In all patients, UVA phototherapy led to a significant improvement of skin symptoms, as assessed by a clinical scoring system (total score before UVA phototherapy: 65.4 ± 6.2; total score after UVA phototherapy: 18.2 ± 3.4; P
). Skin specimens were analyzed for apoptotic cells using the TdT-mediated dUTP labeling (TUNEL) assay, followed by a anti-CD4 staining to detect T helper cells. Before therapy, numerous CD4+
cells were present intradermally in eczematous skin (3
) (Fig. A
). This cell population did not contain a significant number of apoptotic cells, and this was in sharp contrast to specimens obtained from the same skin areas after UVA radiation therapy had been started. Already after the 1st UVA radiation exposure, CD4+
, apoptotic cells were detected (Fig. B
). During subsequent UVA treatments, the number of double positive cells was further increased, whereas the total number of CD4+
cells decreased (Fig. , C
and Fig. ). After 10 exposures, the total number of intradermally located, CD4+
T cells had been significantly diminished, and most of the remaining cells showed signs of apoptosis (Fig. ). No apoptotic cells were detected in the epidermal compartment (data not shown).
Figure 1 Qualitative analysis of UVA phototherapy induced apoptosis in CD4+ cells present in lesional skin of a patient with atopic dermatitis. Biopsy specimens were obtained from lesional skin (flexural creases of the left elbow) of a patient with (more ...)
Figure 2 Semiquantitative assessment of apoptotic (○) and CD4+ (□) cells in lesional skin of five patients with atopic dermatitis before, during and after UVA phototherapy. Each time point was taken from each patient and the standard (more ...)
In atopen-specific human T helper cells, in vitro UVA irradiation induced apoptosis (Fig. , see also Figs. , ). Significant apoptosis was already detectable 4 h after exposure, reaching a maximum 24 h after irradiation with 30 J/cm2 UVA radiation (Fig. and data not shown).
Figure 3 Ultraviolet A radiation-induced apoptosis in atopen-specific, human T helper cells. UVA-irradiated T cells were analyzed for induction of apoptosis by assessing DNA laddering 2 h (lane 3), 4 h (lane 4), 8 h (lane 5), 24 h (lane 6) after UVA (30 (more ...)
Figure 5 UVA radiation and NDPO2 induced T cell apoptosis. T cells were preincubated in the presence of anti-Fas antibody ZB4 (1 μg/ml) (open bar) or an isotype control antibody (solid bar) for 1 h at 37°C. Cells were then exposed to UVA radiation (more ...)
Figure 6 UVA radiation- induced apoptosis and FASL expression in human T cells. Human T helper cells were exposed to increasing doses of UVA radiation (0–30 J/cm2) in the presence (open bar) or absence (solid bar) of sodium azide (50 mM) or deuterium (more ...)
Before UVA radiation exposure, FASL molecules were not present on the cell surface (Fig. ), but significant FASL surface expression was detected in UVA-irradiated cells already 4 h after exposure. Ultraviolet A radiation-induced surface FASL expression was dose-dependent and maximal upon exposure of cells to 30 J/cm2 UVA. In contrast to FASL, FAS surface expression remained essentially unaltered upon UVA irradiation (Fig. ).
Figure 4 FAS and FASL expression in UVA-irradiated T helper cells. T cells were exposed to increasing doses of UVA radiation (0–30 J/cm2). 4 (A) and 16 (B) h after exposure, cells were analyzed for FAS and FASL surface expression by FACS® analysis (more ...)
Addition of the blocking anti-FAS antibody ZB4 (13
), but not of equivalent concentrations of an isotype control antibody (Fig. ) or an isotype-matched anti-CD4 mAb (data not shown), significantly lowered UVA radiation-induced human T helper cell apoptosis (Fig. ).
In the following experiments, reagents capable of quenching (sodium azide) or enhancing (deuterium oxide) singlet oxygen effects were assessed for their capacity to modulate UVA radiation-induced human T helper cell apoptosis (14
). Irradiation of cells in the presence of sodium azide significantly inhibited UVA radiation-induced FASL surface expression as well as apoptosis in human T helper cells, whereas irradiation of cells in the presence of deuterium oxide resulted in a slight, but consistent increase in the percentage of FASL expressing as well as apoptotic cells (Fig. ).
We next assessed whether UVA radiation-induced apoptosis could be mimicked by stimulating unirradiated human T helper cells with singlet oxygen. Singlet oxygen was generated by thermal decomposition of NDPO2
). As shown in Fig. , singlet oxygen increased FASL surface expression in unirradiated T cells to an extent similar to that observed in UVA-irradiated cells. Similar to FASL surface expression, singlet oxygen generation also induced apoptosis in unirradiated cells (Fig. ). NDPO2
-induced FASL surface expression as well as apoptosis were significantly enhanced, if T cells were stimulated in the presence of deuterium oxide. Treatment of cells with NDP did not induce FASL surface expression or apoptosis. Addition of anti-FAS antibody ZB4 significantly inhibited apoptosis in unirradiated cells, which had been exposed to NDPO2
Figure 7 Singlet oxygen-induced apoptosis and FASL expression in human T cells. Human T helper cells were stimulated with increasing concentration of NDPO2 (0–30 mM) or NDP (30 mM) in the absence or presence of deuterium oxide (90%). After 4 h, the percentage (more ...)