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G Berke

Publications and source records attributed to G Berke.

At least 19 recordsLinked to original sources
PubMed1992

Peritoneal exudate lymphocyte and mixed lymphocyte culture hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin.

We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.

Animals↗
PubMed1992

Immunological rejection of heart transplant: how lytic granules from cytotoxic T lymphocytes damage guinea pig ventricular myocytes.

We investigated the mechanism by which lytic granules extracted from cytotoxic T lymphocytes (CTL) damage guinea pig ventricular myocytes in order to determine whether their actions can be related to the overall immunological rejection of the transplanted heart. Granule-induced myocyte morphological changes and final destruction were preceded by shortening of action potential duration (APD) and reductions of the resting potential and the action potential amplitude. APD shortening was probably caused by a granule-induced increase in outward current (most likely non-specific). Ryanodine, which blocks Ca2+ release from the sarcoplasmic reticulum, did not interfere with the morphological and electrophysiological effects of lytic granules. Fura-2 imaging indicated that [Ca2+]i initially increased about 2-fold from 90.0 +/- 11.5 nM, while cell length decreased less than 5% from a mean value of 99.0 +/- 9.0 microns. A further increase in [Ca2+]i (greater than 10 fold) was associated with progressive contracture and destruction, suggesting that the structural damage inflicted by lytic granules is caused by [Ca2+]i overload. The results indicate that the cytocidal action of CTL-derived lytic granules may be involved in immunologically induced damage, even to the extent of rejection of the transplanted heart.

Action Potentials↗
PubMed1992

Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). I. Cell detachment and lysis induced by encephalitogenic CD4+ T lymphocytes.

Myelin basic protein (MBP) reactive CD4+ T lymphocytes, capable of inducing experimental autoimmune encephalomyelitis (EAE), were examined for their ability to damage target cells of central nervous system (CNS) origin. Damage was assessed by monitoring detachment of adherent astrocytes from substratum and astrocyte lysis. MBP-specific, but non-encephalitogenic CD4+ T cells mediated astrocyte detachment but not lysis. However, encephalitogenic CD4+ T cell lines were more efficient in causing astrocyte detachment and could also cause astrocyte lysis. The detachment and lytic activities of the MBP-reactive T cell lines tested were often independent of the presence of specific antigen, and were not restricted to syngeneic major histocompatibility (MHC) antigens. MBP often augmented the detaching and, if lytic, lytic activities of these T cells. The encephalitogenic CD4+ T cells also detached and lysed allogeneic 'bystander' fibroblasts in the presence of unlabelled syngeneic astrocytes, suggesting the involvement of a soluble mediator(s). Although MBP is essential for the initiation of EAE, the presence of MBP on cells of CNS origin, such as astrocytes and oligodendrocytes, does not appear to be necessary for their damage by MBP-specific CD4+ T cells. Immune CD4+ T cells, which penetrate the CNS, may disorganize brain tissue structure by lysing astrocytes directly and by damaging other brain cells indirectly by soluble mediators. Thus cellular detachment, in addition to cell lysis, mediated by MBP-specific CD4+ cells may contribute to EAE pathogenesis.

Animals↗
PubMed1992

Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). II. Characteristics of astrocyte detachment mediated by myelin basic protein (MBP) specific CD4+ T lymphocytes.

We have shown that encephalitogenic, myelin basic protein (MBP)-specific CD4+ T cells can cause astrocyte and oligodendrocyte detachment in vitro. Similar processes may damage the central nervous system (CNS) in vivo by causing disorganization and destruction of brain tissue structure. The finding that 'bystander' allogeneic fibrosarcoma cells were detached by MBP-specific CD4+ T cells only when syngeneic astrocytes were present, suggested that a soluble cell-detaching factor (CDF) is released during the specific astrocyte-CD4+ effector interaction. In this study, CDF activity was detected in the supernatants of MBP-reactive CD4+ T cells incubated with concanavalin A or astrocytes. Lymphocyte-induced astrocyte lysis, but not detachment, was inhibited by the protein synthesis inhibitors, cycloheximide and puromycin, indicating that de novo protein synthesis is required for this type of lysis, but not for detachment. Astrocyte detachment was not inhibited, but rather augmented, by the trypsin inhibitors, soybean trypsin inhibitor (SBTI) and alpha-1-antitrypsin (alpha 1), suggesting that the CDF activity is not due to tryptic serine proteases, although it may be protease susceptible. The heparanase inhibitor, heparin, inhibited CD4+ T cell-mediated astrocyte detachment at low doses, but augmented detachment at higher doses, indicating that detaching activity is not due to heparanases. The actin microfilament disrupting agent, cytochalasin B (CB), inhibited astrocyte detachment induced by MBP-specific CD4+ T cells. CB pretreatment of the target astrocytes, but not of the effector CD4+ T cells, inhibited astrocyte detachment, suggesting that the integrity of the target's, but not the effector's, cytoskeleton is required for astrocyte detachment. The results herein suggest that during astrocyte interaction with MBP-specific CD4+ T cells, soluble factors are released that trigger an intrinsic astrocyte detachment mechanism.

Animals↗
PubMed1991

T-cell-mediated cytotoxicity.

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.

Animals↗
PubMed1990

The lysis of cytotoxic T lymphocytes and their blasts by cytotoxic T lymphocytes.

After binding to their targets, cytotoxic T lymphocytes (CTL) deliver a lethal hit signal, ultimately leading to target cell lysis, and then can recycle to lyse additional targets, without themselves being destroyed. If non-specific secreted lytic mediators are involved in such lysis. CTL survival would not be expected unless the effectors are immune to CTL-mediated lysis. Therefore the lytic susceptibilities of alloimmune peritoneal exudate lymphocytes (PEL), containing up to 50% CTL, and of the cytolytic PEL blasts (PEB), obtained by culturing with interleukin-2 (IL-2), were examined. 51Cr-labelled BALB/c (H-2d) anti-EL4 (H-2b) (d alpha b) PEL were lysed 88%, 78%, and 48% by C3H/eb (H-2k) anti-P815 (H-2d) (k alpha d) PEL, C57BL/6 (H-2b) anti-P815 (b alpha d) PEL and b alpha d PEB, respectively. Similarly, b alpha d PEL were lysed 82% and 21% by d alpha b PEL and PEB, respectively. b alpha d PEB were lysed 82%, 28-39% and 39-51% by k alpha d PEL, b alpha d PEL and b alpha d PEB, respectively, b alpha d PEB were lysed 29-55% by d alpha b PEL. Furthermore, the CTL-containing populations were no less susceptible to lysis than normal lymphocytes. Since the majority (80-90%) of cells in these two types of CTL-containing populations can be directly and specifically lysed by appropriately immunized PEL CTL, we conclude that both the lytic granule and perforin lacking (PEL) and containing (PEB) CTL are not a priori immune to CTL-mediated lysis. These findings are in accord with theories proposing lysis to be induced by receptor-mediated contact between effector CTL and target cells, and challenge those suggesting the involvement of secreted lytic mediators.

Animals↗
PubMed1989

T cell activation: independent induction of killing activity and interleukin 2 secretion in cytolytic hybridomas.

Memory-like cytotoxic T lymphocytes (CTL) hybridomas exhibiting inducible killing activity and IL2 production were used to analyze the anamnestic response of CTL. Four activating agents were examined; anti-Thy-1 monoclonal antibody G7, staphylococcal enterotoxin B, interferon (IFN)-alpha/beta and IFN-gamma. These agents seemed to affect CTL activities in three distinct ways. Anti-Thy-1 monoclonal antibody, like specific antigen, was found to be a potent inducer of specific killing and IL2 production, whereas staphylococcal enterotoxin B induced IL2 production, but not cytolytic activity. On the other hand, IFN-alpha/beta and IFN-gamma effectively stimulated cytotoxicity without inducing IL2 production. The independent triggering of specific killing and IL2 secretion in the monoclonal cytolytic hybridomas suggests that in CTL distinct signals stimulate killing activity and IL2 production. The results also suggest that IFN-alpha/beta and IFN-gamma trigger the cytolytic program through an alternative activation pathway which does not involve the T cell receptor.

Animals↗
PubMed1989

The cytolytic T lymphocyte and its mode of action.

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.

Animals↗
PubMed1988

Highly lytic in vivo primed cytolytic T lymphocytes devoid of lytic granules and BLT-esterase activity acquire these constituents in the presence of T cell growth factors upon blast transformation in vitro.

Demonstration of C-like "rings," lytic granules, and the Ca2+-dependent lytic proteins--perforin/cytolysin--thereof, in certain cytocidal lymphocytes has led to the hypothesis of a mechanism of lytic granule-exocytosis and a common terminal lytic step in lymphocyte and C-induced lysis. However, neither cytolytic granules, nor formation of C-like rings during lysis have been detected in mature, highly potent, peritoneal exudate CTL (PEL) derived directly from the site of allograft rejection or in cytocidal hybridomas derived from them (PEL hybridomas). We now report that when stimulated in vitro in the presence of Con A supernatant, as a source of T cell growth factors (TCGF) or rIL-2, small in vivo primed PEL transform into large, dividing cytolytic T cells (PEL blasts) that express the same lytic specificity of the original PEL in short term lytic assays. The PEL blasts, in contrast to PEL, possess massive quantities of lytic granules, and protease (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester esterase) (BLT-esterase) activity as well as non-specific, cell-mediated cytolytic activity in a long term (4-h) assay. These results suggest that the proposed lytic mechanism involving exocytosis of lytic granules, perforin, and BLT-esterases and the formation of 10 to 20-nm lesions may apply to lysis induced by granule-containing effectors such as large granular lymphocytes and TCGF-dependent CTL lines, such as PEL blasts. However, killing by mature, in vivo primed CTL, such as PEL or their hybridomas, appears to be effected through an alternative, contact-induced, self-destruction process(es) of the target not involving secretory lytic granules or the above lesions. Hence, although the expression of lytic granules and BLT-esterase activities in cytolytic lymphocytes devoid of these components is induced by TCGF, these cellular constituents are not necessary for the expression of CTL-mediated target cell lysis by mature effector cells.

Animals↗
PubMed1987

CTLA-1 and CTLA-3 serine esterase transcripts are detected mostly in cytotoxic T cells, but not only and not always.

We and other investigators previously reported the cloning of CTLA-1 (or CCP-1) and CTLA-3 (or H Factor) serine esterase-related transcripts preferentially expressed in cytolytic T lymphocytes. We extended the survey of the tissue specificity of these molecules. Two main sets of results were obtained. First, both CTLA-1 and CTLA-3 transcripts could be found in the various cytolytic T cells tested, although in widely different amounts, and in some cases just at the threshold of detection. Secondly, these transcripts were not found in most of the other cells tested, including in some natural cytotoxic cells and in activated cytotoxic macrophages; however, they could be detected in mast cells for CTLA-1 and in some noncytotoxic lymphocytes for CTLA-3. Thus, the CTLA-1 and CTLA-3 serine esterase products are most probably not required for macrophage or natural cytotoxicity; their presence cannot be taken as characteristic of cytotoxic T cells; and a discussion about their relevance to T cell-mediated cytotoxicity should take into account their widely different amounts from one cytotoxic T cell to another.

Animals↗
PubMed1987

Effect of oxidation on the conformation of cell surface H-2 antigens detected by monoclonal antibodies.

Periodate treatment of lymphoid cells is known to render them susceptible to nonspecific lysis by cytotoxic T lymphocytes. To detect alterations in cell surface molecules as a result of such treatment, we investigated the binding of antibodies to oxidized and nonoxidized lymphoid cells and found that oxidation selectively decreases the binding of some anti-H-2 and anti-beta 2m antibodies. This effect was not caused by loss of H-2 molecules from the cells, since the rate of exchange of cell surface beta 2m by exogenously added beta 2m was unchanged, and neither H-2 nor beta 2m molecules could be detected in the supernates of oxidized cells. Our findings suggest that oxidation alters the conformation of H-2 antigens in such a way as to reduce antibody binding. Such conformational changes may further affect the recognition of histocompatibility antigens by cytotoxic T cells.

Animals↗
PubMed1987

Are lytic granules and perforin 1 involved in lysis induced by in vivo-primed peritoneal exudate cytolytic T lymphocytes?

We examined whether in vivo-primed, alloimmune, peritoneal exudate CTL obtained directly from the animal kill TC by perforating their membranes with C-like lesions produced by P1 or P2 originating in cytolytic granules of the CTL. In control experiments lytic granules were isolated from the formerly lytic CTLL-2 line as well as from Con A-activated lymphoblasts but not from the in vivo-primed CTL. The use of anti-P1 of CTLL-2 origin supports the above results in that immunoblots were positive with CTLL-2 extracts and isolated granules thereof but negative with extracts and isolated (nonlytic) granules from in vivo primed CTL; immunofluorescence of permeabilized cells gave similar results. We observed typical "ring structures" on EL4 membranes attacked by Ab + C as well as after exposure to lytic granules extracted from the formerly lytic CTLL-2 line; however, no such "structures" were observed after attack by the in vivo-primed CTL. These results are at variance with the recent suggestion of a common lytic mechanism--employed by CTL--of secreted lytic granules containing P1 (and P2) thereof that acts analogously to C9. An alternative mechanism for CTL-induced lysis not involving initial perforation of the TC membrane by 160A "holes" (Poly P1) has been presented elsewhere. It is important to note that our results with in vivo-primed CTL do not preclude involvement of a secreted, pore (channel)-forming agent(s) that is not observed by negative staining EM.

Animals↗
PubMed1986

The mitogenic activity of staphylococcal enterotoxin B (SEB): a monovalent T cell mitogen that stimulates cytolytic T lymphocytes but cannot mediate their lytic interaction.

Staphylococcal enterotoxin B (SEB), a monovalent T cell mitogen and inducer of T suppressor cells, was found to be a potent polyclonal activator of cytolytic T lymphocytes (CTL) effective against concanavalin A (Con A)-treated target cells. In addition to polyclonal stimulation of CTL, SEB could reactivate "memory" CTL, alloimmunized 60 to 90 days earlier, into "secondary" CTL detectable as early as 24 hr after onset of stimulation and specific for the original priming target cells. Optimal cytolytic activity was induced at 0.5 to 10 micrograms/ml SEB; optimal priming time was 3 days, correlating well with the proliferative activity and morphologic transformation of small lymphocytes into large T lymphoblasts. Long-term cultures of splenocytes, stimulated by SEB, continued to express high cytolytic activity. It is noteworthy that although SEB and Con A are comparable CTL inducers, SEB, unlike Con A, is an ineffective mediator of nonspecific, CTL/target cell interactions. To the best of our knowledge this is the first example of a CTL inducer unable to mediate CTL-target interaction and lysis. The latter observations suggests that different receptors are involved in CTL activation and in CTL-target interaction resulting in lysis.

Animals↗