Urticaria pigmentosa

ABSTRACT

UV irradiation is widely used for the treatment of atopic eczema. In recent years, UVA1 phototherapy has gained increasing attention. This study analyzed the influence of different UV wavelengths-especially UVA1-on histamine release from human basophils and mast cells. The modulation of this parameter might be responsible for some of the therapeutic effects of UV irradiation. Enriched human basophils and human mast cells (HMC1 cell line) were irradiated with increasing doses of UVB, UVA and UVA1 in vitro. After irradiation, different stimulants were added to induce histamine release. In additional experiments, basophils were preincubated with superoxide dismutase, ascorbate or trolox to study the role of antioxidants in the modulation of histamine release after UV irradiation. UVA and UVA1 significantly inhibited histamine release from basophils and mast cells. UVB only had an inhibitory effect on mast cells. Preincubation with superoxide dismutase and ascorbate did not influence the inhibitory effect of UVA1 on basophil histamine release, whereas trolox decreased significantly the histamine release from nonirradiated basophils.

Abbreviations: EDTA, ethylenediaminetetraacetic acid; fmlp, f-met-leu-phe; IMDM, Isocove's modified Dulbecco's medium; PIPES, 1,4piperazinebis-(ethanesulphonic acid); SCORAD, Severity Scoring of Atopic Dermatitis.

INTRODUCTION

UV radiation has been used with great success in the therapy of skin diseases (1,2). In recent years, UVA1 phototherapy has gained much interest. High-dose UVA1 therapy is effective in the treatment of atopic eczema (3) and urticaria pigmentosa (4). UVA1 radiation can penetrate the dermis and can therefore affect mast cells and granulocytes (2). In vitro tests showed that UV irradiation can inhibit the stimulated histamine release from human basophils and rat mast cells (5-8). An altered membrane function and changes of the intracellular Ca^sup +^ content are thought to be involved in this mechanism. Probably, some of lhe positive effects of phototherapy, like the suppression of itch, are due to the inhibition on the histamine-releasing cells. Danno et al. (9,10) showed that UV irradiation inhibits the stimulated mast cell degranulation and ear swelling in mice. In previous studies (7,8,11-13), the UV-induced inhibition of histamine release was tested only on unpurified human basophils and on rat mast cells. In vitro studies with UVA1 irradiation are not available. This study was designed to analyze the effect of different UV wavelength regions on in vitro histamine release from enriched human basophils and human mast cells (HMC1 cell line) with special emphasis on UVA1 irradiation. Furthermore, we investigated the influence of antioxidants on basophil histamine release under UVA1 irradiation to assess the role of reactive oxygen species in the modulation of histamine release.

MATERIALS AND METHODS

Volunteers. Freshly drawn blood of 32 healthy donors (11 males and 21 females aged between 20 and 71 years, mean 35 years) was used for the basophil enrichment. The volunteers' history was free of atopy, and they showed a negative standard prick test.

Reagents. Percoll density gradient contained Percoll stock solution (= 1,4-piperazinebis-(ethanesulphonic acid) [PIPES] concentrate [250 mM PIPES, Sigma, Deisenhofen, Germany; 1100 mM NaCl and 50 mM KCl], Percoll [Pharmacia, Uppsala, Sweden] 1:10) and PIPES-A-buffer (25 mM PIPES, 110 mM NaCl, 5 mM KCl). PIPES-B-buffer contained CaCl^sub 2^ 0.0147 g, MgCl^sub 2^ 0.0246 g and 100 mL PIPES-A-buffer, PIPES-Agethylenediaminetetraacetic acid (EDTA) contained human serum albumin 0.006 g, glucose 0.2 g, Na^sub 2^EDTA 0.289 g and 200 mL PIPES-A-buffer, NaCl-EDTA (= 1.488 g Na^sub 2^EDTA and 1000 mL 0.9% NaCl). All buffers were at pH 7.3 and 300 mOsm. The stimulants calcium ionophore A23187, f-met-leu-phe (fmlp), substance P and compound 48/80 and the antioxidants ascorbate and superoxide dismutase were from Sigma. Anti-IgE was from DAKO (Glostrup, Denmark), and trolox was from Aldrich (Deisenhofen, Germany).

Enrichment of basophils. Basophils were enriched (29% purity, with lymphocytes as contaminants) by Percoll density gradient centrifugation. Fifty milliliters of EDTA-anticoagulated peripheral venous blood was centrifuged through a two-layer Percoll density gradient. The basophil-containing band was aspirated, and the basophils were washed with NaClEDTA and PIPES-Ag-EDTA. The washed cells were suspended (5 x 10^sup 4^ cells/mL) in PIPES-B-buffer, put in 24 well plates (Nunc, Wiesbaden, Germany; 400 [mu] per well) and exposed to the UV irradiation.

Human mast cell line. The immortalized human mast cell line HMCl was used, which has been established from the peripheral blood of a patient with mast cell leukemia (14). The cells were cultured in Isocove's modified Dulbecco's medium (IMDM, Sigma) supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, 1% L-glutamine and 1,2 mM [alpha]-monothioglycerol (all from Life Technologies, Eggenstein, Germany). The cells were washed twice, suspended (10^sup 6^ cells/mL) in colorless medium (IMDM, Life Technologies), put into 24 well plates (450 [mu]L per well) and exposed to the UV irradiation.

UV irradiation. Basophils and mast cells were exposed up to 500 mJ/cm^sup 2^ UVB (Philips TL 20W/12, Hamburg, Germany; main emission between 275 and 365 nm, maximum emission at about 315 nm, 1.4 mW/ cm^sup 2^ at a distance of 20 cm, intensity measured with the UV meter from Waldmann, Schwenningen, Germany); up to 100 J/cm^sup 2^ UVA (Waldmann UVA 700; main emission between 340 and 440 nm, maximum emission at about 365 nm, 55.6 mW/cm^sup 2^ at a distance of 20 cm, intensity measured with the UV meter from Waldmann); or up to 130 J/cm^sup 2^ UVA1 (HOnle Dermalight ultrA1, Planegg, Germany; main emission between 352 and 400 nm, maximum emission at about 384 nm, 80.3 mW/cm^sup 2^ at a distance of 70 cm, intensity measured by the manufacturer). During irradiation, cells were shaken carefully in a water bath at 37[degrees]C. After irradiation, cell viability was determined by trypan blue staining. During all experiments, basophils were more than 92% viable and HMCl cells more than 91%.

Stimulation of basophils and mast cells. After irradiation, basophils were stimulated for 25 min with calcium ionophore A23187 (final concentration 2x10^sup -3^ mg/mL), anti-IgE (final concentration 7.7 x 10^sup -2^ g/L) or fmlp (final concentration 2 x 10^sup -5^ mol/L). Basophils from six donors were additionally preincubated for 10 min with antioxidants (superoxide dismutase 1000 U, trolox 10 mM and ascorbate 1 [mu]g/mL) before the UVA1 irradiation. Mast cells were stimulated for 25 min with calcium ionophore A23187 (10^sup -5^ mg/mL), substance P (10^sup -6^ mol/L) or compound 48/80 (0.1 [mu]g/mL). During incubation with stimulants, the cells were shaken carefully in a water bath at 37[degrees]C. The optimal stimulation concentrations were chosen through preceding dose-response relationship experiments.

Measurement of histamine. After centrifugation, histamine content in the supernatant was measured spectrofluorometrically with an autoanalyzer (AutoAnalyzer II, Bran+Luebbe, Norderstedt, Germany). The corrected release of histamine was expressed as percentage of total histamine content according to the formula % histamine release = ([histamine content of the sample - histamine content of the blank in ng/mL]:total histamine content in ng/mL) x 100%.

Statistical analysis. The exact Wilcoxon test was used for comparisons between irradiated and nonirradiated samples. In each figure, the mean and the standard deviation of the mean are represented.

RESULTS

Effect of UVB on basophils and mast cells

Histamine release from basophils was unaffected by UVB irradiation (Fig. 1A). In contrast to basophils, the calcium ionophore-stimulated histamine release from HMC1 cells was significantly (P

Effect of UVA on basophils and mast cells

Preirradiation with UVA had a dose-dependent inhibitory effect on basophil histamine release (Fig. 2A). The amount of released histamine stimulated by calcium ionophore (mean = 32.1%), anti-IgE 7.7 x 10^sup -2^ g/L (12.0%) and fmlp 2 x 10^sup -5^ mol/L (25.4%) decreased down to 9.2, 7.1 and 7.6%, respectively, at 100 J/cm^sup 2^. The maximum inhibition in HMC1 cells was seen at lower doses (6-50 J/cm^sup 2^ UVA) compared with the inhibition in basophils. At 100 J/cm^sup 2^, the histamine release of the mast cells was increased (Fig. 2B).

Effect of UVA1 on basophils and mast cells

UVA1 inhibited the histamine release from basophils in a dose-dependent manner (Fig. 3A). The stimulated histamine release from nonirradiated basophils was 30.9% (calcium ionophore), 20.5% (anti-IgE 7.7 x 10^sup -2^ g/L) and 23.9% (fmlp 2 x 10^sup -5^ mol/L). At 130 J/cm^sup 2^, it decreased to 11.3, 11.8 and 8.6%, respectively. At low doses (6-50 J/cm^sup 2^), UVA1 irradiation showed a more pronounced inhibitory effect on histamine release from HMC1 cells than did UVA irradiation (Fig. 3B). At high doses, UVA1 irradiation yielded variable results.

Effect of UVA1 on basophils in the presence of antioxidants

Preincubation with superoxide dismutase and ascorbate had no effect on the inhibition of histamine release by UVA1 irradiation (Fig. 4). The antioxidant trolox caused a significant (P

Spontaneous and total histamine release

We saw no effect of UVB, UVA and UVA1 irradiation on the histamine release of basophils and mast cells without stimulants.

The mean spontaneous histamine release and the total histamine content of the basophils were 3.7/81.2 ng/mL (UVB), 17.2/62.7 ng/mL (UVA), 17.5/80.3 ng/mL (UVA1) and 5.0/64.9 ng/mL (UVA1 with antioxidants). The mean spontaneous histamine release and total histamine content of the HMC1 cells were 35.9/79.9 ng/mL (UVB), 59.6/108.1 ng/mL (UVA) and 45.1/103.3 ng/mL (UVA1).

DISCUSSION

This study demonstrates a significant inhibitory effect of UVA1 irradiation on histamine release from human basophils and mast cells.

Treatment of enriched basophils and HMC1 cells with UVA and UVA1 significantly inhibited the histamine release, whereas UVB only had an inhibitory effect on mast cells. HMC1 cells seemed to be more sensitive to UVA irradiation than basophils because the maximal inhibitory effect was obtained at lower doses. At higher doses, the histamine release from mast cells was increased. Contrary to the mast cells, basophil histamine release was decreased by UVA in a dose-dependent manner. The results regarding basophils are in accordance with previous results (8). Inhibitory effects of UVA and UVB irradiation were only tested on rat mast cells so far (5,7,11). Until now, no clear model exists to explain this phenomenon.

The mechanism by which UV irradiation inhibits the histamine release is still unclear. Yen et al. (13) reported changes in the plasma membrane ultrastructure from rat peritoneal mast cells after UVA irradiation plus protoporphyrin that suppressed the extrusion of histamine granules. They supposed that UVA influenced the membrane fluidity. To elucidate the mechanism of UV irradiation on the histamine release, we preincubated the basophils of some donors before UVA1 irradiation with the antioxidants superoxide dismutase, ascorbate and trolox.

The primary antioxidant defense mechanisms of human beings can be divided into two major groups: enzymatic (e.g. superoxide dismutase, catalase) and nonenzymatic antioxidants. The nonenzymatic antioxidants consist of lipophilic (e.g. [alpha]-tocopherol, ubiquinol-10) and hydrophilic (e.g. ascorbate, gluthatione) agents (15). We chose one antioxidant from each group by using the water-soluble trolox instead of [alpha]-tocopherol. Furthermore, we wanted to cover different reacting mechanisms and different targets of the antioxidants. Superoxide dismutase is also called a preventive antioxidant because it reduces the general rate of radical formation, whereas ascorbate and trolox act by breaking the radical chain process (16). Superoxide dismutase catalyzes the reduction of superoxide radicals (O2^sup -^), ascorbate directly reacts with singlet oxygen, hydroxyl radicals and superoxide radicals and trolox is a scavenger of lipid peroxide radicals (15,17). The concentrations of our antioxidants were in the same range as in previous investigations (8,18,19).

We wanted to study the role of antioxidants in the modulation of histamine release after UV irradiation. In a previous work (8), the UVA-induced inhibition of basophil histamine release was abolished in the presence of superoxide dismutase. A comparable mechanism was demonstrated by Iwai et al. (20). They irradiated an epidermal cell suspension with UVA in the presence and absence of the antioxidant glutathione. The UV-induced immune suppression was significantly prevented by glutathione. Therefore, addition of antioxidants in our investigation was expected to increase the histamine release. But we saw no effects of the antioxidants on the UV-induced inhibition of histamine release. On the contrary, trolox significantly inhibited the histamine release of the nonirradiated basophils. This result corresponds to the investigation of Strenzke et al. (21), in which vitamin E reduced the anti-IgE-induced basophil histamine secretion.

It is known that free radicals that arose from excessive UV irradiation lead to oxidative damage of the skin, and they are thought to be an important cause of skin cancer, immunosuppression and premature skin aging (17). Mio et al. (12) showed that the high-dose-UVB-induced (UVB > 780 mJ/cm^sup 2^) histamine release from rat peritoneal mast cells was inhibited by the antioxidant ascorbic acid. At high doses, UV-induced free radicals are most likely involved in the histamine releasing process. But it could be possible that UV at low doses has a mechanism different from that at high doses. At low doses, UV inhibits the histamine release, and according to our experiments free radicals would not be involved in the reaction. It would be interesting to measure directly the presence of free radicals after low- and high-dose UV irradiation in basophil and mast cell suspensions.

At present, three concepts of the mode of action of UV phototherapy on the skin are discussed (2): effects on production of soluble mediators, induction of apoptosis and modulation of the expression of cell-surface-associated molecules. UV-induced changes at the level of the cell membrane or of the cell surface most likely cause the decrease in basophil and mast cell histamine release. Amano et al. (7) showed that inhibition of the increase in intracellular Ca^sup +^ could be a possible mechanism for the inhibitory effect of UVA plus a photosensitizer on substance P-induced histamine release from rat peritoneal mast cells, Graevskaya et al. (11) found similar results with rat mast cells and UVB irradiation. They supposed an UV-induced impairment of membrane-dependent function of mast cells to be the reason for the inhibited degranulation. Though the mechanism of UV irradiation is not yet clear, the efficacy of UV phototherapy is clinically evident (3,4,22). Especially the suppression of itch by UVA and UVA1 phototherapy might be explained by modulation of the histamine-releasing cells. Stege et al. (4) reported the successful high-dose UVA1 treatment of patients with urticaria pigmentosa: itching disappeared after treatment, and histamine levels in urine were decreased. Abeck et al. (22) measured the therapeutic effectiveness of UVA1 irradiation in patients with atopic dermatitis by means of Severity Scoring of Atopic Dermatitis (SCORAD). The SCORAD index, which includes the assessment of itching, was significantly reduced after the UVA1 therapy.

Further investigations are needed to clarify the mode of action of UV phototherapy and the possibility of its modification.

Acknowledgements-This study was supported by a grant from the Technical University Munich (KKF; H 20-98).

[para]Posted on the website on 6 March 2003

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Christine Kronauer1,2, Bernadette Eberlein-Konig*1,2, Johannes Ring2 and Heidrun Behrendt1

1Division of Environmental Dermatology and Allergology, GSF/TUM, Neuherberg-Munich, Germany and

2Department of Dermatology and Allergy Biederstein, Technical University, Munich, Germany

Received 6 November 2002; accepted 18 February 2003

*To whom correspondence should be addressed at: Department of Dermatology and Allergy Biederstein, Technical University Munich, Biedersteiner Str. 29, 80802 Munich, Germany. Fax: 49-89-4140-3526; e-mail: eberlein-koenig@lrz,.tu-muenchen.de

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