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PubMed1989

Cytotoxicity mediated by tumor necrosis factor in variant subclones of the ME-180 cervical carcinoma line: modulation by specific inhibitors of DNA topoisomerase II.

The mechanism of tumor necrosis factor (TNF)-induced cytotoxicity has been investigated using two clonal variants of the ME-180 human cervical carcinoma cell line. The clonal lines were characterized with respect to their expression of TNF receptors, kinetics of cell death, and their ability to communicate intercellularly through gap junctions. The ME-180.4 and ME-180.8 clones were identified by their relative sensitivity to TNF induced lysis in a 24-h assay. The dose of TNF required to kill 50% of the target cells was 60 pM for the sensitive ME-180.4 and 2.5 nM for the ME-180.8. However, when assay times were extended, the dose response for both clones was the same, indicating that a difference in the kinetics of cell death and not absolute TNF sensitivity existed between the ME-180.4 and ME-180.8 clones. Both clones were gap junction deficient as judged by their inability to transfer Lucifer yellow or 6-carboxyfluorescein, a characteristic phenotype of cells sensitive to cytotoxicity by TNF. The level of surface receptor expressed on these clones was nearly identical with a Kd = 0.3 nM and 5,000 binding sites per cell. Measurement of the kinetics of cell death revealed that the time between the addition of TNF and the onset of observed cell death (induction phase) was much shorter for the ME-180.4 (32-55 h) than for the resistant ME-180.8 (55-80 h). Mitomycin C, a DNA alkylating agent, significantly reduced the length of the induction phase for both clones, although the kinetic difference between the clones remained unchanged. Two epipodophyllotoxins, VP-16 and VM-26, which specifically inhibit the rejoining activity of DNA topoisomerase II, showed a 10-100-fold synergistic effect when combined with TNF as shown by isobologram analysis. VM-26 when added to the resistant ME-180.8 clones decreased the length of induction phase and abolished the kinetic difference observed with the ME-180.4 clone. These results indicate that the variance in the TNF response of these two clones was closely associated with DNA topoisomerase II, and suggest that this enzyme may play an important role in TNF mediated cytotoxicity.

Cell Communication↗
PubMed1990

Mechanism of human lymphotoxin and tumor necrosis factor induced destruction of cells in vitro: phospholipase activation and deacylation of specific-membrane phospholipids.

The role of phospholipase (PLase) activation and lipid metabolism in lymphotoxin (LT)- and tumor necrosis factor (TNF)-mediated destruction of murine L929 cells was examined. At the levels of LT and TNF employed, cell destruction began at 4-6 h and was 99% complete by 30 h. Cell membrane phospholipids (PL), labelled in situ at the C2 position with 14C arachidonic acid, were analyzed by two-dimensional thin-layer chromatography and quantitated over a 30 h time course after LT or TNF treatment. The ratio of radiolabel incorporation relative to the actual amount of each PL present was determined by inorganic phosphate analysis. Radiolabelled arachidonic acid, eicosanoids, and neutral lipids were released into the medium prior to the onset of cell death (4-6 h) and continued to accumulate linearly throughout the destructive reaction. There was a quantitative relationship between the appearance of radiolabelled metabolites in the media and the loss of radiolabelled cellular PL. Cellular phosphatidylethanolamine was the primary PL deacylated by PLase action, showing a 75% reduction in radiolabel. The PLase inhibitors--quinacrine, hydrocortisone, dexamethasone, and indomethacin--were potent inhibitors of LT- and TNF-mediated cell destruction, suggesting that selective deacylation of specific membrane PL by PLase activation is an important step in the events that lead to LT- and TNF-mediated cellular destruction in vitro.

Animals↗
PubMed1990

Lymphotoxin induces secretion of granule proteins from adherent neutrophils: possible role of intracellular free calcium.

Lymphotoxin (LT) can activate human neutrophils. Using a hemolytic plaque assay to detect secretion of lactoferrin and myeloperoxidase (MPO) from single adherent neutrophils, we showed that LT induced secretion from both primary and secondary granules. Incubation of cells with cytochalasin B was required for MPO secretion, and it enhanced lactoferrin secretion. Pertussis toxin, which blocks a G-protein in the plasma membrane, inhibited LT-induced exocytosis of MPO, but not of lactoferrin. Incubation with LT did not induce any detectable changes of the cytoplasmic free [Ca2+] in neutrophils. On the other hand, secretion of granule proteins from adherent neutrophils in response to LT was blocked by loading neutrophils with quin-2 in order to increase the intracellular calcium buffering capacity. This was achieved at a concentration of quin-2, at which the secretion induced by the phorbol ester PMA and the chemotactic peptide FMLP was unaffected. Trifluoroperazine (TFP), a dual protein kinase C and calmodulin inhibitor, significantly inhibited the LT-mediated secretion of lactoferrin from adherent granulocytes. The PMA effect was unaltered by TFP under these conditions, suggesting that the inhibitory effect was on a calcium-calmodulin dependent step. The secretion induced by TNF and GM-CSF was also blocked by buffering changes in the intracellular [Ca2+] and inhibited to a similar extent by TFP. Our results suggest that calmodulin and minute changes in the cytoplasmic free [Ca2+] may be involved in a common signal transduction pathway engaged in activation of adherent neutrophils by several cytokines.

Calcium↗
PubMed1991

Effect of tumor necrosis factor on the generation of chlorinated oxidants by adherent human neutrophils.

Human neutrophils adherent to simulated biologic surfaces undergo significant activation of the respiratory burst over prolonged periods of time in response to stimulation with the cytokines tumor necrosis factor-alpha (TNF alpha) or tumor necrosis factor-beta (TNF beta) or with the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP). In this study, neutrophils were examined for their ability to generate the highly reactive and powerful oxidant hypochlorous acid (HOCl) and the longer-lived, less reactive endogenous nitrogen-chlorine (N-Cl) derivatives in response to these stimuli either alone or when exposed to recombinant human TNF alpha (rTNF alpha) or beta (rTNF beta) prior to addition of FMLP. Neutrophils adherent to fetal bovine serum-coated polystyrene tissue culture wells were able to generate only small quantities of HOCl when incubated with rTNF alpha, rTNF beta, or FMLP individually. However, when neutrophils were first incubated with either rTNF alpha or rTNF beta prior to addition of FMLP, there was a marked increase in HOCl generation. Neutrophils stimulated in such a manner consumed approximately 18% of the HOCl generated in the formation of N-Cl derivatives. Further scrutiny of the response to the combination of rTNF alpha and FMLP revealed that HOCl release was rapid, with 80% of total HOCl accumulation occurring within 15 min after FMLP addition. The amount of HOCl generated was dependent on the number of cells added and on the concentration of both rTNF alpha and FMLP. Comparison of HOCl generation with superoxide anion and myeloperoxidase release showed that the amount of HOCl generated was limited primarily by the amount of myeloperoxidase released rather than by the degree of respiratory burst activation. These results demonstrate that human neutrophils stimulated with FMLP after a brief incubation with rTNF alpha or rTNF beta can generate cytotoxic and microbicidal concentrations of chlorinated oxidants.

Cell Adhesion↗
PubMed1992

Type I interferons (IFN-alpha and -beta) suppress cytotoxin (tumor necrosis factor-alpha and lymphotoxin) production by mitogen-stimulated human peripheral blood mononuclear cell.

We studied the effect of the different types of interferons on the production of cytotoxin by human peripheral blood mononuclear cells (PBMCs) stimulated with the mitogen phytohemagglutinin (PHA). Maximum secreted levels of cytotoxin were observed at day 3 in culture and consisted of both tumor necrosis factor alpha (TNF-alpha) and lymphotoxin as determined by specific antibodies. Type I interferons (IFN-alpha and IFN-beta) consistently suppressed cytotoxin production. Both TNF-alpha and lymphotoxin were significantly suppressed. Mean suppression by IFN-alpha and IFN-beta (1000 U/ml) was 56 and 66%, respectively, in PBMCs from 18 different donors. The suppressive effects of IFN-alpha and IFN-beta on cytotoxin production were dose responsive over a range of 10 to 1000 U/ml. Type II interferon (IFN-gamma) did not have consistent significant effects. Pretreatment with IFN-alpha or IFN-beta for 24 or 48 h prior to PHA stimulation also resulted in significant suppression. Supplementation with interleukin-2 (10 U/ml) or IFN-gamma (1000 U/ml) did not overcome cytotoxin suppression by IFN-alpha or IFN-beta. Cytotoxin suppression by IFN-alpha and IFN-beta together appeared to be noninteractive. Suppression appeared not to be due to blockade of the cytotoxin release, since both cell-associated cytotoxin and secreted cytotoxin were suppressed to the same level. These results demonstrated that cytotoxin and lymphotoxin production by PHA-stimulated PBMCs could be down-regulated by type I interferons and that there is a substantial difference between the action of type I interferons and type II interferons (IFN-gamma) in modulating the biosynthesis of cytotoxins.

Cells, Cultured↗
PubMed1990

27 amino acid residues can be deleted from the N-terminus of human lymphotoxin without impairment of its cytotoxic activity.

In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N-terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LT delta 37N, in which 37 residues were absent from the N-terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LT delta 28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LT delta 27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.

Amino Acid Sequence↗
PubMed1980

In vitro lymphocyte activity: its relation to the stage of gastric cancer.

Lymphocyte activity of patients with gastric cancer and its relation to the stage of cancer were studied. PHA response of lymphocytes from patients with gastric cancer was decreased markedly even in early stage of cancer. The PHA-stimulated lympho-cytotoxicity was not impaired in all stages of the disease. Spontaneous lymphocyte-mediated cytotoxicity showed a tendency to decrease as the cancer stage advanced. The cytotoxic activity of the supernatants from lymphocytes cultured without PHA was significantly high in the advanced stage of gastric cancer.

Cytotoxicity, Immunologic↗
PubMed1991

Lymphotoxin in human middle ear cholesteatoma.

The progressive growth of epidermis appears to be a crucial factor in the pathogenesis of cholesteatoma. How this growth is encouraged is still not well understood. In the present study, lymphotoxin was found in human middle ear cholesteatoma tissues by the immunoperoxidase method using rabbit anti-human lymphotoxin immunoglobulin G (IgG). Lymphotoxin was localized in the epithelium and connective tissues of the cholesteatomas studied. The epithelium of normal external ear canal skin was lightly stained, but the stain was much weaker than that of the cholesteatomas. In vitro, recombinant human lymphotoxin stimulated proliferation, protein synthesis, and terminal differentiation of basal keratinocytes. These findings suggest that lymphotoxin may be one of the mediators involved in the development of cholesteatoma.

Animals↗
PubMed1975

The effect of a lymphotoxic factor from patients with multiple sclerosis on acute lymphosarcoma cell leukemia and acute lymphoblastic leukemia.

Eight patients with acute lymphosarcoma cell leukemia or acute lymphoblastic leukemia received infusions of plasma containing a lymphotoxic factor. The lymphotoxic factor induced a transient fall in the number of "blast" cell forms in the peripheral blood in six of the eight patients but did not produce sustained remissions. Previous investigations have demonstrated that this lymphotoxic factor, found in the plasma of patients with active multiple sclerosis, is of low molecular weight and does not have the properties normally associated with an antibody. This factor appears to selectively interfere with ribonucleic acid synthesis in the thymus-derived lymphocyte. Lymphotoxic factor activity was demonstrated in the serum of one patient during a remission in leukemia induced by a viral illness.

Adolescent↗
PubMed1989

Overview of biological response modifiers.

The immune system is regulated by a variety of lymphokines, monokines, and colony-stimulating factors. Most have now been cloned and it is clear that these cytokines have a broad range of overlapping immunological, inflammatory, and physiological properties. Some of these cytokines might favorably influence the biological therapy of cancer and they have been collectively referred to by clinicians as "biological response modifiers." This review summarizes current knowledge about the molecular biology and biological properties of most of the common cytokines and provides a brief survey of their therapeutic potential in patients with cancer.

Antineoplastic Agents↗
PubMed1993

Brain vessels near muscle autografts are sites for entry of isogeneic macrophages into brain.

An autograft of skeletal muscle on rat dorsal medulla is a permanent opening in the blood-brain barrier to solutes. Is the graft also a site for the entry of exogenous, isogeneic leukocytes? Five weeks after inserting the graft, peritoneal macrophages (M phi) from inbred Fischer rats were activated by phorbol myristate acetate, labeled with a fluorescent dye, and infused as a bolus of about 2 x 10(6) cells into the axillary artery of Fischer hosts. The cells circulated for 2 h. The brains were then fixed, frozen, and sectioned. Only when M phi had been activated and a muscle autograft inserted did appreciable numbers of M phi enter the medulla. Nonactivated M phi invaded the grafts but very few entered the brain at 2 h. In rats with gel foam grafts, only a few activated M phi invaded gel and brain. Before entering tissues, M phi must adhere to the lumenal face of vessels. Cell adhesion molecules, e.g., I-CAM-1 and its ligand adhesion molecule, leukocyte function antigen (LFA-1), are known to mediate adhesion. I-CAM-1, detected immunohistochemically, increased in graft vessels and in nearby brain vessels. The rise may have been mediated by cytokines, interleukin-6, and tumor necrosis factor-beta, found in the grafts. LFA-1, however, assayed by fluorescence-activated cell sorting, was on both activated and nonactivated, exogenous M phi. Thus, M phi-endothelial attachment may have involved other adhesion molecules, e.g., selectins. The autograft also induced major histocompatibility complex class I on microglia and classes I and II on brain vessels near the graft. These vessels, by expressing adhesion molecules, are entry routes into brain for activated, isogeneic leukocytes that can then migrate for a limited distance of 1-2 mm in an otherwise intact brain.

Animals↗
PubMed1984

Lymphotoxins - a multicomponent system of growth inhibitory and cell-lytic glycoproteins.

Activated lymphocytes from experimental animals and man can release materials, termed lymphotoxins , which cause growth inhibition and cell lysis in vitro. These molecules, from human lymphocytes, are glycoproteins which can be divided into five molecular weight classes. These forms are heterogeneous, for each MW class can be further subdivided into multiple charge subclasses. It is now clear certain MW classes are interrelated and form a system of cell toxins. The larger classes (greater than 140,000 d) are associated with nonclassical antigen-binding receptors (R), which can be of T cell origin. The smaller forms (less than 90,000 d) do not express R function and are derived from the larger forms, possibly by enzymatic action. Two MW classes, one receptor-associated and one non-receptor associated, have been purified to homogeneity and their peptide composition is being studied. Functional studies reveal the larger MW forms derived from alloimmune cell populations; can induce selective and nonselective destruction of cells in vitro. Antibodies which inhibit the in vitro cell lytic ability of various human LT forms can block different classes of human lymphocyte cell killing reactions in vitro.

Animals↗
PubMed1983

Immunoregulation by lymphokines: immune interferon and lymphotoxin induction of lymphokine activity in human peripheral blood leukocyte cultures.

Supernatants of mitogen-induced human PBL cultures contain several lymphokine activities. Analysis of kinetic studies has suggested a correlation in the appearance in culture supernatants of lymphotoxin (LT) and immune interferon (Im-IF). We investigated this interaction between LT and Im-IF in the serum-free human PBL culture system. Partially purified LT was derived from a normal cell line, RPMI 1788. This LT, which had no contaminating IF activity, induced production of significant levels of IF activity as early as 15 hours after initiation of culture. This induced IF activity was identified as Im-IF by criteria of pH 2, SDS and heat sensitivity, as well as neutralization by AB to Im-IF. LT activity, in excess of the units added to the culture, was also measured in the LT-induced cultures. In another group of experiments, partially purified human Im-IF, produced by expression of a cloned gene in monkey COS-7 cells, was added to human PBL cultures. This Im-IF, at concentrations of 160 and 320 units/ml, caused induction of LT activity. The induced LT activity was neutralized by AB raised to 1788-derived LT. Im-IF treated cultures also contained IF activity in excess of the titers added to the cultures. These results would suggest an interaction between LT and Im-IF at the level of production by lymphoid cells.

Adjuvants, Immunologic↗