Research Of The Role Of TRAIL In Keratinocytes’ Apoptosis In Severe Bullous Drug Eruptions

Background:Keratinocyte apoptosis is a critical event in the early phase of Severe Bullous Drug Eruptions (SBDE, including SJS and TEN), which is partly due to the immune mechanism Involving cytotoxicity CD8+ T cells, NK cells, monocytes, etc.; while TNF-a, sFasL, granulysin and other cytokines participate in the amplification of apoptosis, which is now considered to be more important. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and its receptor DR (death receptor) 4, DR5 are regarded as an important pathway in apoptosis, but few studies associated them have been done in this disease. Further study helps to clarify the pathogenesis of SJS/TEN.Purpose:From three levels including skin tissue, blister fluid and peripheral blood, we detected TRAIL and its receptors’ expression of Keratinocytes as well as TRAIL’s expression of blister fluid cells and peripheral blood mononuclear cells; analyzed the concentration of sTRAIL (soluble TRAIL) and other apoptosis-associated cytokines including IFN-y, TNF-a, sFasL, granulysin in blister fluid and peripheral blood; Furthermore, in HaCaT cells we investigated their effect on sTRAIL inducing keratinocyte apoptosis. Clarification of the role of TRAIL in SJS/TEN may provide help to pathogenesis of this disease.Method:1. Immunohistochemically detected TRAIL and its receptors’ expression of Keratinocytes in skin tissue of SJS/TEN.2. Western-Blot and RT-PCR were applied to detect TRAIL’s expression in blister fluid cells (protein and mRNA); Flow cytometric analysis of blister fluid cell phenotype were applied to correlate with the expression of TRAIL.3. Similarly,western-Blot and RT-PCR were applied to detect TRAIL’s expression of peripheral blood mononuclear cells in patients with SJS/TEN, the expression were compared to those in blister fluid cells.4. ELISA was applied to detect concentrations of sTRAIL, IFN-γ, TNF-α, sFasL, granulysin in blister fluid and peripheral blood.5.Flow cytometry was applied to detect apoptosis of cultured HaCaT cells by AnnexinV-FITC/PI assay, the effect of sTRAIL alone and combination with IFN-γ, TNF-α, sFasL, granulysin were detected and compared.6. By cells seeded and immunohistochemical analysis, impact of these cytokines on the expression of TRAIL and DR4/5 in HaCaT cells were investigated.Results:1. In 20 patients with SJS/TEN, elevated expression of TRAIL and DR4/5 in keratinocytes were found, there were significantly different when compared with 10 patients with exanthematous drug eruption as control. Infiltrating inflammatory cells in SJS/TEN skin tissue also expressed TRAIL.2.In 10 cases of SJS/TEN, blister fluid cells were detected with high expression of TRAIL (protein and mRNA), compared with six cases of control (bullae in skin graft donor sites of patients with vitiligo), there were significantly different, but the expression exhibited a gradually downward trend; The percentages of CD8+ T cells and NK cells in blister fluid cells decreased gradually accompanying with the increase of monocytes percentage, through multiple regression analysis it revealed that the expression of TRAIL mainly correlated with CD8+ T cells.3 In 15 cases of SJS/TEN, TRAIL expression in peripheral blood mononuclear cells were lower than those in the blister fluid cells, but higher than those in 10 cases of normal people and patients with exanthematous drug eruption as control. The expression of peripheral blood mononuclear cells were relatively stabler than those of blister fluid cells.4. Concentrations of sTRAIL, IFN-γ, TNF-α, sFasL, granulysin in blister fluid were higher than those in peripheral blood, but sTRAIL, IFN-γ, TNF-α concentrations were relatively stable while sFasL, granulysin concentrations decreased rapidly.5. HaCaT cells apoptosis were weak when treated with sTRAIL alone, only TNF-α showed strong induction of apoptosis. However, IFN-γ, TNF-α facilitated the apoptotic role of sTRAIL while sFasL, granulysin showed no correlation with sTRAIL.6. IFN-γ, TNF-α upregulated HaCaT cells’ TRAIL and DR4/5 expressions, but the former might be more powerful in terms of DR5.Conclusion:TRAIL activations were found in skin tissue, blister fluid and peripheral blood of SJS/ TEN. Peripheral blood mononuclear cells highly expressed TRAIL, also increased the concentration of sTRAIL, which meant TRAIL also involved in the systemic response of the disease. The blister fluid cells were migrated to SJS/TEN skin tissue by chemotaxis, especially CD8+ T cells in early phase elevated TRAIL expression, possibly through direct binding or sTRAIL, these cells induced apoptosis of keratinocytes with high expression of DR4/5; keratinocytes cells also highly expressed TRAIL and DR4/5 simultaneously, which indicated their did not simply present as "victim" in this disease. By HaCaT cell experiments, this phenomenon may be related to the role of IFN-y, TNF-a, in addition, these two cytokines synergistically enhanced the effect of inducing apoptosis of sTRAIL.Therefore, we believe that TRAIL has strong connection with the apoptosis of keratinocytes in this disease, which is under the context of immune disorders and as one of many complex mechanisms of keratinocytes apoptosis like network. However, how drug cause the immune disorders including TRAIL and How death ligand including TRAIL induce apoptosis of keratinocytes still need to be clarified.