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Heng B, Ding H, Ren H, Shi L, Chen J, Wu X, Lai C, Yu G, Xu Y, Su Z. Diagnostic Performance of Fas Ligand mRNA Expression for Acute Rejection after Kidney Transplantation: A Systematic Review and Meta-Analysis. PLoS One 2016; 11:e0165628. [PMID: 27812144 PMCID: PMC5094747 DOI: 10.1371/journal.pone.0165628] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2016] [Accepted: 10/15/2016] [Indexed: 01/25/2023] Open
Abstract
Background The value of Fas ligand (FASL) as a diagnostic immune marker for acute renal rejection is controversial; this meta-analysis aimed to clarify the role of FASL in acute renal rejection. Methods The relevant literature was included by systematic searching the MEDLINE, EMBASE, and Cochrane Library databases. Accuracy data for acute rejection (AR) and potential confounding variables (the year of publication, area, sample source, quantitative techniques, housekeeping genes, fluorescence staining, sample collection time post-renal transplantation, and clinical classification of AR) were extracted after carefully reviewing the studies. Data were analyzed by Meta-DiSc 1.4, RevMan 5.0, and the Midas module in Stata 11.0 software. Results Twelve relevant studies involving 496 subjects were included. The overall pooled sensitivity, specificity, positive likelihood ratio (LR), negative LR, and diagnostic odds ratio, together with the 95% CI were 0.64 (0.57–0.70), 0.90 (0.85–0.93), 5.66 (3.51–9.11), 0.30 (0.16–0.54), and 30.63 (14.67–63.92), respectively. The area under the summary receiver operating characteristic curve (AUC) was 0.9389. Fagan’s nomogram showed that the probability of AR episodes in the kidney transplant recipient increased from 15% to 69% when FASL was positive, and was reduced to 4% when FASL was negative. No threshold effect, sensitivity analyses, meta-regression, and subgroup analyses based on the potential variables had a significant statistical change for heterogeneity. Conclusions Current evidence suggests the diagnostic potential for FASL mRNA detection as a reliable immune marker for AR in renal allograft recipients. Further large, multicenter, prospective studies are needed to validate the power of this test marker in the non-invasive diagnosis of AR after renal transplantation.
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Affiliation(s)
- Baoli Heng
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Hongwen Ding
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Haolin Ren
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Liping Shi
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Jie Chen
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Xun Wu
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Caiyong Lai
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Ganshen Yu
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Yin Xu
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Zexuan Su
- Department of Urology, the First Affiliated Hospital of Jinan University, Guangzhou, China
- * E-mail:
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Nusair S, Gincberg G, Almogi-Hazan O, Breuer R, Or R, Wallach-Dayan SB. Failure of chimerism formation and tolerance induction from Fas ligand mutant bone marrow donors after nonmyeloablative conditioning. Transpl Immunol 2012; 27:184-8. [PMID: 22801052 DOI: 10.1016/j.trim.2012.07.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2012] [Accepted: 07/06/2012] [Indexed: 11/28/2022]
Abstract
Formation of donor-recipient mixed chimerism after nonmyeloablative conditioning allows co-existence of donor and recipient hematopoietic stem cells, with solid organ allograft tolerance and less likeliness of graft versus host development. Using a post-transplant bronchiolitis obliterans murine model, we aimed to test the hypothesis that allograft preservation after mixed chimerism formation is dependent on the presence of a functional Fas ligand (FasL) on donor hematopoietic cells. To form mixed chimerism, two aliquots of 30 × 10(6) whole bone marrow cells (BMC) from either wild-type C57BL/6 in one group, or transgenic gld mice with mutant FasL (d = 0 and 2+) in the other were used, with both groups receiving intravenous busulfan (10mg/kg) on d-1 and intraperitoneal cyclophosphamide (200mg/kg) on d+1. Tracheal allografts obtained from C57BL/6 mice were implanted into recipient BALB/c mice subcutaneously on d = 0. Tracheal allografts were harvested at d+28 post-transplant and were evaluated by histopathology. Mixed chimerism formation was detected in wild type C57BL/6 whole BMC recipients, which was accompanied by tracheal allograft acceptance with near normal structure at d+28 post implantation. However, in recipients of FasL mutant whole BMC, neither mixed chimerism formation nor tracheal allograft acceptance was obtained. We thus conclude that bone marrow cells lacking functional FasL molecules could not be engrafted in allogeneic recipients to form stable mixed chimerism after nonmyeloablative conditioning, thus not allowing tracheal allograft acceptance.
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Affiliation(s)
- Samir Nusair
- The Lung Cellular & Molecular Biology Laboratory, Institute of Pulmonary Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
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Acute cardiac allograft rejection by directly cytotoxic CD4 T cells: parallel requirements for Fas and perforin. Transplantation 2010; 89:33-9. [PMID: 20061916 DOI: 10.1097/tp.0b013e3181be6bc7] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
BACKGROUND CD4 T cells can suffice as effector cells to mediate primary acute cardiac allograft rejection. Although CD4 T cells can readily kill appropriate target cells in vitro, the corresponding role of such cytolytic activity for mediating allograft rejection in vivo is unknown. Therefore, we determined whether the cytolytic effector molecules perforin (PFP) and/or FasL (CD95L) were necessary for CD4 T cell-mediated rejection in vivo. METHODS Wild-type C3H(H-2) or Fas (CD95)-deficient C3Hlpr (H-2) hearts were transplanted into immune-deficient C57B6rag (H-2) mice. Then, recipients were reconstituted with naïve purified CD4 T cells from wild-type, PFP-deficient, or FasL (gld)-deficient T-cell donors. RESULTS In vitro, alloreactive CD4 T cells were competent to lyse donor major histocompatibility complex class II+ target cells, largely by a Fas-dependent mechanism. In vivo, the individual disruption of donor Fas expression (lpr) or CD4 T-cell-derived PFP had no significant impact on acute rejection. However, FasL-deficient (gld) CD4 T cells demonstrated delayed allograft rejection. Importantly, the simultaneous removal of both donor Fas expression and CD4 T-cell PFP completely abrogated acute rejection, despite the persistence of CD4 T cells within the graft. CONCLUSIONS Results demonstrate that the direct rejection of cardiac allografts by CD4 effector T cells requires the alternative contribution of graft Fas expression and T cell PFP expression. To our knowledge, this is the first demonstration that cytolytic activity by CD4 T cells can play an obligate role for primary acute allograft rejection in vivo.
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Shen Y, Young B, Lipman ML. Suppression of the cell-mediated immune response by a Fas-immunoglobulin fusion protein. Transplantation 2006; 81:1041-8. [PMID: 16612282 DOI: 10.1097/01.tp.0000164289.18470.28] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
INTRODUCTION Immunosuppressive agents must not only be effective in impairing the host's allo-immune response, but should also be selective in targeting only those elements of the immune system activated by the allograft. The fact that allo-activated T cells express surface protein molecules that are not typically present on resting T cells can be exploited to specifically target this population. Fas ligand is one such molecule whose cell surface expression on T cells is dramatically up-regulated upon activation. METHODS We constructed a murine fusion protein by linking the extracellular domain of Fas to the Fc region of IgG2a. The rationale being to selectively target activated T cells via binding of its Fas moiety to cell surface Fas ligand, and then to allow the Fc moiety to invoke its usual effector mechanisms resulting in the destruction of the allo-activated T cell. Here, we describe the design and expression of Fas-IgG2a and characterize several key in vitro and in vivo properties of this fusion protein including its ability to impact on both cell-mediated immune responses and cellular apoptotic activity using a murine model of delayed-type hypersensitivity. RESULTS In vitro, our Fas-IgG2a construct bound activated T cells via FasL and invoked cytotoxicity. In vivo, it demonstrated a prolonged half-life characteristic of an immunoglobulin-like molecule. Most significantly, it suppressed the cell-mediated immune response and diminished cellular apoptotic activity in lymphoid tissue in our murine model. CONCLUSION Fas-IgG2a is a novel agent for delivering target-specific immunosuppression with potential applicability in the transplant setting.
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Affiliation(s)
- Yingnian Shen
- Yingnian Shen, Genomics and Proteomics Science, GlaxoSmithKline, Research Triangle Park, North Carolina, USA
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Niederkorn JY, Stevens C, Mellon J, Mayhew E. Differential roles of CD8+ and CD8- T lymphocytes in corneal allograft rejection in 'high-risk' hosts. Am J Transplant 2006; 6:705-13. [PMID: 16539627 DOI: 10.1111/j.1600-6143.2006.01237.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
We examined the role of perforin and FasL in corneal allograft rejection mediated by CD8+ and CD8 T cells. BALB/c corneas were transplanted orthotopically into vascularized, 'high-risk' graft beds in C57BL/6 mice, perforin knockout mice and FasL-defective gld/gld mice. CD8+ and CD8 T cells were collected following graft rejection and adoptively transferred to SCID mice, which were then challenged with BALB/c corneal allografts. In every case, CD8 T cells could mediate graft rejection when adoptively transferred to SCID mice that received BALB/c corneal allografts. Although CD8+ T cells also mediated graft rejection, the tempo was slower. Moreover, CD8+ T cells collected FasL-defective donors that had rejected corneal allografts, mediated corneal allograft rejection in only 50% of the SCID mice that received the adoptively transferred cells. In some cases, CD8+ T-cell-mediated rejection occurred in the absence of delayed-type hypersensitivity and cytotoxic T-lymphocyte activity, but was associated with CD8+ T-cell-mediated apoptosis of BALB/c corneal cells in vitro. The results demonstrate the redundancy in immune mechanisms of corneal allograft rejection. Either CD8+ or CD8 T cells can produce corneal allograft rejection, however functional FasL is necessary for optimal rejection, even in a high-risk setting.
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Affiliation(s)
- J Y Niederkorn
- Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
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Gomez L, Raisky O, Chalabreysse L, Verschelde C, Bonnefoy-Berard N, Ovize M. Link between immune cell infiltration and mitochondria-induced cardiomyocyte death during acute cardiac graft rejection. Am J Transplant 2006; 6:487-95. [PMID: 16468957 DOI: 10.1111/j.1600-6143.2005.01219.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Acute cardiac graft rejection (ACGR) is associated with cardiomyocyte apoptosis. We investigated the respective role of the Fas/FasL and mitochondrial permeability transition pore (mPTP) pathways in cardiomyocyte apoptosis accompanying ACGR. Heterotopic cardiac transplantations were performed in 7-9-week old C57BL6 or C3H mice. Wild type or Fas-deficient (lpr) mice underwent syngeneic (GS) or allogeneic (GA) transplantation, and received either saline or NIM811, a specific inhibitor of the mPTP. At day 5, we assessed ACGR by histology, cardiomyocyte apoptosis by caspase-3 activity and cytochrome c release, Ca(2+)-induced mPTP opening by a potentiometric approach, and expression of Fas, FasL, TNFalpha, perforin, granzyme using RT-PCR. Myocardial infiltration of CD8(+) T lymphocytes was performed by immunohistochemistry. Allogenic transplantation increased infiltration of inflammatory cells, upregulated FasL, perforin, granzyme, and TNFalpha, favored Ca(2+)-induced mPTP opening and increased caspase-3 activity and cytochrome c release in WT grafts. NIM811, but not Fas-deficiency, significantly reduced all these effects. NIM811 also limited infiltration of CD8(+) into WT and lpr transplants. These data suggest that the mPTP pathway plays a major role in cardiomyocyte apoptosis associated with ACGR. Inhibition of mPTP opening may attenuate cardiomyocyte apoptosis either directly or indirectly via a limitation of CD8(+) T-cell activation.
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Affiliation(s)
- L Gomez
- INSERM E 0226, Université Claude Bernard Lyon I, Lyon, France
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Wang YL, Zhang YY, Li G, Tang ZQ, Zhou YL, Zhu ZJ, Yao Z. Correlation of CD95 and soluble CD95 expression with acute rejection status of liver transplantation. World J Gastroenterol 2005; 11:1700-4. [PMID: 15786554 PMCID: PMC4305958 DOI: 10.3748/wjg.v11.i11.1700] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To analyze the expression levels of soluble form of CD95, CD95 ligand (sCD95 and sCD95L, respectively) in plasma and CD95 expression on CD3+ cells in liver-transplanted recipients with acute rejection (AR).
METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 30 clinically liver transplanted recipients. CD95 expression on CD3+ cells was quantitatively measured by two-color fluorescence activated cell sorter (FACS) analysis. Lymphocyte surface phenotypes of CD4, CD8, CD16 and CD56 were determined by flow cytometry. Plasma levels of sCD95 and sCD95L were detected by Enzyme Linked-Immuno-Sorbent Assay (ELISA). The results were compared with that from normal healthy volunteers (n = 15 individuals).
RESULTS: FACS analysis showed that CD95 expression on CD3+ T cells was significantly increased in liver transplanted recipients with AR compared to that in stable recipients without rejection and infection or healthy individuals who did not undergo transplantation (18676.93±11588.34/molecule, 6848.20±1712.96/molecule, 6418.01±2001.95/molecule, respectively, P<0.01). Whereas no significant difference was seen between liver-transplanted stable recipients and healthy individuals. Furthermore, no significant differences were detected between each group with CD4/CD8 ratio or the percentage of CD16+56+ cells. Plasma levels of sCD95 were significantly higher in transplanted recipients with AR compared to that in stable recipients or healthy individuals (391.88±196.00, 201.37±30.30, 148.83±58.25 pg/mL, respectively, P<0.01). In contrast, the plasma levels of sCD95L in liver- transplanted recipients were not significantly different from that in healthy individuals.
CONCLUSION: The present results indicate that the increased CD95 expression on CD3+ cells and the increased levels of sCD95 in plasma may modify the immunological situation of the recipients after transplantation or represent the ongoing graft rejection.
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Affiliation(s)
- Yu-Liang Wang
- Tianjin Institute of Thrombosis and Hemostasis, Laboratory Center, Tianjin First Central Hospital, 24 FuKang Road, Tianjin 300192, China.
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Hegde S, Beauregard C, Mayhew E, Niederkorn JY. CD4(+) T-cell-mediated mechanisms of corneal allograft rejection: role of Fas-induced apoptosis. Transplantation 2005; 79:23-31. [PMID: 15714165 DOI: 10.1097/01.tp.0000147196.79546.69] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
BACKGROUND The role of CD4(+) T cells as effector cells in corneal allograft rejection is poorly understood. We investigated the role of CD4(+) T cells as helper cells in the generation of allospecific effector macrophages in corneal graft rejection and the role of CD4(+) T cells as apoptosis-inducing effector cells. METHODS Corneal allografts were transplanted to CD4 knockout, FasL-deficient, and macrophage-depleted hosts. An Annexin-V binding assay was used to evaluate the susceptibility of corneal cells to both Fas-dependent and CD4 T-cell-mediated apoptosis in vitro. RESULTS Macrophages were essential for graft rejection, but not as effector cells. Anti-BALB/c CD4(+) T cells from immunized C57BL/6 mice induced apoptosis of BALB/c corneal epithelial and endothelial cells. However, anti-BALB/c CD4(+) T cells from FasL-deficient gld/gld mice did not induce apoptosis of BALB/c corneal endothelial cells. Moreover, gld/gld mice had a reduced capacity to reject BALB/c corneal allografts. Although the initial results suggested a role for Fas-induced apoptosis in corneal graft rejection, additional experiments indicated otherwise. The incidence and tempo of immune rejection of Fas-deficient lpr/lpr corneal allografts were no different than those for corneal grafts from Fas-bearing C57BL/6 donors. Moreover, CD4(+) T-cell-mediated apoptosis of corneal cells could not be blocked with either Fas-Fc fusion protein or anti-FasL blocking antibody. CONCLUSIONS The results suggest that CD4(+) T cells function directly as effector cells and not as helper cells in the rejection of corneal allografts. Although the corneal endothelium is highly susceptible to Fas-induced apoptosis, this is apparently not the primary mechanism of CD4(+) T-cell-dependent rejection.
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Affiliation(s)
- Sushma Hegde
- Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA
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Schnickel GT, Whiting D, Hsieh GR, Yun JJ, Fischbein MP, Fishbein MC, Yao W, Shfizadeh A, Ardehali A. CD8 Lymphocytes are Sufficient for the Development of Chronic Rejection. Transplantation 2004; 78:1634-9. [PMID: 15591952 DOI: 10.1097/01.tp.0000141362.33931.40] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND The role of CD8 lymphocytes, in chronic rejection or cardiac allograft vasculopathy (CAV), is incompletely understood. The purposes of this study were to determine whether CD8 lymphocytes, in the absence of CD4 lymphocytes, are capable of causing the intimal lesions of CAV; and if so, to define the effector mechanism(s) of CD8 lymphocytes. METHODS We modified a previously characterized major histocompatibility complex class II mismatched murine model of CAV. Wild-type CD8 lymphocytes were transferred to nude mice followed by heterotopic heart transplantation. Recipient mice were then treated with a CD40 activating antibody, which is known to provide help for CD8 lymphocyte activation, in the absence of CD4 lymphocytes. Donor hearts were harvested on day 40 posttransplantation and analyzed for cellular infiltrates and intimal thickening. In separate experiments, isolated perforin -/-, Fas ligand (FasL) -/-, and interferon (IFN)-gamma -/- CD8 lymphocytes were transferred to nude mice followed by identical experimented protocol. RESULTS With adaptive transfer of wild-type CD8 lymphocytes, the donor hearts were infiltrated with activated CD8 lymphocytes and displayed significant intimal lesions. Adoptive transfer of perforin -/- and FasL -/- CD8 lymphocytes to nude mice resulted in similar patterns of CD8 lymphocyte infiltration and similar severity of intimal lesions. The donor hearts from IFN-gamma -/- CD8 lymphocyte reconstituted recipients displayed minimal intimal lesions, although CD8 lymphocytes were present in the allografts. CONCLUSIONS Unprimed CD8 lymphocytes in the absence of CD4 lymphocytes can cause intimal lesions of CAV. CD8 lymphocytes production of IFN-gamma, but not the perforin or the FasL-mediated cytotoxicity, is the critical step in the development of intimal lesions.
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Affiliation(s)
- Gabriel T Schnickel
- Division of Cardiothoracic Surgery, Department of Surgery, David Geffen School of Medicine at University of California, Los Angeles, CA 90095, USA
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10
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Abstract
Transplantation has been performed clinically for four decades and has become the standard of care for end-stage organ failure. Understanding of the immunobiology of transplantation has made tremendous advances, but knowledge still lags behind the clinical use. As a result, nonspecific immunosuppression remains the standard therapy. This article presents an overview of current knowledge of the immunobiology of solid organ transplantation, with emphasis on T-cell activation (antigen presentation, CoS) and cellular allograft (transplantation) immunity. The molecular events of T-cell activation, with some emphasis on the sites of action of modern immunosuppression, are reviewed. A simplified approach to understanding the immunobiology and strategy of maintenance immunosuppression is discussed. Key early and late steps in T-cell activation and the sites of action of immunosuppressive agents are reviewed. The required cellular interactions for the alloresponse and the targets of biologic agents used in transplants are reviewed. Special considerations for the immunology in neonates, infants, and children as recipients are provided. Understanding the immunobiology of transplantation is key to making decisions about children with transplants, developing better protocols, and creating tolerance in the future.
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Affiliation(s)
- Biagio A Pietra
- Division of Cardiology, Department of Pediatrics, The Children's Hospital, 1056 East 19th Avenue, Box B-100, Denver, CO 80212, USA.
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Kown MH, Miniati DN, Jahncke CL, Lijkwan MA, Murata S, Koransky ML, Blankenberg FG, Strauss HW, Robbins RC. Bcl-2-mediated inhibition of apoptosis in rat cardiac allografts worsens development of graft coronary artery disease. J Heart Lung Transplant 2003; 22:986-92. [PMID: 12957608 DOI: 10.1016/s1053-2498(02)01187-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
BACKGROUND We hypothesized that adenovirally mediated Bcl-2 transfection of donor hearts would reduce the apoptosis that occurs during acute rejection while worsening the development of chronic graft coronary artery disease (GCAD). METHODS PVG donor hearts were treated with either AdvBcl-2 or AdvNull virus before heterotopic transplantation into ACI rats. Bcl-2 expression was assessed on post-operative day 4 (POD) 4 by western blot. Apoptosis was measured using (99m)Technetium-bound-annexin V imaging and caspase 3 activity assay. Allograft survival was determined in a separate cohort of animals. Long-term-treated animals were then assessed for measures of GCAD on POD 90. RESULTS Western blot analysis showed upregulation of Bcl-2 expression in AdvBcl-2-treated hearts. (99m)Tc-annexin V images demonstrated decreased uptake in the AdvBcl-2 group (1.41 +/- 0.33% vs 1.94 +/- 0.37%, p = 0.026). Caspase 3 activity was also significantly lower in this treatment group (0.112 +/- 0.032 vs 0.204 +/- 0.096, p = 0.049). Allograft survival was similar in both groups, respectively (7.7 +/- 1.2 vs 6.8 +/- 1.5 days, p = 0.340). GCAD, as determined by percent luminal narrowing (5.9 +/- 6.1% vs 1.6 +/- 1.5%, p = 0.039), intima-to-media ratio (5.1 +/- 5.1% vs 1.5 +/- 1.7%, p = 0.040) and percent of affected vessels (15.1 +/- 9.9% vs 5.3 +/- 4.4%, p = 0.009), was higher for the AdvBcl-2 group. CONCLUSION Treatment of cardiac allografts with AdvBcl-2 resulted in a reduction of apoptosis that did not significantly improve short-term graft survival, but worsened chronic GCAD.
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Affiliation(s)
- Murray H Kown
- Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California, USA
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Lee P, Sata M, Lefer DJ, Factor SM, Walsh K, Kitsis RN. Fas pathway is a critical mediator of cardiac myocyte death and MI during ischemia-reperfusion in vivo. Am J Physiol Heart Circ Physiol 2003; 284:H456-63. [PMID: 12414449 DOI: 10.1152/ajpheart.00777.2002] [Citation(s) in RCA: 156] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Fas is a widely expressed cell surface receptor that can initiate apoptosis when activated by its ligand (FasL). Whereas Fas abundance on cardiac myocytes increases in response to multiple pathological stimuli, direct evidence supporting its role in the pathogenesis of heart disease is lacking. Moreover, controversy exists even as to whether Fas activation induces apoptosis in cardiac myocytes. In this study, we show that adenoviral overexpression of FasL, but not beta-galactosidase, results in marked apoptosis both in cultures of primary neonatal cardiac myocytes and in the myocardium of intact adult rats. Myocyte killing by FasL is a specific event, because it does not occur in lpr (lymphoproliferative) mice that lack functional Fas. To assess the contribution of the Fas pathway to myocardial infarction (MI) in vivo, lpr mice were subjected to 30 min of ischemia followed by 24 h of reperfusion. Compared with wild-type mice, lpr mice exhibited infarcts that were 62.3% smaller with 63.8% less myocyte apoptosis. These data provide direct evidence that activation of Fas can induce apoptosis in cardiac myocytes and that Fas is a critical mediator of MI due to ischemia-reperfusion in vivo.
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Affiliation(s)
- Peiyee Lee
- Department of Medicine (Molecular Cardiology), Albert Einstein College of Medicine, Bronx, New York 10461, USA
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Krupnick AS, Kreisel D, Popma SH, Balsara KR, Szeto WY, Krasinskas AM, Riha M, Wells AD, Turka LA, Rosengard BR. Mechanism of T cell-mediated endothelial apoptosis. Transplantation 2002; 74:871-6. [PMID: 12364869 DOI: 10.1097/00007890-200209270-00022] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Cytotoxic T lymphocyte (CTL)-mediated destruction of allogeneic vascular endothelium is important in the pathogenesis of both acute and chronic allograft rejection. Despite the importance of this phenomenon, the effector mechanisms responsible for endothelial cell killing are not well defined, and conflicting conclusions have been reached based on variation in experimental methodology. METHODS We used a recently described method for isolating mouse vascular endothelium to evaluate endothelial cell lysis by CTLs. Endothelial cell destruction was assessed in vitro both by 51Cr release and DNA fragmentation using wild-type and lpr (Fas deficient) endothelium of C3H/HeJ (H2(k)) mice by MHC alloantigen-specific T cells of wild-type, gld (Fas ligand deficient), and perforin-deficient mice on a C57BL/6 (H2(b)) background. RESULTS Although maximal lysis of 56.6+/-0.8% was seen when using wild-type targets and effectors, only a moderate decrease in apoptosis to 37.6+/-4.0% was detected when the Fas/Fas ligand death receptor pathway was eliminated. This decrease in cytotoxicity occurred despite the preserved functional capacity of this pathway. Alternatively, a significant decrease in cytotoxicity to 17.4+/-4.7% was seen when the perforin/granzyme exocytosis pathway was eliminated. CONCLUSIONS These data indicate that CTLs destroy vascular endothelium primarily by the perforin/granzyme exocytosis pathway with only a minor contribution to apoptosis by the Fas/Fas ligand death receptor pathway. These data are critical for the proper interpretation of studies evaluating acute and chronic allograft rejection and for the design of rational strategies to ameliorate vascular injury concomitant to the rejection process.
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Affiliation(s)
- Alexander S Krupnick
- Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA
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Yang CW, Faulkner GR, Wahba IM, Christianson TA, Bagby GC, Jin DC, Abboud HE, Andoh TF, Bennett WM. Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice. Am J Transplant 2002; 2:391-9. [PMID: 12123203 DOI: 10.1034/j.1600-6143.2002.20501.x] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
To define the mechanism of cyclosporine (CsA)-induced apoptosis, we investigated the expression of apoptosis-related genes in experimental chronic CsA nephrotoxicity. Mice on a low-salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30 mg/kg/day), and sacrificed at 1 and 4 weeks. Apoptosis was detected with deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain, and the expressions of apoptosis-related genes were evaluated by reverse transcription-polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54 +/- 41 vs. 3 +/- 3, p < 0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p < 0.01). The CsA group showed a significant increase in Fas-ligand mRNA (0.20 vs. 0.02 amol/microgram total RNA, p < 0.05) and Fas protein expression (146% vs. 95%, p < 0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05) and CPP32 mRNA (0.18 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 rho mol/microgram/h, p < 0.05) and CPP32 protease (15.6 vs. 2.7 rho mol/microgram/h, p < 0.05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl-2 protein increased significantly in the CsA group (5.3-fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas-ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis-related genes is associated with CsA-induced apoptotic cell death.
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Affiliation(s)
- Chul Woo Yang
- Division of Nephrology, Catholic University of Korea, Seoul, Korea.
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15
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Uchiyama H, Kishihara K, Minagawa R, Hashimoto K, Sugimachi K, Nomoto K. Crucial Fas-Fas ligand interaction in spontaneous acceptance of hepatic allografts in mice. Immunology 2002; 105:450-7. [PMID: 11985665 PMCID: PMC1782688 DOI: 10.1046/j.1365-2567.2002.01388.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The Fas/Fas ligand (FasL) system plays important roles in the immune system, including host immunoregulation and cytotoxicity. In this study, we investigated the involvement of Fas-FasL interactions in spontaneous acceptance of hepatic allografts in murine orthotopic liver transplantation. Liver transplantation between the C57BL/6 (B6, H-2b) donor and the MRL/Mp (MRL, H-2k) recipient was performed in various combinations of donor and recipient mice with wild type (+/+), Fas-mutant (lpr) or FasL-mutant (gld) genotypes. The prolongation and spontaneous acceptance of the fully allogeneic grafts in recipients was not observed in either MRL-lpr recipients with B6+/+ livers or MRL+/+ recipients with B6-gld livers. Moreover, the serum alanine aminotransferase (ALT) levels and the degree of cell infiltration into hepatic allografts on day 7 after transplantation were inversely correlated with the recipient survival time (in days). The donor-specific cytotoxic T-lymphocyte (CTL) activities of the graft-infiltrating cells (GICs) from MRL-gld recipients with B6+/+ livers were much lower than those from MRL+/+ or -lpr recipients on days 5 and 10 after transplantation. However, the CTL activities of the GICs from MRL+/+ and -gld recipients predominantly disappeared by day 15 after transplantation. Furthermore, the anti-donor CTL activities induced in MRL+/+ recipients were ascribed to CD8+ cells, and were not mediated by Fas-FasL interactions. These results strongly suggest that the Fas/FasL system plays a critical role for recipient immunoregulation, enabling recipients in accepting hepatic allografts by deletion of the donor-specific T cells, but not for CTL/target cell interaction in MRL+/+ recipients.
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Affiliation(s)
- Hideaki Uchiyama
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
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16
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Wiseman AC, Pietra BA, Kelly BP, Rayat GR, Rizeq M, Gill RG. Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2001; 167:5457-63. [PMID: 11673565 DOI: 10.4049/jimmunol.167.9.5457] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Recent studies using mouse models demonstrate that CD4(+) T cells are sufficient to mediate acute cardiac allograft rejection in the absence of CD8(+) T cells and B cells. However, the mechanistic basis of CD4-mediated rejection is unclear. One potential mechanism of CD4-mediated rejection is via elaboration of proinflammatory cytokines such as IFN-gamma. To determine whether IFN-gamma is a critical cytokine in CD4-mediated acute cardiac allograft rejection, we studied whether the expression of IFN-gamma receptors on the donor heart was required for CD4-mediated rejection. To investigate this possibility, purified CD4(+) T cells were transferred into immune-deficient mice bearing heterotopic cardiac allografts from IFN-gamma receptor-deficient (GRKO) donors. While CD4(+) T cells triggered acute rejection of wild-type heart allografts, they failed to trigger rejection of GRKO heart allografts. The impairment in CD4-mediated rejection of GRKO hearts appeared to primarily involve the efferent phase of the immune response. This conclusion was based on the findings that GRKO stimulator cells provoked normal CD4 proliferation in vitro and that intentional in vivo challenge of CD4 cells with wild-type donor APC or the adoptive transfer of in vitro primed CD4 T cells failed to provoke acute rejection of GRKO allografts. In contrast, unseparated lymph node cells acutely rejected both GRKO and wild-type hearts with similar time courses, illustrating the existence of both IFN-gamma-dependent and IFN-gamma-independent mechanisms of acute allograft rejection.
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Affiliation(s)
- A C Wiseman
- Division of Nephrology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA
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17
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Oh SI, Kim IW, Jung HC, Seo JW, Chae IH, Kim HS, Oh BH. CORRELATION OF FAS AND FAS LIGAND EXPRESSION WITH REJECTION STATUS OF TRANSPLANTED HEART IN HUMAN1. Transplantation 2001; 71:906-9. [PMID: 11349725 DOI: 10.1097/00007890-200104150-00015] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Activation of pro-apoptotic systems has been proven in rejection model of animal heart transplantation. The role of Fas and Fas ligand (FasL) in graft rejection is not fully understood, and the expression changes of these genes in human transplanted heart have not been elucidated. METHODS Endomyocardial biopsy samples were taken from 13 consecutive patients undergoing heart transplantation at various times, and they were classified into rejection (REJ, grade 3A or more) and lack of rejection (TOL, grade 1B or less) by International Society of Heart and Lung Transplantation rejection grade. Semiquantitative reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the status of Fas and FasL expression in each sample. RESULTS Fas was constitutively expressed both in REJ and TOL specimens (expression levels normalized by glyceraldehyde-3-phosphate dehydrogenase expression in semiquantitative reverse transcription-polymerase chain reaction of REJ vs. TOL, 0.842+/-0.096 vs. 0.848+/-0.103, P=0.776); however, FasL expression was detected in 66% of REJ samples and 40% of TOL samples. Normalized levels of FasL expression were 0.591+/-0.494 (REJ) and 0.383+/-0.507 (TOL) (P<0.05). FasL was expressed by cardiomyocytes as well as graft-infiltrating cells. CONCLUSIONS This up-regulation of FasL may be one of possible mechanisms of apoptosis in rejection process of human cardiac allograft.
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Affiliation(s)
- S I Oh
- Heart Research Institute, Department of Internal Medicine, Seoul National University College of Medicine, Korea
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18
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Trambley J, Lin A, Elwood E, Bingaman AW, Lakkis F, Corbascio M, Pearson TC, Larsen CP. FasL is important in costimulation blockade-resistant skin graft rejection. Transplantation 2001; 71:537-43. [PMID: 11258433 DOI: 10.1097/00007890-200102270-00009] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Simultaneous blockade of the CD40 and CD28 costimulatory pathways is effective in prolonging allograft survival in murine and primate models. Recent data suggest that intact apoptotic pathways are crucial for the induction of hyporesponsiveness by costimulation blockade. We have studied the impact of fas/fasL signaling, an important T cell apoptotic pathway, on the effects of costimulation blockade. Methods. Wild type, lpr (fas deficient), and gld (fasL deficient), mice were used as donors and recipients in the murine skin graft model. Allograft survival was compared in untreated and costimulation blockade (500 microg anti-CD40L and 500 microg CTLA4-Ig, days 0, 2, 4, 6) treated recipients. In some recipients, CD4+ T cells were depleted using rat anti-murine CD4 (100 microg day -3, -2, -1, and weekly). RESULTS gld mice treated with costimulation blockade enjoy a significantly greater increase in skin allograft survival than do wild-type mice. This effect is not replicated using lpr donors or recipients. Experiments in which CD4+ cells were depleted demonstrate that fasL is not necessary for CD8-mediated allograft rejection, and that depletion of CD4+ cells eliminates some of the survival advantage induced by costimulation blockade. CONCLUSIONS FasL is not required for the establishment of costimulation blockade induced hyporesponsiveness, but rather appears to be required for normal costimulation blockade resistant rejection. Fas expression is not critical for costimulation blockade resistant rejection, suggesting that fasL may be interacting with other receptors. Further, it appears that CD4+ cells are important in the maintenance of allograft protection induced by costimulation blockade in this model.
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Affiliation(s)
- J Trambley
- The Carlos and Marguerite Mason Transplantation Research Center, Department of Surgery, Emory University School of Medicine, Atlanta, GA 30322, USA
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19
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Watzlik A, Dufter C, Jung M, Opelz G, Terness P. Generation of FASL-expressing antigen-presenting cells for suppression of alloreactive T cells in the rat. Transplant Proc 2001; 33:599-601. [PMID: 11266975 DOI: 10.1016/s0041-1345(00)02159-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Affiliation(s)
- A Watzlik
- Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany
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20
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Abe R, Peng T, Sailors J, Bucala R, Metz CN. Regulation of the CTL response by macrophage migration inhibitory factor. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2001; 166:747-53. [PMID: 11145646 DOI: 10.4049/jimmunol.166.2.747] [Citation(s) in RCA: 96] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Macrophage migration inhibitory factor (MIF) has been shown to be a pivotal cytokine that mediates host inflammatory and immune responses. Recently, immunoneutralization of MIF has been found to inhibit tumor growth in mice; however, the contributing mechanisms underlying this effect have not been well defined. We investigated whether MIF plays a regulatory role in the expression of CTL activity. In a mouse model of the CTL response using the OVA-transfected tumor cell line EL4 (EG.7), we found that cultures of splenocytes obtained from EG.7-primed mice secrete high levels of MIF following Ag stimulation in vitro. Notably, parallel splenocyte cultures treated with neutralizing anti-MIF mAb showed a significant increase in the CTL response directed against EG.7 cells compared with control mAb-treated cultures. This effect was accompanied by elevated expression of IFN-gamma. Histological examination of the EG. 7 tumors from anti-MIF-treated animals showed a prominent increase in both CD4(+) and CD8(+) T cells as well as apoptotic tumor cells, consistent with the observed augmentation of CTL activity in vivo by anti-MIF. This increased CTL activity was associated with enhanced expression of the common gamma(c)-chain of the IL-2R that mediates CD8(+) T cell survival. Finally, CD8(+) T lymphocytes obtained from the spleens of anti-MIF-treated EG.7 tumor-bearing mice, when transferred into recipient tumor-bearing mice, showed increased accumulation in the tumor tissue. These data provide the first evidence of an important role for MIF in the regulation and trafficking of anti-tumor T lymphocytes in vivo.
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MESH Headings
- Adjuvants, Immunologic/administration & dosage
- Adoptive Transfer
- Animals
- Antibodies, Monoclonal/administration & dosage
- Antibodies, Monoclonal/pharmacology
- Antibodies, Monoclonal/therapeutic use
- CD8-Positive T-Lymphocytes/immunology
- Cell Movement/immunology
- Cells, Cultured
- Cytotoxicity Tests, Immunologic
- Cytotoxicity, Immunologic/immunology
- Female
- Injections, Intraperitoneal
- Lymphocytes, Tumor-Infiltrating/immunology
- Macrophage Migration-Inhibitory Factors/immunology
- Macrophage Migration-Inhibitory Factors/physiology
- Mice
- Mice, Inbred C57BL
- Neoplasm Transplantation
- T-Lymphocytes, Cytotoxic/immunology
- Thymoma/immunology
- Thymoma/pathology
- Thymoma/prevention & control
- Tumor Cells, Cultured/transplantation
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Affiliation(s)
- R Abe
- Laboratories of. Vascular Biology and Medical Biochemistry, The Picower Institute for Medical Research, Manhasset, NY 11030, USA
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21
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Diamond AS, Gill RG. An essential contribution by IFN-gamma to CD8+ T cell-mediated rejection of pancreatic islet allografts. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2000; 165:247-55. [PMID: 10861058 DOI: 10.4049/jimmunol.165.1.247] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
CD8+ T cells have long been considered to be the prototypical cytotoxic lymphocyte subpopulation. However, whether alloreactive CD8+ T cells require traditional cytolytic pathways such as perforin and Fas ligand (FasL) to mediate graft rejection has been a controversial issue. In the present studies, we examined the role of varied effector pathways in CD8+ T cell-mediated rejection of pancreatic islet allografts. Our goal was to systematically determine the relative requirements, if any, of perforin and FasL as well as the proinflammatory cytokine IFN-gamma in triggering graft destruction. To study CD8+ T cell effector pathways independently of other lymphocyte populations, purified alloreactive CD8+ T cells were adoptively transferred into severe combined immune-deficient (SCID) recipients bearing established islet allografts. Results indicate that to reject established islet allografts, primed CD8+ T cells do not require the individual action of the conventional cytotoxic effectors perforin and Fas ligand. In contrast, the ability to produce IFN-gamma is critical for efficient CD8+ T cell-mediated rejection of established islet allografts. Furthermore, alloreactive CD8+ TCR transgenic T cells (2C) also show IFN-gamma dependence for mediating islet allograft rejection in vivo. We speculate from these results that the production of IFN-gamma by alloreactive CD8+ T cells is a rate-limiting step in the process of islet allograft rejection.
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Affiliation(s)
- A S Diamond
- Department of Immunology and Medicine, Childhood Diabetes, University of Colorado Health Sciences Center, Denver 80262, USA
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22
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Wagener ME, Konieczny BT, Dai Z, Ring GH, Lakkis FG. Alloantigen-driven T cell death mediated by Fas ligand and tumor necrosis factor-alpha is not essential for the induction of allograft acceptance. Transplantation 2000; 69:2428-32. [PMID: 10868653 DOI: 10.1097/00007890-200006150-00037] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Fas ligand (FasL)-Fas and tumor necrosis factor alpha (TNFalpha)-tumor necrosis factor receptor (TNFR) interactions regulate immune responses and contribute to self-tolerance by mediating antigen-driven T cell apoptosis. It is not known whether FasL and TNFalpha, expressed by the recipient's lymphoid or nonlymphoid cells, are essential for the apoptosis of alloreactive T lymphocytes and the induction of allograft acceptance. METHODS We compared the survival of fully allogeneic vascularized cardiac allografts between wild-type (wt) and FasL-mutant (gld) recipient mice. In addition, we studied cardiac allograft survival in gld mice injected with TNFalpha-neutralizing antibody. Allograft acceptance (graft survival >100 days) was induced by treating the recipients with CTLA4Ig, a recombinant fusion protein that blocks B7-CD28 T cell costimulation. In vivo alloantigen-driven apoptosis of mature CD4+ and CD8+ T lymphocytes was analyzed in mice repeatedly stimulated with allogeneic splenocytes. RESULTS We found that CTLA4Ig induces 100% long-term acceptance of cardiac allografts in wt and gld mice. Similarly, CTLA4Ig induced 100% allograft acceptance in gld recipients injected with TNFalpha-neutralizing antibody. In vivo alloantigen-driven apoptosis of mature CD4+ and CD8+ T cells was significantly reduced in gld mice and in wt mice treated with anti-TNFalpha antibody. However, neutralizing TNFalpha activity in gld mice failed to abrogate alloantigen-driven T cell apoptosis. CONCLUSIONS These data indicate that: (1) FasL and TNFalpha expression are not obligatory for the induction of long-term allograft acceptance by CTLA4Ig and (2) FasL- and TNFalpha-independent death pathways contribute to alloantigen-driven T cell apoptosis.
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Affiliation(s)
- M E Wagener
- The Carlos and Marguerite Mason Transplantation Research Center, Department of Medicine, Emory University and the Medical and Research Services, Veterans Affairs Medical Center, Atlanta, GA 30033, USA
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23
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Soccal PM, Doyle RL, Jani A, Chang S, Akindipe OA, Poirier C, Pavlakis M. Quantification of cytotoxic T-cell gene transcripts in human lung transplantation. Transplantation 2000; 69:1923-7. [PMID: 10830232 DOI: 10.1097/00007890-200005150-00030] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Differentiating between acute rejection and cytomegalovirus (CMV) infection is one of the major challenges of lung transplantation. The aims of this study were to: (1) quantify the transcription of the cytotoxic T lymphocyte (CTL) effector molecules in the bronchoalveolar lavage (BAL) of lung transplant recipients and (2) evaluate the clinical usefulness of this technique. METHODS Sixty-six single-lung, double-lung, or heart-lung transplant patients were prospectively enrolled in the study. BAL was performed either for routine surveillance or for acute graft dysfunction. RNA was extracted from BAL cell pellets and underwent competitive reverse transcription-assisted polymerase chain reaction (RT-PCR) for perforin, granzyme B, granulysin, and Fas ligand. Gene transcript analysis was compared to clinical diagnosis established by conventional methods [BAL microbiological and transbronchial biopsy (TBB) analyses]. RESULTS After exclusion of several BAL according to the study criteria, 62 BAL were submitted for data analysis. Significantly higher expression of all the analyzed transcripts was found during CMV infection, compared with each of the other defined diagnostic categories, namely nonsignificant pathology, acute rejection, and nonviral pulmonary infection. CONCLUSION Quantification by competitive RT-PCR of the CTL effector molecule transcripts (perforin, granzyme B, granulysin, and Fas ligand) could represent a valuable tool for the differential diagnosis of graft dysfunction in lung transplantation.
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Affiliation(s)
- P M Soccal
- Department of Internal Medicine, Stanford University Medical Center, California 94305-5236, USA
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24
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Abstract
Based on early studies, it was hypothesized that expression of Fas ligand (FasL) by tumor cells enabled them to counterattack the immune system, and that transplant rejection could be prevented by expressing FasL on transplanted organs. More recent studies have indicated that the notion of FasL as a mediator of immune privilege needed to be reconsidered, and taught a valuable lesson about making broad conclusions based on small amounts of data.
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Affiliation(s)
- N P Restifo
- National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
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25
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Ke B, Coito AJ, Kato H, Zhai Y, Wang T, Sawitzki B, Seu P, Busuttil RW, Kupiec-Weglinski JW. Fas ligand gene transfer prolongs rat renal allograft survival and down-regulates anti-apoptotic Bag-1 in parallel with enhanced Th2-type cytokine expression. Transplantation 2000; 69:1690-4. [PMID: 10836382 DOI: 10.1097/00007890-200004270-00027] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Fas ligand (FasL) induces apoptosis of cells bearing Fas receptor, and may play a role in the acquisition of immune privilege. We have previously shown that adenovirus (Ad)-mediated FasL gene transfer significantly prolongs survival in a strongly major histocompatibility complex-incompatible rat kidney allograft model. This study analyzes putative mechanisms of FasL-mediated effects, with particular emphasis on Th1 and Th2 immune activation and Bag-1 expression, a Bcl-2-binding anti-apoptotic protein. METHODS Kidney transplants were performed in Wistar-Furth to Lewis rat combination. Donor kidneys were perfused in situ with Ad-FasL or Ad-beta-Gal, and then transplanted. Kidney allografts were harvested at days 2, 7, and 56 and were evaluated by hematoxylin and eosin and immunohistochemical staining. The expression of FasL, Bag-1, and Th1/Th2 cytokine genes was assessed by Northern blots, Western blots, and competitive template reverse-transcriptase polymerase chain reaction, respectively. RESULTS Intragraft expression of FasL was enhanced, whereas that of anti-apoptotic Bag-1 gene was diminished throughout, in Ad-FasL-transduced well-functioning renal allografts, compared with Ad-beta-Gal-treated rejecting controls. In parallel, the expression of mRNA coding for IL-2 and IFN-gamma remained depressed, whereas that of IL-4 and IL-10 reciprocally and progressively increased in the Ad-FasL animal group. CONCLUSIONS Prolonged survival in Ad-FasL-transduced rat renal allograft model correlates with down-regulation of Bag-1, a novel anti-apoptotic gene, and preferential Th2-type cytokine elaboration profile at the graft site. Because Th1-like cells are sensitive to FasL-mediated cytotoxic effects, T-cell apoptosis may preferentially spare Th2-like cells, with resultant prolonged graft survival.
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Affiliation(s)
- B Ke
- The Dumont-UCLA Transplant Center, Department of Surgery, UCLA School of Medicine, Los Angeles, California 90095, USA
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26
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Abstract
The Fas/Fas ligand (FasL) pathway has been shown to be important in T lymphocyte-mediated cell death and is a key peripheral immunoregulatory mechanism that limits expansion of antigen-activated lymphocytes. The expression of Fas by commonly transplanted organs such as the heart, lung, kidney, and liver suggests that these tissues may be targets of FasL-expressing allospecific cytotoxic T lymphocytes. In this review the current literature examining the Fas/FasL system as a potential cellular effector pathway in tissue injury is discussed. In addition to a deleterious role in destruction of graft tissue, Fas/FasL interactions may have a beneficial role in transplantation. Recent studies suggest that modulation of FasL in target tissue leads to deletion, via apoptosis, of graft infiltrating lymphoid cells. However, an equally compelling series of reports indicate that overexpression of FasL can lead to a heightened immune response. These data are reviewed in the context of strategies to achieve long term allograft survival.
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Affiliation(s)
- O M Martinez
- Department of Surgery, Stanford University School of Medicine, CA 94305, USA.
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27
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Gray DW. Observations regarding the effect of targeted gene deletions (knockouts) on graft rejection. Transplantation 2000; 69:694-8. [PMID: 10708137 DOI: 10.1097/00007890-200002270-00042] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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28
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Furukawa Y, Matsumori A, Hwang MW, Hirozane T, Ono K, Shioi T, Sasayama S. Cytokine gene expression during the development of graft coronary artery disease in mice. JAPANESE CIRCULATION JOURNAL 1999; 63:775-82. [PMID: 10553920 DOI: 10.1253/jcj.63.775] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Immunologic injury to heart allografts is an initial and essential event in the pathogenesis of graft coronary artery disease (GAD). A variety of cytokines expressed in heart allografts modify both acute rejection and chronic inflammation, and could contribute to the development of GAD. The present study investigated the gene expression of interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and Fas ligand in chronically rejecting DBA/2-to-B 10.D2 mouse heart allografts at defined intervals of 7, 14, 28, or 70 days after transplantation by semiquantitative reverse transcriptase-polymerase chain reaction. GAD developed gradually, showing the highest value for mean intima/media ratio at day 70. Fas ligand, and the Th1 cytokines IL-2 and IFN-gamma, were vigorously induced in allografts at day 7, when histology showed pronounced parenchymal rejection, and rapidly decreased by day 28. However, the level of mRNA expression of Th2 cytokines, IL-6 and IL-10, and other inflammatory cytokines, TNF-alpha and IL-1beta, were still elevated on day 28. The persistent expression of specific cytokines suggests an important role in chronic inflammation. Thus, a persistently high level expression of inflammatory cytokines could be associated with chronic inflammation in the allografts, which promotes the development of GAD.
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Affiliation(s)
- Y Furukawa
- Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Japan
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29
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Sieg S, Smith D, Kaplan D. Differential activity of soluble versus cellular Fas ligand: regulation by an accessory molecule. Cell Immunol 1999; 195:89-95. [PMID: 10448008 DOI: 10.1006/cimm.1999.1530] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Fas ligand induces apoptosis by binding to its receptor Fas. This process has been shown to be important for activation-induced cell death of T lymphocytes, homeostasis of T cell numbers, cytotoxicity, and the maintenance of immunological privilege. Fas ligand is a type II membrane protein that is cleaved by a metalloproteinase to produce an active, soluble molecule. It has been found that a variety of target cells are differentially sensitive to soluble and membrane-associated forms of Fas ligand. However, the explanation for this differential activity has not been determined. One proposed explanation for this differential activity is that membrane-associated Fas ligand is more efficiently aggregated than soluble Fas ligand. Another possibility that we have investigated is that accessory molecules may act to enhance the activity of cellular Fas ligand. We have transfected cells to express membrane-associated Fas ligand and have characterized clones of these transfected cells in terms of Fas ligand and ICAM-1 surface expression. Enhanced activity was associated with enhanced levels of both Fas ligand and ICAM-1. Moreover, inhibition of ICAM-1 modulated the activity of membrane-associated Fas ligand so that its cellular specificity was similar to that of soluble Fas ligand. Thus, ICAM-1 plays a significant role in regulating Fas ligand activity, and this role explains, at least in part, the different functional attributes of the soluble versus the cell-associated molecule.
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Affiliation(s)
- S Sieg
- Case Western Reserve University, 2109 Adelbert Road, Cleveland, Ohio 44106-4943, USA
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Ito A, Minagawa M, Tomiyama K, Ito M, Kawai K. Cytotoxic pathways in the skin allograft rejection by CD4+ T cells. Transplantation 1999; 68:97-100. [PMID: 10428275 DOI: 10.1097/00007890-199907150-00019] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Two major mechanisms of T cell-mediated cytotoxicity are known: perforin-dependent and Fas-dependent cytotoxic pathways. Previous studies in vitro demonstrated that CD4+ cytotoxic T lymphocytes use the Fas pathway as a primary cytotoxic mechanism, but the cytotoxic mechanisms used by CD4+ T cells in vivo are unclear. METHODS We examined the cytotoxic pathways of CD4+ T cells in vivo using a skin allograft model, in which athymic nu/nu mice were transplanted with skin allografts and reconstituted with purified CD4+T cells. Fas-deficient and perforin-deficient mice and anti-tumor necrosis factor (TNF)-alpha monoclonal antibody were used for inactivating each cytotoxic pathway in vivo. RESULTS The skin allografts from Fas-deficient mice were readily rejected by the athymic mice reconstituted with purified CD4+ T cells. Perforin-deficient CD4+ T cells could also reject Fas-deficient skin allografts. Furthermore, in vivo treatment with anti-TNF-alpha monoclonal antibody did not prevent the allograft rejection by CD4+ T cells in the absence of both Fas and perforin pathways. CONCLUSIONS These results indicate participation of undefined mechanisms other than Fas, perforin, and TNF-alpha pathways in CD4+ T cell-mediated cytotoxicity in vivo.
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Affiliation(s)
- A Ito
- Department of Dermatology, Niigata University School of Medicine, Japan
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Affiliation(s)
- J C Reed
- Burnham Institute, La Jolla, California 92037, USA
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El Ghalbzouri A, Drénou B, Blancheteau V, Choqueux C, Fauchet R, Charron D, Mooney N. An in vitro model of allogeneic stimulation of cord blood: induction of Fas independent apoptosis. Hum Immunol 1999; 60:598-607. [PMID: 10426277 DOI: 10.1016/s0198-8859(99)00040-3] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Cord blood is increasingly used in transplantation as it is a readily available source of progenitor cells and is reputed to generate less severe graft-versus-host disease (GVHD) than adult bone marrow. We have compared apoptosis of cord blood lymphocytes (CB) and adult lymphocytes (PBMC) after stimulation via HLA class I, HLA class II or CD3 in order to reproduce in vitro some of the stimuli occurring after allotransplantation. CB spontaneously apoptose more than PBMC ex vivo, stimulation via HLA class I dramatically increased CB apoptosis without altering viability of PBMC. Expression of Fas was markedly lower on CB than on PBMC and this difference was maintained even after activation. Fas ligand was expressed in CB and in PBMC. CB were activated via either HLA class I or class II molecules although proliferation was not observed. Only phorbol ester pre-activation allowed Fas to subsequently induce a death signal. Proliferation of PBMC via CD3 led to enhanced Fas signals. CB therefore differ from PBMC with regard to both spontaneous and activation induced apoptosis and either allo- or CD3 mediated stimulation. Finally, the apoptosis of CB via HLA-class I could have an important role in the moderation of graft-versus-host disease.
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Affiliation(s)
- A El Ghalbzouri
- INSERM U396, Labo. d'Immunogénétique Moléculaire, Institut Biomedical des Cordeliers, Paris, France
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Graft-Versus-Leukemia Effect and Graft-Versus-Host Disease Can Be Differentiated by Cytotoxic Mechanisms in a Murine Model of Allogeneic Bone Marrow Transplantation. Blood 1999. [DOI: 10.1182/blood.v93.8.2738.408k30_2738_2747] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Allogeneic bone marrow transplantation (allo-BMT) is associated with both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect. In the present study, we examined the contribution of cytotoxic effector mechanisms, which are mediated by tumor necrosis factor- (TNF-), Fas ligand (FasL), or perforin, to GVHD and GVL effect in a murine BMT model. Bone marrow cells plus spleen cells (BMS) from wild-type, FasL-defective, or perforin-deficient donors were transferred into lethally irradiated recipients in the parent (C57BL/6) to F1 (C57BL/6 × DBA/2) BMT model with or without prior inoculation of DBA/2 leukemia L1210 or P815 mast cytoma cells. The effect of anti–TNF- antibody administration was also examined. Whereas the defect or blockade of each cytotoxic pathway could ameliorate lethal acute GVHD, the GVL effect was differentially affected. The wild-type BMS recipients died of acute GVHD within 50 days without residual leukemia cells. The FasL-defective BMS recipients showed 60%< survival over 80 days without acute GVHD or residual leukemia cells. Administration of anti–TNF- antibody resulted in early leukemia relapse and the recipients died within 25 days with massive leukemia infiltration in the liver. The perforin-deficient BMS recipients died within 60 days with residual leukemia cells. These results suggest that blockade of the Fas/FasL pathway could be used for ameliorating GVHD without impairing GVL effect in allo-BMT.
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Graft-Versus-Leukemia Effect and Graft-Versus-Host Disease Can Be Differentiated by Cytotoxic Mechanisms in a Murine Model of Allogeneic Bone Marrow Transplantation. Blood 1999. [DOI: 10.1182/blood.v93.8.2738] [Citation(s) in RCA: 104] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Abstract
Allogeneic bone marrow transplantation (allo-BMT) is associated with both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect. In the present study, we examined the contribution of cytotoxic effector mechanisms, which are mediated by tumor necrosis factor- (TNF-), Fas ligand (FasL), or perforin, to GVHD and GVL effect in a murine BMT model. Bone marrow cells plus spleen cells (BMS) from wild-type, FasL-defective, or perforin-deficient donors were transferred into lethally irradiated recipients in the parent (C57BL/6) to F1 (C57BL/6 × DBA/2) BMT model with or without prior inoculation of DBA/2 leukemia L1210 or P815 mast cytoma cells. The effect of anti–TNF- antibody administration was also examined. Whereas the defect or blockade of each cytotoxic pathway could ameliorate lethal acute GVHD, the GVL effect was differentially affected. The wild-type BMS recipients died of acute GVHD within 50 days without residual leukemia cells. The FasL-defective BMS recipients showed 60%< survival over 80 days without acute GVHD or residual leukemia cells. Administration of anti–TNF- antibody resulted in early leukemia relapse and the recipients died within 25 days with massive leukemia infiltration in the liver. The perforin-deficient BMS recipients died within 60 days with residual leukemia cells. These results suggest that blockade of the Fas/FasL pathway could be used for ameliorating GVHD without impairing GVL effect in allo-BMT.
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Borson ND, Strausbauch MA, Kennedy RB, Oda RP, Landers JP, Wettstein PJ. Temporal sequence of transcription of perforin, Fas ligand, and tumor necrosis factor-alpha genes in rejecting skin allografts. Transplantation 1999; 67:672-80. [PMID: 10096521 DOI: 10.1097/00007890-199903150-00006] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Perforin, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha) have been implicated in cytolytic T lymphocyte (CTL) effector function. However, the relative roles of these effector molecules in allograft rejection are unclear, and there has been no rigorous quantitation of transcription of the respective genes throughout the period from transplantation to rejection. METHODS We orthotopically transplanted mouse tail skin allografts and estimated the numbers of transcripts of these genes expressed by graft-infiltrating T cells with rigorous quantitative, competitive reverse transcribed PCR (QC-RT-PCR) that enabled the comparison of transcription of different genes. RESULTS Perforin and FasL mRNA levels correlated closely with the rejection of allografts by normal hosts over the 4 days preceding rejection. Antibody-mediated depletion of host CD4+ T cells retarded perforin transcription and significantly suppressed FasL transcription, suggesting FasL was not required for allograft rejection. TNF-alpha transcription was the highest of these genes in this time period, but these levels were dwarfed by TNF-alpha transcription at 24 hr posttransplant when transcription in both autografts and allografts was 30-fold higher than in allografts on the day before rejection. Elimination of the function of these single or paired genes through genetic mutation or antibody treatment had no significant effect on the speed of rejection. CONCLUSIONS The levels of perforin and FasL transcription appeared to be related to the process of allograft rejection in normal hosts. However, TNF-alpha transcription was highest during the posttransplant period suggesting that the principal role of TNF-alpha is in wound-healing rather than the effector phase of rejection.
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Affiliation(s)
- N D Borson
- Department of Immunology, Mayo Foundation, Rochester, Minnesota 55905, USA
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Kayagaki N, Yamaguchi N, Nakayama M, Kawasaki A, Akiba H, Okumura K, Yagita H. Involvement of TNF-Related Apoptosis-Inducing Ligand in Human CD4+ T Cell-Mediated Cytotoxicity. THE JOURNAL OF IMMUNOLOGY 1999. [DOI: 10.4049/jimmunol.162.5.2639] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Abstract
TNF-related apoptosis-inducing ligand (TRAIL) has been identified as a member of the TNF family that induces apoptosis in a variety of tumor cells, but its physiological functions are largely unknown. In the present study, we examined the expression and function of TRAIL in human CD4+ T cell clones by utilizing newly established anti-human TRAIL mAbs. Human CD4+ T cell clones, HK12 and 4HM1, exhibited perforin-independent and Fas ligand (FasL)-independent cytotoxicity against certain target cells, including T lymphoma (Jurkat) and keratinocyte (HaCaT) cell lines, which are susceptible to TRAIL-mediated cytotoxicity. In contrast to FasL, the expression of which was inducible upon anti-CD3 stimulation, TRAIL was constitutively expressed on HK12 and 4HM1 cells, and no further increase was observed after anti-CD3 stimulation. Spontaneous cytotoxic activities of resting HK12 and 4HM1 cells against Jurkat and HaCaT cells were blocked by anti-TRAIL mAb but not by anti-FasL mAb, and bystander cytotoxic activities of anti-CD3-stimulated HK12 and 4HM1 cells were abolished by the combination of anti-TRAIL and anti-FasL mAbs. These results indicate a differential regulation of TRAIL and FasL expression on human CD4+ T cell clones and that TRAIL constitutes an additional pathway of T cell-mediated cytotoxicity.
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Affiliation(s)
- Nobuhiko Kayagaki
- *Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and
| | - Noriko Yamaguchi
- *Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and
- †Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corp., Tokyo, Japan
| | - Masafumi Nakayama
- *Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and
| | - Akemi Kawasaki
- *Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and
| | - Hisaya Akiba
- *Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and
- †Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corp., Tokyo, Japan
| | - Ko Okumura
- *Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and
- †Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corp., Tokyo, Japan
| | - Hideo Yagita
- *Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and
- †Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corp., Tokyo, Japan
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Seino K, Ogino T, Ju ST, Hamada H, Yagita H, Okumura K, Fukao K. Biological factors that affect CD95 ligand-mediated inflammation. Transplant Proc 1999; 31:893-5. [PMID: 10083393 DOI: 10.1016/s0041-1345(98)01824-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
MESH Headings
- Animals
- Antigens, Surface/genetics
- Antigens, Surface/immunology
- Fas Ligand Protein
- Inflammation/immunology
- Liver Neoplasms, Experimental/immunology
- Liver Neoplasms, Experimental/pathology
- Male
- Membrane Glycoproteins/genetics
- Membrane Glycoproteins/immunology
- Mice
- Mice, Inbred C3H
- Mice, Inbred C57BL
- Mice, Inbred MRL lpr
- Mice, Inbred Strains
- Neoplasm Transplantation
- Recombinant Proteins/immunology
- Transfection
- Transplantation, Homologous
- Transplantation, Isogeneic
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Affiliation(s)
- K Seino
- Department of Surgery, University of Tsukuba School of Medicine, Ibaraki, Japan
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38
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Djamali A, Odorico JS. Fas-mediated cytotoxicity is not required for rejection of murine nonvascularized heterotopic cardiac allografts. Transplantation 1998; 66:1793-801. [PMID: 9884278 DOI: 10.1097/00007890-199812270-00038] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
BACKGROUND Using mice with loss-of-function mutations in the Fas and Fas ligand (FasL) genes (lpr and gld, respectively) in transplantation experiments has resulted in contradictory findings concerning the role of Fas/FasL-mediated cytotoxicity in allograft rejection. The observation that these mutant mice develop an abnormal lymphocyte phenotype with increasing age that is hyporesponsive in vitro led us to examine the possibility that this characteristic might explain seemingly discordant observations in the literature. Therefore, to distinguish between the effects of Fas/FasL pathway disruption and the effects of immune senescence on in vivo cytotoxicity and allograft rejection, we evaluated the survival of cardiac allografts in gld, lpr, and wild-type mice of varying ages. METHODS Six- to 21-week-old C3H, C3H/HeJ-Fasl(gld), C57B1/6, and B6.MRL-Fas(lpr) recipients were transplanted with heterotopic, nonvascularized cardiac allografts from neonatal Balb/c, C3H, C57Bl/6, and B6.MRL-Fas(lpr) donors. Mixed lymphocyte reactions were performed in naive gld, lpr, and wild-type animals, 6 and 12 weeks of age. Rejected allografts in gld, lpr, and wild-type recipients and functioning syngeneic transplants were evaluated for intragraft apoptosis by a DNA fragmentation detection assay. RESULTS Graft survival was not significantly different between 6-week-old gld and lpr recipients and their respective wild-type controls. However, allograft rejection was delayed significantly in older (13-week) gld mice compared with age-matched wild-type mice (P=0.02) or young (6-week) gld animals (P=0.04). Similarly, 21-week-old lpr mice exhibited prolonged graft survival compared with 6-week-old lpr animals (P=0.01). Reduced alloreactive proliferative responses in 12-week-old gld and lpr mice were observed when compared with age-matched wild-type strains. Rejecting allografts displayed a similar level of intragraft apoptotic cells regardless of mutant or wild-type phenotype or age of recipient. CONCLUSIONS The findings of this study confirm that Fas/FasL-mediated cytotoxicity is not required for murine cardiac allograft rejection. Our findings also demonstrate that the observed delayed graft rejection in lpr and gld mice is a consequence of an age-related alteration of the immune system, specific to gld and lpr mice and associated with an in vivo and in vitro hyporeactivity to alloantigens.
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Affiliation(s)
- A Djamali
- Department of Surgery, University of Wisconsin Hospitals and Clinics, Madison 53792-7375, USA
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Josien R, Müschen M, Gilbert E, Douillard P, Heslan JM, Soulillou JP, Cuturi MC. Fas ligand, tumor necrosis factor-alpha expression, and apoptosis during allograft rejection and tolerance. Transplantation 1998; 66:887-93. [PMID: 9798699 DOI: 10.1097/00007890-199810150-00013] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
BACKGROUND Cytotoxic T cells can induce target cell lysis and apoptosis by different pathways. The interactions of CD95 antigen (Fas) with its ligand (CD95L) and of tumor necrosis factor (TNF)-alpha with its receptor (TNF-R1) lead to apoptotic cell death. Recently, conflicting studies have been published concerning the expression and the role of CD95L in allograft rejection and tolerance. METHODS In this study, the intragraft expression of CD95/CD95L and TNF-alpha and the frequency and distribution of apoptotic cells were compared in a model of heterotopic cardiac allograft in the rat in which recipients were either not treated (acute rejection) or pretreated with donor-specific blood transfusion (tolerant). RESULTS In the acutely rejected allografts, a peak in the expression of CD95L and TNF-alpha and in the number of apoptotic cells was observed during the first week after transplantation; apoptotic cells were confined to graft-infiltrating cells. In the tolerated allografts, however, levels of graft-infiltrating cell apoptosis and CD95L and TNF-alpha expression during the same period of time were dramatically lower. The expression of Fas was constitutive and was not modulated during acute rejection or tolerance. CONCLUSION This down-regulation of CD95L and TNF-alpha in allografts rendered tolerant by donor-specific transfusion suggests a role for apoptosis-inducing pathways in acute allograft rejection.
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Affiliation(s)
- R Josien
- INSERM U437 Immunointervention dans les Allo et Xénotransplantations, Nantes, France
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40
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Hong NM, Masuko-Hongo K, Sasakawa H, Kato T, Shirai T, Okumura K, Nishioka K, Kobata T. Amelioration of lymphoid hyperplasia and hypergammaglobulinemia in lupus-prone mice (gld) by Fas-ligand gene transfer. J Autoimmun 1998; 11:301-7. [PMID: 9776707 DOI: 10.1006/jaut.1998.0204] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We recently demonstrated that the transplantation of wild-type bone marrow cells into lupus-prone mice (gld), resulted in the normalization of autoimmune syndromes due to induction of direct elimination of pathogenic cells by apoptosis via Fas/Fas ligand (L) interactions. This finding supports the beneficial therapeutic effect of Fas-mediated apoptosis on autoimmunity in gld mice. To further establish the therapeutic effect of Fas-mediated apoptosis on autoimmunity, we investigated the effect of cells transfected with the FasL gene on autoimmune symptoms in gld mice. The FasL transfectants exhibited cytotoxic activity against gld splenocytes via the Fas/FasL system in vitro. In vivo administration of irradiated-FasL transfectants induced a reduction in hypergammaglobulinemia, the disappearance of lymphoid hyperplasia and of the accumulation of gld cells (B220+ T-cells). Furthermore, in situ nick end labelling analysis revealed that cells in the spleen and lymph nodes frequently underwent apoptosis. These results clearly indicate that FasL transfectants induce the apoptosis of the pathogenic cells responsible for hypergammaglobulinemia and lymphoid hyperplasia in gld mice by cell/cell interaction via the Fas/FasL system. Thus, ex vivo gene transfer of FasL may represent a new therapeutic strategy for autoimmunity caused by the FasL dysfunction.
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Affiliation(s)
- N M Hong
- Institute of Medical Science, St Marianna University School of Medicine, Kawasaki, 216, Japan
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Wever PC, Boonstra JG, Laterveer JC, Hack CE, van der Woude FJ, Daha MR, ten Berge IJ. Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection. Transplantation 1998; 66:259-64. [PMID: 9701275 DOI: 10.1097/00007890-199807270-00021] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Graft-infiltrating T-cell (GIC) lines cultured from biopsies obtained during acute renal allograft rejection exhibit donor-specific cytotoxicity toward proximal tubular epithelial cell (PTEC) lines cultured from corresponding biopsies. This system allows for study of the relative contributions of perforin/granzyme B (GrB)- and Fas ligand (FasL)-based cytotoxicity to killing of PTEC. METHODS Expression of perforin, GrB and FasL was analyzed by immunocytochemical staining of cytocentrifuge preparations of GIC lines cultured from 10 renal allograft biopsies. Specific inhibitors of the perforin/GrB- and FasL-based pathways were used in 51Cr release and apoptosis assays to determine their relative contributions to cytotoxicity of GIC lines toward corresponding donor PTEC lines. RESULTS Cells with a strong granular pattern were observed upon immunocytochemical staining of GIC lines with anti-perforin or anti-GrB monoclonal antibodies. A diffuse staining pattern was observed upon staining with anti-FasL polyclonal antibodies. Six of eight GIC lines cultured from biopsies with acute rejection showed cytotoxicity toward corresponding donor PTEC lines, whereas two GIC lines cultured from biopsies without rejection did not. Preincubation of cytotoxic GIC lines with concanamycin A, an inhibitor of the perforin/GrB-based pathway, caused inhibition of both lysis and apoptosis of PTEC. Inhibition was not observed upon incubation with monoclonal antibodies that inhibit Fas. CONCLUSIONS The perforin/GrB-based pathway is mainly responsible for cytotoxicity of GIC lines toward corresponding donor PTEC lines, suggesting that this pathway predominates in tubular epithelial cell destruction by cytotoxic T lymphocytes during acute renal allograft rejection in vivo.
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Affiliation(s)
- P C Wever
- Renal Transplant Unit, Academic Medical Center, University of Amsterdam, The Netherlands
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Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K, Kopiwoda S, Abrams MJ, Darkes M, Robbins RC, Maecker HT, Strauss HW. In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proc Natl Acad Sci U S A 1998; 95:6349-54. [PMID: 9600968 PMCID: PMC27696 DOI: 10.1073/pnas.95.11.6349] [Citation(s) in RCA: 370] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.
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Affiliation(s)
- F G Blankenberg
- Department of Radiology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5105, USA
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Holtz-Heppelmann CJ, Algeciras A, Badley AD, Paya CV. Transcriptional regulation of the human FasL promoter-enhancer region. J Biol Chem 1998; 273:4416-23. [PMID: 9468493 DOI: 10.1074/jbc.273.8.4416] [Citation(s) in RCA: 131] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The human FasL enhancer region was cloned and functionally characterized in transformed and primary T cells. Within the 2.3 kilobase pairs of the FasL untranslated region, the distal 3' 300-base pair portion contains a single transcription initiation site and confers basal and inducible transcriptional activity. Stimuli that increase [Ca2+]i such as CD3 cross-linking or ionomycin, but not activation of protein kinase C, were found to induce FasL enhancer transcription in a cyclosporin-sensitive manner. Moreover, calcineurin and NFAT, but not AP1, were identified as necessary and sufficient effectors in driving FasL transcription through an NFAT cis-acting motif (GGAAA). Additional modes of T cell activation such as CD4 cross-linking were also found to induce NFAT binding to the FasL enhancer region and to functionally transactivate its transcription. These results indicate that the induction of FasL gene transcription in T cells after CD3 or CD4 activation is selectively mediated by calcineurin and NFAT.
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Affiliation(s)
- C J Holtz-Heppelmann
- Department of Experimental Pathology, Mayo Clinic, Rochester, Minnesota 55905, USA
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44
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Boonstra JG, Wever PC, Laterveer JC, Bruijn JA, van der Woude FJ, ten Berge IJ, Daha MR. Apoptosis of acinar cells in pancreas allograft rejection. Transplantation 1997; 64:1211-3. [PMID: 9355845 DOI: 10.1097/00007890-199710270-00025] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
BACKGROUND Recently it has been recognized that apoptosis of target cells may occur during liver and kidney allograft rejection and is probably induced by infiltrating cells. Pancreas rejection is also characterized by a cellular infiltrate, however, the occurrence of apoptosis has not been investigated. We assessed whether pancreas rejection was associated with apoptosis of target cells and an influx of granzyme B (GrB)-positive or CD68-positive cells. METHODS Eighteen pancreas biopsies (10 of 18 with rejection) from 15 patients with a pancreas-kidney transplantation were stained with the in situ end-labeling method for apoptosis, and for CD3, GrB, and CD68. RESULTS Significantly more apoptotic acinar cells were found in biopsies with rejection when compared with biopsies without rejection. No difference was observed between the groups for GrB+ or CD68+ cells. CONCLUSION We conclude that pancreas rejection is associated with apoptosis of acinar cells, but not with an increased influx of GrB+ cells or macrophages.
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Affiliation(s)
- J G Boonstra
- Department of Nephrology, Leiden University Hospital, The Netherlands
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Kobata T, Takasaki K, Asahara H, Hong NM, Masuko-Hongo K, Kato T, Hirose S, Shirai T, Kayagaki N, Yagita H, Okumura K, Nishioka K. Apoptosis with FasL+ cell infiltration in the periphery and thymus of corrected autoimmune mice. Immunology 1997; 92:206-13. [PMID: 9415028 PMCID: PMC1364060 DOI: 10.1046/j.1365-2567.1997.00347.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Fas (CD95) ligand (L) is a death factor that binds to its receptor, Fas, and induces apoptotic cell death, a crucial process in immunological tolerance. gld (generalized lymphoproliferative disorder) mice, which have a point mutation in the FasL gene, develop spontaneous systemic autoimmune syndromes characterized by hypergammaglobulinaemia and lymphoid hyperplasia owing to accumulation of abnormal B220+ CD3+ cells. Transplantation of wild-type (wt) bone marrow cells into old gld mice on the same strain background results in normalization of autoimmune syndromes. We characterized the cellular mechanisms (functionally and histologically) of the above phenomena in gld mice after bone marrow transplantation (BMT) to determine the role of apoptosis via Fas/FasL interactions in inducing and maintaining self-tolerance in vivo. Activated splenocytes from wt and BMT (wt to gld) mice showed significant cytotoxic activity against Fas transfectant cells while those from BMT (gld to gld) mice did not. Cells in the thymus, spleen and lymph nodes of gld mice uniformly upregulated Fas expression and were sensitive to Fas-mediated apoptosis compared with those in wt mice. Cells sensitive to Fas-mediated apoptosis in gld mice resided not only among abnormal B220+ CD3+ cells but also among conventional lymphocytes. More importantly, histological analysis revealed that cells in the spleen, lymph nodes and thymus frequently underwent apoptosis with infiltration of FasL+ cells in BMT (wt to gld) mice compared with BMT (gld to gld) mice. Our results indicated that apoptosis via Fas/FasL interactions can directly eliminate pathogenic cells responsible for autoimmunity in the periphery and possibly in the thymus in vivo.
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Affiliation(s)
- T Kobata
- Rheumatology Program, St Marianna University School of Medicine, Kawasaki, Japan
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Kayagaki N, Yamaguchi N, Nagao F, Matsuo S, Maeda H, Okumura K, Yagita H. Polymorphism of murine Fas ligand that affects the biological activity. Proc Natl Acad Sci U S A 1997; 94:3914-9. [PMID: 9108079 PMCID: PMC20542 DOI: 10.1073/pnas.94.8.3914] [Citation(s) in RCA: 158] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Fas ligand (FasL) is a member of the tumor necrosis factor family and induces apoptosis in Fas (CD95)-bearing target cells. In this study, we generated several mAbs that react with mouse FasL (mFasL) and characterized their functional properties. One of these mAbs, K10, specifically reacted with mFasL derived from C57BL/6 (B6) mice, but not that from BALB/c mice as estimated by surface staining and blocking of cytotoxic activities of mFasL transfectants, suggesting a polymorphism of mFasL. Sequence analysis of mFasL cDNA from several strains revealed that BALB/c and DBA/2 mice have three nucleotide differences from the known B6 and C3H sequences, which result in two amino acid substitutions (Thr-184 --> Ala-184 and Glu-218 --> Gly-218) in the extracellular region. Analysis of the K10 reactivity and genotyping by PCR-restriction fragment length polymorphism revealed that inbred mice segregate into the following two allotypes: mFasL.1 (B6, C3H, MRL, SJL, NOD, NZB, NZW) and mFasL.2 (BALB/c, DBA/1, DBA/2). Interestingly, COS7 cells expressing BALB/c FasL lysed Fas-bearing target cells more efficiently than those expressing B6 FasL. Furthermore, BALB/c-derived CD8-FasL fusion protein, which is composed of the extracellular domains of human CD8alpha and mFasL, exhibited 9-fold higher specific activity than did B6-derived CD8-FasL. These results suggest that in mFasL.2 mice the Fas/FasL system works more effectively than in mFasL.1 mice.
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Affiliation(s)
- N Kayagaki
- Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
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Abstract
Activation of the cell-surface antigen CD95 induces apoptosis of CD95-bearing tumor cells. In this study, we investigated the antitumor effect of locally produced CD95 ligand (CD95L) on CD95-negative tumor cells in vivo. Introduction of CD95L cDNA into murine tumor cells did not affect growth in vitro but caused rejection in vivo. Neutrophils were primarily responsible for this rejection. A CD8 T cell-mediated protective immunity against subsequent challenge with parental tumor cells was also elicited. These results provide evidence for the potential utility of CD95L in tumor eradication and also reveal a proinflammatory function of CD95L.
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Affiliation(s)
- K Seino
- Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
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