An improved In Vitro assay for Lymphotoxin

Kramer, J. J.; Granger, Gale A.

doi:10.1016/0008-8749(72)90235-3

Cited by 13 publications

(4 citation statements)

References 6 publications

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“…These results may explain the negative reports of various authors who have attempted to find evidence of toxic factors active in in vitro cellular immune reactions (35,36). In the case of lymphoid cells obtained from immune animals activated ivz vitro with antigens, it has been calculated that a very small number, perhaps 2-5 % of the cells, actually respond to the antigen (37)) and from tests in our laboratory, we know that the amounts of LT released into the cell-free medium under these conditions is low and detectable only with sensitive test systems such as the microplate method (38). In situations in which cells are activated with strong activating agents, such as phytohemagglutinin, the activity in the undiluted medium can be detected with a wide variety of target cells, but even then could be missed with a relatively resistant indicator cell, such as the bovine kidney cell.…”

Section: Discussionmentioning

confidence: 99%

Lymphocyte in vitro cytotoxicity: Mechanism of human lymphotoxin-induced target cell destruction

Williams

Granger

1973

Cellular Immunology

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These in. vitro studies were conducted in an attempt to elucidate the mechanism of how cell-free supernatant fluids obtained from PHA-stimulated human lymphocytes cause destruction of cells. The undiluted supernatant fluids with high activity exerted a nonspecific cytotoxic effect on many different continuous cell lines. However, upon dilution, a wide spectrum of cell sensitivities was observed. These studies suggest human lymphotoxin acts by first absorbing to receptors on the target cell plasma membrane. The next effect is shut-down of cellular DNA synthesis, followed later by a decrease in cell numbers and finally, cellular destruction. Once sufficient LT has bound to the target cell surface, the cytopathic effect is irreversible. A role for LT in lymphocyte-mediated tissue destruction is discussed.

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Section: Discussionmentioning

confidence: 99%

Lymphocyte in vitro cytotoxicity: Mechanism of human lymphotoxin-induced target cell destruction

Williams

Granger

1973

Cellular Immunology

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“…It has been demonstrated that changing the levels of cyclic AMP and GMP in fibroblasts has an effect on the growth rate (2,10,17). This is not a factor involved in the cytotoxic assay since: (1) LT activity on target cells is independent of DNA synthesis (12,20); (2) target cell growth is inhibited in our assays by adding mitomycin C. The actual mechanism involved in the protection is not clear and needs further investigation. It could be because of affecting numbers of LT receptors on the cell surface or in some way modifying the cellular repair mechanism involved in resistance to cytolysis.…”

mentioning

confidence: 88%

The Effect of Agents Which Modulate Levels of the Cyclic Nucleotides on Human Lymphotoxin Secretion and Activity in Vitro

Prieur¹,

Granger²

1975

Transplantation

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The ability of concanavalin-A (Con-A) to activate lymphocytes to secrete human lymphotoxin (LT) was tested in the presence of agents known to modify the intracellular levels of cyclic nucleotides (cAMP and cGMP). Lymphocytes were treated with these agents at different stages of activation: (1) during the first encounter with mitogens, and (2) after they had been fully activated and were restimulated. Two agents, Dibutyryl cAMP and theophylline, dramatically inhibit the amount of LT secreted during both "primary" and "secondary" activation by Con-A. In contrast, DL-isoproterenol had a weak effect during primary activation, but greatly reduced the level of LT secreted during secondary activation. Agents which affect the intracellular level of cGMP were also tested. Imidazole had no effect on LT secretion by either primary or secondary Con-A activated cells. In contrast, carbamyl choline greatly reduced LT secretion to a level comparable to Dibutyryl cAMP and theophylline. All agents tested protected, to some degree, the sensitive a-L cell against LT-induced destruction in vitro. Agents which affect the levels of cyclic nucleotides affect both the effectiveness of the aggressor cell and the sensitivity of the target cells in vitro.Cyclic nucleotides have been shown to have an important role in the regulation of cellular biosynthesis and metabolism (19) in inflammation and immunity (1). They appear to be important in both immediate and delayed hypersensitivity reactions. Production and/or secretion of antibodies by B lymphocytes is reduced when intracellular levels of cyclic adenosine 3',5'-monophosphate are raised (16,23). Destruction of cells by immune lymphocytes activated by contact with target cell antigens, and in vitro measure of cell-mediated immunity, is suppressed when the effector lymphocytes are treated with pharmacologic drugs which increase the adenosine 3',5'-monophosphate (cAMP) level (14,21,22) . On the other

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“…Assays for TNF. TNF was assayed by several methods which are similar to those developed to study the actions of other cytotoxins, most notably lymphotoxin (11,13,21). In the morphological microassay, cultures of cells were established in 96-well flat-bottomed trays (3040; Falcon Plastics, Oxnard, Calif.) at 50,000 cells per well and incubated in a humidified atmosphere at 37°C with 5% C02, unless noted otherwise.…”

Section: Cells L-929 Is a Transformed Cell Line Originallymentioning

confidence: 99%

Rabbit tumor necrosis factor: mechanism of action

Ruff¹,

Gifford²

1981

Infect Immun

180

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Rabbit tumor necrosis factor (TNF) was examined for effects on normal and transformed cells in culture. Several assays for killing of L-929 cell targets were developed, and their sensitivities were compared. Normnal celLs were not killed by TNF, and the discrimination between normal and transformed cells was shown not to be due to a cell cycle-dependent mechanism. TNF killing of L-929 cells was delayed for 10 to 12 h and thereafter showed concentration and time-dependent increases in cytolysis. Actinomycin D or cycloheximide treatment of L-929 cells resulted in an enhancement of the rate of cell killing as well as a shortening of the preceding lag period. TNF killing of L-929 cells was temperature dependent; cells were considerably more resistant to lysis at 250C and showed enhanced killing at 39°C as compared to 370C controls. The slope of the dose curve showed less than single-hit kinetics. A model for cell killing whose general features incorporate both the specificity and catalytic properties of an enzymatic reaction is proposed for TNF action.

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An improved In Vitro assay for Lymphotoxin

Cited by 13 publications

References 6 publications

Lymphocyte in vitro cytotoxicity: Mechanism of human lymphotoxin-induced target cell destruction

Lymphocyte in vitro cytotoxicity: Mechanism of human lymphotoxin-induced target cell destruction

The Effect of Agents Which Modulate Levels of the Cyclic Nucleotides on Human Lymphotoxin Secretion and Activity in Vitro

Rabbit tumor necrosis factor: mechanism of action

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