retinol), although a recent study demonstrated the conversion of beta-carotene into retinol in human cultured epidermal cells. In this study, we addressed the question of the possible bioconversion of topical beta-carotene into vitamin A or derivatives by human and mouse skin. Surgically excised human abdominal skin was mounted on Franz perfusion chambers to assess the cutaneous penetration of topical beta-carotene as well as its metabolism, after a 24-h incubation period, whereas hairless mice received topical beta-carotene 24 h before assaying epidermal beta-carotene and retinoid concentrations. Epidermal retinoid and beta-carotene concentrations were determined by high-pressure liquid chromatography. Topical beta-carotene penetrated well into human and mouse epidermis and induced a 10-fold (human) and a threefold (mouse) increase of epidermal retinyl esters, which demonstrates that topical beta-carotene is converted into retinyl esters by human and mouse epidermis and thus appears as a precursor of epidermal vitamin A.     

Distribution of ATPase-positive Langerhans cells in normal adult human skin

The distribution of ATPase-positive Langerhans cells (LC) was investigated in 117 specimens of normal adult human skin and mucosa taken from different areas of the body. Although there were significant variations in the numbers of LC in each area examined, skin from the face and neck contained the highest density of cells (976 +/- 30.93/mm2). The densities of LC in trunk skin (740 +/- 28.97/mm2), scalp (693 +/- 69.56/mm2) and arm or leg skin (640 +/- 40.95/mm2) were similar. Buccal mucosa had significantly fewer LC (567 +/- 42.94/mm2) than trunk skin, and sacrococcyx skin and palm and sole skin displayed the smallest number of these cells (267 +/- 56.14/mm2 and, 189 +/- 19.15/mm2 respectively). No ATPase-positive LC were detected in the centre of two corneal specimens.     

Culture of human epidermal Langerhans cells in a skin equivalent

Langerhans cells (LCs) have been cultured in a skin equivalent (SE). Seventy-two SEs were produced by inserting skin biopsies from nine subjects into dermal equivalents consisting of fibroblasts in a collagen matrix. The SEs were cultured in a serum-free medium containing 2-mercaptoethanol with or without 5 ng/mL granulocyte–monocyte colony-stimulating factor (GM-CSF). The SEs were cultured for 12 or 15 days. In the latter case, 0, 1 or 10 μg/mL cyclosporin A (CyA) was added for the last 3 days. The SEs were then snap frozen for immunohistochemistry. The migration of LCs was evaluated by measuring the distances from the inserted skin biopsy in the SEs to the HLA-DR + and CD1a+ dendritic cells localized at the longest distance from the biopsy in the epidermal outgrowth on both sides of the biopsy. The density of these cells was estimated in 15-day-old SEs by counting them on both sides of the inserted skin biopsy and dividing the number of positive cells by the migrated distances. All epidermal outgrowths (range 0.6–3.7 mm) were well differentiated and displayed HLA-DR+, CD1a+ and Lag+ dendritic cells. Only occasionally were CD83+ cells observed. In the 15-day-old SEs cultured with GM-CSF, a few CD86+ cells were seen in the epidermal outgrowths and occasionally CD80+ cells. The median ( n   =  4) density of CD1a+ and HLA-DR+ cells in the epidermal outgrowths at day 15 was 5.2 and 9.1 cells/mm, respectively. GM-CSF did not influence migration in 12-day-old SEs, but there was a tendency to increased migration of HLA-DR+ dendritic cells in 15-day-old SEs. CyA did not affect migration or density. We conclude that LCs can be cultured with an in vivo -like density in a SE. They express the phenotype of immature antigen-presenting cells efficient in capturing and processing antigen. This model may be suitable for studies of the initial phase of contact allergic reactions.     

Differential distribution of Langerhans cells in organ culture of human skin

Epidermal Langerhans cells (LCs) function as the antigen-presenting cells in such cutaneous cell-mediated immune responses as contact hypersensitivity and in the mixed epidermal cell-lymphocyte reaction. They have also been implicated in the immune response in skin allograft rejection. Since organ culture of thyroid and pancreas has been shown to prolong allograft survival, presumably through the loss of antigen-presenting cells, we examined the effect of skin explant culture on LC survival. Human skin explants were placed in organ culture and examined serially as whole mounts of epidermis for the presence of LCs as judged by ATPase activity, and OKT-6 and HLA-DR antigens. Although we observed morphologic changes and an absolute reduction in the number of positively stained cells, culture for up to 28 days failed to deplete explants of these cells. Langerhans cells were also sought in the epidermal outgrowths that develop peripheral to the original explants. They were never seen in the area beyond 0.3 mm from the explant edge. Organ culture of skin thus provides a means to explore the contribution of LCs to skin allograft rejection by comparing the immunogenicity of epidermal portions of the explant with the epidermal outgrowth.     

Further evidence for the self-reproducing capacity of Langerhans cells in human skin

The limited number of Langerhans cells (LC) in the epidermis is one of the main reasons for the technical difficulties in resolving the question of LC kinetics. In the present paper, we describe a method to evaluate the LC replication potential in epidermis. The procedure is based on the specific incorporation of bromodeoxyuridine (BrdU), a thymidine analogue, into the DNA during the S-phase of the cell cycle. Mice, bearing human skin grafts, were injected s.c. every 6 h for up to 17 days with BrdU. At different times, the incorporated BrdU as well as the human epidermal LC were revealed on skin sections using anti-BrdU and OKT-6 monoclonal antibodies, respectively. After 6 h, 4.9% of the LC were labeled with BrdU. Then, the number of OKT-6(+) BrdU(+) cells increased in a linear manner and achieved 34% at 120 h, 67% at 240 h, and 94% at 400 h during the course of continuous labeling procedures. Based on this result we calculated a total cell cycle time of 392 h (16.3 days) and 12 h for the S-phase for human epidermal LC. Applying this technique, we were able to show also that 48 h after local treatment with 12-O-tetradecanoylphorbol-13-acetate or after stripping, the number of BrdU-labeled LC was considerably increased. Furthermore, after i.p. injection of colchicine in the nude mouse, human epidermal LC undergoing mitosis were evidenced by electron microscopy in the graft. From these results we conclude that the LC are actively cycling--therewith a self-reproducing cell population in human epidermis.     

Distributional changes of Langerhans cells in human skin during irritant contact dermatitis

We used light and electron microscopic immunocytochemistry to study distributional changes in the human Langerhans cell (LC) system during the first 14 days of a mild irritancy caused by sodium lauryl sulphate (SLS). A marked initial decrease in epidermal LC was noted possibly resulting from migration from the epidermis to the dermis and from irreversible cell damage. Several studies have previously found an unchanged number of LC in SLS-induced contact irritant dermatitis, but these studies may not have taken into account the fact that SLS is effectively absorbed from the test chamber. Unless certain precautions are taken the SLS concentration rapidly falls to topical levels that have no effect on the LC system. Simultaneously with the decrease in the epidermis we observed an increase in dermal CD1a+ cells, confirming an often reported finding. There is, however, no consensus as to the identity of these cells, and several authors have reported that such cells lack LC granules and thus these cells have often been classed as indeterminate cells. We found that, during irritant contact dermatitis, provided an adequate number of sections were scrutinized in the electron microscope, all dermal CD1+ cells contained Birbeck granules.     

Sun exposure rapidly reduces plasmacytoid dendritic cells and inflammatory dermal dendritic cells in psoriatic skin

Background Interferon (IFN)‐α‐producing plasmacytoid dendritic cells (pDCs), inflammatory CD11c+CD1c? myeloid dendritic cells (mDCs) and macrophages have been found to contribute to the pathogenesis of psoriasis. Heliotherapy is a well‐established treatment modality of this disease, although the details of how the effects are mediated are unknown. Objectives To test the hypothesis that exposure to natural sun affects pathogenic DC subsets in lesional skin. Methods Skin biopsies were obtained from lesional and nonlesional skin in 10 patients with moderate to severe psoriasis subjected to controlled sun exposure on Gran Canaria. Biopsies were obtained at baseline, day 2 and day 16 and examined by immunohistochemistry. Results  Sixteen days of heliotherapy had excellent clinical effect on patients with psoriasis, with significant reductions in Psoriasis Area and Severity Index (PASI) scores. In lesional skin pDC numbers and expression of MxA, a surrogate marker for IFN‐α, were rapidly reduced. Inflammatory CD11c+CD1c? mDCs were significantly reduced whereas resident dermal CD11c+CD1c+ mDCs were unaffected. Expression levels of the maturation marker DC‐LAMP (CD208) on mDCs were significantly reduced after sun exposure, as were the numbers of lesional dermal macrophages. A decrease of dermal DC subsets and macrophages was already observed after 1 day of sun exposure. An additional finding was that DC‐SIGN (CD209) is primarily expressed on CD163+ macrophages and not DCs. Conclusions The clinical improvement in psoriasis following sun exposure is associated with rapid changes in dermal DC populations and macrophages in lesional skin, preceding the clinical effect. These findings support the concept that these DC subsets are involved in the pathogenesis of psoriasis and suggest that sun‐induced clinical benefit may partly be explained by its effect on dermal DCs.     

Inhibition of MAPK signaling pathways enhances cell death induced by 5-Aminolevulinic acid-photodynamic therapy in skin squamous carcinoma cells

Background

Combination of a photosensitizer, 5-aminolevulinic acid (ALA), with photodynamic therapy (PDT) has been widely used to treat skin squamous cell carcinoma (SCC). However, a portion of SCC patients do not respond well to PDT. The molecular reason for this resistance is not clear. We hypothesize that mitogen-activated phosphorylation kinase (MAPK) plays a key role in mediating SCC resistance to PDT.

Objectives

To determine whether inhibition of MAPK signaling enhances the anti-tumor effect of ALA-PDT in SCC.

Material and methods

The human squamous carcinoma cell line, SCL-1, was either untreated or treated with various combinations of ALA, PDT light source and inhibitors of MAPK signaling components.

Results

ALA-PDT treatment significantly decreased cell viability, increased the percentage of annexin-V positive cells and resulted in formation of apoptotic bodies. ALA-PDT treated cells showed increased levels of p-MEK, p-ERK1/2, p-p38, p-Elk-1, p-JNK and p-c-Jun. Addition of inhibitors for ERK1/2 (PD98059), p38 (SB203580) and JNK (SP60125) reversed the changes and led to a more dramatic decrease in SCL-1 cell viability than seen with ALA-PDT alone.

Conclusion

Inhibition of the MAPK pathway enhances the cytotoxic effect of ALA–PDT on SCL-1.