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J Tschopp

Publications and source records attributed to J Tschopp.

At least 199 records · Page 11Linked to original sources
PubMed1991

Immunohistochemical localization of granzyme B antigen in cytotoxic cells in human tissues.

Human granzyme B antigen is expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme B was generated using a prokaryotic expression vector under the control of T7 transcription and translation signals. The 25-kd recombinant protein (granzyme B) was used to develop a rabbit polyclonal antiserum. Purified anti-granzyme B antibodies were used to detect the antigen expression in cytotoxic cells in human tissues. Using the avidin-biotin-complex peroxidase technique, formalin-fixed, paraffin-embedded tissue sections from patients with acute mild or moderate allograft cardiac rejection were stained. A constant cytoplasmic staining of the lymphocytic allograft infiltrate was observed. These results provide a basis for using the anti-granzyme B antibodies for detection of cytotoxic cells in human tissues. The detection and quantitative analysis of the granzyme-associated cytotoxic cells may help to evaluate the significance of these functionally distinct cytotoxic cells in human tissues associated with increased expression of cytoplasmic granule effector molecules.

Antibodies↗
PubMed1990

Interaction of chondroitin sulfate with perforin and granzymes of cytolytic T-cells is dependent on pH.

Cytolytic T-lymphocytes (CTL) harbor cytoplasmic granules containing the lytic, pore-forming protein perforin, a family of serine proteases designated granzymes, and proteoglycans as major constituents. Growth of CTL lines in the presence of PNP-xyloside completely inhibited the glycosylation of the granule-associated chondroitin sulfate A type proteoglycans. Only short glycosaminoglycan molecules were detected. The absence of intact proteoglycans neither altered the sorting of the granule-associated proteins perforin or granzyme A nor influenced their secretion into the extracellular milieu upon T-cell receptor complex stimulation. With a weak base, the pH of the granules was determined to be acidic. At pH 5.2, granzyme A and perforin formed complexes with chondroitin sulfate A. At neutral pH, perforin and only a minor fraction of granzyme A dissociated from the proteoglycan. Upon secretion of the granule contents induced by immobilized anti-CD3 antibodies, most granzyme A molecules remained complexed with the chondroitin sulfate A glycosaminoglycans, even if synthesis of intact proteoglycans was inhibited. We suggest that granule-associated molecules complex with proteoglycans under the acidic conditions prevailing in the trans Golgi and cytolytic granules. A possible pH shift occurring during exocytosis would cause perforin, but only a minor fraction of granzyme A, to dissociate from the proteoglycans.

Animals↗
PubMed1990

Cell specificity of granzyme gene expression.

Granzymes are serine proteases present in secretory granules of cytolytic T lymphocyte lines. We have studied the expression of the granzyme family (granzyme A, B, C, D, E, F, and G) in different lymphoid cell populations and cell lines as well as in nonlymphoid cells and tissues. Our data show that with few exceptions expression of granzyme genes is restricted to T cells and their thymic precursors. In mature T cells granzymes are expressed only upon activation. The same is true for thymocytes, with the exception of grazyme A that is expressed also in non-stimulated cells. In T cells and thymocytes the distribution of mRNAs coding for different granzymes depends on the subpopulation tested and the activation protocol. Highly cytolytic PEL express granzymes A and B but none of the other granzymes.

Animals↗
PubMed1990

Domain structure of the endothelial cell receptor thrombomodulin as deduced from modulation of its anticoagulant functions. Evidence for a glycosaminoglycan-dependent secondary binding site for thrombin.

Rabbit thrombomodulin (TM) influences blood coagulation by serving as a cofactor for thrombin-induced protein C activation (activity a), by directly affecting the procoagulant activity of thrombin (activity b) and by accelerating the inhibition of thrombin by antithrombin III (AT III) (activity c). Although high molecular weight cationic compounds, such as poly-L-lysine and the ionophore-releasate from human platelets, only partly affected activity a in a concentration-dependent manner, activities b and c, however, were almost totally inhibited by these cationic compounds. Likewise, a heparin- and dermatan sulfate-binding peptide which represents a portion of the glycosaminoglycan-binding domain of vitronectin (VN) selectively inhibited activities b and c, indicating the presence of clustered acidic domain(s) in TM responsible for these activities. While heparinase or heparitinase did not affect rabbit TM function at all, digestion of rabbit TM with chondroitin ABC-lyase abolished activities b and c, whereas activity a remained unaffected. Modification of rabbit TM with chondroitin ABC-lyase was associated with a decrease in molecular mass of the receptor by about 10 kDa and a 2- to 3-fold decrease in affinity to thrombin as deduced from direct binding studies. These results suggest that at least two acidic thrombin binding domains are present in rabbit TM, whereby a dermatan sulfate-like glycosaminoglycan moiety constitutes the secondary binding domain for thrombin, eliciting both the direct as well as the AT III-dependent anticoagulant function of rabbit TM (activities b and c) but not protein C activation (activity a). In contrast to rabbit TM, human TM isolated from placenta only showed weak activities b and c. These differences in reactivity of TM from different sources appeared to be due to the masking (or absence) of the proposed secondary thrombin binding site in human TM, since VN could be identified as a major contamination in the human TM preparation as revealed by enzyme-linked immunosorbent assay and Western blot analysis. In addition, the major part of human TM could be immunoprecipitated by monospecific antibodies to VN. These findings indicate a possible modulatory function for VN in the human thrombin-TM system.

Amino Acid Sequence↗
PubMed1990

Transients of perforin pore formation observed by fluorescence microscopic single channel recording.

A new type of single channel recording is described. Large pores were generated in the membranes of resealed human erythrocyte ghosts by incubation with perforin (cytolysin). The flux of the polar fluorescent probe Lucifer Yellow was measured in single ghosts by the fluorescence microphotolysis (photobleaching) technique. The distribution of flux rates for ghosts treated with a limiting perforin concentration showed equidistantly spaced peaks suggesting that subpopulations of ghosts with 0, 1 and 2 pores were resolved. Furthermore, distributions obtained for very different perforin concentrations could be well simulated by using one common value for the flux rate of the single pore (k = 4.65 x 10(-3) s) and assuming a Poisson distribution of pores among ghosts. The flux rate of the single pore corresponds to a pore radius of approximately 50 A, a value which is much smaller than that obtained previously by electron microscopic studies but which agrees well with recent electrical single channel recordings. Mature perforin pores were observed to be very stable. No closing events were detected at a time resolution of 0.2 s for a wide range of temperatures and Ca2+ concentrations. However, the formation of new pores was an unexpectedly slow process. Fluorescence microscopic single channel recording as introduced by this study is applicable to a variety of cellular systems and fluorescent probes and thus may complement the information obtainable by electrical single channel recording of anorganic ion fluxes.

Erythrocyte Membrane↗
PubMed1990

Inhibition of lymphocyte mediated cytotoxicity by perforin antisense oligonucleotides.

The granule/perforin exocytosis model of CTL mediated cytolysis proposes that CTL, upon recognition of the specific targets, release the cytolytic, pore-forming protein perforin into the intercellular space which then mediates the cytotoxic effect. However, direct evidence for the involvement of perforin is still lacking, and indeed, recent results even seem incompatible with the model. To determine directly the role of perforin in CTL cytotoxicity, perforin antisense oligonucleotides were exogenously added during the stimulation of mouse spleen derived T cells and human peripheral blood lymphocytes (PBL), respectively. Perforin protein expression in lymphocytes was reduced by up to 65%, and cytotoxicity of stimulated T cells by as much as 69% (5.7-fold). These results provide the first experimental evidence for a crucial role of perforin in lymphocyte mediated cytotoxicity.

Animals↗
PubMed1990

Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and perforin.

Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.

Affinity Labels↗
PubMed1990

Complement activation by pulsed tunable dye laser in normal skin and hemangioma.

Pulsed tunable dye laser (577 nm) (PTDL) therapy induces hemoglobin coagulation and tissue necrosis, which is mainly limited to blood vessels. To define whether this treatment activates complement in normal skin and senile hemangioma, we analyzed complement deposition in blood vessels by immunofluorescence. C3 fragments, C8, and C9 were detected with specific polyclonal antibodies. The membrane attack complex of complement (MAC) was demonstrated with a monoclonal antibody which reacts only with a neoantigen of MAC. Amplification of C3 deposition by the alternative pathway was determined on cryostat sections by indirect immunofluorescence with use of C4 deficient guinea pig (GP) serum. Normal skin and hemangiomas from three individuals were studied. In PTLD-irradiated normal skin, the main findings were as follows: 1) C3 fragments, C8, C9, and MAC were deposited in vessel walls; 2) these deposits were not due to denaturation of the proteins since they became apparent only 7 min after irradiation, contrary to immediate deposition of transferrin at the sites of erythrocyte coagulates; 3) the C3 deposits were shown to amplify complement activation by the alternative pathway, a reaction which was specific since tissue necrosis itself did not lead to such amplification; 4) these reactions preceded the local accumulation of polymorphonuclear leucocytes. Tissue necrosis was more pronounced in the hemangiomas. The larger angiomatous vessels in the center of the necrosis did not fix complement significantly. By contrast, complement deposition in the vessels situated at the periphery was similar to that observed in normal skin with one exception: C8, C9, and MAC were detected in some blood vessels immediately after laser treatment, a finding consistent with assembly of the MAC occurring directly without the formation of a C5 convertase. These results indicate that complement is activated in PTDL-induced vascular necrosis, and might be responsible for the ensuing inflammatory response.

Coloring Agents↗
PubMed1990

Involvement of granule proteins in T-cell-mediated cytolysis.

Cytolytic T lymphocytes (CTL) and large granular lymphocytes contain dense cytoplasmic granules which, when isolated, are lytic for a variety of target cells. Granule proteins are released from the effector cell upon target cell interaction, further suggesting that they play a role in the cytolytic mechanism. Major proteins in CTL granules are a family of serine esterases (granzymes) and a pore-forming protein called perforin (cytolysin). Despite structural similarities between functionally conserved regions of perforin and the ninth component of complement (C9), these two lytic molecules are clearly distinct in their mode of target cell recognition. Perforin, unlike C9, is not dependent on a protein receptor molecule but binds to the target cell membrane via phosphorylcholine in a Ca2(+)-dependent manner. Here, we discuss the stimulus-secretion model for T-cell-mediated cytotoxicity with respect to our current understanding of perforin and the granzyme proteases.

Animals↗
PubMed1989

Complete primary structure and functional characterization of the sixth component of the human complement system. Identification of the C5b-binding domain in complement C6.

Complement C6 is one of five plasma proteins that are incorporated into the lytic terminal complement complex on lipid membranes (C5b-9m) upon activation of the complement cascade. Oligonucleotide probes derived from partial amino acid sequences of purified C6 were used to isolate cDNA clones from a human liver cDNA library. The complete polypeptide structure of mature C6 deduced from the cDNA sequence consists of 913 amino acid residues preceded by a typical 21-residue signal peptide. C6 is most similar in structure to complement C7, sharing 33.5% identical residues with C7 including all 56 cysteine residues. The low density lipoprotein receptor class A and B modules, the thrombospondin type I module at the carboxyl terminus, and the two short consensus repeat modules are arranged in the same way as in C7. In contrast to C7 and other terminal complement proteins, the thrombospondin type I module at the amino terminus occurs as a tandem repeat in C6. The last tandem repeat at the carboxyl terminus of C6 and C7 has been identified as a new distinct module (factor I module), which is closely related to a segment in the heavy chain of complement control factor I. Binding studies with filter-bound C6 fragments generated by proteolysis showed that the C5b-binding domain of C6 was located in the 34-kDa carboxyl terminal fragment consisting of two short consensus repeats and two factor I modules.

Amino Acid Sequence↗
PubMed1989

Isolation and complete structure of the lymphocyte serine protease granzyme G, a novel member of the granzyme multigene family in murine cytolytic T lymphocytes. Evolutionary origin of lymphocyte proteases.

A cDNA clone that is closely related to the granule-associated serine proteases of cytolytic T lymphocytes (CTL), called granzymes A-F, was isolated from a CTL expression library. The encoded serine protease, granzyme G, shows 70%-89% nucleotide identities to the granzymes C-F and, like those, consists of 228 amino acids preceded by the short propeptide Glu-Glu and a 18 residue long signal peptide. Granzyme G was identified by amino-terminal sequence analysis as a correctly processed and sorted protein stored in lysosome-like granules. The phylogenetic history of the granzyme multigene family was reconstructed by two tree-making methods and by Southern blot analyses of human, rat, and mouse DNA. Our results indicate differences in the evolutionary pathway between these species. The murine granzymes C-G descended from a progenitor present at the time of mammalian radiation. Granzyme C branched off first after the primate-rodent split and was involved in a recombination event with granzyme B before the rat-mouse divergence. Granzymes D and E have diverged after the mouse-rat speciation. However, no experimental evidence for the existence of a granzyme C-D-E-F-G equivalent was found in humans, and loss of the ancestral gene in the primate lineage is discussed. In view of the species differences in the number of granzyme gene copies during recent evolution, we propose that the murine granzymes B-G play several distinct roles in CTL-mediated effector functions as a response to quite recent changes of the biochemical environment.

Amino Acid Sequence↗
PubMed1989

Phosphorylcholine acts as a Ca2+-dependent receptor molecule for lymphocyte perforin.

Large granular lymphocytes and cytolytic T-lymphocytes (CTL) contain numerous cytoplasmic granules thought to be responsible, at least in part, for the cytolytic activity of these effector cells. Isolated granules are lytic for a variety of target cells and the granule proteins are specifically released upon target-cell interaction. Major proteins in mouse CTL granules are a family of seven serine proteases designated granzymes A to G, and a pore-forming protein called perforin (cytolysin). Purified perforin is cytolytic in the presence of Ca2+ and shows ultrastructural, immunological and amino-acid sequence similarities to complement component C9. Despite these similarities, perforin and C9 are clearly distinct in their mode of target-cell recognition. Whereas C9 insertion is absolutely dependent on a receptor moiety assembled from the complement proteins C5b, C6, C7, and C8 on the target-cell membrane, no requirement for a receptor molecule has been reported for perforin. Here, we demonstrate that phosphorylcholine acts as a specific, Ca2+-dependent receptor molecule for perforin.

Animals↗
PubMed1989

Absence of homologous restriction factor does not affect CTL-mediated cytolysis.

Homologous restriction factor (HRF) is a membrane protein of erythrocytes and leukocytes that inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. HRF has also been reported to inhibit perforin-mediated cytolysis and postulated to play a role in cytotoxic T lymphocyte (CTL) self-protection. We show that paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, lacking HRF, are no more sensitive to Ca2+-dependent human CTL-mediated lysis than normal erythrocytes. Furthermore, mouse and normal human erythrocytes, as well as PNH erythrocytes, are similarly lysed by isolated murine perforin-containing granules. We conclude that HRF does not inhibit perforin-mediated lysis and therefore is not likely to play a role in CTL self-protection.

Blood Proteins↗