Inhibition of antigen and mitogen-induced lymphocyte transformation by fetal calf serum.

Use of fetal calf serum as a serum supplement in whole blood microcultures of human lymphocytes resulted in a significant suppression of in vitro stimulation with herpes simplex virus (type 1) antigen, purified protein derivative, and phytohemagglutinin. If the response to viral antigen is weak in autologous serum it may be completely missed if cultures are carried out in fetal calf serum.     

Inhibition of mitogen-induced lymphocyte transformation by local anesthetics.

Chlorpromazine (CPZ) and lidocaine were added to cultures of mouse spleen cells stimulated by concanvalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). Concentrations of CPZ greater than 5 x 10(-6)M and concentrations of lidocaine greater than 2 x 10(-3)M totally inhibited the mitogenic responses to all four mitogens. Minimal inhibitory concentrations of neither drug interferred with cell viability as determined by trypan blue uptake or 51Cr release. The effects were totally reversed by the removal of the drugs from the culture. Addition of the drug at intervals after mitogen exposure demonstrated that the inhibited event occurred relatively soon after exposure to the mitogen. For example, the addition of lidocaine or CPZ more than 24 hr after Con A stimulation had no effect on tritiated thymidine incorporation. Elevated concentrations of cyclic AMP, cyclic GMP (or their derivatives) or calciunown membrane active actions of these drugs and the rapid reversibility of the effect strongly support the idea that the local anesthetics act on the surface membrane of lymphocytes. Binding of radiolabeled Con A or LPS to lymphocyte membranes in the presence of lidocaine or CPZ was not inhibited. The possibility exists that CPZ and lidocaine disorganized cell membranes so as to interfere with the surface membrane elaboration or action of a second messenger, or interfere with cell-cell interactions.     

Effect of quinidine on mitogen-induced human lymphocyte transformation

The effect of quinidine on mitogen-induced lymphocyte transformation was examined using a microculture technique. The results showed that, at concentrations attainable during therapy, this drug exerted an inhibitory effect on lymphocyte proliferation. This inhibitory effect could be partially reversed by washing. The inhibitory effect was quite substantial when the drug was added 48 hours after the initiation of cultures. The clinical implications of this immunosuppressive property of quinidine are discussed.     

Rapid loss of sensitivity of mitogen-induced lymphocyte activation to inhibition by cyclosporin A

The ability of the immunosuppressive drug cyclosporin A (CS-A) to inhibit the activation of lymphocytes by phytohaemagglutinin (PHA) and concanavalin A (Con A) is progressively lost over the 8-hr period following mitogen addition. This process is dependent on the presence of Ca²⁺ in the culture medium and is complete at a time when activation still requires the continued presence of the mitogen. While inhibition by CS-A is reduced to some extent by lymphokines produced by mitogen-activated cultures, the initial loss of sensitivity to CS-A is too complete and too rapid to be accounted for in this way. We conclude that CS-A inhibits an early Ca²⁺-dependent step in mitogen-induced activation that is not in itself sufficient to commit the cells to initiate proliferation, but is required for later steps in the activation process, including lymphokine production.     

Suppression of mitogen-induced lymphocyte proliferation by syringomycin-E

Abstract Syringomycin E (SR-E) is low molecular weight bacterial lipodepsipeptide with antifungal properties. Owing to immunosuppressive activities of such compounds as cyclosporine, FK506 and rapamycin, we studied the effect of SR-E on proliferation of human blood lymphocytes in vitro. SR-E, by itself, had no effect but the mitogen-induced lymphocyte proliferation was significantly suppressed. The suppressive effect was more pronounced with pokeweed mitogen (PWM) as compared to phytohemagglutinin (PHA) or monoclonal antibody to CD3 (anti-CD3). Since these mitogens induce cellular immunity (T cell-dependent), SR-E may potentially be a novel immunosuppressive compound.     

Potentiation of mitogen-induced lymphocyte stimulation by polyethylene glycols

The effect of polyethylene glycols of different molecular weights on mitogen-induced lymphocyte stimulation was investigated. The stimulation obtained by neuraminidase/galactose oxidase treatment, and by addition of several plant lectin mitogens, was markedly enhanced in the presence of polyethylene glycols of molecular weights ranging from 6 000 to 40 000. Optimal potentiation of stimulation was found for polyethylene glycol concentrations up to about 5 %.     

Relation of 8-ketotrichothecene and zearalenone analog structure to inhibition of mitogen-induced human lymphocyte blastogenesis.

The 50% effective doses of fusarenon X, nivalenol, deoxynivalenol, and 15-acetyldeoxynivalenol required to reduce [3H]thymidine uptake in mitogen-stimulated human lymphocytes by 50% were 18, 72, 140, and 240 ng/ml, respectively. These results indicated that lymphotoxicity of 8-ketotrichothecenes decreased according to the C-4 substituent order acetyl greater than hydroxyl greater than hydrogen, whereas acetylation of position C-15 of deoxynivalenol caused a slight decrease in in vitro toxicity. The 50% effective doses for zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol were 3,500, 6,300, 36,000, 3,750, and 33,000 ng/ml, respectively, suggesting that a keto group or alpha-hydroxyl at the position C-6' contributed to the lymphotoxicity of the parent molecule. The inhibitory effects of zearalenone analogs observed in the blastogenesis assay did not correlate with the estrogenic potencies of these compounds. All 8-ketotrichothecenes and zearalenone analogs tested were capable of inhibiting B- and T-cell subsets stimulated by a mitogen panel of leukoagglutinin, concanavalin A, and pokeweed mitogen.     

Induction and inhibition of human lymphocyte transformation by the lectin from the red marine alga Amansia multifida

Lectins are powerful stimulants of quiescent peripheral blood lymphocytes. They can induce blast transformation leading to mitosis of these cells in vitro. We report here the dose-dependent proliferative curve for human peripheral blood monouclear cells (PBMC) stimulated by the lectin amansin, from Amansia multifida. Amansin stimulated proliferation of (PBMC) at relatively low concentrations (3.12 to 12.5 μg mL^-1). We observed also a gradual reduction in mitogenic capacity with progressive increase in the lectin concentration above 12.5 μg mL^-1. This decrease in the mitogenic potential did not result from a toxic effect on the cells, and was predominant at a lectin concentration above 50 μg mL^-1. This decrease in lymphocyte proliferation could be blocked by avidin and could not be overcome by IL-2 or another lectin (Con Br) at stimulatory concentrations. Additionally, we observed that cells incubated at stimulatory concentrations of amansin produced IFN-γ. Analysis of the culture supernatants established a direct correlation between the IFN-γ and the mitogenic and anti-mitogenic capacity of amansin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhancement of antigen- and mitogen-induced human T lymphocyte proliferation by tumor necrosis factor-alpha

The capacity of human recombinant tumor necrosis factor-alpha (rTNF alpha) to modulate human T cell proliferation was examined. To examine the effect of rTNF alpha on the responding T cell directly, T cell activation was studied in the absence of viable accessory cells (AC). Highly purified AC-depleted peripheral blood T4 or T8 cells were stimulated with immobilized monoclonal antibodies to the cluster of differentiation (CD)3 molecular complex, an AC-independent stimulus. rTNF alpha augmented anti-CD3-stimulated T4 and T8 cell proliferation. The capacity of rTNF alpha to enhance T cell proliferation varied inversely with the density of immobilized anti-CD3 and the number of responding cells in each culture. The capacity of rTNF alpha to enhance antigen-induced T4 cell proliferation was also examined. Antigen-bearing paraformaldehyde-fixed antigen-presenting cells induced modest T4 cell proliferation when cultured in flat-bottomed wells; this response was enhanced by rTNF alpha. The results demonstrate that rTNF alpha has direct effects on T cells, facilitating their capacity to proliferate in response to mitogens and antigens. These data indicate that rTNF alpha may play an immunoregulatory role, enhancing the proliferation of T lymphocytes.     

The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of adenosine deaminase deficiency

The biochemical mechanism of lymphocyte dysfunction with adenosine deaminase deficiency has been investigated using cultured phytohemagglutinin stimulated normal peripheral blood lymphocytes and the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin. The addition of deoxyadenosine to ADA-inhibited (but not to uninhibited) cells generated increased dATP pools (up to 50-fold greater than controls) and depressed the mitogen response. dATP Accumulation was accompanied by depletion of the other three deoxynucleoside triphosphate (dNTP) pools (dTTP, dCTP, and dGTP). Suppression of the mitogen response could be prevented ("reversed") to 90% of control levels by the addition of deoxynucleoside precursors for the depleted dNTPs at the initiation of mitogen stimulation. "Reversal" restored the dTTP and possibly the dGTP pools. Thus the mechanism of toxicity in this model appears to be inhibition of ribonucleotide reductase by massive accumulation of dATP, resulting in starvation for the other three deoxyribonucleoside triphosphates. "Reversibility" of this toxicity by providing sources for the missing three deoxynucleoside triphosphates argues for ribonucleotide reductase inhibition rather than other mechanisms of deoxyadenosine toxicity in this model.     

The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of purine-nucleoside phosphorylase deficiency

Purine-nucleoside phosphorylase (PNP) is a purine degradative enzyme that catalyzes the phosphorolysis of (deoxy) inosine or (deoxy) guanosine to their respective bases and (deoxy) ribose 1-phosphate. A severe T-cell immune deficiency syndrome with hypouricemia is associated with impaired PNP function. To study the biochemical basis for this syndrome we created an in vitro model of PNP deficiency in mitogen (phytohemagglutinin)-stimulated normal human peripheral blood lymphocytes using guanosine to competitively inhibit deoxyguanosine phosphorolysis. Guanosine-induced guanine toxicity was reversed by adenine. Under these conditions, deoxyguanosine (5-45 microM) diminished mitogen stimulation to 30% of control while increasing the deoxyguanosine triphosphate pool (dGTP) by over 20-fold. Deoxycytidine reversed deoxyguanosine toxicity with a diminution of dGTP accumulation, but no significant change in the deoxycytidine triphosphate pool. Thymidine reversed the deoxyguanosine toxicity, repleted the thymidine triphosphate (dTTP) pool, and caused an even further increase in the accumulation of dGTP. These data support a model of lymphotoxicity in PNP deficiency based on dGTP accumulation with inhibition of ribonucleotide reductase and depletion of the thymidine triphosphate pool. Thymidine triphosphate depletion is reversed by either deoxycytidine or thymidine; however, the former diminishes dGTP accumulation (probably by competition for phosphorylation) and the latter potentiates dGTP accumulation (probably through feedback augmentation of guanosine diphosphate (GDP) reduction by ribonucleotide reductase secondary to an increased dTTP pool).