Inhibition by interferon of Sendai virus cytolysis.

Treatment of HeLa cells with human interferons inhibited 51Cr release from cells induced by ultraviolet-inactivated Sendai virus. The inhibitory effect became apparent about 6 h after interferon treatment and persisted for 24 to 48 h. In the interferon-treated cells, the cytolysis was inhibited within 10 min after adding virus and the inhibitory action was suppressed by the treatment of the cells with cycloheximide. Mock interferon and mouse interferon did not inhibit the cytolysis and antiinterferon serum neutralized the effect of interferon. All these findings indicate that Sendai virus-induced cytolysis is inhibited by interferon per se. However, interferon did not have any influence on Sendai virus hemolysis.     

Effect of exogenous interferon on virus-specific immune cytolysis

Virus-specified immune cytolysis has been shown in mouse cell cultures infected with vesicular stomatitis virus under the action of specific virus antibody and active complement. The exogenous mouse interferon failed to enhance the cellular plasmalemma permeability or to inhibit cellular adsorption of infectious and inactivated virus as well as the lysis of the cells in the presence of the antiviral serum and complement. The interferon localization was studied by the method of fluorescent antibodies. In vital preparations the exogenous species-specific interferon was revealed on the surface of most cells in the treated cultures; in the fixed preparations of the same cultures the interferon was found only in the form of small lumps in the cytoplasm of few cells. A decrease in immune cytolysis was observed in the cultures treated with the interferon 20-24 hours before the infection, that can be accounted for by the inhibitory action of the interferon on the viral replication and the reduction of the number of the virus-inducing antigens in the cellular plasmalemma.     

Protective effect of enterosgel on rat liver lysosomes during cytostatic treatment

Polychemotherapy with a complex of cytostatics (cyclophosphamide, doxorubicin, vincristine, prednisolone) induces progressive damage to hepatocyte membranes, which manifested in labilization of lysosomes and activation of lysosomal enzymes and serum transaminases. Enterosgel stabilized liver lysosomes and reduced manifestation of hepatocyte cytolysis.     

T-cell mediated cytolysis: evidence for target-cell suicide.

The mechanism by which cytotoxic T-lymphocytes (Tc) induce the death of specific target cells is still controversial. We have used quantitative cytochemical methods to distinguish the metabolic activities of the target cells from those of the Tc, even when they are attached to each other. Early events following Tc-P8(15) target cell interaction were first, increased glucose 6-phosphate dehydrogenase activity and second, labilization of the lysosomes within the target cell: these changes could be mimicked, in part, by polyamines and could be inhibited by inhibiting ornithine decarboxylase (ODC) activity. The crucial role of ODC in the chain of events that led to cytolysis in this particular experimental system was shown first, by measuring ODC activity directly and secondly, by the inhibition of cytolysis by the presence of a selective inhibitor of ODC activity.     

Natural killing (NK) of cytomegalovirus (CMV)-infected fibroblasts: a comparison between two strains of CMV, uninfected fibroblasts, and K562 cells

Spontaneous cytolysis of uninfected human fibroblasts (HF), K562 cells, and HF cells infected with cytomegalovirus (CMV) was associated with nonadherent peripheral blood leukocytes (PBL) that passed through a nylon wool column, and rosetted with sheep erythrocytes at low affinity; cytolysis was further associated with enriched preparations of large granular lymphocytes (LGL). The Leu-7 marker did not correlate as a cell phenotype with functional activity in normal donors. The HF cells infected with the AD169 strain of CMV were as sensitive to cytolysis as K562 cells, although the kinetics of cytolysis differed; HF cells infected with the Davis strain of CMV were generally less sensitive to cytolysis than uninfected fibroblasts. Equivalent amounts of interferon were detected in NK assays containing Davis targets, AD169 targets, or K562 cells; interferon was not detected in NK assays of HF cells. Neither uninfected nor infected HF cells competed effectively against 51Cr-labeled K562 cells in cold competition experiments; K562 cells, however, competed effectively against 51Cr-labeled AD169-CMV targets. Uninfected HF cells were efficient competitors of AD169-CMV targets. Davis-CMV-infected cells were less effective, however, than uninfected HF cells in competition experiments, suggesting that fewer receptors for NK cells were present on these targets.     

Hairy cell leukemia cells are relatively NK-insensitive targets

The hypothesis that interferon alpha (IFN alpha) has its beneficial effects in hairy cell leukemia by activating natural killer cells against hairy cells was examined. Leukemic cells from patients with hairy cell leukemia were tested for their susceptibility to lysis by fresh and IFN alpha activated peripheral blood mononuclear (PBMC) cells from normal donors. All hairy cells tested were relatively insensitive to cytolysis by PBMC and IFN alpha activated PBMC. The low levels of 51Cr release obtained with a few donors was due to lysis of leukemic cells, not residual normal cells, and was mediated by a natural killer cell (T cell receptor independent) mechanism. Chronic lymphatic leukemic cells before and after treatment with phorbol ester were also resistant to cytolysis. Hairy cells were not susceptible to lymphokine activated killer (LAK) cells but were sensitive to lysis by antibody and complement. The insensitivity to cell mediated cytolysis against hairy cells was shown by cold target inhibition to be a lack of target recognition by NK cells.     

Role of age-related serum factors in the mechanism of interferon production

The activity (free and total) of cathepsin D and acid phosphatase was studied in cells of peritoneal exudate of mice of different ages in the process of interferon production in the presence of sera from newborn and adult animals. Cathepsin D release in newborn mice upon interferon induction is actively stimulated by serum factors of newborn animals altering lysosome permeability selectively for this enzyme alone. Another lysosomal enzyme, acid phosphatase, was more strongly bound to the structures and showed no such features. With an increased age of the donor the amount of stimulating factors in the serum decreases and that of inhibiting factors increases. Inhibition of cathepsin release from lysosomes by the serum factor of adult animals protects the synthesized interferon molecules from degradation and facilitates intensive interferon production in adult mice. A higher susceptibility of children to virus infections and inefficiency of the earliest defence system, interferon, may be due to degradation of newly synthesized interferon molecules by lysosomal cathepsin D.     

Activation of a heat-stable cytolytic protein associated with the surface membrane of Naegleria fowleri.

Surface membrane-enriched fractions of Naegleria fowleri obtained after isopycnic centrifugation experiments contain a potent cytolytic activity as determined by hemolysis and 51Cr release assays. This surface membrane cytolysin was unaffected by a treatment at 75 degrees C for 30 min and accounted for 70 to 90% of cytolysis by whole-cell lysates of amoebae. This heat resistance as well as intimate membrane association distinguished the surface membrane cytolytic activity from a second heat-labile cytolytic activity which appears to be latent within lysosomes. The surface membrane cytolysin was found to be specifically activated by diluted samples of lysosomal fractions. The possible role of this surface membrane cytotoxin in the pathogenicity of N. fowleri is discussed.     

Effector cytolotic function but not IFN-gamma production in cytotoxic T cells triggered by virus-infected target cells in vitro

Cytolysis and interferon(IFN)-gamma production are two independent effector functions of activated cytotoxic T (Tc) cells. We have used the Tc-cell response against the flavivirus, Murray Valley encephalitis virus (MVE), to investigate the requirements for inducing these two functions with regard to antigen-concentration and CD8 coreceptor involvement. Cognate peptide-pulsed target cells triggered cytolysis by primary ex vivo MVE-immune as well as in vitro peptide-restimulated splenocytes at lower peptide concentrations than IFNgamma-production (100-fold lower in the case of primary ex vivo effectors). Little difference was observed in CD8 dependency. Importantly, neither of the effector populations were triggered to produce IFN-gamma by virus-infected target cells, although cytolysis occurred. This result raises the possibility that the levels of presentation of cognate antigen on virus-infected cells in vivo may be below the threshold required for the IFN-gamma production.     

Phenotypically and functionally distinct T-cell subsets in anti-tumor responses

Helper T cells and cytolytic T cells have both been implicated in tumor cell rejection in vivo but their roles and relative importance are still controversial. Here, Toshiyuki Hamaoka and Hiromi Fujiwara discuss recent experiments which suggest that tumor cell growth can be inhibited nonspecifically by the action of gamma interferon as well as by cytolysis and that immunized cells of both ‘cytotoxic cell’ and ‘helper cell’ phenotype can produce this lymphokine.     

Alterations in leukocytes induced by virus and interferon

Alterations occurring in cells under an influence of interferon or viral interferon inducer were observed under the fluorescence microscope. The experiments were carried out with human leukocytes stained intravitally with acridine orange. From our observations it follows that both interferon as well as interferon inducer evoke morphological changes in leukocytes causing partial or complete disappearance of lysosomes in cytoplasm.     

Tumor cell killing and cytostasis by C3H/HeJ macrophages activated in vitro by lipid A-associated protein and interferon gamma

Experiments have been carried out to assess the ability of purified protein-free lipopolysaccharide (LPS) and lipid A-associated protein (LAP) containing LPS to activate macrophages from C3H/HeJ endotoxin-unresponsive mice. Assays for in vitro activation have included cytotoxic and cytostatic effects on a simian virus 40 (SV40)-transformed fibroblast cell line. While neither preparation of LPS would effect C3H/HeJ macrophage activation for either cytostasis or cytolysis, the addition of murine interferon gamma to cultures of LAP-LPS stimulated C3H/HeJ macrophages resulted in cells which were both cytotoxic and cytostatic. These results suggest that LAP can provide one component of the triggering mechanism but, of itself, is insufficient to effect full activation. A second signal, which can be provided by murine interferon gamma, appears also to be required.     

Gamma interferon reverses inhibition of leukocyte bactericidal activity by a 25-kilodalton fraction from Mycobacterium tuberculosis.

In this study we examined the effects of Mycobacterium tuberculosis cell extracts on the phagocytic activity of polymorphonuclear leukocytes and cultured peripheral blood monocytes. M. tuberculosis cell extracts were fractionated on Sephacryl S-200 columns, and a 25-kilodalton glycolipoprotein was shown to inhibit the intracellular killing ability of these leukocytes but had no effect on their phagocytic potential. This same fraction inhibited fusion of phagosomes with lysosomes, as assessed by noting the transfer of acridine orange from lysosomes to phagosomes. This fraction was shown to have a maximal inhibitory effect when it was in the form of an intact carbohydrate-lipid-protein complex. Gamma interferon (IFN-gamma), but not IFN-alpha, reversed the inhibitory effect of the mycobacterial component on bactericidal activity and on fusion of phagosomes and lysosomes. Thus, this 25-kilodalton fraction of M. tuberculosis cell extract may be important in protecting organisms against phagocytic degradation, an effect which can be reversed by IFN-gamma.     

Natural Cytotoxicity to Murine Cytomegalovirus-Infected Cells Mediated by Mouse Lymphoid Cells: Role of Interferon in the Endogenous Natural Cytotoxicity Reaction

Lymphoid cells from unstimulated normal C57BL/6J mice were shown to lyse murine cytomegalovirus (MCMV)-infected syngeneic mouse embryo fibroblasts but not uninfected mouse embryo fibroblasts. This cytotoxicity by mouse effector cells was not restricted to MCMV-infected syngeneic cells since MCMV-infected xenogeneic rat heart fibroblasts were also lysed. Characterization of the effector cells mediating this cytotoxicity against MCMV-infected cells indicated that the effector cells are similar to described natural killer (NK) cells mediating lysis of tumor cells and virus-infected cells. Because of the described augmentation of NK activity by interferon, we examined the role of interferon in the NK reaction. Although low levels of virus-induced interferon were detectable in supernatants of MCMV-infected mouse embryo fibroblasts, no interferon was detectable in supernatants of MCMV-infected rat heart fibroblasts, a target significantly more sensitive to NK cytolysis than infected mouse embryo fibroblasts. We were able to augment the NK reaction against MCMV-infected cells by in vitro treatments with interferon. However, the amounts of interferon required for augmentation were significantly greater than the amounts generated by infected target cells. In vitro interferon-stimulated NK cells retained selective cytotoxic activity since they continued to remain incapable of lysing uninfected target cells. MCMV-infected rat heart fibroblasts induced more interferon and were also more susceptible to NK activity than MCMV-infected mouse embryo fibroblasts. In spite of this difference in interferon-inducing capacity, there was no augmentation of cytotoxicity of MCMV-infected mouse embryo fibroblasts when mouse splenocytes were cocultivated with both target cells. Finally, when production of interferon in the NK reaction was inhibited by the addition of actinomycin D, no reduction of NK activity was seen. Our findings suggest that native mouse NK cells can discriminate between MCMV-infected cells and uninfected cells, this ability leading to the selective lysis of the virus-infected cells. Furthermore, although we could demonstrate augmentation of NK activity by interferon, interferon activation of NK cells may not be a necessary precondition for the development of endogenous NK activity.     

Inhibition of T cell-mediated cytolysis by 2-deoxy-D-glucose:dissociation of the inhibitory effect from glycoprotein synthesis.

Previous studies have established that T cell-mediated cytolysis can be reversibly inhibited by the hexose analogue 2-deoxy-D-glucose (2-DG) by a mechanism which is apparently unrelated to energy depletion. The possibility that the inhibitory effect of 2-DG on cytolysis was linked to its known inhibitory effect on glycoprotein synthesis was therefore investigated. In contrast to the results obtained with 2-DG, no inhibition of cytolysis was observed in the presence of tunicamycin, a potent and specific inhibitor of lipid carrier-dependent protein glycosylation. Furthermore, populations of cytolytic cells which had been pretreated with doses of tunicamycin sufficient to block the incorporation of mannose (or 2-DG) into glycoproteins were still fully susceptible to inhibition by 2-DG. Other known inhibitors of viral protein glycosylation, such as glucosamine and galactosamine, inhibited cytolysis only weakly under conditions where 2-DG was highly effective. Kinetic studies revealed that the inhibitory effect of 2-DG on cytolysis could be reversed within minutes by the addition of exogenous glucose. Furthermore, suggestive evidence was obtained that inhibition cytolysis by 2-DG was linked to a parallel inhibition of effector: target cell binding. Taken together, these results strongly suggest that the inhibitory effect of 2-DG on cytolysis can be dissociated from its effect on protein glycosylation. An alternative mechanism of action of 2-DG is suggested.     

Genetic defects affecting lymphocyte cytotoxicity

Cytolytic lymphocytes kill virus-infected cells as well as tumor cells by the exocytosis of the content of specialized secretory lysosomes at the immunological synapse (IS). Perforin and granzymes are the molecular effectors that induce rapid target cell death. Cytolytic T cells are activated by specific antigen recognition whereas the cytolytic activity of natural killer cells is initiated by specific activating receptors or combinations thereof and is inhibited by self MHC class I recognition. The cytolytic process has received considerable attention and can now be described as a multi-step process including cell activation, polarization of specialized lysosomes -- lytic granules -- toward the IS, tethering of the lytic granules to the plasma membrane, priming for fusion with the plasma membrane, effective fusion and release of granule content in the IS cleft, and death of the target. This is a highly flexible system that could enable a cytolytic cell to subsequently kill target cells bound at different sites around the effector cell. Cytolytic cells exert a second effector function consisting of the secretion of cytokines, notably interferon gamma. The latter secretory process functions independently from the exocytic pathway of the lytic granules.     

Differential effects of interferon on the MHC expression of human lymphocytes. Enhanced expression of HLA without effect on Ia.

HLA antigens are the principal serologically detectable products of the major histocompatibility complex (MHC), and they function as targets for antibody-mediated and cell-mediated cytolysis. Anti-HLA sera were used in a quantitative absorption procedure to study the effect of interferon (IF) treatment on HLA expression. This study was undertaken since IF has been shown to play an important role in the regulation of cell division and function. We found that IF enhanced the expression of HLA antigens on human peripheral blood lymphocytes by 8-fold. This increase in HLA expression was specific for both the HLA-A and HLA-B regions of the MHC. There was no increase in the expression of the Ia region after IF treatment as opposed to the HLA region. Since IF is an antiviral agent and it also enhances the expression the HLA-A and HLA-B regions, it may be involved in the elimination of virus-infected cells by A and B identical effector cells.     

Strain dependence of rat macrophage natural tumoricidal capacity and its stimulation by endotoxins

Without known stimulation in vivo and in vitro, resident peritoneal macrophages from 5 conventional or specific pathogen-free (SPF) rat strains [Hairless (H), BDIX, Wistar (W), Sprague-Dawley (SD) and Long-Evans (LE)] exhibited an in vitro strain-dependent cytolysis against DHD-K12/TS cancer cells. This natural cytolysis was also observed when polymyxin B was added to the culture medium. The percentage of natural cytolysis varied from one rat to another but was significantly different according to the strain. In the presence of 10 micrograms endotoxin/ml, macrophages from BDIX, W, SD and LE rats were always cytolytic, whilst those of H rats were irregularly cytolytic. Endotoxins induced or increased macrophage-mediated cytolysis from H, BDIX, W and SD rats, but they were without effect for LE rats. The endotoxin effect depended on the level of natural cytolysis. In contrast to mouse resident peritoneal macrophages, which were not naturally cytolytic and not activated in vitro by endotoxins, these results show that rat resident peritoneal macrophages can be naturally cytolytic. This cytolysis can be enhanced by endotoxins as the sole in vitro stimulus. Rat macrophage natural cytolytic activity is strain-dependent.     

Cytolysis by Cloned Helper T Cells: Induction by Specific Antigen or by Anti-CD3 Hybrid Antibodies

We have explored the use of hybrid antibodies--prepared by covalently linking anti-CD3 to an antibody specific for a monomorphic major histocompatibility complex (MHC) class II determinant using N-succinimidyl 3-(2-pyridyldithio)proprionate/succinimidyl 4-(N-malcimidomethyl)cyclohexane-1-carboxylate (SPDP/SMCC) as coupling reagent--in inducing cytolysis in human tuberculin (PPD)-specific T helper (TH) clones. These clones have been shown to lyse PPD-bound Epstein-Barr virus (EBV)-transformed B-cell lines (B-EBV) in an MHC class II-restricted manner. In this paper anti-CD4-induced cytolysis is compared with antigen/MHC-induced cytolysis with the same clones. Cytolysis induced by the hybrid antibodies was highly efficient, with killing of both syngeneic and allogeneic tumour cells positive for MHC class II. Conjugate-induced cytolysis was maxima within 4 h; that of antigen-positive targets at 16 h. Killing of bystander cells was seen only when cytolysis was triggered by antigen/MHC, suggesting that the mechanism of cytolysis in the two systems may be distinct. Targets treated simultaneously with hybrid antibody and with antigen, thereby providing both activation signals to the clones, are lysed less efficiently than those treated with either PPD or hybrid antibody alone. Evidence is presented showing that this inhibition is most marked against syngeneic PPD-coated cells treated with hybrid antibody, suggesting that two signals independently capable of activating cytolytic function in the clones, when presented simultaneously, interfere with the induction of the cytolytic process.     

Pasteurella (Mannheimia) haemolytica leukotoxin-induced cytolysis of bovine leukocytes: role of arachidonic acid and its regulation

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis only in ruminant leukocytes and platelets. Cytolysis of many cell types is mediated by arachidonic acid (AA) and its generation by phospholipases is regulated by G-protein-coupled receptors. However, the contribution of Lkt-induced AA generation to cytolysis and the signalling cascade underlying AA generation in bovine leukocytes have not been determined. We have determined whether AA mediates Lkt-induced cytolysis and delineated the signalling mechanisms underlying AA generation in bovine leukocytes. Bovine lymphoma cells were used as an experimental system to investigate the Lkt-induced [(3)H] AA release, an index of AA generation and lactate dehydrogenase release, an index of cytolysis. The results indicate that Lkt induces AA release and cytolysis in a concentration- and time-dependent fashion. The AA analog, 5,8,11,14-eicosatetraynoic acid inhibited Lkt-induced cytolysis, but not AA release. Lkt-induced AA release and cytolysis were inhibited by pertussis toxin, inhibitors of cytosolic phospholipase A(2)(cPLA(2)), phospholipase C and protein kinase C (PKC), and by chelation of intracellular calcium. Furthermore, Western blot analysis revealed the presence of G(i), G(s)and G(q)type G-proteins. These results demonstrate that AA metabolites from cPLA(2)activation contribute to Lkt-induced cytolysis and G(i)type G-proteins, Ca(2+)and PKC, regulate the cPLA(2)activity.