Cytokine expression in psoriatic skin lesions during PUVA therapy

To determine whether an improvement in skin lesions as a result of PUVA therapy may be correlated with changes in cytokine patterns, RT-PCR amplification was used to compare the levels of IL-2, IL-6, IL-8, IL-10, TNF-α and IFN-γ cytokine mRNA expression in serial biopsies from three chronic plaque psoriatic patients. In each case, 3-mm punch biopsies were taken from lesional skin before and during 2–28 days of treatment with PUVA. Total mRNA was extracted from each biopsy, cDNA synthesized, and then amplified by 35 cycles of PCR using cytokine-specific primers. The specificity of the PCR products was confirmed by the Southern blot technique. Substantial levels of specific mRNA for each of the cytokines studied was present in the lesions prior to treatment. In two of the three patients who responded well to PUVA, a reduction in all the cytokines including IL-10 was observed compared with baseline levels. In contrast, PUVA proved to be ineffective in clearing the psoriasis of the third patient whose skin lesions worsened during the course of treatment. This was accompanied by an increase in IFN-γ but not of the other cytokines investigated, above the pretreatment level. This study showed an association between PUVA-induced resolution and decreases in the levels of various cytokines highly expressed in psoriatic lesions. Received:14 August 1995     

Mast cell tryptase and chymase in developing and mature psoriatic lesions

The number and distribution of mast cells in nonlesional and lesional skin samples from 13 psoriatic patients were analyzed enzyme- and immunohistochemically. Mast cell tryptase was stained with the sensitive substrate Z-Gly-Pro-Arg-4-methoxy-2-naphthylamide, and chymase with Suc-Val-Pro-Phe-MNA and monoclonal B7 anti-chymase antibody. In addition, healthy-looking skin from 27 psoriatic patients was tape-stripped resulting in induction of the Köbner response in 9 patients. Sequential biopsies were taken before and after (7, 14 and 21 days) tape-stripping, and both tryptase and chymase were stained enzyme-histochemically. In nonlesional psoriatic skin, 70 ± 24% (mean ± SD) of the mast cells contained chymase enzyme activity, and 78 ± 18% chymase immunoreactivity. About 10% of the chymase-immunoreactive cells lacked chymase activity. In lesional psoriatic skin, tryptase-positive cells were increased in number throughout the dermis but especially beneath the epidermis. Chymase immunoreactivity paralleled the tryptase activity, whereas chymase activity was strongly diminished both in terms of mast cell numbers and in staining intensity in the papillary dermis. The apparent inactivation of chymase may be due to the action of the chymase inhibitors, ₁-antitrypsin and ₁-antichymotrypsin, localized immunohistochemically in mast cells of lesional and nonlesional psoriatic skin. In the developing psoriatic lesion, mast cells displaying chymase activity were already 27–38% decreased in number in the upper dermis on day 7 after tape-stripping, along with the first clinical signs of psoriasis. Earliest alterations in tryptase-positive cells were observed on day 14 as increased mast cell contacts with the epidermis combined with only a slight increase in mast cell numbers in the upper dermis. During the development of a psoriatic lesion, TC mast cells (tryptase⁺, chymase⁺) increase in number in the upper dermis, but chymase becomes inactive at an early stage. The abundant presence of active trypase but inactive chymase in the upper dermis may have a potential role in psoriasis since both of these enzymes can process several biologically active peptides and proteins.     

PUVA therapy damages psoriatic and normal lymphoid cells within milliseconds

Summary Results of previous investigations have indicated that photochemotherapy (PUVA) attacks membranes of target cells. Using the combination of a stopped-flow technique and laser irradiation we were able to prove that the fast PUVA effect is explainable solely by the membrane damage. Lymphoid cells of healthy persons or psoriatics were taken, within 1 ms mixed with 8-methoxy-psoralen (8-MOP) at concentrations of 1.0, 0.1, and 0.05 g/ml, and then irradiated by a 337-nm laser pulse (0.5 mJ/cm²) lasting some picoseconds. Approximately 1 ms after administration of 8-MOP to the cell surface at least 10% of the cells were damaged, as could be judged using the standard trypan blue exclusion test. This happened at 8-MOP concentrations of 1.0 or 0.1 g/ml plus laser irradiation, but a concentration of 0.05 g/ml 8-MOP plus laser exposure did not cause any effect within 8 ms after mixing. There was no difference between using lymphoid cells from healthy persons or from psoriatics. The fact that only a very short time is necessary before cell damage occurs means that, as far as the fast PUVA effect is concerned, a photochemical reaction involving nuclear DNA can be discounted.     

Melanin granula distribution and phagocytosis in psoriasis vulgaris after PUVA therapy

Summary Melanin-containing basal cells of the epidermis, melanin-containing macrophages, mast cells, eosinophilic granulocytes and plasma cells were quantitatively investigated with the purpose of gaining an understanding of the quantitative changes in these cell systems under PUVA therapy. This patients have been exposed to solar radiation some weeks ore months before the begin of the PUVA-treatment. Different dying-processes were used to investigate biopsy samples of psoriatically healthy and psoriatically affected skin, from 28 patients before, and 39 patients after PUVA therapy, using a 2 d m with a field of view of 0.1 mm². Altogether more than 9,000 fields of view have been analysed. The average radiation amount was 12 irradiations with an average total energy of 21.5 J/cm². It was found that the count of granula-containing basal layer cells decreases in the psoriatic healthy region due to pigment incontinence and increase in the psoriatically affected region. The subepidermal melanincontaining phagocytes increase in both regions to a similar extent. In the case of the mast cells there was no trend to degranulation. The count of eosinophilic granulocytes and plasma cells was unchanged.     

The presence of tryptase-positive and bikunin-negative mast cells in psoriatic skin lesions

Human mast cells are well known to produce a serine protease, tryptase, which appears to play a pathogenic role in various skin inflammations. It was previously reported that a rat homologue of bikunin may inhibit tryptase activity. Various type of cells (i.e. keratinocytes) are able to produce this protein inhibitor, it still remains unclear if bikunin is present in dermal inflammatory milieu, in which mast cells, through secretion of tryptase, play an inflammatory role. Therefore, the purpose of the present study was to exploit expression and production of bikunin in dermis and dermal constituents. We first compared the dermal mast cells in psoriatic lesions with those in lesional skin of atopic dermatitis or of chronic eczema by use of immunoelectron microscopy and immunohistochemical analyses using antibodies to bikunin and tryptase. Then, we tested what kinds of cytokines may regulate the de novo synthesis of bikunin. To do so, RNA was extracted from a human mastocytic cell line, HMC-1, reverse-transcribed, and semiquantitative RT-PCR was performed using primers specific for bikunin. With immunoelectron microscopy, bikunin was found to localize on the cell membrane, while tryptase was in the secretary granules of the mast cells. In psoriatic lesions, around 70% of dermal mast cells were positive for both tryptase and bikunin, and the remaining was mostly positive for tryptase, but the expression of bikunin was under the detection limit of the experimental setting. This observation was seen in only psoriatic lesions, even in almost cured lesions, while in atopic dermatitis or chronic eczema only mast cells doubly positive for bikuin and tryptase were seen. In HMC-1, bikunin was constitutively expressed at an mRNA level, which was upregulated by stimulation with interleukine-4, but was suppressed by interferon-γ. Bearing in mind the concept that in psoriasis local cytokine milieu is shifted toward a Th1 pattern (predominant secretion of interferon-γ), tryptase-positive, bikunin-negative mast cells may be induced.     

Immunologic implications of PUVA therapy in psoriasis vulgaris

Summary During the combined effects of psoralen and UVA irradiation (PUVA therapy) a significant decrease (P<0.005 in T cells has been noted in 10 psoriasis patients and 10 healthy controls especially after four exposures. Based on the fact that the total number of circulating lymphocytes of the patients and the PUVA-treated healthy controls remained within the normal range, this decrease may be due to temporary physicochemical changes of the membranes of these cells but not to T cell lysis. After eight exposures these decreased T cell values returned to starting range. The starting T cell range in psoriasis patients is significantly lower (P<0.005) compared to that of healthy controls.It is of importance that before PUVA therapy in all the patients antibodies reactive with a basal cell nuclear antigen belonging to the four main Ig classes (IgM, IgD, IgE, and IgA) could be removed from the membrane of circulating lymphocytes by means of acid elution. In contrast, mainly the IgA antinuclear basal cell antibody could be eluted from circulating PMN leukocytes in the patients under investigation. After eight PUVA exposures, however, corresponding antibodies, belonging to the three main Ig classes (IgM, IgD and IgE) could also be eluted from the membranes of circulating PMN-leukocytes of the same patients. This implies an exchange of molecules during photochemotherapy. Finally, it could be shown that after effective PUVA therapy antinuclear basal cell antibodies of the psoriasis patients under study were reactive not only with the nuclei of the basal cell layer but also with almost all the nucleic of the epidermis of the uninvolved and lesional skin. The latter finding points to the fact that PUVA treatment causes at least antigenic changes of nuclear proteins in all the nuclei of the epidermis of the PUVA treated skin. Moreover, inflammatory cells with Fab fragments within the cells present in the lesional skin before PUVA disappear during this treatment.Based on a poster session presented at the occasion of the seventh joint meeting of the ESDR, Amsterdam, The Netherlands, May 1977     

Mast cells and macrophages in early relapsing psoriasis

Summary Five patients with widespread plaque-type psoriasis were treated continuously with clobetasol under occlusion. Clinical healing was seen after 6–10 days of treatment. All plaques treated in this way clinically relapsed approximately 12 days later. During the period of remission, sequential biopsies were taken and prepared for light and electron microscopy. Histologically, the earliest indications of relapse were endothelial alterations (swelling, intercellular widening) followed by the appearance of mast cells around the postcapillary venules; these mast cells showed signs of degranulation. Hours later, activated macrophages showing pericellular edema were present, and these migrated into the epidermis soon after. Associated with the presence of macrophages, there was a complete loss of desmosome-tonofilament complexes. Later, lymphocytes and neutrophils were seen. Under these experimental conditions, the psoriatic-tissue alterations appear to have been initiated by degranulating mast cells as well as by macrophages which later invaded the epidermis.This paper was presented at the 14th Meeting of the ESDR (Amsterdam, April 1984)     

Therapy of psoriasis with retinoid plus PUVA: Clinical and histologic data

Summary In a group of 40 patients suffering from wide-spread psoriasis oral administration of a retinoid (Ro 10-9359) was followed by PUVA therapy. The clearance rate was increased by 30% as compared to PUVA alone. Except for cheilitis no side effects were seen. Histological analysis in 20 patients before, during and after therapy revealed an intensification of psoriatic tissue changes after retinoid treatment. Loss of corneal layers, massive exoserosis, and neutrophil migration were prominent features. Mitotic counts were not increased by the pretreatment. The increased susceptibility of diseased skin to PUVA as produced by this drug appears to be based on several factors related to the tissue changes revealed by histology.Presented at the Niels-Stenson-Symposion, Appenrade, Denmark, October 28, 1978     

Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-α and their degranulation is associated with expression of ICAM-1 in the epidermis

Abstract The release of cytokines from cutaneous cells may be of major importance in the initiation and development of many inflammatory skin disorders. For example, tumor necrosis factor-alpha (TNF-α), which in healthy skin is found preformed only in mast cells, is able to induce the expression of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1). Increased expression of ICAM-1 occurs in keratinocytes in lesional skin of psoriasis and atopic dermatitis (AD) and it is considered to be an important initiator of leucocyte/keratinocyte interactions in skin inflammation. We counted the mast cells showing TNF-α immunoreactivity using a double-staining method in nonlesional and lesional skin sections from 12 patients with AD and 12 patients with psoriasis. The percentage of TNF-α⁺ mast cells in lesional and nonlesional AD skin was 36 ± 22% and 21 ± 15% (P < 0.018, paired t-test), respectively, and in psoriatic skin was 16 ± 25% and 15 ± 15%, respectively (P < 0.89, paired t-test). We also cultured whole skin biopsies taken from the healthy-looking skin of psoriatic and AD patients in the presence of mast cell degranulator compound 48/80, which resulted in focal expression of ICAM-1 in the epidermis. In cultured keratinocytes, both histamine and an extract of a human mast-cell line (HMC-1) induced ICAM-1 immunostaining only in occasional cells, but the combination of histamine and the HMC-1 extract resulted in intense ICAM-1 staining in numerous cells. This enhancement of ICAM-1 staining was abolished by preincubation of the HMC-1 extract with anti-TNF-α antibody. These results suggest that the degranulation of mast cells induces the expression of ICAM-1 in keratinocytes probably via TNF-α and histamine. Received: 8 August 1997     

Mast cell density and IL-8 expression in nonlesional and lesional psoriatic skin

BACKGROUND: An important cellular aberration at sites of psoriatic inflammation is an increase in the number of dermal mast cells. Being multifactorial immune effector cells, it is believed that mast cells play an essential role in perpetuating the inflammatory process of psoriasis. However, factors responsible for the infiltration and accumulation of mast cells in psoriatic lesions are largely unknown. Recent studies have demonstrated that Interleukin-8 (IL-8) exerts strong chemotactic effects on mast cells in vitro. Overexpression of IL-8 has also been reported in psoriatic lesions. In this study, we have found a correlation between the expression of IL-8 and dermal mast cell density in lesional psoriatic skin as compared to nonlesional psoriatic skin. METHODS: Four-mm punch biopsies were taken from 14 psoriatic patients and eight healthy volunteers. Using immunohistochemical techniques, 8 microm sections of lesional psoriatic, nonlesional psoriatic, and normal control samples were evaluated for dermal mast cell density and the density of IL-8 expressing keratinocytes. RESULTS: It was found that dermal mast cell density in lesional psoriatic, nonlesional psoriatic, and normal skin was 105.4 +/- 71.2, 42.3 +/- 30.1, and 47.5 +/- 32.5 mast cells/mm(2), respectively. IL-8+ keratinocyte density in lesional psoriatic, non lesional psoriatic, and normal skin was 171.5 +/- 67.1, 25.4 +/- 14.9 and 20.6 +/- 8.7 IL-8+ Keratinocytes/mm(2), respectively. CONCLUSIONS: The results of this study suggest that increased levels of IL-8 in the keratinocytes of psoriatic plaques play a contributing role in the migration of mast cells to lesion sites.     

Mast cell proteinases and cytokines in skin inflammation

The role of mast cells in provoking immediate-type hypersensitivity reactions is well established, but their involvement in chronic inflammation and immune reactions is not so clear. Mast cells synthesize and secrete large amounts of active proteinases, including tryptase, chymase, carboxypeptidase and cathepsin G, which can rapidly process numerous biologically active peptides and proteins or their precursors. Furthermore, mast cells are able to produce a variety of cytokines such as interleukin-4 (IL-4), IL-5, IL-6, tumour necrosis factor- (TNF-) and interferon- (IFN-) which are known to be intensively involved in modulating and directing inflammatory responses in the skin. In this review, the role of mast cell proteinases and cytokines in skin inflammation is discussed.     

PUVA treatment in chromium hypersensitivity: Effect on skin reactivity and lymphocyte functions

Summary Two male patients with longstanding contact sensitivity to chromium were treated with PUVA. One patient, suffering from concomitant photosensitivity, reacted very favorably; his skin lesions cleared and light tolerance increased. This was paralleled by a decrease in the photopatch test reactivity and by the extinction of the patch-test reactivity on PUVA-exposed (pigmented) skin. Patch and photopatch tests on PUVA-shielded skin showed no decrease in skin test reactivity. PUVA-treatment caused a decrease in the number of rosette-forming T cells and an increase in lymphocyte stimulation in both patients. In one patient, abnormally high PHA-induced suppressor cell activities were recorded prior to treatment; after PUVA therapy the values were back to normal. In both patients, the PPD-induced suppressor cell activity of PWM response was clearly increased by PUVA-therapy. Other suppressor cell functions were not much affected. It is concluded that while PUVA-therapy may produce some systemic immunological effects, its abating effect on contact sensitivity and photosensitivity is mainly mediated through local mechanisms in the skin.     

Squamous cell carcinoma in vitiligo lesion after long-term PUVA therapy

Photochemotherapy using psoralen and ultraviolet (UV)A irradiation (PUVA) is a useful treatment method for vitiligo. However, long-term PUVA therapy may cause several adverse effects including skin cancer, especially squamous cell carcinoma (SCC). We describe a case of SCC in vitiligo lesion after long-term PUVA therapy.     

Immunohistochemical analysis of sensory nerves and neuropeptides,and their contacts with mast cells in developing and mature psoriatic lesions

The distribution of the neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) was studied immunohistochemically in psoriatic skin during the Koebner response (6 h, 2 days, 7 days, 14 days, 21 days), and in mature psoriatic plaques, of 37 psoriatic patients. The morphological association of sensory nerves, SP and VIP with papillary mast cells was also monitored. The nerves containing SP, VIP or CGRP were very scanty in control skin, and in non-lesional and Koebner-negative psoriatic skin. The first psoriatic lesions were seen 7 days after tape stripping the symptomless psoriatic skin. SP- and VIP-containing nerves were slightly increased in Koebner-positive specimens, but the increase was very prominent in dermal papillae of mature psoriatic plaques. In the plaques, nerve-mast cell contacts were significantly increased (p<0.001) compared with non-lesional psoriatic skin. Only SP-positive fibres were detected in the epidermis and in contact with papillary mast cells. VIP was mainly located around capillaries where SP was also found. No change was noted in CGRP-positive fibres between lesional and non-lesional specimens. The appearance of SP and VIP in the capillary walls is morphological evidence for their function as vasodilators in psoriatic lesion. A slight increase in SP- and VIP-positive fibres in Koebner-positive specimens suggests that these neuropeptides may participate in the inflammatory reaction at an early stage. Their prominence in mature psoriatic plaques in turn indicates a role for them in the maintenance of psoriatic lesions. Morphological contacts between mast cells and SP-containing nerves give further evidence to the view that SP is capable of amplifying the inflammatory reaction also through the axon-reflex mechanism.Part of this work was presented at the meeting of the European Society for Dermatological Research, London, UK, 4–7 April 1992     

Comparison of turbo‐PUVA and conventional American‐style PUVA in the treatment of psoriatic patients

Background: Ultraviolet (UV)A protective properties of dihydroxyacetone (DHA) have been used as a topical UV‐resisting barrier to optimize psoralens and UVA (turbo‐PUVA). Starting doses and increments were based on the DHA diffuse reflectance spectroscopy‐derived protection factor. Objective: To evaluate the efficacy of turbo‐PUVA in psoriatic patients using a simpler method for determining starting doses and increments, in comparison to the conventional American‐style PUVA photochemotherapy. Methods: Thirty psoriasis patients (15 on American‐style PUVA and 15 on turbo‐PUVA) were evaluated, each receiving PUVA twice weekly. Starting UVA dose was determined according to skin phototype for the American‐style PUVA group and according to the patient's skin phototype × DHA SPF 3 in turbo‐PUVA group. UVA increments used were 0.5–1.5 J/cm² per treatment in American‐style PUVA and 25% of the previous dose in turbo‐PUVA. Results: Turbo‐PUVA group showed a significantly lower mean cumulative dose, a significantly higher psoriasis area and severity index score reduction, lesser mean number of treatment sessions, and less duration of treatment till remission (188.44±106.2 J/cm², 92.164±1.975%, 11.2±3.52 session, and 1.4±0.44 months, respectively) than conventional American‐style PUVA group (255.13±18.304 J/cm², 74.725±10.976%, 30±0.00 sessions, and 3.75±0.00 months, respectively). Conclusion: Turbo‐PUVA is more effective and time convenient for the treatment of psoriasis with less cumulative dose than the conventional American‐style PUVA.     

Raised galactose plus galactose-1-phosphate blood levels in patients under PUVA therapy

Background PUVA has become a preferred method of treatment for vitiligo and psoriasis. However, 8-methoxypsoralen (8-MOP) as well as a high galactose diet may cause liver and kidney function damage and enhance cataract formation. Patients and Methods Twenty one patients aged 32 ± 2.6 yrs with vitiligo (N= 11) or psoriasis (N= 10) underwent eye-examination, liver and kidney function tests and galactose + galactose 1-phosphate (gal + gal P) blood evaluation using Porton-Cambridge Ltd reagents, before and after the 12th PUVA treatment during a 35–40 day study. Results All these functional tests including eye-examination were normal before and after commencing therapy. However, the mean gal + gal P blood levels (1.29 ± 0.41 mg/dl) were statistically elevated (2.95 ± 0.42 mg/dl) at the end of the study. It is suggested that 8-MOP either impairs liver function or acts as an inhibitor of the enzymes responsible for galactose metabolism. We propose gal + gal P blood estimation in all patients before and, at intervals, after the initiation of PUVA therapy in order to prevent as early as possible an additional risk factor for liver or kidney dysfunction as well as cataract formation.     

Coculture of mast cells with psoriatic fibroblasts: an experimental system for studying the two cell interactions

In this study we established a coculture system for rat peritoneal mast cells (MC) with psoriatic (PSO) dermal fibroblasts. Rat MC adhered within five minutes to the fibroblasts monolayer and their attachment and viability was maintained for at least 2-3 days. Cocultured MC could be activated to release high percentages of histamine with compound 48/80 indicating that they retained their full functional activity. Attachment, viability and functional activity were similar for MC seeded on PSO fibroblasts or on normal human fibroblasts (NOR) used as a control. This would indicate that PSO fibroblasts altered biochemical characteristics do not interfere with these MC properties. We suggest that this coculture system is a suitable in vitro defined model to study mutual effects of MC and fibroblasts in psoriasis.     

Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin

Stereological quantification of mast cell numbers was applied to sections of punch biopsies from lesional and nonlesional skin of atopic dermatitis patients and skin of healthy volunteers. We also investigated whether the method of staining and/or the fixative influenced the results of the determination of the mast cell profile numbers. The punch biopsies were taken from the same four locations in both atopic dermatitis patients and normal individuals. The locations were the scalp, neck and flexure of the elbow (lesional skin), and nates (nonlesional skin). Clinical scoring was carried out at the site of each biopsy. After fixation and plastic embedding, the biopsies were cut into 2 μm serial sections. Ten sections, 30 μm apart, from each biopsy were examined and stained alternately with either toluidine blue or Giemsa stain and mast cell profile numbers were determined. The study yielded the following results: (1) in atopic dermatitis lesional skin an increased number of mast cell profiles was found as compared with nonlesional skin, (2) comparing atopic dermatitis skin with normal skin, a significantly increased number of mast cell profiles per millimetre squared was found in specimens from the neck, (3) staining with toluidine blue yielded a lower number of mast cell profiles than Giemsa staining, (4) the use of Carnoy’s fixative resulted in a lower mast cell profile count than the use of formaldehyde, and (5) there was no statistically significant correlation between the clinical score and the number of mast cell profiles per millimetre squared. Using stereological techniques, this study indicated that mast cells might participate in the inflammatory process in skin leading to atopic dermatitis. Received: 17 April 1996     

PUVA suppresses the expression of cell adhesion molecules of lymphocytes

Abstract To determine the therapeutic mechanism of PUVA in psoriasis vulgaris, the effects of PUVA on activated T lymphocytes were investigated in vitro. Peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers were activated with Con A stimulation (Con A blasts). Both untreated PBMC and Con A blasts were irradiated with UVA light in the presence of 8-methoxypsoralen (8-MOP). The expressions of CD4, CDS, VLA-4 and LFA-1 of PBMC and Con A blasts were stained with each monoclonal antibody and the intensity of fluorescence was analyzed by FACScan. PUVA-treated PBMC showed decreased response to both Con A and PHA stimulation. PUVA treatment also suppressed the IL-2 production of Con A blasts and IL-2 response of PBMC with increasing UVA fluence. The expressions of LFA-I, VLA-4, CD4, CDS and CD25 (IL-2R) molecules were decreased in PUVA-treated Con A blasts. Con A blasts were more sensitive than untreated PBMC to PUVA treatment. These results suggest that the therapeutic effects of PUVA on psoriasis vulgaris can be induced by suppression of the expression of cell surface molecules of activated T lymphocytes.