TNF-α and IFN-γ Are Potential Inducers of Fas-Mediated Keratinocyte Apoptosis through Activation of Inducible Nitric Oxide Synthase in Toxic Epidermal Necrolysis  Isabelle Viard-Leveugle, Olivier Gaide, Dragana Jankovic, Laurence Feldmeyer, Katrin Kerl, Chris Pickard, Stéphanie Roques, Peter S. Friedmann, Emmanuel Contassot, Lars E. French  Journal of Investigative Dermatology  Volume 133, Issue 2, Pages 489-498 (February 2013) DOI: 10.1038/jid.2012.330 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Nitric oxide (NO) donors increase membrane FasL expression and cell apoptosis in keratinocytes. (a) Immunohistological detection of inducible nitric oxide synthase (iNOS) and FasL in toxic epidermal necrolysis (TEN) and normal skin biopsies. Bar=200μm. (b) Total RNA was extracted from lesional skin from patients with maculo-papular rash (MPR, n=10) or TEN (n=5), and iNOS quantitative RT–PCR was performed (*P<0.01). (c) Cell-surface FasL expression on keratinocytes by FACS analysis of HaCaT cells treated or not treated (Cont) with NO donors (SNP and NOC18). Results from one of four representative experiments. SNP- and NOC18-induced keratinocyte apoptosis detected by Annexin-FITC labeling (d) was completely inhibited by ZVAD and Fas:Fc. Results from one of four representative experiments. (e) Percentage of apoptotic keratinocytes (early plus late) in HaCaT cells treated or not treated (blank histogram) with ZVAD (black histogram) or Fas:Fc (gray histogram) in addition of NO donors. Data shown are mean±standard deviation, n=3. Journal of Investigative Dermatology 2013 133, 489-498DOI: (10.1038/jid.2012.330) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Nitric oxide (NO) donors induce FasL expression and apoptosis in keratinocytes of EpiDerm. (a) Quantification of apoptotic keratinocytes counted per cm of EpiDerm pretreated with different apoptotic inhibitors and then treated (+NO) or not treated (-NO) with NOC18. Apoptotic keratinocytes are detected by active caspase-3 staining. Results are mean±standard deviation, n=5–9. *P<0.05 and **P<0.01: we compared each result with control/+NO and, in addition, Z-IETD-FMK/+NO with its own control Z-FA-FMK/+NO. (b) Representative slides used for counting active caspase-3-stained keratinocytes in panel (a). Journal of Investigative Dermatology 2013 133, 489-498DOI: (10.1038/jid.2012.330) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cytokines induce inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide (NO) production in human interfollicular epidermal keratinocyte (HEK) cells. (a) iNOS and FasL mRNA expression in keratinocytes treated with different combinations of IFN-γ, tumor necrosis factor-α (TNF-α), and IL-1β. (b) iNOS and FasL expression in keratinocytes was inhibited by L-NMMA treatment, *P<0.005. (c) NO production in cytokine-treated HEK cells relative to untreated cells detected by DAF–FM–DA fluorescence (mean±standard deviation (SD)), n=5. *P<0.005 and **P<0.001 compared with untreated cells. ◊P<0.001, IFN-γ versus TNF-α. •P<0.001, IFN-γ plus TNF-α, and IFN-γ plus IL-1β versus IFN-γ. ▴P<0.001, IFN-γ plus TNF-α versus the three cytokines. (d) NO production in HEK cells upon exposure to IFN-γ and TNF-α. Left panel shows the DAF–FM–DA fluorescence in HEK cells treated with IFN-γ plus TNF-α (bold line) and untreated cells (thin line). One representative experiment of four. Right panel shows L-NMMA inhibition of cytokine-induced NO production in HEK cells relative to untreated cells as detected by DAF–FM–DA NO dye fluorescence (mean±SD, n=5–8). *P<0.001 compared with untreated control cells; ◊P<0.001 compared with L-NMMA-treated cells. L-NMMA, L-NG-monomethyl-arginine citrate. Journal of Investigative Dermatology 2013 133, 489-498DOI: (10.1038/jid.2012.330) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Conditioned medium of T cells induce inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide (NO) production that is dependent on IFN-γ and tumor necrosis factor-α (TNF-α). (a) iNOS RT–PCR analysis of keratinocytes treated with medium of CD3-activated (+) or nonactivated (-) peripheral blood mononuclear cells (PBMCs; top). iNOS versus β-actin PCR products are indicated (bottom). (b) DAF–FM–DA fluorescence in human interfollicular epidermal keratinocyte (HEK) cells treated with the medium of CD3-activated (bold line) or nonactivated PBMCs (thin line). One representative experiment. Right panel: L-NMMA inhibition of NO production induced in HEK cells incubated with CD3-activated (+) relative to nonactivated (-) PBMC-conditioned medium. NO production is detected by DAF–FM–DA fluorescence (mean±SD); n=4–10 (+CD3 vs. -CD3, •P<0.001; +CD3 vs. +CD3+L-NMMA, ▴P<0.001). (c) Representative FACS analysis of DAF–FM–DA fluorescence in HEK cells exposed to conditioned medium of a phenytoin-activated T-cell clone (bold line) compared with conditioned medium of the same T-cell clone not activated by phenytoin (thin line) (left panel). NO production induced in HEK cells incubated with phenytoin-activated T-cell clone–conditioned medium relative to nonactivated T-cell clone–conditioned medium. NO production was determined by DAF–FM–DA fluorescence (mean±standard deviation); n=10 different T-cell clones from the same patient with phenytoin-induced toxic epidermal necrolysis (TEN; right panel, *P<0.01). L-NMMA, L-NG--monomethyl-arginine citrate. Journal of Investigative Dermatology 2013 133, 489-498DOI: (10.1038/jid.2012.330) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Conditioned medium of T cells induce inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, FasL, and subsequent apoptosis in an IFN-γ- and tumor necrosis factor-α (TNF-α)-dependent manner. (a) Quantitative reverse transcriptase (RT)–PCR analysis of FasL and iNOS in human interfollicular epidermal keratinocyte (HEK) cells exposed to the supernatant from activated peripheral blood mononuclear cells (PBMCs) with or without specific IFN-γ and TNF-α blockers (mean±standard deviation (SD) of three independent experiments, **P<0.001). (b) Specific blockade of iNOS mRNA induction and NO production by HEK cells with neutralizing IFN-γ antibodies and TNF-receptor:Fc. (mean±SD, n=3). (c) Viability (annexin-V−/PI− cells) of HEK cells exposed to conditioned medium from activated PBMCs in the presence or absence of specific IFN-γ and TNF-α blockers (mean±SD, n=3, *P<0.01). For all these experiments control isotypes and ectodysplasin A receptor (EDAR):Fc were used as irrelevant controls. Journal of Investigative Dermatology 2013 133, 489-498DOI: (10.1038/jid.2012.330) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions