Platelet-activating factor is crucial in psoralen and ultraviolet A-induced immune suppression, inflammation, and apoptosis

Abstract

Psoralen plus UVA (PUVA) is used as a very effective treatment modality for various diseases, including psoriasis and cutaneous T-cell lymphoma. PUVA-induced immune suppression and/or apoptosis are thought to be responsible for the therapeutic action. However, the molecular mechanisms by which PUVA acts are not well understood. We have previously identified platelet-activating factor (PAF), a potent phospholipid mediator, as a crucial substance triggering ultraviolet B radiation-induced immune suppression. In this study, we used PAF receptor knockout mice, a selective PAF receptor antagonist, a COX-2 inhibitor (presumably blocking downstream effects of PAF), and PAF-like molecules to test the role of PAF receptor binding in PUVA treatment. We found that activation of the PAF pathway is crucial for PUVA-induced immune suppression (as measured by suppression of delayed type hypersensitivity to Candida albicans) and that it plays a role in skin inflammation and apoptosis. Downstream of PAF, interleukin-10 was involved in PUVA-induced immune suppression but not inflammation. Better understanding of PUVA's mechanisms may offer the opportunity to dissect the therapeutic from the detrimental (ie, carcinogenic) effects and/or to develop new drugs (eg, using the PAF pathway) that act like PUVA but have fewer side effects.

Figures

Figure 1

PUVA-induced PAF receptor activation is crucial for PUVA-induced immune suppression. PUVA-induced systemic immune suppression was studied in the model of DTH to C. albicans. Groups of C3H, C57BL/6, or PAF receptor knockout mice (n = 5 to 6 per group) were left untreated or treated with topical 8-MOP and different doses of UVA, ranging from 6.25 to 50 kJ/m2, solar-simulated UV radiation (15 kJ/m2), or were injected with PUVA-treated, UVA-treated, or untreated PC 5 days before immunization with C. albicans. Nine days later the mice were challenged with Candida antigen. DTH was measured 24 hours after challenge. The background response (negative control) was measured in mice that were not immunized but challenged. Mice that were immunized (but not PUVA-treated) and challenged served as positive control group. PUVA exposure was highly immunosuppressive and suppressed DTH by up to more than 90% at the PUVA dose of 50 kJ/m2. a: The intraperitoneal injection of C3H mice immediately before PUVA exposure with either the PAF receptor antagonist PCA-4248 or the COX-2 inhibitor SC-236 totally blocked PUVA-induced immune suppression. *P < 0.0001; +P = 0.0005; #P < 0.01 versus positive control group. b: C57BL/6 (WT) but not PAF receptor knockout (PAFR−/−) mice were susceptible to immune suppression induced by either PUVA at a dose of 50 kJ/m2 or solar-simulated UV at 15 kJ/m2. *P < 0.005; +P < 0.05 versus respective positive control group. c: The injection of C3H mice with PUVA-treated PC 5 days before sensitization with C. albicans resulted in significant immune suppression, in a dose-dependent manner similar to that in the in vivo PUVA experiments. The injection of UVA-treated PC (without psoralen) also led to immune suppression in a very similar dose-dependent manner than PUVA-treated PC. *P < 0.0001; +P < 0.005; #P < 0.01 versus positive control group.

Figure 2

IL-10 is essential downstream of PAF receptor activation for PUVA-induced immune suppression. a: PUVA-induced IL-10 protein expression in the skin of C3H mice was studied by immunohistochemical anti-IL-10 staining. Note that there is diffuse brownish staining (indicating the presence of IL-10 protein) of keratinocytes and dermal cells in PUVA-treated skin. The intraperitoneal injection of mice immediately before PUVA exposure either with the PAF receptor antagonist PCA-4248 or the COX-2 inhibitor SC-236 inhibited PUVA-induced IL-10 up-regulation in the skin. Only patchy epidermal staining and occasional IL-10-positive dermal cells were present in the skin of mice injected with either the PAF receptor antagonist or the COX-2 inhibitor, similar to the staining pattern observed in untreated control mice (none) or UVA- or 8-MOP-treated mice. Photographs shown are from skin samples taken 24 hours after PUVA exposure to 50 kJ/m2. b: There was significant p53 up-regulation of IL-10 protein (as measured by ELISA) in the serum at 120 hours after PUVA exposure to 50 kJ/m2. This IL-10 up-regulation was not found in serum of mice injected immediately before PUVA exposure with either the PAF receptor antagonist or the COX-2 inhibitor. n = 2 to 3 mice per group. c: Immune function was studied in the model of DTH to C. albicans (for description of positive and negative control group see legend of Figure 1). The intraperitoneal injection of mice immediately before PUVA exposure with an anti-IL-10 antibody abrogated PUVA-induced immune suppression, whereas an isotype control antibody had no significant effect. n = 5 mice per group. *P < 0.0001; +P = 0.05 versus positive control group.

Figure 3

PAF receptor activation is involved in PUVA-induced apoptosis. Apoptosis was determined by TUNEL staining. Results shown are from skin samples taken 24 hours (time point of maximum effect) after PUVA exposure to 50 kJ/m2. PUVA-induced apoptosis (green nuclear staining; right) was diminished in the skin of mice injected intraperitoneally immediately before PUVA exposure with either the PAF receptor antagonist PCA-4248 or the COX-2 inhibitor SC-236. The left panel (red nuclear staining) shows the propidium iodide controls. Treatment with UVA or 8-MOP alone did not lead to significant levels of apoptosis.

Figure 4

PAF receptor activation is also involved in PUVA-induced p53 up-regulation. PUVA-induced p53 protein expression in the skin of mice was studied by immunohistochemical staining. Results shown are from skin samples taken 24 hours (time point of maximum effect) after PUVA exposure to 50 kJ/m2. a: PUVA-induced p53 up-regulation (brownish nuclear staining) was reduced in the skin of mice injected intraperitoneally immediately before PUVA exposure with either the PAF receptor antagonist PCA-4248 or the COX-2 inhibitor SC-236. Treatment with UVA or 8-MOP alone did not lead to significant apoptosis. b: Computerized image analysis quantifying the optical density of nuclear staining of epidermal cells revealed that the effect of the PAF receptor antagonist and the COX-2 inhibitor on PUVA-induced p53 expression was stronger than evident from the differences in the numbers of p53-positive epidermal cells in a. n = 2 to 3 mice per group. *P < 0.05 versus 8-MOP- or UVA-treated mice.

Figure 5

PAF pathway blockade does reduce skin inflammation on the macroscopic level. a: Groups of C3H mice (n = 5 per group) were treated with topical 8-MOP and/or different doses of UVA, ranging from 6.25 to 50 kJ/m2. Skin swelling was determined by measuring double skin-fold thickness of dorsal skin of the mice before and at 48 hours after PUVA exposure. The skin swelling data are from the DTH experiment presented in Figure 1a. The skin swelling response was greatly diminished in the skin of mice injected intraperitoneally immediately before PUVA exposure with either he PAF receptor antagonist PCA-4248 or the COX-2 inhibitor SC-236. Note that treatment with UVA or 8-MOP alone did not lead to significant skin swelling. *P < 0.05 versus none (no treatment). b: Both the PAF antagonist PCA-4248 and the COX-2 inhibitor SC-236 more consistently protected in repeat experiments against PUVA-induced immune suppression, but they gave only variable protection against inflammation (at the dose of 50 kJ/m2). This is evident from the plotted graphs showing mean skin swelling (top graph) versus mean percent immune suppression (bottom graph). Results are means from several independent experiments (n = 2 to 7 per treatment with at least five mice per experimental group per experiment). *P < 0.01 versus 8-MOP- or UVA-treated mice (top graph). *P < 0.0005 versus 8-MOP- or UVA-treated mice (bottom graph). c: Swelling of dorsal skin was also determined in PAF receptor knockout mice (PAFR−/−) and their C57BL/6 littermates (WT) at 48 hours after exposure to PUVA at a dose of 50 kJ/m2 or solar-simulated UV (ssUV) radiation at a dose of 15 kJ/m2. The presented skin swelling data are means from three independent experiments with at least five mice per experimental group per experiment. *P < 0.01; +P < 0.05 versus respective untreated control group (none).

Figure 6

PAF pathway blockade does reduce PUVA-induced skin inflammation and other alterations on the microscopic level. Like macroscopic skin inflammation (ie, skin swelling), the inflammatory skin infiltration was also maximum at 48 hours after PUVA exposure (data shown), as examined on the microscopic level in H&E stainings. PUVA-induced skin infiltration and hyperplasia were greatly diminished in C3H mice injected intraperitoneally immediately before PUVA exposure with the PAF receptor antagonist PCA-4248 or the COX-2 inhibitor SC-236. The drug effects are quantified in Table 1. Treatment of mice with UVA or 8-MOP alone did not lead to significant levels of inflammation or hyperplasia, neither on the macroscopic nor on the microscopic level.