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J Tschopp

Publications and source records attributed to J Tschopp.

At least 145 records · Page 8Linked to original sources
PubMed1995

Demonstration of granzyme A and perforin messenger RNA in the synovium of patients with rheumatoid arthritis.

OBJECTIVE: To examine the gene expression of 2 highly specific markers of cytotoxic T lymphocyte (CTL) activation, the serine protease granzyme A and the pore-forming protein perforin, in synovial tissue of patients with rheumatoid arthritis (RA), and to compare the findings with those in osteoarthritis (OA) synovial tissue. METHODS: Snap-frozen synovial tissue specimens from 9 patients with RA and 5 patients with OA were examined. The number of CTL that expressed granzyme A or perforin messenger RNA was determined by in situ hybridization using nonradioactive riboprobes for granzyme A and perforin, and by a novel in situ reverse transcriptase technique. The signals were visualized by an immunogold-silver immunohistochemistry technique and compared with immunohistochemical labeling of T and B cells. Additional double-labeling was achieved using anti-type IV collagen, anti-macrophage (anti-CD68), anti-T lymphocyte (anti-CD45RO), anti-B lymphocyte (anti-CD20), and anti-natural killer cell (anti-CD56) antibodies in an alkaline phosphatase-anti-alkaline phosphatase assay. RESULTS: Granzyme A and perforin messenger RNA (mRNA) was observed in CTL in synovial specimens from all of the RA patients, whereas in specimens from OA patients only a few, single cells with a positive mRNA signal for these molecules could be detected. In the RA specimens, the number of lymphocytes showing a positive mRNA signal for granzyme A or perforin varied from 10% to 50%, reflecting the recent findings of other investigators studying synovial fluid. CONCLUSION: Our results demonstrate that gene expression of at least 2 CTL products, granzyme A and perforin, is up-regulated in the synovium of patients with RA compared with that in the synovium of patients with OA. These molecules presumably play an important role not only in lymphocyte-mediated cytotoxicity, but also in facilitating the migration of blood-borne mononuclear cells through the vascular basement membrane into the rheumatoid synovium.

Adult↗
PubMed1995

Down-modulation of CD4+ T helper type 2 and type 0 cells by T helper type 1 cells via Fas/Fas-ligand interaction.

Fas was recently demonstrated to be the major target molecule engaged by CD4+ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4+ or CD8+ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4+ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4+ CTL and a CD8+ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4+ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.

Animals↗
PubMed1995

Regulation of Fas(Apo-1/CD95)- and perforin-mediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2.

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.

Animals↗
PubMed1995

Comparative molecular modelling of the Fas-ligand and other members of the TNF family.

A number of proteins with significant similarity to the tumour necrosis factor (TNF) have been identified over the last years. Upon interaction with their cognate receptor (members of the TNF-receptor family), all members of this protein family induce either cell death or proliferation/differentiation of the receptor-bearing cells. One of the last identified members of the TNF family is the apoptosis-inducing ligand of the Fas-receptor, termed Fas-ligand (FasL). Here we report the cloning and sequencing of the mouse cDNA for the FasL. Using knowledge-based protein modelling, we demonstrate that all members of the TNF family form trimeric complexes, and define the residues located at the subunit interfaces. The resulting structurally corrected multiple sequence alignment allows the identification of residues potentially involved in receptor recognition, and should help design mutagenesis experiments for structure-function relationship studies.

Amino Acid Sequence↗
PubMed1995

Characterization of the non-functional Fas ligand of gld mice.

Mice homozygous for either the gld or lpr mutation develop autoimmune diseases and progressive lymphadenopathy. The lpr mutation is characterized by the absence of functional Fas, whereas gld mice exhibit an inactive FasL due to a point mutation proximal to the extracellular C-terminus. The structural repercussions of this amino acid substitution remain unknown. Here we report that FasL is expressed at similar levels on the surface of activated T lymphocytes from gld and wild-type mice. Using a polyclonal anti-FasL antibody, indistinguishable amounts of a 40 kDa protein are detected in both gld and wild-type splenocytes. The molecular model of FasL, based on the known structure of TNF-alpha, predicts that the Phe --> Leu gld mutation is located at the protomer interface which is close to the FasR interaction site. We conclude that the gld mutation allows normal FasL biosynthesis, surface expression and oligomerization, but induces structural alterations to the Fas binding region leading to the phenotypic changes observed.

Amino Acid Sequence↗
PubMed1994

A null mutation in the perforin gene impairs cytolytic T lymphocyte- and natural killer cell-mediated cytotoxicity.

Lymphocyte-mediated cytotoxicity has been proposed to consist of the polarized secretion of granule-stored perforin leading to target-cell lysis. Nevertheless, perforin-independent pathways were postulated to explain the cytolytic activity of apparently perforin-free lymphocytes and the DNA degradation found in dying target cells. To evaluate the role of perforin, we used gene targeting in embryonic stem cells to produce mice lacking perforin. Mice homozygous for the disrupted gene have no perforin mRNA. The mice are healthy. Activation and granzyme A secretion of perforin-free cytolytic T cells are unaltered. The killing activity of cytolytic T cells as well as natural killer (NK) cells, however, is impaired but not abolished. Approximately one-third of the killing activity remains when lysis of 3T3 fibroblast targets and the apoptotic cell death of YAC-1 NK targets are analyzed. We conclude that perforin is a crucial effector molecule in T cell- and NK cell-mediated cytolysis. However, alternative perforin-independent lytic mechanisms also exist.

Animals↗
PubMed1994

Resistance of CTL to perforin-mediated lysis. Evidence for a lymphocyte membrane protein interacting with perforin.

Cytotoxic T lymphocytes are highly resistant to killing by their own cytolytic protein perforin. We have investigated the molecular basis of this self-protection mechanism. CTL withstood high dosages of perforin, when complete lysis was obtained with various tumor target cell lines. A peptide-specific CTL clone readily lysed tumor targets presenting the peptide, but was unable to kill the peptide-presenting CTL in spite of equal degranulation. With streptolysin O, a pore-forming lytic protein of bacterial origin, no difference in susceptibility between the various targets was detected. Perforin was radioactively labeled by using the Bolton and Hunter method without any loss of its lytic activity. Experiments performed at 4 degrees C revealed that, on all of the cells studied, binding of the labeled perforin to the membrane is reversible and strictly Ca(2+)-dependent. After a short exposure to 37 degrees C, perforin was no longer dissociated from cell membranes by EDTA. Although no correlation between susceptibility and the extent of perforin binding was detected, differences in the conformation of membrane-bound perforin were observed. Perforin adsorbed to resistant cells was cleaved by trypsin into a 55-kDa (C-terminal) and a 10- to 15-kDa (N-terminal) fragment, whereas this cleavage was not found on tumor cell-bound perforin. A portion of perforin was included in the Triton X-114 detergent phase at 4 degrees C on resistant CTLs only. Our results are compatible with the notion that a protective molecule, specifically expressed on CTLs, interacts with perforin, thereby rendering it lytically inactive.

Animals↗
PubMed1994

Bcl-2 protects from oxidative damage and apoptotic cell death without interfering with activation of NF-kappa B by TNF.

The recent demonstration of the anti-oxidant properties of the Bcl-2 gene product suggested that expression of Bcl-2 may interfere with the nuclear migration of the NF-kappa B transcription factor, which is thought to depend on the presence of reactive oxygen intermediates. In mouse L cells, overexpression of Bcl-2 interfered with the activation of NF-kappa B by H2O2. However, Bcl-2 had no effect on the activation of NF-kappa B by TNF, even though it protected cells from TNF-induced apoptosis. The effects of exogenous pyrrolidine dithiocarbamate were very similar to those of Bcl-2 overexpression. We conclude that the protective effects of anti-oxidants against induced apoptotic cell death are unrelated to their ability to interfere with NF-kappa B activation.

Animals↗
PubMed1994

Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways.

The recent generation of perforin knock-out mice has demonstrated a crucial role for the pore-forming perforin in cytolytic T-lymphocyte (CTL)-mediated cytolysis. Perforin-deficient mice failed to clear lymphocytic choriomeningitis virus in vivo, yet substantial killing activity still remained in perforin-free CTLs in vitro, indicating the presence of (a) further lytic pathway(s). Fas is an apoptosis-signalling receptor molecule on the surface of a number of different cells. Here we report that both perforin-deficient and Fas-ligand-deficient CTLs show impaired lytic activity on all target cells tested. The killing activity was completely abolished when both pathways were inactivated by using target cells from Fas-receptor-deficient lpr mice and perforin-free CTL effector cells. Fas-ligand-based killing activity was triggered upon T-cell receptor occupancy and was directed to the cognate target cell. Thus, two complementary, specific cytotoxic mechanisms are functional in CTLs, one based on the secretion of lytic proteins and one which depends on cell-surface ligand-receptor interaction.

Animals↗
PubMed1994

Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes.

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg-Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As brain-infiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.

Amino Acid Sequence↗
PubMed1994

Macrophage precursor cells produce perforin and perform Yac-1 lytic activity in response to stimulation with interleukin-2.

Macrophage precursor cells, derived from mouse bone marrow culture with granulocyte-macrophage colony-stimulating factor or colony-stimulating factor 1 (CSF-1) as growth factor and interleukin-2 (IL-2) as stimulating factor, were activated by IL-2 to exert strong cytolytic activity against Yac-1 cells. In response to IL-2 stimulation these bone marrow macrophage precursor cells produced perforin as lytic molecules. The purity of the precursor cells for the study was proved as homogeneous positivity for Mac-1, NK-1.1 and negativity for Lyt 1 and 2. The cells express CSF-1 receptors on their surface, are able to proliferate and differentiate into typical macrophages when stimulated with CSF-1, and are therefore members of the macrophage lineage. Perforin transcripts were identified by Northern blot analysis of IL-2-treated macrophage precursor cells, and the presence of perforin protein in the cytoplasmic granules was demonstrated by immunohistochemical staining using a monoclonal antiperforin antibody. In addition, the biological activity of the perforin contained in the macrophage precursor's granules could be documented as calcium-dependent lytic activity using Yac-1 and sheep red blood cells as targets. The results presented in this paper imply the existence of a bipotent precursor cell, which can mature into a typical macrophage if CSF-1 or phorbol 12-myristate 13-acetate is supplied as differentiation stimulating factor but develops into an NK/LAK cell when early activation with IL-2 is provided.

Animals↗
PubMed1994

Granzyme B.

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Amino Acid Sequence↗
PubMed1994

The apoptosis endonucleases: cleaning up after cell death?

The term apoptosis describes the predictable structural changes associated with many forms of programmed cell death. One of the first visible events of apoptosis is the collapse of the nucleus. Nuclear degradation is manifested by digestion of chromatin into nucleosome-sized fragments or multiples of these. This digestion of DNA is enzymatic, and several attempts have been made to characterize apoptosis-specific endodeoxyribonucleases. Although there are strong candidates for such enzymes, direct evidence for their role in apoptosis is yet to be provided.

Journal Article↗
PubMed1994

The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.

Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

Amino Acid Sequence↗
PubMed1994

Effects of purified perforin and granzyme A from cytotoxic T lymphocytes on guinea pig ventricular myocytes.

OBJECTIVE: Involvement of cytotoxic T lymphocytes (CTL) in heart transplant rejection as well as in viral myocarditis is well established, but the precise mechanisms whereby infiltrating CTL damage the myocardium are unknown. The aim of the study was to investigate how CTL derived perforin, the serine protease granzyme A, and the combination of both, damage guinea pig ventricular myocytes. METHODS: Action potentials and membrane currents were recorded by means of the whole cell configuration from guinea pig ventricular myocytes. RESULTS: Resembling the effects of CTL derived lytic granules, perforin caused gradual myocyte shortening and contracture, leading to complete loss of the rod shaped morphology and to cell destruction. These changes were preceded by shortening of action potential duration and reduction of resting potential and action potential amplitude, followed by complete inexcitability. Granzyme A alone was ineffective, but accelerated the deleterious effects of perforin on the morphological and electrophysiological properties of myocytes. The effects of perforin were further evaluated by measuring membrane currents by means of the whole cell voltage clamp. Perforin induced discrete changes in membrane current, reminiscent of single ion channels, with large conductance and open time of up to several seconds. Linear regression analysis of the channel I-V relations resulted in a conductance of 890 pS and a reversal potential of -7.6 mV. These results suggest that perforin induces large non-selective channels, which can account for most of the observed adverse effects. CONCLUSIONS: As CTL participate in the immunological rejection of the transplanted heart, it is conceivable, but remains to be shown, that part of this damage is inflicted by perforin containing lytic granules.

Action Potentials↗
PubMed1994

Human clusterin gene expression is confined to surviving cells during in vitro programmed cell death.

Clusterin is a serum glycoprotein endowed with cell aggregating, complement inhibitory, and lipid binding properties, and is also considered as a specific marker of dying cells, its expression being increased in various tissues undergoing programmed cell death (PCD). However, no study has so far directly shown that cells expressing clusterin in these tissues are actually apoptotic as defined by morphological and biochemical criteria. We have studied cellular clusterin gene expression in vitro using three different models of PCD: (a) ultraviolet B (UV-B) irradiation of human U937, HeLa, and A431 cell lines, (b) in vitro aging of human peripheral blood neutrophils (PMNs), and (c) dexamethasone-induced cell death of the human lymphoblastoid cell line CEM-C7. In all three models, the classical morphological and biochemical features of PCD observed did not correlate with an increase, but with either a marked decrease or an absence of clusterin gene expression as assessed by Northern blot analysis. In situ hybridization of U937 and A431 cells after UV-B irradiation revealed, in addition, that only morphologically normal cells that are surviving continue to express the clusterin gene. Our results demonstrate that in the human myeloid, lymphoid, and epithelial cell types studied, clusterin gene expression is not a prerequisite to their death by apoptosis. In addition, and most interestingly, in situ hybridization of U937 and A431 cells revealed that only surviving cells express the clusterin gene after the induction of PCD, thus providing novel evidence suggesting that clusterin may be associated with cell survival within tissues regressing as a consequence of PCD.

Apoptosis↗