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J Tschopp

Publications and source records attributed to J Tschopp.

At least 127 records · Page 7Linked to original sources
PubMed1996

Expression of the CTL-associated protein TIA-1 during murine embryogenesis.

TIA-1 is a T cell-associated protein that binds poly(A) in vitro and induces apoptosis in permeabilized thymocytes. It may be involved in the induction of apoptosis in target cells during lymphocyte attack. To elucidate the role of TIA-1 in mammalian development, a cDNA-encoding mouse TIA-1 was cloned. The predicted mouse TIA-1 protein contains three RNA binding domains at the amino terminus and a putative lysosomal targeting sequence at the carboxyl terminus. The mouse sequence shows 96% overall identity with the human TIA-1 homologue. During murine embryogenesis, abundant mouse TIA-1 mRNA is detectable from 12.5 days of development onward in the brain and the retina, where it is selectively expressed within neuronal cells. Transcripts are also found in the lung, kidney, and thymus. TIA-1 in the adult mouse is expressed mainly in T cells and NK cells. The expression of TIA-1 during mouse embryogenesis is endogenous to tissues in which apoptotic cell death occurs. The conservation of this RNA-binding protein throughout evolution implicates its importance in embryogenesis, and in particular neuronal development.

Amino Acid Sequence↗
PubMed1996

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.

Acetylcysteine↗
PubMed1996

Differential expression of perforin in muscle-infiltrating T cells in polymyositis and dermatomyositis.

Polymyositis (PM) and dermatomyositis (DM) are the prototypical inflammatory diseases of skeletal muscle. In PM, CD8+ T cells invade and destroy muscle fibers, whereas humoral effector mechanisms prevail in DM. We studied the expression of the cytotoxic mediator perforin in inflammatory cells in PM and DM muscle by semiquantitative PCR, immunohistochemistry and confocal laser microscopy. Similar levels of perforin mRNA were expressed in PM and DM, and abundant perforin-expressing CD3+CD8+ and CD3+ CD4+ T cells were observed in both diseases. However, there was a striking difference in the intracellular localization of perforin. In DM, perforin was distributed randomly in the cytoplasm of the inflammatory T cells. In contrast, 43% of the CD8+ T cells that contacted a muscle fiber in PM showed perforin located vectorially towards the target muscle fiber. The results suggest (a) that the random distribution of perforin in the cytoplasm of muscle-infiltrating T cells observed in DM reflects nonspecific activation, and (b) that the vectorial orientation observed only in PM reflects the specific recognition via the T cell receptor of an antigen on the muscle fiber surface, pointing to a perforin- and secretion-dependent mechanism of muscle fiber injury.

Base Sequence↗
PubMed1996

The serine protease granzyme A does not induce platelet aggregation but inhibits responses triggered by thrombin.

Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.

Amino Acid Sequence↗
PubMed1996

Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen (SAg) that predominantly interacts with V(beta)8+ T cells. In vivo treatment of mice with SEB leads to an initial increase in the percentage of V(beta)8+ T cells, followed by a decrease in the numbers of these cells, eventually reaching lower levels than those found before treatment with the SAg. This decrease is due to apoptosis of the SEB-responding cells. In the present study, we use the distinct light scattering characteristics of apoptotic cells to characterize T cells that are being deleted in response to SEB in vivo. We show that dying, SEB-reactive T cells express high levels of Fas and Fas ligand (Fas-L), which are implicated in apoptotic cell death. In addition, the B cell marker B220 is upregulated on apoptotic cells. Moreover, we show that the generation of cells with an apoptotic phenotype is severely impaired in response to SEB in functional Fas-L-deficient mutant gld mice, confirming the role of the Fas pathway in SAg mediated peripheral deletion in vivo.

Animals↗
PubMed1996

Cytotoxic T cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is the main complication after allogeneic bone marrow transplantation. Although the tissue damage and subsequent patient mortality are clearly dependent on T lymphocytes present in the grafted inoculum, the lethal effector molecules are unknown. Here, we show that acute lethal GVHD, induced by the transfer of splenocytes from C57BL/6 mice into sensitive BALB/c recipients, is dependent on both perforin and Fas ligand (FasL)-mediated lytic pathways. When spleen cells from mutant mice lacking both effector molecules were transferred to sublethally irradiated allogeneic recipients, mice survived. Delayed mortality was observed with grafted cells deficient in only one lytic mediator. In contrast, protection from lethal acute GVHD in resistant mice was exclusively perforin dependent. Perforin-FasL-deficient T cells failed to lyse most target cells in vitro. However, they still efficiently killed tumor necrosis factor alpha-sensitive fibroblasts, demonstrating that cytotoxic T cells possess a third lytic pathway.

Animals↗
PubMed1996

Functional expression of Fas and Fas ligand on human gut lamina propria T lymphocytes. A potential role for the acidic sphingomyelinase pathway in normal immunoregulation.

The expression and function of Fas (CD95/APO-1), a cell surface receptor directly responsible for triggering cell death by apoptosis, was investigated on human T lymphocytes resident within the intestinal lamina propria, a major site of antigen challenge and persistent lymphocyte activation. Three color immunofluorescence and FACS analysis indicated that virtually all freshly isolated human gut lamina propria T lymphocytes (T-LPL) express Fas, together with the marker of progress activation CD45R0. A discrete fraction of freshly isolated T-LPL also constitutively expressed Fas ligand (FasL), perhaps as a result of recent in vivo activation. Importantly, whereas Fas cross-linking did not result in apoptosis induction in peripheral blood T lymphocytes (T-PBL), Fas was found to be fully effective in generating the apoptotic signal in T-LPL. This was associated with the activation of an acidic sphingomyelinase and with ceramide generation, early events known to be involved in Fas-mediated apoptotic signaling. By contrast, acidic sphingomyelinase activation and ceramide production were not detectable in T-PBL after Fas cross-linking. However C2-ceramide, a cell permeant synthetic analog of ceramide, could efficiently induce apoptosis in T-LPL and T-PBL when added exogenously. These data indicate that T-LPL constitutively express both Fas and FasL and that Fas cross-linking generates signals resulting in sphingomyelin hydrolysis and apoptosis, outlining a potential mechanism involved in intestinal tolerance. Moreover, they provide the first evidence of a role for ceramide-mediated pathways in normal immunoregulation.

Apoptosis↗
PubMed1996

Activated B cells express functional Fas ligand.

Fas ligand (FasL, Apo-1L) is a member of the tumor necrosis factor protein family and binding to its receptor (Fas, Apo-1, CD95) triggers cell death through apoptosis. Ligand expression is restricted to cells with known cytolytic activity and found on hematopoietic cells of the T cell and natural killer lineage. Here we provide evidence that B lymphocytes can express FasL. Flow cytometric analysis revealed that FasL is expressed on the surface of B cells upon stimulation with either lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin. FasL expression on activated B cells was confirmed by western blot and reverse transcriptase polymerase chain reaction analysis. FasL on B cells is functional since lipopolysaccharide-activated B lymphocytes derived from wild type, but not from gld mutant mice, were able to kill Fas-sensitive target cells. Our data suggest that the Fas system may contribute to the control of B cell homeostasis.

Amino Acid Sequence↗
PubMed1996

Neurons induced to express major histocompatibility complex class I antigen are killed via the perforin and not the Fas (APO-1/CD95) pathway.

Cytotoxic T lymphocytes (CTL) kill target cells by perforin-mediated pore formation, induction of apoptosis by the Fas ligand, or both. It has been demonstrated that depolarized neurons can be induced to express major histocompatibility complex (MHC) class I antigens by interferon-gamma. Evidence for antigen-dependent CTL-mediated killing was obtained by transfecting neurons with MHC class I cDNA. The present study was designed to investigate the mechanisms of killing of cerebellar granule neurons depolarized by high K+ concentrations and thereby inducible for MHC class I antigen expression. We found that neurons express only low levels of Fas (APO-1/CD95) and are resistant to Fas ligand-mediated killing even when pretreated with cytokines. However, granules extracted from CTL as well as purified perforin induce almost complete lysis of neurons. These data suggest that CTL-mediated elimination of neurons involves the perforin, but not the Fas pathway of target cell killing.

Animals↗
PubMed1996

Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.

Animals↗
PubMed1996

Comparison of Fas(Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lymphocytes.

Cytolytic T lymphocytes kill target cells by two independent cytolytic mechanisms. One pathway depends on the polarized secretion of granule-stored proteins including perforin and granzymes, causing target cell death through membrane and DNA damage. The second cytolytic effector system relies on the interaction of the Fas ligand (Fasl) on the effector cell with its receptor (Fas) on the target cell, leading to apoptotic cell death. Using mixed lymphocyte culture (MLC)-derived primary T lymphocytes of perforin-knockout and gld (with non-functional FasL) mice, the molecular basis of the two killing mechanisms was compared. The activity of both pathways was dependent on extracellular Ca2+. Incubation of MLC-stimulated primary T cells with protein synthesis inhibitors prior to TCR triggering impaired FasL cell surface expression and abolished cytolytic activity, although the cells exhibited an intracellular pool of FasL. The perforin-dependent mechanism induced cell death more rapidly, although both pathways ultimately showed similar killing efficiencies. Both pathways induced comparable levels of DNA degradation, but Fas-induced membrane damage was less pronounced. We conclude that upon TCR triggering FasL may be recruited in part from pre-existing intracellular stores. However, efficient induction of target cell death still depends on the continuous biosynthesis of FasL molecules.

Animals↗
PubMed1996

Increased p34cdc2-dependent kinase activity during apoptosis: a possible activation mechanism of DNase I leading to DNA breakdown.

Cells undergoing apoptosis typically exhibit distinctive morphological characteristics. Early events include the rounding up of the cell, chromatin condensation, nuclear membrane breakdown and blebbing of the cellular membrane. Strikingly similar changes take place in the cell cycle progression, at the entry into mitosis, suggesting a link between mitosis and apoptosis. Here we show that expression of active p34cdc2 at inappropriate phases during the cell cycle leads to morphological changes reminiscent of apoptosis, including DNA degradation. Cells cotransfected with the active mutant of p34cdc2 and DNase I displayed degraded DNA, which was absent in p34cdc2 wild-type and DNase I-transfected cells, in spite of similar DNase activities. Upon induction of apoptosis in thymocytes, transient p34cdc2 activation was detected prior to lamina breakdown and nuclease activation. P34cdc2 activation was also observed during APO-1 (Fas/CD95)-induced apoptosis in a B lymphoblastoma cell line. Our results suggest that unscheduled activation of p34cdc2 may participate in the initiation of the typical apoptotic phenotype.

Animals↗
PubMed1996

Constitutive Fas ligand expression in several non-lymphoid mouse tissues: implications for immune-protection and cell turnover.

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis which have been shown to be implicated in peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. Analysis of FasL expression during mouse embryogenesis and in adult tissues reveals that FasL, although initially thought to be restricted to lymphoid cells, is constitutively expressed in a wide array of non lymphoid tissues. FasL mRNA is detectable in mouse embryos from 16.5-d onwards in epithelial cells of the submaxillary gland, and neurons of the developing nervous system. In general, FasL mRNA was not detectable in characteristic sites of embryonic programmed cell death. In the adult mouse, by RNase protection analysis, FasL mRNA is detectable in all 20 tissues tested except for the heart and pancreas. Similar analysis performed simultaneously for Fas indicates that several tissues, including the thymus, lung, spleen, small intestine, liver, seminal vesicle, prostate and uterus co-express the two genes. Most tissues constitutively co-expressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune-privileged. The pattern of FasL expression in mice suggests that FasL may be implicated in the regulation of physiological cell turnover, and the protection of tissues against potential lymphocyte mediated damage.

Animals↗
PubMed1995

Interaction of peptides derived from the Fas ligand with the Fyn-SH3 domain.

Interaction of the widely expressed Fas with its membrane-bound ligand (FasL) leads to rapid cell death via apoptosis. To avoid pathological tissue damage, the activity of FasL requires tight regulation. Here, we report that the Src homology 3 (SH3) domain of Fyn binds to the proline-rich cytoplasmic region of FasL. Binding of the SH3 domain occurs between amino acid residues 44-71 which contains several potential SH3 interaction sites. This binding is specific, as SH3 domains of Lck, Grb2 and ras-GAP bind only weakly or not at all. We suggest that FasL activity may be modulated by SH3 domains of the src-like Fyn kinase.

Amino Acid Sequence↗
PubMed1995

The death domain motif found in Fas (Apo-1) and TNF receptor is present in proteins involved in apoptosis and axonal guidance.

The interaction of Fas (Apo-1) and TNF receptor-1 with their respective ligands can lead to cell death. The so-called death domain, a sequence motif present in the cytoplasmic portion of the two receptors, has been identified as a critical structural element involved in signal transduction that leads to apoptosis. Here we describe several additional proteins which contain a death domain. Novel members of this family include proteins known to be implicated not only in apoptosis but also in neuron guidance.

Amino Acid Sequence↗
PubMed1995

Granzyme A is an interleukin 1 beta-converting enzyme.

Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.

Amino Acid Sequence↗
PubMed1995

Genomic organisation and expression of mouse deoxyribonuclease I.

Deoxyribonuclease I (DNase I) has recently been implicated in cell death by apoptosis, a process which is frequently accompanied by chromatin DNA degradation. Despite extensive studies on DNase I, its genomic organisation remained unknown. Here we report for the first time on the intron-exon structure of the DNase I gene. The coding region of mouse DNase I is composed of eight introns and eight exons, spanning 2315 base pairs. The deduced protein sequence is 91.5% identical to its rat counterpart, but does not carry the two mutations (Glu13 to D and V67 to I) responsible for the decrease in actin-binding of rat DNase I. The enzymatic activity of mouse DNase I is found in striated muscle, kidney, intestine, liver, lymphnodes, but not in the heart, spleen or pancreas.

Amino Acid Sequence↗