Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the ...mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene-knockout (P-/-) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, Cai transients, and contractions of myocytes conjugated to P-/- PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P-/- PELs were, respectively, as followsVm, -73.2 +/- 1.5 and -53.6 +/- 6.4 mV (mean +/- SEM); action potential amplitude, 117.9 +/- 3.9 and 74.3 +/- 21.2 mV; and action potential duration at 80% repolarization, 17 +/- 6 and 42 +/- 13 milliseconds (all P<.05). P-/- PELs also induced early and delayed afterdepolarizations as well as arrhythmogenic activity. Diastolic Cai increased during the cytocidal interaction with P-/- PELs, from a fluorescence ratio of 0.82 +/- 0.05 (n=7) to 1.98 +/- 0.09 (n=13) (P<.05). All of the effects caused by P-/- PELs were reproduced by incubating the myocytes with Jo2. Heparin (50 micro signg/mL), an antagonist of inositol trisphosphate (IP3)-operated sarcoplasmic reticulum Ca channels, or U-73122 (2 micro signmol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP3 analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P-/- PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP (3). We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy. (Circ Res. 1998;82:438-450.)
T-cell-mediated cytotoxicity Berke, G
Current opinion in immunology,
06/1991, Letnik:
3, Številka:
3
Journal Article
Recenzirano
There are two competing theories to explain the mechanism(s) by which cytolytic T lymphocytes kill target cells: granule exocytosis of a pore-forming protein, and contact-induced internal ...disintegration. Accumulated evidence supports alternative pathways in lymphocytoxicity, possibly reflecting distinct effector functions expressed by different killer cells and cells at different stages of activation.
The central theme of this work has been the roles of the CTL receptor and of MHC-proteins in CTL recognition and lysis. A major conclusion that may be deduced from the work presented here is that one ...CTL receptor is responsible for both target cell recognition and lysis. Although their function as recognitive structures is well established, involvement of MHC-proteins in the events that follow recognition has not been investigated in detail. We have proposed that MHC-proteins are molecular mediators whereby CTL receptors transmit signals ultimately leading to lysis of the target cell. I see future work on CTL-mediated lysis proceeding in the following directions: 1. Verification and analysis of the precise role of MHC proteins in CTL recognition and lysis by use of cell and vesicle systems of defined composition and structure. 2. Study of CTL-mediated 'lethal hit' in systems enabling analysis of early events (millisecond level) preceding lysis. 3. Grafting of CTL receptor(s) activity onto naive cells, using liposomes or other vehicles, and 4. Production of idiotypic reagents such as monoclonal antibodies specific for the combining site/effector mechanism of CTL.
Excessive urea excretion associated with a negative nitrogen balance and massive loss of skeletal muscle mass (cachexia) is a frequent life threatening complication in malignancies and HIV infection. ...As these patients have often elevated interleukin-6 (IL-6) and abnormally low cystine levels, we have now determined the intracellular levels of glutathione and other cysteine derivatives in the liver and muscle tissue of IL-6-treated or tumor-bearing C57BL/6 mice. IL-6 treatment or inoculation of the MCA-105 fibrosarcoma caused a significant increase in hepatic gamma-glutamyl-cysteine synthetase activity and a decrease in the sulfate level, glutamine/urea ratio, and glutamine/glutamate ratio, suggesting that a decrease of the proton generating cysteine catabolism in the liver may increase carbamoyl-phosphate synthesis and urea formation at the expense of net glutamine synthesis. Treatment with cysteine, conversely, caused an increase in sulfate, glutamine/urea ratios, and glutamine/glutamate ratios and may thus be a useful therapeutic tool in clinical medicine. In contrast to the liver, muscle tissue of tumor-bearing mice showed decreased glutathione and increased sulfate levels, suggesting that the cysteine pool may be drained by an increased cysteine catabolism in this tissue. The findings indicate that tumor cachexia is triggered initially by IL-6 and is later sustained by processes driven by an abnormal cysteine metabolism in different organs.-Hack, V., Gross, A., Kinscherf, R., Bockstette, M., Fiers, W., Berke, G., and Dröge, W. Abnormal glutathione and sulfate levels after interleukin 6 treatment and in tumor-induced cachexia.
Ionic Basis of Fas Receptor Effects on Ventricular Myocytes.
Introduction: Experimental evidence suggests a major role for Fas receptor activation in a wide range of myocardial pathologies. Because ...clinical situations, which are likely to be associated with Fas activation, are accompanied by a variety of ventricular arrhythmias, the major goal of this study was to investigate the ionic mechanisms responsible for these phenomena.
Methods and Results: To delineate the origin of Fas‐mediated electrophysiologic perturbations, the transient outward K+ current Ito and the L‐type Ca2+ current ICa,L were studied in murine ventricular myocytes treated with the Fas‐activating monoclonal antibody Jo2. Jo2 decreased Ito (4.36 ± 1.2 pA/pF vs 17.48 ± 2.36 pA/pF in control, VM=+50 mV; P < 0.001) and increased ICa,L (− 13.17 ± 1.38 pA/pF vs − 3.94 ± 0.78 pA/pF in control, VM= 0 mV; P < 0.001). Pretreatment of ventricular myocytes with ryanodine or thapsigargin prevented the electrophysiologic effects of Jo2, suggesting that Ca2+i elevation is important for Fas‐mediated action. In agreement with our previous studies demonstrating dependence of Fas‐based myocyte dysfunction on an intact inositol trisphosphate (1,4,5‐IP3) pathway, the effects of Jo2 on Ito and ICa,L were prevented by the phospholipase C (generates 1,4,5‐IP3) blocker U73122, and by xestospongin C (tested with Ito), a specific blocker of IP3‐operated sarcoplasmic reticulum Ca2+ release channels. Furthermore, intracellular perfusion with 1,4,5‐IP3, but not with 1,3,4‐IP3, caused electrophysiologic effects resembling those of Jo2.
Conclusion: Decreased Ito and increased ICa,L underlie Fas‐induced action potential alterations and arrhythmias in murine ventricular myocytes, effects that appear to be mediated by 1,4,5‐IP3‐induced intracellular calcium release.
We used the CellScan, a novel static cytometer, to monitor changes induced by anti-neoplastic drugs in the fluorescence intensity and polarization of fluorescently-labeled tumor cells.
T47D and T80 ...human breast cancer cell lines were exposed to navelbine and to 5-fluorouracil and the fluorescence properties of the treated cells, stained with fluorescein diacetate and rhodamine 123, were measured by the CellScan.
A strong correlation was found between the inhibition of cell growth induced by the two drugs, as estimated from cell counts, and the resulting changes in fluorescence intensity and polarization, as monitored by the CellScan. Fluorescence hyperpolarization of the labeled cells occurred in conjunction with AnnexinV binding and propidium iodide exclusion, indicating that such hyperpolarization, resulting from drug action, reflects an early stage of apoptosis, as previously proposed.
The system presented here could serve as the basis for assessing drug sensitivity or resistance of cancer cells derived from small biopsies of solid human tumors, thus eliminating prior tumor culturing and time-consuming assays.
While the binding step of cytolytic T lymphocyte (CTL) target cell interaction resulting in conjugate formation is a well-characterized event, there seems to be more than one mechanism whereby ...lymphocytes kill the target. In recent years, infliction of complement (C)-like "holes" (I.D. 10-20 nm) on the target cell membrane, believed to be produced by the Ca2+-dependent lytic protein(s) perforin/cytolysin of secretory lytic granule origin has been proposed to be the mechanism of lymphocytotoxicity. More recent evidence, however, suggests that Ca2+-dependent exocytosis of lytic granules (where detectable) is not involved in lymphocyte-mediated cytolysis. Furthermore, neither formation of C-like "holes" in targets exposed to CTL, nor higher-than-background levels of lytic granules, perforin or BLT-esterases, have been detected in highly potent, peritoneal exudate CTL (PEL) derived directly from the animal or in cytocidal PEL-hybridomas. Hence exocytosis of perforin and formation of the above pores may apply to certain effector cells, particularly those grown in vitro in IL-2, but not to in vivo primed CTL such as PEL. On the other hand, work from this laboratory with Ca2+ probes has shown that lysis induced by CTL such as PEL-not involving lytic granules, perforin or formation of the above "holes"-is preceded by a marked prelytic elevation of cytosolic Ca2+ in the target. CTL-induced target cell membrane perturbation--a direct result of receptor-mediated effector-to-target interaction or through a membrane-bound or secreted effector component(s)--may be responsible for triggering the prelytic influx of Ca2+ from external sources, or its mobilization from internal stores in the target. We propose that CTL-induced, persistent elevation of cytosolic Ca2+, above a critical level, rather than formation of 10-20 nm pores, is responsible for the catastrophic prelytic events observed in the target, such as bleb formation, metabolic exhaustion and DNA degradation, ultimately leading to lysis.
The recent advent of peptide–MHC tetramers has provided a new and effective tool for studying antigen-specific T cell populations through monitoring tetramer binding to T cells by flow cytometry. Yet ...information regarding T cell activation induced by the bound tetramers cannot be deduced from binding studies alone; complementary methods are needed to bridge this gap. To this end, we have developed a new approach that now enables monitoring both binding to and activation of T cells by peptide–MHC tetramers at the single-cell level. For this purpose, we have employed the CellScan, a non-flow cytometer designed for repetitive measurements of optical parameters (e.g., fluorescence intensity and polarization) of individual living cells. A melanoma-specific MART1 CTL line and a gp100-specific CTL clone were incubated with specific and control single-chain peptide–MHC tetramers for 45 min. Subsequently, the fluorescence intensity and polarization were measured by the CellScan. Specific binding of fluorescently labeled peptide–MHC tetramers to CTLs, recorded by the CellScan, was comparable to that measured by flow cytometry. CellScan monitoring of the degree of fluorescence polarization of fluorescein diacetate-labeled CTLs that were reacted with tetramers revealed specific activation of the CTLs, which was confirmed by cytokine (INF γ) production. These results provide a new means of monitoring both the binding to and activation of T lymphocytes by cognate peptide–MHC complexes at the single-cell level, which can now be applied to distinguish between cognate responding and anergic T cells.
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