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Koga S, Takazono T, Namie H, Okuno D, Ito Y, Nakada N, Hirayama T, Takeda K, Ide S, Iwanaga N, Tashiro M, Sakamoto N, Watanabe A, Izumikawa K, Yanagihara K, Tanaka Y, Mukae H. Human Vγ9Vδ2 T cells exhibit antifungal activity against Aspergillus fumigatus and other filamentous fungi. Microbiol Spectr 2024; 12:e0361423. [PMID: 38426765 PMCID: PMC10986472 DOI: 10.1128/spectrum.03614-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Accepted: 02/11/2024] [Indexed: 03/02/2024] Open
Abstract
Invasive aspergillosis (IA) and mucormycosis are life-threatening diseases, especially among immunocompromised patients. Drug-resistant Aspergillus fumigatus strains have been isolated worldwide, which can pose a serious clinical problem. As IA mainly occurs in patients with compromised immune systems, the ideal therapeutic approach should aim to bolster the immune system. In this study, we focused on Vγ9Vδ2 T cells that exhibit immune effector functions and examined the possibility of harnessing this unconventional T cell subset as a novel therapeutic modality for IA. A potent antifungal effect was observed when A. fumigatus (Af293) hyphae were challenged by Vγ9Vδ2 T cells derived from peripheral blood. In addition, Vγ9Vδ2 T cells exhibited antifungal activity against hyphae of all Aspergillus spp., Cunninghamella bertholletiae, and Rhizopus microsporus but not against their conidia. Furthermore, Vγ9Vδ2 T cells also exhibited antifungal activity against azole-resistant A. fumigatus, indicating that Vγ9Vδ2 T cells could be used for treating drug-resistant A. fumigatus. The antifungal activity of Vγ9Vδ2 T cells depended on cell-to-cell contact with A. fumigatus hyphae, and degranulation characterized by CD107a mobilization seems essential for this activity against A. fumigatus. Vγ9Vδ2 T cells could be developed as a novel modality for treating IA or mucormycosis. IMPORTANCE Invasive aspergillosis (IA) and mucormycosis are often resistant to treatment with conventional antifungal agents and have a high mortality rate. Additionally, effective antifungal treatment is hindered by drug toxicity, given that both fungal and human cells are eukaryotic, and antifungal agents are also likely to act on human cells, resulting in adverse effects. Therefore, the development of novel therapeutic agents specifically targeting fungi is challenging. This study demonstrated the antifungal activity of Vγ9Vδ2 T cells against various Aspergillus spp. and several Mucorales in vitro and discussed the mechanism underlying their antifungal activity. We indicate that adoptive immunotherapy using Vγ9Vδ2 T cells may offer a new therapeutic approach to IA.
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Affiliation(s)
- Satoru Koga
- Department of Respiratory Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
| | - Takahiro Takazono
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
- Department of Infectious Diseases, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Hodaka Namie
- Department of Infectious Diseases, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Daisuke Okuno
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
| | - Yuya Ito
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
| | - Nana Nakada
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
- Health Center, Nagasaki University, Nagasaki, Japan
| | - Tatsuro Hirayama
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
- Department of Pharmacotherapeutics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Kazuaki Takeda
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
| | - Shotaro Ide
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
- Infectious Diseases Experts Training Center, Nagasaki University Hospital, Nagasaki, Japan
| | - Naoki Iwanaga
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
| | - Masato Tashiro
- Department of Infectious Diseases, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Noriho Sakamoto
- Department of Respiratory Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
| | - Akira Watanabe
- Medical Mycology Research Center, Chiba University, Chiba, Japan
| | - Koichi Izumikawa
- Department of Infectious Diseases, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Katsunori Yanagihara
- Department of Laboratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
| | - Yoshimasa Tanaka
- Center for Medical Innovation, Nagasaki University, Nagasaki, Japan
| | - Hiroshi Mukae
- Department of Respiratory Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
- Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan
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Use of the Human Granulysin Transgenic Mice To Evaluate the Role of Granulysin Expression by CD8 T Cells in Immunity To Mycobacterium tuberculosis. mBio 2022; 13:e0302022. [PMID: 36409085 PMCID: PMC9765553 DOI: 10.1128/mbio.03020-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
The cytotoxic granules of human NK and CD8 T cells contain the effector molecule granulysin. Although in vitro studies indicate that granulysin is bactericidal to Mycobacterium tuberculosis and human CD8 T cells restrict intracellular M. tuberculosis by granule exocytosis, the role of granulysin in cell-mediated immunity against infection is incompletely understood, in part because a granulysin gene ortholog is absent in mice. Transgenic mice that express human granulysin (GNLY-Tg) under the control of human regulatory DNA sequences permit the study of granulysin in vivo. We assessed whether granulysin expression by murine CD8 T cells enhances their control of M. tuberculosis infection. GNLY-Tg mice did not control pulmonary M. tuberculosis infection better than non-Tg control mice, and purified GNLY-Tg and non-Tg CD8 T cells had a similar ability to transfer protection to T cell deficient mice. Lung CD8 T cells from infected control and GNLY-transgenic mice similarly controlled intracellular M. tuberculosis growth in macrophages in vitro. Importantly, after M. tuberculosis infection of GNLY-Tg mice, granulysin was detected in NK cells but not in CD8 T cells. Only after prolonged in vitro stimulation could granulysin expression be detected in antigen-specific CD8 T cells. GNLY-Tg mice are an imperfect model to determine whether granulysin expression by CD8 T cells enhances immunity against M. tuberculosis. Better models expressing granulysin are needed to explore the role of this antimicrobial effector molecule in vivo. IMPORTANCE Human CD8 T cells express the antimicrobial peptide granulysin in their cytotoxic granules, and in vitro analysis suggest that it restricts growth of Mycobacterium tuberculosis and other intracellular pathogens. The murine model of tuberculosis cannot assess granulysin's role in vivo, as rodents lack the granulysin gene. A long-held hypothesis is that murine CD8 T cells inefficiently control M. tuberculosis infection because they lack granulysin. We used human granulysin transgenic (GNLY-Tg) mice to test this hypothesis. GNLY-Tg mice did not differ in their susceptibility to tuberculosis. However, granulysin expression by pulmonary CD8 T cells could not be detected after M. tuberculosis infection. As the pattern of granulysin expression in human CD8 T cells and GNLY-Tg mice seem to differ, GNLY-Tg mice are an imperfect model to study the role of granulysin. An improved model is needed to answer the importance of granulysin expression by CD8 T cells in different diseases.
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Iweha C, Graham A, Cui W, Marsh C, Nothnick WB. The uterine natural killer cell, cytotoxic T lymphocyte, and granulysin levels are elevated in the endometrium of women with nonstructural abnormal uterine bleeding. F&S SCIENCE 2022; 3:246-254. [PMID: 35654738 DOI: 10.1016/j.xfss.2022.04.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Revised: 03/30/2022] [Accepted: 04/14/2022] [Indexed: 06/15/2023]
Abstract
OBJECTIVE To examine the expression of uterine natural killer (uNK) cells and cytotoxic T lymphocytes (CTLs) in endometrial biopsies from reproductive-age women with and without nonstructural abnormal uterine bleeding (AUB) and evaluate the expression of granulysin within these cell populations and potential modulation of matrix metalloproteinase (MMP) expression. DESIGN Experimental study, retrospective design. SETTING Academic research laboratory. PATIENT(S) Patients with nonstructural AUB with no other gynecological pathologies and control patients without AUB. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Immunohistochemical analysis of granulysin, CD56 (uNK cell marker), and CD8 (CTL marker) expression as well as granulysin messenger ribonucleic acid (mRNA) expression levels in endometrial biopsy samples. Assessment of granulysin regulation of human endometrial stromal cell MMP-1 and MMP-3 mRNA expression. RESULT(S) The numbers of uNK cells and CTLs were significantly greater in endometrial biopsy tissue from women with AUB than those from controls. In accord with the increased expression of uNK cells and CTLs, granulysin expression was significantly greater in endometrial biopsies from patients with AUB than in from controls and colocalized to both cell types but not endometrial stromal or epithelial cells. The increased granulysin protein expression was associated with the increased granulysin mRNA expression in adjacent serial sections from these same samples. The treatment of the human endometrial stromal cell line t-HESC with granulysin resulted in a significant increase in MMP-1 and MMP-3 mRNA expression. CONCLUSION(S) In the current study, immunohistochemistry showed an increased expression of uNK cells, CTLs, and granulysin among subjects with AUB compared with that of subjects without AUB, leading to conclusions that disturbances in the balance of immune cells and an increase in granulysin expression may have implications in the pathophysiology of AUB and include enhanced MMP-1 and MMP-3 expression.
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Affiliation(s)
- Chidinma Iweha
- Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas
| | - Amanda Graham
- Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas
| | - Wei Cui
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas; Institute for Reproduction and Perinatal Research, Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, Kansas
| | - Courtney Marsh
- Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas; Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas; Institute for Reproduction and Perinatal Research, Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, Kansas
| | - Warren B Nothnick
- Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas; Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas; Institute for Reproduction and Perinatal Research, Center for Reproductive Sciences, University of Kansas Medical Center, Kansas City, Kansas.
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Ham H, Medlyn M, Billadeau DD. Locked and Loaded: Mechanisms Regulating Natural Killer Cell Lytic Granule Biogenesis and Release. Front Immunol 2022; 13:871106. [PMID: 35558071 PMCID: PMC9088006 DOI: 10.3389/fimmu.2022.871106] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 03/29/2022] [Indexed: 11/22/2022] Open
Abstract
NK cell-mediated cytotoxicity is a critical element of our immune system required for protection from microbial infections and cancer. NK cells bind to and eliminate infected or cancerous cells via direct secretion of cytotoxic molecules toward the bound target cells. In this review, we summarize the current understanding of the molecular regulations of NK cell cytotoxicity, focusing on lytic granule development and degranulation processes. NK cells synthesize apoptosis-inducing proteins and package them into specialized organelles known as lytic granules (LGs). Upon activation of NK cells, LGs converge with the microtubule organizing center through dynein-dependent movement along microtubules, ultimately polarizing to the cytotoxic synapse where they subsequently fuse with the NK plasma membrane. From LGs biogenesis to degranulation, NK cells utilize several strategies to protect themselves from their own cytotoxic molecules. Additionally, molecular pathways that enable NK cells to perform serial killing are beginning to be elucidated. These advances in the understanding of the molecular pathways behind NK cell cytotoxicity will be important to not only improve current NK cell-based anti-cancer therapies but also to support the discovery of additional therapeutic opportunities.
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Affiliation(s)
- Hyoungjun Ham
- Division of Oncology Research, Mayo Clinic, Rochester, MN, United States
| | - Michael Medlyn
- Department of Immunology College of Medicine, Mayo Clinic, Rochester, MN, United States
| | - Daniel D Billadeau
- Division of Oncology Research, Mayo Clinic, Rochester, MN, United States.,Department of Immunology College of Medicine, Mayo Clinic, Rochester, MN, United States
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5
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Lettau M, Janssen O. Intra- and Extracellular Effector Vesicles From Human T And NK Cells: Same-Same, but Different? Front Immunol 2022; 12:804895. [PMID: 35003134 PMCID: PMC8733945 DOI: 10.3389/fimmu.2021.804895] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2021] [Accepted: 12/07/2021] [Indexed: 12/13/2022] Open
Abstract
Cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells utilize an overlapping effector arsenal for the elimination of target cells. It was initially proposed that all cytotoxic effector proteins are stored in lysosome-related effector vesicles (LREV) termed "secretory lysosomes" as a common storage compartment and are only released into the immunological synapse formed between the effector and target cell. The analysis of enriched LREV, however, revealed an uneven distribution of individual effectors in morphologically distinct vesicular entities. Two major populations of LREV were distinguished based on their protein content and signal requirements for degranulation. Light vesicles carrying FasL and 15 kDa granulysin are released in a PKC-dependent and Ca2+-independent manner, whereas dense granules containing perforin, granzymes and 9 kDa granulysin require Ca2+-signaling as a hallmark of classical degranulation. Notably, both types of LREV do not only contain the mentioned cytolytic effectors, but also store and transport diverse other immunomodulatory proteins including MHC class I and II, costimulatory and adhesion molecules, enzymes (i.e. CD26/DPP4) or cytokines. Interestingly, the recent analyses of CTL- or NK cell-derived extracellular vesicles (EV) revealed the presence of a related mixture of proteins in microvesicles or exosomes that in fact resemble fingerprints of the cells of origin. This overlapping protein profile indicates a direct relation of intra- and extracellular vesicles. Since EV potentially also interact with cells at distant sites (apart from the IS), they might act as additional effector vesicles or intercellular communicators in a more systemic fashion.
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Affiliation(s)
- Marcus Lettau
- Molecular Immunology, Institute of Immunology, University Hospital Schleswig-Holstein, Kiel, Germany.,Department of Internal Medicine II, Unit for Hematological Diagnostics, University Hospital Schleswig-Holstein, Kiel, Germany
| | - Ottmar Janssen
- Molecular Immunology, Institute of Immunology, University Hospital Schleswig-Holstein, Kiel, Germany
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6
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Sparrow EL, Fowler DW, Fenn J, Caron J, Copier J, Dalgleish AG, Bodman-Smith MD. The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for Vδ2 + γδ T cells in the modulation of immune response to tumour? Immunology 2020; 161:245-258. [PMID: 32794189 PMCID: PMC7576882 DOI: 10.1111/imm.13248] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2019] [Revised: 07/09/2020] [Accepted: 07/23/2020] [Indexed: 12/12/2022] Open
Abstract
Release of granulysin by γδ T cells contributes to tumour cell killing. A cytolytic 9000 MW isoform of granulysin kills tumour cells directly, whereas a 15 000 MW precursor has been hypothesized to cause both the maturation and migration of dendritic cell (DC) populations. Recruiting DC to a tumour is beneficial as these cells initiate adaptive immune responses, which contribute to the eradication of malignancies. In this study, Vδ2+ γδ T cells were activated by stimulation of peripheral blood mononuclear cells with zoledronic acid or Bacillus Calmette-Guérin (BCG), or were isolated and cultured with tumour targets. Although a large proportion of resting Vδ2+ γδ T cells expressed 15 000 MW granulysin, 9000 MW granulysin expression was induced only after stimulation with BCG. Increased levels of activation and granulysin secretion were also observed when Vδ2+ γδ T cells were cultured with the human B-cell lymphoma line Daudi. High concentrations of recombinant 15 000 MW granulysin caused migration and maturation of immature DC, and also initiated fugetaxis in mature DC. Conversely, low concentrations of recombinant 15 000 MW granulysin resulted in migration of mature DC, but not immature DC. Our data therefore support the hypothesis that Vδ2+ γδ T cells can release granulysin, which may modulate recruitment of DC, initiating adaptive immune responses.
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Affiliation(s)
- Emma L Sparrow
- Infection and Immunity Research Institute, St. George's University of London SW17 0RE, London, UK.,Antibody and Vaccine Group, Centre of Cancer Immunotherapy, Southampton General Hospital, University of Southampton, Southampton, UK
| | - Daniel W Fowler
- Infection and Immunity Research Institute, St. George's University of London SW17 0RE, London, UK
| | - Joe Fenn
- Infection and Immunity Research Institute, St. George's University of London SW17 0RE, London, UK
| | - Jonathan Caron
- Infection and Immunity Research Institute, St. George's University of London SW17 0RE, London, UK
| | - John Copier
- Infection and Immunity Research Institute, St. George's University of London SW17 0RE, London, UK
| | - Angus G Dalgleish
- Infection and Immunity Research Institute, St. George's University of London SW17 0RE, London, UK
| | - Mark D Bodman-Smith
- Infection and Immunity Research Institute, St. George's University of London SW17 0RE, London, UK
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Li SS, Saleh M, Xiang RF, Ogbomo H, Stack D, Huston SH, Mody CH. Natural killer cells kill Burkholderia cepacia complex via a contact-dependent and cytolytic mechanism. Int Immunol 2020; 31:385-396. [PMID: 31051036 DOI: 10.1093/intimm/dxz016] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Accepted: 04/19/2019] [Indexed: 12/15/2022] Open
Abstract
Burkholderia cepacia complex (Bcc), which includes B. cenocepacia and B. multivorans, pose a life-threatening risk to patients with cystic fibrosis. Eradication of Bcc is difficult due to the high level of intrinsic resistance to antibiotics, and failure of many innate immune cells to control the infection. Because of the pathogenesis of Bcc infections, we wondered if a novel mechanism of microbial host defense involving direct antibacterial activity by natural killer (NK) cells might play a role in the control of Bcc. We demonstrate that NK cells bound Burkholderia, resulting in Src family kinase activation as measured by protein tyrosine phosphorylation, granule release of effector proteins such as perforin and contact-dependent killing of the bacteria. These studies provide a means by which NK cells could play a role in host defense against Bcc infection.
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Affiliation(s)
- Shu Shun Li
- Department of Microbiology, Immunology and Infectious Diseases, Alberta, Canada.,The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Alberta, Canada
| | - Marwah Saleh
- Department of Microbiology, Immunology and Infectious Diseases, Alberta, Canada
| | - Richard F Xiang
- Department of Microbiology, Immunology and Infectious Diseases, Alberta, Canada.,The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Alberta, Canada
| | - Henry Ogbomo
- Department of Microbiology, Immunology and Infectious Diseases, Alberta, Canada.,The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Alberta, Canada
| | - Danuta Stack
- Department of Microbiology, Immunology and Infectious Diseases, Alberta, Canada.,The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Alberta, Canada
| | - Shaunna H Huston
- The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Alberta, Canada
| | - Christopher H Mody
- Department of Microbiology, Immunology and Infectious Diseases, Alberta, Canada.,The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Alberta, Canada.,Department of Medicine, University of Calgary, Calgary, Alberta, Canada
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Kathamuthu GR, Moideen K, Sridhar R, Baskaran D, Subash Babu. Diminished systemic levels of antimicrobial peptides in tuberculous lymphadenitis and their reversal after anti-tuberculosis treatment. Tuberculosis (Edinb) 2020; 122:101934. [PMID: 32275232 DOI: 10.1016/j.tube.2020.101934] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2019] [Revised: 03/09/2020] [Accepted: 03/29/2020] [Indexed: 11/26/2022]
Abstract
Pulmonary tuberculosis is associated with higher plasma levels of antimicrobial peptides (AMPs) and lower granulysin levels. However, the association of AMPs with tuberculous lymphadenitis (TBL) is not well studied. Hence, we measured the plasma levels of human beta defensin-2 (HBD2), granulysin, human neutrophil peptides 1-3 (HNP1-3) and cathelicidin (LL37) in TBL compared to latent tuberculosis (LTB) and healthy controls (HC) and in TBL individuals upon completion of anti-tuberculosis treatment (ATT). We examined the association of AMPs with TBL lymph node culture grade or lymph node involvement. Finally, the discriminatory potential of these proteins was assessed using receiver operating characteristic (ROC) analysis. TBL individuals display significantly diminished circulating levels of AMPs (granulysin and HNP1-3) but not HBD-2 and LL-37 in comparison to LTB and HCs. Similarly, after ATT, both HBD-2 and HNP1-3 were significantly elevated and LL-37 was significantly reduced in TBL individuals. Granulysin and HNP1-3 discriminates TBL from LTB and HC individuals upon ROC analysis. AMPs did not exhibit significant correlation either with lymph node culture grades or lymph node involvement. Overall, TBL individuals show decreased AMPs and their reversal after ATT suggesting their association with underlying immune alteration in this poorly studied form of TB disease.
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Affiliation(s)
- Gokul Raj Kathamuthu
- National Institutes of Health-NIRT-International Center for Excellence in Research, Chennai, India; National Institute for Research in Tuberculosis (NIRT), Chennai, India.
| | - Kadar Moideen
- National Institutes of Health-NIRT-International Center for Excellence in Research, Chennai, India
| | | | - Dhanaraj Baskaran
- National Institute for Research in Tuberculosis (NIRT), Chennai, India
| | - Subash Babu
- National Institutes of Health-NIRT-International Center for Excellence in Research, Chennai, India; Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
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9
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Sparrow E, Bodman-Smith M. Granulysin: The attractive side of a natural born killer. Immunol Lett 2020; 217:126-132. [DOI: 10.1016/j.imlet.2019.11.005] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Revised: 10/29/2019] [Accepted: 11/10/2019] [Indexed: 12/16/2022]
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10
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Mutant GNLY is linked to Stevens-Johnson syndrome and toxic epidermal necrolysis. Hum Genet 2019; 138:1267-1274. [PMID: 31642954 DOI: 10.1007/s00439-019-02066-w] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2019] [Accepted: 09/25/2019] [Indexed: 12/19/2022]
Abstract
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe cutaneous adverse reactions to drugs. Granulysin (GNLY) plays a key role in keratinocyte apoptosis during SJS/TEN pathophysiology. To determine if GNLY-encoding mutations might be related to the protein's functional disturbances, contributing to SJS/TEN pathogenesis, we performed direct sequencing of GNLY's coding region in a group of 19 Colombian SJS/TEN patients. A GNLY genetic screening was implemented in a group of 249 healthy individuals. We identified the c.11G > A heterozygous sequence variant in a TEN case, which creates a premature termination codon (PTC) (p.Trp4Ter). We show that a mutant protein is synthesised, possibly due to a PTC-readthrough mechanism. Functional assays demonstrated that the mutant protein was abnormally located in the nuclear compartment, potentially leading to a toxic effect. Our results argue in favour of GNLY non-synonymous sequence variants contributing to SJS/TEN pathophysiology, thereby constituting a promising, clinically useful molecular biomarker.
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Prager I, Watzl C. Mechanisms of natural killer cell-mediated cellular cytotoxicity. J Leukoc Biol 2019; 105:1319-1329. [PMID: 31107565 DOI: 10.1002/jlb.mr0718-269r] [Citation(s) in RCA: 364] [Impact Index Per Article: 60.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2019] [Revised: 03/22/2019] [Accepted: 04/14/2019] [Indexed: 12/29/2022] Open
Abstract
Cellular cytotoxicity, the ability to kill other cells, is an important effector mechanism of the immune system to combat viral infections and cancer. Cytotoxic T cells and natural killer (NK) cells are the major mediators of this activity. Here, we summarize the cytotoxic mechanisms of NK cells. NK cells can kill virally infected of transformed cells via the directed release of lytic granules or by inducing death receptor-mediated apoptosis via the expression of Fas ligand or TRAIL. The biogenesis of perforin and granzymes, the major components of lytic granules, is a highly regulated process to prevent damage during the synthesis of these cytotoxic molecules. Additionally, NK cells have developed several strategies to protect themselves from the cytotoxic activity of granular content upon degranulation. While granule-mediated apoptosis is a fast process, death receptor-mediated cytotoxicity requires more time. Current data suggest that these 2 cytotoxic mechanisms are regulated during the serial killing activity of NK cells. As many modern approaches of cancer immunotherapy rely on cellular cytotoxicity for their effectiveness, unraveling these pathways will be important to further progress these therapeutic strategies.
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Affiliation(s)
- Isabel Prager
- Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo), Dortmund, Germany
| | - Carsten Watzl
- Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo), Dortmund, Germany
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12
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Lettau M, Dietz M, Dohmen K, Leippe M, Kabelitz D, Janssen O. Granulysin species segregate to different lysosome-related effector vesicles (LREV) and get mobilized by either classical or non-classical degranulation. Mol Immunol 2019; 107:44-53. [DOI: 10.1016/j.molimm.2018.12.031] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2018] [Revised: 12/12/2018] [Accepted: 12/29/2018] [Indexed: 01/06/2023]
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Granulysin as a novel factor for the prognosis of the clinical course of chickenpox. Epidemiol Infect 2018; 146:854-857. [PMID: 29633679 DOI: 10.1017/s0950268818000717] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
Abstract
Granulysin is a recently discovered cytolytic protein of natural killer (NK) cells and cytotoxic T lymphocytes. Studies of healthy and immunocompromised patients with primary or recurrent varicella-zoster infections demonstrate the importance of virus-specific cellular immunity in controlling viral replication, but also some studies presented granulysin as a molecule that can play a role in chickenpox immunopathogenesis. This study investigated possible correlation between serum granulysin levels and clinical course of chickenpox. A total of 69 patients with chickenpox were included in the study. We measured the levels of granulysin and percentage count for CD4+, CD8+ and NK cells in serum for all patients and healthy controls. For detection and quantification of granulysin in sera, we performed ELISA test and flow cytometry for detection, identification and percentage measurement of T and B lymphocytes. Descriptive methods, analysis of variance and multivariate logistic regression were used for statistical data analysis. We found respective correlation between serum granulysin level and severity of clinical presentation. These findings can be a good input for further studies, since there is no relevant prognostic parameter of chickenpox in everyday clinical practice. Granulysin, as a therapeutic, also deserves to be a point of interests in the future. If we prove its potential to stop dissemination of human herpes viruses, possibilities to use it in some life-threatening forms of viral disease can be very valuable.
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Abstract
It is well known that natural killer (NK) cells are involved in defense against viruses and some tumors. NK cells kill target cells by the directed release of cytolytic granules that contain perforin, granzymes, and granulysin. It is increasingly important to evaluate NK cell function in immunotoxicity testing. NK cell function can be evaluated by determining cytolytic activity against target tumor cells by the 51Cr-release assay and also by determining the number of NK cells in peripheral blood in humans and in the spleen in animals using flow cytometry. Recently, the intracellular levels of perforin, granzymes, and granulysin determined by flow cytometry have also been used in the evaluation of NK cell function. In addition, chemical-induced apoptosis in NK cells also has been applied to evaluate the immunotoxicity of chemicals. This chapter will describe the methods for NK cell assays in immunotoxicity testing.
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15
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Expression of KIR2DS1 by decidual natural killer cells increases their ability to control placental HCMV infection. Proc Natl Acad Sci U S A 2016; 113:15072-15077. [PMID: 27956621 DOI: 10.1073/pnas.1617927114] [Citation(s) in RCA: 65] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The combination of the activating killer cell Ig-like receptor 2DS1 (KIR2DS1) expressed by maternal decidual natural killer cells (dNK) and the presence of its ligand, the HLA-C allotype HLA-C2, expressed by fetal trophoblasts, reduces the risk of developing pregnancy complications. However, no molecular or cellular mechanism explains this genetic correlation. Here we demonstrate that KIR2DS1+ dNK acquired higher cytotoxic function than KIR2DS1- dNK when exposed to human cytomegalovirus (HCMV)-infected decidual stromal cells (DSC), particularly when DSCs express HLA-C2. Furthermore, dNK were unable to degranulate or secrete cytokines in response to HCMV-infected primary fetal extravillous trophoblasts. This emphasizes the immunological challenge to clear placental viral infections within the immune-privileged placenta. Activation of dNK through KIR2DS1/HLA-C2 interaction increases their ability to respond to placental HCMV infection and may limit subsequent virus-induced placental pathology. This mechanism is directly related to how KIR2DS1 expressed by dNK reduces development of severe pregnancy complications such as miscarriages and preterm delivery.
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16
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Hsiao YW, Lai TC, Lin YH, Su CY, Lee JJ, Liao AT, Lin YF, Hsieh SC, Wu ATH, Hsiao M. Granulysin expressed in a humanized mouse model induces apoptotic cell death and suppresses tumorigenicity. Oncotarget 2016; 8:83495-83508. [PMID: 29137359 PMCID: PMC5663531 DOI: 10.18632/oncotarget.11473] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2016] [Accepted: 08/09/2016] [Indexed: 12/21/2022] Open
Abstract
Granulysin (GNLY) is a cytolytic and proinflammatory protein expressed in activated human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Conventional mouse models cannot adequately address the triggering mechanism and immunopathological pathways in GNLY-associated diseases due to lack of the GNLY gene in the mouse genome. Therefore, we generated a humanized immune system (HIS) mouse model by transplanting human umbilical cord blood mononuclear cells into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice after sublethally irradiation. We examined the GNLY expression and its effects on tumor growth using this system. Our HIS mice expressed human CD45+, CD4+, CD8+ and CD56+ cells in the peripheral blood and spleen. A high expression level of human Th1/Th2 and NK cytokines was detected, indicating the activation of both T and NK cells. Importantly, we found an elevated level of GNLY in the serum and it was produced by human CTLs and NK cells obtained from the peripheral blood mononuclear cells and spleen cells in the HIS mice. The serum level of GNLY was negatively correlated with the proliferation of transplanted tumor cells in HIS mice. Collectively, our findings strongly supported that HIS mouse as a valuable model for studying human cancer under an intact immune system and the role of GNLY in tumorigenesis.
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Affiliation(s)
- Ya-Wen Hsiao
- Genomics Research Center, Academia Sinica, Taipei, Taiwan
| | | | - Yu-Hsiang Lin
- Department of Obstetrics and Gynecology, Chung Shan Medical University Hospital, Taichung, Taiwan
| | - Chia-Yi Su
- Genomics Research Center, Academia Sinica, Taipei, Taiwan
| | - Jih-Jong Lee
- School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan
| | | | - Yuan-Feng Lin
- Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Shu-Chen Hsieh
- Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan
| | - Alexander T H Wu
- Ph.D. Program for Translational Medicine, College of Medical Sciences and Technology, Taipei Medical University, Taipei, Taiwan
| | - Michael Hsiao
- Genomics Research Center, Academia Sinica, Taipei, Taiwan.,Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
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17
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Dominovic M, Laskarin G, Glavan Gacanin L, Haller H, Rukavina D. Colocalization of Granulysin Protein Forms with Perforin and LAMP-1 in Decidual Lymphocytes During Early Pregnancy. Am J Reprod Immunol 2016; 75:619-30. [PMID: 26972359 DOI: 10.1111/aji.12503] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2015] [Accepted: 02/02/2016] [Indexed: 12/11/2022] Open
Abstract
PROBLEM Granulysin (GNLY) occurs in two forms, which have molecular weights of 9 and 15 kDa. We analyzed the cytotoxic potential of decidual lymphocytes (DLs) and peripheral blood lymphocytes (PBLs) based on the forms of GNLY that colocalizes with perforin (PER) and LAMP-1 following activation. METHODS The forms of GNLY were detected by using confocal microscopy. We investigated the colocalization with PER and LAMP-1 in freshly isolated and activated DLs and PBLs. RESULTS Activation of DLs and PBLs by K-562 cells increased the colocalization of 9 kDa GNLY with PER and LAMP-1. K-562 cells transfected with HLA-C decreased 9 kDa GNLY colocalization with PER in DLs only. IL-15 in DLs decreased 9 kDa GNLY and LAMP-1 colocalization, but increased both 15 kDa GNLY and LAMP-1, and PER and LAMP-1 colocalization. CONCLUSION Activated DLs and PBLs show greater cytotoxic potential based on increased colocalization of 9 kDa GNLY and PER. HLA-C and IL-15 affect DLs, indicating their role in maintaining the pregnancy tolerance.
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Affiliation(s)
- Marin Dominovic
- Department of Physiology and Immunology, Medical Faculty, University of Rijeka, Rijeka, Croatia
| | - Gordana Laskarin
- Department of Physiology and Immunology, Medical Faculty, University of Rijeka, Rijeka, Croatia
- Division of Cardiology, Hospital for Medical Rehabilitation of the Hearth and Lung Diseases and Rheumatism "Thalassotherapia" Opatija, Opatija, Croatia
| | - Lana Glavan Gacanin
- Department of Obstetrics and Gynecology, Clinical Hospital Center Rijeka, Rijeka, Croatia
| | - Herman Haller
- Department of Obstetrics and Gynecology, Clinical Hospital Center Rijeka, Rijeka, Croatia
| | - Daniel Rukavina
- Department of Physiology and Immunology, Medical Faculty, University of Rijeka, Rijeka, Croatia
- Department of Clinical and Transplantation Immunology and Molecular Medicine in Rijeka, Croatian Academy of Sciences and Arts, Rijeka, Croatia
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18
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Martinez-Lostao L, de Miguel D, Al-Wasaby S, Gallego-Lleyda A, Anel A. Death ligands and granulysin: mechanisms of tumor cell death induction and therapeutic opportunities. Immunotherapy 2015; 7:883-2. [PMID: 26314314 DOI: 10.2217/imt.15.56] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
The immune system plays a key role in cancer immune surveillance to control tumor development. The final goal is recognizing and killing transformed cells and consequently the elimination of the tumor. The main effector cell types exerting cytotoxicity against tumors are natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although the mechanism of activation of NK cells and CTLs are quite different, both cell types share common antitumor effector mechanisms of cytotoxicity which lead to induction of cell death of tumor cells by apoptosis. Among these mechanisms are the death ligand- and granulysin-mediated cell deaths. In this review, we summarize the main concepts of these effector cytotoxic mechanisms against cancer cells, how NK cells and CTLs use them to control tumor development and the therapeutic approaches currently developed based on these molecules.
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Affiliation(s)
- Luis Martinez-Lostao
- Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, C/Pedro Cerbuna 12, Zaragoza, 50009, Spain.,Instituto de Nanociencia de Aragón, Zaragoza Spain
| | - Diego de Miguel
- Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, C/Pedro Cerbuna 12, Zaragoza, 50009, Spain
| | - Sameer Al-Wasaby
- Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, C/Pedro Cerbuna 12, Zaragoza, 50009, Spain
| | - Ana Gallego-Lleyda
- Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, C/Pedro Cerbuna 12, Zaragoza, 50009, Spain
| | - Alberto Anel
- Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, C/Pedro Cerbuna 12, Zaragoza, 50009, Spain
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19
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Lim HS, Chun SM, Soung MG, Kim J, Kim SJ. Antimicrobial efficacy of granulysin-derived synthetic peptides in acne vulgaris. Int J Dermatol 2015; 54:853-62. [PMID: 25601314 DOI: 10.1111/ijd.12756] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/09/2013] [Revised: 04/30/2014] [Accepted: 05/03/2014] [Indexed: 12/27/2022]
Abstract
BACKGROUND Antimicrobial peptides are considered as a potential alternative to antibiotic treatment in acne vulgaris because the development of a resistant strain of Propionibacterium acnes is problematic. Granulysin can be regarded as an ideal substance with which to treat acne because it has antimicrobial and anti-inflammatory effects. OBJECTIVES This study was performed to explore the effectiveness of granulysin-derived peptides (GDPs) in killing P. acnes in vitro under a standard microbiologic assay and to evaluate their potential use in a topical agent for the treatment of acne vulgaris. METHODS Twenty different peptides based on the known sequence of a GDP were synthesized and tested in vitro for antimicrobial activity. Thirty patients with facial acne vulgaris were instructed to apply a topical formulation containing synthetic GDP to acne lesions twice per day for 12 weeks. RESULTS A newly synthesized peptide in which aspartic acid was substituted with arginine, and methionine was substituted with cysteine, showed the highest antimicrobial activity against P. acnes. Moreover, it was effective against both Gram-positive and Gram-negative bacteria in vitro. After treatment with the topical formulation containing 50 ppm of synthetic peptide for 12 weeks, a significant reduction in the number of pustules was observed, regardless of the increase in the number of comedones. In addition, a significant reduction in the clinical grade of acne based on the Korean Acne Grading System (KAGS) was evident. CONCLUSIONS Synthesized GDP shows strong antimicrobial activity against P. acnes in vitro. The clinical improvement observed suggests a topical formulation containing the GDP has therapeutic potential for the improvement of inflammatory-type acne vulgaris by its antimicrobial activity.
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Affiliation(s)
- Hee-Sun Lim
- Department of Dermatology, Chonnam National University Medical School, Gwangju, South Korea
| | - Seung-Min Chun
- Department of Dermatology, Chonnam National University Medical School, Gwangju, South Korea
| | - Min-Gyu Soung
- Department of Applied Biology and Chemistry, College of Agriculture and Life Science, Chungnam National University, Daejeon, South Korea.,Cellicon Laboratory, Hannam University, Daejeon, South Korea
| | - Jenny Kim
- Division of Dermatology, David Geffen School of Medicine, UCLA
| | - Seong-Jin Kim
- Department of Dermatology, Chonnam National University Medical School, Gwangju, South Korea
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20
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Nagasawa M, Ogawa K, Nagata K, Shimizu N. Granulysin and its clinical significance as a biomarker of immune response and NK cell related neoplasms. World J Hematol 2014; 3:128-137. [DOI: 10.5315/wjh.v3.i4.128] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/19/2014] [Accepted: 02/20/2014] [Indexed: 02/05/2023] Open
Abstract
Granulysin is a cytotoxic granular protein that was identified from human T cells by using the gene subtraction method in 1987. Based on its amino acid homology, granulysin belongs to the saposin-like protein family. The bioactive 9-kDa form of granulysin is processed from the 15-kDa pro-product in the cytoplasmic granules. It is expressed in CD8-positive αβT cells 5 d after mitogenic stimulation and constitutively in natural killer (NK) cells and γδT cells, although regulation of its expression has not yet been precisely determined. The 9-kDa granulysin form has anti-microbial activity against microorganisms such as bacteria, fungi, mycobacteria and parasites, as well as tumoricidal activity against some tumors at 1-10 μmol/L concentrations. Granulysin is secreted in both Ca-dependent and -independent manners. In sera, only the 15-kDa form is detectable and is expected to be a biomarker for immune potency, acute viral infection, anti-tumor immune reaction, acute graft vs host disease, and NK cell associated neoplasm.
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21
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Su SC, Chung WH. Cytotoxic proteins and therapeutic targets in severe cutaneous adverse reactions. Toxins (Basel) 2014; 6:194-210. [PMID: 24394640 PMCID: PMC3920257 DOI: 10.3390/toxins6010194] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2013] [Revised: 12/20/2013] [Accepted: 12/27/2013] [Indexed: 11/16/2022] Open
Abstract
Severe cutaneous adverse reactions (SCARs), such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN), are rare but life-threatening conditions induced mainly by a variety of drugs. Until now, an effective treatment for SJS/TEN still remains unavailable. Current studies have suggested that the pathobiology of drug-mediated SJS and TEN involves major histocompatibility class (MHC) I-restricted activation of cytotoxic T lymphocytes (CTLs) response. This CTLs response requires several cytotoxic signals or mediators, including granulysin, perforin/granzyme B, and Fas/Fas ligand, to trigger extensive keratinocyte death. In this article, we will discuss the cytotoxic mechanisms of severe cutaneous adverse reactions and their potential applications on therapeutics for this disease.
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Affiliation(s)
- Shih-Chi Su
- Department of Dermatology, Drug Hypersensitivity Clinical and Research Center, Chang Gung Memorial Hospitals, Taipei, Linkou, and Keelung, 33305, Taiwan.
| | - Wen-Hung Chung
- Department of Dermatology, Drug Hypersensitivity Clinical and Research Center, Chang Gung Memorial Hospitals, Taipei, Linkou, and Keelung, 33305, Taiwan.
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22
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Granule exocytosis of granulysin and granzyme B as a potential key mechanism in vaccine-induced immunity in cattle against the nematode Ostertagia ostertagi. Infect Immun 2013; 81:1798-809. [PMID: 23478322 DOI: 10.1128/iai.01298-12] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Ostertagia ostertagi is considered one of the most economically important bovine parasites. As an alternative to anthelmintic treatment, an experimental host-protective vaccine was previously developed on the basis of ASP proteins derived from adult worms. Intramuscular injection of this vaccine, combined with QuilA as an adjuvant, significantly reduced fecal egg counts by 59%. However, the immunological mechanisms triggered by the vaccine are still unclear. Therefore, in this study, the differences in immune responses at the site of infection, i.e., the abomasal mucosa, between ASP-QuilA-vaccinated animals and QuilA-vaccinated control animals were investigated on a transcriptomic level by using a whole-genome bovine microarray combined with histological analysis. Sixty-nine genes were significantly impacted in animals protected by the vaccine, 48 of which were upregulated. A correlation study between the parasitological parameters and gene transcription levels showed that the transcription levels of two of the upregulated genes, those for granulysin (GNLY) and granzyme B (GZMB), were negatively correlated with cumulative fecal egg counts and total worm counts, respectively. Both genes were also positively correlated with each other and with another upregulated gene, that for the IgE receptor subunit (FCER1A). Surprisingly, these three genes were also correlated significantly with CMA1, which encodes a mast cell marker, and with counts of mast cells and cells previously described as globule leukocytes. Furthermore, immunohistochemical data showed that GNLY was present in the granules of globule leukocytes and that it was secreted in mucus. Overall, the results suggest a potential role for granule exocytosis by globule leukocytes, potentially IgE mediated, in vaccine-induced protection against O. ostertagi.
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23
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Pitabut N, Sakurada S, Tanaka T, Ridruechai C, Tanuma J, Aoki T, Kantipong P, Piyaworawong S, Kobayashi N, Dhepakson P, Yanai H, Yamada N, Oka S, Okada M, Khusmith S, Keicho N. Potential function of granulysin, other related effector molecules and lymphocyte subsets in patients with TB and HIV/TB coinfection. Int J Med Sci 2013; 10:1003-14. [PMID: 23801887 PMCID: PMC3691799 DOI: 10.7150/ijms.6437] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/10/2013] [Accepted: 05/30/2013] [Indexed: 11/14/2022] Open
Abstract
BACKGROUND Host effector mechanism against Mycobacterium tuberculosis (Mtb) infection is dependent on innate immune response by macrophages and neutrophils and the alterations in balanced adaptive immunity. Coordinated release of cytolytic effector molecules from NK cells and effector T cells and the subsequent granule-associated killing of infected cells have been documented; however, their role in clinical tuberculosis (TB) is still controversy. OBJECTIVE To investigate whether circulating granulysin and other effector molecules are associated with the number of NK cells, iNKT cells, Vγ9(+)Vδ2(+) T cells, CD4(+) T cells and CD8(+) T cells, and such association influences the clinical outcome of the disease in patients with pulmonary TB and HIV/TB coinfection. METHODS Circulating granulysin, perforin, granzyme-B and IFN-γ levels were determined by ELISA. The isoforms of granulysin were analyzed by Western blot analysis. The effector cells were analyzed by flow cytometry. RESULTS Circulating granulysin and perforin levels in TB patients were lower than healthy controls, whereas the granulysin levels in HIV/TB coinfection were much higher than in any other groups, TB and HIV with or without receiving HAART, which corresponded to the number of CD8(+) T cells which kept high, but not with NK cells and other possible cellular sources of granulysin. In addition, the 17kDa, 15kDa and 9kDa isoforms of granulysin were recognized in plasma of HIV/TB coinfection. Increased granulysin and decreased IFN-γ levels in HIV/TB coinfection and TB after completion of anti-TB therapy were observed. CONCLUSION The results suggested that the alteration of circulating granulysin has potential function in host immune response against TB and HIV/TB coinfection. This is the first demonstration so far of granulysin in HIV/TB coinfection.
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Affiliation(s)
- Nada Pitabut
- Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
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24
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Guo Y, Luan G, Shen G, Wu L, Jia H, Zhong Y, Li R, Li G, Shen Y, Sun J, Hu S, Xiao W. Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris. Appl Microbiol Biotechnol 2012; 97:7669-77. [PMID: 23224405 DOI: 10.1007/s00253-012-4602-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2012] [Revised: 10/22/2012] [Accepted: 11/17/2012] [Indexed: 01/11/2023]
Abstract
Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.
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Affiliation(s)
- Yugang Guo
- Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China
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Qiu Y, Hu AB, Wei H, Liao H, Li S, Chen CY, Zhong W, Huang D, Cai J, Jiang L, Zeng G, Chen ZW. An atomic-force basis for the bacteriolytic effects of granulysin. Colloids Surf B Biointerfaces 2012; 100:163-8. [PMID: 22766293 DOI: 10.1016/j.colsurfb.2012.05.011] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2012] [Revised: 05/10/2012] [Accepted: 05/11/2012] [Indexed: 12/12/2022]
Abstract
While granulysin has been suggested to play an important role in adaptive immune responses against bacterial infections by killing pathogens, and molecular force for protein-protein interaction or protein-bacteria interaction may designate the specific functions of a protein, the molecular-force basis underlying the bacteriolytic effects of granulysin at single-molecule level remains unknown. Here, we produced and purified bactericidal domain of macaque granulysin (GNL). Our bacterial lysis assays suggested that GNL could efficiently kill bacteria such as Listeria monocytogenes. Furthermore, we found that the interaction force between GNL and L. monocytogenes measured by an atomic force microscopy (AFM) was about 22.5 pN. Importantly, our AFM-based single molecular analysis suggested that granulysin might lyse the bacteria not only through electrostatic interactions but also by hydrogen bonding and van der Waals interaction. Thus, this work provides a previous unknown mechanism for bacteriolytic effects of granulysin.
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Affiliation(s)
- Yueqin Qiu
- Department of Chemistry, College of Life Science and Technology, Jinan University, Guangzhou 510632, China
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26
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Clayberger C, Finn MW, Wang T, Saini R, Wilson C, Barr VA, Sabatino M, Castiello L, Stroncek D, Krensky AM. 15 kDa granulysin causes differentiation of monocytes to dendritic cells but lacks cytotoxic activity. THE JOURNAL OF IMMUNOLOGY 2012; 188:6119-26. [PMID: 22586033 DOI: 10.4049/jimmunol.1200570] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Granulysin is expressed as two isoforms by human cytotoxic cells: a single mRNA gives rise to 15 kDa granulysin, a portion of which is cleaved to a 9 kDa protein. Studies with recombinant 9 kDa granulysin have demonstrated its cytolytic and proinflammatory properties, but much less is known about the biologic function of the 15 kDa isoform. In this study, we show that the subcellular localization and functions of 9 and 15 kDa granulysin are largely distinct. Nine kilodalton granulysin is confined to cytolytic granules that are directionally released following target cell recognition. In contrast, 15 kDa granulysin is located in distinct granules that lack perforin and granzyme B and that are released by activated cytolytic cells. Although recombinant 9 kDa granulysin is cytolytic against a variety of tumors and microbes, recombinant 15 kDa granulysin is not. The 15 kDa isoform is a potent inducer of monocytic differentiation to dendritic cells, but the 9 kDa isoform is not. In vivo, mice expressing granulysin show markedly improved antitumor responses, with increased numbers of activated dendritic cells and cytokine-producing T cells. Thus, the distinct functions of granulysin isoforms have major implications for diagnosis and potential new therapies for human disease.
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Affiliation(s)
- Carol Clayberger
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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27
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Iwai S, Sueki H, Watanabe H, Sasaki Y, Suzuki T, Iijima M. Distinguishing between erythema multiforme major and Stevens-Johnson syndrome/toxic epidermal necrolysis immunopathologically. J Dermatol 2012; 39:781-6. [PMID: 22458564 DOI: 10.1111/j.1346-8138.2012.01532.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The early clinical presentations of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are similar to that of erythema multiforme major (EMM). Cytotoxic molecules, especially granulysin, are expressed in the skin lesions of SJS/TEN and cause extensive keratinocyte death. It is postulated that the function of regulatory T cells (Treg) in SJS/TEN is inadequate. This study examined whether an immunohistological examination of cytotoxic molecules and the immunophenotype of Treg is useful for discriminating SJS from EMM in the early period. Over the past 9 years, the lesional skin of 14 patients with SJS/TEN and 16 patients with EMM was biopsied. Double immunofluorescence labeling of CD8 and granulysin, perforin, or granzyme B was performed, and immunohistochemical analyses of granulysin, perforin, granzyme B, CD1a, CD3, CD4, CD8, CD68 and Foxp3 were conducted using a highly sensitive indirect immunoperoxidase technique. The number of cells positive for each antibody per five high-power fields was counted. The proportions of granulysin(+) cells/CD8(+) cells (P = 0.012) and perforin(+) cells/CD8(+) cells (P = 0.037) in SJS/TEN were significantly higher than in EMM. The number of Foxp3(+) cells/five high-power fields in SJS/TEN was significantly lower than in EMM (P = 0.004). Similarly, the number of CD4(+) cells/five high-power fields in SJS/TEN was significantly lower than in EMM (P = 0.0017). These data suggest that these panels of antibodies for labeling cytotoxic molecules, CD4 and Treg are useful for discriminating early SJS/TEN and EMM with a skin biopsy.
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Affiliation(s)
- Shinsaku Iwai
- Department of Dermatology, Showa University School of Medicine, Tokyo, Japan.
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Abstract
Granulysin is a cytotoxic granule expressed in cytotoxic T cells and natural killer cells. Although its cytotoxic effect against a number of tumor cell lines has been demonstrated in vitro, recent studies with transgenic mice, and a number of clinical studies, have further established its significance in cancer immunology. Furthermore, granulysin-induced in vitro chemotaxis and activation of both human and mouse dendritic cells have been reported. Given the results in recent clinical studies, granulysin may offer a useful indicator in the prognosis of cancer. Taken together, an understanding of the mechanism by which granulysin destroys target cells would provide vital information in the development of new therapies for the treatment of this disease.
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Affiliation(s)
- Satoshi Okada
- Department of Internal Medicine, Ichikawa General Hospital, Tokyo Dental College, 5-11-13 Sugano, Ichikawa 272-8513, Japan
| | - Tetsuo Morishita
- Department of Internal Medicine, Ichikawa General Hospital, Tokyo Dental College, 5-11-13 Sugano, Ichikawa 272-8513, Japan
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Cell death mechanisms at the maternal-fetal interface: insights into the role of granulysin. Clin Dev Immunol 2011; 2012:180272. [PMID: 21912564 PMCID: PMC3170798 DOI: 10.1155/2012/180272] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2011] [Revised: 07/12/2011] [Accepted: 07/12/2011] [Indexed: 01/11/2023]
Abstract
During mammal pregnancy, a sensitive balance between hormones, cytokines, humoral factors, and local cellular interactions must be established. Cytotoxic cells infiltrating the decidua are heavily equipped with cytolytic molecules, in particular perforin and granulysin. Granulysin is especially abundant in NK cells which are able to spontaneously secrete high quantities of granulysin. Besides being a potent bactericidal and tumoricidal molecule, granulysin is also found to be a chemoattractant and a proinflammatory molecule. The precise role(s) of granulysin at the maternal-fetal interface has not been elucidated yet. It is possible that it behaves as a double-edged sword simultaneously acting as an immunomodulatory and a host defense molecule protecting both the mother and the fetus from a wide spectrum of pathogens, and on the other hand, in case of an NK cell activation, acting as an effector molecule causing the apoptosis of semiallograft trophoblast cells and consequently leading to various pregnancy disorders or pregnancy loss.
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Vujaklija DV, Gulic T, Sucic S, Nagata K, Ogawa K, Laskarin G, Saito S, Haller H, Rukavina D. First trimester pregnancy decidual natural killer cells contain and spontaneously release high quantities of granulysin. Am J Reprod Immunol 2011; 66:363-72. [PMID: 21623991 DOI: 10.1111/j.1600-0897.2011.01015.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
PROBLEM Granulysin (GNLY) is a novel cytolytic protein lytic against a variety of tumor cells and microbes. The role of GNLY during pregnancy has not been extensively explored. The aim of this study is to examine GNLY expression and distribution in the first trimester pregnancy peripheral blood (PB) and decidua, the ability of decidual and PB natural killer (NK) cells to secrete GNLY spontaneously, and the role of antigen-presenting cells (APC) in the regulation of GNLY expression in decidual NK cells. METHOD OF STUDY GNLY expression was analyzed using cell permeabilization method, flow cytometry, and immunohistochemistry. GNLY secretion by purified NK cells was detected by ELISA method. RESULTS GNLY is abundantly expressed at the maternal-fetal interface in the first trimester pregnancy. Decidual T lymphocytes express significantly higher levels of GNLY (58%) then PB T lymphocytes (11%). Over 85% of decidual CD56(+) cells express GNLY and when cultured spontaneously release high quantities of GNLY. Decidual APC participate in the control of GNLY expression in CD56(+) cells. CONCLUSION Abundant expression of GNLY in the decidual immunocompetent cells and the capacity of decidual CD56(+) cells to spontaneously secrete high quantities of GNLY point to important protective and immunomodulatory role that this molecule could play at the maternal-fetal interface.
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Park Y, Choi YJ, Park SJ, Lee SR, Sung HJ, Park KH, Kim SJ, Choi CW, Jung KY, Kim BS. Pretreatment serum level of 15-kDa granulysin might have a prognostic value in patients with diffuse large B cell lymphoma. Acta Haematol 2011; 126:79-86. [PMID: 21540579 DOI: 10.1159/000327255] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2011] [Accepted: 02/28/2011] [Indexed: 11/19/2022]
Abstract
AIM Granulysin (cytolytic molecules of cytotoxic T lymphocytes and natural killer cells) is synthesized as cytosolic 9-kDa and secretary 15-kDa isoforms. We evaluated the prognostic significance of the pretreatment serum level of 15-kDa granulysin in patients with diffuse large B cell lymphoma (DLBCL). PATIENTS AND METHODS A retrospective analysis was conducted on 88 DLBCL patients treated homogeneously with standard chemotherapy. The granulysin level was quantified in pretreatment samples. RESULTS The granulysin level in DLBCL patients was significantly lower than that in healthy controls (522 ± 496 vs. 1,945 ± 1,696 pg/ml; p < 0.0001), and the level in patients who experienced recurrence within 3 years was significantly lower than that of patients without recurrence (305 ± 337 vs. 720 ± 607 pg/ml; p = 0.001). Patients with granulysin levels higher than the median level showed significantly longer progression-free and overall survival according to univariate analysis (p = 0.031 and p = 0.014, respectively). In multivariate analysis, the granulysin level was an independently significant prognostic factor of overall survival (p = 0.018; hazard ratio, 0.521; 95% confidence interval, 0.188-0.841). CONCLUSIONS Pretreatment serum level of 15-kDa granulysin may be a valuable prognostic marker in DLBCL patients treated with standard chemotherapy.
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MESH Headings
- Antibodies, Monoclonal, Murine-Derived/administration & dosage
- Antibodies, Monoclonal, Murine-Derived/therapeutic use
- Antigens, Differentiation, T-Lymphocyte/blood
- Antigens, Differentiation, T-Lymphocyte/chemistry
- Antineoplastic Agents/administration & dosage
- Antineoplastic Agents/therapeutic use
- Antineoplastic Combined Chemotherapy Protocols/administration & dosage
- Antineoplastic Combined Chemotherapy Protocols/therapeutic use
- Cisplatin/administration & dosage
- Cisplatin/therapeutic use
- Cyclophosphamide/administration & dosage
- Cyclophosphamide/therapeutic use
- Cytarabine/administration & dosage
- Cytarabine/therapeutic use
- Doxorubicin/administration & dosage
- Doxorubicin/therapeutic use
- Etoposide/administration & dosage
- Etoposide/therapeutic use
- Female
- Humans
- Lymphoma, Large B-Cell, Diffuse/blood
- Lymphoma, Large B-Cell, Diffuse/diagnosis
- Lymphoma, Large B-Cell, Diffuse/drug therapy
- Male
- Methylprednisolone/administration & dosage
- Methylprednisolone/therapeutic use
- Middle Aged
- Molecular Weight
- Prednisone/administration & dosage
- Prednisone/therapeutic use
- Prognosis
- Protein Isoforms/blood
- Protein Isoforms/chemistry
- Recurrence
- Reproducibility of Results
- Retrospective Studies
- Rituximab
- Salvage Therapy
- Survival Analysis
- Vincristine/administration & dosage
- Vincristine/therapeutic use
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Affiliation(s)
- Yong Park
- Division of Hematology/Oncology, Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea
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Castiello L, Stroncek DF, Finn MW, Wang E, Marincola FM, Clayberger C, Krensky AM, Sabatino M. 15 kDa Granulysin versus GM-CSF for monocytes differentiation: analogies and differences at the transcriptome level. J Transl Med 2011; 9:41. [PMID: 21501511 PMCID: PMC3094223 DOI: 10.1186/1479-5876-9-41] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2011] [Accepted: 04/18/2011] [Indexed: 02/06/2023] Open
Abstract
Background Granulysin is an antimicrobial and proinflammatory protein with several isoforms. While the 9 kDa isoform is a well described cytolytic molecule with pro-inflammatory activity, the functions of the 15 kDa isoform is less well understood. Recently it was shown that 15 kDa Granulysin can act as an alarmin that is able to activate monocytes and immature dendritic cells. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) is a growth factor widely used in immunotherapy both for in vivo and ex vivo applications, especially for its proliferative effects. Methods We analyzed gene expression profiles of monocytes cultured with 15 kDa Granulysin or GM-CSF for 4, 12, 24 and 48 hours to unravel both similarities and differences between the effects of these stimulators. Results The analysis revealed a common signature induced by both factors at each time point, but over time, a more specific signature for each factor became evident. At all time points, 15 kDa Granulysin induced immune response, chemotaxis and cell adhesion genes. In addition, only 15 kDa Granulsyin induced the activation of pathways related to fundamental dendritic cell functions, such as co-stimulation of T-cell activation and Th1 development. GM-CSF specifically down-regulated genes related to cell cycle arrest and the immune response. More specifically, cytokine production, lymphocyte mediated immunity and humoral immune response were down-regulated at late time points. Conclusion This study provides important insights on the effects of a novel agent, 15 kDa granulysin, that holds promise for therapeutic applications aimed at the activation of the immune response.
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Affiliation(s)
- Luciano Castiello
- Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA
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Finn MW, Clayberger C, Krensky AM. Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system. Protein Expr Purif 2011; 75:70-4. [PMID: 20674748 PMCID: PMC2966544 DOI: 10.1016/j.pep.2010.07.009] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2010] [Revised: 07/26/2010] [Accepted: 07/26/2010] [Indexed: 01/09/2023]
Abstract
Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.
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Affiliation(s)
- Michael W. Finn
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 2016, Bethesda, MD 20892-4256
| | - Carol Clayberger
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 2016, Bethesda, MD 20892-4256
| | - Alan M. Krensky
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 2016, Bethesda, MD 20892-4256
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Chung WH, Hung SI. Genetic markers and danger signals in stevens-johnson syndrome and toxic epidermal necrolysis. Allergol Int 2010; 59:325-32. [PMID: 20962567 DOI: 10.2332/allergolint.10-rai-0261] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2010] [Indexed: 12/26/2022] Open
Abstract
Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse reactions, which could be induced by a variety of drugs. It was proposed that human leukocyte antigen (HLA)-restricted presentation of antigens (drugs or their metabolites) to T lymphocytes initiates the immune reactions of SJS/TEN. However, the genetic susceptibility and the exact pathogenesis were not clear until the recent studies. We first identified that HLA-B*1502 is strongly associated with carbamazepine (CBZ)-induced SJS/TEN and HLA-B*5801 with allopurinol-SJS/TEN in Han Chinese. The same associations had been validated across different human populations. For the downstream danger signals, Fas-Fas ligand (FasL) and perforin/granzyme B had been advocated as cytotoxic mediators for keratinocyte death in SJS/TEN. However, expression levels of these cytotoxic proteins from the skin lesions were too low to explain the distinct and extensive epidermal necrosis. Our recent study identified that the granulysin, a cytotoxic protein released from cytotoxic T cells or natural killer (NK) cells, is a key mediator for disseminated keratinocyte death in SJS/TEN. This article aims to provide an overview of both of the genomic and immunologic perspectives of SJS/TEN. These studies give us a better understanding of the immune mechanisms, biomarkers for disease prevention and early diagnosis, as well as providing the therapeutic targets for the treatments of SJS/TEN.
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Affiliation(s)
- Wen-Hung Chung
- Department of Dermatology, Chang Gung Memorial Hospital, Chang Gung University, Taipei, Taiwan.
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Tewary P, Yang D, de la Rosa G, Li Y, Finn MW, Krensky AM, Clayberger C, Oppenheim JJ. Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin. Blood 2010; 116:3465-74. [PMID: 20660289 PMCID: PMC2981473 DOI: 10.1182/blood-2010-03-273953] [Citation(s) in RCA: 109] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2010] [Accepted: 07/11/2010] [Indexed: 11/20/2022] Open
Abstract
Granulysin (GNLY), an antimicrobial protein present in the granules of human cytotoxic T lymphocytes and natural killer (NK) cells, is produced as an intact 15-kDa form that is cleaved to yield a 9-kDa form. Alarmins are endogenous mediators that can induce recruitment and activation of antigen-presenting cells (APCs) and consequently promote the generation of immune response. We hypothesized that GNLY might function as an alarmin. Here, we report that both 9- and 15-kDa forms of recombinant GNLY-induced in vitro chemotaxis and activation of both human and mouse dendritic cells (DCs), recruited inflammatory leucocytes, including APCs in mice, and promoted antigen-specific immune responses upon coadministration with an antigen. GNLY-induced APC recruitment and activation required the presence of Toll-like receptor 4. The observed activity of recombinant GNLY was not due to endotoxin contamination. The capability of the supernatant of GNLY-expressing HuT78 cells to activate DC was blocked by anti-GNLY antibodies. Finally we present evidence that supernatants of degranulated human NK92 or primary NK cells also activated DCs in a GNLY- and Toll-like receptor 4-dependent manner, indicating the physiologic relevance of our findings. Thus, GNLY is the first identified lymphocyte-derived alarmin capable of promoting APC recruitment, activation, and antigen-specific immune response.
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Affiliation(s)
- Poonam Tewary
- Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute- Frederick/NIH, Frederick, MD, USA
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Nagy N, McGrath JA. Blistering skin diseases: a bridge between dermatopathology and molecular biology. Histopathology 2010; 56:91-9. [PMID: 20055907 DOI: 10.1111/j.1365-2559.2009.03442.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Although dermatopathology and molecular biology are often considered to be separate laboratory disciplines, the respective approaches are far from mutually exclusive. This is certainly the case for understanding the pathology of blistering skin diseases, both acquired and inherited. For example, in toxic epidermal necrolysis, dermatopathology in isolation may provide few clues to disease pathogenesis. There is widespread keratinocyte apoptosis and a variable infiltrate of cytotoxic T cells, but morphology alone offers little insight into what causes the epidermal destruction. In contrast, molecular biology studies have revealed several key processes that help explain the keratinocyte death, including increased expression of death receptors and their ligands on keratinocyte cell membranes as well as the presence of local or systemic immunocyte-derived cytolytic granules. For some inherited blistering diseases, however, such as epidermolysis bullosa, the molecular pathology is complex and difficult to unravel in isolation, yet the addition of dermatopathology is helpful in focusing molecular investigations. Notably, immunolabelling of cell adhesion proteins using specific antibody probes can identify reduced or absent immunoreactivity for candidate genes/proteins. Bridging dermatopathology and molecular biology investigations facilitates a greater understanding of disease processes, improves diagnostic accuracy, and provides a basis for the development and appraisal of new treatments.
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Affiliation(s)
- Nikoletta Nagy
- St John's Institute of Dermatology, King's College London, Guy's Campus, London, UK
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Abstract
It is well known that natural killer (NK) cells are involved in defense against viruses and some tumors. NK cells kill target cells by the directed release of cytolytic granules that contain perforin, granzymes, and granulysin. It is increasingly important to evaluate NK cell function in immunotoxicity testing. NK cell function can be evaluated by determining cytolytic activity against target tumor cells by the (51)Cr-release assay and also by determining the number of NK cells in peripheral blood in humans and in the spleen in animals using flow cytometry. Recently, the intracellular levels of perforin, granzymes, and granulysin determined by flow cytometry have also been used in the evaluation of NK cell function. This chapter will describe the methods for NK cell assays in immunotoxicity testing.
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Zhang H, Zhong C, Shi L, Guo Y, Fan Z. Granulysin induces cathepsin B release from lysosomes of target tumor cells to attack mitochondria through processing of bid leading to Necroptosis. THE JOURNAL OF IMMUNOLOGY 2009; 182:6993-7000. [PMID: 19454696 DOI: 10.4049/jimmunol.0802502] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Granulysin is a killer effector molecule localized in cytolytic granules of human NK and CTL cells. Granulysin exhibits broad antimicrobial activity and potent cytotoxic action against tumor cells. However, the molecular mechanism of granulysin-induced tumor lysis is poorly understood. In this study, we found that granulysin causes a novel cell death termed necroptosis. Granulysin can target lysosomes of target tumor cells and induce partial release of lysosomal contents into the cytosol. Relocalized lysosomal cathepsin B can process Bid to active tBid to cause cytochrome c and apoptosis-activating factor release from mitochondria. Cathepsin B silencing and Bid or Bax/Bak deficiency resists granulysin-induced cytochrome c and apoptosis-activating factor release and is less susceptible to cytolysis against target tumor cells.
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Affiliation(s)
- Honglian Zhang
- National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
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Characterization and expression profile of complete functional domain of granulysin/NK-lysin homologue (buffalo-lysin) gene of water buffalo (Bubalus bubalis). Vet Immunol Immunopathol 2009; 128:413-7. [DOI: 10.1016/j.vetimm.2008.11.029] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2008] [Revised: 11/02/2008] [Accepted: 11/28/2008] [Indexed: 11/23/2022]
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Capinos Scherer CF, Endsley JJ, de Aguiar JB, Jacobs WR, Larsen MH, Palmer MV, Nonnecke BJ, Ray Waters W, Mark Estes D. Evaluation of granulysin and perforin as candidate biomarkers for protection following vaccination with Mycobacterium bovis BCG or M. bovisDeltaRD1. Transbound Emerg Dis 2009; 56:228-39. [PMID: 19389081 DOI: 10.1111/j.1865-1682.2008.01058.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
The development of improved vaccines against tuberculosis (TB) is directly linked to the investigation of new and better correlates of protection after vaccination against TB. Cloning and characterization of bovine homologues of the antimicrobial protein granulysin (Bo-lysin) and perforin by our group could be used as potential biomarkers for TB vaccination efficacy. In the present study, we examined the kinetics of granulysin, perforin, IFNgamma and Fas-L responses to Mycobacterium bovis purified protein derivative (PPD) stimulation by peripheral blood mononuclear cells from M. bovisDeltaRD1-, BCG- and non-vaccinated cattle. Gene expression profiles following PPD stimulation showed significant increases in transcripts for granulysin and IFNgamma in both CD4(+) and CD8(+) T cells in BCG-vaccinated as compared with non-vaccinated animals. Perforin and IFNgamma examined by flow cytometry, showed a difference of 1-2% more PPD-specific cells in BCG-vaccinated than non-vaccinated animals. In the vaccine trial, granulysin and perforin were significantly increased in both vaccine groups as compared with control after vaccination and challenge. IFNgamma expression was increased only after vaccination and secretion was higher in the control, non-protected group as compared with both vaccine groups demonstrating no correlation with protection upon vaccination. In summary, results shown here provide evidence that granulysin and perforin are prospective candidates as biomarkers of protection after vaccination against TB.
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Abstract
Granulysin is a cytolytic and proinflammatory molecule first identified by a screen for genes expressed 'late' (3-5 days) after activation of human peripheral blood mononuclear cells. Granulysin is present in cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Granulysin is made in a 15-kDa form that is cleaved into a 9-kDa form at both the amino and the carboxy termini. The 15-kDa form is constitutively secreted, and its function remains poorly understood. The 9-kDa form is released by receptor-mediated granule exocytosis. Nine kiloDalton granulysin is broadly cytolytic against tumors and microbes, including gram-positive and gram-negative bacteria, fungi/yeast and parasites. It kills the causative agents of both tuberculosis and malaria. Granulysin is also a chemoattractant for T lymphocytes, monocytes and other inflammatory cells and activates the expression of a number of cytokines, including regulated upon activation T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1 alpha, interleukin (IL)-10, IL-1, IL-6 and interferon (IFN)-alpha. Granulysin is implicated in a myriad of diseases including infection, cancer, transplantation, autoimmunity, skin and reproductive maladies. Small synthetic forms of granulysin are being developed as novel antibiotics. Studies of the full-length forms may give rise to new diagnostics and therapeutics for use in a wide variety of diseases.
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Affiliation(s)
- A M Krensky
- Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4256, USA.
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Chung WH, Hung SI, Yang JY, Su SC, Huang SP, Wei CY, Chin SW, Chiou CC, Chu SC, Ho HC, Yang CH, Lu CF, Wu JY, Liao YD, Chen YT. Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis. Nat Med 2008; 14:1343-50. [PMID: 19029983 DOI: 10.1038/nm.1884] [Citation(s) in RCA: 540] [Impact Index Per Article: 31.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2008] [Accepted: 09/30/2008] [Indexed: 12/22/2022]
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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Nakashima A, Shiozaki A, Myojo S, Ito M, Tatematsu M, Sakai M, Takamori Y, Ogawa K, Nagata K, Saito S. Granulysin produced by uterine natural killer cells induces apoptosis of extravillous trophoblasts in spontaneous abortion. THE AMERICAN JOURNAL OF PATHOLOGY 2008; 173:653-64. [PMID: 18688023 DOI: 10.2353/ajpath.2008.071169] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Immune changes are known to occur in recurrent spontaneous abortion, but it is unclear whether either maternal natural killer (NK) cells or T cells attack fetus-derived trophoblasts. To clarify the immunological causes of spontaneous abortion, we examined the relationship between cytotoxic granule proteins in decidual lymphocytes, such as granulysin, granzyme B, and perforin, and the induction of apoptosis in extravillous trophoblasts (EVTs). The number of granulysin-positive CD56(bright) NK cells increased significantly in the decidua basalis during spontaneous abortion compared with normal pregnancy; however, granzyme B- and perforin-positive cells did not change. Interestingly, the expression of granulysin was also detected in the nuclei of EVTs in spontaneous abortion samples. When IL-2-stimulated CD56(bright) NK cells were cocultured with EVT cells (HTR-8/SV40neo), granulysin was found initially in the cytoplasm and then accumulated in the nuclei of the HTR-8/SV40neo cells. Furthermore, transfected cells expressing a GFP-granulysin fusion protein induced apoptosis in HTR-8/SV40neo cells independently of caspases. Our results suggest that granulysin-positive uterine NK cells attack EVTs; subsequently, the uNK-derived granulysin actively accumulates in the nuclei of EVTs, causing the death of EVTs due to apoptosis. These data support a new apoptosis pathway for trophoblasts via uNK-derived granulysin, suggesting that granulysin is involved in spontaneous abortion.
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Affiliation(s)
- Akitoshi Nakashima
- Department of Obstetrics and Gynecology, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan
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Li Q, Morimoto K, Nakadai A, Inagaki H, Katsumata M, Shimizu T, Hirata Y, Hirata K, Suzuki H, Miyazaki Y, Kagawa T, Koyama Y, Ohira T, Takayama N, Krensky AM, Kawada T. Forest bathing enhances human natural killer activity and expression of anti-cancer proteins. Int J Immunopathol Pharmacol 2007; 20:3-8. [PMID: 17903349 DOI: 10.1177/03946320070200s202] [Citation(s) in RCA: 137] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37-55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50 percent increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.
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Affiliation(s)
- Q Li
- Department of Hygiene and Public Health, Nippon Medical School, Tokyo.
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Di Liberto D, Buccheri S, Caccamo N, Meraviglia S, Romano A, Di Carlo P, Titone L, Dieli F, Krensky AM, Salerno A. Decreased serum granulysin levels in childhood tuberculosis which reverse after therapy. Tuberculosis (Edinb) 2007; 87:322-8. [PMID: 17379576 PMCID: PMC2692947 DOI: 10.1016/j.tube.2007.01.003] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2006] [Revised: 01/09/2007] [Accepted: 01/18/2007] [Indexed: 10/23/2022]
Abstract
Granulysin is a cytolytic protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Serum levels of granulysin are related to host cellular immunity. We used an ELISA to quantify granulysin serum levels in children with tuberculosis (TB), before and after chemotherapy. The study involved children affected by different clinical forms of TB (n=72) and healthy control children (n=150) from the same geographical area and of similar socio-economic background. Serum granulysin levels before the initiation of TB therapy were significantly lower in children with TB compared to controls, with the lowest levels being found in TB patients who were PPD skin test negative. No statistically significant differences were found between serum granulysin levels and clinical severity (mild/moderate or advanced pulmonary TB) or the clinical form (pulmonary or extra-pulmonary) of TB. At four months after completion of therapy, serum granulysin levels in children treated for TB were not significantly different to those observed in control children. This finding was paralleled by the increased in vitro mycobactericidal activity of sera from TB patients after completion of therapy. We propose that serum granulysin levels may provide a marker of disease activity in childhood TB and might be useful for monitoring improvement after chemotherapy.
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Affiliation(s)
- Diana Di Liberto
- Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy
| | - Simona Buccheri
- Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy
| | - Nadia Caccamo
- Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy
| | - Serena Meraviglia
- Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy
| | - Amelia Romano
- Istituto di Malattie Infettive, Università di Palermo, 90134 Palermo, Italy
| | - Paola Di Carlo
- Istituto di Malattie Infettive, Università di Palermo, 90134 Palermo, Italy
| | - Lucina Titone
- Istituto di Malattie Infettive, Università di Palermo, 90134 Palermo, Italy
| | - Francesco Dieli
- Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy
| | - Alan M. Krensky
- Department of Pediatrics, Stanford University School of Medicine, CA, USA
| | - Alfredo Salerno
- Dipartimento di Biopatologia e Metodologie Biomediche, Università di Palermo, Corso Tukory 211, 90134 Palermo, Italy
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Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes. BMC Immunol 2007; 8:9. [PMID: 17596262 PMCID: PMC1914365 DOI: 10.1186/1471-2172-8-9] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2006] [Accepted: 06/27/2007] [Indexed: 01/18/2023] Open
Abstract
Background Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. Results In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation. Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. Conclusion This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing.
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Wiseman JCD, Ma LL, Marr KJ, Jones GJ, Mody CH. Perforin-dependent cryptococcal microbicidal activity in NK cells requires PI3K-dependent ERK1/2 signaling. THE JOURNAL OF IMMUNOLOGY 2007; 178:6456-64. [PMID: 17475875 DOI: 10.4049/jimmunol.178.10.6456] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Previously, NK cells have been reported to kill the opportunistic fungal pathogen Cryptococcus neoformans through a perforin-dependent mechanism; however, the receptor and signaling involved are unknown. In this report we sought to identify the signaling pathways activated and required for direct perforin-mediated killing of microbes. In this study, using the NK-like cell line YT and primary peripheral blood NK cells, it is demonstrated that YT cells kill C. neoformans and that the killing is accompanied by the activation of PI3K. We demonstrate that inhibition of either the catalytic subunit (using a pharmacological inhibitor) or the alpha-regulatory subunit (using small interfering RNA knockdown) of PI3K significantly inhibited the killing of C. neoformans. Downstream of PI3K, ERK1/2 was activated in a PI3K-dependent fashion and was required for cryptococcal killing. Furthermore, we demonstrate that perforin release from YT cells can be detected by 4 h after contact of the YT cells with C. neoformans and that the release of perforin is blocked by pharmacological inhibition of either PI3K or ERK1/2. Defective degranulation is rooted in the inability to polarize perforin-containing granules toward the target. Finally, we demonstrate that PI3K-ERK1/2-dependent signaling is activated and required for the killing of C. neoformans by primary NK cells. Taken together, these data identify a conserved PI3K-ERK1/2 pathway that is used by NK cells during the direct killing of C. neoformans and demonstrate that the pathway is essential in the formation and activation of the microbicidal mechanism.
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Sahiratmadja E, Alisjahbana B, Buccheri S, Di Liberto D, de Boer T, Adnan I, van Crevel R, Klein MR, van Meijgaarden KE, Nelwan RHH, van de Vosse E, Dieli F, Ottenhoff THM. Plasma granulysin levels and cellular interferon-gamma production correlate with curative host responses in tuberculosis, while plasma interferon-gamma levels correlate with tuberculosis disease activity in adults. Tuberculosis (Edinb) 2007; 87:312-21. [PMID: 17382591 DOI: 10.1016/j.tube.2007.01.002] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2006] [Revised: 01/22/2007] [Accepted: 01/22/2007] [Indexed: 10/23/2022]
Abstract
Granulysin is a recently identified cytolytic protein which is expressed by human cytotoxic T-lymphocytes and natural killer (NK)-cells, and has broad antimicrobial and tumoricidal activity. Circulating granulysin levels are associated with T- and NK-cell activity, and may thus reflect protection-associated cellular immune responses. In a case-control study in Indonesia, a highly tuberculosis (TB)-endemic country, we therefore determined plasma granulysin levels in adults with active pulmonary TB before, during, and after TB treatment, both in mild/moderate-TB and advanced-TB patients, and compared these to healthy neighbourhood controls. Adults with active pulmonary TB had significantly lower plasma granulysin levels compared to controls. After 2 months of anti-TB therapy, levels in TB patients had significantly increased, reaching values similar to those in controls. Plasma granulysin levels further increased after completion of TB therapy, being significantly higher than those in controls. Plasma granulysin levels correlated inversely with TB disease activity but not with TB disease severity. In contrast, plasma interferon-gamma (IFN-gamma) levels were significantly higher in active TB cases than in controls, normalised during treatment and correlated with both TB disease activity and TB disease severity. At the cellular level, granulysin and IFN-gamma expression both correlated inversely with disease activity. Interestingly, granulysin was predominantly expressed by IFN-gamma negative T-cells, suggesting that the cellular sources of IFN-gamma and granulysin in TB are partly non-overlapping. The observation that plasma granulysin levels and cellular IFN-gamma production correlate with curative host responses in pulmonary tuberculosis points to a potentially important role of granulysin, next to IFN-gamma, in host defence against M. tuberculosis.
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Affiliation(s)
- E Sahiratmadja
- Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands
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Li Q, Morimoto K, Nakadai A, Qu T, Matsushima H, Katsumata M, Shimizu T, Inagaki H, Hirata Y, Hirata K, Kawada T, Lu Y, Nakayama K, Krensky AM. Healthy lifestyles are associated with higher levels of perforin, granulysin and granzymes A/B-expressing cells in peripheral blood lymphocytes. Prev Med 2007; 44:117-23. [PMID: 17030356 DOI: 10.1016/j.ypmed.2006.08.017] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/09/2006] [Revised: 08/22/2006] [Accepted: 08/22/2006] [Indexed: 11/27/2022]
Abstract
OBJECTIVE It is well documented that natural killer (NK) cells provide host defense against tumors and viruses. We previously showed that lifestyle affects human NK and LAK activities. In order to explore the underlying mechanism, we investigated the effect of lifestyle on intracellular perforin, granulysin, and granzymes A/B in peripheral blood lymphocytes (PBL). METHODS 114 healthy male subjects, aged 20-59 years, from a large company in Osaka, Japan were selected with informed consent. The subjects were divided into groups reporting good, moderate, and poor lifestyles according to their responses on a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, sleeping hours, working hours, physical exercise, eating breakfast, balanced nutrition, and mental stress). Peripheral blood was taken, and numbers of NK, T, perforin, granulysin, and granzymes A/B-expressing cells in PBL were measured by flow cytometry. RESULTS Subjects with good or moderate lifestyle showed significantly higher numbers of NK, and perforin, granulysin, and granzymes A/B-expressing cells and a significantly lower number of T cells in PBL than subjects with poor lifestyle. Among the eight health practices, cigarette smoking, physical exercise, eating breakfast, and balanced nutrition significantly affect the numbers of NK, T cells, perforin, granulysin, and/or granzymes A/B-expressing cells, and alcohol consumption significantly affects the number of granzyme A-expressing cells. On the other hand, mental stress, sleeping, and working hours had no effect on those parameters. CONCLUSIONS Taken together, these findings indicate that poor lifestyle significantly decreases the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells in PBL.
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Affiliation(s)
- Qing Li
- Department of Hygiene and Public Health, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
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Barman H, Walch M, Latinovic-Golic S, Dumrese C, Dolder M, Groscurth P, Ziegler U. Cholesterol in negatively charged lipid bilayers modulates the effect of the antimicrobial protein granulysin. J Membr Biol 2007; 212:29-39. [PMID: 17206515 DOI: 10.1007/s00232-006-0040-3] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2006] [Revised: 09/05/2006] [Indexed: 10/23/2022]
Abstract
The release of granulysin, a 9-kDa cationic protein, from lysosomal granules of cytotoxic T lymphocytes and natural killer cells plays an important role in host defense against microbial pathogens. Granulysin is endocytosed by the infected target cell via lipid rafts and kills subsequently intracellular bacteria. The mechanism by which granulysin binds to eukaryotic and prokaryotic cells but lyses only the latter is not well understood. We have studied the effect of granulysin on large unilamellar vesicles (LUVs) and supported bilayers with prokaryotic and eukaryotic lipid mixtures or model membranes with various lipid compositions and charges. Binding of granulysin to bilayers with negative charges, as typically found in bacteria and lipid rafts of eukaryotic cells, was shown by immunoblotting. Fluorescence release assays using LUV revealed an increase in permeability of prokaryotic, negatively charged and lipid raft-like bilayers devoid of cholesterol. Changes in permeability of these bilayers could be correlated to defects of various sizes penetrating supported bilayers as shown by atomic force microscopy. Based on these results, we conclude that granulysin causes defects in negatively charged cholesterol-free membranes, a membrane composition typically found in bacteria. In contrast, granulysin is able to bind to lipid rafts in eukaryotic cell membranes, where it is taken up by the endocytotic pathway, leaving the cell intact.
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Affiliation(s)
- Hanna Barman
- Division of Cell Biology, Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland
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