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Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1deficiency ameliorated hepatocellular PMR induced with TNF plus d-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia–reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia–reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1–3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4–8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1−/−macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1–/–macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1–/–mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

Intracellular LPS sensing by caspase-4/5/11 triggers proteolytic activation of pore-forming gasdermin D (GSDMD), leading to pyroptotic cell death in Gram-negative bacteria-infected cells. Involvement of caspase-4/5/11 and GSDMD in inflammatory responses, such as lethal sepsis, makes them highly desirable drug targets. Using knock-in (KI) mouse strains, we herein provide genetic evidence to show that caspase-11 auto-cleavage at the inter-subunit linker is essential for optimal catalytic activity and subsequent proteolytic cleavage of GSDMD. Macrophages from caspase-11–processing dead KI mice (Casp11Prc D285A/D285A) exhibit defective caspase-11 auto-processing and phenocopy Casp11−/− and caspase-11 enzymatically dead KI (Casp11Enz C254A/C254A) macrophages in attenuating responses to cytoplasmic LPS or Gram-negative bacteria infection. GsdmdD276A/D276A KI macrophages also fail to cleave GSDMD and are hypo-responsive to inflammasome stimuli, confirming that the GSDMD Asp276 residue is a nonredundant and indispensable site for proteolytic activation of GSDMD. Our data highlight the role of caspase-11 self-cleavage as a critical regulatory step for GSDMD processing and response against Gram-negative bacteria.

Glioblastoma is the most malignant form of primary brain tumor in adults, with no effective therapy and a low survival rate. TRAIL is a member of the TNF family, which selectively induces apoptosis in certain neoplastic cells, but not normal cells. In this study, we investigated the sensitivity of 7 human glioblastoma cell lines to TRAIL and the expression in them of TRAIL receptors. TRAIL exhibited significant cytotoxicity in 5 of 7 glioma cell lines. These glioblastoma cell lines expressed TRAIL‐R2, but not TRAIL‐R1, R3, or R4. However, no correlation was observed between the TRAIL sensitivity and the TRAIL‐R2 expression level, suggesting that there is an additional determinant of TRAIL sensitivity. Treatments with NF‐κB inhibitors, such as LLnL, MG132, and SN50, significantly increased the sensitivity of glioma cells to TRAIL. These results suggested that activation of NF‐κB is a protective mechanism against TRAIL‐induced cell death in some glioma cells, and thus NF‐κB inhibitors may be useful to improve the clinical treatment of glioblastoma with TRAIL.

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) have been implicated in antitumor immunity and therapy. In the present study, we investigated the sensitivity of Philadelphia chromosome (Ph1)–positive leukemia cell lines to TRAIL- or FasL-induced cell death to explore the possible contribution of these molecules to immunotherapy against Ph1-positive leukemias. TRAIL, but not FasL, effectively induced apoptotic cell death in most of 5 chronic myelogenous leukemia–derived and 7 acute leukemia–derived Ph1-positive cell lines. The sensitivity to TRAIL was correlated with cell-surface expression of death-inducing receptors DR4 and/or DR5. The TRAIL-induced cell death was caspase-dependent and enhanced by nuclear factor κB inhibitors. Moreover, primary leukemia cells from Ph1-positive acute lymphoblastic leukemia patients were also sensitive to TRAIL, but not to FasL, depending on DR4/DR5 expression. Fas-associated death domain protein (FADD) and caspase-8, components of death-inducing signaling complex (DISC), as well as FLIP (FLICE [Fas-associating protein with death domain–like interleukin-1–converting enzyme]/caspase-8 inhibitory protein), a negative regulator of caspase-8, were expressed ubiquitously in Ph1-positive leukemia cell lines irrespective of their differential sensitivities to TRAIL and FasL. Notably, TRAIL could induce cell death in the Ph1-positive leukemia cell lines that were refractory to a BCR-ABL–specific tyrosine kinase inhibitor imatinib mesylate (STI571; Novartis Pharma, Basel, Switzerland). These results suggested the potential utility of recombinant TRAIL as a novel therapeutic agent and the possible contribution of endogenously expressed TRAIL to immunotherapy against Ph1-positive leukemias.

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) have been implicated in antitumor immunity and therapy. In the present study, we investigated the sensitivity of Philadelphia chromosome (Ph1)–positive leukemia cell lines to TRAIL- or FasL-induced cell death to explore the possible contribution of these molecules to immunotherapy against Ph1-positive leukemias. TRAIL, but not FasL, effectively induced apoptotic cell death in most of 5 chronic myelogenous leukemia–derived and 7 acute leukemia–derived Ph1-positive cell lines. The sensitivity to TRAIL was correlated with cell-surface expression of death-inducing receptors DR4 and/or DR5. The TRAIL-induced cell death was caspase-dependent and enhanced by nuclear factor κB inhibitors. Moreover, primary leukemia cells from Ph1-positive acute lymphoblastic leukemia patients were also sensitive to TRAIL, but not to FasL, depending on DR4/DR5 expression. Fas-associated death domain protein (FADD) and caspase-8, components of death-inducing signaling complex (DISC), as well as FLIP (FLICE [Fas-associating protein with death domain–like interleukin-1–converting enzyme]/caspase-8 inhibitory protein), a negative regulator of caspase-8, were expressed ubiquitously in Ph1-positive leukemia cell lines irrespective of their differential sensitivities to TRAIL and FasL. Notably, TRAIL could induce cell death in the Ph1-positive leukemia cell lines that were refractory to a BCR-ABL–specific tyrosine kinase inhibitor imatinib mesylate (STI571; Novartis Pharma, Basel, Switzerland). These results suggested the potential utility of recombinant TRAIL as a novel therapeutic agent and the possible contribution of endogenously expressed TRAIL to immunotherapy against Ph1-positive leukemias.

To define the normal physiological role for the TRAIL/Apo2L in vivo, we generated TRAIL/Apo2L gene-targeted mice. These mice develop normally and show no defects in lymphoid or myeloid cell homeostasis or function. Although TRAIL/Apo2L kills transformed cells in vitro, TRAIL/Apo2L^–/– mice do not spontaneously develop overt tumors at an early age. However, in the A20 B cell lymphoma-transferred tumor model, TRAIL/Apo2L^–/– mice are clearly more susceptible to death from overwhelming tumor burden, due to increased lymphoma load in the liver. A20 tumors are susceptible to TRAIL/Apo2L killing in vitro, indicating that TRAIL/Apo2L may act directly to control A20 cells in vivo. Despite the fact that TRAIL binds osteoprotegerin and osteoprotegerin-transgenic mice are osteopetrotic, TRAIL/Apo2L^–/– mice show no evidence of altered gross bone density, and no alterations in frequency or in vitro differentiationof bone marrow precursor osteoclasts. Moreover, leucine zipper TRAIL has no toxicity when repeatedly administered to osteoprotegerin^–/– mice. Thus, TRAIL/Apo2L is important in controlling tumors in vivo, but is not an essential regulator of osteoprotegerin-mediated biology, under normal physiological conditions.

Natural killer (NK) cells and interferon (IFN)-γ have been implicated in immune surveillance against tumor development. Here we show that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) plays a critical role in the NK cell–mediated and IFN-γ–dependent tumor surveillance. Administration of neutralizing monoclonal antibody against TRAIL promoted tumor development in mice subcutaneously inoculated with a chemical carcinogen methylcholanthrene (MCA). This protective effect of TRAIL was at least partly mediated by NK cells and totally dependent on IFN-γ. In the absence of TRAIL, NK cells, or IFN-γ, TRAIL-sensitive sarcomas preferentially emerged in MCA-inoculated mice. Moreover, development of spontaneous tumors in p53+/− mice was also promoted by neutralization of TRAIL. These results indicated a substantial role of TRAIL as an effector molecule that eliminates developing tumors.

Natural killer (NK) cells play an important role in early defense against viral infection. The cytotoxic activity of NK cells is increased by interferon-α/β (IFN-α/β), produceden masse in virally infected cells. However, the mechanism(s) by which IFN-α/β contribute to the NK-cell-mediated antiviral response is not well understood. Here we provide evidence that the cytotoxicity of NK cells is enhanced by IFN-α/β through induction of TNF-related apoptosis-inducing ligand (TRAIL). Isolation and analysis of the murine TRAIL promoter revealed the presence of an IFN-stimulated response element (ISRE), which binds to the transcription factor ISGF3 (interferon stimulated gene factor-3). This promoter is indeed activated by IFN-β in ISGF3-dependent manner. We also show that virally infected cells, but not uninfected cells, are susceptible to TRAIL-mediated cytotoxicity in vitro, and that the TRAIL expressed in NK cells is indeed crucial in limiting virus replication in vivo. Thus, our study reveals a new molecular link between IFN-α/β signaling and activation of NK cells in antiviral response of the host.

To elucidate the molecular mechanism(s) involved in the TRAIL-induced apoptosis sensitivity, we conducted the following experiments utilizing TRAIL-sensitive and -resistant glioma cells. We examined the expression of TRAIL receptors mRNA, but no significant differences were detected in those cells. TRAIL-resistant cells were sensitized to TRAIL-induced apoptosis by staurosporine pretreatment and preferentially expressed PKCε. Since several lines of evidence suggest that PKC may play a protective role for apoptosis, we analyzed the involvement of PKCε in TRAIL-induced apoptosis by an adenovirus vector expression system. We found that TRAIL susceptibility was augmented by the expression of a dominant negative PKCε in TRAIL-resistant cells. Conversely, PKCε introduction in TRAIL-sensitive cells resulted in the reduction of TRAIL-induced apoptosis. Taken together, these data suggest that PKCε may be a regulator of susceptibility to TRAIL-induced apoptosis in gliomas and probably other malignancies.

Interferon-α (IFN-α) exerts the anti-tumour effect on various tumours at least partly through induction of apoptosis. Apoptosis is induced by members of the tumour necrosis factor (TNF) family, including Fas (CD95) and TNF-related apoptosis-inducing ligand (TRAIL). In the present study, we examined whether the TRAIL/TRAIL-R system is involved in IFN-α-induced apoptosis using Daudi B lymphoma cells. IFN-α upregulated the expression of TRAIL within 12h, as assessed by flow cytometry and RT-PCR, and the level increased with time until 72h. The levels of both TRAIL-R1 and TRAIL-R2, low in Daudi cells, were enhanced by IFN-α. The enhanced TRAIL-R1/-R2 appeared to function as a death-inducing molecule since IFN-α-stimulated cells were more susceptible to TRAIL-induced cell death. The IFN-α-stimulated Daudi cells or their derived culture supernatants displayed cytotoxicity against TRAIL-sensitive, but not resistant lines. Moreover, the IFN-α-induced reduction in mitochondrial membrane potential preceding the induction of apoptosis was substantially prevented by neutralizing anti-TRAIL monoclonal antibody. Taken together, IFN-α-induced apoptosis appears to be mediated by the autocrine and/or paracrine loop involving TRAIL/TRAIL-R.

Human Vα24NKT cells are activated by α-galactosylceramide (α-GalCer)-pulsed dendritic cells in a CD1d-dependent and a T-cell receptor–mediated manner. Here, we demonstrate that CD4+Vα24NKT cells derived from a patient with acute myeloid leukemia (AML) M4 are phenotypically similar to those of healthy donors and, in common with those derived from healthy donors, express tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) when the cells are activated by α-GalCer–pulsed dendritic cells but not prior to activation. We also show that myeloid leukemia cells from patients with AML M4, but not from patients with AML M0 or M1, undergo apoptosis following culture with TRAIL-expressing autologous or allogeneic healthy donor Vα24NKT cells. Apoptosis of AML M4 leukemia cells from patient peripheral blood was almost completely blocked by a neutralizing monoclonal antibody against TRAIL, indicating that TRAIL on Vα24NKT cells is essential for the induction of apoptosis in AML M4 leukemia cells. A nonobese diabetic–severe combined immunodeficient human leukemia (AML M4) model showed that human activated CD4+Vα24NKT cells induced apoptosis of human leukemia cells in vivo. This is the first evidence that activated Vα24NKT cells express TRAIL and that TRAIL causes apoptosis of monocytic leukemia cells from patients with AML M4 in vitro and in vivo. Adoptive immune therapy with activated Vα24NKT cells, or other strategies to increase activated Vα24NKT cells in vivo, may be of benefit to patients with AML M4.

Human Vα24NKT cells are activated by α-galactosylceramide (α-GalCer)-pulsed dendritic cells in a CD1d-dependent and a T-cell receptor–mediated manner. Here, we demonstrate that CD4+Vα24NKT cells derived from a patient with acute myeloid leukemia (AML) M4 are phenotypically similar to those of healthy donors and, in common with those derived from healthy donors, express tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) when the cells are activated by α-GalCer–pulsed dendritic cells but not prior to activation. We also show that myeloid leukemia cells from patients with AML M4, but not from patients with AML M0 or M1, undergo apoptosis following culture with TRAIL-expressing autologous or allogeneic healthy donor Vα24NKT cells. Apoptosis of AML M4 leukemia cells from patient peripheral blood was almost completely blocked by a neutralizing monoclonal antibody against TRAIL, indicating that TRAIL on Vα24NKT cells is essential for the induction of apoptosis in AML M4 leukemia cells. A nonobese diabetic–severe combined immunodeficient human leukemia (AML M4) model showed that human activated CD4+Vα24NKT cells induced apoptosis of human leukemia cells in vivo. This is the first evidence that activated Vα24NKT cells express TRAIL and that TRAIL causes apoptosis of monocytic leukemia cells from patients with AML M4 in vitro and in vivo. Adoptive immune therapy with activated Vα24NKT cells, or other strategies to increase activated Vα24NKT cells in vivo, may be of benefit to patients with AML M4.

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-γ production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, α-galactosylceramide (α-GalCer), a powerful inducer of NKT cell IFN-γ and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-γ–deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and α-GalCer were strictly IFN-γ dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-γ–mediated antimetastatic effects of IL-12 and α-GalCer.

Apoptosis is a key for CD4+ T cell destruction in HIV-1–infected patients. In this study, human peripheral blood lymphocyte (PBL)-transplanted nonobese diabetic (NOD)-severe combined immunodeficient (SCID) (hu-PBL-NOD-SCID) mice were used to examine in vivo apoptosis after HIV-1 infection. As the hu-PBL-NOD-SCID mouse model allowed us to see extensive infection with HIV-1 and to analyze apoptosis in human cells in combination with immunohistological methods, we were able to quantify the number of apoptotic cells with HIV-1 infection. As demonstrated by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL), massive apoptosis was predominantly observed in virus-uninfected CD4+ T cells in the spleens of HIV-1–infected mice. A combination of TUNEL and immunostaining for death-inducing tumor necrosis factor (TNF) family molecules indicated that the apoptotic cells were frequently found in conjugation with TNF-related apoptosis-inducing ligand (TRAIL)-expressing CD3+CD4+ human T cells. Administration of a neutralizing anti-TRAIL mAb in HIV-1–infected mice markedly inhibited the development of CD4+ T cell apoptosis. These results suggest that a large number of HIV-1–uninfected CD4+ T cells undergo TRAIL-mediated apoptosis in HIV-infected lymphoid organs.

TWEAK, a new member of the tumor necrosis factor (TNF) family, induces cell death in some tumor cell lines, but its physiological functions are largely unknown. In this study, we investigated the expression and function of TWEAK in human peripheral blood mononuclear cells (PBMCs) by using newly generated anti–human TWEAK mAbs. Although freshly isolated PBMCs expressed no detectable level of TWEAK on their surfaces, a remarkable TWEAK expression was rapidly observed on monocytes upon stimulation with interferon (IFN)-γ but not with IFN-α or lipopolysaccharide. Cytotoxic activity of IFN-γ–stimulated monocytes against human squamous carcinoma cell line HSC3 was inhibited partially by anti-TWEAK mAb alone and almost completely by combination with anti-TRAIL (TNF-related apoptosis-inducing ligand) mAb. These results revealed a novel pathway of monocyte cytotoxicity against tumor cells that is mediated by TWEAK and potentiated by IFN-γ.

Since Fas ligand (FasL) can induce apoptosis of Fas-bearing cells, Fas/FasL interactions can play a critical role in maintaining self-tolerance. Fas/FasL interactions in lupus-like autoimmune disease have been well characterized in studies using either Fas or FasL mutant mice. However, the effect of the defective FasL-mediated signaling on the establishment of lupus in other mouse strains, such as NZB/W (B/W) F1, remains uncertain. In the present study, we examined the effect of anti-FasL monoclonal antibody (mAb) on the development of lupus. Treatment of B/W F1 mice with anti-FasL mAb augmented IgG1- and IgG2a-type anti-dsDNA Ab production. However, treatment of B/W F1 mice with anti-FasL mAb also significantly prevented the development of lupus nephritis. These results indicate that Fas/FasL interactions not only regulate IgG-type autoantibody production, but also influence the development of lupus nephritis in B/W F1 mice.

Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1β. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora.

Autoreactive T lymphocytes probably cause pancreatic β-cell death by inducing apoptosis. To visualize apoptotic β-cells in vivo, we accelerated diabetes of NOD mice with cyclophosphamide (CY) or adoptive transfer. We also studied whether Fas-mediated apoptosis is involved in the development of diabetes by administrating anti-Fas ligand (FasL) Ab and by grafting Fas-deficient neonatal pancreas from NOD- lpr/lpr mice. Apoptotic cells were clearly shown 8 days after CY treatment. β-cell apoptosis was also observed after adoptive transfer but in a different kinetic pattern. Anti-FasL Ab administration failed to inhibit diabetes after CY treatment or adoptive transfer, while it inhibited Con A-induced hepatitis. Fas-deficient neonatal pancreata were destroyed by lymphocytic infiltration in diabetic NOD mice. Our results clearly demonstrate apoptosis of β-cells in accelerated diabetes and indicate that Fas-FasL interaction is not involved in diabetes of NOD mice, contrary to the previous reports.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a proapoptotic member of the TNF family of type II membrane proteins, which constitutes one component of T cell cytotoxicity. In this study, we investigated the expression and function of TRAIL in human peripheral blood T (PBT) cells. Although freshly isolated PBT cells did not express a detectable level of TRAIL on their surface, a remarkable TRAIL expression was rapidly induced on the surface of both CD4+ and CD8+ PBT cells upon stimulation with anti-CD3 monoclonal antibody and type I interferons (IFNs). This enhancement of TRAIL expression was a unique feature of type I IFNs (IFN-α and IFN-β), and neither type II IFN (IFN-γ) nor various other cytokines enhanced TRAIL expression on anti-CD3–stimulated PBT cells. Type I IFNs have been used for clinical treatment of renal cell carcinomas (RCCs), and we found that most RCC cell lines were susceptible to TRAIL-induced apoptosis. Type I IFNs substantially augmented cytotoxic activity of anti-CD3–stimulated PBT cells against RCC cell lines in a TRAIL-dependent manner. These results indicate a unique feature of type I IFNs to regulate TRAIL-mediated T cell cytotoxicity, which may be involved in the antitumor effects of type I IFNs against various tumors.

Both tumor necrosis factor a (TNFa) and Fas ligand (FasL) have been implicated in the pathogenesis of graft-versus-host disease (GVHD). In this study, we examined the ameliorating effects of neutralizing anti-FasL and/or anti-TNFa monoclonal antibody (MoAb) in a lethal acute GVHD model in mice. Whereas the treatment with either anti-FasL or anti-TNFa MoAb alone significantly delayed the mortality and improved the body weight, a complete protection was achieved by the administration of both MoAbs. Pathological examination indicated differential effects of anti-FasL or anti-TNFa MoAb on GVHD-associated pathologies. Hepatic lesion was improved by anti-FasL but not anti-TNFa MoAb. In contrast, intestinal lesion was improved by anti-TNFa but not anti-FasL MoAb. Cutaneous and splenic lesions were improved by either MoAb. The combination of both MoAbs improved all these lesions. These results indicate that FasL and TNFa differentially contribute to the GVHD pathologies and a complete protection from mortality can be achieved by neutralization of both FasL and TNFa.

Fas ligand (FasL), a cell surface molecule belonging to the tumour necrosis family, induces apoptosis through its receptor, Fas antigen (Fas). Germinal centre B cells strongly express Fas, but the role of the Fas–FasL system in B‐cell selection in the germinal centre remains unclear. In the present study, FasLmRNA in the tonsils was examined by RNA in situhybridization. FasLmRNA was detected in the lymphocytes of both germinal centres and interfollicular areas, but much more intensively in the former. The distribution of cells strongly expressing FasLmRNA in the germinal centres was quite similar to that of CD45RO‐positive T cells. Immunohistochemically, however, most of the germinal centre cells were positive for FasL. Flow cytometric analysis demonstrated that FasL‐positive cells of the tonsils included not only CD3‐positive/CD45RO‐positive T cells, but also CD19‐positive B cells. This finding therefore suggests either that germinal centre B cells can produce FasL, although the level of mRNA was equivocal, or that the soluble form of FasL may be released from FasL‐positive T cells in the germinal centres and then bind to Fas‐positive germinal centre B cells. Thus, the Fas–FasL system may participate in the positive selection of B cells. © 1997 by John Wiley & Sons, Ltd.

Fas ligand (FasL) has been shown to be processed by the action of certain metalloproteinase and released from the cell surface. However, it is unclear whether death of Fas-sensitive target cells is mediated by a membrane-bound form of FasL (mFasL) or by a soluble form of FasL (sFasL). In the present study, we demonstrated that JCaM, a p56lck-deficient mutant of Jurkat, underwent Fas-dependent apoptosis only upon physical contact with anti-CD3-stimulated Jurkat cells or with human FasL- expressing transfectant (hFas/L5178Y). Recombinant FasL or sFasL-containing supernatant failed to induce apoptosis in both Jurkat and JCaM. Moreover, addition of a metalloproteinase inhibitor, which led to the accumulation of mFasL in hFas/L5178Y, was found to augment apoptosis in both Jurkat and JCaM. These findings indicate that, in a physiologic setting represented by the activation-induced cell death in Jurkat T cells, cell–cell contact appears to be required for the induction of Fas-mediated killing.

Fas ligand (FasL) is a type II membrane protein which belongs to the tumor necrosis factor family. Ligation of its receptor (Fas/APO-1/CD95) by FasL induces apoptosis of Fas-expressing cells. However, thein vivofunction of these molecules in cutaneous immunity is presently unknown. In the present study, we investigated the involvement of Fas and FasL in the pathogenesis of cutaneous lupus by immunohistochemical methods. In normal skin, expression of Fas was observed on keratinocytes in the basal to granular layers. Unexpectedly, FasL was constitutively expressed on histiocytes in the dermis. In specimens of cutaneous lupus, Fas was expressed on infiltrating lymphocytes, as well as on keratinocytes as observed in normal skin. FasL was expressed on a portion of infiltrating CD4+T cells and on histiocytes more frequently than those in normal skin. Double staining indicated that these FasL-expressing histiocytes were CD68 positive macrophages. Especially, FasL-expressing macrophages were distributed around appendages such as hair follicles. These results suggest the Fas/FasL interaction may be involved in the destruction of hair follicles which is a characteristic feature of cutaneous lupus.

Tumor necrosis factor (TNF ) and Fas ligand (FasL) have been implicated in the pathogenesis of graft-versus-host disease (GVHD), which is a major complication after allogeneic bone marrow transplantation. We examined here the ameliorating effect of a metalloproteinase inhibitor (KB-R7785) that inhibits TNF-a and FasL release in a lethal acute GVHD model in mice. Administration of KB-R7785 into (BALB/c × C57BL/6) F1 that received C57BL/6 spleen cells markedly reduced the mortality and weight loss in association with minimal signs of GVHD pathology in the liver, intestine, and hematopoietic tissues. The ameliorating effect of KB-R7785 was superior to that of anti–TNF-a antibody. Our results suggest that KB-R7785 could be a potent therapeutic agent for GVHD.

Fas ligand (FasL), a cell surface molecule belonging to the tumour necrosis family, induces apoptosis through its receptor, Fas antigen (Fas). Germinal centre B cells strongly express Fas, but the role of the Fas–FasL system in B-cell selection in the germinal centre remains unclear. In the present study, FasL mRNA in the tonsils was examined by RNA in situ hybridization. FasL mRNA was detected in the lymphocytes of both germinal centres and interfollicular areas, but much more intensively in the former. The distribution of cells strongly expressing FasL mRNA in the germinal centres was quite similar to that of CD45RO-positive T cells. Immunohistochemically, however, most of the germinal centre cells were positive for FasL. Flow cytometric analysis demonstrated that FasL-positive cells of the tonsils included not only CD3-positive/CD45RO-positive T cells, but also CD19-positive B cells. This finding therefore suggests either that germinal centre B cells can produce FasL, although the level of mRNA was equivocal, or that the soluble form of FasL may be released from FasL-positive T cells in the germinal centres and then bind to Fas-positive germinal centre B cells. Thus, the Fas–FasL system may participate in the positive selection of B cells. © 1997 by John Wiley & Sons, Ltd.

Both tumor necrosis factor α (TNFα) and Fas ligand (FasL) have been implicated in the pathogenesis of graft-versus-host disease (GVHD). In this study, we examined the ameliorating effects of neutralizing anti-FasL and/or anti-TNFα monoclonal antibody (MoAb) in a lethal acute GVHD model in mice. Whereas the treatment with either anti-FasL or anti-TNFα MoAb alone significantly delayed the mortality and improved the body weight, a complete protection was achieved by the administration of both MoAbs. Pathological examination indicated differential effects of anti-FasL or anti-TNFα MoAb on GVHD-associated pathologies. Hepatic lesion was improved by anti-FasL but not anti-TNFα MoAb. In contrast, intestinal lesion was improved by anti-TNFα but not anti-FasL MoAb. Cutaneous and splenic lesions were improved by either MoAb. The combination of both MoAbs improved all these lesions. These results indicate that FasL and TNFα differentially contribute to the GVHD pathologies and a complete protection from mortality can be achieved by neutralization of both FasL and TNFα.

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.

The serum level of soluble Fas (sFas) molecules in 35 patients with SLE was determined by enzyme-linked immunosorbent assay (ELISA) and its relation to other lymphocyte activation markers and clinical parameters was examined. The level of sFas increased significantly compared to that in normal subjects, consistent with previous reports. There was a significant correlation between the level of sFas and that of sCD4, suggesting some relation between sFas and activation of CD4+ T cells. Patients with lymphopenia tended to have low levels of sFas, making it possible to hypothesize that sFas protects against apoptosis. Although the change in the level of sFas with steroid therapy was variable, some relation to the differential activation of T cell subsets was suggested.

Soluble forms of CD95 and CD95 ligand (sCD95 and sCD95L, respectively) can increase in the serum of patients with some inflammatory disease. In this study, we investigated the serum levels of sCD95 and sCD95L in liver transplantation recipients.

Pyroptosis requires the induction of gasdermin D expression by the transcription factor IRF2.