Effects of amino and imino acridines on tumor necrosis factor production by human leukocytes.

Tumor necrosis factor (TNF) is a multifunctional cytokine with diverse effects on different cells and tissues. The biological activity of TNF is described on the basis of its cytotoxic action in vivo and in vitro. Different acridines were systematically synthesized and their effects were tested on endotoxin and Staphylococcus aureus-induced TNF production by human leukocytes. 9-aminobutylacridine and 9-ethylaminoacridine totally abrogated the TNF production of leucocytes at a concentration of 3.5 microM, whereas 9-imino -10-butylacridine and 9-imino-10-ethylacridine exerted only a 50% inhibition in the same concentration. Derivatives designated as 9-amino-(2-dimethylamino-ethyl)-acridine and 9-imino-10-(2-dimethylamino-ethyl)-acridine in a concentration of 7 microM exerted only a 30% and a 10% inhibition respectively. A significant modulation of TNF production was not observed when other alkylated derivatives in this series were applied. The TNF-mediated cytotoxic effect of monocytes against WEHI cells was also reduced by the most effective compounds. The acridines did not interfere with the expression of CD 14 molecules on monocytes. The exact mechanism of the suppression of TNF synthesis by acridines remains to be elucidated, but might be useful in the screening and evaluation of their anticancer properties and antimalarial effects.

The inhibitory effects of allopurinol on the production and cytotoxicity of tumor necrosis factor.

Allopurinol, a xanthine oxidase inhibitor, impaired the cytotoxic effect of human recombinant tumor necrosis factor (TNF) against WEHI cells. Actinomycin D abolished the inhibition of cytotoxicity by allopurinol. Allopurinol also exerted an inhibitory effect on the production of TNF by human mononuclear cells stimulated by either heat-killed Staphylococcus aureus or E. coli lipopolysaccharide. It is suggested that allopurinol inhibits TNF cytotoxicity by decreasing the level of oxygen free radicals generated (among other mechanisms) by the action of xanthine oxidase. Whatever the mechanism, the fact that allopurinol counteracts the toxicity of TNF can help towards an understanding of the complex nature of TNF toxicity.

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.

The inhibitory effect of interferon-alpha on the serotonin-induced impairment of human NK cell activity in whole blood.

Serotonin (5-HT) at a concentration of 10(-4) to 10(-10) M impaired the cytotoxicity of human natural killer cells in whole blood. 5-HT added at the onset of the short-term cytotoxic assay had a pronounced inhibitory effect. It is very likely that the 5-HT2 receptor is involved in the inhibition of cytotoxicity because ketanserin, an inhibitor of the 5-HT2 receptor, partially prevented the effect of 5-HT. Treatment with 10(2)-10(4) IU/ml of interferon-alpha before or after the application of 5-HT decreased its inhibitory effect on the cytotoxicity. Radioligand binding studies revealed that the antagonistic effect of interferon was not due to the competition for the 5-HT2 receptors. Cycloheximide, an inhibitor of protein synthesis, did not block the suppression of natural killer activity by 5-HT, but it exerted a blocking effect on the acquisition of resistance to 5-HT induced by interferon.

Inhibition of biological effects of endotoxins by phenothiazines.

The importance of change transfer reaction involving endotoxins of bacteria and interactions between endotoxin-induced tumor necrosis factor (TNF) were investigated both in vitro and in vivo in the presence of several phenothiazines. Complex formation between endotoxins and ring-substituted phenothiazines, benzodiazepines, amantadine and promethazine was measured using spectrophotometric methods. The endotoxin-induced hypotensive effect was prevented by phenothiazine pretreatment of the dogs; however, the endotoxin-phenothiazine complex had the same effect as the endotoxin itself. The tumor necrosis factor-inducing ability of endotoxin in human monocytes was prevented by promethazine. The TNF induction by endotoxin was inhibited by promethazine in vivo.

[Activity of natural killer cells and granulocytes in peripheral blood and separated cells]. / Természetes ölö sejtek és granulocyták aktivitásának vizsgálata perifériás vérben és szeparált sejtekkel.

The natural killing of K 562 cells by whole blood from normal subjects was comparable with that shown by separated mononuclear cells. In order to establish the conditions for a reliable natural killer assay by using very small numbers of effector cells in whole blood, the isotope uptake of target cells was increased by a modified labelling method, which permitted the use of fewer target cells in the assay. The natural cytotoxicity of whole blood was augmented by interferon to the same extent as observed with separated mononuclear cells. The chemiluminescence of granulocytes in whole blood comparable with that of separated granulocytes. Taken together, these methods are considerably less tedious than the conventional methods, technique is also economical, and the results may reflect in vivo cytolytic processes much better.

Tumor necrosis factor production by human granulocytes.

Human polymorphonuclear leukocytes kill WEHI 164 cells in an 18-hour 51Cr release assay. Antibody to human tumor necrosis factor (TNF) blocks the lysis of targets mediated by human granulocytes. Resting granulocytes produce an undetectable amount of TNF, if any. Granulocytes stimulated with Staphylococcus aureus release 250-500 U/ml TNF alpha. The specificity of the released TNF in the WEHI 164 cytotoxicity assay was confirmed by using neutralizing anti-TNF alpha monoclonal antibodies. The thymidine uptake of endothelial cells was inhibited by granulocyte-derived TNF. The identity of TNF alpha was further confirmed by molecular weight determination, by gel filtration on Sephacryl S-200, with a result of approximately 44,000. Besides their antimicrobial capacity, therefore, granulocytes may contribute to tumor rejection, inflammation and septic infections by releasing TNF.