Ilona Béládi

Are Granulocytes the Main Effector Cells of Natural Cytotoxicity in Chickens

Purified peripheral blood granulocytes from chicken were tested for cytotoxic activity against two types of virus-transformed chicken cell line, LSCC-H32 and LSCC-RP9. Strong cytotoxicity could be demonstrated, as measured in a 4-hr 51Cr-release assay, especially against the fibroblastoid LSCC-H32 cells. The degree of cytotoxicity was dependent on the E:T ratio. Normal CEF cells were completely resistant to the cytotoxicity. No cytotoxicity of human granulocytes could be observed against a variety of adherent and non-adherent target cells, as measured by the same microcytotoxicity technique. The priority of granulocytes in the natural cytotoxicity in the avian system is, therefore, suggested.

Inhibition of cytotoxicity of chicken granulocytes by serotonin and ketanserin

Serotonin (5-HT) has been observed to impair the cytotoxicity of human natural killer (NK) cells. A study has now been made of the effect of 5-HT on the cytotoxic activity of chicken granulocytes. 5-HT at concentrations of 10(-3) to 10(-6) M inhibited the cytotoxicity of chicken granulocytes when added at the onset of the short-term cytotoxicity assay. Ketanserin, a 5-HT2 receptor antagonist, did not reverse the inhibitory effect of 5-HT on chicken granulocyte cytotoxicity. Moreover, ketanserin at concentrations of 5 x 10(-4) to 5 x 10(-7) M inhibited the cytotoxicity mediated by chicken granulocytes. Pretreatment of the effector cells for 2 h with chick fibroblast interferon reduced the inhibition of chicken NK cytotoxicity induced by 5-HT and by ketanserin. These data indicate that, in birds, the neurotransmitter 5-HT serves as a link between the central nervous system and the immune system, and that interferon can modulate the inhibitory effect of 5-HT on the function of cytotoxic granulocytes.

Comparison of roles of serine esterase in chicken and human natural cytotoxicity.

The requirements for serine esterase activity in the spontaneous cell-mediated cytotoxicity of human lymphocytes and chicken granulocytes have been compared. The lysis of K-562 target cells and of LSCC-H32 chicken target cells was prevented by the serine esterase inhibitor TPCK. ATEE, the substrate of chymotrypsin, impaired both cytotoxic reactions, but to a lesser degree the cytotoxicity of chicken granulocytes. TPCK inhibited the trigger mechanism in an early calcium-dependent step and later calcium-independent events in both systems. However, calcium-independent lysis was depressed by serine esterase inhibitor only in the avian cytotoxicity. These findings suggest that avian target cell cytolysis consists of similar sequential phases to those already demonstrated in the human NK cell reaction, and serine esterase is required during several stages of cytotoxicity in the avian system.

Effect of the Platelet-Activating Factor Antagonist BN 52021 on Human Natural Killer Cell Cytotoxicity

The possible role of platelet-activating factor (PAF) in natural killer (NK) cell cytotoxicity was investigated by examining the effect of the PAF antagonist BN 52021 in NK cytotoxicity towards 51Cr-labelled K 562 target cells. When BN 52021 (30-120 microM) was added during the assay, a dose-dependent inhibition of NK activity was observed. The inhibition of cytotoxicity by BN 52021 was not due to an alteration of the binding of lymphocytes to K 562 cells. When lymphocytes were preincubated with BN 52021 (60 microM) for 60 min before the target cells were added, the inhibitory effect of the drug was similar to that observed when it was added at the start of the reaction. Inhibition was more pronounced when the target cells were pretreated for 60 min before the start of the assay. BN 52021 (60 microM) also inhibited gamma interferon induced NK activity. These studies provide indirect evidence that NK cells can generate PAF and that this mediator is involved in cytotoxic processes.

Involvement of Tumor Necrosis Factor in Human Granulocyte-Mediated Killing of WEHI 164 Cells

Human polymorphonuclear leukocytes (PMNLs) kill WEHI 164 clone 13 cells in an 18-hour 51Cr release assay. Antibody to human tumor necrosis factor (TNF) blocks the lysis of targets mediated by human granulocytes. PMNLs triggered by sensitive targets not only displayed cytotoxic activity, but also released a soluble factor capable of selectively lysing WEHI 164 cells. The killing of these cells by supernatants of triggered granulocytes was totally inhibited by anti-TNF antibody. These experiments suggest that the killing of WEHI 164 sarcoma cells by human PMNLs involves TNF or TNF-like molecules.

Cross‐reactivity between human adenoviruses in delayed‐type hypersensitivity

The aim of this study was to determine the antigen responsible for the induction of delayed‐type hypersensitivity (DTH) by human adenoviruses (Ads). The estimation of DTH was based on measurement of the extent of swelling of the hind footpads of mice. CsCl density gradient‐purified human Ad serotype 6 (Ad6) induced DTH in a dose‐dependent manner. In Ad6‐sensitized mice, DTH could be elicited by serotypes belonging to the same species of human Ads (types 1 and 5) and by a serotype (type 3) belonging to another species. Latex particles coated with purified hexon antigen prepared from Ad5 had the capacity to sensitize mice and elicit a DTH reaction. We suggest that, for serotypes belonging to species C, the cross‐reactive highly conserved T cell epitope of the hexon protein might be responsible for the DTH induction, and furthermore the same epitope might result in the cross‐reactivity between serotypes 3 and 6. The possible importance of these data is discussed in relation to human gene therapy through the application of Ad vectors.

Effect of human adenovirus on antibody-dependent cellular cytotoxicity (ADCC) in chickens.

The influence of human adenovirus type 6 on antibody-dependent cellular cytotoxicity (ADCC) in chickens has been investigated. The cytotoxic effect of peripheral blood mono-nuclear cells of chickens was studied on sheep red blood cells (SRBC) coated with chicken anti-SRBC serum. Cytotoxicity was estimated using a 51Cr release assay system. A single intravenous injection of the virus enhanced ADCC. ADCC was enhanced 14 to 24 hr after the virus injection, but then decreased and the preinjection level was reached after 36 hr. The capacity for virus-augmented activity was not removed with phagocytic cell depletion. The possible role of interferon induced by the virus in chickens in augmenting ADCC is discussed.

The role of interferon in the adenovirus-induced augmentation of granulocyte-mediated cytotoxicity in chicken.

The effects of human adenoviruses on the granulocyte-mediated natural cytotoxicity of chicken leukocytes were investigated. A significant, but transient augmentation of granulocyte cytotoxicity was observed 24 h after virus injection, followed by a relatively long period of its suppression. A good correlation was found between the augmented cytotoxicity and interferon induction. The interferon-inducing capacity of adenovirus type 6 and type 12 in vitro similarly ran parallel with their ability to stimulate granulocyte-mediated cytotoxicity. An adenovirus-induced elevation of cytotoxicity was not observed when IFN production was inhibited by pretreatment of the leukocytes with monoclonal antibody specific for bursal cells and monocytes. In addition, anti-IFN antibody abrogated the stimulation of cytotoxicity as well. During the in vitro experiments in which granulocyte-specific monoclonal antibody was applied, evidence was found that the effector cell activity is associated with the granulocytes. These results suggest that both the in vitro and the in vivo adenovirus-induced augmentation of granulocyte-mediated cytotoxicity is due to the IFN-inducing capacity of the virus. In chickens, the rapid augmentation of the granulocyte cytotoxicity may be important in the acute stage of infection, increasing the resistance to the virus in question and also to bacterial infections.