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Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis

Nature Medicine volume 14, pages 1343–1350 (2008)Cite this article

Abstract

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse drug reactions characterized by massive epidermal necrosis, in which the specific danger signals involved remain unclear. Here we show that blister cells from skin lesions of SJS-TEN primarily consist of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and both blister fluids and cells were cytotoxic. Gene expression profiling identified granulysin as the most highly expressed cytotoxic molecule, confirmed by quantitative PCR and immunohistochemistry. Granulysin concentrations in the blister fluids were two to four orders of magnitude higher than perforin, granzyme B or soluble Fas ligand concentrations, and depleting granulysin reduced the cytotoxicity. Granulysin in the blister fluids was a 15-kDa secretory form, and injection of it into mouse skin resulted in features mimicking SJS-TEN. Our findings demonstrate that secretory granulysin is a key molecule responsible for the disseminated keratinocyte death in SJS-TEN and highlight a mechanism for CTL- or NK cell—mediated cytotoxicity that does not require direct cellular contact.

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Figure 1: Cytotoxicity of blister fluids and blister cells of subjects with SJS-TEN.
Figure 2: Gene expression analyses of blister cells (BCs) and PBMCs of subjects with SJS-TEN.
Figure 3: Immunohistochemistry staining of cytotoxic proteins in skin biopsies.
Figure 4: Analyses of cytotoxic proteins in the blister fluids.
Figure 5: In vitro cytotoxicity of granulysin, perforin, granzyme B and sFasL against keratinocytes (KERTr cells).
Figure 6: Intradermal injection of 15-kDa granulysin in mice.

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Acknowledgements

DH4 monoclonal antibody was a gift from A.M. Krensky (Stanford University). We thank H.-J. Lin, S.-J. Li, K.-H. Chen, L.-H. Lu of the Institute of Biomedical Sciences, Academia Sinica and members of the National Microarray & Gene Expression Analysis Core Facility, National Yang-Ming University, Taiwan, for excellent technical assistance. This work was supported by grants from the National Science Council, Taiwan (NSC95-2314-B-182A-048, 95-2314-B-010-096, 96-2320-B-010-021-MY2 and 96-2628-B-182A-065-MY2), Chang-Gung Memorial Hospital, Taiwan Ministry of Education (Aim for the Top University Plan, National Yang-Ming University), National Research Program for Genomic Medicine, Taiwan (National Clinical Core, NSC-95-3112-B-001-010 and National Genotyping Core, NSC-95-3112-B-001-011) and the Genomics and Proteomics Program, Academia Sinica.

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Author notes

  1. Wen-Hung Chung and Shuen-Iu Hung: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Dermatology, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, 199 Tunghwa North Road, Taipei, 105, Taiwan

    Wen-Hung Chung, Chien-Chun Chiou, Hsin-Chun Ho & Chih-Hsun Yang

  2. Taiwan International Graduate Program, Molecular Medicine Program, Institute of Biomedical Sciences, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan

    Wen-Hung Chung, Shuen-Iu Hung, Shih-Chi Su, Shien-Ping Huang, Chun-Yu Wei, Jer-Yuarn Wu, You-Di Liao & Yuan-Tsong Chen

  3. Institute of Biochemistry and Molecular Biology, School of Life Sciences, 155 Section 2, Linong Street, Taipei, 112, Taiwan

    Wen-Hung Chung

  4. Institute of Pharmacology, School of Medicine, National Yang-Ming University, 155 Section 2, Linong Street, Taipei, 112, Taiwan

    Shuen-Iu Hung & See-Wen Chin

  5. Linkou Burn Center & Department of Plastic Surgery, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, 199 Tunghwa North Road, Taipei, 105, Taiwan

    Jui-Yung Yang

  6. Division of Oncology and Hematology, Buddhist Tzu Chi General Hospital, 707 Section 3, Chungyang Road, Hualien, 971, Taiwan

    Sung-Chao Chu

  7. Department of Dermatology, Chung Shan Hospital, 11, Lane 112, Section 4, Jenai Road, Taipei, 105, Taiwan

    Chi-Fang Lu

  8. Department of Pediatrics, Duke University Medical Center, Box 103856, 595 LaSalle Street, Durham, 27710, North Carolina, USA

    Yuan-Tsong Chen

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Contributions

W.-H.C. and S.-I.H. designed and conducted the experiments and data analysis and wrote the manuscript. J.-Y.Y., C.-C.C., S.-C.C., H.-C.H., C.-H.Y. and C.-F.L. cared for the involved human subjects and provided clinical samples. S.-C.S., S.-P.H., C.-Y.W. and S.-W.C. performed experiments. J.-Y.W. and Y.-D.L. helped design the expression of protein. Y.-T.C. supervised the entire project and wrote the manuscript.

Corresponding author

Correspondence to Yuan-Tsong Chen.

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Competing interests

W.-H.C., S.-I.H. amd Y.-T.C. have filed a patent application to the Taiwan and US patent offices on granulysin and uses thereof.

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Supplementary Figs. 1 and 2, Supplementary Methods, Supplementary References and Supplementary Tables 1–3 (PDF 635 kb)

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Chung, WH., Hung, SI., Yang, JY. et al. Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis. Nat Med 14, 1343–1350 (2008). https://doi.org/10.1038/nm.1884

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